IOBC/WPRS

Working Group

"Fruit Flies of Economic Importance"

OILB/SROP

Groupe de Travail

"Mouches des Fruits d'lmportance Economique"

and

lnstituto de lnvestiga�lo Cientifica Tropical

II International Open Meeting

at/ a

Lisbon, Portugal 22 -24 September 1997

Edited by J. Piedade-Guerreiro

IOBC / wprs Bulletin Bulletin OILB / srop Vol. 20(8)1997 The IOBC/WPRS Bulletin is published by the International Organization for Biological and Integrated Control of Noxious Animals and Plants, West Palaearctic Regional Section (IOBC/WPRS)

Le Bulletin OILB/SROP est publie par !'Organisation lntemationale de Lutte Biologique et lntegree contra les Animaux et les Plantes Nuisibles, section Regionale Quest Palearctique (OILB/SROP)

Copyright IOBC/WPRS 1997

Address General Secretariat: !NRA - Centre de Recherches de Dijon Laboratoire de Recherches sur la Flore Pathogene dans le sol 17, Rue Sully - BV 1540 F-21034 DIJON CEDEX France

ISBN 92-9067-093-2 6'II INTERNATIONAL OPEN MEETING" "WORKING GROUP "FRUIT FLY OF ECONOMIC IMPORTANCE" Reunion Pleniere du Groupe de Travail "Mooches des Fruits d'lmportance Economique"

� -·-·-"·CIMIAM Tropical �

INSTITUTO DE INVESTIGA<;AOCIENTIFICA TROPICAL

ORGANISAT. INTERNAT. DE LUTTE BIOLOGIQUE ET INTEGREE SECTION REGIONALE OUEST PALEARCTIQUE OILB- SROP

22, 23 e 24 de Setembro de 1997 Lisboa - Portugal - i -

Foreword

The enormous spreading and the increasing losses due to fruit flies of economic importance all over the world and particularly in the Mediterranean Region, lead the InternationalOrganization for Biological Control/ West Paleartic Regional section to mobilize it's means for the implementation of actions that enable the controlof these pests. In this context the IOBC/WPRS Working Group "Fruit Flies ofEconomic Importance" organized the "II International Open Meeting", under the high patronages of the Instituto de Investigayao CientificaTropical (IICT) and the mentioned InternationalOrganization. This meeting usually takes place every fouryears and has forpurpose the: - Establishment of contacts and exchange of knowledge between specialists in this area of science and also to enhance effortsfor a wider cooperation among the differentsgroups. - Evaluation of the progress made in the improvement of the control techniques and identificationof new areas forfuture programmes involving laboratories in different countries, aiming forthe setting ofan effectiveprotection without negative consequences not only forman but also the environment. - We hope that the friendly spirit and enthusiasm that animated that "II International Open Meeting" in wich participated more than forty scientists of eleven countries, that presented twenty six communications, maybe materialized in concrete and effectivecontrol of one of the great nuisances ofagriculture: Fruit flies. Finally we must not forget the cooperation and support not only of the IOBC/WPRS, namely of it's President Dr. D. Royle and the SecretaryGeneral Dr. S. Poitout, but also ofthe IICT Presidence, especially of the Vice-President Prof.A. Reffega,whose confidence, support and incentive greatly contributedto the success ofthis Meeting. Finally we must not forget the cooperation of the Vice-President Dr. Inacio Guerreiro, who presided to the open Session, representing the President ofIICT who was absent in an official mission. - ii - ORGANIZING COMMITTEE

- J. Piedade-Guerreiro, Convenor W.G. - IICT / Portugal - Carlos Matias - ENFVN - Portugal - Afellah Mohamed - Morocco - Pedro Ros - INIA - Spain -Dhouibi M. Habib - INA T - Tunis - Salah Oukil - SRPV - Algeria - M. Isabel Guardado - IICT/ Portugal - Catarina Alves IHMT- Portugal - Claudia R. C. Pereira - Portugal

SESSION'S STRUCTURE Chairman Secretaries

Opening Session I. Guerreiro J. Piedade-Guerreiro Ecological Aspects and Controlof Fruit Flies C. Matias S. Quilici/ A. Del Pino Populations

Biotechnical Aspects of Management of Fruit Flies P.Ros M. Mui!.is/J.P.Coutinho Populations

Genetic Aspects and Microrganisms of Fruit R. Mitani S. Oukil/ D.Ochando Flies Populations

Integrated Control Programme A.M. Wakid M. Affelah/ M.H.Dhouibi

ORGANIZING SECRETARIAT

M. Isabel Guardado Instituto de Investiga,;:aoCientifica Tropical Centrode Zoologia Rua da Junqueira 14 - 1300 Lisboa - Portugal Fax: 351 - 1 - 362 26 26 - iii -

TABLE OF CONTENTS

Foreword ......

OPENING SESSION

Welcome address by Dr.Inacio Guerreiro, Vice-President oflICT ...... vii

Welcome address by Prof. RicardoMilani - Univ.Pavia ...... •...... ix

Welcome address by Dr. Afellah Mohamed,Conseil d'OILB/SROP ...... x

Welcome address by Doutor J.Piedade-Guerreiro ,Convenor ofthe Working Group ...... xi

SESSION 1 ECOLOGICAL ASPECTS AND BIOLOGICAL CONTROL OF FRUIT FLIES POPULATIONS ( Carlos Matias - Serge Quilici - A. Del Pino)

Effectof variable exposition to threshold temperature on egg development ofCeratitis capitata (Wiedemann) ...... M.T.G. Putruele;A. Garrido; A. A. Del Pino

Influence de la temperature sur le developpement preimaginal de Psyttaliajletcheri silvestri (Hymenoptera: Braconidae) parasitoide de Bactrocera cucurbitae Coquillett (Diptera: Tephritidae) ...... 7 B. Hurtrel;S. Quilici

Modeliza�aoda evolu�ao da mosca-da-azeitona Bactrocera oleae (Gmelin) pelo metodo do somat6rio de temperaturas, para a regiao de Castelo Branco,Portugal ...... 14 J. Coutinho; M. Sequeira; C. Veiga

Field population survey of fruitflies (Diptera, Tephritidae) by use of attractive trapsin citrus orchardsin Piracicaba, State of Sao Paulo, Brazil ...... 21 M. C.R. Dias; V. Arthur

Recherches sur la multiplication de Ceratitis Capitata(Wied. ) dans la foretd'Arganier au Maroc et sur Jes potentialites de son developpement experimental sur deux hotes de relais ...... 28 K. Naamani;C. Herbaut; A. Ait El Koch; A. Bakri; M. Chemsseddine - iv-

Effect of soil moisture and texture on pupal survival of the Mediterranean fruit fly,Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) ...... 38 A. A. DelPino; M. T. G. Putruele; A. Garrido

Relationship between the photoperiod and the presence of mature eggs in Bactrocera oleae (Gmel.) (Diptera: Tephritidae) ...... 46 A. Raspi; A. Canale; A. Felicioli

Developmental rate and number of generation estimates for Ceratitis capitata (Wiedemann)in fruitgrowing regions of California ...... 55 M. Mufliz; F. G. Zalom

Strategiede lutte optimisee selon Jes criteres bio-ecologiques de Bactrocera oleae (Dipt., Tephritidae) dans la region de Tlemcen (Quest Algerien) ...... 67 Gaouar Nassira

Repartition de !'infestationde la mouche de !'olive Bactrocera oleae (Gmel.) (Dipt.,Teph.) en verger oleicole dans le Sais au Maroc ...... 76 Afellah, M.; Ben Hamadi, I.; Smaili, C.; Hila!, A.; Chemseddine,M

SESSION 2 ASPECTS BIOTECHNIQUESDE LA GESTION DES POPULATIONS DES MOOCHES DES FRUITS ( Pedro Ros - Mariano Munis - Pereira_ Coutinho )

Application of the Cobalt-60 gamma radiation at greatvolumes of fruit flies Ceratitiscapitata (Wied.) pupae ...... 87 Piedade-Guerreiro,J.; Gomes Da Silva, J.; Guardado,M.I.; Alves, C.

Evolution de l'attaque des fruitsd'ete parla mouche des fruitsCeratitis capitata (Wied.) en fonctionde la succession des plantes hotes ...... 92 H. Fellah ; M. H. Dhouibi

A general view of recent advances on attractantsand trapsagainst Ceratitis capitata (Wied.) (Diptera: Tephritidae) ...... 102 J.P. Ros

Rela\:aoentre as capturas de adultos de Bactrocera oleae (Gmelin) em diferentessistemas de armadilhas e a infesta�aodos frutos...... 113 M. I.Patanita; M. Cardoso; A. Mexia

Utilization of low cost media for rearing the Mediterraneanfruit fly ...... 122 A. G. Manoukas; E. N. Zografou

Monitoriza\:ao da mosca-da-azeitona Bactrocera oleae na regiao de Castelo Branco, Portugal ...... 128 J. Coutinho; M. Sequeira; C. Veiga -v-

Capture des femellesde la mouche Mediterraneenne des fruits, Ceratitis capitata, dans les agrumes:evaluation de nouveaux pieges et appats ...... 137 A. Bakri; H. Hadis

Capturas de adultos de Bactrocera oleae (Gmel.) mediante annadilhas cromotr6picase ferorm6nicaspara a previsao da infesta9ao ...... 145 C. Pucci; A. F. Spanedda; S. Speranza; F. Lipizzi

Utiliz.ation de la pheromone de marquage dansla Jutte contrela mouche des fruits,Ceratitis capita/a (Wied.), (Diptera, Tephritidae) ...... 156 M. H. Dhouibi; H. Fellah

Eficaciade dos insecticidas en aplicaci6n aerea contrala mosca de! MediterraneoCeratitis capitata (Wied.) em citricos .Alicante (Espana) 168 Llorens Climent, Jose M.; M. Gilabert Artigues

SESSION 3 ASPECTS GENETIQUES ET MiCRORGANISMES SUR LES POPULATIONS DES MOUCHES DES FRUITS (R. Milani - Salah Ouk.ii- Dolores Ochando)

Fitnessof mitochondrial DNAhaplotypes in Ceratitis capitata ...... 175 A. Reyes; M. D. Ochando

Recent advances in Ceratitis capitata genetics.Research programsdeveloped at the department of animal biologyof the University of Pavia ...... 186 R. Milani

Action d'une dose substerilisante des irradiationsaux rayons gamma sur la descendance de Ceratitis capitata (Wiedemann) (Diptera, Trypetidae) ...... 193 S. Oulcil; R. Causse

Etude de la variabilite genetique chez 6 populations de Ceratitis capitata dans differenteszones geographiquesdu Bassin Mediterraneen ...... 203 S. Oulcil;R. Bues; R. Causse ; J. F. Toubon

SESSION 4 INTEGRATEDCONTROLPROGRA1\1ME (A. Wak.id- M.Afellah - H. Dhouibi)

Madeira-med, SIT programagainst medflyin Madeira, Portugal ...... 211 R. Pereira; A. Barbosa; A. Brazao; N. Silva; J. Pacheco; A. Rodrigues; J. Caldeira; L. Dantas; J.P. Carvalho; D. Lindquist

Controlof Mediterranean fruitfly with Vienna-43/44 � sterile males in Tunisian oases ...... 219 J.P. Cayo!; E. J. Buyckx; F. Loussaief; M. Zarai; M. Boukhari; T. Arfaoui -Vi -

Effects of two modem pesticides:·Azadirachtin and Tebufenozide on the parasitoid Opius concolor (Szepligeti) ...... 233 M. Gonzalez; E. Villuela

List of participants ...... 241

Index of authors ...... 245 OPENING SESSION

Chairman: Inacio J. Guerreiro Secretary: J. Piedade-Guerreiro - vii -

Welcome address Inacio Guerreiro Vice-Presidente do Instituto de Investiga9ao Cientifica Tropical

Na qualidade de Vice-Presidente do Instituto de Investiga9ao Cientifica Tropical, e por motivo da ausencia do Senhor Presidente, Prof. Doutor Joaquim Alberto da Cruz e Silva, que se desloca a Republica de Cabo Verde em missao oficial, cumpre-me o honroso ensejo de dirijir aos ilustres congressistas aqui presentes umas breves palavras, o que fac;ocorn muita satisfa<;ao. Anfitriao e promotor desta Reuniao, motivo de grande honra e muita satisfac;ao poder dirigir estas breves palavras a tao ilustres congressistas aquando da abertura solene deste "Il InternationalOpen Meeting". Como julgo ser do conhecimento de V. Exas este e o segundo a ter lugar em Portugal ligado ao Grupo de Trabalho "Mouches des Fruits d'Importance Economique" da Organiw;:ao Internacional de Luta Biol6gica e Integrada - Sec9ao Regional Oeste Palearctica, o qua!, obviamente, constitui para este Instituto e para este Pais motivo de fundamentadoe saudavel orgulho. Na verdade, a concretizac;ao desta Reuniao muito dignifica a Comunidade Cientifica Nacional e Internacional e particularmente os especialistas em Luta Biol6gica, a qua! se vem afirmando cada vez mais como um instrumento poderoso na luta contra pragas das culturas. Naturalmente que nao me quero alongar nestas minhas breves palavras. Todavia nao posso deixar de tecer algumas considera95es, obviamente sucintas, acerca das moscas da fruta de importancia econ6mica. Estas sao fundamentalmente consideradas como uma das grandes pragas das fruteiras a nivel mundial, nomeadamente na bacia mediterranica onde a sua ac9ao se reflecte de modo muito nefastoprovocando anualmente elevados prejuizos. Os temas que irao ser debatidos neste Encontro Internacional ternpor fim fazer o ponto da situa9ao dos conhecimentos actuais, e encontrar novas orienta96es no combate as moscas da frutade importancia econ6mica. Desta fonna,pretende-se estabelecer, nao somente estrategias convenientes para o seu combate, mas tambem definir novas areas de investiga9ao ligadas ao campo da luta biol6gica e da protec9ao integrada. Gostaria ainda de referir muito sucintamente, que foi a partir da decada de cinquenta que na Europa a luta contra insectos nocivos se come9ou a orientar no sentido da luta biol6gica e integrada, principalmente corn o apoio dado pela Organiza9ao Internacional de Luta Biol6gica e Integrada, fundada em 1956, tendo o Instituto, ao qua! tenho a honra de presidir, sido um dos seus Membros Fundadores. - viii -

Foi com o incentivo dado por esta Instituic;ao que o IICT emprendeu na decada de cinquenta as primeiras ac96es de luta biol6gica realizadas no continente africano, numa altura em que grande parte dos especialistas mantinha uma atitude bastante passiva ou mesmo de inaceitavel cepticismo em relayao a estas materias. As campanhas de luta biol6gica, as primeiras realizadas em Africa com grande sucesso, revelaram bem a necessidade de se reverem os metodos de aplica9ao empirica de insecticidas orientando a luta no sentido de uma protecc;ao integrada, e onde o estudo da entomofauna das biocenoses relativamente aos insectos nocivos e dos seus parasitas e predadores devera ter um papel decisivo. E neste contexto que a Sec9ao Regional Oeste Palearctica tern desenvolvido uma ac9ao importante de coordenac;ao no campo da investiga9ao e das tecnicas de combate, atraves de diferentes Grupos de Trabalho os quais tern contribuido, de forma muito not6ria, para o desenvolvimento de uma agriculturta sustentada, capaz de preservar a nossa qualidade de vida contribuindo para uma gestao racional dos precarios ecossistemas naturais e antr6picos. Foi nesta 6ptica que se formou em 1978 o Grupo de Trabalho "Mouches des Fruits d'Importance Economique" o qua! teve como primeiro Responsavel o Dr. E. Boller da Sui9a, sucedendo-lhe o Prof R. Cavalloro e o Prof G. Delrio, ambos de Italia. Presentemente, e desde ha sete anos, a responsabilidade do Grupo cabe a um investigador do Instituto de Investigac;ao Cientifica Tropical, Doutor Piedade­ Guerreiro, o que muito me apraz registar. Estou certo de que todos n6s estamos cientes da grande importancia desta Reuniao respeitante a uma area do conhecimento cientifico que embora restrita e no entanto de enorme alcance do ponto de vista econ6mico e cuja orienta9ao sera decisiva para a protecc;ao das fruteiras dentro da area de influencia da Sec9ao Regional Oeste Palearctica. Nao tenho duvida de que este "Il International Open Meeting" sera enriquecedor em termos cientificos e humanos, tendo em atenc;ao a elevada e bem conhecida especializac;ao dos seus participantes pertencentes a doze paises que passo a referir por ordem alfabetica: Argelia, Brasil Egipto, Espanha, Fran9a, Grecia, Italia, Libia, Marrocos, Portugal, Suic;ae Tunisia. Termino minhas Senhoras e meus Senhores desejando a esta vasta pleiade de Congressistas cuja presen9a muito nos honra um trabalho proficuo e uma boa estada entre n6s e deixando uma palavra de estimulo ·e de reconhecimento a todos aqueles que deram o melhor do seu esfor90 na prepara9ao deste "II International Open Meeting" que nao tenho duvidas constituira um enorme exito cientifico. - ix -

Welcome address Riccardo Milani ProfessorEmeritus, University of Pavia Italy

Professor Piedade-Guerreiro, Members of the Organizing Committee, Authorities, Ladies and Gentlemen,

It is a great honour for me both to thank you for having organized the present meeting, and to welcome all the participants, so highly qualified, from so many countries. Your countries represent a great part of the world, and reflect the dimension of the problems which need a solution. I take great pleasure from being with you all again, after a gap of some years. But I greatly miss Professor Raffaele Cavallaro, who played such a leading part in the recognition of the socioeconomical importance of agricultural pest insects, and of the need to develop control methods that are compatible both with the development of agriculture and with respect for the environment; our understanding of need for international open coordination is especially indebted to Professor Cavallaro. His scientific contributions to both basic and applied research are quite familiar to the participants at the present meeting, and do not need enunciation here. But I can't refrain from recalling the great humanity of Professor Caval!oro, his enheartening optismism towards people and problems, and his entertaining colloquial capacity to find common interests with other people, interests that touched all sides of art, culture and life. In my opinion, one of his greatest attributes was his unswerving amenability to cooperation, his search for common interests and for levels of interaction between apparently remote fields of activity. And now let us welcome Professor Cavalloro among us again, in the form of a passage of the inaugural adress which he read as President of the CEC/IOBC International Symposium, ten years ago in Rome: "Fruit flies play an important economic role in the world: the very great damage which they cause to fruits and vegetable crops requires vigilant and constant attention and close collaboration between reserchers, with frequent exchange of information to gain ever greater knowledge of the various species, of their behaviour and mode of action and of ways of controlling them. As far as this last point is concerned, recurse to methods and means which disturb natural equilibria in agroecosystems as little as possible, respecting the environment and the communities living in it, is now undoubtedly necessary. We must set about the work of our Symposium with this task in mind". In the intervening years a good many steps forward have been accomplished. We are now here to evaluate the progress made, and to look ahead. I earnestly hope that the present meeting meets the expectation of each participant, and stands comparison with the successes of all the previous ones. Thank you. -x-

Welcome address Afellah Mohamed Membre du Conseil d'OILB/SROP Responsable de liaison entre le Conseil et le Groupe Mouches des Fruits

Monsieur le representant des autorites locales, Monsieur le President de l'lnstitut, honorable assistance, chers collegues. Au nom du President de l'OILB/SROP, je tiens a souhaiter la bien venue a tout le monde et bon succes a nos travaux. Je remercie egalement les collegues qui se sont deplaces de loin notamment du Bresil et des Etats-Unis. Le Groupe "Mouches des Fruits d'lmportance Economique" de l'OILB/SROP se reunit aujourd'hui pour son "11 International Open Meeting" pour evaluer l'etat d'avancement des recherches et tracer un plan de travail pour l'avenir. Le dynamisme et la reussite du groupe passent imperativement par la collaboration de tous, la concertation et la coordination avec d'autres organisations travaillant dans le meme domaine, notamment l'AIEA et la FAO. Je termine mon allocution en rendant un grand hommage au Responsable du Groupe Doutor l Piedade-Guerreiro pour les efforts deployes durant ces huit annees de coordination et pour )'organisation de ce Meeting. Sa responsabilite arrive a son terme aujourd'hui et ii va certainement nous manquer car ii a toujours ete l'ami de tout le monde. Encore bien venue a tous et merci - xi -

Welcome address J. Piedade-Guerreiro Convenor of the Working Group

For statutory reasons the Chairman of the Working Group "Fruit Flies of Economic Importance" of the IOBC/WPRS has the competence of organizing every three or four years a plenary Reunion that congregate all the experts involved in the fruit flies of economic importance problematic. We have the joy of having this event taking place for the second time in Portugal and we hope that "it occurs with enthusiasm allowing a close and fraternal collaboration between all the experts present here to whom we wish a pleasant stay. We also wish to express our gratitude to those that made this "II International Open Meeting" possible: To the Instituto de Investigac,:ao Cientifica Tropical Presidence and to the International Organisation for Biological Control our recognition for the facilities given. To the Sintra Town-Hall and the Forest General Board we testifyour regard for the support rendered. To the World Exposition - Expo-98 we thankfully acknowledge not only for receiving the congressmen in the intervention area but also for providing the Secretariat material that greatly contributed for the Meeting's success. And also a finalword of thanks for those that anonymously collaborated in the organizing and implementation of this Meeting. We hope that this "II InternationalOpen Meeting" may contribute efficaciously towards an adequate fight against the fruit flies of economic importance in it's zone of influence- West Palearctic Region. Session 1 ECOLOGICAL ASPECTS AND BIOLOGICAL CONTROL OF FRUIT FLIES POPULATIONS

Chairman: Carlos Matias Secretaries: Serge Quilici A. Del Pino - 1 -

EFFECT OF VARIABLE EXPOSITION TO THRESHOLD TEMPERATURE ON EGG DEVELOPMENT OF Ceratiti.� capitata (Wiedemann)

1 2 2 M.T.G. Putruele , A. Garrido y A. A. Del Pino • 11nstituto Nacional de Tecnologia Agropecuaria (!NTA) Estacion Experimental Agropecuaria Concordia CC 1-l (}200) Concordia. ARGENTINA 'lnstilllto Valenciano de lnYestigaciones Agrarias (!VlA) Dcpartamcnto de Protecci6n Vegetal \' Biotecnologia -!6113 Moncada (Valencia. ESPANA)

Summary

Development and survival of Ceratitis capita/a (Wiedemann) eggs were studied at threshold temperatures determined previously in laboratory. When eggs were exposed to lower threshold temperature (9°C) during 7 days and then transferred to 26°C, survival rate was close to 50%. One hundred percent mortality was reached after I 7 � to 18- days of exposition When survival at upper threshold (39°C) was studied, 32 hours killed nearly 50% of eggs and I 00% mortality occurred after 48- to 56- hours at 39°C. Thus, ( '. capirala eggs can survive during a variable period of time. to continuous exposition at threshold temperatures, which corroborate the high adaptation of this insect to unfavorable conditions. Related words: Cerati1is rnpilala, medtly, egg, temperature, threshold.

I - Introduction

The most important abiotic factor influencing the developing time in most insects is temperature. As a result, numerous studies have been undertaken to determine the relationship between temperature and survival of different life stages of C. capita/a. (Bodenheimer, 1951; Messenger & Flitters, 1958; Shrouky & Hafez, 1979; Muniz & Gil, 1984; Del Rio et al, 1986; Crovetti et al, 1986; Laborda et al, 1990; Conti, 1990; Putruele et al, 1996)

Survival percentage per unit of time or biological stage may vary with temperatures in the interval that allows complete pest development and the continuity of its generations

The temperature outside of this interval of normal development is a temperature threshold associated to a higher mortality rate forone or more stages of the life cycle.

It has been demonstrated that exposition to threshold temperatures (zero development) or lower, can be tolerated for a brief period of time. The more this temperature is separated from the developing threshold; the shorter this period will be.

In this study the times of maximum exposition of C. C'apitata eggs to threshold temperatures (9 and 39°C) were identified, as well as, its prediction with mathematical models. - 2.

2 - Materials and Method

Eggs were obtained from a continuous culture maintained in the Entomological Laboratory of the IVIA (lnstituto Valenciana de Investigaciones Agrarias), as described by Arroyo et al. ( l 967) and Albajes & Santiago Alvarez ( l 980) A know number of eggs of C. r.:apilala, 3 to 5 hours old were placed with a fine brush on a solid layer of agar in distilled waken (I%. 3 mm thick) in Petri dishes

Experiments were carried out under the temperature thresholds previously established (Putruele, M et al. 1996) of 9 ± 0.5°C and 39 ± 0 5°(, during a determined period of time. Afterwards eggs were placed under the optimum growth temperature (26 ± 0.8°() measuring the percentage of maximum hatching. The photoperiod used was 12. l 2 h (LD) and a humidity higher than 90% R.H. Three assays with 4 - 15 replicates of I00 eggs each were done for each temperature

The number of hatched eggs for the lower temperature was recorded every 24 hours, and every 8 hours for the maximum temperature until no more births took place. Eggs were considered hatched when the first instar had abandoned the chorion. Some larvae were capable to rupture the chorion but not to emerge from it and were considered dead. The experiment was finished when no hatching was recorded during one week.

3 - Results

Table l.. Effect of variable exposition to threshold temperature (9/26°() on egg deve l opment ofC .. capilata. Number of eggs Exposition time at Number of eggs Survival 9"C (days) hatched rate(%) 2000 5 1535 76.75 1898 6 1140 60.06 1925 7 930 48.31 2036 8 835 41.0 I 2030 9 684 33.69 1980 10 400 20.20 2120 11 318 15.00 2513 12 230 9.15 2780 13 116 4.17 2562 14 97 3.78 2917 15 82 2.81 3018 16 67 2.22 2560 17 14 0.54 2500 18 0 0 00 - 3 -

It was observed that as exposition periods increased hatching was reduced. One hundred percent mortality was reached after 18 d�ys of exposition when the possibility of recuperation is irreversible.

The relation between the hatching percentage and the number of days at the minimum threshold temperature (9°C) was adjusted to different mathematical models, obtaining a correlation coefficientofR 2 = 0.99, with the functiony = 0.61*Days 2 - 19.55 * Days+ 157.29 (Fig. l ).

In Table 2 are shown the results obtained with variable exposition periods between the maximum threshold (39°C) and a temperature of optimum growth (26 ± 0. 8°C) determined by checking every eight hours until I 00% mortality was reached

Table 2: Effect of variable exposition to threshold temperature (39/26°C) on egg d eve I ooment o f ('. c:ap,tat a. Number of eggs Exposition time at Number of egg Survival 39"C (hours) hatched ,ate(%) 2180 8 1680 7706 2070 16 1350 65.21 2870 24 1050 57.69 1120 32 482 43.00 3940 40 900 29.60 4410 48 250 5.66 4118 56 0 0 00

After 32 hours exposition at 39°C, the survival rate was lower than 50% and l 00% mortality occurred after 56 hours at 3 9°C. Eggs submitted to this extreme temperature resist long exposition times, after 8 hours survival rate was 77 06% This demonstrates the importance of temperature variation on egg development, since an excellent recovery was obtained when placed at 26°C.

In Figure 2 the percentage of eggs hatched is related to the hours the eggs wer� submitted to the maximum threshold and adjusted to a polynomic regression (y = 86.85 1.11 *Hours - 0 008*Hours2; R 2 = 0 98)

4 - Discussion

Egg development of C .c:apitata, when exposed to extreme temperatures, is very different from that of other T ephritidae. The survival interval for eggs of Darns oleae (GmeL) is between 9 y 32°C. For C. capitata the lower threshold is the same (9°C), but the upper threshold is 7°C higher, reaching 39°C

Besides this behavior it must be taken into consideration that 18 days of continuous exposition at 9°C were needed to inhibit egg hatching and 56 h at 39°C are necessary to obtain I 00% mortality -4-

---·--····------· ------·------Figure l. Egg development ofC. capitata. Surnvalrate at variable temperatures (9°-26°C). 100�----,------,-----,----,--,------,---, 90 -----·---·--·...... -+-- --j 80 y = 0.61x2 -19.55x + 157.29 R2 = 0.99 "' 70 60 _(_ 50 - ;;; - � ·� 40 30 ,_ 20 10 -+-+- 0 4 6 7 8 9 10 11 12 13 14 15 16 17 18 19 I Exposition at 9 •c (days)

1-- ·-··---·------, 1 Figure 2. Egg development of C.cap/tata. Survival rate at variable temperatures (39° - 26 °C). 100 T------,------, ! 90 I 80 "1--��---,-----+-----+------+--Y =:.0.008x2 - 1.11x + 86.85 2 70 R = 0.98 l 60 ..i 50 40 30 i I ] 20 10 + '.---+-----"""'"-'-+------1 0 ---+----+------4 12 20 28 36 44 52 60 Exposlllon al 39 •c (hours) - 5 -

Acknowledgments

We thank at M.J Verdu (IVIA) for providing access to chambers and other facilitiesduring the course of this project, E. Carbonell (IVIA) forhis assistance and MI PLATA (TNTA) for her review of the manuscript..

References

ALBAJES, R, and S ALVAREZ, C. 1980 Influencia de la temperatura en el r desarrollo de ( 'erc,/ilis rnpila/a(Wied ). An !NIA Ser. Ag ic. 13 183-190. ARROYO, M, MELLADO, L, GIMENEZ, A. and CABALLERO, F. 1967. Ensayos sobre erradicaci6n de Ceratitis wpilala Wied. por el metodo de los machos esteriles en la isla de Tenerife. Ro/. Pat. l'eg Ent. AgTic:. 30 233-249. BODENHEIMER, F. S. 1951 Citrus Entomology in the Middle East. W Junk. The Hage, Netherlands. 87-160. CONTI, B. 1990. Effects of abiotic factors on Ceratilis capita/a (Wied ) (Diptera Tephritidae). 3- Larval and total development under constant temperatures Fmstula F,11101110/ogicu, Nuova Serie, Vol. XI (XXIV) 157-170. CROVETTI, A, CONTI, B. and DEL RIO, G. 1986. Effect of abiotic factorson Cera/iii., capita/a (Wied ) (Diptera: Tephritidae). 2- Pupal development under constant temperatures. Proceeding of the CEC/IOBC, Hamburg 23 agosto 1984. Ed. Cavallaro, pp 141-147. DEL RIO, G, CONTI, B and CROVETTI. A. 1986. Effect of abiotic factors on Ceralitis rnpilala (Wied.) (Diptera Tephritidae). 1- Eggs development under constant temperatures. Proceeding of the CEC/IOBC, Hamburg 23 agosto 1984. Ed. Cavallaro, pp 133-139. FLETCHER, B.S. and ET KAPATOS. 1982. An evaluation of different yemperature-development rate models for predicting the phenology of the olive fly Darns oleae. Proceeding of the CEC/IOBC, Athens nov 1982. Ed Cavalloro, 321-329 pp HARRIS, E.J, CAREY, JR. KRANACKER, DA and LEE, CY.L 1991 Life history of ( 'era111is capiiala (Diptera. Tephriliclaej reared from mock orange in Hawaii E111'/lrn1. Enwmol 20( 4) l 048- l 052. LA.BORDA, R, SANT.A.BALLA, E. and F. GARCIA MARI 1990. Evoluci6n y desarrollo de la mosca de la fruta C'eratitis capitata Wied. sabre limones espafioles. Bo/. San r·eg Plagm, 16 613-618. MEATS, A 1984. Thermal constraints to successful development of the Qu·eensland fruit fly in regimes of constant and fluctuating temperature. Emomol. exp. appl. 36 55-59 - 6 -

MEATS, A 1989. En Fruit Flies Their Biology, Natural Enemies and Control. Ed. Robinson, Netherlands, Hooper, Australia; Elsevier, Amsterdam. Oxford, New York, Tokyo Vol. A 213-221 MESSENGER, P and FLITTERS, N. 1958 Effect of constant temperature environments of the egg stage of three species of Hawaiian fruit flies. Ann. Em. Soc. Amer. 51(2) 109-119. MUNIZ, M and GIL, A 1984. Desarrollo y reproducci6n de Ceratitis capita/a (Wied ) en condiciones artificiales. Roletin Jel Servtcio contra l'lagas e l/1.\peccuin ° Fitopalolrigica. Ministerio de Agricultura, Pesca y Alimentaci6n Fuera de Serie. N 2. PUTRUELE, M.T.G. DEL PINO, A.A and GARRIDO. A 1996. Effect of field and constant temperatures on egg development of Ceratitts capilalu (Wied.)(J)1p1era:

Tephrilidae). IOBC Congress, in press SHOUKRY, A and HAFEZ, M. I 979. Studies on the Biology of the Mediterranean fruit fly Ceratitis capita/a. £111. exp. & appl. 26 33-39. TSITSIPIS, l A 1979 Effect of constant temperatures on the eggs of the fruit fly, ]Jarn.1·oleae (Diptera, Tephritidae). Ann. Zoo/. },'col. Anim. 9 (1): 133-139 - 7 -

INFLUENCE DE LA TEMPERATURE SUR LE DEVELOPPEMENT PREIMAGINAL DE PSYTTALIA FLETCHER/ SILVESTRI (HYMENOPTERA: BRACONIDAE), PARASITOIDE DE BACTROCERA CVCURBITAE COQUILLETT (DIPTERA: TEPHRITIDAE)

B. HURTREL (1) et S. QUILICI (2) (1): CIRAD-FLHOR Reunion, Laboratoire d' Entomologie, B. P. 20, 97408 St Denis Mcssagcrie C:edex09, France. (2) : CIRAD-FLHOR Reunion, Laboratoire d'Entomologic, Station de Bassin Martin, B.P. 180, 97455, St Pierre Cedex, France

Resume Les stades de developpement successifs de Psyttalia jletcheri Silvestri ont ete etudies a 25±1 °C. En outre, la duree totale du developpement preimaginal (de l'oeuf a !'emergence de l'adulte) a ete precisee dans une gamme de temperatures de 15 a 27°C. Nous avons constate la presence de 4 stades larvaires parmi lesquels les stades 2 et 3, qui sont tres similaires, ne durent que 1,25 jours a 25°C. Au contraire, les derniers stades preimaginaux, larve de 4eme stade et nymphe, presentent une duree beaucoup plus longue (respectivement 4,5 et 5,75 jours a 25°C). Dans la gamme de temperatures testees (15 a 27°C), la vitesse de developpement croit de faconlineaire (R2=0,97) avec la temperature. La temperature seuil de developpement se situe a l l,02°C et la somme des degresdours.necessaire au developpement preimaginal est de 231,44 ± 19,53°C.

1 - fotroduction: La mouche du melon, Bactrocera (Zeugodacus) cucurbitae Coquillett constitue un ravageur important des Cucurbitaceae dans sa zone d'origine asiatique, ainsi que dans differents pays d' Afrique et du Pacifique. Dans !'Ocean Indien, I'espece est notamment presente a La Reunion· oir elle s'avere principalement nuisible dans Jes zones de basse et moyenne altitude (Etienne, 1972).

Le parasitisme observe sur les populations de B. cucurbitae reste limite et semble essentiellement du a un Eulophidae, Tetrastichus giffardianus Silvestri (Quilici et al., non pub!.). Cette espece avait ete importee dans les annees 70 dans le cadre d'operations anterieures de lutte biologique (Etienne, 1973). Aussi, un nouveau programme de Jutte biologique a-t-il ete engage, avec !'importation d'Hawaii* en juin 1995, d'un endoparasitoi:de larvo-pupal solitaire, Psytta/ia jletcheri Silvestri (Hymenoptera: Braconidae: Opiinae). L'elevage de cet auxiliaire est maintenant bien mai"trise, et ii a fait l'objet depuis deux ans de liichersdans de nombreuses zones de l'ile (Quilici et al., non pub!.).

Une connaissance fine de la biologie du parasitoi:de s'avere toutefois indispensable pour optimiser les differentes etapes de cette operation de lutte biologique, dans sa realisation (production, liichers), comme dans son evaluation.

Aussi, Jes objectifs de la presente etude sont-iJs de connaitre avec precision Jes differentes etapes du developpement preimaginal de P. jletcheri et !'influence de Ja temperature sur ce developpement. De meme, nous nous sommes proposes de determiner le seuil thermique de developpement et la constante thermique de l'espece.

*: en collaboration avec nos collegues de l'USDA-ARS (Dr M. Ramadan) et de !'Universityof Hawaii (Dr R. Messing), que nous tenons ici a remercier vivement - 8 -

2 - Materiels et Methodes:

EleJ•age de l'hote et du parasitoide: En conditions semi-controlees (T=25±1 °C, HR=60-70%, photoperiode naturelle = 12: 12 ), dans des cages ( 100 x 50 x 50 cm), les femelles de B. cucurbitae sont mises a pondre sur des courgettes pendant 24 heures. Apres ce delai, le fruit est retire de la cage et place dans une boite en Plexiglas de 355 x 235 x 130 mm sur un lit de flocons de pomme-de-terre. Trois jours plus tard, les larves nourries de la pulpe de courgette ont atteint le stade L3 preferentiellement parasite par P. fletcheri (Wong & Ramadan, 1993). Les larves sont done extraites de la courgette, rincees, essorees et emmaillotees dans une mousseline afin de former une « galette » bien compacte et tout juste humide. La galette est ensuite presentee pour la ponte aux femelles de P.fletcheri fecondees et ayant atteint leur maturation ovarienne (apartir de !'age de 5 jours) pour une duree de 6 heures pendant la photophase. Apres ce delai, la galette est retiree de la cage et placee dans une boite sur un lit de sciure de bois pour permettre le saut des larves et leur pupaison. Trois jours plus tard, la sciure contenant Jes pupes est tamisee et les pupes sont placees dans des petites boites rondes (diam. 80, haut. 50 mm) dans lesquelles se deroulera le developpement des parasitoides.

Etude du developpement larvaire et nymphalaux conditions de l'elevage: Quotidiennement, a partir de 24 heures post-infestation, 20 larves ou pupes de mouches soot dissequees dans une solution physiologique (solution de chlorure de sodium 6%o). Pour chaque parasite trouve, on note son stade de developpement et sa longueur. L'experimentation est arretee lorsque au moins 30 individus de chaque stade ont ete mesures.

Influence de la temperature sur le developpement pre-imaginal: lei, nous nous proposons d'etudier !'influence de la temperature sur le developpement (duree totale, mortalite)et sur le sex-ratio de la progeniture. Les experimentations ont lieu dans 4 enceintes en conditions de temperature, d'humidite relative et de photoperiode controlees: T = 15, 20, 25 et 27°C; HR= 80±20%; photoperiode naturelle = 12:12 h Les gaieties parasitees pendant 24·heures---sont·placees·dans 'Cles boites'de pupaison reparties entre Jes 4 temperatures. Une fois la pupaison effectuee, 100 pupes de chaque galette sont transferees dans des petites boites rondes. A chaque temperature, on note la date d'emergence des males et des fernelles de P. jletcheri. On denornbre le pourcentage de rnortalite des pupes (saines et parasitees confondues) et on calcule le sex-ratio (fernelles/fernelles+mfiles) de la descendance.

3 - Resultats:

Etude du developpement larvaire etnymphal aux conditions de l'elevage: Description des differentes etapes du developpement: Les observations realisees confirrnent Jes descriptions faites par Willard (1920): le developpernent preirnaginal de P. fletcheri cornporte 1 stade oeuf, 4 stades larvaires (notes LI, L2, L3, L4) et I stade nyrnphal (a partir duquel on· peut differencier les sexes). L'oeuf est pondu par la fernellejuste sous le tegurnent de la larve-hote ou ii reste solidement attache. De cet oeuf sort la larve L1 tres rernarquable par sa capsule cephalique chitinisee dotee de fortes rnandibules. En cas de superparasitisrne, c'est ace stade que les larves Ll surnurneraires soot eliminees par la larve la plus forte. Le stade L2 est tres different du stade LI; la capsule cephalique a disparu et Jes mandibules sont reduites a leur plus simple expression, au point d'etre difficilernent visibles a la loupe binoculaire, rnerne au plus fort grossissernent. Le stade L3 etant different du stade L2 uniquernent par la taille et par la forrne des mandibules - 9 -

minuscules, nous avons choisi de regrouper ces deux stades en un seul, note L2-3. Le stade L4, beaucoup plus gros, se distingue par la presence d'epines aigues sur certaines parties du corps, notamment sur les parties dorsales et laterales des segments 2 et 3, ainsi que sur Jes parties laterales des segments 4 a 12 (inclus). On remarquera egalement la presence de globules lipidiques juste sous le tegument. Le passage au stade nymphe est progressif, debutant par une contraction de la partie anterieure du corps, qui fonnera la tete et le thorax. Au debut de sa metamorphose, la nymphe est entierement blanche sauf les yeux qui sont orange. Mais, en quelques heures, elle commencera a acquerir une teinte de plus en plus jaune, puis orangee, et ceci jusqu'a !'emergence de !'imago. C'est a partir du debut du stade nymphe que !'on peut diiferencierJes femelles des males, l'ovipositeur entourant !'abdomen entre les 2 antennes.

Chronologie du developpement preimaginal: Les dissections d'hotes parasites nous ont permis de suivre la succession des diiferents stades immatures de P. jletcheriau cours du temps (Tableau I et Figure I). Tableau I: Evolution du pourcentage de chaque stade de developpement de Psyttalia jletcheridans Jes hates disseques (Bactrocera cucurbitae) en fonction du nombre de jours post-infestation (PI), a 25°C. jours PI Oeuf Lt L2-3 IA Nymphe male Nympllefemelle 1 100.00 0.00 0.00 0.00 0.00 0.00 2 35.00 65.00 0.00 0.00 0.00 0.00 3 0.00 89.36 10.64 0.00 0.00 0.00 4 0.00 7.69 89.74 2.56 0.00 0.00 5 0.00 0.00 15.38 84.62 0.00 0.00 6 0.00 8.33 0.00 91.67 0.00 0.00 7 0.00 0.00 16.67 75.00 8.33 0.00 8 0.00 0.00 4.00 84.00 8.00 4.00 9 0.00 0.00 0.00 64.29 14.29 21.43 10 0.00 0.00 0.00 0.00 44.44 55.56 11 0.00 0.00 0.00 8.11 37.84 54.05

/,/· 100%.

80%-; jDocuf 'O" i � " 60%� IL,JLJCL2-3 l"!lU l!ln}mphcmfilc "' 40%)j Dnymphe femelle " i -::, ' .� 20%� i

2 7 joun post-infestation 9 JO 11

Figure I: Pourcentages relatifs des differentsstades de developpement de Psyttaliajletcherien fonctiondu nombre de jours apres !'infestation, a 25°C. - 10 - D'ou les durees respectives (en jours) des stades de developpement preimaginal stade oeuf: 1,75 stade LI: 1,75 stade L2-3: 1,25 stade L4: 4,5 stade nymphe: 5,75. A partir de 358 heures post-infestation (c'est-a-dire 15 jours), ii y a emergence des premiers males suivie de celle des premieres femelles 24 heures plus tard.

Les mesures de la longueur des stades preimaginaux nous ont egalement permis de suivre !'evolution de ce parametre en fonction des jours post-infestation et du stade de developpement (Tableau II; Figures 2 et 3).

Tableau II: Evolution de la longueur des parasitordes (en mm) en fonction du jour post­ infestation (PI) et du stade considere, a 25°C.

.ioursPI Oeuf Ll L2-3 lA Nymphemale Nymphe femelle 1 0.67 2 0.98 1.00 3 1.43 1.58 4 1.69 2.96 4.67 5 2.95 4.85 6 5.08 7 2.28 4.94 8 4.33 9 3.77 4.23 5.20 10 3.76 4.18 11 4.06 4.21

10 II jours post-infestation Figure 2: Evolution de la longueur du parasitoide au cours de son developpement preimaginal: A: longueur du parasitorde en fonctiondes jours de developpement Gours post-infestation) B: longueur du parasitoide en fonctiondu stade de developpement - 11 -

6 T ------... -- --o-12-3 , f ---IA Nymphe male --+-Oeuf"' - N}mphe femellj . r---o..__ ··O ··LI f: I i .s 2�i . J,/ · -ii- Dd. · __ .. a··••. ·• I .. -··· • • C ,

2 6 8 10 11 jours post-infestation Figure 3: Longuer des differentsstades de developpement preimaginal • • 0 de Psyttalia jletcheri en fonctiondu nombre de jours apres !'infestation. 4 7 9

Nous constatons qu'il y a un accroissement exponentiel de la longueur des larves jusqu'au stade L4 apres lequel va debuter la nymphose. Entre les stades L4 et nymphe, on observe une phase de transition (prenymphe) qui commence par une reduction de la longueur du parasitoi:de due a une constriction au niveaudu thorax.

Influence de la temperature sur le developpement pre-imaginal:

A partir des donnees de duree de developpement en fonctionde la temperature, nous avons pu calculer deux variables, la temperature seuil de developpement (notee t) et la constante thermique (notee K). La theorie de la constante thermique est basee sur !'observation generale que la vitesse de developpement d'un insecte est proportionnelle a la temperature du developpement a l'interieur d'un intervalle de temperatures. Cette constante est calculee par la fonnule suivante: K = D (T - t), ou D est le temps de developpement en jours, T est la temperature du developpement et t est la temperature seuil de developpement (Morris & Fulton, 1970).

Afin de calculer ces deux variables, nous avons represente la vitesse de developpement en fonction de la temperature testee (Figure 4). Nous avons obtenu une regression lineaire et la valeur d'abscisse a laquelle la droite de regression coupe I' axe des abscisses nous donne le seuil theorique de developpement, que !'on a calcule etre l l,02°C. Cette valeur nous permet, grace a la formule ci-dessus, de calculer la constante thermique K, qui est egale a 231,44 ± l9,53 °C. - 12 - 0.08

0.07 l •

I 006 t .:,. I � O.OS f � I i 0.04 f y = 0.00427x - 0.04705 � i R' = 0.96730 i 0.0 3 t

;; � 0.02 l I 0.01 + i II.OZ 0.00 -r--'-----�--�----�--�----�--�----�- 10 15 20 25 30 temperature {°C)

Figure 4: Relation entre la vitesse de developpement de Psyttaliajletcheri et la temperature de developpement

D'autre part, si on represente Jes dynamiques d'emergence des parasitoi:des pour chaque temperature (Figure 5), on constate que plus la temperature est elevee, plus Jes emergences soot synchrones. En effet, a 15°C, la totalite des emergences est realisee en 10 jours alors qu'a 27°C, tous Jes parasitoi:des ont emerges dans un intervalle de 4 jours. De plus, on peut constater que la protandrie diminue avec l'accroissement de la temperature jusqu'a une presque totale synchronisation des emergences males et femellesa 27°C.

--%males 15°C 20°c - - - -% femelles 30 45 .tl 40 25 i. 35 20 .. .f E 30 , - ... 25 , !9 15 !9 , , ' , ' ... -= 20 , ·5: 10 , ·5: , -� '6 15 , ] .s 10 ; /--' ' , 5 , '- '$. ,, '$. --J ,_ 0 0 25 46 48 50 52 54 56 27 29 31

duree developpementde cmreede deft)ORJl'meDt

50 90 "' ... T .:} 80 40 � 70 S 60 .5 30 t 20 ] !� ·;: :E-= 30 .5 20 .5 10 10 '$. --- "'#, 0 14 16 18 20 14 16 wree dedevelo(111'ment wreede developpement

Figure 5: Dynamiques d'emergences des males et femelles de Psyttaliafletcheriaux 4 temperatures testees. (La duree de developpement est exprimee enjours). - 13 -

4 - Discussion - Conclusion:

L'etude de la succession des stades preimaginaux de P. jletcheri nous confirme que cette espece, comme la plupart des Opiinae parasito1des de Tephritidae (Pemberton, 1918; Willard, 1920; N'Guetta, 1990), presente quatre stades larvaires. Willard (1920) a bien montre que la duree du stade LI est fortement dependante de la physiologie de l'hote. En effet, les LI de P. jletcheri n'effectuent la premiere mue larvaire qu' apres la pupaison de Ieur hote. Aussi, en conditions adverses retardant la pupaison de l'hote, la duree du stade LI peut-elle atteindre 10-12 jours. Globalement, les durees de developpement que nous avons enregistre a 25°C, pour Jes stades oeuf, Ll et nymphe sont proches de celles mentionnees par Willard (1920), en conditions de temperatures favorables. Nous avons en outre precise la duree cumulee des deux stades suivants, L2 et L3, qui sont particulierement fugaces(1,25 jours a 25°C) et celle du stade L4. La valeur du seuil thermique de developpement (ll,02), obtenue par calcul, demande a etre confirmeeexperimentalement.

Les connaissances acquises au cours de cette etude trouveront une application dans !'optimisation des methodes de production de l'espece, dans le cadre des programmes de Jutte biologique en cours. La connaissance de !'influence de la temperature sur le developpement preirnaginal de P. jletcheri sera egalement tres utile pour !'interpretation de la distribution et des fluctuations d'abondance de l'auxiliaire, dans l'hypothese d'une confirmation de son acclimatation dans l 'ile.

References: Etienne, J. (1972). Les principales Trypetides nuisibles a l'ile de la Reunion. Ann. Soc. ent. Fr.,8: 485-491.

Etienne, J. (1973 ). Lutte biologique et aperyu sur les etudes entomologiques diverses effectueesces dernieres annees a La Reunion. L 'Agron. trop., 28 (6-7) : 683-687. Morris, R. F. et W.C.Fulton, (1970) . Models for the development and survival of Hyphantria cunea in relation to temperature and humidity. Entomol Soc. Can.Mem., 70: 1-60.

N'Guetta, K. (1990) . Effets des basses temperatures, appliquees aux formes preimaginales, sur le maintien du potentiel biotique du couple Ceratitis capitata Wied. (Dipt. Trypetidae) Opius longicaudatus Ashm. (Hymenopt. Braconidae). These de !'Ecole Nationale Superieure Agronomique;specialite: biologie appliquee a l'agronomie, protection des cultures. Rennes. 92 pp.

Pemberton, C. E. (1918) A contribution to the biology of fruit-flyparasites in Hawaii. J.Agr. Res., !5: 419-465.

Willard, H. F. (1920) Opiusjletcheri as a parasite of the melon fly in Hawaii. J. Agr. Res.,20: 423-438.

Wong, T. T. Y. and M.M.Ramadan. (1993) . Mass rearing biology of larval parasitoids (Hymenoptera: Braconidae: Opiinae) of tephritid flies (Diptera: Tephritidae) in Hawaii. In Advances in insect rearing for research and pest management. T.E. Anderson & N.C. Leppla (eds.). Westview, Boulder.: 405-426. - 14 -

MODELIZA<;AODA EVOLU<;AODA MOSCA-DA-AZEITONA (BACTROCERA OLEAE (GMELIN)) PELO M.ETODODO SOMATORIO DE TEMPERATURAS, PARA A REGIAO DE CASTELO BRANCO, PORTUGAL

J. Coutinho, M. Sequeira* & C. Veiga Escola Superior Agrariade Castelo Branco, Qt"Sr" Mercules, 1'6000-CasteloBranco. Portugal *Dir�o Regional Agricultura Beira Interior, R. Amato Lusitano, L3 - P6000-Castelo Branco. Portugal

Summary

Moddeling oi the evolution of the olive-fly (Bactrocera oleae (Gmelin)) using the temperature sum method for the Castelo Branco region, Portugal.

The modelling of the evolution of the olive-fly (Bactrocera oleae (Gmelin)) using the temperatures sum method (growing degree days) for the Castelo Branco region, Portugal, from 1995 to 1996 is presented. It was observed, in these years, that the olive fly (Bactrocera oleae (Gmelin)) developed only one complete generation while the second generation has not completed its life cycle.

Resumo

Apresenta-se a modeliza9ao da evolu9ao da mosca-da-azeitona (Bactroceraoleae (Gmelin)) pelo metodo do somat6rio de temperaturas, para a regiao de Castelo Branco, Portugal, nos anos de 1995 e de 1996. Verificou-se que nos anos referidos, em Castelo Branco, a mosca-da-azeitona (Bactrocera oleae (Gmelin)) desenvolveu apenas uma gera9ao completa, nao tendo terminado a segunda gera9ao. A especie, nesta regiao, hiberna no estado de larva ou pupa, terminando o ciclo no ano seguinte.

1. Introdu\:lio

Uma boa tecnica que possa prever os estados de desenvolvimento do insecto e da cultura, baseada em pariimetros simples, em conjunto corn um metodo apropriado para calcular as datas em que o risco surge, pode ajudar os produtores a combater a praga, rninimizando os estragos e evitando prejuizos (Pitzalis, 1985). Neste trabalho foi aplicado o metodo do somat6rio de temperaturas para prever a emergencia dos adultos e para estimar o numero passive! de gera96es de Bactrocera oleae (Gmelin) para a regiao de Castelo Branco, Portugal, nos anos de 1995 e de 1996. -15-

2. Material e Metodos

0 metodo utilizado neste trabalho, para determinar o ciclo de vida de B. oleae (Gmelin), foi o descrito por Pitzalis (l 985), que relaciona o somatorio de temperaturas medias diarias corno desenvolvimento da especie, desde as primeiras posturas. Com base nas temperaturas medias diarias, registadas no posto meteorologico da Escola Superior Agraria de Castelo Branco, nos anos de 1995 e de 1996, efectuaram-se as somas das temperaturas medias diarias a partir da data em que se verificaram as prirneiras posturas. Foram considerados validos apenas os dias cujas temperaturas medias estiio situadas entre os 9.5°C e os 30°C, conforme o modelo proposto por Pitzalis (1985) e utilizado por Patanita (1995) para a regiiio de Moura. A temperatura media diaria (graus-dia 0D) foi calculada segundo a formula de Newman (1968) e adaptada por Pitzalis (1985):

T = T max. + T min. - TI - *(T max. - 30) 2 TI - temperaturalimite (9.5°C) * Quando a temperatura maxima e superior a 30°C

Os valores da constante termica K (°D), o limite minimo de temperatura (°C) e o limite maximo de temperatura (°C), usados para a determinayiio do desenvolvimento dos diferentes estados de Bactrocera oleae (Gmelin), siio os obtidos por Crovetti et al. ( 1979; 1981 e 1982), segundo o metodo das temperaturas, constantes no Quadro 2.1.

Quadro 2.1 • Valores da constante termica e temperaturas limite para o desenvolvimento dos diferentes estados de Bactrocera oleae (Gmelin) (Crovetti et al., 1979, 1981, 1982)

Ovo Larva(-) Pupa Total

Constante termica K (0D) 50 130 200 380

Limite minimo de temperatura (°C) 9-10 10-11 9-10 9-10

Limite maximo de temperatura (°C) 31-33 31 * O resultado da duracao do desenvolvimento larvar foi obtido por diferen1,3.

Nas Figuras 2.1 e 2.2 apresenta-se a evoluyao das capturas de B. oleae e a evoluyao dos frutos atacados corndesenvolvimento larvar, respectivamente em 1995 e em 1996 - 16 -

Capturas medias de B. oleae em dois tipos de armadilhas e percentagem de frutos atacados cl evolu,;:ao larvar ESA-Qt• Sr'. de Mercules- C. Branco 1995 160 45

- 140 +-Armad. l'vtAlail 40

120 -t.-Armad. Cromotr6pica Cl "' Feromona "' 100 ----*- %de Frutos Atacados Cl Evolu,;:ao g 80 :%QI QI"' 60 z

20 5

0 0 :i .:i :i 0 0 0 0 0 ai ai ai > > > . C) C) C) C) :l :l0 :l :l 0 0 fi 0 fi B'J ...M "' 'l' 9 C C C C aN.. N...... ;'!. tZ j cl, 6 :Y. '1' ! cbN "' N cJ, :i, cl, 6 -;!. NJ N � "' M a ;;; a a a a N a Datas de observa,;:ao

Fig. 2.1 - Capturas medias de Bactrocera oleae (Gmelin) em dois tipos de armadilhas e a percentagem de frutos atacados corn evolu�ao larvar, na regiao de Castelo Branco, em 1995.

Capturas medias de 8. o/eae em dais tipos de armadilhas e percentagem de frutos atacados cl evolu,;:ao larvar ESA - Sr'. de Mercules- Branco 1996 160 or. c. 70

140 -+-Armad. McPhail 60 0 120 -lr-Armad. Cromotr6pica Cl Feromona ..C: .. -*-de % Frutos Atacados C/ 100 Evolu9ao

20 10

Datas de observa,;:ao

Fig. 2.2 - Capturas medias de Bactrocera o/eae (Gmelin) em dais tipos de armadilhas e a percentagem de frutos atacados corn evolu�o larvar, na regiao de Castelo Branco, em 1996. - 17 -

3. Resultados

Nos Quadros 3.1 e 3.2 sao apresentadas as datas em que se registaram os 380°D, necessarios para se completar uma gerayao de Bactrocera oleae (Gmelin), e o numero de dias requerido para os atingir, respectivamente no ano de 1995 e de 1996, em Castelo Branco.

Quadro 3.1 - Oatas de emergenciados adultos e dura9ao do desenvolvimento, em dias, desde a fase de ovo ate ao adulto, baseado no somat6rio de temperaturas de 380°0. Castelo Branco, em 1995.

Primeiras posturas Primeira Gerayao

Emergencia de adu!tos N° de dias

31-08-95 11-10-95 42

07-09-95 20-10-95 43

14-09-95 29-10-95 45

Quadro 3.2 - Oatas de emergencia dos adultos e durayao do desenvolvimento, em dias, desde a fase de ovo ate ao adulto, baseado no somat6rio de temperaturas de 380°0. Castelo Branco, em 1996.

Primeiras posturas Primeira Gerayao

Emergencia de adultos N° de dias

16-08-96 20-09-96 36

26-08-96 03-10-96 39

09-09-96 27-10-96 49

Nos Quadros 3 .3 e 3 .4 constam a durayao do desenvolvimento embrionario, larvar e pupal e as datas do fim de desenvolvimento, corn base nos somat6rios termicos propostos por Crovetti et al. (1982). - 18 -

Quadro 3.3 - Dados referentes a durai;;ao do desenvolvimento embrionario, larvar e pupal e datas da sua finalizai;;ao, baseado no somat6rio termico de 50, 130 e 200 °D respectivamente. Castelo Branco, 1995.

Posturas Embriao Larva Pupa

Data ND de Data ND de Data ND de dias dias dias

1• Gera!;ao

lnicio 31-08-95 03-09-95 4 19-09-95 16 11-10-95 22

lnicio 07-09-95 11-09-95 4 29-09-95 18 20-10-95 21

lnicio 14-09-95 20-09-95 6 04-10-95 14 29-10-95 25

2• Gera!;ao

lnicio 20-10-95 27-10-95 7 21-11-95 25

lnicio 28-10-95 03-11-95 6

lnicio 06-11-95 19-11-95 13

Quadro 3.4 - Dados referentes a durai;;ao do desenvolvimento embrionario, larvar e pupal e datas da sua finalizai;;ao, baseado no somat6rio termico de 50, 130 e 200 DD respectivamente. Castelo Branco, 1996.

Posturas Embriilo Larva Pupa

Data ND de Data ND de Data ND de dias dias dias

1• Gera!;ilo

lnicio 16-08-96 20-08-96 5 31-08-96 11 20-09-96 20

lnicio 26-08-96 29-08-96 4 10-09-96 12 03-10-96 23

lnicio 09-09-96 14-09-96 6 29-09-96 15 27-10-96 28

2• Gera!;ilO

lnicio 30-09-96 05-10-96 6 24-10-96 19

lnicio 13-10-96 21-10-96 9 25-11-96 32

lnicio 07-11-96 01-12-96 25 - 19 -

4. Discusslio

No ano de 1995, no calculo te6rico do numero de geracoes, as datas consideradas para o inicio da I" geracao foram 31 de Agosto, 7 de Setembro e 14 de Setembro. As · datas te6ricas de previsao da eclosao dos adultos da Ia geracao sao, respectivamente, 11 de Outubro, 20 de Outubro e 29 de Outubro, datas que correspondem ao periodo em que se verificou o maior numero de individuos capturados em armadilhas, como podemos constatar na Fig. 2.1. Em 1996 o inicio das posturas comec;:ou mais cedo que em 1995. Consideraram-se, no calculo te6rico do numero de geracoes, as datas de inicio da I• gerac;:ao de 16 de Agosto, 26 de Agosto e de 9 de Setembro. As datas te6ricas de previsao da eclosao dos adultos da 1• geracao foram, respectivamente, 20 de Setembro, 3 de Outubro e 27 de Outubro. Verificamos, ta! como em 1995, que estas datas correspondem ao periodo de maior captura de adultos nas armadilhas, como se pode verificar pela Fig. 2.2. Nos dois anos considerados as datas de eclosao dos adultos, calculadas teoricamente, correspondem ao periodo de maxima eclosao de adultos na natureza. Tenda em consideracao que a mosca-da-azeitona leva cerca de 1 semana a atingir a maturidade sexual e a acasalar, a segunda geracao tern inicio, em func;:ao das datas consideradas para a 1• geracao, respectivamente, em 20 de Outubro, em 28 de Outubro e em 6 de Novembro no ano de 1995 e em 30 de Setembro, em 13 de Outubro e em 7 de Novembro no ano de 1996. Em qualquer dos anos nao houve um somat6rio de temperaturas suficiente para completar a 2• geracao. Sendo assim e provavel que a especie hiberne na regiao de Castelo Branco no estado de pupa, completando-se a gerac;:ao no ano seguinte.

Referencias bibliognificas

CROVETTI, A, LOI, G., QUAGLIA, F. & RASP!, A. (1979). Ricerche etoecologiche sul Dacus oleae (Gmel.). Durata dello svillupo pupale a temperature costanti. Notiziario su!le malattie delle piante, JOO. Ill serie, 27. Pavia (cit. PITZALIS, 1985).

CROVETTI, A, LOI, G, QUAGLIA, F. & BELCARI, A. (1981). Durata dello svillupo dell'uovo di Dacus oleae (Gmelin) a temperature costante. Fntstula Ent., IV (XVII): 83-91. (cit. PATANITA, 1995).

CROVETTI, A, QUAGLIA, F., LOI, G., ROSSI, E., MALFATTI, P., CHESI, F., LONTI, B., BELCARI, A., RASP!, A. & PAPARATTI, B. (1982). Influenza di temperatura e umidata sullo svillupo degli stadi preiimmaginali di Dacus oleae (Gmelin). Fmstula Ent., V (XVIII): 133-166. (cit. PATANITA, 1995).

NEWMAN, J. (1968). Growing degree days. A new system of rating crop maturities could help you predict harvest dates more accurately. Crop and soils, vol. 21 (cit. PITZALIS, 1985).

PATANITA, M. I. (1995). Esh,do sabre a mosca da azeitona - Bactrocera oleae (Gmelin) ea tra9a da oliveira Prays oleae Bernard na regii:io de Moura numa perspectiva de protec9i:io integrada. Dissert. Mestrado Protec�ao Integrada,ISA!UTL. Lisboa. - 20 -

PITZALIS, M. (1985). Bioclimatology and insect development forecast: Degree days and phenophases of Dacus oleae Gmel.. ln CAVALLORO, R & CROVETTT, A. (Eds.). Integrated pest control in olive-groves - Proc. of the CEC/FAO/IOBC InternationalJoint Meeting, Pisa 3- 6 April /984: 84-93. (cit. PATANITA, 1995).

SEQUEIRA, M. (1977). lnfluencia da mosca da azeitona Bactrocera oleae (Gmelin) na qualidade do azeite. Relat. Fim Curso Eng. Prod. Oleos Alimentares (CESE). Escola Superior Agraria. Castelo Branco.

VEIGA, C. (1977). A mosca da azeitona na regiiio de Castelo Branco. Relat. Fim Curso Eng. Prod. Agricola (Relat. Preliminar). Escola Superior Agraria. Castelo Branco. - 21 -

FIELD POPULATION SURVEY OF FRUIT FLIES (DIPTERA, TEPHRITIDAE) BY USE OF ATTRACTIVE TRAPS IN CITRUS ORCHARDS IN PIRACICABA, STATE OF SAO PAULO, BRAZIL

M. C. R. Dias & V. Arthur Center for Nuclear Energy in Agriculture, University of Sao Paulo. P.O.Box 96 - Piracicaba,SP, 13400-970, Brazil, e.mail: [email protected]

Summary

A survey of fruitflies from the family Tephritidae was carried out from Febrnary 1990 until February 1991 in a Citrus sinensis orchard in Piracicaba, State of Sao Paulo, Brazil. The flieswere weekly captured, on traps baited with Vegamine™ plus amonium sulphate at the proportion of seven to one; sugar cane syrup plus amonium sulphate at the proportion of three to one; a ten percent solution of Vegamine™ and a twenty five percent solution of concentrated maracock juice. As results, a capture of 1004 flies was achieved. Out of these, 72.9% were Ceratitis capitata Wied, and 27.9% were eight species of the genus Anastrepha, where the most common was A. fraterculus. The most effective attractant was the maracock juice, which captured respectively 43.3% of C. capitata and 46.1% oftheAnastrepha flies. Key Words: Fruit flies, field survey insects attractants, Citrus sinensis, Ceratitis capitata, Anastrepha spp.

1. Introduction

In Brazil, orange orchards are a common aspect of agriculture. As an important international trade, the. export of concentrated orange juice brings to the country the foreign.currency to develop the social trends of the nation. Fruit flies like Ceratitis capita/a and many Anastrepha species are the most important pests in citrus. The use of traps baited with attractives is an important weapon against the severe losses caused by these flies. Monitoring their population and determining the proper time to spray the adequate insecticides in the orchards is one of the great deals to control infestations and keep losses under controlled conditions, with the aditional advantage to maintain spraying low and keeping natural enemies effective. - 22 -

In the early beginning of this centmy, LIMA (1926) related the need to control C. capitata and A. fraterculus in citrus plantations. In the mid centmy, PUZZI et al. (1955) related losses of 30% of the expected yield of oranges from the variety "Pera", in the region of Bebedouro, State of Sao Paulo. In the eighties, MALAVASI & MORGANTE ( 1980) related an infestation in oranges of 43.05% of C. capitata, 13.71% of Anastrepha spp. and 43.24% of Silba spp. with a mean of 1.32 larvae per fruit. BRESSAN & TELLES ( 1991) related that they found an infestation rate of 1 % of orange fruits, with the presence of 61.8% of A. obliqua, and 38.2% of C. capitata. But RAGA et al. (1994) related an infestation rate of 44% of the orange fruits, with 1.28% larvae per fruit in orchards of the region of Atibaia, SP, identifying 98.5% as A. fraterculus and 1.5% as C. capitata. Early in the fortieths, GOMES (1942) performed experiments using extractions of orange juice at a dilution of 17.5%, vinegar at 25%, wheat bran extract at 7.5%, sugar solutions at 10 and 20%, and soap water at 15%, added to kerosene. He observed that the most effective attractant was orange juice, fermented from 24 to 48 hours after extraction, and then the wheat bran extract. PUZZI & ORLANDO (1957) using brown sugar, observed that this could be an usefull attractive substance but economically the use of sugar cane melasse was more suitable forextensive use. In peach orchards, FEHN (1977) observed that the use of sugar cane melasse and peach juice were the most attractive substances. BLEILCHER et al. (1978) observed that grape juice at the concentration of 25% was a good attractant to A. fraterculus. Similarly, TAMASHIRO et al. (1984) using sugar cane melasse in common house-fly traps was the most effective to attract fruit flies. Otherwise, ENGELSTEIN & AMARAL (1987) observed that a mixture of amonium sulphate and sugar cane melasse at the proportion of 7:1 was the most effective attractant to all fruit flies. In a study CALZA et al. (1988) found the following Anastrepha species: A. fraterculus (73.84%), A. sororcula (10.76%), A. pseudoparalella (8.84%), A. obliqua (3.46%), A. montei (1.52%), A. grandis (0.76%), A. manhoti (0.38%), and A. distincta (0.38%). Also NASCIMENTO & ZUCCHI (1981) in a study in the State of Bahia, found twenty different species of Anastrepha, where the most important were A. obliqua, A. fraterculus, A. sororcula, A. distincta, A. serpentina, and A. bahiensis. But in Mexico, HERNANDEZ & ORTIZ (1992) determined that only A. ludens and A. serpentina were harmful to citrusfruits. As an overall, the literature and the practice showed that monitoring fruit flies populations in the field is an effective tool to determine - 23 - the most important species in a given area besides their degree of infestation. Only so it would be able to predict the adequate moment to apply proper controlmeasures in the fields.

2. Methodology

The experiments were conduced at the Agricultural Experiment Station, in Piracicaba, State of Sao Paulo. The orchard, with an area of about 20.500 square meters and with various varieties of Citrus sinensis, such as cv. Pera; Natal; Valencia; Baianinha and Baia, bordered with plants known as attractiveto fruitflies. The traps in number of 12, were distributed in the area of about 15 meters distant each one from the other, made from liter perforated plastic bottle containing 200 mililiters of attractive substance, and installed about 1. 7 meters from the ground level. The traps were baited with Vegamine™1 (Protein-h.ydrolisa1e) plµs ampnium sulphate at the proportion of seven to one; sugar cane syrup plus amonium sulphate at theproportion of three to one; a ten percent solution of Vegamine™ and a twenty five percent solution of concentrated maracock juice. The flies were captured during seven days and counted weekly. All traps were substituted weekly and baited with a new solution. The flies were separated in the laboratory and their species determined withthe use of thespecies key fromZUCCHI (1978).

3. Results and Discussion

In Table I it could be observed that 1004 individuals of fruit flies were captured. From these, 72.9% were C. capitata and 27.1% from the genus Anastrepha. The highest number of C. capita/a was collected from June to October, coinciding with thedryest months of the year and the yield of oranges. These results were similar to those of RAGA (1996). The Anastrephas were mostly collected from February until April, and from September until November. In Table II we can observe that the percentage of males of C. capitata was 38.7% and the rest was females, and to genus Anastrepha 48,9% were males and therest was females. In Table III it could be observed that the flies of the genus Anastrepha and the highest percentage was from A. fraterculus (63.3%). A.

1 TM =Trade Mark - 24 - bistrigata was 17.7%, A. obliqua was 15.1%, A. sororcula 1. 1 % and the species A. serpentina, A. barbiellini, A. pseudoparalella and A. distincta had the sum of0.8%. These results confinned those of NASCIMENTO & ZUCCHI ( 198 1), NASCIMENTO & ZUCCHI (1982), ARAUJO et al. ( 1996) and RAGA et al. ( 1996). In Table IV we can observe that the most efficient attractive was the concentrated solution of maracock juice at 25%, which has captured 42,3% of C.capitata and 46, 1 % of Anastrepha spp.

Table I - Number of monthlycaptures of C. capitata and Anastrepha spp . .in Piracicaba, State of Sao Paulo, from February i990 until February 199 1.

MONTH C. capitata Anastrepha spp.

February 0 28 March 0 44 April 0 40 May 16 3 June 123 0 July 239 5 August 110 8 September 1 15 34 October 94 4 1 November 14 16 December 17 11 January 4 7 February 0 35

TOTAL 732 272 - 25 -

Table II - Percentage of males and females of C. capitata and Anastrepha spp. captured in Piracicaba, State of Sao Paulo, from February 1990 until February 1991.

C. capitata Anastrepha spp.

males females males females 38.7% 61.3% 48.9% 51.1%

Table III - Species of the genus Anastrepha spp. captured in Piracicaba, State of Sao Paulo, fromFebruary 1990 until February 1991.

Species Percentage A. fraterculus 63.3 A. bistrigata 17.3 A. obliqua 15.1 A. sororcula 1.1 A. serpentina 0.8 A. barbiellini 0.8 A. pseudoparalella 0.8 A. distincta 0.8

Table IV - Percentual efficiencyof attractiveson thecapture of C. capitata and Anastrepha spp. in Piracicaba, State of Sao Paulo, from February 1990 until February 1991.

Attractant C. capitata Anastrepha

Sugar cane syrup plus amonium sulphate at the proportion of three to one 4.6% 6.0%

Ten percent solution of VegamineT M 24.4% 20.5%

Vegamine T M plus amonium sulphate at the proportion of seven to one 28.7% 27.5%

Twenty five solution of concentrated maracock juice 42.3% 46.1% - 26

References

ARAUJO, E.L.; ZUCCHI, R.A; MALAVASI, A; NEGREIROS, J. (1996) - Levantamento de moscas-das frutas do genero Anastrepha Schiner, 1868 (Dip., Tephritidae) nos municipios de Mossor6 e Assu, RN. Revista de Agricultura, 71(2):225-232. BRESSAN, S. & TELES, M.C. (1991) - Lista de hospedeiros e indices de infestayao de algumas especies do genero Anastrepha Schiner, 1868 (Diptera: Tephritidae) na regiao de Ribeirao Preto - SP. Anais Sociedade Entomologica do Brasil, 20(1):5-15. BLEICHER, E.; SCHROEDER, AL.; BLEICHER, J. (1978) - Efeito de modelo de frasco "caya-mosca" a atratividade na captura de moscas-das­ frutas Anastrepha fraterculus (Wied., 1930). EMPASC, Comunicado Tecnico, 14, 9p. CALZA, R.; SUPLICY FILHO, N.; RAGA, A; RAMOS, M.R.K. (1988) - Levantamento populacional· de moscas-das-frutas do genero Anastrepha em varios municipios de Sao Paulo. Arquivos do Instituto Biol6gico, 55(1/4):55-60. ENGELSTEN, N. & AMARAL, P.M. (1987) - Uso de atrativos alirnentares na captura de Tefritideos (Diptera). Congresso Brasileiro de Entomologia, 9(2):501. FEHN, L.M. (1977) - Levantamento da ocorrencia das moscas-das-frutas, Diptera, Tephritidae, em pessegueiro na area metropolitana de Curitiba e regiao de Arati, Parana. Universidade Federal do Parana, Tese de Mestrado, 172p. GOMES, S.J. (1942) - Moscas-das-frutasespecies capturadas em frascos "caya moscas". Relayiio das especies dos generos Anastrepha e Lucamophyla no Brasil. Boletim da Sociedade Brasileira de Agronomia, 5(1):25-37. HERNANDEZ-ORTIZ, V. (1992) - El genero Anastrepha Schiner en Mexico (Diptera: Tephritidae) - taxonomia, distribucion y sus plantas huespedes. Xalapa, Sociedade Mexicana de Entomologia, 162p. LIMA, AC. (1926) - Sohre as moscas-das-frutas que vivem no Brasil. Chacaras e Quintaes, 34(1):21-24. NASCIMENTO, AS. & ZUCCHI, R.A (1981) - Dinfunica populacional das moscas-das-frutas (Diptera, Tephritidae) no Reconcavo Baiano. I - Levantamento das especies. Pesquisa Agropecuaria Brasileira, 16(6):763- 767. - 27 -

NASCIMENTO, A.S.; ZUCCHI; R.A.; MAORGANTE, J.S.; MALAVASI, A. (1982) - Dinamica populacional das moscas-das-frutas do genero Anastrepha (Dip., Tephritidae) no Reconcavo Baiano. 11 - Flutuayao populacional. Pesquisa Agropecuaria Brasileira, 17(7):969-980. MALAVASI, A. & MORGANTE, J.S. (1980) - Biologia de "moscas-das­ frutas"(Diptera: Tephritidae). I. Lista de hospedeiros e oconencia. Revista Brasileira de Biologia, 40(1):9-16. PUZZI, D. & ORLANDO, A. (1957) - Ensaios para seleyao de substancias, sob forma liquida como atraente para a "mosca do Meditenaneo" Ceratitis capitata (Wied., 1924). Arquivos do Jnstituto Biol6gico, 24(10): 137-161. PUZZI, D.; ANDRADE, A.D.; CAMARGO, J.C. (1955) - Experiencia de controlea mosca das frutasem 1955. Biologico, 21(1): 185-188. TAMASHIRO, E.; MENEZES, E.B.; OGAWA, E.S. (1984) - FlutuayaO populacional de "moscas-das-frutas" Anastrepha spp. (Wied., 1839) (Diptera: Tephritidae). I. Lista de hospedeiros e oconencias na baixada (R.J.). Congresso Brasileiro de Entomologia, 9(1):97. RAGA, A.; BATISTA FILHO, A.; SOUZA FILHO, M.F. DE; MACHADO, L.A. (1994) - Grau de infestayao e incidencia de moscas-das-frutas (Diptera, Tephritidae) em laranja doce Citrus sinensis. Cngresso Brasileiro de Fruticultura, 13(2):386. RAGA, A.; BATISTA FILHO, A.; SOUZA FILHO, M.F. (1996) - Dinamica populacional de adultos de moscas-das-frutas (Diptera, Tephritidae) em pomar de citros de Presidente Prudente. Arquivos Jnstituto Biol6gico (no prelo ), lOp. ZUCCHI, R.A. (1978) - Taxonomia das especies de Anastrepha Schiner 1868 (Diptera, Tephritidae) assinaladas no Brasil. Piracicaba, ESALQ/USP, Tese de Doutorado, 105p. - 28-

RECHERCHESSUR LA MULTIPLICATION DE CERATITIS CAPITATA WIED. DANS LA FORET D' ARGANIER AU MAROC ET SUR LES POTENTIALITES DE SON DEVELOPPEMENT EXPERIMENTAL SUR DEUX HOTES DE RELAIS.

1 1 1 K. NAAMANI ; C.HERBAUT 2; A. AIT EL KOCH 1; A. BAKRI & M. CHEMSSEDDINE. (I): Fae. Sc. Semlalia, Departement de Biologie., B.P.S 15, Av. Pr. My Abd Allah, Marrakech, Maroc. (2): Centre Univ. de Polynesie Fran9aise, B.P. 6570 FAAA- AEROPORT, TAHffi- Polynesie Fran',aise.

Resume Ceratitis capitata est un seneux ravageur de nombreuses cultures fruitieres. L' Arganier (Argania spinosa: Sapotaceae), arbre endemique du Maroc, constitue l'un des sites naturels pour la proliferation de Ja ceratite. L'etude de la dynamique de population de ce diptere sur Jes fruits d' Arganier dans la region d'Essaouira (Sud-Ouest marocain), a montre qu'il attaque au debut du mois de Mai Jes fruits mfirissants ou mfirs trouves chutes, surtout des cotes Est et Sud (respectivement: 50% et 25%), avant qu'elle ne se generalise a toutes Jes expositions ou elle atteint 100% vers le mois de Juin. Les fruits non chutes - moins mfirs- sont moins infestes, surtout du cote Nord. En !'absence de traitement, le seul facteur pouvant limiter Ja proliferation des larves, est Ja chaleur estivale qui irradie toute larve vivante. Le taux de fertilite des oeufs a temperature optimale, se situe entre 14% et 94%. En parallele, on a experimente au laboratoire !'infestation des poivrons et des tomates cultives dans Jes zones avoisinantes. Il en resulte un taux d'eclosion de l'ordre de 54% pour le poivron, alors qu'il ne depasse pas 16% a Ja premiere generation (et 44% a Ja seconde) pour Ja tomate.

1 - Introduction : La mouche mediterraneenne de fruits Ceratitis capitata W.( Diptere:Tephritidae ) est le plus grand ennemi de J'arborictilture. Onie-signalepresque dans tous Jes pays ayant un clirnat ternpere, grace a sa grande polyphagie. Dans le rnonde, ii attaque plus de 200 varietes de fruits cultives ou sauvages ( Bodenheimer, 1951; Mitchell, 1977 ), ou, 353 selon Liquido et al. ( 1991 ). Au Maroc, cette espece est presente sur Jes arbres fruitiers et ptillule dans les·fruits de l'Arganier, ce qui a conduit Sacantanis ( 1957 ), a qualifier la foret d'Arganier cornrne etant .. le plus grand foyer de Ja ceratite connu au rnonde ··. Les etudes concernantla relation de ce diptere avec l'Arganier sont rares et incornpletes, d'ou s'avere la necessite de ces investigations. L'Arganier couvre au Maroc environ 700 OOO ha, dont 150 OOO ha dans Ja region d'Essaouira ( site d'etude ), ou il constitue un element stabilisateur pour Jes habitants. Le fruit est une baie ayant une forme ovale de 3 a 4 cm. de longueur; une pulpe externe ( ou se developpe Jes Jarves ) et un noyau dur contenant au centre une amande d'ou est extraite l'huile d' Argan qui possede d'enormes valeurs nutritives. Le developpement de la ceratite sur !' Arganier se poursuit durant la periode de fructification ( Avril - Aofit, parfois, il y a une fructification automnale ). Au laboratoire, on a pratique des essais d'infestation d'autres fruits: tomate et poivron, cultives dans Jes zones voisines, pour avoir une idee preliminaire sur leur potentialite d'intervention comme hotes de substitution pour le maintient de la population de la ceratite jusqu'a Ja fructification suivante. - 29 2 - Materiels et Methodes: 2 -1 : Sur Arganier: L'etude a ete entamee dans la region d'Essaouira ( Sud- Guest marocain ) a 150 Km. au sud- Ouest de Marrak:ech. La foret est representee par des pieds d' Arganier de 3 a 6 m. de hauteur et espaces de 5 a 60 m. La zone est classee dans le semi aride, beneficiant d'une moyenne de pluviometrie de l'ordre de 300 mm. Durant l'etude, Jes variations climatiques enregistrees a partir du poste d'Igrounzar avoisinant sont representees sur la figure I. Les prelevements sont effectues tous Jes 20 jours. On preleve 100 fruits pour Jes deux types chutes et non chutes, par orientation. Au Iabo, les fruits sont analyses pour denombrer les piqGres et Jes oeufs, puis disseques pour recueillir les Iarves dont on note le nombre par stade larvaire. 2- 2 : Sur la tomate et le poivron: Les mouches maintenues en elevage a partir de pupes recueillies des fruits d'Arganier, sont mises dans des cages (32 x 32 x 32 cm.). On Ieur expose pendant 2 a 3 h. une tornate ou un poivron ou les femelles effectuent leur ponte. Les fruits sont mis par la suite chacun sur une portion de grillage surrnontant une petite cuve ( pour proteger les larves de se noyer dans l 'eau secretee par le fruit ), le tout est mis dans un bac contenant du sable et recouvert par un tissu a mailles tres fines.

3 - Resultats: 3 - 1 - Arganier: 3 -1 - 1 - La ponte: L'etude a montre qu'elle debute des !'apparition des premieres fructifications surtout du cote Est dont les fruits apparaissent et mfirissent precocement ( fig. 2 et 3 ) et qui seraient plus receptives aux attaques de la ceratite. Par la suite, la frequence des attaques augmente au fur et a mesure que la population de la ceratite et les fruits recepteurs deviennent plus importants, jusqu'au debut du mois de Juillet, surtout des cotes Est et Ouest, pour les deux types de fruits. Mai� cette activite de ponte connalt une forte chute au cours de ce rneme mois sous l'effet des fortes chaleurs estivales. La reprise est lente une fois que Ies conditions climatiques se sont ameliorees au mois d' Aofit et avant la recolte totale des fruits. La fertilite des oeufs de la ceratite debute importante du cote Sud pour Ies fruits chutes, puis ii s'y ajoute l'Est par la suite. Pour ceux non chutes, l'eclosion ne devienne importante que vers fin Mai au sud et a I'Ouest (Tab.I), avant que la chaleur du mois de Juillet ne vienne alterer leur developpement. II reprend au mois d' Aofit particulierement au Nord. 3 -1 - 2 - Les Larves: Elles apparaissent surtout dans Ies fruits chutes et aussi du cote Est. Ces Iarves sont representees par Ies stades 1 et 2 essentiellement, puis par le stade 3 au couts du mois de Mai et Juin. L'infestation atteint son maximum au cours du mois de Juin ou t;us Ies fruits chutes recoltes sont attaques. Pour Jes fruits non chutes, !'infestation est mains importante en Mai, puis s'amplifie en Juin surtout a l'Ouest; tandis que le cote Nord, reste le mains touche. Pour Jes deux types de fruits, Tes larves vivantes connaissent une disparition soudaine au debut du mois de Juillet ou elles sont trouvees toutes a l'etat mort - essentiellement aux stades I et 2 - ( fig. 6 et 7; Tab!. I) sous l'effet de la chaleur estivale. Cela s'observe particulierement du cote Est pour Jes fruits chutes et Ouest pour Jes non chutes. Le faible effectif de larves qui survivent au mois d'Aofit, se retrouve du cote Nord le mains expose aux radiations solaires et ou la maturite des fruits est plus Iente. Pour Jes non chutes, la reprise s'effectue essentiellement du cote Sud. 3 -1 - 3- L'infestation: Le taux d'infestation des fruits chutes ( Tab. I ), debute assez eleve du cote Est, augrnentesensiblement au cours du mois de Mai avant de se generaliser en Juin a toutes Jes - 30 - expositions avant de chuter sous !'action des fortes chaleurs. Pour ceux non chutes, !'infestation debute faible pour toutes Jes expositions, augmente sensiblement du cote Est et Ouest.

Tableau : I : Evolution du developpement larvaire de la ceratite et ses potentialites d'infester Ies fruits !ors d'un periode de fructification de I'Arganier dans la region d'Essaouira.

Dates Expositions Nord Sud Est Ouest chutes non chutes non chutes non chutes non Fruits chutes chutes chutes chutes 1 I % 7,14 0 40 0 22,5 0 33,33 0 d'eclosion 5/ % de 0 0 100 - 66,66 - 100 - mortalite 94 % 30 5 25 5 50 5 5 10 d'infestation 20 / % 37,5 0 43,33 14,25 67,37 3,33 36 100 d'eclosion 5 I % de 6,66 0 0 0 20,31 75 5,55 0 mortalite 94 % 65 5 65 5 80 30 50 5 d' infestation 12/ % 46,57 8,7 36,72 39,77 59,1 15,1 38,8 43,6 d'eclosion 6/ % de 7,35 0 3,07 22,85 12,30 6,25 18,9 14,63 mortalite 94 % 100 40 100 50 100 70 100 65 d' infestation 2 / % 27,33 12,8 25,43 14,6 32 24,05 27,45 42,30 d'eclosion 7 / % de 100 100 100 100 100 100 100 100 mortalite 94 % 85 30 70 25 100 40 80 65 d'infestation 21 / % 2,5 0 4 2,77 5,4 42,85 7,7 0 d'eclosion 7/ % de 66,66 - 16,66 0 0 0 100 - mortalite 1Cr 94 % 70 5 70 30 65 10 10 d' infestation 12 / % 54,26 94,44 40,64 56,57 18 43,47 41,33 69,23 d'eclosion 8/ % de 37,07 41,17 38,83 27,9 70,83 26,66 61,43 44,44 mortalite 94 % 100 20 90 70 85 60 95 15 d' infestation

3 -2 - Tomate et poivron : Le developpement moyen du Stade o.euf au stade pupe vivante ( donnant par la suite des adultes), est presque voisin dans Jes deux types de fruits ( 58,24 % pour le poivron et 63 - 31 -

.fi.g.__1_;_Les variations climatiques enregistrees dans la region lors de la periode d'etude ( poste d'lgrounzar ). 140 1 - Les precipitations

120

100

80

60

40

20 o�-...... --.--+--.-- ...... --r- --'�-�-�-+---e---

2 - Les temperatures �T.max( °C 50 �t.min(°C) 45 40 35 30 25 20 15 10 5 0 +---.---.----"'¥.,---,t,,-=jl----r---,.--,----r---,.--,---,----, sept' J/94 mai-94 sept- Dates 93 94

.Ei.g,_2 :Evolution de l'activite de ponte par la ceratite dans les fruits chutes de l'Arganier selon !'exposition.

1800

1600 -+-Nord

1400 -0-Sud -lr-Est 0 1200 . �Quest - en 1000 en '5 � Q)::, •0 .c u 800 600

400

200 Dates 0 01/05/94 20/05/94 12/06/94 02/07/94 21/07/94 12/08/94 • 32 •

Fig. 3 : Evolution de l'activite de ponte de la ceratite dans les fruits non chutes de l'Arganier selon !'exposition. 700

C 0 600

500 -+-Nord -0-Sud � .., ui 400 -+-Est -II) :i --*"-Ouest "fi 300 i0

200 .c� E z 100 Dates 0 01/05/94 20/05/94 12/06/94 02/07/94 21/07/94 12/08/94

Fig. 4 : Evolution de !'infestation des fruits chutes de l'Arganier par les differents stades larvaires vivantsde la ceratite selon !'exposition.

400

!!l 350

300 � 250 -II) ui -+-Nord >QI.., - � ::, 200 -0-Sud .!! '£ -+-Est 150 -*-Ouest .c� E 100

50

0 Dates 01/05/94 20/05/94 12/06/94 02/07/94 21/07/94 12/08/94 - 33 -

Fig. 5 : Evolution de !'infestation des fruits non chutes de l'Arganier par les differents stades larvaires vivants de la ceratite selon !'exposition. 180

160 !!l ] 140 �Nord -0-Sud � ,120 '°'*""Est -�g: ";5100 �Quest >� � � 80 Q) 0 .c:: c: 60 E 40 0 z 20 Dates 0 �:.....==::::::f!!j..,:::::::::..__ ...... ;;;;;;;;===�:::::::::------,__ ...:::::�...... 01/05/94 20/05/94 12/06/94 02/07/94 21/07/94 12/08/94

Fig. 6 : Evolution du taux de mortalite des larvesde la ceratite a 600 differents stades dans les fruits chutes de l'Aganier selon !'exposition.

!!! 500 � �Nord 0 0 400 -0-Sud

-II).; ....,.._Est Q)> - ... � :, 300 -*-Quest -;"� u

Q) 200

0 100

Dates 0 01/05/94 20/05/94 12/06/94 02/07/94 21/07/94 12/08/94

Fjg. 7 : Evolution du taux de mortalite des larvesde la ceratite a 300 differents stades dans les fruits non chutes de l'Arganier selon l'exp sition.

250 !!! -+-Nord g 200 -0-Sud - II) "'� �Est � �50 ....;+-Quest -" C: Ol 0 "O C: .c., 100 0 z 50 Dates 0 iL-====:::2�===��::::=-�-=:::::::��::'::::===: 01/05/94 20/05/94 12/06/94 02/07/94 21/07/94 12/08/94 - 34 - % pour la tomate ), ( Tab. II), mais , ii y a plus de larves recueillies a partir du poivron (53% d'eclosion) que de la tomate ( 16,98 % ) en Gl, alors que la moyenne de ponte est plus importante sur la tomate que sur le poivron. La mortalite des larves au stade 3 et au stade pupe peut connaJtre une legere hausse chez le poivron. Pour la G 2, l'eclosion s'est amelioree chez la tomate et la mortalite des larves a diminue chez le poivron. Le leger retard de la periode du developpement est sfirement du au ralentissement du rythme en Automne ( periode du deroulement de ]'experience)

Tableau II : Donnees sur !'infestation de la ceratite experimentee sur le poivron et la tomate. ( moyenne recueillie iipartir de 13 poivrons en G 1; 8 en G 2 et 12 tomates en G 1 et 8 en G 2 ). ( L 2= larves au stade 2; L 3= larves au stade 3; P = pupe, Moy. = moyenne, ± = ecart type)

Generations Gl G2 Poivron Tomate Poivron Tomate Moy.de la duree du 20 ± 2,01 19 ± 2,85 24 ± 3,38 28,5 2,95 develovvement. ± Moy.d'oeufs pondus 135 ± 58,8 238,61± 94,5 42,42 ± 20 156,5± 41,41 Moy. de L 2 mortes 8,91± 3,58 8,46 ± 3,12 0 37,5 ± 13 Moy. de L 3 mortes 10,16 ± 3,66 3,7 ± 2,9 1,14 ± 0,34 2,5 ± 1,2 Moy. de P. vivantes 42,41 ± 25,76 25,38 ± 7,95 17,14 ± 0,14 29 ± 7,4 Moy. de P. mortes 11,33± 5,15 2,77 ± 1,96 6,57 ± 2,82 1,25 ± 0,43 % d'eclosion 53 16,89 58,58 44,73 % de viabilite des 58,24 63 69 41,07 larves. Nombre de males 327 114 Nombre de femelles 294 121

4 - Discussion : Le rythme d'activite de la ceratite sur l'Arganier, paralt etre tributaire du degre de maturite des fruits et des conditions clitnatiques qui peuvent influencer a leur tour le degre d' infestation des differentesexpositions. Ainsi, aux premieres fructifications, ou on a une dominance de fruits verts et mfirissants, l'attaque de la ceratite ne connaH qu'une activite de ponte, Jes fruits n'etant pas encore infestes. Au fur et a mesure, de la maturation croissante des fruits, l' attaque se generalise et s'amplifie pour n'epargner aucun fruit ( Tab. I ). Puis ce ne sont que Jes conditions climatiques et la recolte des fruits qui parviennent a stopper cet afflux. Pour les fruits chutes, !'infestation se declenche au mois de Mai, surtout du cote Est ou les premiers fruits mfirissent, ce qui rejoint ]'observation faite sur Valencia late en Tunisie ( Dhouibi et al., 1995) ou ]'exposition Sud - Est, suivie du sud - Ouest qui sont les plus attaquees car les fruits mfirs sont plus riches en sucre et seront plus aptes a provoquer l'attaque des insectes. Une autre etude anterieure menee par Bohlen ( in Bateman, 1976 ), a montree aussi que J'exposition Sud - Est est celle ou Jes captures de la ceratite sur agrumes dans Jes conditions du Souss ( Sud - Ouest marocain) sont Jes plus elevees. D'autre part, ces fruits piques peuvent constituer un site favorable a d'autres piqfires se Jocalisant stir celles anciennes ou a leur proximite ( Papaj, 1989 ). Par ailleurs, selon certains auteurs, la femelle pondrait durant les heures chaudes de la joumee ( Bodenheimer, 1951; Feron, 1962) et le pie d'oviposition se situerait a 12 h. ( Hendrichs et al., 1989); le cote de J'arbre - 35 -

le plus ensoleille a ce moment et le plus eclaire est celui expose au Sud- Est, ii est par consequent le plus favorable pour la ponte. Les autres expositions, Nord et Ouest sont egalement frequentees par la mouche, mais avec une activite moindre ( Feron, 1957; Drummond et al., 1984; Zervas, 1986; Hendrichs et al., 1990). De meme la couleur jaune indiquant le degre de maturite du fruit, peut jouer un role pour l'attraction des femelles de la ceratite ou Jes deux sexes comme a montre Katsoyannos ( 1987) !ors de J'experimentation des spheres jaunes assimilees a des fruits. Cette couleur jaune, constitue un ··supernormal"stimuli des Tephritidae pour la recherche des sources de nourritures ( Nakagawa et al., 1978 ). Une autre etude sur I' Arganier dans la region du Souss, entamee par Mazih( 1996 ), n'a pas montre de cote preferentiel pour la ceratite; l'attaque parait homogene. Ceci ne se realise dans la region d'Essaouira, que vers le mois de Juin ou tous les fruits sont a l'etat mfiret I' attaque atteint 100% dans Jes fruits recoltes. Lors de la periode de la haute chaleur estivale au mois de Jui!let ( 45°C et plus ), l'activite de la ceratite se trouve reduite. On observe une nette chute de l'activite de ponte ( Fig. 2 et 3), surtout du cote Ouest. Les larves se trouvent toutes a l'etat mort ( Fig. 4, 5, 6 et 7). L'etude realisee par Shoukry et al. ( 1979), a montre qu'une exposition des oeufs de la ceratite a 30 % d'H.R. durant 6 h. reduit l'eclosion de 98 % a 12 %; apres 12 h., aucune eclosion n'est possible. A la temperature de 35°C, l'eclosion est de 86 %, a!ors que Jes larves deviennent tres foibles et ne peuvent plus quitter le milieu d'elevage, tandis que pour Jes nymphes, cette temperature leur est fatale.De meme, Bateman ( 1972 ), a montre que !'exposition a 45°C pendant quelques heures est fatale pour le developpement des !arves. Une fois l'effet de la chaleur est surmonte au mois d'Aofit, l'activite de la ceratite reprend surtout du cote Nord le moins expose aux radiations solaires. Pour Jes fruits non chutes et qui sont pour la plus part a l'etat vert ou mfirissants, l'attaque se differencie elle aussi du cote.Est en Mai ( Fig. 3), mais Jes larves issues de ces oeufs connaissent une mortalite pouvant atteindre 75 % ( Tab. I), alors qu'a cette periode, on note en revanche une bonne croissance des larves des cotes Ouest et Sud. Cela s'expliquerait par le fait que ces fruits de !'Est sont pour la plupart des fruits verts ( ceux mfirs sont deja chutes ), et ne contenant pas Jes elements nutritifs essentiels pour la croissance des Jarves. Parmi ces elements, on peut citer le sucre dont Ja teneur augmente au fur et a mesure de Ja maturite des fruits tel est le cas de Valencia late ( Dhouibi et al., 1979). Les hotes de substitutions tels la tomate et le poivron ( Solanaceae ), l'etude experimentale montre que c'est la tomate qui re�oit en moyenne un nombre d'oeufs presque double a celui du poivron en G 1 et 4 fois plus en G 2. Cela s'explique par le fait que Ja peau de la tomate est plus facile pour la femel!e pour effectuer la ponte. Mais Ja moyenne de developpement des oeufs sur le poivron est plus importante que sur la tomate. Compare a I'Arganier, le pourcentage d'eclosion dans ce dernier parait plus favorable a la ceratite, ou Jes valeurs peuvent atteindre Jes 94 % ( Tab. I ). D'autres experiences anterieures sur Ja tomate ont montres des resultats plus ou moins importants. Ainsi, Leonard ( 1925), a obtenu 1 larve a partir d'une tomate, Liquido et al. ( 1990) a Hawaii a obtenu 22,54 adultes /Kg.de tomate infeste; Back et Pemberton ( 1918 ) a expose devant 200 ceratites des tomates, il a obtenu 1 a 16 adultes par fruit. Nakagawa et al. ( 1958 ), a obtenu 386 pupes dont 205 adultes ont emerges sur un total de 11,429 tomates. D'autres auteurs ont travaille de meme sur le poivron tels ( Marlatt ( 1931 ), Silvestri ( 1914), Anonymous ( 1975)) in Liquido et al. ( 1991 ). Dans notre present essai, on a obtenu 621 adules a partir de 13 poivrons et 12 tomates en G I, soit 24,84 adulte par fruit; en 02, on a obtenu 235 adultes a partir de 16 fruits, soit 14,69 adultes par fruit. - 36 - 5 - Conclusion : Cette etude preliminaire sur la ceratite et sa relation avec Jes fruits d' Arganier a montre la grande adaptabilite de ce diptere .a cet arbre. Cependant, cette infestation differe quand le fruit est chute ou non : elle est plus importante dans les fruits chutes mfirs ou mfirissants, alors qu'elle reste faible dans ceux verts generalement non encore chutes. Cette infestation debute surtout a partir du cote Sud et Est, avant qu'elle ne se generalise a toutes les expositions avec un degre moindre du cote Nord. Durant cette haute population on peut recueillir un effectif de 30 larves et 110 oeufs par fruit. Le facteur pouvant limiter la proliferation de la oeratite est la temperature ( en plus d'une faible H.R ). En dehors de la periode de fructification de l'Arganier, !'experimentation sur le poivron et la tomate cultives dans les regions voisines pourraient constiti.i�r un hote de relaie pour la ceratite, surtout le poivron. L' Arganier constitue ainsi un enorme reservoir naturel pour la proliferation de la ceratite, toute etude concernantla relation de cet arbre avec ce diptere pourrait apporter des elements complementaires pour le controle de l'espece.

References : BACK, E. A. et PEMBERTON, C. E ( 1918 ) - The Mediterranean fruit fly in Hawaii. USDA. Bulletin 536. BATEMAN, M . A. ( 1972) - The ecology of fruit flies.Annual Review of Entomology, 17: 493 - 518. BATEMAN, M . A. ( 1976) - .Fruit .flies. In: V..LDelucchi (ed ), .. Studies in Biolo..gical control. Cambridge University Press, Cambridge, pp. 11 - 49 .BODENHEIMER, F.S. ( 1951 ) - The Mediterranean fruit fly ( Ceratitis capitata Wied). In : F. S. Bodenheimer ( ed. ), Citrus Entomology, in the Middle East. Dr. W Junk, Den Haag. pp. 87 -160. DRUMMOND, F.; GRODEN, D.; PRO:{(OPY, R. J. ( 1984) - Comparative efficacity and optimal positioning of traps for monitoring apple maggot flies ( Diptera : Tephritidae). Environ. Entomol : 13 : 232 - 235. DHOUIBI, M . H.; GAHBICH, H.; SAAIDIA, B . ( 1995 ) - Evolution de l'attaque des fruits par Ceratitis capitata en fonttion de la position de l'arbre et du degre de maturite des oranges. Fruits, vol. 50, n° 1, p. 39 - 49. FERON, M . ( 1957 ) - Le comportementde ponte de Ceratitis capitata Wied. Influence de la lumiere. Revue de Pathologie Vegetate et d'Entomologie Agricole de France 36 : 127 - 144. FERON, M . ( 1962 ) - L'instinct de reproduction chez la mouche mediterraneenne des fruits Ceratitis capitata Wied. Comportement sexuel, comportement de ponte. Rev. Pathol. Veget. -Entomol. Agric. Fr. 41 : 1 - 129. FIMIAMI,P. ( 1989) - Pest status - Mediterranean region. In: A.S. Robinson et G. Hooper ( ed. ), Fruit Flies, their Biology, Natural Ennemies and Control. Vol. 3 A. Elsevier, Amesterdam,pp. 37 - 53. HENDRICHS, J .; REYS, J .; ALUJA, M . ( 1989 ) - Behaviour of female and male Mediterranean fruit flies, Ceratitis capitata in and around Jackson traps placed on fruiting host trees. Insect Sci.Appl. 10(3): 285 - 294. HENDRICHS, J .; HENDRICHS, M . A . ( 1990 ) - Mediterranean fruit fly, Ceratitis capitata (Diptera : Tephritidae ) in nature : location and diel pattern of feeding and other activities on fruiting and non fruiting hosts and not hosts. Ann. Entomol. Soc. Am. 83: 632 - 641. KATSOYANNOS, B. I. ( 1987) - Field response of Mediterranean fruit flies to colored spheres suspended in Fies, Citrus and Olive trees. Insect - Plants. I.S.B.N. 90 - 6193 - 642 - X. Netherlands pp. 167- 172. - 37 - LEONARD, M . D. ( 1925 ) - Notes on the embargo of grapes from Almeria, Spain, on account of the Mediterranean fruit fly ( Ceratitis capitata Wied ). J. Econ. Entomol. 18 : 257 - 265. LIQUIDO, N. J.; CUNNINGHAM, R. T. et NAKAGAWA, S.( 1990 ) - Host plants of Mediterranean fruit fly on the island of Hawaii ( 1949 - 1985 survey ). J. Econ. Entomol. 83 : 1863 - 1878. LIQUIDO,N. J.; SHINODA, L .A. et CUNNINGHAM,R. T. ( 1991 ) - Host plants of the Mediterranean fruit fly ( Diptera : Tephritidae ) : An annotated world review. MPPEAL, 77 : 1 - 52 MAZIH, A. et DEBOUZIE, D. ( 1996 ) - Infestation rate fruit ( Argania spinosa) by the Mediterranean fruit fly ( Ceratitis capitata ) in relation to phenology and maturation of the fruit. Entomologia Experimentaliset Applicata. 81 : 31 - 38. MITCHELL, W .C.; ANDREW, K. S.; HAGEN, R. A.; HAMil..,TON, E. J.; HARRIS, K. L.; MAEHLER and RHODE, R.H. ( 1977 ): The Mediterranean fruit fly and its Economic Impact in central America Countries. U.C, A.I.D. Pest Management Project Report. Berkley, Calif. Univ. of Calif.Pub. 189 pp. NAKAGAWA, S .; YAMADA, T .; FARIAS, G.; URAGO, T. et BALOCK, J.W. (1958)- The status of cucumber, tomato, eggplant and green pepper as Mediterranean fruit fly host in Hawaii. Special Report HO - 21, 25 April 1958. USDA, ARS, Entomology Research Division, Honolulu. NAK AGAWA, A .S.; PROKOPY, R. J.; WONS, T .T .Y.; ZIEGLER, J.R.; MITCHELL, S . M. ; URAGO, T. and HARRIS, J . ( 1978 ) - Visual Orientation of flies to fruit models. Ent. Exp. Appl. 24: 193 - 198 p. PAPAJ, D . R. ; KATSOYANNOS, B . I. and HENDRICHS, J . ( 1989 ) - Use of fruit wounds in oviposition by Mediterranean fruit flies. Entomol. Exp. Appl. 53 : 203 - 209. SACANTANIS, K . B. ( 1957 ) - La foret d'Arganier, le plus grand foyer de Ceratitis capitata connu au monde. Bolletino Laboratorio Entomologia Agraria ·· Filippo Silvestri·· di Portici 15 : l - 53. SHOUKRY, A. et HAFEZ, M.( 1979 ) - Studies on the biology of the Mediterranean fruit fly Ceratitis capitata. Ent. Exp.et Appl. 26 ( 1979) : 33 - 39. ZERVAS, G . A . ( 1986 ) - Trapping Mediterranean fruit flies in delta and plastic Mc Phail traps. iin Fruit flies. Proceeding of the second international symposium of fruit flies, 16 - 21 Semptember,1986. Colymbari, Crete, Greece. Amesterdam, Pays - Bas, Elsevier, Economopoulos ed., p 475 - 481. - 38 -

EFFECT OF SOIL MOISTURE AND TEXTURE ON PUPAL SURVIVAL OF THE MEDITERRANEAN FRUIT FLY, Ceratitis capitata(Wiedemann) (DIPTERA: TEPRHITiDAE)

1 2 1 A. A. Del Pino , M.T.G. Putruele & A. Garrido 1 • Dpto. de Protecci6n Vegetal y Biolecnologia. lnstitnlo Valenciano de Jm·estigaciones Agrarias (IVIAJ -+6113-Moncada. Valencia. SPAIN 2Instituto Nacional de Tccnologia Agropccuaria ([NT A) Estacion Experimental Agropccuana Concordia CC. 3-+ (3200) Concordia. ARGENTINA

Summary The survival of the pupal stage of Cerafifi.1· captlata (Wied.) was studied in several soil textures, irrigated at different times during pupal development. Additional experiments were conducted to determine the cause of mortality. Results showed that survival depended on soil type and moment of irrigation. For sandy soil, the highest emergence of adults occurred when the soil was irrigated early during the pupal development period. When it was irrigated at the end of the period, most of the adults emerged from the puparium, but only a few of them (less than 15%) were able to leave out of the soil. When the clay loam soil was irrigated during the first part of pupal development, no adults emerged, but when it was irrigated at the end of the period, the highest emergence was registered (77 %) When the soils were irrigated twice, the adult emergence in the sandy clay loam soil was similar to that obtained when irrigated at the end of the pupation period. The lowest emergence rate was registered in the clay loam soil. Results showed that mortality occurred because adults were unable to quit their puparia ( in clay soils), or dig out of sandy soils. Related words: Ceratitis capita/a, soil, moisture, irrigation, survival rate.

I - Introduction

The Mediterranean fruit fly, Ceratitis capitara (Wied.), is a major orchard pest in the Mediterranean area. Fluctuations of its population are strongly affected by rainfall. Thus, a period of time with high precipitation may negatively affect adult behavior (Zapien et al, 1982; Prokopy and Hendrichs, 1979) and increase pupal mortality (Santaballa. l 996 ), thus reducing the population Several authors have shown the negative effect of precipitation on fruit fly pupal survival especially when combined with low temperatures (Bodenheimer. 1951; - Trottier and Townshend, 1979; Neuenschwander et al, 1981) Interaction of soil texture and moisture, and temperature 1s important to determine the survival rate of C. capita/a ·pupae. Eskafi and Fernandez (1990), suggested that anoxia may cause pupa to die during the rainy season in soils with high bulk densities Thus, pupal mortality in the soil is not affected by drought, but, by - 39

excessive moisture Milward-de-Azevedo and Postali Parra ( 1989) reported that in clay soil, higher survival was obtained under ·dry conditions, while in sandy soil, the higher values were obtained with intermediate moisture levels. Another element affecting pupal survival is the depth at which the third larval instar buries itself, because of its interaction with several factors as predation, compactness of the soil, pathogens, etc. Moisture and texture determine pupation depth of fruit flies (Milward-de-Azevedo and Postali Parra, 1989; Cavallaro and Del Rio, 1975; Hennessey, I 994) Working on C. c.:apitata. Gomez Clemente (in Ruiz Castro, 1945) reported that if the third larval stage buries itself more than 7 cm. deep, although pupa completes development, most of the adults are unable to get out of the soil. Most of the laboratory studies dealing with pupal development at different soil moistures, have been carried out using constant levels of water content during the whole pup-atiorr period, but in nature, rain may affect pupae at different ages and humidity may persist only foran interval of time. Soil compactness and water content may change during the pupation period and affect both pupal development and final adult emergence. Thus, the objective of this study was to measure the influence of soil moisture and texture and pupal age on adult emergence in a laboratory experiment.

2 - Materials and Method Soils were obtained at 3 different locations, representing different cultivated areas of Valencia two of them from citrus orchards and the other one from a stone fruit area. The three locations are usually affected by C. c.:apilala. For all experiments, dry top soils, 10 cm. deep, were sifted through a screen of 2 mm mesh. Additional experiments were carried out with river sand sifted through the same mesh. The samples were analyzed for soil properties: The pH was determined by suspending l O g. of dry soil in 20 ml of distilled water and measuring immediately with a bench-top pH meter (Hennessey, 1994) Texture, bulk density and field capacity were determined following the method of Ingelmo and Cuadrado ( 1986) Field capacity of the soil is defined as the percentage of water retained by the soil in a metastable condition against the downward pull of gravity over a long period of time Thus, field capacity was determined by placing the soil in a metal cylinder (5 cm. high by 5 cm. diameter) with a fine mesh bottom and then immersed in 4 cm. deep water while water was added from the top. Sample was left 24 hours in water, after that, the cylinder was lifted and the excess water was left to drain during 24 hours more. Field capacity was determined by the difference between the wet and the oven-dried weight soil Insects used in our assays were obtained from a laboratory colony reared at. the IVIA during the last three years on an artificial medium as described by Putruele et al ( 1996). Mean pupal weight was 1 I .7 mg.(SD = 0 24) at age 0. - 40 -

° ° Experiments were carried out under laboratory conditions, at 26 C (±2 ), 60% RH (±10%), 12 12 L:D. Under these conditions, the pupal stage lasted from 9 to 12 days

To carry out the experiments, 6 plastic cylindrical containers (9.5 cm. diameter. 4,0 cm. high) with bottom perforate to provide drainage, were half-filled (2 cm.) with each soil. Then, 25 newly formed pupae (0-5 hour old), were picked with a small soft-tipped forceps and distributed regularly over the surface of the soils. After that, containers were filledto top. Thus, pupae were buried 2 cm. deep. Percentage data were transformed by arcsin('1p), previous to ANOVA analysis. Means were compared, when the F value was significant, by the Student­ Newman-Keuls procedure (level of signification 95%) Software used was the Statgraphics Vers 7.0 (1993) program. Irrigation at different ages. Soils were wetted only once by adding water until field capacity was reached, then allowed to dry under laboratory conditions. River sand was irrigated with double soil capacity, to get a homogeneous soil humidity. Irrigation was applied at different pupal ages, when they were 0, 2, 4, 6 and 8 days old. Thus, 5 differenttreatments were made with six replicates each. Samples were covered with screened plastic cylinders, to make an aerated emergence system. In each treatment, four days after the end of adult emergence, a count of normally emerged flies, dead buried adults, as well as eclosed and uneclosed pupae was carried out. Samples were weighted daily to register the loss of water content by evaporation Irrigation at beginning and end of pupation. This experiment was made in order to test if irrigation facilitated adult emergence from a dry, compact soil. Thus, each sample was irrigated at two different times: first when pupae were I day old, and again 7 days later, near the end of pupal development. Under laboratory conditions, at the time of the second irrigation, soils were compact, dry, and water had to be· added very carefully, to wet uniformly the whole volume of soil.

Testing for pupae survival. Clay0loam soil was chosen to . make this experiment because this soil is easily compressed when dry, and has a brick-like consistency. Twenty samples were arranged as above, and irrigated the first day of pupal development. Then, each time 4 samples were removed at the 6, 7, 8, 9 and 10th days, and pupae were gently recovered and transferred to Petri dishes. These were placed under laboratory conditions until adult emergence occurred. Thus, we tested if the low adult emergence was due to 'pupal mortality or because adults were ·unable to emerge

3 - Results Table l shows soil properties All soils were slightly alkaline and of similar pH. therefore pH was not considered a factor in the experiments. - 41 -

Table I. Soil properties of the samples considered. Texturea clay silt sand Bulk density pH Field capacity %, 4�{, 1x. ('.X,water)b Sand O 5 0 5 99 U,9 7 55 6 75 Sandy-loam I(, + 80 1.19 7.99 20 00 SandY-cl,n-loam 23 7 70 125 7.52 39.6+ ClaY-loam 37 37 26 117 7.83 ++ I+ ausoA standards for soil bFollowing lngelmo and Cuadrado ( 1986). weight percentage.

Figure I shows loss of water due to evaporation under laboratory conditions. When soils were irrigated at the beginning of pupal development. they were almost dry 10 days later. which coincide with the time of adult emergence. The sandy-loam soil had a hard top layer of approximately I cm. deep. while the clay-loam soil acquired a brick-like consistency Sand was completely dry and loose. Figure 2 shows at the end of the assay. the percentage of adult emergence. died buried adults. and non eclosed pupae recovered when soil samples were removed. Both the soil type and the moment when irrigation was made significantly influenced the number of adults emerged. Most flies emerged from sand when irrigated at the beginning of pupation (figure 2a) From the remaining soil samples (all of them containing clay particles). the highest emergence rate occurred when soils were irrigated at the end of pupal development (figure 2b. 2c and 2d). Table 2 shows the percentage of adult emergence when soils were irrigated twice. The effect on loamy soil was similar to that obtained when samples were wetted only the 8th day Clay soil was very difficult to wet. and it conserved the compactness at least during 2 more days after being wetted.

Tahle 2: C. t:apitata adult emerged at two different irrigation regimes Soil texture x ± SO adult emerged x ± SO adult emerged Significativ from soils irrigated from soils irrigated at e difference twice the end of pupation Sand 34.5 ± 117 14.7 ± 9.3 ** Sandy-loam 72 (, ± 84 713 ± 8.9 Sandy-clay-loam 318 ± 8.7 76.7 ± 18.8 ** Clay-loam 13 ± 2.1 50 7 ± 28.9 *** ** Significative difference (p < 0. 0 5) *** Significative difference (p<0.01)

Adult emergence of pupae recovered from the clay soil are showed in figure 3. Some of these pupae were damaged when recovered. thus. actual survival rates were underestimated. In any case. similar survival rate were recorded in all samples. though. because of the mortality caused when removed. no statistical comparison was made - 42

j----6- Sandy-clay-loam i--e-clav-loam - I i�Sand j-o-sandy-loam

6 10 12

Days afterirrigation

Figure 1: Loss of water after irrigated at laboratory conditions

Fig 2a: Sand Fig 2b: Sandy-clay-loam 100% T 9 80%0% 70% i 60% 40%50% t

30% + 20% + 10% 0% 0 4 6 0 6 Pupalage at the irrigation time Pupal age at the irrigationtime

Fig 2c: Sandy-loam Fig 2d: Clay-loam

20%

10% 0% 4 6 4

Pupal age at the irrigationtime Pupal age at the irrigationtime

I O adults emerged [;Iadults buried launeclosed pupae ! Fig. 2: Percentage of normally emerged adults, adults which died while emerging, and uneclosed pupae. - 43 -

7 JO Days afterirrigation

Figure 3: % of adult emerged from pupa recovered after irrigation on day soil

4 - Discussion Soil type caused a different patternof adult emergence. In the three soil types considered, the number of adults which could not emerge was higher when samples were irrigated at the beginning of the pupation period. Thus, a slight proportion of clay in soil composition was enough to make a hard upper layer difficultto break by newly emerged adults on their way up to the soil surface. When soils were completely dry and compacted, adults were even unable to emerge from their puparia. When soils were partially wetted at the moment of the emergence (soils irrigated at days 6 and 8), a part of the freshlyemerged adults were able to reach the surface. Pupal mortality is increased in wet conditions, especially in soils with high clay proportion (Eskafi and Fernandez, 1990; Milward-de-Azevedo and Postali Parra, 1989). But our results have showed that a short period of humidity does not directly affectpupal survival. In this case, a part of the failed adult emergence was caused by the increment of hardness and weight of soil. The importance of loose vs. packed dry soils, as they affect the emergence of adult stage from pupation site, has been studied on Bactrocera oleae by Neuenschwander et al (1981). They showed that, upon emergence, teneral adults suffered a higher mortality in soils which, following inundation, had hardened to a brick-like consistency. A similar pattern was reported by Roach and Campbell (1983) working on Heliothis spp., moth adult emergence was limited by the combination of water addition and compaction of soil. Results obtained in this study suggest that, under field conditions, rain may have a very different effect on adult emergence depending on soil texture, pupal age, - 44 -

and posterior climatic conditions. Results confirm that after a dry period, rainfall may let adults emerge, as reported previously by other authors (Aluja, 1993; Vadora et al, 1990), though adults will survive within the puparium only a few days. Irrigation towards the end of pupal development will reduce adult emergence on sand, while in soils with a clay percentage, it does not reduce drastically adult emergence and may actually help emerging adults when soils are dry and compacted. We have obtained a remarkable difference between the river sand and the others soil-types tested. This may indicate that experiments carried out using this medium are far from natural conditions. Presence of weeds, litter cover, tillage, cracks, arthropod burrows and other factors, which can make the soil looser, may have an important effect on pupation depth and then, on the subsequent facilityemergence of new adults.

Acknowledgment We thank F. Ingelmo and M. Estela, Soil Laboratory, IVIA, for their help with the soil analyses, and J Jacas and R. Arouni, Entomology Laboratory, IVIA for their critical review of the manuscript.

References ALUJA, M. (1993) - Manejo integrado de la mosca de la fruta. Trillas, Mexico, 251 pp. BODENHEIMER,FS. (1951) "Citrus Entomology in the. Middle East W Junk. The Hage, Netherlands. pp. 87-160. ESKAF!, FM . and FERNANDEZ, A (1990) - Larval-pupal mortality of Mettiterranean Fruit Fly (Diptera Tepbritidae) from interaction of soil, moisture, and temperature Environ. Entomo!. 19: 1666-1670. HENNESSEY, MK (1994) - Depth of pupation of Caribbean Fruit Fly (Diptera Tephritidae) in soils in the laboratory. Environ. Entomo!. 23 1119-1123. INGELMO. F. and CUADRADO, S. (1986) - El Agua y el medio fisico del suelo. Centro de Edafologia y Biologia Aplicada (CS I.C), Salamanca. 101 pp. MILWARD-DE-AZEVEDO, E.M.V. and POST ALI-PARRA. JR. (1989) - lnfliencia da umidade em dois tipos de solo, na emergencia de Ceratitis capitata. Pesq. Agropec. Bras., Brasilia. 24: 321-327. NEUENSCHWANDER. P, MICHELAKIS, S. and BIGLER. F (1981) - Abiotic factors affecting mortality of Dacus oleae larvae and pupae in the soiL Ent. exp & appl. 30 1-9 PROKOPY. R.J and HENDRICHS, J ( 1979) - Mating behaviour of Ceratitis c:apilata on a field-caged host tree. Ann. Ent. Soc. Am., 72 462-468. - 45-

ROACH, SH and CAl\1PBELL, R.B. (1983) - Effects of soil compaction on Bollworm (Lepidoptera Noctuidae) Moth emergence. Environ. Entomol. 12 1883-1886. RUIZ CASTRO, A (l 945) - Fauna entomol6gica de la vid en Espana. Estudio sistematico-biol6gico de !as especies de mayor importancia econ6mica III (DIPTER.A) Trabajos de! Instituto Espanol de Entomologia (CSI C) Madrid, IOI pp SA.NTABALLA, E. ( 1995) - La mosca de !as frutas Ceratitis capitata (Wiedemann) en el cultivo de los citricos Phytoma E.17,m1a. 72: 53-61. TROTTIER, R and TOWNSHEND, lL (1979) - Influence of soil moisture on apple maggot emergence, Rhago!etis ponome!!a (Diptera: Tephritidae). Can. Entomol. 111. 975-976. VADOR.A, M.L, SALVARR.EY, L and RODRIGUEZ. (1990) - Anexos Relativos al Documento de ldentificaci6n de! Proyecto de Erradicaci6n de la Mosca de] Mediterraneo de! Litoral del Rio Uruguay. Proyecto Regional Argentina­ Uruguay. Organizaci6n de !as Naciones Unidas Para la Agricultura y la Alimentaci6n Tomo 2. FAO. ZAPIEN, G, HENDRICHS, l, LIEDO, P and CISNEROS, A (1983) - Comparative mating behaviour of wild and mass-reared sterile medfly Ceratitis capitata (Wied.) on a field cage host tree - II. Female mate choice. In R. Cavallaro (Ed.), Fruit Flies of Economic Importance. Balkema, Rotterdam, pp. 397-409. - 46

RELATIONSHIPBETWEEN THE PHOTOPERIOD AND THE PRESENCE OF MATURE EGGS INBACTROCERA OLEAE (GMEL.) (DIPTERA: TEPHRITIDAE)

A. Rasoi, A. Canale & A. FelidoU Universita di Pisa, Dipartiment;di Coltivazionee Difesa delle Specie Legnose,Sezione Entomologia Agraria, via S. Michele degli Scalzi, 2 - 56124 Pisa.

SUMMARY The Authors report results on the presence of Bactrocera oleae (Gmelin) with mature eggs captured by yellow chromotropic traps in three localities during six years of investigation in Tyrrhenian coastal areas of Central Italy (44th parallel of latitude N). Traps were replaced and captures examined every ten days throughout the year. Preliminary experimental data concerning the relationship between the photoperiod and the presence of B. oleae with mature eggs in a laboratory reared population are also reported. Results obtained from field captures show a lack of mature eggs in the ovaries of B. oleae during late spring and early summer, as previously reported in the literature, while 63% of females reared under constant 16:8 (L:D) photoperiod show the presence of mature eggs. The Authors discuss the results obtained from both the field and laboratory experiments on the basis of data available in literature. Earlier and new evidence, associated with a revision of the available literature on this topic, allow the Authors to speculate that the photoperiod plays a key role in accounting for the lack of mature eggs in B. oleae adults in during late spring and early summer, when the photophase is undergoing continuous increase from the end of April (day-length 13.30) to June 21 (day-length 15.16). The Authors also point out that with a roughly 12: 12 (L:D) photoperiod (March-April and September-October) two peaks of females with eggs are observed, suggesting that, at this value, the photoperiod likewise plays a role in favoring oogenesisin B. oleae.

1 - INTRODUCTION The olive fly, Bactrocera oleae (Gmel.), is the key phytophage of the olive grove agro-ecosystem throughout Mediterranean basic countries. In this area the fly lives by feeding on the stone-fruits of cultivated olive (Olea europea sativa Hoffin. et Lk.) and wild olive (Olea europea oleaster Hoffin. et Lk.). The annual trend and extent of B. o/eae infestation are influenced mainly by abiotic factors (temperature and humidity), which regulate development time, preimaginal mortality and the fertility of females (Delrio & Prota, 1975-76; Delrio & Cavalloro, 1977; Kapatos & Fletcher, 1984). Ovarian ripening is modulated by climatic factors but also by the food supply (presence/absence of olives and/or sources of protein) (Fletcher, 1978; Fletcher & Kapatos, 1983; Kapatos & Fletcher, 1984; Neuenschawander et al., 1986). Tzanakakis & Koveos (1986) showed by laboratory experiments that B. o/eae adults raised under long photoperiod (16:8 (L:D)) and high temperature (26°C) conditions presented an elevated percentage of females with immature ovaries if their respective pre­ imaginal stages were maintained under a short photoperiod (12: 12 (L:D)) at moderately low temperature (20°C). According to Tzanakakis (1987) the observed ovarian immaturity" has the attributes of a facultative reproductive diapause "; he therefore states that " the olive fruit fly should no longer be considered a homodynamic insect ". This hypothesis is widely supported by experimental data recorded in the field in different geographic areas showing that olive fly females have no mature eggs in late spring - early summer (Baranov, 1937; Ayoutantis et al., 1954; Stavrakis, 1973; Delrio e Prota, 1975-76; Delrio e Cavalloro, 1977; - 47 -

Economopoulos et al., 1977; McFadden et al., 1977; Fletcher et al., 1978; Neuenschwander e Michelakis, 1979; Stavrakis e Fytizas, 1980; Ballatori et al., 1981; Girolami et al., 1983; Neuenschwander et al., 1986; Tzanakakis et al., 1986; Canale, 1994; Solari, 1995; Raspi et al., 1996), in some case even in the presence of olive fruits (Fletcher et al., 1978; Tzanakakis & Koveos, 1986). The aim of this study was therefore to investigate the effect of the photoperiod as a primary factor inhibiting egg ripening in B. oleae in late spring-early summer.

2 • MATERIALSAND METHODS The data utilized in this study are the re-elaboration ofresults obtained by the Authors over 6 years of observations carried out in three different olive growing areas, two in Tuscany and one in Liguria. Observations were performed in Diano Calderina (IM, Liguria) for the years 1989-1991, Montopoli Valdarno (PI, Tuscany) for the years 1992-1993, and Alberese (GR, Tuscany) for 1995 (Crovetti et al, 1992; Canale, 1994; Raspi et al., 1996). These olive groves are located between the 44th and 42nd parallel N, in the climatic band of annual mean temperature of 16-17 °C (IM) and 15-16 °C ( PI, GR). The predominant cultivar are Taggiasca in Liguria and Leccino, Frantoio e Moraiolo in Tuscany. B. oleae adults were captured by means of rectangular (15cm x 20cm) yellow chromotropic plexigrastraps smeared with entomological glue, using three traps per hectare. Traps were replaced every l O days. B. oleae females were removed from the chromotropic traps by means of an organic solvent, placed in physiological solution for 12 hours, and dissected under the stereomicroscope to search for mature eggs. Mean number of eggs per female was calculated by dividing the total number of eggs collected by total number of femaleswith mature eggs. The climatic trend of this area was analyzed by referring to mean monthly temperatures over the thirty-year period 1926-1955 for the climatic bands having a mean annual temperature of l 6-l 7°C (Imperia) and l 5-l 6°C (Pisa) (Publication of the Ministero dei Lavori Pubblici, 1966). Observations were also carried out on mature eggs in flies reared in the laboratory in various different conditions. Preimaginal instars were reared on a substrate of olive stone­ fruits with a 10:14 (L:D) photoperiod and mean temperature of 20 °C. Pupae that were close to hatching were then placed in a climatized chamber at 26 ± 1 °C, with 45% relative humidity and constant photoperiod of 12:12 (L:D) (experiment A) or 16:8 (L:D) ( experiment B). Adults emerged and reared in this environment were fed with a dry diet composed of10 parts oficing sugar and 1 part of yeast extract. Water was made available in a purpose-designed drinking container (Raspi & Loni, 1994). Determinations were carried out after 14 days to investigate the presence of any mature eggs in females. Data obtained were subjected to';( test.

3- RESULTS Graphics 1 and 2 show percent presence of females with eggs at different times ofthe year in relation to day-length, and mean number of eggs, respectively. For each of the six trial years and for all three localities examined, there was total absence of females with mature eggs in January. In the second ten-day period of February (day-length 10.27), a small percentage (3%) was observed, with a mean number of 14 eggs. From the second ten-day period ofMarch (day-length 11.57), the presence offemales with mature eggs rose to 38%, with a mean number of 11 eggs. Starting from early April (day-length 12.33 - 13.57), the percentage decreased from 34% to 23% in the first ten-day period of May, with mean - 48-

Jrl..PDEROF CAPTURED FS.MALES i 10 19 2 45 DB 65 69 24 77 99 10i 24 34 II 15 7 29 32 15 4$ 92 52 52 70 1'12 133 240 572 924 1830901637147 70 27 7 "------,''·"

....

'""

0112

OHO

orn

0000 � l � M � a � � iii OCT. NOV. DEC. !ml%of females with mature eggs ....,.photophase

Fig. 1 - Bactrocera oleae - Percentage of females with mature eggs captured over six years in three different locations. The continuous curve represents photophase on a daily basis. At the top, total numbers offemales observed in each ten-dayperiod are also indicated.

20 20 .. l" ,s •c § 'lS, o 10

! 5

1 1 1 1 i , i � � , l • • r r r 1 1 1 � 2 2 r r r I i i 1 1 1 § § � i i � 0 � � � 2 � * � � � � � � � � � � � � - � � � � � � � � � � � 2 � � 2 � � --Meannumber of eggs/females -Mean monthly temperature of Pisa -+-Mean monthly temperature of lmperia

Fig. 2 - Bactrocera oleae - Mean number of eggs over six years of observations, and thirty-year mean monthly temperatures in Pisa and lmperia. - 49 number of eggs ranging from 12 to 25. By May 10 (day-length 14.22) no further females with mature eggs were captured until the end of the first ten-day period of July ( day-length 15.05). Only starting fromthe second ten-day period of July (day-length 14.52) were fairly elevated percentages again observed(31 % ), with a mean number of 21. 8 eggs (fig. 2). From the end of August (day-length 13.14) to the end of October (day-length 10.08), large numbers of females with mature eggs were captured (Fig.1). In addition, mean egg number was also high, peaking at 23 eggs/femaleat the end of September (Fig.2). The percentage of females with eggs peaked in the first ten-day period of November (27.3%), but from this date until December both this percentage and the mean number of eggs decreased drastically. Our preliminary laboratory tests (Tab. I) showed that B. oleae adults emerged and maintained at constant 12: 12 (L:D) and 16:8 (L:D) did not differ statistically (P < 0.01) in percentage of mature eggs (74% and 63% respectively).

TAB.I I TEST EGG TEROUGIIT PUPA-ADULT FEMAL�SWITH MATURE LAQ.VA EEGS = A 10:14 (L:D) 20 °C 12:12 (L:D)26 °C 74% a (n 27) = B 10:14 (L:D) 20 °C 16:8 (L:D) 26 °C 63% a (n SJ)

4 - DISCUSSION According to theoretical calculation of generations (Belcari et al., 1987; Crovetti et al., 1992; Canale 1994, 1996; Raspi et al., 1996), the presence, and elevated percentage, of females with eggs between the second ten-day period of March and the first ten-day period of May provides evidence of the emergence of a generation that overwintered in the ground as a pupa. This probably indicates that the newly emerged females were the ones with eggs. Mean egg number was elevated despite the fact that mean thirty-year monthly temperatures for March and April in the trial areas range between a minimum of 10.6 °C (in March in ° Pisa) to a maximum of 14.7 C (in April in lmperia) (fig.2). It should be kept in mind that cv Taggiasca stone-fruitsmay still be present on trees in March-April, while this is not the case for other cv in Tuscan olive groves. During this period females developed mature eggs although temperatures· are very close to the reproduction threshold of this species, which is reported by Azzi (1967) to lie at a mean temperature of roughly 13.5 °C. In November­ December, on the other hand, when mean temperatures are little different from those of February-March, there was a sharp drop in mean number of eggs per female despite the presence of stone-fruits (Fig.2). But in March-April and September -October the percentage of females with eggs and the mean number of eggs were similar. This patternis reported by Delrio & Prota (1975-76) for Sardinia and Ballatori et al. (1991) for Umbria. Field data suggest that olive fly oogenesis may be stimulated by the photoperiod at values ranging between 10:14 (L:D) (early February and late October) and 13.30:10.30 (L:D) (late April and early September). In particular, the fly shows a cyclical trend with two reproduction peaks around 12:12 (L:D) in spring and in autumn. Only in the latter case, however, are stone-fruits also present and the temperature and humidity conditions favorable, suggesting that B. oleae may originally have been linked to other hosts that bear fruitat differenttimes, some in spring and others in autumn, or to a reflowering host. However, the adult fly's sensitivity to the photoperiod allows cyclic repetition of peak egg production, at two different times of the year. - 50 -

In our view, these data also suggest that the photoperiod may have an inhibitory effect on olive fly oogenesis. Inhibition seems to occur between the end of April (13,30 : 10,30 (L:D))and June 21, in the period when the photophase increases and reaches maximum day­ length (15.16). The absence of mature eggs during this period is shown by our data and confirmed by numerous studies (Baranov, 1937; Ayoutantis et al., 1954; Stavrakis, 1973; Delrio e Prota, 1975-76; Delrio e Cavalloro, 1977; Economopoulos et al., 1977; McFadden et al., 1977; Fletcher et al., 1978; Neuenschwander and Michelakis, 1979; Stavrakis and Fytizas, 1980; Ballatori et al., 1981; Girolami et al., 1983; Neuenschwander et al., 1986; Tzanakakis et al., 1986; Canale, 1994; Solari, 1995; Raspi et al., 1996) performed in various climatically distinct Mediterranean areas and in the presence or absence of stone-fiuits (Fletcher et al., 1978; Tzanakakis & Koveos, 1986). After June 21 day-length begins to decrease and a portion of the females, probably the younger ones, succeed in developing a new batch of mature eggs, partly by virtue of the presence of olives. A fewof these females then succeed in laying eggs in July. But only in the first or second subsequent generation, i.e. in September, when the photoperiod is optimal in the trial area latitudes, do females reach maximum reproductive capacity. Inhibition by the photoperiod is confirmed, in our interpretation, by Fletcher's experiment (1978) using cages placed in the open field and B. oleae adults captured in June: according to Fletcher, these flies do not develop mature eggs in July even when an adequate diet is available. According to our experience, in certain years and in locations included within the l 6-l 7°C climatic band, eggs may at times be found inside olives, and may even reach substantial percentages, as early as the first ten-day period of July; the generation that begins in this period can be completed between the end of the month and early August, although the higher maximum temperatures generally provoke pronounced mortality of pre-imaginal instars. In general, however, it would appear that quantitatively significant olive fly attacks on cultivated olive begin roughly 40 days after maximum day-length. This estimation is supported by the finding that in Tuscany attacks usually begin between late July and early August (Belcari et al., 1987), in Algeria (Tlemcen region) at the 30u,. parallel Nin August, (Gaouar & Debouzie, 1991) (maximum day-length on 21 June, 13.46) and in south Africa (Cape Town) (34u, parallel S) around February (Hancock; 1989) (maximum day-length on 22 December, 14.16). In our interpretation, as early as 1986 Tzanakakis & Koveos provided laboratory evidence that B. oleae adults enter into reproductive diapause when passing from a 12:12 (L:D) to a 16:8 (L:D) photoperiod. As also shown by these authors, such a phenomenon does not occur if adults emerged at 12: 12 (L:D) or at 16:8 (L:D) are maintained at the same constantphotoperiod, or when adults emerged at 16:8 (L:D) are subsequently transferred to 12:12 (L:D). Analogously, our laboratory tests showed that if adults were maintained at the same photoperiod as that at which they emerged, whether it be 12:12 (L:D) or 16:8 (L:D), mature eggs were developed. It can therefore be hypothesized that in nature diapause does not occur in long photophase conditions, but rather when the photophase is increasing more than- at certain value of day-lenght (13.30). The constant 16:8 (L:D) photoperiod does not induce diapause, as testified by the laboratory rearing technique of Koveos & Tzanakakis (1990, 1993), in which adult flies were maintained at 16:8 precisely in order to obtain eggs. Eggs maturation in March - April, documented in the Mediterranean area, is unlikely to have the pourpose of laying in the few stone-fiuits still present on wild or cultivated olive trees. For this reason, we believe that in the Mediterranean area the olive fly is not synchronized with the only host on which it can live in this geographic range. Instead, we suggest it may use such fiuits simply in order to free itself of the eggs that it would in any case not be able to reabsorb. According to some authors (Delrio & Prota, 1975-76; Delrio & - 51 -

Cavalloro, 1977; Kapatos & Fletcher, 1984), there may even be one or two spring generations completed on these fiuits. In Tuscany, however, the flies born in the spring can survive until egg-laying in July-August and thus become more numerous in the following generations. The above reflections raise a number of questions concerning the origin of the olive fly. Firstly, it seems unlikely that monophagism could exist in nature as an original condition if this is not justified at least by perfect synchronism between the insect's biology and host phenology. Furthermore, in the Mediterranean area B. oleae has no specific parasites, since its main parasites are polyphagous ectophagous Chalcidoidea parasites (Silvestri, 1913, 1914; Raspi, 1993). Effectively, the only factor of ecoresistance that limits population levels is temperature, above all low winter temperatures and dry summer heat. In this perspective, it is worth pointing out that Silvestri suggested as early as 1914 that the fly was not original to the Mediterranean area, and indicated that its parasites should be sought both in Africa and in Asia. Ifwe analyze the current world-wide distribution ofB. Oleae (White and Elson­ Harris, 1992), we note that it is distributed throughout the coastal areas of all Mediterranean countries, but in Africa it is also present in Sudan, Ethiopia (Eritrea) and Kenia, in addition to the Canary Islands and South Africa. In Europe it is present in Georgia, and, in the Middle East, also in Syria and throughout Turkey. In Asia it is also present, but in a patch clearly separated from its other areas of distribution, between Northern India and North­ westemPakistan (Silvestri, 1916; Kapoor, 1989). We owe the first evidence ofthis presence of the olive fly in the Indian sub-continent to a flash of intuition by Silvestri, who in 1915 wrote to the entomologist Th. B. Fletcher asking for information on the presence of the olive flyin Indian provinces where wild olive was known to grow. In a letter penned on 18 December 1915, Fletcher replied with the following statement: "Regarding Dacus oleae, I had some parcels of olive fiuits sent down fromNorth-West India but they arrived here in bad condition and no insects have been bred out of them. So far as we know at present, then, Dacus oleae has not been found in India ". But Silvestri continues his report by commenting " but he visited the North-Western provinces personally and on 22 May he informed me that he had found some worm-eaten olive fiuits (possibly fromOlea cuspidata) [now 0. ferrngineaRoyle] and that he had reared adults of Dacus oleae and of a type of Opius acting as a parasite ofDacus oleae, and would send me a few specimens to study on his return to Pusa, which in fact he very kindly did in September " (Silvestri, 1916). The Opius was subsequently described by Silvestri as Opius ponerophagus. From an ecological point of view, it is unrealistic to think that a fly with such a broad distribution in olive­ growing areas throughout the Mediterraean and in South Africa would have settled in a restricted isolated area of the Indian subcontinent, where until not long ago the only Oleaceae present were wild types. In recent years, a very important revision of Tephritidae has been carried out (Drew, 1989; White & Elson-Harris, 1992), and the olive fly has been included in the genus Bactrocera. This has in our view allowed a better systematic account of this fly. White & Helson-Harris ( 1992) showed that "Bactroceraspp. (formerly included in Dacus) are native to tropical Asia, Australia and the South Pacific region, with a fewspecies found in Africa and warm-temperate areas of Europe and Asia ". In contrast, "Dacus spp. are almost all associated with the flowers and fiuits of Cucurbitaceae or with the pods of Asclepiadaceae, and most species are found in Africa". Indeed, the olive flyis the only Bactrocera recorded in the Mediterranean area among the 440 known species, but we know it is also present on the Indian subcontinent. Only a few species ofBactrocera are present in Africa, but two of these, B. cucurbitae (Coquillet) and B. zonata (Saunders), are considered adventive (White and Elson-Harris, 1992). Thus there remain only B. o/eae and B. biguttula (Bezzi), foundin - 52 -

South Africa. The firstof these attacks cultivated olive, but also lives on Olea ajricana Mill. (of which Olea verrucosa and Olea chrysophylla are synonyms) (Munro, 1984). In contrast, B. biguttula (Bezzi) attacks only wild species (Chionanthus foveolata and Olea spp.) with stone fruit infestation occurring from May to September (Munro, 1924, 1925, 1984). The few data available in the literature seem to show that the distribution in South Africa of B. oleae is different fromthat of B. biguttula. The former lives in the Western Cape provinces (in a Mediterranean climate) and the latter lives along the coastal bush on the eastern coast (in a humid subtropical climate). Finally, there are another two Bactrocera that attack Oleaceae in Australia. These are the two polyphages, B. tryoni (Froggatt) and B. nigra (Tryon). The former attacks both cultivated and wild olive, while the latter has been recorded from a wild olive in Eastern Australia (Olea paniculata R. Br.) (White and Helson-Harris, 1992). But both of these Bactrocera were originally distributed in the eastern part of Australia that has a humid subtropical climate (Pinna, 1977). Therefore, if one leaves aside Munromya nudiseta Bezzi (Diptera: Tephritidae) (provisionally left in the Adraroinae by Munro (1984)), which in South Africalives on the host Chionanthusfoveolata (Munro, 1924; 1984), it appears clear that in a world-wide perspective there are only a few species of Bactrocera whose host is represented by the genus 0/eae and related genera. B. o/eae seems to be distinguished fromcongeneric fliesby the factthat 1t is bound by a climatic range between a subtropical seasonal climate with arid summers (Mediterranean climate) and the hot semi-arid climate bordering on Mediterranean climate areas (Pinna, 1977). Thus throughout virtually all ofits distribution in Europe, Africa,Asia Minorand the restricted area within the Indian subcontinent, it is only foundjust above and just below the 34th parallel in both the Northernand Southernhemisphere, even though in East Africa, from the coast of the Red Sea to South Africathere is an extensive and uninterrupted distribution of wild species of the genus Olea (Levinson, 1984; Zohary, 1994). Therefore,B .. o/eae lives in a seasonal climate with an arid summer; the reproductive diapause from May to June, induced primarily by the photoperiod and associated with the fly's characteristic change in behavior (preceding the diapause) (Masaki, 1980) which is manifested as a tendency to "migration" (Grandi, 1951), allows tbe olive fly to avoid both the elevated'"temperatures, to which it is extremely susceptible, and a period in which fruitsare unavailable.

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NEUENSCHWANDER, P., MICHELAKIS, S., KAPATOS, E. (1986) - Dacus oleae (Gmel.). In Y. Arambourg"Entomologie oleicole", Conseil Oleicole International,Madrid: 115-159. ORPHANIDIS,P. A. (1967) - The problem "Dacus of the olive". Nea Agrotiki Epitheorissis, July 1%7: 1- 14 (in Greek). PINNA, M. (1977) - Climatologia. Utet, Torino, 442 pp. RACCAUD-SCHOELLER, J. (1980) - Les insectes physiologie developpement. Masson, Paris. RASPI, A., MALFATTI,P., ANTONELLI R. (1983) - Ricerche perl'applicazione di metodologiestatistiche nella lotta integrata in olivicoltura. Analisi dei dati ottenuti con la sperimentazione eseguita ad Asciano (Pisa)nel 1983. Frustula Entomologica, n. s. VI (XIX):395-406. RASPI, A., CONTI, B., CROVETTI, A. (1993) - Verifica di un modello previsionale dell'andamento delle generazioni daciche in oliveti dei Monti Pisani. Convegno Nazionale di Protezione delle Colture, Pescara, 7-8 ottobre, pp: 433-454. RASPI, A., LONI, A. (1994) - Alcune note sull'allevamento di Opius concolor Szepl. (Hymenoptera Braconidae) e su recentitentativi di introduzionedella speciein Liguria. Frustula Entomologica, n.s. XVII (XXX):133-145. RASPI, A., CANOVAI, R., ANTONELLI, R. (19%) - Andarnento della infestazione di Bactrocera oleae (Gmelin) in oliveti del Parco Regionaledella Maremma. Frustula Entomologica, n. sJCIX (XXXII), 10 pp. (in press). SACANTANIS, K. (1957) - The scientific bases of a rational control of the olive fruit fly (Dacus oleae Gmel.). Geoponica 3:219-225; 304-310; 340-345 (in Greek) SILVESTRI, F. (1913) - Viaggio in Africa percercare i parassiti di mosche dei frutti.Boll. Lab. Zoo/. Gen. e Agr., Portici, VIII: 1-164. SILVESTRI, F. (1914) - Viaggio in Eritreaper cercare i parassiti della mosca delle olive. Boll. Lab. Zoo/. Gen. e Agr., Portici, IX: 186-226. SILVESTRI, F. (1916) - Prima notizia sulla presenza della mosca delle olive e di un parassita di essa in India. Atti della Reale Accademia dei Lincei, anno CCCXIII,serie quinta, volume XXV: 424-427. SOLARI, L. (1995) - Andarnentodella infestazione e dei voli della mosca delle olive nel tricnnio 1992-1994 in oliveti del Monte Pisano. Tesi di laurea, Facolta di Agraria, Univcrsita degli Studi di Pisa, a.a. 1994-95. STAVRAKIS, G. N. (1973) - Observationsur I' etat des organes reproducteurs de fcmelles de Dacus oleae (Diptera, Trypetidae), captureeen gobe-mouches pendantune annee.Ann. Zoo/. Ecol. Anim., 5: 111- 117. STAVRAKIS, G. N., FYTIZAS, E. (1980) - Observations sur I' etat des organes reproducteurs de fcmelles de Dacus oleae (Diptera, Trypetidae), capturees en gobe-mouches pendant les annees 1970-1980. Meded. Fae. Landbouwwet.Rijksuniv. Gent, 45: 587-592. TZANAKAKIS, M. E. (1987) - Summer diapause in the olive fruit fly and its .signifiance.In: A. P. Economopoulos,"Fruit flies": 383-386. TZANAKAKIS, M.E., KOVEOS D. S. (1986) - Inhibition of ovarian maturation in the olive fruitfly Dacus oleae (])iptera: Tephritidae), under long photophase and an increase of temperature.Ann. Ent. Soc. Am., 79: 15-18. WJilTE, I. M., ELSON-HARRIS M. M. (1992) - Fruit Flies of Economic Signifiance: their identification and bionomics. CAB International,Wallingford, pp. 601. ZANGHERI, V., CAVALLORO R., DELRIO G., GIROLAMI V., PROTA U., RICCI C. (1978) - Osservazioni su1 Dacus oleae Gmelin in varie regioni italiane nell'ambito di un programma coordinato.Atti XI Congresso Nazionale Italiano di Entomologia, Portici-Sorrento, 429-436. ZOHARY, D. (1994) - The wild genetic resources of the cultivated olive. Acta Horticolturae, 356: 62-65. - 55-

DEVELOPMENTAL RATE AND NUMBER OF GENERATION ESTIMATES FOR CERATITIS CAPITATA. (WIEDEMANN) IN FRUIT GROWING REGIONS OF CALIFO-RNIA

M. MUNIZ1 & F.G. ZALOM2

1 Departamento de Protecci6n Vegetal. Centro de Ciencias Medioambientales (CSIC). c I Serrano 115 Dpdo. 28006 Madrid (Spain) 2 Department of Entomology. University of California. Davis, CA. 95616. USA

Abstract - Phenology models for preoviposition, egg, larval, pupal, egg-adult and adult-adult periods of Ceratitis capitata (Wiedemann) were elaborated. Results were compared and contrasted to other presented in the literature. The minimum number of times C. capitata generations can occur in a given number of days was then estimated for 11 distinct sites in California fruit growing areas for the period 1992-1993 using observed temperatures for this period·and 30 year average temperature data. Although not presently detected in California, this analysis shows that C. capitata could survive in all of the fruit growing areas if they were introduced and suitable host plants were present.

Key-words - Ceratitis capitata, Medfly, developmental thresholds, phenology,

Introduction

California is the largest agricultural state of the United States, with total farm revenue exceeding $18 billion annually. California also leads the United States in fruit production ranking in the top 3 of states nationally in citrus, grapes, apples, pears, stonefruits (apricots, cherries, nectarines, peaches, plums), strawberries and tomatoes among other fruit crops. The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) is a multivoltine, poliphagous species, widely considered to be the most harmful fruit pest in tropical and subtropical regions where it occurs because of its high level of adaptability and reproductive potential. Both biotic and abiotic factors influence its developmental and reproductive rates. Specific understanding of the actions of biotic and abiotic factors on the life cycle of C. capitata in fruit growing regions is an important goal of their effect on population dynamics (Debouzie, 1989). Among the abiotic factors, temperature likely has the greatest impact on C. capitata phenology, directly limiting its development and reproduction and the continuity of its generations (Fletcher, 1989). C. capitata has been introduced into California and elsewhere in the continental United States on several occasions, and there are numerous studies describing these introductions or speculating on the possibility for its successful establishment (e.g. Messenger and Flitters, 1954; Gjullin, 1931; Cunningham, 1989; Cunningham and Couey, 1986; Mangel et a/., 1984). Following its detection, each of these infestations was immediately followed by an eradication effort. In every instance, regulatory officials declared the infestations to be eradicated. Detections of C. capitata have occurred more frequently in California since 1975. - 56 -

These introductions may be due to increased immigration and travel into urban areas of California, where all flies have been detected, although it has been suggested that C. capitata may be established in the Los Angeles area (Carey, 1991 ). In any case, there remains a threat to California's fruit production s�ould C. capitata become established in primary fruit growing areas. Recently the University of California inaugurated the Center for Exotic Pest Research to systematize information about present and potential exotic pests of California agriculture and to ·identify research needs at the state and federal level. An important initial effort of the Center will focus on "surveying current scientific information about C. capitata from a worldwide viewpoint and developing a cohesive plan for research directed toward improved methodology for dealing with this vexatious exotic pest of California" (Metcalf, 1995). The influence of temperature on fruit fly growth and survival was among the priorities for research from a California Department of Food and Agriculture perspective (Dowell, 1994). Comprehensive information about C. capitata biology and ecology is necessary to predict its potential distribution and abundance in California (Carey, 1992) and to apply effective biological control programs. A measure of infestation potential is the generation index, or the potential number of generations possible per season that can be calculated using phenology and associated temperature data (Meats, 1989). In this study we estimate the minimum number of generations possible for C. capitata assuming this pest was introduced into fruit growing regions of California.

Material and methods

Eleven locations (Figure 1) were selected which represent ·somewhat distinct areas of California fruit production, all of which had complete records of maximum and minimum temperatures for the period 1965 through 1994 available from the meteorology database of the IMPACT computer system (University of Californ.ia, 1993). Five of the sites were in the Central Valley (Parlier, Merced, Waterford, Lindsay, Marysville), which comprises the majority of California's fruit production. Four of the sites were in southern California (Santa Ana, Riverside, Santa Paula, Oxnard) and two were in the San Francisco Bay area (Livermore, San Jose). Santa Ana·and San Jos€ are +argely urban .areas .at present .and sites of multiple C. capitata detections over the past 20 years. Citrus is a major crop grown in the vicinity of Lindsay, Riverside, Santa Paula, and Oxnard. Thirty year average temperatures calculated from the IMPACT database, and actual data from 1992 and 1993 at each location were used for the analysis. Degree-day accumulations for each developmental stage or period were calculated using the single sine method with a horizontal upper cutoff (Baskerville and Emin 1969) on the IMPACT computer system (University of California, 1993). In Table 1 are presented the

lower (T min) and upper (Tmax) thresholds for each stage or period of C. capitata development. Values were obtained using Muniz & Gil"s method (Muniz & Gil, 1984). - 57 -

N LOCATION COUNTY

1 Lindsay Tulare 2 Livermore Alameda 3 Marysville Yuba 4 Merced Merced 5 Oxnard Ventura @] 6 Parlier Fresno 7 Riverside Riverside 8 San Jose Santa Clara 9 Santa Ana Orange 10 Santa Paula Ventura � Waterford Stanislaus QJ

Fig.1.- Locations of weather data in California from which Ceratitis capitata generation estimates were made - 58 -

Table 1 Lower and upper development thresholds (0C) for Ceratitis capitata.

Lower Upper --- Preoviposition period 4.544 28.688 Eggs 12.713 36.910 Larvae 12.294 37.605 Egg-adult 12.564 35.092 Adult-adult 11.975 39.336

If it is satisfied that development can occur; then the number of times (N) that a certain developmental stage or period can occur in a given number of days (d) is

N = d/t where t = K/(T-c) is the duration (days) of each developmental stage or period. It follows then, that

N = d(T- c)/K = (- dc/K) + dT/K

If (dc/K) = a and d/K = b, there is a linear relationship between N and T:

N = - a+ bT.

The accumulated number of times a given developmental period can occur in a given number of days can be calculated by the following expression:

i=n i=n

i=1 i=1 We used this formula to estimate the number of C. capitata generations that could occur at each of the 11 sites.

Results and discussion

Fletcher (1989) reported other studies of developmental thresholds for C. capitata by severa1 .. authors. Delrio et al. (1986) calculated the lower developmental threshold for eggs of C. capitata with data in the literature from several authors. applying three different methods to each set of data (thermal summation, linear regression and sigmoid curve). The lower thresholds they calculated varied from 4.5 to 11.6°C. Meats (1989) reported the lower developmental threshold for eggs to be 9.?°C (Fares, 1973; McBride, 1935), for larvae to be - 59

9.7°C (McBride, 1935; Shoukry and Hafez, 1979), and the lower and upper thresholds for pupae to be 13 °C and 35°C (Shoukry and Hafez, 1979; El Gazzar, 1979). Also, the cumulative number of degree-days necessary to complete each immature stage, developmental period and the adult-adult life cycle were calculated from observed and estimated data obtained by MUNIZ and Gil (1984). These data are presented on Table 2.

Table 2 Degree-day accumulation required for the development of C. capitata using observed and estimated data for the duration of each developmental stage or period from Muniz & Gil (1984).(Figures in parenthesis indicate duration in days of each stage or period).

OBSERVED DATA ESTIMATED DATA

Temperature (±SEM) Temperature (±SEM ) (DC (DC) 19 ±1 22 ± 1 25 ± 1 28 ± 1 31 ± 1 19 ± 1 22 ± 1 25 ± 1 28 ± 1 31 ± 1 Preov. 74.61 60.24 61.38 76.25 72.42 69.55 69.49 69.56 69.68 63.49 Period (5.16) (3.45) (3.00) (3.25) (3.00) (4.81) (3.98) (3.40) (2.97) (2.63)

Eggs 31.45 27.87 30.73 30.58 36.58 30.76 30.75 30.73 30.73 30.73 (5.00) (3.00) (2.50) (2.00) (2.00) (4.89) (3.31) (2.50) (2.01) (1.68)

Larvae 102.66 92.83 96.72 102.59 117.87 100.58 100.60 100.54 100.54 100.47 (15.30) (9.56) (7.61) (6.53) (6.30) (14.99) (10.36) (7.91) (6.40) (5.37)

Pupae 135.24 132.16 124.40 138.96 247.52 134.21 134.14 134.10 134.17 134.06 (21.00) (14.00) (10.00) (9.00) (8.00) (20.84) (1421) (10.78) (8.69) (7.27)

Egg- 268.93 252.59 251.58 271.90 301.71 265.09 265.14 265.21 265.22 265.25 adult (41.31) (26.56) (20.11) (17.53) (16.30) (40.72) (27.88) (21.20) (17.10) (14.33)

Adult- 326.22 300.70 300.89 332.90 367 09 319.97 320.04 320.03 320.08 320.11 adult (46.47) (30.01) (23.11) (20.78)(19.30) (45.58) (31.94) (24.58) (19.98) (16.83) - 60 -

The mean (±SEM) observed and estimated degree-day requirements for development of C. capitata calculated from Table 2 (n = 5) are: preoviposition period, 68.98 ± 3.40 and 68.36 ± 1.22; eggs. 31.44 ± 1.42 and 30.74 ± 0.01; larvae, 102.54 ± 4.26 and 100.55 ± 0.02; pupae, 135.66 ± 3.81 and 134.14 ± 0.03; egg-adult period, 269.34 ± 9.09 and 265.18 ± 0.03, and adult-adult period, 325.56 ± 12.27 and 320.05 ± 0.02. These data are very close to those previously reported by Muniz and Gil (1984). Differences in C. capitata developmental thresholds between authors occasionally leads to different degree-day accumulations for specific life stages or periods. Tassan et al., (1983) obtained a K value of 142.8 degree-days using a lower developmental threshold of 9.7°C. Messenger and Flitters (1958), using a lower threshold of 11.7 °C and an upper threshold of 35.6°C for eggs in Hawaii, calculated the accumulated degree-days required for eggs to be 29.1, which is very close to our estimation in this study (30. 75). The study of Delrio et al. (1986), which resulted in variable lower developmental thresholds, resulted in calculated values for the thermal constant of eggs ranging from 22.4 to 35.9 degree-days. To be conservative in our estimate of the minimum number of C. capitata generations that are possible at the 11 sites, the lowest maximum temperatures for each month were used. The number of generations for each site are calculated using expression (1 ), with observed temperatures for the period 1992 and 1993 [Tm(1)], and average temperatures [Tm( 2)] from 30 years (1965-1994) (Table 3). The minimum number of cumulative generations, through 1992-1993, are shown on Figure 2. The results show that the decreasing order of sites in terms of minimum number of generations using average temperatures is: Riverside > Lindsay > Santa Paula = Marysville > Merced > Livermore = Oxnard > San Jose > Parlier> Santa Ana = Waterford. These results are based on the assumption that development is not impacted by biotic factors (for example, biological controls are not impacting populations, and suitable fruit hosts are present) and that temperature is the primary abiotic influencing development (assuming lack of winter mortality due to cold conditio.nsor.rainfall). Given our assumptions, it is possible that C. capitata could survive and develop in these primary fruit growing areas of California, even where the insect has never been introduced to date. This underscores the significance of this insect should it become introduced into these regions. More extensive ecological studies would need to be conducted using models which consider other biotic and abiotic factors in order to validate the relative significance of temperature alone as a variable in predicting C. capitata development. However, such studies are not possible in California because of the quarantine status of C. capitata which requires regulatory agencies to conduct eradication efforts whenever the pest is detected. - 61 -

Table 3 Lowest maximum temperatures (Tm: °C) and estimated minimum number (N) of generations (adult- adult) per month of C. capitata in 11 California fruit areas. Results were obtained using a minimum threshold of 11.975 and K = 320.05. (1 ): Estimated were based upon (1 ): observed temperatures during 1992-1993 and (2): 30 years average temperatures for the period (1965-1994).

LIVERMOR.C PARLIER.A MERCED.C ST A-ANA.A Tm Tm Tm Tm Tm Tm Tm Tm Month 1992 1'!_ 1993 lL 1992 lL 1993 1'l_ 1992 lL 1993 JL 1992 lL 1993 lL Jan (1) 2.8 0 4.4 0 3.3 0 4.4 0 2.8 0 3.9 0 11.7 0 11.7 0 (2) 12.2 0 12.2 0 10.0 0 10.0 0

Feb (1) 11.1 0 7.8 0 10.6 0 13.9 0.2 14.4 0.2 12.2 0 12.8 0.1 12.8 0.1 (2) 15.0 0.3 15.0 0.3 - 13.9 0.2 13.9 0.2 -

Mar (1) 14.4 0.2 12.2 0 15.6 0.4 13.9 0.2 16.7 0.5 14.4 0.2 15.6 0.4 15.0 0.3 (2) 16.1 0.4 16.1 0.4 17.2 0.5 17.2 0.5 -

Apr (1) 19.4 0.7 17.2 0.5 21 1 0.9 18.3 0.6 21.1 0.9 18.3 0.6 18.3 0.6 17.8 0.5 (2) 18.9 0.6 18.9 0.6 20.6 0.8 20.6 0.8 -

May (1) 23.3 1.1 19.4 0.7 25.6 1.3 21.7 0.9 26.7 1.4 23.3 1.1 19.4. 0.7 18.3 0.6 (2) 22.8 1.0 22.8 1.0 24.4 1.2 24.4 1.2 -

Jun (1) 20.6 0.8 18.3 0.6 23.3 1.1 18.9 0.6 25.0 1.2 23.3 1.1 19.4 0.7 17.2 0.5 (2) 26.1 1.3 26.1 1.3 30.6 1.7 30.6 1.7 -

Jui (1) 23.3 1.2 25.0 1.3 27.8 1.5 28.9 1.6 30.0 1.7 31.7 1.9 23.3 1.1 22.2 1.0 (2) 30.0 1.7 30.0 1.7 34.4 2.2 33.9 2.1 -

Aug (1) 23.9 1.2 23.3 1.1 27.8 1.5 28.9 1.6 32.2 1.9 30.0 1.7 23.8 11 22.8 1.0 (2) 30.0 1.7 30.0 1.7 33.3 2.0 33.3 2.1 -

Sep (1) 22.8 1.0 20.6 0.8 26.1 1.3 22.2 1.0 29.4 1.6 26.7 1.4 24.4 1.2 19.4 0.7 (2) 28.3 1.5 28.3 1.5 30.0 1.7 30.0 1.7 -

Oct (1) 16.1 0.4 17.8 0.6 17.2 0.5 21.1 0.9 18.9 0.7 22.8 1.0 18.9 0.7 20.0 0.8 (2) 21.7 0.9 21.7 0.9 22.2 1.0 22.2 1.0 -

Nov (1) 11.7 0 11.7 0 13.3 0.1 12.2 0 13.9 0.2 13.9 0.2 18.9 O.f3 16.7 0.4 (2) 15.0 0.3 15.0 0.3 15.0 0.3 15.6 0.3

Dec (1) 5.0 0 5.0 0 2.2 0 2.2 0 5.0 0 5.0 0 15.0 0.3 15.0 0.3 (2) 12.2 0 12.2 0 11.1 0 11 1 0 - 62 -

Table 3 (Cont.)

SANJOSE.C RIVRSIDE.C STAPAULA.C WATRFORD.T Tm Tm Tm Tm Tm Tm Tm Tm Month 1992 _l:L1993 _l:L 1992 N 1993 N 1992 N 1993 N 1992 N 1993 N Jan (1) 8.9 0 6. 7 0 10.6 0 11 .1 0 15.0 0.3 12 .2 0 2.2 0 2.2 0 (2) 12 .8 0.1 12 .8 0.1 17.2 0.5 17.2 0.5 18.3 0.6 18.3 0.6 -

Feb (1) 12 .8 0.1 10.6 0 11 .1 0 11.7 0 144 0.2 144 0.2 10.0 0 7.8 0 (2) 15.6 0.3 15.6 0.3 18.3 0.6 18.3 0.6 18.9 0.6 18.9 0.6 -

Mar (1) 13.9 0. 2 13.9 0. 2 15.0 0.3 13 .9 0. 2 15.6 04 16.7 0.5 144 0.2 13.9 0. 2 (2) 16.1 04 16.1 04 18.3 0.6 18.3 0.6 194 0.7 194 0.7 -

Apr (1) 189 0. 6 17.8 0.5 189 0. 6 18.9 0.6 20.0 0.8 20.0 0.8 17.8 0.5 16.7 04 (2) 189 0. 6 189 0.6 21 .1 09 21.1 0. 9 21 .7 09 21.7 09

May (1) 21.7 09 20.0 0.8 21 .7 09 21.7 09 21 .7 09 20.6 0.8 25.0 1.3 21 .1 0.9 (2) 21.7 09 21 .7 09 22 .8 1.0 22 .8 1.0 21.7 0.9 21 .7 09 -

Jun (1) 20.6 0.8 17.8 0.5 21.1 0. 9 16.7 04 21 .1 0.9 194 0.7 20.0 0.8 15.6 0. 3 (2) 23 .9 1.1 23.9 1.1 27.2 14 26.7 14 23 .3 1.1 22.8 1.0 -

Jui (1) 244 1.2 244 1.2 25.6 1.3 26.7 14 244 1.2 23.9 1.2 26.7 14 28.3 1.6 (2) 26.7 14 26.7 14 33.3 2.1 32 .8 2.0 26.1 14 26.1 14 -

Aug (1) 26.7 14 22 .8 1.0 26.7 14 27.8 1.5 25.6 1.3 23 .9 1.2 26.7 14 26.7 14 (2) 26.7 14 26.7 14 32.2 2.0 32 .2 2.0 26.7 14 26.7 14 -

Sep (1) 23.9 1.1 194 1.1 27.8 1.5 22 .8 1.0 244 1.2 22 .8 1.0 25.0 1 2 25.0 1.2 - (2) 25.6 1.3 25.6 1.3 28.3 1.5 28.3 1.5 25.6 1.3 25.6 1.3 Oct (1) 17.2 0.5 194 0. 7 17.2 0. 5 21 .7 0.9 22 .2 1.0 22.8 1.0 15.0 0.3 20.0 0.8 (2) 20.6 0. 8 20.6 0.8 25.0 1.3 25.0 1.3 23.9 1.2 23.9 1.2 -

Nov (1) 13 .9 0.2 15.6 0. 3 17.8 0. 5 194 0.7 20.6 0.8 20.0 0.8 12 .2 0 12.2 0 (2) 15.6 0.3 15.6 0.3 194 0.7 194 0. 7 20.0 0.8 20.0 0.8 -

Dec (1) 10 0 0 12 .8 0 1 11 1 0 17 2 0.5 15 0 0.3 17.8 06 20 0 20 0 (2) 12.8 0.1 12 8 0 1 17 2 0.5 ff2 0.5 17 8 0.6 17.8 06 - - 63

Table 3 (Cont.)

LINDSAY.C OXNARD.C MARYSVLE.C Tm Tm Tm Tm Tm Tm Month 1992 1i 1993 1i 1992 1i 1993 JL 1992 1i 1993 li Jan (1) 5.0 0 6.7 0 11.1 0 11.1 0 2.8 0 6.1 0 (2) 11.1 0 11.1 0 17.8 0.6 17.8 0.6 10.0 0 10.0 0

Feb (1) 13.9 0.2 12.8 0.1 14.4 0.2 14.4 0.2 12.8 0 11.7 0 (2) 14.4 0.2 14.4 0.2 18.3 0.6 18.3 0.6 13.9 0.2 13.9 0.2

Mar (1) 14.4 0.2 14.4 0.2 14.4 0.2 14.4 0.2 13.9 0.2 13.9 0.2 (2) 17.8 0.6 17.8 0.6 17.8 0.6 17.8 0.6 16.7 0.5 16.7 0.5

Apr (1) 23.3 1.1 18.3 0.6 18.3 0.6 17.2 0.5 16.1 0.4 16.7 0.4 (2) 21.7 0.9 21.7 0.9 18.9 0.6 18.9 0.6 21.1 0.9 25.0 1.2

May (1) 26.7 1.4 24.4 1.2 18.9 0.7 17.8 0.6 27.2 1.5 21.7 0.9 (2) 26.1 1.4 26.1 1.4 20.0 0.8 20.0 0.8 25.0 1.3 25.0 1.3

Jun (1) 23.9 1.1 21.7 0.9 18.3 0.6 18.3 0.6 22.2 1.0 16.7 0.4 (2) 31.1 1.8 31.1 1.8 20.6 0.8 20.6 0.8 30.0 1.7 29.4 1.6

Jui (1) 28.9 1.6 30.6 1.8 20.6 0.8 21.7 0.9 33.9 2.1 30.6 1.8 (2) 35.0 2.2 35.0 2.2 22.8 1.0 22.8 1.0 33.9 2.1 33.3 2.1

Aug (1) 30.6 1.8 30.0 1.7 22.2 1.0 21.1 0.9 26.7 1.4 27.8 1.5 (2) 33.9 2.1 34.4 2.2 23.9 1.2 23.9 1.2 32.2 2.0 32.2 2.0

Sep (1) 29.4 1.6 26.7 1.4 21.1 0.9 21.1 0.9 28.3 1.5 24.4 1.2 (2) 30.0 1.7 30.0 1.7 23.3 1.1 23.3 1.1 29.4 1.6 29.4 1.6

Oct (1) 18.9 0.7 21.1 0.9 18.9 0.7 20.0 0.8 18.3 0.6 19.4 0.7 (2) 22.8 1.0 22.8 1.0 22.2 1.0 22.2 1.0 21.1 0.9 21.1 0.9

Nov (1) 12.2 0 15.6 0.3 18.3 0.6 19.4 0.7 11.7 0 13.9 0.2 (2) 15.6 0.3 15.6 0.3 19.4 0.7 19.4 0.7 13.9 0.2 13.9 0.2

Dec (1) 11. 7 0 11.7 0 13.9 0.2 17.8 0.6 11.7 0 10.6 0 (2) 11.7 0 11.7 0 17.8 0.6 17.8 0.6 10.6 0 10.6 0 28 28 28 Merced.C (Merced County) 24 Livermor.C (Alameda County) 24 ,..1992-93. Parlier.A (Fresno (Ob.) County) 24 .,..1992-93(Ob) -1992-93(Av.) .,..1992-93(Obs.) -1992-93 (Av). 1 I 20 20 20 16 16 16 12 12 12 8 8 8 4 4 4 Cl) C 0 0 0 28 28 28 :.;:::; Stapaula.C (Ventura County) Sanjose.C (Santa Clara County) 24 CU 24 .,..1992-93 (Ob.) -1992-93 (Av.) 24 .,..1992-93 (Ob). -1992-93 (Av.) ,._ 20 20 Q.) 20 C 16 16 16 12 12 12 Q.) 8 8 8

4 4 4 CU 0 0 0 :::::i 28 28 28 E Stapaula.C (Ventura County) Watrford.T(Stanislaus County) 24 24 Lindsay.C (Tulare County) .,..1992-93 (Ob). -1992-93 (Av.) .,..1992-93 (Ob). 24 0) (..) 1 '*-1992-93 (Ob.}'--19921993 (Av.) / .i:,.. 20 20 20 16 E 16 16 12 12 E 12 8 8 C 8 4 � 4 4 0 0 0 C 0. > C >- - 0. > 28 28 ro a; lo' Q) 0 ro a; ro ::, Q) 0 Oxnard.C (Ventura Count ) --, :::a :::a'S --, Cl) z --, :::a :::a --, (/) z 24 24 *1992-93 (Ob.}'--1992-93 (Av.) 20 20 16 16 12 12 8 8 4 4

0 C 0. > C 0. > 0 C 0. > C 0. > ro a; fa' "S Q) 0 ro a; fo' "5 Q) 0 ro a; fo' "5 Q) 0 ro a; fo' "5 Q) :::a :::a --, (/) z --, :::a :::a --, (/) z --, :;f; :::a --, (/) z --, :::a :::a --, (/) z Fig. 2.- Minimum number of cumulative generations of Ceratitis capitata in different fruit production regions of California through 1992-1993, based on observed and average temperature data. - 65

Acknowledgements

This research was conducted while M. Muniz was a sabbatic scholar in the Department of Entomology at the University of California, Davis with support from the Direcci6n General de lnvestigaci6n Cientifica y Tecnica of the Ministerio de Educacion y Ciencia of Spain. We thank Chia Thao for her assistance in this analysis.

References

Baskerville, G.L. & Emin, P. (1969) - Rapid estimations of heat accumulation from maximum and minimum temperatures. Ecology 50: 514-517. Carey, J. R. (1991) - Establishment of the Mediterranean fruit fly in California. Science 25 3:1369 -1373. Carey, J. R. (1992) - The Mediterranean fruit fly in California: taking stock. California Agriculture 46(1): 13-17. Cunningham, R. T. (1989) - Population detection. In: Fruit Flies. Their Biology, Natural Enemies and Control (AS. Robinson & G. Hooper, eds.). Elsevier, Amsterdam-Oxford­ New York, Tokyo.Vo!. 38. pp. 169-173, Cunningham, R. T. & Couey, H.M. (1986) - The Mediterranean fruit fly (Diptera: Tephritidae) distance-response curves to trimedlure to measure trapping efficiency. Environ. Entomol. 15: 71-74. Debouzie, D. (1989) - Biotic mortality factors in Tephritid populations. In: Fruit Flies. Their Biology, Natural Enemies and Control (A. S. Robinson & G. Hooper, eds.). Elsevier, Amsterdam-Oxford-New York-Tokyo.Vol.38. pp. 221-227 Delrio G., Conti, B & Crovetti, BA (1986) - Effect of abiotic factors on Ceratitis capitata (Wied.) (Diptera:Tephritidae). 1. Egg development under constant temperatures. In: Fruit Flies of Economic Importance 84. (R. Cavalloro, ed.). Balkema, Rotterdam. pp. 133-141. Dowell, R. V. (1994) - Priorities for research from a California Department of Food and Agriculture perspective. In: The Mediterranean Fruit Fly in California: Defining critical research. University of California. Center for Exotic Pest Research. J. G. Morse, R. L. Metcalf & J. R. Carey, eds.). College of Natural and Agricultural Sciences. 318 pp. EI-Gazzar, L. M. (1979) - Induced sterility of the Medfly using heat treatments. Zeitschrift fur Angewandte Entomologie 88: 436-439. Fares. F. M. (1973) - Effect of cold storage on the hatchability of the Mediterranean fruit fly eggs. Agricultural Research Reviews 51: 57-58. Fletcher. B. S. (1989) - Temperature-development rate relationships of the immature stages and adults of Tephritid fruit flies. In: Fruit Flies. Their Biology, Natural Enemies and Control (A. S. Robinson & G. Hooper. eds.). Elsevier, Amsterdam-Oxford-New York­ Tokyo.Vol.3A. pp. 273-289. Fletcher, B. S. (1989) - Life history strategies of Tephritid fruit flies. In: Fruit Flies. Their Biology, Natural Enemies and Control (A. S. Robinson & G. Hooper. eds.). - Elsevier. Amsterdam Oxford-New York-Tokyo.Vol.38. pp. 195-208. - 66

Gjullin, C. M. (1931) - Probable distribution of the Mediterranean fruit fly (Ceratitis capitata Wied.) in the United States. Ecology 12: 248-258. Mangel, M., Plant, R.E. & Carey, J.R. (1984) - Rapid delimiting of pest infestations: a case study of the Mediterranean fruit fly. Journal of Applied Ecology 21: 563-579. McBride, 0. (1935) - Response of the Mediterranean fruit fly to its environmental factors. Proceedings of the Hawaiian Entomological Society 9: 99-108. Meats, A (1989) - Abiotic mortality factors - temperature. In: Fruit Flies. Their Biology, Natural Enemies and Control (A. S. Robinson & G. Hooper, eds.). Elsevier, Amsterdam­ Oxford, New York-TokyoVol.3B. pp. 273-289. Messenger, P. S. & Flitters. ['- JE. (1954) - Bicclimatic studies of three species of fruit flies in Hawaii. Journal of Economic Entomology 47: 756-765. Messenger, P. S. & Flitters, N.E. (1958) - Effect of constant temperature environments on the egg stage of three species of Hawaiian fruit flies. Annals of the Entomological Society of America 51: 109-119. Metcalf, R. L. (1995) - Invasion of California by exotic pests. California Agriculture 49(1 ): 2. Muniz, M. & Gil, A. (1984) - Desarrollo y reproduccion de Ceratitis capitata (Wied.) en condiciones artificiales. Bol. Serv. Plagas. Fuera de serie 2, 139 pp. Shoukry, A. & Hafez, M. (1979) - Studies on the biology of the Mediterranean fruit fly, Ceratitis capitata. Entomologia Experimentalis et Applicata 26: 33-39. Tassan, R.L, Hagen, K.S , Cheng, A., Palmer, T.K., Feliciano, G. & Blong, T.L. (1983) - Mediterranean fruit fly life cycle estimations for the California erradication program. In: Fruit Flies of Economic Importance 84. (R. Cavallero, ed ). Balkema, Rotterdam. pp. 564- 570. University of California. (1993) - IMPACT Users·s Manual. A Guide to use of the Statewide 1PM Project's Computer System and IMPACT Program. UC 1PM Publication 15. University of California Division of Agriculture and Natural Resources. Davis, CA. - 67 - STRATEGIEDE LOTTE OPTIMISEE SELON LES CRITERES BIO-ECOLOGI­ QUES DE BACTROCERA OLEAE(DIPT.:TEPHRITIDAE) DANS LA REGION DE TLEMCEN (OUEST ALGERIEN) GAOUAR Nassira Institut de Biologie Universite de Tlemcen BP. 358 R.P. 13 OOO Tlemcen - Algerie .

Resume : A partir de certains parametres biologiques et ecologiques de B. oleae specifiques a !:!Otreregion, notamment la repartition spatio- temporelle de ses attaques, nous avons eta:,li une nouvelle strategie de lutte dont nous avons teste l'efficacite. Les traitements insecticides optimises ont reduit de 5 foisles infestations. Les soins culturaux ont reduit de 3.5 foisles attaques du ravageur. Avec un taux de parasitisme variant de 9 a 46 %, la combinaison de ces differents types de Jutte donnera d'excellents resultats.

Mots cles: Bactrocera.oleae - Opius concolor - Lutteintegree •

1- INTRODUCTION Les raisons a l'appui de la lutte integree et qui ont conduit a son application en oleiculture sont a rechercher clans !'incidence de l'emploi, croissant et indiscrimine des traitements chimiques telle que la resurgence d'autres ravageurs, !'apparition des phenomenes de resistance, le cofrt eleve des traitements, la pollutionde I' environnement et la presence de residusde pesticidestant dans l'huile que dans l' olive de table ( Kapatos et Fletcher,1982; Claridge et Walton , 1992; Katsoyannos, 1992). Dans la Jutte contre Jes ravageurs, nous devons savoir comment se dispersent Jes insectes clans une region et definir " Jes pointschauds " de I 'infestation pourmoduler la dose des pesticides; ii est done importantde trouver. Jes structures spatiales et temporalesainsi que la dynamiquede leurs populations ( Debouzieet Thioulouse, 1986 ). Nous nous sommes interesses a l'etude du ravageur le plus notoire de !'olive Bactrocera o/eae en raison des degats importants qu'il provoque ( soit 50 a 80 % de la production) particulierement a Tlemcen ou aucun traitement insecticide n'a ete pratique depuis de nombreuses annees. Les travaux menes de 1987 a 1989 ont permis de montrer l'influence de certains facteurstels que le bioclimat,la variete et la date d'observation sur les infestations de B .0/eae alors que !'orientation n'a aucune incidence significative( Gaouar et Debouzie,1991) . De 1990 ii1994, nous avons considere d'autres aspects du cycle et la biologie des populations de ce ravageur, notamment la repartition spatio-temporelle des attaques ( Gaouar et Debouzie, 1995 ), qui ont permisd' etablir une strategie de Jutte dont nous avons teste l'efficacite

2 - MATERIELET METHODES Les observations ont ete effectuees dans la region de Tlemcen,caracterisee par un climat mediterraneen.L 'hiver est froidavec une rnoyenne de 9°C en Janvier, I' ete est chaud et sec avec une moyenne de 26° C en aofrt. La pluviometrie annuelle varie de 260 ii637 mm. De 1987 ii l989 les prelevements ont ere effectuesdans toute la region de Tlemcen selon les regles de l'echantillonnage aleatoire,clans 5 sites appartenant iides bioclimatsdifferents . De 1990 ii 1994 toutes les observations ont ere faites a Mansourah, situee ii 830 m d'altitude et appartenant au sub-humide tempere, ii a ete retenu trois (03) parcelles A, B et C comportant respectivement 423 , 240 et 259 oliviers de la variere Sigoise. Ils ont ete echantionnees13, 16 et 16 fois respectivementselon un echantillonnage systematique , plus apte iidetecter les structuresspatiales. En juillet et aout , quand les infestations sont faibles, les prelevements sont effectues tous les quinze jours. De septembre iidecembre, les prelevements sont hebdomadaires. En 1990, troisstrates par arbre ont ete definies : moins de. 2 m ( S1 ), entre 2 et 3 m ( S2) et au deli!de 3m ( S3). Trois lots d'olives ont ete preleves par arbre . Chaque echantill. Jn de 100 olives est structure en 4 sous-unites de 25 fruits, correspondantaux 4 orientations cardinales. L 'etude des variations au cours du temps des structures spatiales de B. oleae est effectuee a partir des deux indices Jes plus consideres pour la commercialisationdes olives : Jes piqiireset le nombretotal de larves( larves, pupes et trous de sortie) . La repartition horizontale des infestations a ete etudiee a partir des donnees recueillies de 1990 a 1992, alors que la repartition verticale n'a ete etudieequ'en 1990 . Toutes les observations ont ete effectuees a Tlemcen, pourla seule variete "Sigoise "dans le verger A en 1990 et dans le B, en 1991 et 1992. - 68 Pour connaitre la date des premieres applications d'insecticiele, les adultes ont ete captures par des pieges Mc Phail appiites au diphosphate d'ammoniwn a 3 % ; Jes appats sont renouveles chaque semaine. Dans la parcelleC tous Jes arbres ont ete traitesa la main parun insecticidele !er et le 20 aofrt puis le 4 septembre 1992. Chaque pulverisationcomporte 100 I/ ha d'une solution contenant 6 % de Fenthion, elle a ete appliquee au niveau de la strate basse de I' arbre( S1 ), la plus attaquee, en etant plus accentuee auxbords du verger. Les piegeages et Jes 46 elevages effectues de 1987 a 1992 ont permis d'obtenir un certain nombre de parasites appartenant a l'especeOpius concolor. Nous avons evalue I' impact des travaux culturauxtels que la taille, le labourage, I'irrig;ition . . . surJes attaquesde B. oleae en comparant,en 1991, lesinfestations de deuxvergers oleicoles, l'un etant entretenu

( le verger C) et l'autre pas( le verger B ), aucun d'euxn' ayant subi detraitement insecticide. Les taux d'infestationsont estimes par deux indices - La frequence desfruits attaques, - Le nombre moyen des piqures,d' oeufs, de larves ou de pupespar fruit. Les resultats obtenus sont testes par des analyses de variance a un ou deux criteres de classification selon Jes besoins, en completant par des comparaisons multiples de moyennes ainsi que par l'etude des correlations ( indicede Geary)•

3-RESULTATS: 1. Structuresspatio-temporelles 1.1 Structureshorizontales des infestations 1.1.1 - Repartition liespigares

En 1990, de la mi-juillet a la finaofrt, Jes infestations defaible intensite, varient peu selonJes arbres (Fig .I) Debut Septembre, Jes infestations sontplus importantes vers la partie Sud du verger co=e le confirme le test de Geary (IG = 2,88 ,P = 0,002 . Le taux moyen de piqw:es estde 2,47 pour la moitie Sud du verger alors qu'il est de 1,45 pourla moitie Nord. liusqu'au debut octobre, aucune structure particuliere n'est apparue.Le 9 octobre, les memes repartitionsque celles dudebut Septembresont reapparues ( IG= 2,81; P = 0,002 ). Les moyennes de piqfrres etaient de 2,61 clans la partie Sud du verger et seulement de 1,04 clans la partie Nord. Aux autres dates d'observation,aucune structureparticuliere n'estrelevee.

IS 30 IS 30 ·uillet :uillcr •••DQ• ••DUD ...... ••D•• eeO•• a• ••DD DD•••• ••••D nm 1

II TI 9 sc tcmbrc o:::1obre •OD•• .....D•••• . . •a•••a••••...... :vi OD••• :·iw D•••• iji

19 29 12 19 26 octobrc octoblc novcmbrc novcmbrc novcmbrc ,aaa "V

a88�•DD• ••U• Ui :··:o· 1 ..."" .. DOD : :6: !Vi:,; ODO � •D• ==, :v;: D1J·• Ui ..•.... . OD . . �H ...... Fig. 1 : Distribution spatiale desattaques par B.oleae . Cartographie du nombre de piqures parolive. Le carre le plus grandcorrespond a 6,5 piqfrrespar fruit . Donneesde 1990 - 69 -

1.1.2 Repartitiondes larves En 1990, quand Jes effectifs de larves etaient trop faibJes a certaines dates, nous Ies avons regroupes pour mieux Jes visualiser ( Fig . 2 ). Les infestations larvaires, tres faibles jusqu' en finseptembre, ne presentent aucune structure spatiale particuliere. Le 9 octobre, la partie Nord du verger etait moins infestee que la partie Sud ( I0 = 6,02; P < 0,000) avec des taux rnoyens de Jarves de 0,09 et 0,29 respectivement . La meme oppositionNord-Sud est observeele 19 Octobre(lo = 5,39; P < 0,0001) avec des rnoyennesfaibles au Nord ( 3,17 et 1,64 ) et fortesau sud ( 6,44 et 10,38) . Cette repartition corresponda celle de piqiiresaux memes dates. Pour Jes prelevernentsulterieurs, le test de Gearyn'a detecteaucune repartitionspatiale.

i i I •I I ' . . I ' ' I i I � ' I • I g . 0 ' ' 0 : ' g . . ! .I ·1·fI1 . . f. llau 27/09/90 29110 et 12111190 19 et 26/11190 u '. : ." . . .' ! . i ! . i � e . . •g C . !i G . . l . . 9110/90 ! .!

�

a. [] 19110/90

Fig.2 : Distribution spatiale des attaquespar B. Oleae. Cartographie du nombre delarvespar olive .. Le carrele plus grandcorrespond a I, I Iarvespar fruit.Donneesde 1990.

1.2 Structure verticale des infestations.

La repartitionverticale des attaques de B. oleae est etudiee en 1990, dans la parcelle A .. Trois strates ont ete considerees : basse (Si ),moyenne (S2) et haute (S3) ( cf Materiel et Methodes ). Pour toutes Jes variables, le taux d'infestation decroit progressivement de la strate basse vers la strate haute de l'arbre (Tab.I), comme l'illustre la Figure 3. - 70 -

Tableau. I: Taux moyensd'infestation par olive, pardate et par strate. Le facteur arbre n'est pas considere. Resultats des analysesde variance( ANOVA)

Trousde Total des �Strate pjm1TPC Oeufs Larves Punes Sortie Larves S1 2,12 0,26 0,12 0,021 0,046 0,19

S2 l,76 0,22 0,09 0,023 0,035 0,14

S3 1,54 0,20 0,07 0,015 0,032 0,11

F = = 2 = 2 = 2,87 = = anova F •.., 159,25 F'.., 43,69 F .., 22,89 F .., F'.., 6,87 F •.., 18,67

p <0,001 < 0,0001 <0,058 <0,001 <0,001 < 0,001

Lesanalyses de variancea deux facteurs ( strate et date) indiquent des variationstres significatives des taux moyens ( P < 0,001 ) , selon la hauteur pour toutes les variations a J'exceptiondes pupes dont la variabilite est plusfaible ( P = 0,058 ; Tab. I)

T,mx • S1ra1c has.\l' d'infl'slali S1ra1c ha111c IU

0,2

0.1

�40 250 260 270 280 29() 3

Fig. 3:Taux moyend'infestation parolive , pardate et parstrate. Donneesde 1990. Chaquc moyenne est accornpagneede sonintervalle df' confiance ( calculesur une IPoyennede 320 fruits. )

2. Etude de l'efficacitedu traitement insecticide

A partir des donnees acquises de 1987 a 1991 sur la bio-ecologie de Battrocera oleae dans notre region , notamment sur sa repartition spatio-temporelle, nous avons etabli une strategie de Jutte basee sur l'emploi d'un insecticide organophosphore, le Fenthion ( a 6 %.). - 71 -

2.1 Apparition des premieres mouches et dates des traitements

Les premiersadultes ont ete capture� le 7 Juillet 1992, ils etaient au nombre de 3 dans le verger B et 2 dans le verger C .L'insecticide a ete applique dans le verger C, plus petit, des que les pieges ont capture 4 a 5 mouches en moyenne par semaine, ce qui correspondrait a !'apparition d'une nouvelle generation. Trois applicationsont ete effectuees : le ler aoiit, le 20 aoiit puis le 4 septembre. 2.2 - Variation du nombre d'adultes

En juillet, avant que le premier traitement ne soit applique, Jes infestations etaient similaires avec une moyenne de 1,23 mouches/ piege / sernaine dans le vergerBet 1,17 mouches / piege/ sernainedans le vergerC (Tab. II).

Tableau II : Nombre moyen d'adultes par piege et par semaine. Les dates correspondent aux releves de pieges. Les premiers pieges ont ete installes le 15 Juin 1992.

Date Ve rgerB VergerC 01/07 0,13 0,17 15/07 0,42 0,5 30/07 3,13 2,84

01/08 Premier traitement insecticide 08/08 4,92 2,00 15/08 1,08 5,17 20/08 Deuxieme traitement insecticide 22/08 5,25 1,00 30/08 1 0,67 04/08 Troisieme traitement insecticide 08/09 0,75 00

Apres le premier traitement, Jes captures furertreduites, mais a la deuxieme semaine nous avons note une recrudescence dans le verger traiteC , probablement due a un deplacement de la populationdu vergerB ou d'autres oliveraies voisines ; cette dispersion a probablement ete favorisee par le sirocco qui a sevi Jes jours precedents ( du 16 au 18 aoiit). Apres le deuxieme traitement, effectue le 20 aoiit, Jes attaques ont imrnediatement baisse dans le verger traite alors qu'elle etaient importantes dans le verger non traite, notamment entre lel5 et le 22 aoiit. Le troisieme traitement futle plus efficacepuisqu'il a permis!'eradication totale des adultes (Tab.II).

2.3 - Vari&tion des stades lmmatures

En juillet avant que le premier traitement ne soit applique, Jes taux de piqiires n' etaient pas significativementdifferents ( Tab. III )

Tableau . ID : Nombre moyen de piqiirespar olive, par mois et par verger

Mois Verger non traite B Verger traite c Test .T l'robabilite

Juillet 0,19 0,26 1,07 0,29

Aout 6,34 2,91 2,92 0,004

Septembre 6,48 3,19 4,11 < 0,0001

Octobre 8,78 1,74 8,57 < 0,0001

Novembre 10,20 1,71 10,11 < 0,0001 - 72 -

Les resultats sont similaires pour les oeufs Notons qu'ils etaient particulierement nombreux mais beaucoup etaient noiratre et desseches ; its devaient probablement etre morts En juillet, les infestations ne presentaient aucune difference significative. A partir du mois d'aoilt, les attaques etaient significativement plus importantes dansle verger non traite B que dans le verger traiteC La figure 4B montre que Jes infestations etaient comparables jusqu'a la mi-aoilt, puis elles ont varie fortement dans le verger non traite en presentant trois pies importants en debut septembre, debut et finnovembre elles ont peuvarie dans le verger traite . Les infestations larvaires etaient faibles dans les deux vergers jusqu' a la fin aoilt puis elles ont augmente dans le verger non traite en presentanttrois pies sesituant a la fin septembre, debut puis finnovembre ( Fig . 4C ) . Dans le verger traite C, les attaques ont peu varie a ['exception du debut novembre. En comparant les infestations finales d'octobre et novembre des deux vergers, nous constatons que le verger non traite B est presde 5 fois plus attaque que le vergertraite C

r 14 12 p I Q & u • Avcc imoaicide ll 6 DSans E s 4 2

&o

0 IS.0 E 12.S B u • Awx; insc:Clicidc F DSans s 10,0 7.S s.o 2.S

l&o 200 220 240 260 280 30() 320 340 DATE'(i.part1.r du ler Janvier)

L A 1,4 ll 1.2 V E 1,0 s • Avec. insecticide 0,8 CSaru; 0,6 0.4 --A2.

180 200 220 240 DATE (l partir du !er Janvier) AOUT OCTODllE DECEMBRE

Figure. 4 : Variations temporelles desnombres de piqiires,d'oeufs et delarves par olive, au niveaudes vergers traite et non traite

3 - Etude du panuitisme

Les piegeages et certains elevages ont permis de recolter des parasites appartenant tous a l' espece Opius concolor. Nous avons calcule le taux de parasitisme en rapportant leur nombre a celui du total des emergences ( mouches plus parasites) obtenues lors des 46 elevages effectues de 1987 a 1992 ; les resultats obtenus sont regroupesdans le tableau IV. - 73 -

Tableau IV: Taux de parasitisme de B.o/eae par O.concolor obtenus a partir des elevages effectues au laboratoire, regroupes par annee. Le nombre d'adultes represente Jes mouches et Jes parasitesqui ont emerge.

Annee Nombre de fruits Nombre d'adultes Nombre de parasites Taux de parasitisme en% 1987 1500 27 11 45.7 1988 2500 44 4 9.1 1989 3000 51 13 25.5 1990 3500 43 16 37.2 1991 3000 0 0 - 1992 3000 33 5, 14.7 Le taux deparasitisme, obtenu a partir des elevagessur des effectifsreduits de pupesa varie de 9 a 46 % selon les annees.Toutefois, ii reste faible pourpouvoir contrf>ler a lui seul Jes populationsdu ravageur . Le faible parasitisme de 1988 ( 9 % ) et I' absence de parasites en 1991 semblent directement lies aux conditions climatiques puisque les temperatures furent tres elevees en ete avec une pluviometrie pratiquement nulle, ce qui implique une hygrometrie tres faible . Or , le developpement larvaired'Opius conco/or necessite une hygrometrie importante pour eviter la dessication des tissus contenus dans le puparium ( Delanoue et Pralavorio, 1977).

4 . Evaluation de !'impact des soins culturaux

Nous avons juge interessant d'evaluer !'importance des soins culturaux, en comparant en 1991, Jes infestations de deux vergers oleicoles , l'un etant entretenu, l'autre pas. Ces deuxvergers n'ont subi aucun traitement parinsecticide Le taux moyen de piqures varie d'aoiita septembre en etant significativement plus important dans le verger C non entretenu que dansle verger B entretenu, a !'exception du mois d'aoiitou la differenceentre les deux vergers n' etait pas significative . En regroupant octobre et novembre, on peut estimer I' efficacite de l' entretien, notamment pour Jes larves qui representent le mieux les infestations ; les attaques larvaires sont en moyenne de 3,5 fois plus importantesdans le verger sansentretien C quedans le verger entretenu B ( Fig . 5 )

A 3 SANS ENTRETlEN ! 2.S

£°I..S AVEC ENTREI'IEN

320 340

! ,4 �) .2 ,I, .\VEC ENTRETIEN ( llO 240 260 21<1 300 320 Dile

C ! .2 t" ,!

,05

AOUT DECEMDRE

Figure. 5: Variations temporelles desnombres de piqilres,d'oeufs et de Iarvespar olive, au niveau des vergersentretenu et non entretenu • 74 - 4 - mscussmN Les premieres attaques efficaces, provoquant des degats supeneursau sew! de tolerance fixea 10 % ( soit d'apres nos estimations , 0, 12 piqtlres / olive ) ont lieu en juin dans Jes zones du littoral;en juillet a Tlemcen et seulementen aoiit pour Jes regions situees a plus de I OOO m d'altitude ; le bioclimat a une incidence particulierement significative ( Gaouar et Debouzie, 1991 ).L'epaisseur et le degre de maturation des fruits semblent egalement influencer le declenchement des premieres infestations (Jerraya et al., 1982).Par ailleurs Longo et Benfatto (1982) constatent que Jes attaques commencent finjuin en Sicile, alors que la teneur en huile des olives atteint 2% . Les infestations deviennent maximales quand le taux d'huile est de 40 %. A Corfou, Jes fruits deviennent receptifs en juillet, ce qui pennetJes premieres attaquesefficaces (Kapatos et Fletcher, 1984 ). Dans la Crete, plus au Nord, Jes premi(!resinfestations ont lieu a la mi-aoiit(Louskas et al., 1980 ) . La structure horizontale des infestations revele qu'il existe une forte heterogeneite entre Jes arbres d'un meme verger . Elle serait probablement due aux differences phenologiques existant entre Jes oliviers, ce qui induit des emanations d'intensite variable, done de receptivite variable ( Prokopy, 1977 ) . Les piqtlres et Jes larves suivent generalement le meme type de repartition a une date donnee, en etant plus importantes vers la partie Sud du verger , la partie Ouest etant la moins infestee. Dans Jes trois vergers etudies, leurs parties Sud jouxtent d'autres oliveraies, des populations de B. oleae ont pu se deplacer en creant un effet classique de bordure, signale dans de nombreux travaux ( Fletcher et Kapatos, 1981 ; Debouzieet Thioulouse, 1986 ). En ce qui concerne la repartition verticale des attaques, la partie basse de I'arbre est plus infestee que la strate haute. Ce gradient a ete observe du debut des infestations , enjuilletjusqu'a la recolte, finnovembre . Nos resultats differentde ceux de Montiel Bueno ( 1984 ) qui avail observe des differencessignificatives entre Jes deux parties de l'arbre seulement fin septembre, debut Octobre. 11 avait note egalement que le secteur Sud de J'arbre etait plus infeste que Jes trois autres, alors que nous n'avons trouve aucune incidence de !'orientation ( Gaouar et Debouzie, 1991 ) . Par contre, nos resultats concordentavec ceux de Mustapha et Al Zaghal (1987) en Jordanie qui avaient observe que Ja partie basse de l'arbre etait la plus infestee, quelle que soit la variete consideree . Ces resultats suggerent que Jes imagos qui emergent du sol, interceptent la prernierestrate qui offre des conditions de temperature et d'hygrometrie favorables, jusqu'a saturation des fruits avant d'attaquer les strates superieures( Fletcher et Kapatos, 1982 ; Fletcher, 1987 ). Les pulverisations d'insecticides doivent etre effectuees des que Jes pieges Mc Phail capturent en moyenne 4 a 5 mouches par piege ( Broumas et al ., 1982 ) ; elles seront nettement plus accentuees a la strate basse de l'arbre, la plus infestee, des I'apparition d'une nouvelle generation d'adultes .Cette strategie pennet la reduction des infestations de 5 fois en moyenne, ainsi que celle de I'emploi des insecticides dont la nocivite a fait l'.objet de nombreux travaux (Argentesi et al , 1982 ; Kapatos et Fletcher, 1982 ; Haniotakis , 1986 ; Claridge et Walton, 1992; Katsoyannos, 1992; Jacas et Vifiuela,1994 ) Le seul parasite de B. oleae que nous ayons trouve a Tlemcen est le Braconide O.concolor, a des taux variant de 9 a 46 %, selon Jes annees. Son role est tres variable d'une annee a l'autre, mais ii demeure toujours insuffisant ..pour limiter effectivement I'accroissement des populations des phytophages. Selon Delrio et Cavallorci( 1977 ) et Debouzie ( 1989 ), un parasitisme de 90 % ne suffit pas a 'Jui seul, pour detruire une population de mouches . II faut en outre intervenir au printemps, sur Jes fruits residuels pour obtenir des resultats acceptables ( Kapatoset al . , 1977 ; Liaropouloset al. , 1977 ) . Plusieurs travaux ont signale !'importance des pratiques culturales ( la taille, le labourage, !'irrigation ... ) comme moyen de prevention, voir de Jutte visant a reduire Jes infestations ( Bonnet, 19.60 ;. Pagnol, 1979 ; Loussert et Brousse, 1978 ; Amouretti et Comet , 1989 ). En revanche , aucune evaluation quantitative n 'a ete fournie, pennettantde mesurer a quel pointl'entretien premunit le verger des attaques de B . oleae . L'etude comparative de deux vergers, dont l'un a subi Jes differents soins culturaux conseilles en oleiculture et l'autre pas , a montre qu'il existe une difference significative entre Jes infestatins par B.oleae . En effet, le verger entretenu est de trois fois moins infeste que celui qui ne I'est pas : ce resultat doit etre considere comme positif.

5 - CONCLUSION

Les resultats que nous avons obtenus ont perrnisd'etablir une strategie de Jutte appropriee aux criteres bio-ecologiquesde la mouche, specifiquesa la region de Tlemcen. Nous proposons d'effectuer trois traitements d'insecticides a la fin juillet, vers le 20 aout puisle debut septembre en insistant sur Ja strate basse de I' arbre , situeea moins de 2 m de haul . Les oliveraies doivent subir Jes soins culturaux classiques pour favoriser !'action des parasites ainsi que celle des pulverisations d'insecticides . Nous sommes convaincu que la combinaison des trois types de Jutte donne de bons resultats puisque meme en Jes separant, ils ont pu reduireJes infestations . Actuellement, ce schema de Jutte que nous preconisons semble etre le plus efficacepuisqu'il pennetde !utter contrele ravageur en reduisant!'utilisation des insecticidesce qui protege I'entomofaune, notamment Jes parasites naturels qui aideront a reduire Jes populationsdu phytophage - 75-

6 - BIBLIOGRAPHIE

• ArnomettiM., Comet G.( 1985)- Livre de l'olivier, 161 p • ArgentesiF. , Cavaller R ., Di Cola G. (1982)- A New theoretical approach lo the control and management offruit fly systems. CEC/ IOBC Symposium - Athenes . pp . 297-305 • Bonnet P. (1960) - La reconstitution des oliveraies fran�aises detruites par le gel de Fevrier 1956 Service Regional de l'Oleiculturede Marseille . 82 p. • BroumasT ., Katsoyannos P., Yamvrias S., Liaropoulos C., Haniotakis G., StrongF. ( 1982) - Controlof the olive fruit fly in a pest managementtrial in olive culture. CEC/IOBC Symposium- Athens , pp . 584-59 I . * Claridge M.F ., Walton M.P ( 1992)-The European olive and its pests management strategies. BCPC Mono-research collaboration in European . IPM Systeme, 5_2:3 - 12 • DebouzieD. ( 1989) - Biotic mortality faclorsin Tephritide populations . In : Fruit fly.Their biology, natural enemis and control . Ed . byA.S . Robinson and G.Hooper . Amsterdam,Elsevier, 38 : 221 - 227. • Debouzie D. ,Thioulouse J. ( 1986) - Statistics to find spatial and temporalstructures in populations . In : Pest control operations and systems analysis in fruit fly management. Nato AS! Series G : Ecological Sciences , Springer Vcrlag, Berlin, 11: 263 - 282. • DelanoueP. Pralavorio R . ( 1977) -Besoins en eau comparesde Ceratitis capita/a Wied et de son parasite inteme Opius concolor Szepl. durant la phase pupale de l'hote. Bull. Soc. Zoo] .Agric., 1: I -7. * DelrioG, Cavalloro-R ( 1977}· - Repertisul ciclo biologicoc sulla dinamica di popolazionc de! Dacus oleae Gmelin in Liguria ., 60:211 - 253 • Fletcher B.S . ( 1987) -The biology ofDacine fruit fly. Ann. Rev . Entomol., 32: l 15 -144. • Fletcher B.S .Kapatoo E.T ( i 981) -Dispersul of ihe oiive fly Dacus oleae, during thesummer period in Corfu. Entomol Exp. Appl., 29: l -8. • Fletcher RS . Kapalos E.T ( 1982) - An evaluation of different temperature-development rate modelsfor predicting thephenology ofthe fly Dacus oleae. CEC/IOBC Symposium -Athens, pp. 321-329 • Gaouar N., DebouzieD. ( 1991) -Olive fruitfly, Dacus o/eae Gmel. ( Dipt . Tephritidae) damage in Tlemcen region , Algeria.J. Appl . Ent ., 112: 288 - 297. • Gaouar N., Debouzie D. ( 1995)- Whithin treevertical patternin Bactrocera oleae Gmel ( Dipt., Tephritidae) infestation and optimization ofinsecticide applications. J. Appl Ent ., 119: 262 -289 • Haniotakis G.E. ( 1986) • The role of sex pheromones in the control ofthe olive fruitfly Dacus oleae: Present status prospects. In: Pest controloperations andsystems analysisin fruit fly management. Nato AS! Series G: Ecological Sciences, Springer verlag, Berlin , 11: 147 -15 8. • Jacas J.A., ViiiuelaE. ( 1994 ) -Analysis of a laboratory methodlo test the effectsof pesticides on adult femalesof Opius conco/or( Hym.,.Braconidac), a parasitoid ofthe olive fruit fly ,B. oleae ( Dip., Tephritidac) . Biocontrol Science and Technology, 4: 147. 154. • Kapatos E.T Fletcher B.S. ( 1982)- Developmentof a pest management systemfor Dacus o/eae in Corfuby utilising ecologicalcriteria . CEC/IOBCS ymposium- Athens, pp . 593-602. • Kapatos E.T Fletcher B.S. Pappas S.,Laudeho Y.( 1977) -The release of Opius concolor and 0. conco/or var. siculus ( Hym: Braconidae)on Corfu. Entomophage, 22: 265 - 270 . • Kapatos E.T ., Pappas S., McFadden M.W. ( 1977a) -Sampling teclmiques and preparation ofpartial life tables for the olive fly, Dacus 0/eae ( Dipt: Trypetidae)in Corf.i. Ecological Entomology,2: 193 - 196 • KatscyannosP. ( 1992)- Oliv e pestproblems and their control in the near-east.FAO Plant Production. Doc. FAO . • LiaropoulosC., Canard M. LaudcboY. ( 1977) -lachers d "Opius concolor.( Hym., Braconidae) dans des populations de printemps de Dacus oleae ( Dipt , Trypetidae).Entomophaga, 22:259 -264 . * Longo S., Benfatto D . ( 1982) • Observation on olive fly( Dacus oleae Gmel . )population dynamics in Sicily . CEC/!OBC Symposium-Athens ,pp . 612-615. • LouskasC. ,Liaropoulos C., Canard M., Laudeho Y. ( 1980) -• Infestationestivale precoce des olives par Dacus oleae Gmel . ( Dipl., Trypetidae). et role limitant du parasite Eupelmus urozonus Dalm. (Hym.Eupelmidae) dans une oliveraie grecque. Z. ang. Ent. , 90: 473 -481. • Loussert R ., Brousse G. ( 1978)- !'Olivier . Ed. Maisonneuve et Larose, Paris . 464 p. • Montiel-Bueno M.A., ( 1984) - Estudios preliminarde la reparticion de! ataquc de Dacus oleae a nivel de arbol. Bo!. Ser:. Plagas, 10: 59 - 64 . • Mustapha T.M., Al Zaghal K.H . ( 1987) -Frequency of Dacus oleae Gmel innnature stages and their parasites in seven olive varieties , in Jordan . Insect.Sci . Appl ., 8:165 -169 • PagnolJ. ( 1975) -L'olivier. Ed . Aubanel,95p. * Prokopy R.J. ( 1977)- Stimuli influencingtrophic relations in Tephritidae Colloq. int CNRS, Paris, 265: 30 5 -336. - 76 -

REPARTITION DE L'INFESTATION DE. LA MOUCHE DE L'OLIVE BACTROCERA OLEAE (GMEL.) (DIPT., TEPH.) EN VERGER OLEICOLE DANS LE SAIS AU MAROC

1 1 1 2 3 AFELLAH M ., BEN HAMADI 1 ., SMAILJ C ., HILAL A ., CHEMSEDDINE M • 1: I.N.R.A. Laboratoire de Zoologie B.P. 578 Meknes Maroc Fax 5. 51 20 40 2: I.N.R.A. Laboratoire de Zoologie B.P. 533 Marrakech Maroc 3: Faculte des Sciences Semlalia Universite Cadi AyyadB.P. S15 Marrakech Maroc

Resume Bactroce ra oleae Gruel. semble presenter une tendance a infester d avantage Jes parties sud, haute et externe de l'arbre. Le suivi de la grosseur et de la maturation des fiuits a montre que ces parties se distinguent egalement par des fiuits precocement plus gros et receptifs. Une difference d'infestation a egalement ete notee entre les varietes avec un niveau plus eleve pour la Gordale (variete a gros fiuit). Cependant la Meslalla, variete egalement a gros fiuit, m;oit moins d'infestations suite a sa maturation avancee. L'ensoleillement et des facteurs biochimiques semblent jouer un r61e dans la repartition de !'infestation en agissant sur l'activite de l'insecte et sur le developpement du fiuit. Des differences d'attaque ont egalement ete observees entre les vergers situes a altitudes differentes et entre annees Mots cies: Olivier, variete, Bactrocera oleae GmeL; infestation, Sais(Maroc). l- Introduction Dispersees dans la nature, apres la recolte, les populations de la mouche de !'olive B.oleae se concentrent dans Jes oliveraies des que Jes fiuits deviennent receptifs (LUPO, 1973; GIROLAMI, 1978). En ete les adultes se dirigentvers les vergers precoces ou irrigues (NEUENSCHWANDER, et al., 1986) et plus tard les populations attaquent les oliviers situes dans Jes zones oleicoles tardives (MICHELAKIS, 1980) sans parter de migration au sens strict du ter, ne. Depuis plus d'une vingtaine d'annees, la relation entre la plante-hote et l'insecte qui l'infeste et les mecanismes qui entrent en jeu attirent !'attention des chercheurs. La·difficulte est de dist'inguer, parmi la multitude de produits vegetaux secondaires, d'eventuelles traces de substances susceptibles d'etre un signal utilisable par les femelles pour reconna1tre la plante convenant le mieux a la reproduction(GIROLAMI etal., 1981a). La ponte des Tephr itidae peut etre stimulee par des composes chimiques·provenantdes fruits visites, c'est le cas de Rhagoletis cerati L (HAISCH et LEVINSON, 1980). En ce qui concerne B.oleae, la ponte est stimulee par des substances intervenant dans la biochimie de l'oleoeuropeine, glucoside phenolitique de l'olivier(GIROLAMI et al., 1975). B.oleae peut aussi etre attire par la couleur de la plante (GIROLAMI et CAVALLORO, 1973) Les stimulants physiques et chimiques ·contribuent a induire la ponte (PROKOPY et HANIOTAKIS, 1976). CIRIO (1971) et GIROLAMI et al. (1981a) parlent d'un autre cote, de substances inhibitrices qui se degagent des olives blessees suite aux pontes precedentes. L'activite inhibitrice de ces substances est essentiellement liee a des composes liposolubles (GIROLAMI et al., 1981b). Dans cette etude, nous contribuons a la connaissance du mode de repartition de !'infestation de B.oleae au sein de l'arbre, entre varietes et localites a altitudes differentes dans le but de comprendre et de resoudre les problemes que pose encore ce ravageur aux producteurs en zone extensives malgre ]'application de traitements chimiques annuellement et mettre au points des methodes d'echantillonnage adaptees aux objectifs des producteurs et des services des avertissements. agricoles. 2- Materiels et methodes 2-1- Vergers d'etude Le travail a ete mene dans deux zones a altitudes differentes Ain Taoujdate (500m) avec quatre vergers plantes par quatre varietes (deux a huile Picholine marocaine et Picholine de Languedoc - 77 - et deux de table Gordale et Meslalla ) et la zone allant vers Zerhoune (200m) avec un seul verger plante comme toute la zone par la Picholine marocaine. Les soins apportes dans les deux zones sont lirnites a une taille irreguliere et un labour annuel pour !utter contre les mauvaises herbes. 2-2- Techniques d'echantillonnage L'echantillonnage a porte sur quatre arbres par verger dans les deux localites a Ain Taoujdate et a Zerhoune. Chaque arbre est divise en 16 sites de prelevement representant quatre orientations, deux strates (haut et bas) et deux couronnes (exteme et inteme). 5 fruits sont preleves au niveau de chaque site, ce qui donne un echantillon hebdomadaire de 80 olives/arbre et un total de 1280 fruits par semaine Le travail a dure deux annees de 1993 a 1994. Un examen systematique de tous les fruits est realise au laboratoire sous loupe binoculaire pour determiner l'etat sanitaire des fruits en provenance des deux zones, des quatre varietes et des differentssites de l'arbre. Le poids de chaque olive est egalement mesure. 3- Resultats 3-1- Influence de !'orientation sur la repartition de !'infestation Nos resultats montrent que la grande partie des fruits infestes est situee au niveau de !'orientation sud de l'arbre (Fig 1 et 2). Son pourcentage par rapport a !'ensemble des fruits infestes est maximal vers la fin du mois d'octobre au niveau de tous Jes vergers. II varie de 41 a 43 % pour la Picholine de Languedoc, la Picholine marocaine et la Gordale et de 3 l a 37 % pour la Meslalla a Ain Taoujdate et de la Picholine Marocaine a Zerhoune. Le nord d� l'arbre est le moins infeste. Le pourcentage de fruits attaques preleves de ce cote oscille entre 15 et 21 % de !'ensemble des fruits infestes au niveau de tous Jes vergers. 3-2- Influencede la strate sur la repartition de !'infestation Le pourcentage des fruitsinfestes preleves du haut de l'arbre par rapport a !'ensemble des fruits infestes est toujours superieur a celui des fruitspreleves de la strate basse (Fig 3 et 4). Ce pourcentage est maximal vers la findu mois d'octobre, ii varie de 60 a 73 % selon les vergers. Cette differenceentre les taux d'infestation au niveau des strates haute et basse est significative au seuil de I% pendant les prelevements du 21 et du 27 octobre. 3-3- Influence de la profondeursur !'infestation Les resultats obtenus montrent que la couronne exteme de l'arbre a tendance a etre legerernent plus infestee que celle de l'interieur (Fig 5). Les fruits infestes preleves de l'exterieur de l'arbre representent de 51 a 62 % de !'ensemble des fruits infestes quelle que soit la variete On note, cependant, que c'est vers la fin du mois d'octobre que la difference devient significative au seuil de 5%. La constatation faite precedemrnent reste valable pour la profondeur de la couronne. 3-4- Influence de la variete sur !'infestation La figure 6 illustre !'evolution des taux d'infestation entre lcs varietes d'olive a Ain Taoujdate pendant la periode autornnale. En debut de saison, !'infestation des fruits est tres faible sur la Gordale et nulle sur Jes autres varietes. Elle augrnente ensuite progressivement clans tous Jes vergers pour atteindre son pie le 21 octobre sur la Picholine rnarocaine et la Meslalla avec respectivement 37,5 et 13,6 % des fruits infestes et le 27 du rneme mois pour la Gordale et la Picholine de Languedoc avec respectivernent 43 et 35,6 % des fruits infestes. A partir du rnois de novembre,. Jes taux d'infestation pour la Gordale, la Picholine rnarocaine et la Picholine de Languedoc tendent a representer un groupe hornogene, tandis que !'infestation des fruitssur la Meslalla derneure significativernentplus faible. 3-5- Influencede !'altitude sur le taux d'infestation Nos observations ont rnontrela presence de populations irnaginales en debut d'ete a Zerhoune dont Jes captures se sont etalees de la rni-juinjusqu'en debut juillet. Cette presence d'adultes bien que lirnitee clans le temps a perrnis d'avoir les premiers oeufs dans les olives. Ces oeufs, faibles en effectif, s'annulent rapidement par la suite A Ain Taoujdate, aucun stade pre-irnaginal n'a ete trouve a l'interieur des olives avant l'automne. Durant la periode automnale, !'infestation des fruits de la Picholine marocaine est significativement plus faible a Zerhoune par rapport a Ain Taoujdate pour tous Jes prelevernents(Fig 7) - 78 -

Meslalla 1993 Gordale 1993 Effectifs de fruits infestes Effectifs de fruits infestes 80 ll\\'!Notd I 1 •:s11d DE•t Oouett �Nord I '==

00 80

40 40

I 20

21/10 �•nl iS/10 21/10 27/10 10/11 19/11 15/10 27/10 10/11 19/11 Date de prelevement Date de prelevement

P. de Languedoc 1993 Picholine marocaine 1933 Effe.ctifs de fruits infestes Effectifs de .fru.its ,.infestes oo;=-:.===-=-===--====------� 80 �Nord I •eud OE1t DOuHt �Nord I f •a11d DE11 Douee1 I I 00 oof 40 40� ..

20

! 15/10 2V10 27 /10 10/11 19/11 15/10 2·.,10 27/10 10/11 19/11 I Date de prelevement Date de prelevement

Figure 1: Evolution de !'infestation des olives par B.o/eae selon l'orientation a Ain Taoujdate - 79 -

Zerhoune 1993 Effectifs de fruits infestes 80r------·------, I ..Sud DEst L_J Quest &WWW: 60

4 0 r

2oL I I a ab i L I 0 L= •• =r:::-:mI�• '� :::::r--. •·=ra I 15/10 21/10 27 /10 10/11 19/11 Date de prelevement Fig2: Evolution de !'infestation des olives par B.oleae selon !'orientation a Zerhoune

Zerhoune 1993 Effectifs de fruits infestes 160 r------� -----i 4 1 0 �Haut DBas I 120

100

80

60

40

20 t 0 == 15/10 21/10 27/10 10/11 19/11 Date de prelevement Fig 3: Evolution de !'infestation des olives par B.oleae selon la strate a Zerhoune - 80 -

Meslalla 1993 Gordale 1993 Effectifs de fruits intestes Effectifs de fruits infestes 1•0�------�

I �Haut Dau I

'!00

80

80

40

20

15/10 21/10 27/10 10/11 19/11 21/10 27110 10/11 19/11 Date de prelevement Oate de preJevement

P. de Languedoc 1993 Picholine marocaine 1993 , Effectifs de·-fruits infestes Eifectifs 'de fruits infestes ---- -� 1•0�------� ••o�------Den l�Hawt I l�Haut 08-) 140 140

120 120 � 100 '1"00 ,

80 80

60 ••

19/11 15/10 21/10 27110 10/11 19/11 15/10 21/10 27/10 10/11 Date de prelevement Date de prelevement

Figure 4: Evolution de l'infestation des olives par B.oleae selon la strate a Ain Taoujdate - 81 -

Meslalla 1993 Gordale 1993 Eflectifs de fruits infestes Eflectifs de fruits infestes ,00 ,------� 100 � �Ext Dini I �Ext ! ' 140 � 140

12or 120

100� 100

BO� BO

60 BO r

40r 40

20� 20 ol== CJ � l[J 15/10 21/10 21/10 Date de 27/10pretevement 10/11 19/11 15/10 Date de 27/10prelevement 10111 19/11

P. de Languedoc 1993 Picholine marocaine 1993 Effectifs de fruits infestes Effectifs de fruits infestes 160,------� ,00,------�

1,40 � !�Ext Ot11t I 1�Ex1 Olntl 140�

120 � :::� ,oot- •or BO

00 I 40 1 20 ��'-==--- IJ""'------'--11=--ul �L-...llillll.-lJ-'------=1��• 10/11 15/10 Date de 27/10pre1evement 19/11 15/10 21/10Date de 27/10prelevement 10/11 19/11

Figure 5: Evolution de !'infestation des olives par B.oleae selon la profondeur a Ain Taoujdate - 82

Taux d'infestation cumules 140 Gord ale 120 P.marocaine � I !ii 100 P.de Languedoc i Ii Meslalla j jl 80

60 ' 40 I c_

2: Jb==c.a·=1::-1:��-·:=1111_..U�ILLJ, 7/10 15/10 21/10 27/10 10/11 19/11 Date de prelevement Fig6: Evolution des taux d'infestation des olives par B.oleae sur les varietes a Ain Taoujdate

Taux d'infestation cumules 140 m PM Ain Taoujdate 120 D PM Zerhoune

100

80

60

40

20

15/10 21/10 27/10 10/11 19/11 Date de prelevement Fig7: Evolution des taux d'infestation des olives de la P.marocaine par B.oleae dans les deux zones - 83 -

4- Discussions et conclusions D'apres nos resultats, ii semble que la mouche de !'olive presente une preference, bien que non stable durant la periode des prelevements, pour les parties sud, haute et exteme de l'arbre. Cette variabilite du choix des sites de ponte s'explique par le comportement de ponte de la femelle qui ne revient pas sur la meme olive ou sur un fruit deja piquee par une autre femelle (GIROLAMI et al., 1981, GIROLAMI et al., 1982, STRAPAZZON et al., 1984), ce qui conduit a l'elargissement des attaques aux autres parties. Le suivi de la grosseur et de la maturation des fruits a montre que ces parties se distinguent egalement par des fruits en avance dans la taille et precoces dans la maturation. Cette liaison entre !'infestation et le poids des fruits au niveau de l'arbre ou du verger a ete egalement observee par d'autres auteurs MONTIEL et MORENO (I 984) en Espagne, DOMINICIet al. (1986) en Italie et par ZAOUG (1993)et AFELLAH etal.(1996) au Maroc. L'ensoleillement est le facteur essentiel pour le choix des lieux d'infestation et de developpement des fruits dans le meme arbre. Son action peut etre directe sur l'insecte qui pourrait chercher les parties Jes plus ensoleillees et les plus chaudes de l'arbre pendant cette periode de l'annee (octobre et novembre) ou les temperatures commencent a baisser serieusement dans notre region. Cet effet pourrait apparaitre sur des fruits qui evoluent differemment aussi bien au niveau du meme arbre qu'au niveau du merneverger et de la meme.zone .. Lataille, Je poids, la coloration, la receptivite et la biochimie des fruits de ces arbres evoluent dans le temps comme J'a rapporte LAUDEHO et al.( 1978) suite a des mesures de temperatures dans diverses parties de l'arbre et meme dans des fruits a maturation differenteet ce en liaison avec les attaques de B.oleae. Les differences d'infestation notees entre les varietes etudiees ne peuvent que confirrner ces observations. Plusieurs auteurs parlent de l'effet attractif ou repulsif de substances biochimiques des fruits selon leur degre de maturation sur le niveau d'infestation (GIROLAMI et al., 1975; vrrA et BARBERA, 1978; GIROLAMI et al., 1981a et b; CIRIO et GHERADINI, 1981). Des etudes ont egalement montre que si !'infestation est moyenne, B.oleae pond de preference dans des olives de grande taille et si elle est e!evee, Jes fruits de taille et poids inferieurs sont egalement attaques. Ces memes etudes (CHESI et SANDI, 1982; CHESI etal., 1983) rapportent que, la comparaison entre la taille et le poids des olives infestees par les oeufs et Jes larves du premier stade et de celles indemnes d'attaque a explique ce comportement. Le fruit, par le biais de substances biochimiques attire egalement la femelle qui, apres la ponte, blesse !'olive qui degage un jus qui, a son tour devient repulsif pour Jes autres femelles. Ces substances chirniques ont ete etudiees par plusieurs auteurs qui ont confirrne leur effet par des tests au laboratoire et en conditions semi-controlees (GIROLAMI et al., 1981a et b; GIROLAMI et al., 1982; STRAPAZZON etal., 1984). On peut dire que la repartition de !'infestation au sein de l'arbre, dans le verger et entre les varietes est regie par des facteursphysiques et surtout chimiques. L'action des facteurs chimiques suit une chronologie qui commence par !'attraction des mouches vers Jes arbres, peu de temps avant la maturation (STRAPAZZON etal., 1984). Les fruits precoces serom done Jes premiers attaques en degageant des substances biochimiques qui stimulent la ponte des femetles. GIROLAMIet al. (1981 a), parlent d'une substance de nature phenolique liee a la biochimie de J'oleoeuropeine, extraite par le methanol et testee au laboratoire et a J'air libre par piegeage Ces fruits attaques produiront de nouveau des substances repulsives qui sont essentiellement des composes liposolubles (GIROLAMI et al., 1981b). Ces phenomenes perrnettent d'expliquer Jes differences d'infestations enregistrees au sein du meme arbre, entre Jes differentes varietes a Ain Taoujdate et entre cette zone et celle de Zerhoune. II parait alors anorrnalde constater que la zone oleicole allant vers Zerhoune.de faible altitude et a maturation precoce, soit moins attaquee malgre la presence d'adultes en debut d'ete. Ce phenomene s'explique par le comportement de B.oleae qui revient sur Jes vergers precoces en debut d'ete (DOMINICI et al.1986), rriais dont l'activite ovarienne est completement bloquee par Jes chaleurs elevees (MICHELAKIS, 1980; ARAMBOURG, 1984; NEUENSCHWANDER et al., 1986; AFELLAH et al.1995). Apres J'ete, Jes olives de cette zone, se trouvant dans un etat de maturation - 84- avance, sont moins attaquees que celles de la zone de Ain Taoujdate ou Jes fruits· montren� un · retard net clansla maturation. La non receptivite des olives dont la maturation est tres avancee a ete egalement observee a Ain Taoujdate ou, la Meslalla, variete de table a gros fruit, rec;oit moins d'infestations suite a sa precocite nette par rapport aux autres varietes clans cette zone. En automne, les populations de la mouche sembleraient faire le chemin inverse en retoumant sur Jes oliviers tardifs en altitude ou les olives sont plus receptives et permettent le developpement de nouvelles generations de la mouche. MICHELAKIS (1980) et STRAPAZZON et al.(1984) ont constate le meme phenomene en Grece sans parter de vrai phenomene de migration. On a egalement constate que !'infestation varie d'une annee a l'autre. En effet, en automne 1993, Jes attaques sur olives sont importantes alors qu'en 1994 l'infesation est nulle dans nos vergers. La premiere situation s'explique par l'abondance des fruits, les conditions climatiques favorables (automne pluvieux et temperatures entre 15 et 20°C) et un pourcentage de femelles matures eleve. L'autre situation s'explique par un automne sec et chaud et par un faible niveau de populations de femellesmatures. Nos observations ont montre que Jes infestations d'ete sont soit absentes soit tres faibles et disparaissent rapidement sous l'effet des chaleurs elevees. Cependant des infestations importantes peuvent exceptionnellement apparrutre si Jes conditions climatiques estivales sont clementes en presence d'un effectifeleve de femelles matures de B.oleae. L'installation d'un reseau de piegeage des adultes et d'un nombre de pastes meteorologiques depuis le mois de juin s'avere necessaire clansnotre region, en plus de prelevements reguliers d'echantillons. En automne, le producteur dispose d'une periode d'environ 45 jours avant la recolte, pendant laquelle les conditions d'infestation sont favorables en basse et haute altitude, pour suivre et !utter contre B.oleae.. La comprehension.du mode de repartition de !'infestation dans l'arbre permet egalement d'effectuer un echantillonnage adequat selon la date de prelevement ainsi que !'application de traitements localises du cote sud et haut de l'arbre en remplacement de traitement de couverture dans le cadre d'un systeme de lute integree. References bibliographiques AFELLAH M., BENHAMMADI I.; HILAL A., CHEMSEDDINE M. (1995) - Influencxe de l'alimenation et de la temperature sur la longevite et la fecondite de Bactrocera oleaeGmel. Olea. lp. AFELLAH .M., SMAIL! C., BENHAMMADI I.; HILAL A., CHEMSEDDINE M. (1996) - Influencede la variete et du type de piege sur la courbe des veils de la mouche de I 'olive Bactrocera o/eae Gmel. dans la region de Ain Taoujdate au Maroc. Ecologia Mediterranea. !Op.(a paraitre). ARAMBOURG Y. (1984) - La faune entomologique de l'olivier- Dacus oleae (Gmel.), olivae N°1 Avril - 1984. p:37-40. CHESI F. (1982) - Methcidi inteformatici nella ricerca di lotta integrata in olivicoltura. Frost. ent. p:197- 210. CHESIF. MALFATTI P. et FORNASARI L. (1983) - Dipendenza statistica tra infestazione dacica e peso dell drupe. Atti XIIICongr. Naz. It. Ent. Sestrieer-Torino.p:653-660. CHESIF. et SANDI C. (1982) - Ricerca delle variabili intluenzantila probabiliti.di infestazionedelle olive Dacus oleae (Gmel.) .(Diptera, Tripetidae). Esame prelimiminaredi due anni di sperimentazione in Toscana (Asciano, 1980-1981). Frust.ent. pl 17-132. DOMINICIM., PUCCI C. et MONTANARI G.E. (1986) - Dacus oleae (Gmel.) (Diptera..Tephritidae) ovipositing in olive drupes. II lntemSymp.Fruit.Flies.,Crete. p: l 11-120, 315-320. CIRIO U. (1971) - Reperti sul mecanismo stirnolo-riposta nell'ovideposizione del Dacus oleae (Gmel.) (Diptera, Tripetidae). Redia.p: 577-600. CIRIO U. et GHERARDINI P. (1981) - Dispersione dclle uova e intracompetitivia larvale in Dacus oleae (GMEL.) (Diptera, Tripetidae).Atti XII Congr. Naz. ltal. Entomol. Roma 1980. p: 475. RAISCH A., LEVINSON H. Z. (1980)- Influences offruit volatiles and coloration on ovipositional of the cherry fruit fly.Naturwissenschaten. p: 67-544. GIROLAMI V., CAVALLO RO R. (1973) - Metodi cromotropiciper indagina di popolazione delgi adulti di Dacus oleae. Note App. Sper. Ent. Agr. Perugia. p:13-29. - 85 -

GIROLAMI V., PELLIZZARI G., RAGAZZI E., VERNESE G. (1975)- Prospects of increased egg production in the rearing of Dacus oleae ( GmeL). by the use of chemical stimuli. In : Sterility principle for insecte control 1974. FAO/IAAE. Vienna. p:209-217. GIROLAMI V., STRAPAZZON A. et GERLONI P.F. (1981a) - Stimulants de ponte de Dacus oleae G. Reunion fa-perts C.C.E.. Antibes 4-6 Novembre 198 l p:1-13. GIROLAMI V., VIANELLO A., STRAPAZZON A., RAGAZZI E. et VERONESE G. (1981b) - Ovipositional deterrents in Dacus oleae. Ent.exp & appl. p: 29.177-188. GIROLAM! V., STRAPAZZON A. et GERLONI P.F. (1982) - Insect-Plant relationship in olive flies general aspects and new findings. Proseedings of the CEC/IOBC lntemat. S)�11pos. Athens LAUDEHO Y., LIAROPOULOS C. et CANARD M. (1978) - Etude, pendant la periode automnale, du rythme de sortie hors des fruits des larves du demier age de la mouche de !'olive Dacus oleae (Gmel.) (Dipt., Trypetidae).Ann.Zool.Ecol.anim.• 10(1).p: 37-50. LUPO V. (1973) -L'andamento clin1atico, la mosca delle olive e sua migrazione. Boll. Ent. Agr. Portici, XXX:Il.p:137-177. NEUENSCHWANDER P., MICHELAKlS S. et KAPATOS E. (1986) - Tephritidae-Dacus oleae (Gmel.). Entomologie oleicole. C.O.I. Madrid 28006 Espagne,p: 115-159. MICHELAKJS S.(1980) - Contribution a l'etude de la dynanuquc des populations de Dacus oleae (Gmel.).(Diptera, Trypetidae) en Grece. These Doct.Ing.U11.iv.Aix Marseille Fac.SciSt.Jer6me. 133p. MONTIEL BUENO A. et MORENO VASQUEZ R.(1984) - Prelinunary study of distribution of infestation on the olive tree by Dacus oleae (Gmel.) Proceedings of the CEC/F AO/IOBC. Internationaljoint meeting, Pisa 3-6 April. p:63-70. PROKOPY R, HANIOTAKIS G.E. (1976) - Host detection by wild and lab-cultured.olive flies.. In Jemi, S:,mp. Biol. Hw1g., 16, p 209-214. STRAPPAZON A., GIROLAMI V. et MASIA. (1984) - Host plant chemical regulating the reproductive behaviour of olive flies.Proceedings of the CEC/F AO/IOBC. International joint meeting., Pisa 3-6 April. p 122-127. VJT A G., BARBERA F. (1976) - Aspetti biochimici dcl rapporto pianta insetto nel Dacus oleae (Gmel.). Atti XI Congr. Naz. Ital. Entomol. Portici, Italia, 10-15 Mai 1976, p 155-161. ZAOUG .S. (1993) - Contribution a l'etude de la dynamique des populations de la mouche de l'olive Dacus oleae Gmel. (Dip., Teph.) clans la region du Haouz de Marrakech (Maroc). These de D.E.S., Fae. Semlalia Marrakech. IOOp. Session 2 BIOTECHNICAL ASPECTS OF MANAGEMENT OF FRUIT FLIES POPULATIONS

Chairman: Pedro Ros Secretaries: Mariano Muriis J. Pereira Coutinho ·• 87 -

APPLICATION OF THE COBALT-60 GAMMA RADIATION AT GREAT VOLUMES OF FRUIT FUES CERATIT/SCAP!TATA WIED. PUPAE

PIEDADE-GllERREIRO, P. * ; GOMES OA SILVA, J. ** ; GUARDADO, M. I.*; ALVES, C. H* • fnstilu/v de fnvestigartlo Cienlijica Tropical- Centro de Zoologia - Lisboa ** Faculdade de Ciencias A1edicus- Biofisica- Lisboa *** fnstituto de Higiene e Afedicina Tropical

Summary The Authors has focused a beam of cobalt-60 gamma radiation upon a great volume of pupae fruit flies, having verified an accentuated energy lost caused by the radiation interaction with the particles of the absorbent. This absorption quantitative knowledge constitutes a very important parameter as through it, it can be leant the way of the radiation dose distribution absorbed by the biological material in function with the profundity. 1 - Introduction The necessity to obtain a homogeneous distribution of the gamma radiation of the cobalt-60, using great volumes of the pupae Ceratitis capitata, having in mind their sterilization, led us to determine the variation of the referred energy in function of the biological material and also the profundity. This variation is function of the absorption by the biological material and this one with the particles of the absorbent. The quantitative knowledge of the absorption constitutes a very important parameter, as it allows us to know the procedures for the distribution of the absorbed radiation dose by the biological material.

2 - Material and Methods To accomplish the purpose a set of ten trays made of "plexiglass" was made with 200x200x25 mm, con-esponding each one to a volume of I OOO 3 cm which is equivalent to .50000 pupae (Fig. I). The bottom of these trays is of a nylon canvans which mesh of 350 mi era has no influenceat all in the process of radiation transmission (Fig.2). - 88 -

Through the side of each tray a circular opening was made of 10 mm to give entrance to an ionisation chamber, from which the information was carried into a dosimetry unit, which indicated the radiation dose received at different depths (Fig. 3). In the irradiation of biological material a cobalt source was used, having at the time an activity of 3500 Ci. The beam presented a superficial flat and uniformed distribution, being of 60 cm the distance between the source and the surface of the biological material. Through this procedure we tried to determine the absorbency value at different levels from 2,5 to 22,5 cm (Fig. 4). The dosage applied was of 2,04 Gy/min and the measured radiation at 2,5 cm of depth was of 1,73 Gy/min, which corresponds to 84,8% of the total radiation, being therefore the absorved dose of 15,2%. At 22,5 cm of thickness the quantity of measured radiation was only of 35,3% being the absorption of 64, 7%. The analysis of the presented results {Fig.5), leads us to infer that for the measured dose, when time is settled, the absorption follows an exponencial curve in the depth of the type Y=a e �P being a the ordinate in the origine which depends linearly of the time; .p is the gradient of the �urve which is independent from the time. These parameters were adjusted by the least squares method having been established the following equation: ·0• 112 Y=(-17,860 + 197,651 t) e p . r.epr.esentingJ the time and_nthe profundity.

3 - Conclusion We have thus determined the absorption value by the Ceratitis capitata Wied. pupae of the energy gamma of cobalt-60 at differentdepth levels.

4 - References CIRIO, u. - 1977. Control of mediterranean fruit flyby sterile insect technique. Boll. Lab. Ent. Agr. "Filippo Silvestri" Portici, 34: 62-73. CIRIO, u. - 19'82. Use of sterile insect technique to control medflyin the mediterranean area: application, research needs and development. Contribution n° 15 J,-om ENEA Department FARE, Division BIO-AG Laboratory Technologiesfor the Protection of Agro-ecosystem, C. S. Casaccia, Rome, Italy. - 89 -

FERON, M. - 1996. Sterilisation de la mouche mediterraneenne des fruits, Ceratitis capitata Wied., par irradiation des pupes aux rayons gamma. An. Epiphyties, 17 (2) : 229-239. HOOPER, G. H. s. - 1971. Gama sterilization of mediterranean fruitfly. Proc. Symp. Athens 14-18 Septembre 1970, IAEAIFAO: 87-95. LaCHANCE, L. E., SCHMIDT, c. H. AND BUSHLAND, R. c. - 1967. Radiation induced sterilization. Pest Control Biol. Phys., Se!. Chem. Meth. Academic Press, New York. PROVERBS, M. D. - 1969. Induced sterilization and control of insects. Ann. Rev. Entomol., 14: 81-102. PlEDADE-GUERRElRO, J., FREITAS, M. c. and MARTINHO, E. - 1985. Application of NAA in determining the inorganic contente of the fruit fly Ceratitis capitata Wied. and artificial food. Internal. Symp. Nuclear Anal. Chem. Halifax, Canada. PIEDADE-GUERREIRO, J. - 1987. A mosca da fruta Ceratitis capitata Wiedemann, 1824 (Diptera-Tephritidae) e a sua esteriliza9ao por meio de radia9oes ionizantes. Tese de doutoramento em biologia. Univ. dos A9ores. - 90 -

'" ... COVER F

I TRAYS .. zep

BASE

220

Fig. I - Prototype used in the pupae irradiation Fig. 2 - Perspective of the prototype built in acrylic with 5 mm of thickness

// �n FROl"f �!DI:: �I � laf // ,r '.· rt.AN ./.

j ! /' i I

. r-ig. J • Detail of one tray - 91 -

Gama radiation of cobalt-60

Dose variation at different levels � Output 2,04 Gy/mio Time Depth .. ( rn in) (cm) Measured Absorbed

Gy % Gy %

A 1 2.5 1.7 3 8 4.8 0.3 1 1 5.2

B 1 5.0 1,5 5 7 5.9 0.48 2 4.1

C 1 7,5 1,41 6 9,1 0.6 3 3 o. 9

D 1 1 o.o 1.25 6 1.2 0.7 9 3 8.8

E 1 12. 5 1,13 55.4 0.91 4 4.6

F 1 1 5.0 1.01 4 9.5 1.03 50.5

G 1 1 7,5 0.8 9 4 3.6 1. 1 5 5 6.4

H 1 20.0 0,79 3 8.7 1.2 5 61 .3 --- 0,12- - 3 5.3 1,3 2 6 4,7

Fig. 4 - Percentage of the radiation absorbed by the pupae Ceratitis capita/a Wied. relatively to the different considered depths.

Radiation dose Gama radiation of cobalt-60 Dose variation at dif(erent levels 100 90% eo 1i 1 0 2 7 Y = (- 7,860 + 197,651 t) e · ·11 0 p �

GOJ

"l"1O"'il 30 H Trays

20 22.5 Depth

Fig. S - Histogram and the exponential equation of the dose variation since 2,5 cm till the 22,5 cm of depth. - 92 -

EVOLUTION DE L'ATTAQUE DES FRUITS D'ETE PAR LA MOUCHE DES FRUITS CERATITIS CAP/TATA (WIED) EN FONCTION DE LA SUCCESSION DES PLANTES HOTES

H. FELLAH (1) ET M.H. DHOUIBI(1)

RESUME Le suivi de !'evolution de !'infestation des fruits d'ete par la Ceratite, rnontre que l'attaque est etroiternent liee au niveau de la population adulte dans le milieu d'etude. Le taux d'infestation est regi par Ja periode de rnaturiteet sa synchronisation avec i'apparition des generations estivales. D'autre part !'ensemble des especes et des varietes de fruits d'ete olfrea la mouche des fruits des periodes de receptivite chevauchantes tout au long de la periode estivale perrnettantson developpement et sa presence continue pendant l'ete. Le suivi de la charge en individus par fruit a montrer que la rnouche peut rnoduler le volume de sa ponte, en fonctionde l'espece et de la variete disponible, selon les caracteristiques nutritives de cette dernii:re. Ces fruit� olfrent a la Ceratite dilferentes performances pour ce qui est des taux d'ernergence. Alers qu'ils n'affectentpas la sex-ratio et les emergences.

Mots cles: Ceratite,fruits d'ete, tat1x d'infestation, charge, emergence.

INTRODUCTION La mouche mediterraneenne des fruits, diptere, polyvoltine, tres polyphage, se developpe dans le Nord de la Tunisie en sept a huit generations successives (Gahbiche, 1993; Fellah, 1996) dont cinq estivales (Fellah, 1996). De ce fait, l'arboriculture tunisienne lui offre une multitude de plantes hotes rcparties sur toute l'annee. Ainsi en plus des degiits sur agrumes, estimes a 1,6 Millions de Dinars, la Ceratite s'attaque a l'ensembie des fruits d'ete dont fa plupart ont· iine grande ·importance·economique. Les pertes sont estimees sur les fruits a noyaux et a pepins a 5,01 Millions de Dinars. La lutte anti-Ceratite reste encore aleatoire dans la majorite des biotopes a polyculture. Les interventions anarchiques constituent une serieuse menace ·pour la faune et !a tlore en plus de leurs .couts .exorbitants estimes a 0,8 Millions de Dinars (F AO-AIEA, 1995) .. Soria (1962a) et Cheikh et Ben Salah (1975) ont evalue les degats de la Ceratite se developpant au cours de la periode estivale sur les nefles, les abricots, les prunes, les peches, les figues, les pommes, les poires et les figues de barbarie. D 'autre part, la specificite alirnentaire chez la Ceratite est essentiellernent regie par des cornposantesgenetiques (Prokopy et Roitberg, 1984; Cooley et al., 1986; Chapman et Bernays,1989). La mouche choisit les fruits dans lesquels elle pond en fonction de leur valeur nutritive, qu'elle peut evaluer grace aux sensiHes gustatives localisees au niveau de son ovipositeur (Crnjaret Prokopy, 1982). La Ceratite fernelle etant la seule responsable du choix des conditions nutritives des stades immatures, ce qui conditionne la ponte (Zuccoloto, 1987).

(1) Departement de Biologie appliquee. Laboratoire d'entomologie. Institut Agronomique de Tunisie. - 93 -

Nous nous proposons dans ce present travail d'etudier !'evolution temporelle de !'infestation des differents fruits hates d'ete en rapport avec l'activite imaginale de la mouche, le comportement de ponte de la femelleet les caracteristiques de chaque hate vis a vis du ravageur.

MATERIEL ET METHODES L 'etude de !'evolution de !'infestationa ete effectuee dans un verger arboricole il polyculture d'une superficie de 13 ha, situe dans le biotope de Mornag a 16 Km de la ville de Tunis. Ce biotope appartient a l'etage bioclimatique semi-aride superieur, avec des precipitations entre 450 et 650 mm/an concentrees au cours de la periode froide. Les courbes de temperature montrent un hiver doux avec un minimum de 5°C en Janvier et Fevrier et un ete avec des temperatures superieures il26 °C.

Climatogramme de Mornag 1994. ·

100

80

Figure: Climatogramme de Mornagpour !'annee 1994.

Le domaine comporte plusieurs vergers arboricoles (pornmier, pomer, pecher, neflier, vigne, figues et figues de barbarie) et agrumicoles (maltaise et mandarinier). II est protege par des brises vents primaires et secondaires constitues de tarnarix(Tamarix sp) et de cypres (Cyprussp). Quant a la flore de la strate herbacee, elle se compose de plusieurs especes telles que Jes genres:

Amaranthus, Clandu/a, Chenopodium, Cynodon, Chrysantheme, Lupinus, 0/earacus, Oxa/is, Ma/va ... Le sol est argileux sur toute la surface de la ferme. Le suivi de !'evolution du taux d'infestation des fruits-hates se fait par le biais d'echantillonnage perioaique pris au hasard sur des pieds qui n'ont re9u aucun traitement chimique au cours de la periode de veraison et de maturite. Les fruits preleves sont examines sous loupe binoculaire pour determiner le pourcentage des fruits vereux et le nombre des individus pre-imaginaux. Ces derniers seront ensuite niiS, dans des boites remplies d'une couche de !cm de sable fin, pour la collecte des pupes qui seront il leur tour deposees dans des bo1tes d'emergence jusqu 'a leur eclosion. En ce qui conceme la population adulte, elle a ete suivie par un reseau de 5 pieges Rebell a glu et appates a la trimedlure. - 94 -

RESUL TATS ET DISCUSSION:

Evolution de /'infestation des fruits d'ete par la mouche des fruits L'echantillonnage et le suivi de l'attaque ont commence dans le biotope de Momag , le 15 Avril sur neflier du japon (Eriobot.rya japonica. Thumb), variete Arbi au stade veraison, alors que Jes captures des adultes au niveau des pieges soot encore nulles. Le taux d'infestation est nu! jusqu'au demier controle du 19 Mai, date de la recolte finale. Sur la variete de nefles "Tanaka", les premieres piqures n'ont ete enregistrees que le 29 Mai; c'est a dire a la fin de la rnaturite des nefles et avec !'apparition des prernieres rnouches dans Jes pieges. Le taux d'infestation enregistre a cette date n'etait que de 0,8%, ii est passe a 1,71% le 5 Juin. Ainsi Jes nefles echappent a l'attaque de la Ceratite dans le biotope de Momag, a cause du retard de reprise d'activite de la mouche. Ceci, contrairernent au biotope de Hammarnet, ou !'infestation sur Neflier commence des le rnois d'Avril avec des taux d'infestation pouvant atteindre 71,05% (Fellah, 1996). De rnerne,Wong et a/.(1983) trouvent a Hawaii des taux d'infestation allant de 0,9 a 96,4%. Les nefles a Momag echappent a l'attaque de la Ceratite, puisque le niveau de la population de la mouche reste foible a cette epoque de l'annee suite a des applications regulieres d'insecticides sur citrus puis sur fruits d'ete. En plus de L 'effet de la variete et de la periode de receptivite, le biotope joue un role tres important au niveau de l'intensite des degiitssur ce fruit. Les tres faiblcs taux d'infestation des nefles n'ont pas perrnis d'etudier la charge moyenne des fruits en individus pre-imaginaux. Par centre (Masson, 1932) a rnontre la grande preference de la mouche pour ce fruit, ou la charge moyenne d'infestation de 8,7 individus et peut atteindre 50 individus par fruiten cas de forteinfestation (Fellah, 1996). Sur les abricots, Pnmus armeniaca. L, de la variete "chachi", Jes premieres infestations ont ete decelees le 3 Juin avec un taux de 2,36%, alors qu'une semaine avant nous n'avons decele aucun fruit attaque. Le taux d'infestation passe de 8,53% le 10 Juin puis a 9,23% le 17 Juin pour atteindre 11,53% le 24 Juin 1994, en fin de rnaturite. Pendant cette meme periode, on observe sur la courbe de vol un pie qui correspond a la premiere generation estivale et qui coincide ainsi avec des taux d'infestation eleves. Fellah(1996) a montre !'importance de cet hote pour le developpement de la Ceratite puisque les infestations peuvent atteindre de� taux allant de 34,72% a 100% en fonction de la variete et le milieu d'etude. Des taux d'infestation sirnilaires ont ete observes en Italie par Firniani (1972), qui montre que l'attaque peut atteindre parfois Jes 100%. Alers qu'en general les taux d'infestations soot de l'ordre de 5,3 a 16,4%(Cirioetal. 1972). Sur cette espece la charge peut s'elever jusqu'a une quarantaine d'individus resultant de plusieurs pontes, alors qu'une charge de 6 et 10 individus a ete observee pour 30,20% des fruits examines et une charge de 11 a 15 et de 15 a 20 individus a respectivement ete constatee pour 22.91% et 21.87% des fruits. Ceci permet d'avoir une charge moyenne de 15,53 individus par fruit (Tableau 1). Ce resultat est tres proche de celui obtenu par Selim (1967) qui trouve une charge moyenne de 14,2 larves par fruit et un ma-ximurnde 29. Alors qi.leCheikh et al., (1975) obtiennent une moyenne de rnoinsd'une larve par fruit. 100�------"---, --+---; deCourbe vol --m-- NCflearbi ---.!:--Nefle Tanaka --Ill--abricot � PCche Kas.serine --+-- Peche Pde Chine -+- Peche IBI.. --- PCChe Canivale

---Figu ier .. +. ·Vigne -----Figue de barbaric

80

C" t! ·; 60 .S E �"' " ., '.; ol �= .if • :....:e- ., " = (0 "';..:, :'2

20 + +/ ,+ I I ,/

0� A A 8 I I .� 1 1 : I I� 1 1 !I L , 1 1 I -i -i -i -i vi vi vi vi vi vi ,-: ,-: ,-: ,-: z,-: ,-: 00 00 00 00 00 00 00 CO .; 0 0 0 '° '° 0 0 0 0 0 0 "' "' "' 0 0 0 ;g 0 0 0 0 0 � 0 � 0 0 8 0 0 0 0 0 0 0 gj 0 0 s 2 � 00 vi 00 vi vi ,-: -i '.:l 00 00 vi 0 M \0 � � ...; 00 '°' '° N"' 0 N 0 0 '.'.:! g 0'°' 0 '.'.:! N 0 N'° :"'. 0 0 N N 0 l; � � N s � � gj '"' � � N N 0 � 0 � g

FiJ!Ure 1: Evolution de l'infestnfion des fruitsd'cte en Conctiondu niveau des captures de la population adultcs de la Ceratite a Mornagau cours de 1994. • 96 -

Tableau 1 Charge des fruits hotes en individus pre-imaginaux durant Ieur periode de receptivite.(expime en%) Classe individus pre- imaeaux/fruit 1 - 5 6-10 11-15 16-20 21-25 26-30 31-35 36-40 > 41 Abricotier 12,5 30,20 22,91 21,87 4,16 3,12 33,12 0 2,08 Pecher kasserine 9.41 18.81 24.70 23.52 9.41 2.35 3.52 1.17 7.05 Pecher Plate de Chine 6.25 43.75 37.50 6.25 6.25 Pecher JHL 28.94 52.63 7.89 5.26 2.63 2.63 Pecher Camaval 15.85 39.02 35.36 7.31 3.65 1.21 Vigne 79.25 20.17 0.28 0.20

L'etude sur peches, Prunus persica. L, a conceme quatre varietes se succcdant dans Ieur maturite. Les observations ont ete poursuivies du mois de Juin jusqu'au mois de Septembre. Les echantillons etudies ont ete preleves sur des pieds n'ayantrer;u aucun traitement insecticide anti-Ceratite. Sur la premiere variete "Kasserine"; !'infestationa demarre avant le 24 juin, correspondant a une augmentation du niveau de la population adulte au niveau des pieges. A cette date, le taux d'infestation des fruits etait de l'ordre de 13,79%. L'impact de la premiere generation estivale est important sur Ja variete "Kasserine", on obtient lors du second controle du 1•r Juillet, un taux de piqilres de 31,48% qui atteindra le 15 Juillet; 39,81% puis 45,23% le 18 Juillet pour arriver en fin de maturite a 48,48% le 22 Juillet. L'importance de !'infestation est due ·a !'absence -de tout autre fruit hote,, . .ainsi .qu'a une longue periode d'exposition a des mouches avides sous pression ovarienne. L'etude de la charge pour cette varietc a montre que 25% des fruits examines contiennent entre 11 a 15 individus par fruit et 23% entre 16 et 20 individus par fruit. Les charges inferieures a 10 individus par fruit sent les plus frequentes sur cette variete. Quant a la charge maximale peut atteindre 61 individus par fruit. L'examen des echantillons pris sur la deuxieme variete "Plate de chine" a partir du 19 Juillet montre que !'infestation demai'i'ele 26 Juillet avec un niveau de 3,57% qui double apres uniquement trois jours pour atteindre 6,75% le 29 Juillet. Les peches "Plate de chine", ayant une maturite rapide et synchrone, offrent a la Ceratite une courte periode de receptivite. Une semaine apres !'apparition de !'infestation; le taux.atteint 10,81 %. Cette variete est rapidement recoltee a cause de sa maturite precoce et sa grande receptivite. Le temps entre le debut de la veraison et la fin de la maturite coincide .ivec le temp& qui separe la deuxieme de la troisieme generation avec une moyenne de captures allant de 4,4 a 6,8 individus par piege par semaine. Ces controles ont montre une moyenne de 6 a 10 individus clans 42.85% des fruits examines, cette charge ne depassera pas 25 individus par fruit. Sur la variete "J.H.L", Jes piqilres ont commence le 2 Aoilt avec un taux de piqilre de 2,04%. Cette variete offre une longue periode de receptivite correspondant a ('apparition de deux pies dans la courbe de vol de la Ceratite avec un niveau faible au cours de la mi-aoilt; ce qui a favorise le maintien du taux d'infestation a un niveau plus ou moins stable. Nous avons obtenu respectivement 5,00; 6,66 et 8,08% pour le 5; 9 et le 12 Aoilt. Ce taux est passe a 8,51 % le 20 Aoilt, apres quoi et vers la fin de la maturiteune augmentationrapide s'est ressentie a travers Jes echantillons du 26 Aoiltavec 12,96% et du 3 t Aoilt avec 17,64% correspondant a ('augmentation du niveau de la population des adultes. Ceci est engendre par !'apparition d'un pie le 6 Septembre correspondanta la quatrieme generation estivale. - 97 -

Pour cette variete, 71,56% des fruits examines ont une charge de 6 a 15 individus par fruit, dont les 2/3 n'en contenaient que 6 a 10 soit 52,05%. La charge ma"Ximale,comme pour le cas de la "plate de chine", n'a pas atteint les 30 individus par fruit. Au cours de cette meme periode, lcs figues, Ficus carica. L, variete "Zidi" sont receptives. Cependant Jes piqilres ne s 'observent que le 2 Aoilt avec un taux d'infestation tres faible de l'ordre de 0,86%. Il est de 1,50% le 15 Aoilt et n'atteint que 3,25% a la fin de la maturite le 20 Aoilt. Ceci est principalement dil a la courte periode de receptivite des figues qui sont recoltees au fur et a mesure de leur maturite. Nous remarquons une preference des mouches pour les peches "J.H.L" par rapport aux figues "Zidi" dont Jes maturites se chevauchent dans le temps. Cette preference pour Jes peches est due essentiellement a leur couleur jaune tres attractive, et a la recolte periodique des figues mfires. La Ceratite trouvant des difficultes a deposer sa progeniture sur Jes fruits immatures riches en liquide laiteux et acre. Pour Jes Figues le milieu joue un tres grand role selon l'abondance ou !'absence des fruits hotes concurrentiels. Le niveau d'infestation se trouve souvent accru par !'absence d'autres especes hotes au cours de la meme periode. Wong et al. (1983) ont montre que Jes taux d'infestation sur figues varient de 3,3 a 43,3% alors que Cirio et al. (1972) trouvent que ce taux peut atteindre 96,9%. Ceci signifie que Jes figues sont des fruits tres recherches par la Ceratite mais le niveau d'attaque est regie par plusieurs facteurs En ce qui conceme la quatrieme variete la plus tardive de peches "Camivale"; Jes piqiires ont commence le 31 Aoiitavec un taux de 3,6 l %. Cette variete a peau epaisse et de couleur jaune-rouge est tres attractive, avec une periode de maturite qui coincide avec un niveau eleve et croissant de la population adulte de la Ceratite. Son niveau d'infestation double, 3 jours apres !'apparition des piqiires pour atteindre 7,33% le 2 Septembre. Le niveau continue a augmenter rapidement pour atteindre 13,63% le 6 Septembre. Quant a la charge en individus par fruit, elle est la meme que pour Jes varietes "Plate de Chine" et "J.H.L" puisque 39,02% des fruits examines contiennent de 6 a 10 individus. Les peches favorisent le developpement estival de la Ceratite. Le grand nombre de varietes, et la repartition de leur periode de receptivite tout au long de l'ete fontde cette espece, un milieu favorablequi peut etre suffisant pour le developpement et le maintien de la Ceratite au cours de la periode allant du mois de Juin jusqu'au mois de Septembre. Bodenheimer (1951) montre que les peches permettent le deve1oppement de deux a trois generations de la mouche. Les taux d'infestation sont variables en fonctionde la variete et de sa periode de maturite. Cirio et al. (1972) trouvent qu'au cours de la periode allant de Juin a Septembre, Jes taux d'infestation varient de 0, 1% a 99,8% en fonction de la variete. De meme, Wong et al. ( 1983) montrent des taux d'infestation variant de 5,8% a 87,2% ce qui montre !'importance de ce fruit hote clans le developpement de la mouche des fruits. Pour ce qui est de la charge, Selim (1967) presente un nombre ma'Cimum de 76 individus par fruit avec une charge moyenne de 24,6 individus par fruit. Alors que Cheikh et al. (1975) obtiennent a partir de fruits ramasses une moyenne de 13,49 individus par fruit. En ce qui conceme la vigne, la Ceratite ne figure pas parrni les ravageurs potentiels de cette speculation de grande importance en Tunisie, la presence de la mouche passe souvent inapen;:ue. Nous avons remarque des infestations spectaculaires sur la variete "Rich Baba sam". Le 1er septembre 1994, - 98 - on a enregistre 27,79% de fruits attaques. Ce taux augrnente vertigineusement, en l'espace d'une semaine, pour atteindre le niveau de 50,35%. Les niveaux de population eleves et la rarete d'hotes receptifs en cette periode de l'annee en dehors des varietes tardives de peches qui sont abondamment traitees, ont fait que Ja Ceratite se rabat sur la vigne. Des observations sur la variete "Rich baba sam" clans deux autres biotopes, (Fellah, 1996) ont confirmela susceptibilite de ces fruitsa l'attaque de la Ceratite en Tunisie, avec des tam: d'infestation de 9,82% et 7,56% de fruitsattaques. L'observation d'autres varietes tel que Jes muscats dont la maturite coi'ncide avec celle de "Rich Baba sarn" n'a pas revele la presence d'attaque. Ceci pourrait etre explique par la differentes caracteristiques physico-chimiques des varietes (la rigidite de la peau des baies, ia teneur en sucre et le pH du pulpe... ). L'etude de la charge de cette espece montre que 79.25% des fruits examines hebergent de I a 5 ind.ividus, ce qui montre la capacite de la mouche a moduler le volume de sa ponte en fonctiondu milieu present. En.fin, avant de passer sur les agrumes, la Ceratite va avoir pour hotes relais, les fruits residuels du figuier de Barbarie pour toute la periode automnale et hivemale (Soria 1962a, 1962b; Cheikh et Ben Salah,1976). Pour mieux comprendre cette phase du cycle de la Ceratite sur cette plante h6te, nous avons precede a l'examen hebdomadaire des fruitsau cours du mois d'Octobre. L'infestationdes figuesde Barbarie a commence le 1er Octobre avec un taux de 3,38% Ceci apres un echantillon indemne effectue le 26 Aout 1994. Une semaine apres, le 8 Octobre 1994 date d'apparition de la cinquieme generation, on a enregistre un taux d'infestation de 7,93%, puis 26,66% le 14 Octobre pour finir avec 36, 11 % le 30 Octobre. Il faut remarquer qu'au cours de cette periode, les pieges refletent un niveau ele�e de la population adulte ( 42 mouches par piege et par semaine le 14 Octobre, et 39,4 le 28 Octobre 1994). Ii' faut signaler que notre etude a porte sur Jes poires "Williams", sans pour autant deceler l'attaque de la Ceratite et ce malgre le fait que Cirio et al. (1972) montrent que les poiues peuvent etre infestees jusqu'i 71,3% et pouvant meme atteindre les 100% pour Firniani (1972). Nous pouvons expliquer !'absence de la Ceratite sur ce fruit hate, par la recolte precoce avant l'entree en maturite puisque cette variete est destinee au stockage frigorifique.

Le developpement de la Ceratite sur differentsfruits d'ete Le developpement de la Ceratite sur une garnme de fruits hates tres variable Jui procure des conditions de croissance et de developpement tres heterogenes qui ne manquent pas de se repercuter sur la duree de croissance et Jes dates d'emergence. Ainsi Jes caracteristiques physico-chirniques du milieu de ponte et de developpement larvaires conditionnent la duree des stades pre-imaginaux et deterrninentle moment de !'emergence. Pour rnieux comprendre l'effet de la qualite du fruit hote sur le developpement larvaire et la duree d'emergence de l'adulte, nous avons procede au suivi des emergences pour Jes nefles, les abricots, trois varietes de peches et les figues. Differents lots de pupes ont ete collectes a partir de ces fruits, Jes emergences totales males et femelles ont ete enregistrees. Les resultats obtenus sont consignesclans le tableau 2. - 99 -

Tableau 2 Importance et effet de la pi ante hote sur !'emergence des pupes. Peches Plantes Hates Nefles Abricots Kasserine Plate de J.H.L Figues Chine Taux d'emergence 58,35% 79,59% 79,32% 81,69% 78,15% 66,66% Sex-ratio 1,01 1,03 0,95 0,93 1 1 Duree d'emergence (jours) 4il5 4a 5 4 4 4 4a5 Periode nymphale (jours) 6a7 8a9 10 6a7 6 a 7 8

Nous pouvons observer que la plante hate peut affecter, par ses caracteristiques nutritives, le taux d'emergence, la sex ratio et la durP.e et la periode d'emergence. Ainsi Jes Abricots et les Peches, quelques soit la variete, offrent a la Ceratite le meilleur milieu de developpement permettant des taux d'emergences de l'ordre de 80%. Ceci est essentiellement du aux qualites physico-chimiques de ces especes ,(pH, sucres, acides amines ...), procurant des conditions nutritives plus adequates. Alors que pour les ni:fles et les figues ce taux est plus faible. Pour les autres parametres pris en consideration, nous remarquons une grande similitude entre les differentesespi:ces. Ainsi nous obtenons la meme sex-ratio allant de 0,93 pour les Peches "Plate de Chine" a 1,01 pour les Abricots. Les. .differentes--espi:ces...eLvarietes ..n'ont. aucun .effet .sur le sexe de l'adulte a l'emergence. En ce qui concerne la duree des emergences, elle est de 4 a 5 jours pour les differcnts hates etudies. Elle n'est regie que par les conditions climatiques qui sont favorables tout au long de la periode d'etude. Alors que pour la duree de la periode nymphale, elle presente une variabilite inter et intra-specifique. En effet,elle est de 6 a 7 jours pour !cs Ni:fles,8 a 9 jours pour les Abricots et les Figues et elle varie pour les Peches selon les varietes puisqu'elle est de 6 a 7 jours pour la "Plate de Chine" et la "J.H.L" et de 10 jours pour la "Kasserine". Cette.differencc chez Jes Peches est essentiellement..due aux conditions climatiques qui evoluent plus rapidement et deviennent plus favorablespour les deux varietes les plus tardives.

CONCLUSION

L'etude de !'infestation des differents fruits h6tes par la mouche mediterraneenne au cours de la saison estivale dans un biotope du Nord la Tunisie, montre que !'ensemble des espi:cessusceptibles d'etre attaqueGs offrent a la Ceratite des periodes de receptivite chevauchantes et un milieu favorable a un developpement continu jusqu'il la fin du mois d'octobre. Apres quoi la Ceratite continue son developpement sur les d.ifferentes varietes d'agrumes. Le taux d'infestation est etroitement correle au niveau de la population naturelle adulte. Cette meme correlation a ete montree par Gahbiche (1993) sur agrumes. Nous avons pu montrer !'importance des degats causes par la mouche des fruits, ainsi que !'importance de ces h6tes estivaux clans le cycle biologique annuel du ravageur, puisqu'ils lui fournissent un tres bon milieu de developpement en plus des conditions climatiques favorables de cette periode de l'annee. Si la Ceratite epargne au debut de la saison estivale certaines espi:ces, ceci est essentiellement du a son absence au niveau des pieges de contr6le. - 100 -

Pendant Jes periodcs creuses et en absence de plantes hotes, la Ceratite peut s'attaquer a d'autres espi:ces dont Jes relations avec le developpement de la mouche ne peuvent etre suspectees. La Ceratite, tri:s polyphage etpolyvoltine, s'adapte bien a la.qualite de l 'hote qui lui est offertet ri:gle sa ponte en fonction des disponibilites alimentaires offertes.L'etude de la charge des fruits et le suivi des performances de la Ceratite sur ces differents hotes, a en effet montre que la mouche peut s'adapter atLx condition.x offertes en controlant sa ponte pour la regulation.du nombre d'individus par fruit. Le niveau de la population, au moment de la receptivite des fruits, conditionne aussi bien le tau:-C'd'infestation que la charge moyennepar fruit. La plante hote peut de son cote affecter le taux d'emergence par ses caracteristiques nutritives, sans pour autant perturberla sex ratio, la duree et la periode d'emergence des pupes issues de ses fruits.D'autre part, ia mouche des fruits est capable de reguier sa ponte en fonction des caracteristiques nutritives de l'hote ce qui con.firmele resultat de Zuccoloto (1987; 199 l) et Waldbauer ei.F�iedman (1991).

BIBLIOGRAPHIE

BODENHEIMERF.S., 1951 - Citrus entomology in the Middle East; Junk, The Hague, 663 pp. CHAPMAN R.F., AND BERNA YS E.A., 1989 - Insect behavior at the leaf surface and learning as aspect of host plant selection. Experientia 45 : 215-222. CHEIKH M., ET BEN SALAH, H., 1976 - Degres et epoques des infestations des principaux hotes de la mouche mediterraneenne des fruits Ceratitis capitata wied (Dipti:re, Trypetidae). Communication presentee au . colloque .du CLAM {Comite de. ..liaison de fAgrumiculture Mediterraneenne). CHEIKH M., HOWELL J.F., HARRJS E.J., BEN SALAH H., ET SORIA F., 1975- Suppression of the Mediterranean fruit fly in Tunisia with released sterile insects. J.Econ.Entomol., 68 : 237-243. CIRIO U., DE MURTAS I., GOZZO, S., AND ENKERLIN D., 1972 - Preliminary ecological observation of Ceratitis capitata wied or the island of Procida with an attempt to control the species using the sterile male technique. Bollettino Laboratorio Entomologia Agraria Portici, 30;175-188. COOLEY S.S., PROKOPY R.J., MC DONALD R.P., AND WONG T.T.Y., 1986 - Learning in oviposition site selection by Ceratitis capita/a. Entomologia exp appl. 31: 49-56. CRNJAR R.M., AND PROKOPY R.J., 1982 - Morphological and electrophysiological mapping of tarsal -chemoreceptors of'OViposition deterring''Pheromone ·of Rhagoletis ponenella flies, Journal insect,of _ Physiology, 28 : 393-400. FELLAH H., 1996 - Contribution a l'etudede la Bioecologie de la mouche mediterraneenne des fruits Ceratitis capitata Wiedemann (Diptera: Tephritidae) sur fruits d'ete. Memoire de find'etudes du cycle de specialisation de !'INAT. 234pp FIMIANIP., 1972-1973 - Osservazioni biologiche sulla Mosca della Frutta (Ceratitis capitata wied) effettuante nella zona di Monte di Procida (Napoli) negli anni 1968-69. Boll. Lab. Ent. Agraria, portici, 30:71-87. GAHBICHE H., 1993 - Contribution a L'etude de la Bioecologie de la mouche mediterraneenne des fruits Ceratitis capita/a Wiedemann 1829 (Diptera: Tephritidae) dans deux biotopes du Nord de la Tunisie. Memoire de find'etudes du cycle de specialisation de l'INAT. 266pp - 101 -

MASSON A.C., 1932 - The economic importance of the Mediterranean fruit fly to Hawaiian horticulture. Proc Hawaii Entomol. Soc. 8 . 163-178. PROKOPY R.J., AND ROCTBERG B.D., 1984 - Foraging behavior of true fruit flies Ann. Sci. 72: 41-49. QUILICI S , GESLIN P. AND MANIKOM R., 1987 - Utilisation du piegeage clans la lutte contre les mouches des fruits a l'ilc de la Reunion. I. Comparaison de difforents types de pieges. Fruits vol. 42 11°1,P 41-57. SELIM 0.F., 1967 - Studies on the Mediterranean fruit fly, Ceralilis capita/a Wied, in U.A.R.Bull.Soc.Entomol. Egypte. 51: 315-323. SORIA F., 1962a - Epoques d'infestation des divers hates de Ceratitis capitata Wied dans le Nord de la Tunisie Rapport d'activite de l'INRAT 1-2 (reneotype). SORIAF., 1962b - Plantes hates secondaires de Cera__titiscapita ta wied en Tunisie. Ann. de l'INRAT, vo! 35: 51-72. WALDBAUER G.P., AND FRIEDMAN S., 1991 - Self selection of optimal diets by insects. A. Rev. Ent. 36:43-63. WONG T.T.Y., NISHIMOTO J.I., ET MOCHIZUKI N., 1983 - Infestation patterns of Mediterranean fruit fly and the oriental fruit fly (Diptera : Tephritidae) in the Kula area of Maui, Hawaii. Environ. Entomol., 12: 1031-1039. ZUCCOLOTO F.S .. 1987 - Feeding habits of Ceralilis capitata: can larvae recognize a nutrition any effective diet? J. Insect Physiol. 33:349-353. ZUCCOLOTO F.S., 1991 - Effects of flavour and nutritional value of diets selection by Ceratitis capita/a larvae (Diptcra : Tephritidac). J.Insect. Physiolo. 37:21-25. - 102 - A GENERAL VIEWOF RECENT ADVANCES ON ATTRACTANTS AND TRAPS AGAINSTCERATITIS CAP/TATA (WIED.) (DIPTERA: TEPHRITIDAE)

J.P. Ros

lnstituto Nacional Investigaciones Agrarias. C/Jose Abascal 56, Madrid

SUMMARY

The mediterraneanfruit fly C capitata is the most dangerous insect pest forcitrus and bone fruits in the mayority of countries with a warm, tropical or subtropical climate. Its populations are controlledin most countriesby insecticidesused in cover spray, ultra low volume or bait spray; this chemical control has as a consequence that toxic residues remain in the fruit and the destruction of useful insectspecies in theecosystem, apart from all the inherent problems ofthe insecticides. We tried to develop a medfly selective attractant to preserve the rest of the insect fauna in fruit orchards and the same time to improve the trapping systems with innovations in the traps that actuallywe have. In a Mcphail type trap(Tephri Trap)four lateral symetric holes were done. The captures of Ceratitis were increasing greatly with this transformation. Hydrolised protein Nulure (9%) was a goodattractant, like in previous assays, but it was better when amonium acetate(9%) was addedin the solution of hydrolised protein and better if the amoniun acetate is placed in solid form in the basket ofthe trap. A new trial was started on solid attractantsbut Nulure when solid (mi,::edwith caramelized sugar, absorbedin clay, gelatine,etc .. ) is not so attractiveto medflyas when liquid. The following year we assayed Putrescine together -with amonium acetate, ( both in polyetilene menbrannes) getting excelent results, increasing highly the percentage of captured femalesalthough the total value ofcaptures remained thesame as withhydrolised proteins. Next year a big increase in number of flies trapped was achieved withtrimetil amine. The three components, Putrescine, Amonium Acetate and Trimetil Amine, as slow release polietylene membranne,are highly attractive specially forfemales (75'%-80%). Presently it is the most efficient group ofattractants known for medfly.

INTRODUCTION

Many differents substances were used as attactant forthe mediterranean fruit fly from many years ago.. These are some examples: molasses syroup , 25% of vinegar disolvcd with water, the "Clensel" solution (2% ofliquid amonia soap), the solution of 5% ofAmonia or the solution of 3% of cryolite (Amonium fluoride)disolved with water. In those years, the experts already underlined the influence of the temperature on the attractant effectof these substances, They said that, at high temperature the "Clensel "solution captured a high nunber of fliesthan the solution of 5% of Amonia. (Gomez Clemente 1939) The bringingout ofthe Mcphail trap has been of great help forthe fanners. It helped them to fightagainst the plague andto control the populations, so that, if necessary, they could use insecticides(Cafiamas 1972). We have recently been using the Mcphail traps with the amonimn or biamonimn phosphate. The launching ofTrimedlure, the sexual attractant for male Ceratitis flies, caused a revolution in the studies realized by the scientists at the end of60's. This was the strongest attractant substance forthe male fliesof this specie. Even in our days, this substance is still being used. In these years the studies of the populations of Ceratitis started in all those countries where the crops were being destroyed by the plague. The insect flightlines were essential to give the aerial treatments in the citricultural zones. In some of these studies, up to 60,000 traps - 103- with trimedlurc and DDVP were installed in a area of 630 hectares in the zone of Sagunto (Valencia, Spain).(Arroyo 1972) The function of the trap to control the pest was soon forgotcn. In its place, the insecticide was highly effectiveweapon. In the 70's, the trap became the tool the experts used to decide when they should start the insecticide treatment. Each country had a differentsystem of trap batteries to elaborate their own supervision rate. In 1986 the InternationalAtomic Energy Agency coordinated an international research programmto, all around the world, standardizea kind oftrap. So that the captures in differents countries could be referredto one unique name, and what's more, in their projects on sterile insects (S.I.T) © they could use their best trap to this date. Trimedlure, Nulure and Borax combination baiting the InternationalPheromone Mcphail Trap was the star ofthe project . The followingfact could be underlined. The hydrolisate protein Nulure was a selective attractant forfemales of Ceratitis. The social concern for the environment as well as the position taken by the occidental countries with respect to the insecticide residues foundin fruits and vegetables of direct cornsumption, have changed what scientists and experts thought about the fight against the plagues and deseasesthat destroy our crops. The drastic reduction ofinsecticide treatments imposed by national and European laws, as well as the security range periods, makes it necessaryto find alternative ways of lessening the possible damageof causal plague during these periods.. Our markets demand these ecological fruitsmore and more, so that some of them had to develop a parallel economy. This circunstance has changed the criteria ofthose researchers that work on this specie·. Nowadays, we not only need to knowthe population level of this fly in our crops (for what we use Trimedlure that just captures the male flies), but also need to defend the fruits frompl agues in those periods of time in wich they can attack and, no insecticide can be use to cope with them. It is here that the female fly develops an essential role since it is the one that lays her eggs on the fruit and causes its putrification. The highly effectiveresults of the hydrolised protein Nulure in capturing the female flies, (Ros, 1988,1990). as previously mentioned, originated a new intervenction of I.A.E.A and the U.E ( Project AIR3CT920300) to reach the aim of our research studies: to get an effective femaleattractant (liquid in capsule, in a slow release membranne or solid) whose attraction lasts at least two months, when it applied in traps or artificialsticky surfaces. The liquid attractants (protein solution) are expensive for the farmersbecause they require a frequent recharge and thus a lot of man labour.(Ros,1996) Threeexperiment we present in this paper were done consecutivily in the years 1994, 95 and 96. The first was a modification on the mcphail Trap, (Tephri Trap), four S)'metric holes were open in the bodyof this trap makinga synthesis ofa Dry with a Mcphail Trap. The rol of the amonium acetate as attractant was assayed to. The second experiment consisted in a first aproximation to solid or liquid dispenser attractants with long last activity , adapting them to Mcphail Traps and avoiding the continous renewals ofliquid hydrolised proteins. The new attractant putrescine was the substance to be contrasted. (Epsky,1995. Heath,1995) The third experiment was a mayor step to the results of the last year, a new attractant came to help Putrescine and Amonium acetate: Trimetil Amine. The slow release poliethilene membrannes was a very good tool to reach a good trap without assistance forlong time.

MATERIALAND METHODS

Three experiments we have carried out in the last years to improve traps and attractants in order to obtain a easy methodto fight against Ccapitata Wied. in a efficiency way that disminue the use ofinsecticides compatible with a- soustenable agriculture. - 104 -

The first experiment was a comparison of the behaviour oftwo different traps, a standard type of Mcphail trap (Tephri Trap) and the same trap with four simetric lateral holes (a synthesis of Dry and Mcphail Trap). The holes makes it an open trap, so , it is necessary a killing agent to kill the attracted flies. In the same trial we also tested the behaviour of Amonium Acetate mixed with the solution ofNulurc or as solid formin the basket of the trap. Six Mcphail Traps (Tephri Trap), three standard type and three with fourlateral holes were randonly distributed in a peach orchard from19th to 31th of October in thelocality of Mora la Nova (Tarrag-0na,Spain). The six traps were baited as folows:

Table I. Types of traps and baits. Experiment 1994. Spain ------N° ofTrap Type ofTrap Bait Killing Agent

I. Standard 250cc Nulurc 9% Borax 3%

2. With holes 250cc Nulure 9% DDVP Borax 3%

3. Standard 250cc Nulure 9% Borax 3% A.Acet 9%

4. With holes 250cc Nulure 9% DDVP Borax 3% A. Acet 9%

5. Standard 250cc Nulure 9% Borax 3% Solid A. Acet 9g.

6. With holes 250cc Nulure 9% DDVP Borax 3% Solid A. Acet 9g.

The second experiment was desigoed to compare different solid forms of attractants or liquid dispensersto adapt themin Mcphail traps,in orderto avoid the continous renewalsof liquids in these kinds of traps. So, we could offera trappingSystem more available to the funners. The usedtrap was Tephri Trap with holes andthe attractantswere as follows:(TableII) - 105 -

Table II. Types of traps and baits. Experiment 1995. Spain

Trap Kind of Trap Bait Killing Agent

A. Tephri Trap TrimedlurePlug DDVP B. Tephritrap Syntetic Pheromone(!) DDVP c. Tephri Trap Putrescine(2) + Amonium Acetate(3) DDVP D. Tephri Trap Nulure absorbedin clay DDVP E. Tephri Trap Solid Amonium Salt(4) DDVP ------(!)Dr.Howse. Dep. of Biology. University of Southampton (2) Consep (3) Slow release poliethilene membranne. Consep (4) Agrisense Lted

The experiment was carriedout in six localities of Spain \vith different climatic conditions and hosts. (Table ill)

Table III. Localities, Culture and Oimates. Experiment 1995. Spain

Locality Host Climate Date

Fraga Peach Continental Jui-Ago. Alcafiiz Peach Continental Sep-Oct Mora la Nova Peach Continental Jui-Ago. Castellon Citrus Mediterranean Sep-Oct Mallorca Citrus Mediterranean Sep-oct. Malaga Citrus Subtropical Sep-Oct

In all localities the field plot design was a randomized complete block, with 3 blocks consisting of 5 traps each, baitedwith the attactantscited above. Thetest was run at least fortwo months. The attractantswere renovatedeach month.

The third experimentwas carriedout in the nextyear ( 1996), in the same localitiesand with the same extructure that 1995's �-periment, varying the attractants. This experiment was designe to compare the females attractants (specially solid or liquid dispensers) created recently in the world and our apportation to resolve several aspects that make expensive the use of traps to the farmers. We are tI)ing adapt them to Mcphail traps, to give a good and cheap trapping systems to the farmers. The chossen trap was the Tephri trap withholes andthe attractants to assay this year were the follows: (TableIV) - 106 -

Table IV. Attractants, Types of Dispenserand Time of Activity. Medfly 1996 TRAP BAIT FORM DOSE LAST Putrescina Membrane Pacht 28 days AmoniunA. Membrane Paeth 28 days 2 Putrescine Membrane Paeth 28 days AmoniumA. Membrane Paeth 28 days Trimetilam Membrane Paeth 28 days 3 S. Protein Tablet l. T. 28days 4 S. Protein+ Tablet I. T. 28 days Water Nulure Dispenser with 4 cc 28 days 5 AmoniumA. two sponge 4 cc 28 days Salt with Salt 10 grs. 28 days Nulure Perforated 10 cc 28 days 6 Borax dispenser. I gr. 28 days AmoniumA. !Occ Nulure liquid 250cc l week 7 Borax Solid 3% I week Water Liquid 88% l week Nulure Liquid 250cc 1 week 8 Borax Solid 3% l week water liquid 88% 1 week AmoniumA. in dispenser 5 grs. I week 9 Trimedlure solid Plug 28 days

In all localities the field plot design was a randomiz.ed complete block, with 2 blocks consistingof 9 traps per block, baitedwith the attractants cited above. The test was run at leastfor twomonths

ls a roulefor all trials

*Traps withina block were rotatedsequencially after each sample. *Trapswere hung, one to two metres above the ground, in the lower half of south- easternpart of host tree canopy. *All traps were 25-50 m. away each other. *Data collectedtwice a week *All data of captures have beentransformed by thechange of variable:

X'= SQRT ( x+ 0.5 ) x = flies/trap/day

to be treated statistically by an analysis of variance Means were compared using Tuckey's w procedure. - 107 -

RESULTS

Table V show us the mean of captures of each treatment of traps and baits of the first experiment (1994 ). Are better the traps with holes?. IncreaseAmonium Acetate the atraction power of thehydrolised protein?. TheFig 1. Give us a graficinfomiation to these questions.

Ta::ite V. Means (flies/trap/day 211d statistical) of medfly captures by ciifferents traps and baits. Medfly. 1994 ------T,ap Kind of Trap Bait Meanflies/trap/day Statistical Mean ------1. Standard Nulure 9% 0.37 0.85c Borax 3%

2. Withholes Nulure 9% 1.92 1.44 be Borax 3%

3. Standard Nulure 9% 1.42 1.28 be Borax 3% A.Acet 9%

4. Withholes Nulure 9% 5.35 2.17 ab Borax 3% A.Acet 9%

5. Standard Nulure 9% 0.27 .84 C Borax 3% Solid A. Acet 9g.

6. With holes Nulure 9% 8.15 2.47 a borax 3% Solid A. Acet 9g,

Means followof the sameletter are not significantlydifferent (Tuckey's w procedure)!.'(=.05 - 108 -

QI"' � � Cl) *Cl) ii!- ::, 0 Cl) <{ <{ z I �

Fig.1 Meanof captures of Tephri Traps withoutand withholes baited with Nulure 9"/o+ Borax3%,Nulure 9% + Borax 3% + Amonium Acetate9%, andNulure 9% + Borax3% + Amonium Acetatein solid formin thebasket of thetrap.

Theresults of thesecond experimen t (1995) are givenin thetable VI. Both means (statisticaland flies/trap/day)give us thenecessary infonnation of thebehaviour of eachattractant. The Fig 2. Is the graficalwiew ofthese data.

Table VI. Meansof captures(a. statistical and b. flies/trap/day)of the differentsbaits in Tephri Traps with holes tested in 6 localitiesof Spain. Medtly 1995

Locality Mean Trimedlur S.Pherome Put.+A.A S.Nulure Am.Salt

Fraga a 6.58a 0.70c 2.85b 0.76c 0.81c

Alcaiiiz a 6.04a 1.23d 3.72b 1.04d 2.llc

Mallorca a 2.91a 0.96c 2.41b l.Olc 0.89c

Malaga a 2.94a 0.74c 1.42b 0.94c 1.06c

Castellon a 3.19a 0.75d 2.63b 0.76c 1.25c

Mora a 2.06a 0.70b 1.92a 0.80b 0.95b

Mean b 47.7 0.81 18 0.89 2.54

%Females 0.5% 50% 74% 68% 55% - 109 ·

Means of each row follO\Ycdby the same letter are not significantlydifferent (Tuckey·s w procedure)\X =0.05

------·------

25

20

10

5

0 Q) � C + � 'la :, 0 2! � :, "' E z3 � e :, ., :J2 ·c � .c: 'j j 0 0 a. � <( V) E ui <(

Fig.2. Comparison ofmeans ofmedflies captured in Tephri traps with holes with differentsbaits. Trimedlure plug, Syntetic pheromone, Putrescine + Amonium Acetate (slow release poliethylene membrannes), nulure absorbed in clay and solid Amonium salts.

The Table Ill show the values of the mean of captures of each assayed trap in the third experiment (1996). The trials of the locality of Fraga can't be finished due to the insecticide trea1mentthat thefarmer gave in the orchard in which we are working. The mean flies/trap/day shows us thetotal eficacy of the trap but, as the trial was directed to select a effectivefemale attractant it must complete the information with the % of females. The statistical mean shows us, afterthe Tuckey's w procedure, the significant differences between the meanof each trapand the others. To take anidea of the number offlies thatthese typesof traps can catcht we give an example : In the locality of Mora la Nova in a orchard of harvested peaches the medlies captured by thenine trapsin four days, were as follows: (males/females)

I. 286/390 4. 187/114 7. 290/346 2. 882/760 5. 386/350 8. 304/305 3. 7/99 6. 552/590 9. 1334/0

( These data and others of so big populations haven't beenincorporated in the statisticalanalysis due to it desproporcionated size) - 110-

TABLE III. Means of captures of the differents traps involved in the third experiment. a. ( statistical) b. (flies/trap/day).Medfly 1996

Locality Mean 1 2 3 4 5 6 7 8 9 ------Alcafuiz a 2.0bc 3.lb l.Oc l.lc Ube 2.2b I.9bc l.Obc 4.9a Castellon a 2.4b 3.5ab 1.5b 2.0b 1.6b 2. lb 3.0ab 3,0ab 4. 6a Mallorca a 4. 8b 5.0b 1.4d 1.6d 1.8d 3.lc 3.lc 3.3c 6.7a Mora a 1.9b 2.8a l.lbc 1.3bc Ube 0.7bc l.8bc 2.2ab 1.9b Sevilla a Ua I.Sa 0.9b 0.9b 0.8b 0.9b !.lab 1.6a 1.3a Mean b 8.8 13.5 1.5 2.1 2.7 6.0 7.0 8.8 27,6 0/o Females 76 73 73 72 53 70 71 72 0.01 ------Within a row, numbers followed by the. same letter are not significantly different (P= 0,05) w Tuckey's procedure)

1. PU+A.A. 4. SOLID PROT + H20 7. NU+B.+H20 2. PU.+A.A+.TMA 5. NU+A.A.+ Salt 8. NU+B.+H20+A.A. 3. SOLID PROT 6. NU+B.+A.A. 9. TM Plug

30

25

20

15

l!I males/trap/day 10 ClFemales/trap/day

5

0 c, I <( � <( C .. :, ·m � Cl).. f-- J:+ + $ $ $ + ec. C CD :i <( + J:� � a. 31 + $ 0 � ::::>$ CD Cl) c. z + a. 31 ::::> 0 z Cl)

Fig. 3. mean of medflies captured by nine different combinations of attractants baiting Tephri Traps with holes. Putrescine + Amonium Acetate, Putrescine + Amonium acetate + Trimetil Amine, a Solid protein, a Solid protein with water in the trap, Nulure + Amonium Acetate solid dispenser, Nulure + Amonium Acetate + borax liquid dispenser, 250 cc of Nulure 9% + Borax 3%, 250 cc ofNulurc 9% + Borax 3% + 5 g. Amonium Acet,atc in the basket of the trap, Trimedlure Plug. - 111 -

DISCUSSION

The holes in this type of Mcphail Trap (Tephri Trap) have increasedgreatly the medfly captures , perhaps the smell of attractantsgo out more easily and attract fromfarer. TI1e rol of Amonium Acetate was positive helping Nulure , both cases were positivebut, in solid formplaced in the basket ofthe trap was better. The results of the secondexperinlent shows a big difference of capturesbetween trinledlure and the rest of attractants,however thenev.- attractantPutrescine when work withAmonium Acetate baitingtephri Traps with holes capture a big number of femalesdoing this trapthe best for our requirements. It seemsthat the Amonium Acetate patch of Consep Ltedis betterthan the solid formwe tested last year. Both, Putrescine and Amonium Acetatepatchs (slow release poliethilene membranne) glued in the interior of the trap make it a Dry trap with a monthat leastof activity.A piece ofDDVP assecurethe died of the attracted insects. A example ofthe capacity of this kind of trapwas tile captures obtainedin a weekof september in one of the localities of tile experinlent. 294 males and 621 females. The rest of tile attractantassayed didn'twork well. Analysing the results of tile third experinlent we can apreciatc that Trinledlure is, we expected, a excellent male attractantof this espccie, but it isn't a goodinstrument to fightagainst the pest, due to the females could matingwith oilier males and to continue puncture the fruits. We can help our farmersutilizing this male attractant informing tilem of it appareance and the evolutionof it populations in therisk areas in order to take thenecessary measures. In the data of captures in each locality , we can seeclearly that the trap baited with three components (Trinletyl Amine, Putrescine andAmoniun Acetate) alwais rank in the first or second place with respect of the total number of captured flies and is alwais the first capturing fen1ales. (exceptSevilla) The behaviour of hydrolisedprotein Nulure in liquid form(solution 9% + Borax 3%) was positive but it worked better with solid Amonium Acetate in the basket of tile trap. It seems that Amonium Acetate increasethe efficacy of all attractants when they work together. ThePutrescine and tile Amonium Acetateconfirm the previous observations(trials of1995) referent it capacity to trapflies (Mallorca) and it selectivityfor females. Ros et al ( 1996). The solid Autolised protein withor withoutwater in thetrap didn't work well and always was under theother attractants. The climate temperature probably have a determinant rol in the behaviour of each type of attractant.The weatheris a parameterto bein mind at the time of valoratetile efficiency of tile traps. The experinlents of Fraga (SUil1ffier) and Sevilla (September-October-November) are a example of this The results obtained showthat Trinletyl Amine is a excellent additive to enhance tile effects of the Putrescine-AmoniumAcetate combination. The combination of thethree components captured a very high percentageof tileavailable flies, especially females (70-90%). Nulure would be a second alternativeif we could develop an effective liquid dispenser or solid form.

CONCLUSIONS

We can conclude that tile combination of the three attractants included in slow release poliethilene membrannes result an excellent bait for tile medfly, over all for it selectivity to the females. TheTephri trap worked verywell and its handling was easy. We had not problems placing the patchtes of tile syntetic attractants inside tile trap (glued in tlle lateral interior wall). It has capacityfor four pachts. - 112 -

Both, the Tephri Trap trap with holes and the three attractantscould be a goodinstnunent to fight against the pest. The control of the first generations of the insect, these flies that scape of the winter, Papadopoulos ( 1996), would be a goodpractice to avoid thebig populationsthat the farmers must supportin summer andthe fu.11 first months.( the mentioned example of Mora la Nova).

B!BLIOGRAFY

* Arroyo,M. '·"La lucha contrapl agas, pasado, presente y .. ·t,futuro?".Revista de la Universidad de Madrid. Vol 22 pp249-280. 1972

* CafuunasMendoza R. "Evoluci6n de los tratarnientoscontra Ceratitis capitata Wied. en la provincia de Valencia.Bo!. lnf. De plagas 92. Abril 1972

* Gomez ClementeF. "Experienciasde lucha contraCeratitis capitata Wied.con cazamoscas de vidrio. Bol. Pat. Veg.Y Ent. Agricola.Vol. VITI. 1939

* Epsky,N.Hcath,R.Guzman,A.Meyer,W."Visua!Cuc andChemical Interactionin a Dry Trap with Food-Based SyntheticAttractant for Ceratitis capitata and Anastrephaludens ° (Diptera:Tephritidae).Enviromnental Entomol ogyVol.24 n 6 1995

* Heath,R.Epsky,N.Guzman,A.Dueben,B.Manukian,A.Meyer,W. "Developmentof a dry plastic insecttrap with foodbasedsynthetic attractant for the mediterranean and mexican fruit flies ° (Diptera:Tephritidae).Journal of EconomicEntomol ogy. Vol 88 n 5 1995

* Papadopoulos;N.Carey,J.Katsoyanos,B.Kouloussis,N. "Overwinteringof the Mediterraneanfiuit fly(Diptera:T ephritidae)in NorthernGreece. Annals ofEntomological Societyof America Vol.89 n° 4 1996

* Ros,J.P. "£studiode diferentescombinaciones de productosatrayentes en las pulverizacionescebo contracapitata C. Wied.. Bol. San.Veg. Plagas 16: 263-267. - 1990

* Ros,J.P.Garijo,C.Navarro,L.Castillo,E."Ensayos de campo con un nuevo atrayentede hembrasde la mosca mediterraneade la fiutaCeratitis capitataWied. (Diptera, Tephritidae)."Bol. San.Veg. Plagas 22: 151:157, 1996

* Ros,J.P.Moner,P.Roig,V.Castillo,E.Lorite,P."Eficacia del hidrolizado de proteina en !as pulverizaciones-cebocontra Ceratitis capitata Wied." Bol. San.Veg. Pl agas 14:5-9 1988

* Ros,J.P. Alernani,A. Castillo,E. Crespo,}. Latorrc,Y. Moner,P. Sastre,C .Wong,E. "Ensayos para el control de la moscamediterranea de la fiutaCeratitis capitata Wied. mediantetecnicas que limiten los tratamientos insecticidas."Bol. San.Veg. Plagas 22:703-710,1996 - 113 -

RELA(:AOENTRE AS CAPTURAS DE ADULTOS DE BACTROCERA OLEAE (GMELIN) EM DIFERENTES SISTEl\tAS DE ARMADILHAS EA INFESTACAODOS FRUTOS

M. I. Patanita(l>, M. Cardoso<1> & A. Mexia<2> (lllscola Superior Agraria de Beja, Praceta Rainha D. Leonor, 7800 Beja - Portugal <2> lnstituto Superior de Agronomia - SAPI, Tapada da Ajuda, 1300 Lisboa - Portugal

Resumo

A mosca da azeitona - Bactrocera o/eae (Grnelin), e uma praga chave do olival no Alentejo, a principal regiao produtora do pais. 0 combate contra a B. oleae na regiao continua hoje a basear-se na luta quimica classica, cornos seus graves inconvenientes econ6micos, ecol6gicos e toxicol6gicos. Para o desenvolvimento de estrategias de luta mais adequadas, orientadas no sentido da Proteci;ao Integrada, e necessario conhecer melhor a dinamica de populay

RELATION BETWEENTHE CAPTURE OF ADULT BACTROCERA OLEA£ (GMELIN) IN DIFFERENT SYSTEMS OF TRAPS AND THE INFES1'ATI0NOF FRUITS

M. I. Patanita(I>, M. Cardoso<1> & A. Mexia<2> (I)Escola Superior Agraria de Beja, Praceta Rainha D. Leonor, 7800 Beja - Portugal (2) Instituto Superior de Agronomia- SAPI, Tapada da Ajuda, 1300 Lisboa - Portugal

Summary

The olive fly- Bactrocera oleae (Grnelin), is one key pest of the olive grove in Alentejo, the main producing region of the country. The control against B. oleae in the region keeps on being based upon the classic chemical control, with very serious economic, ecologic and toxic drawbacks. For the development of some more adequate control strategies, directed towards the integrated pest manegement, it's necessarya better knowleage of the dynamic of the populations, as well as the opportunity of the treatments. So, the use of traps assumes a very special interest. In this perspective for two years some surveys have been made in olives groves of the Cordovi! cultivar, whose results are introduced here. • 114 •

1 - Introdu�ao

A estimativa do risco resultante dos inimigos das culturas, constitui um dos componentes essenciais da protec,;ao integrada. Para a efectuar, recorre-se a metodos de amostragem que podem ser directos ou indirectos. Os metodos de amostragem directos, que incidem na observa91io directa de um deterrninado numero de org1ios vegetais definidos, sao geralmente muito trabalhosos, o que tern levado os investigadores a interessarem-se cada vez mais pelos metodos indirectos. Nestes metodos de amostragem indirectos, procede-se a captura de artr6podes atraves de dispositivos apropriados (Amaro & Baggiolini, 1982). Os metodos indirectos devem ser rapidos e faceis de executar e e essencial que tenham suficiente precisao para o fim a que se destinam. Com este trabalho pretende-se contribuir para a defini91io de metodos de amostragem a utilizar na protec,;1io integrada da oliveira, relativamente a B. oleae. Neste sentido, estudou-se a possibilidade de utiliza,;1io de armadilhas cromotr6picas corn e sem feromona e garrafas mosqueiras, para a previsao do risco resultante da praga.

2 - Material e Metodos

Os ensaios foram conduzidos durante as epocas de 1993 e 1994, num olival da cultivar Cordovi!, situado a cerca de 4 km da cidade de Moura. 0 olival, corn uma area de 30 hectares, cultura estreme e implantado ha cerca de 100 anos, ternuma densidade de 80 arvores. Para efeitos do trabalho realizado, foi escolhida uma parcela do olival corn cerca de 5 hectares, de topografia mais ou menos plana. Esta parcela foi dividida em cinco sub-parcelas e em cada uma delas foram colocadas, no ano de 1993, duas armadilhas cromotr6picas de plastico amarelo, opacas, verticais, de 15 cm x 20 cm, revestidas de cola de ambos os lados, uma sem feromona e a outra iscada corn uma capsula de feromona de 20 mg de 1,7-dioxaspiro (5,5) undecane e uma garrafa mosqueira de plastico, em que a parte inferior e amarela (Biological Control Systems Limited, Treforest, U.K.), contendo uma solu91io de fosfato de am6nio a 4%. (Patanita, 1995). Em 1994, por razoes que nao conseguimos ultrapassar, nao nos foi possivel obter as capsulas de feromona, pelo que dispusemos em cada sub-parcela duas armadilhas cromotr6picas sem feromona, identicas as usadas no ano anterior, e duas garrafas mosqueiras de vidro (armadilha McPhail) (Cardoso, 1996). Todas as armadilhas foram distribuidas ao acaso em cada uma das sub-parcelas, mas distanciadas de pelo menos 50 metros e colocadas no lado Sul da copa da arvore, a altura de 1,8 a 2,0 metros. As garrafas mosqueiras foram colocadas no interior da copa das arvores, enquantQ que as placas cromotr6picas foram colocadas tangencialmente a copa. As contagens do numero de adultos capturados foram realizadas semanalmente. A infesta91io dos fiutos considerada como a percentagem de fiutos corn ovos viaveis, larvasou pupas, foi avaliada, no ano de 1993, quinzenalmente sobre uma amostra de 25 arvores e 25 fiutos por arvore e no ano de 1994, semanalmente sobre uma amostra de 10 arvores e 10 fiutos por arvore. As arvores amostradas foram escolhidas ao acaso em cada uma das amostragens e os fiutosretirados das varias orienta95es da copa e a diferentesalturas. A rela91io existente entre as capturas das armadilhas e os niveis de infesta91io foi investigada utilizando analises de regressao simples. - 115 -

3 - Resultados e Discussiio

Da analise das Fig. s 1 e 2 podemos observar que as cotas de capturas de 1994 foram superiores cerca de duas vezes as de 1993, revelando maior nivel populacional infestante da mosca da azeitona, naquele ano. No que diz respeito ao tipo de armadilha, a que proporcionou uma curva de maior amplitude foia armadilha cromotr6pica corn feromona no ano de 1993 e a garrafa mosqueirano ano de 1994. Os valores mais elevados de capturas foram obtidos em ambos os anos no periodo entre Setembro e Novembro, tendo-se capturado durante este periodo cerca de 82 % do total de moscas em 1993 e 76 % em 1994. No ano de 1993, as contagens das capturas tiveram inicio em Junho, data em que o nivel populacional de B. o/eae era reduzido. Durante os meses de Junho, Julho e Agosto forampoucos os insectos capturados pelos diferentes tipos de armadilhas, ta! como ja foi verificado por outros autores (Evaristo, 1980, Ramos et al., 1983, Sobreiro, 1989, 1990, Ramos et al., 1989, Calado et al., 1994), embora se pudessem observar a partir das capturas nas garrafas mosqueiras, alguns adultos no campo, se bem que em pequeno numero. A popula9iio diminuiu por uma mortalidade natural dos adultos, ao longo do tempo, niio recuperada, por as remeas estarem impossibilitadas de efectuarem posturas, ja que os olivais, nesta regiiio, apenas permitem viabilidade as posturas efectuadas a partir de meados de Julho nas cultivares mais precoces. Um outro factor abi6tico de importancia relevante e a temperatura e neste caso parece evidente que as temperaturas extremamente elevadas verificadas nos meses de Julho e Agosto, siiolimitativasdo desenvolvimento do insecto (Patanita & Mexia, 1997). Verificou-se um periodo de inactividade sexual ate inicio de Setembro, ta! como ja se verificou em outros trabalhos (Delrio, 1985, Haniotakis & Vassiliou-Waite, 1987, Ramos et al., 1989), sendo as primeiras oviposi9oes observadas nesta data. A esta gera9iio, de grande irnportanciapela oviposi9iio continuada sobre os frutos ja desenvolvidos, segue-se uma outra que se ira sobrepondo a presen9a e actividade infestante da popula9iio anterior, tomando muito dificil a sua distirn;:iio.

200 180 1ro --Arm cronnt.c/ feronnna 140 - · · - · Arm crmrot.s/ ferom:ma 120 · · · · · · · Garrafannsqueira � 100 '" 80 •' C ro ::,::i z 40 .. 20 0 "3 "3 0 0 § § " = " 00 00 " " " ..., =0 � � � '° "' 0 � <"l ..... 0 ..... N"'

Fig. 1 - Curvade voo deBactrocera oleae (Gmelin). Moura, 1993 - 116 -

350 .. 300 - · · - · Anncromot. s/ feromrna .' 250 · · · · · · · Gmafa mos ueiraq

•' 200 •' 1" 150 ·' ' I :,:i JOO ..... \ I 50 " ·-, ··--· .. -.:_ .. _; 0 0 0 .; .; = 5'= .., .., -!!! ] � ..,:,= = 6 6 � or, � � 00 0 N .... � 0 N°' ..,., 0 N s ....N 0

Fig. 2 - Curvade voo Bactrocerade o/eae (Gmelin). Moura, 1994

Em 1994, as capturas das garrafas mosqueiras foram relativamente elevadas nos meses de Julho e Agosto e sofreram grandes flutuayoes. No entanto, as primeiras oviposiyoes verificaram­ se somente em finais de Agosto e de acordo corn o tipo de infestai;:aoobservada sobre os frutos ao longo da epoca, a primeira gerar;:lio tera ficado concluida no inicio de Outubro, seguindo-se a esta uma segunda gerar;:lioque tera dado origem a pupas e adultos hibemantes (Cardoso, 1996). 0 Quadro I assinala as diferenyas entre o numero total de adultos de B. oleae capturados em armadilhas cromotropicas corn feromona (ACF), armadilhas cromotropicas sem feromona (ACSF) e garrafas mosqueiras (GM) durante os tres meses de maior actividade do insecto, no ano de 1993. Podemos observar que durante este periodo, foram as armadilhas cromotropicas corn feromona as mais eficientes, nlio se notando diferenr;:as significativas entre o numero de adultos capturados nas armadilhas cromotropicas sem feromona e nas garrafas mosqueiras. Estes resultados estlio em conforrnidade corn os observados por outros autores (Delrio et al., 1983, Ramos et al., 1989).

Quadro I - Compara� entre as capturas de Bactrocera oleae (Gmelin) em trestipos de annadilhas durante o periodode Setembro a Novembro. Moura, 1993. Armadilha Atractivo N" total de machos Media • e Iemeas capturados Cromotr6pica Feromona sexual 7105 1421,0 a Cromotr6pica Seroferomona 1424 284,8 b Garrafa Mosqueira Fosfafode am6rjo 1599 319,8 b (*) As medias seguidas da mesma letra nao sao significativamente diferentes para p � 0,05, usando o teste de Duncan.

0 Quadro II mostra os valores das capturas efectuadas pelos diferentes tipos de armadilhas e a infestar;:ao dos frutos durante os tres meses de maior actividade da praga (Setembro a Novembro) de 1993 e 1994. - 117 -

Quadro II - Valores das caeturas medias nos tres tie:!s de armadilhas e infesta£!ode Bactrocera o/eae (Gmelin). Data Media das caeturas Somat6rio da media das cae!uras Percentagem GM ACF ACSF ACF ACSF GM de infesta120 1993 07/09 27.4 1.2 15.4 27.4 1,2 15,4 4,0 13/09 47.8 5,2 13.4 75,2 6,4 28.8 19/09 64.8 13,8 9,6 140,0 20,2 38,4 13,9 25/09 91.0 16,6 21,6 231.0 36,8 60,0 02/10 188.0 22.0 20.0 419.0 58,8 80,0 25,9 10/10 190.4 46,6 12.6 609.4 105,4 92,6 16/10 126.4 43.2 6,8 735,8 148,6 99,4 45,0 25/10 156,2 54.4 160,0 892,0 203,0 259,4 02/11 133.2 24,0 35,2 1025,2 227,0 294,6 76,3 07/11 134.2 26,6 2,4 1159,4 253,6 297,0 !3/11 136,6 20,8 8.0 1286.0 274,4 305,0 88,0 20/11 80,8 8,0 13,0 1366,8 282,4 318,0 27/11 44.2 2.4 2.3 1411,0 284,8 320,3 85,9 1994 25/08 5,0 87.4 5,0 87,4 7,0 01/09 6,3 19,7 11.3 107,1 5,0 08/09 5,0 66,8 16,3 173,9 11,0 15/09 5,9 87,0 22,2 260,9 10,0 22/09 8,8 79,8 31,0 340,7 6,0 29/09 22,7 209,3 53,7 550,0 12,0 06/10 19.5 31,4 73,2 581,4 23,0 13/10 23,8 167,8 97,0 749,2 21,0 20/10 12,5 103,1 109,5 852,3 32,0 27/10 127,6 304,7 237,l 1157,0 44,0 03/11 100.9 279,2 338,0 1436,2 92,0 10/11 40,0 258.2 378.0 1694.4 64,0 17/11 37.8 47,7 415.8 1742.1 81,0

Do Quadro III constam as correlac;:oes entre as capturas de B. o/eae em armadilhas cromotropicas sem feromona e a infestac;:ao dos frutos. Os resultados do conjunto dos dois anos de ensaios sao bastante animadores, pois indicam a existencia duma relac;:ao altamente significativa (P:50,00 I) entre o somatorio do numero medio de capturas semanais e a intensidade de ataque da praga. A variiinciaexplicada pelos modelos e elevada, sendo de 86,36% no caso do modelo linear (Fig. 3) e de 86,27% no caso do modelo multiplicativo.

Quadro III - Correla�s entre as capturas de Bactrocera oleae em arrnadiihas cromotr6picas sem feromona e a infestayiio dos frutos. Moura, 1993 e 1994. Ano Modelo a b r 1993 Linear 6,167 0,291 0,996 . ______Mu lti(?licativo ______3.057 ______0 ,571 ______0,989 ______1994 Linear 5,156 0,191 0,960 ______Mu lti(?licativo ______l,320 ______0 ,654 ______0.935 ______19993 e 1994 Linear 7,004 0,217 0,929 Multiplicativo 1,872 0,612 0,929 - 118 -

100 90 80

7 0 Y = 7,004 + 0217X l (,() 0 R' = 86,36 .. 50 P = 0,0000 40 N = 20 30 20 10 0 100 200 0 300 400 500 Somat6riodas capturas

Fig. 3 - Relac,;ao entre as capturas de Bactrocera oleae em armadilhas cromotr6picas sem feromona e a infestac,;ao dos frutos. Recta de regressiio: 1 993 e 1994.

No Quadro IV podemos observar as correlai;:oes entre as capturas de B. oleae nas garrafas mosqueiras e a infestai;:ao dos frutos. A analise dos resultados relativos a 1993 e 1994, isoladamente, permitiu evidenciar a existencia de relai;:oes altamente significativas entre as variaveis estudadas, resultados identicos foram obtidos por Bento (1997). No entanto, a analise conjunta dos dados referentes a 1993 e 1994, resultou numa dirninuii;:ao da relai;:ao e a vatiancia explicada pelos modelos foi de apenas 28,68% no caso do modelo linear e 36,72% no caso do modelo multiplicativo (Fig. 4). Estes resultados nao sao inesperados, pois tambem Delrio (1989) afirma que este tipo de armadilha tern geralmente maior dificuldadeem medir quantitativamente as populai;:oes da mosca da azeitona.

QuadroIV - Correla9(ies entre as capturas de Bactrocera o/eae em garrafas mosqueiras e a infestac,;ao dos frutos. Moura. 1993 e 1994. Ano Modelo a b r 1993 Linear 6,476 0,255 O. 982 ········--····················Multij>licativo ...... 0,362 ...... 0 ,967...... 0,984 ...... 1994 Linear -4,026 0,047 0,934 ...... _ .... _ ...... Mul t!{>licativo ...._. _. _ .._ .. 0,074 ...... 0,899 ...._ ... _ ...... 0.9 13 .. __ ..... 19993 e 1994 Linear 20.524 0.031 0.536 Multiplicativo 1,465 0.491 0.606 - 119 -

100 90 80 .. - 70 Y = 1,465 -t-X0·"' � R2 = 36,72 50 P = 0,0046 40 N = 20 ] 30 20 10 . . 0 . 0 200 400 600 800 HXXl 1200 1400 1600 ISCO Somat6riodas capturas

Fig. 4 - Rela930 entre as capturasde Bactrocera o/eae em garrafas mosqueiras e a infesta930dos frutos. Rccta de regressao: 1993 e 1994. As armadilhas crornotr6picas corn feromona foram apenas utilizadas no ano de 1993 e a analise dos resultados (Quadro V, Fig. 5) sugere a possibilidade da sua utilizai;ao como tecnica de estirnativa do risco da praga uma vez que o sornat6rio do nurnero medio de adultos capturados neste tipo de armadilha se encontra estreitamente correlacionado corn a percentagern de infestai;aodos fiutos.

Quadro V - Correlai;:oes entre as capturas de Bactrocera o/eae em armadilhas cromotr6picas corn feromona e a infesta930dos frutos. Moura, 1993. Ano Modelo a b 1993 Linear 2.607 0.064 0.989 Multiplicativo 0.273 0,792 0,993

100 90 80 70 :i Y = 2,607 + 0,064X � (,() R2= 97,81 50 P = 0.0000 40 N = 7 ] 30 20 10 0 0 200 400 £iCO 800 l(XX) 1200 1400 1600 Somat6riodas capturas

Fig. 5 - Rel3930entre as capturas de Bactrocera o/eae em annadilhas cromotr6picas corn feromona e a infesta930 dos frutos. Recta de regressiio: I 993. • 120 -

4 - Conclusoes

Os resultados das analises efectuadas sobre a possibilidade de utilizaylio de annadilhas cromotr6picas corne sem feromona e garrafas mosqueiras na estimativa do risco resultante de B. oleae, apontam claramente para ta! possibilidade no caso das armadilhas cromotropicas corn e sem feromona, resultando dai um grande avanyo no combate da praga, uma vez que se podera dispor de uma metodologia pratica e segura, a ser utilizada nas tomadas de decislio por parte dos agricultores. Contudo, foram apenas utilizados dados de dois anos de ensaios e para deterrninar a aplicabilidade das correlayoes aqui apresentadas slionecessiuios dados de varios anos de ensaios.

Referencias bibliograficas

AMARO, P. & BAGGIOLINI, M. (Eds.) (1982) - Introdufdo a protecfiio integrada. Vol. 1, Lisboa, F AO/DGPPA BENTO, A (1997) - A mosca da azeitona, Bactrocera oleae (Gmelin) em Tras-Os-Montes (Nordeste de Portugal): ciclo biologico, importancia dos prejuizos e estimativa do risco. II Congresso Iberoamericano e III Congresso Iberico de Ciencias Horticolas, 11 - 15 Mar90, 1997, Vilamoura, Portugal. Actas deHorticultura, 15: 138-144. CALADO, M., SANTOS, L & EVARISTO, F. (1994) - Atracylio comparativa de annadilhas amarelas corn feromonas normais ou de longa duraylio para a Bactrocera (Daculus) o/eae (Gruel.). In PIEDADE-GUERREIRO, J. (Ed), Froit Flies of Economic Importance. Intern Open Meeting, Lisbon, Portugal, 14-16 October 1993. IOBCIWPRSBulletin 17 (6): 130-138. CARDOSO, M. (1996) - Estudo sobre a mosca da azeitona na regiiio de Moura. Trab. Fim Curso Prod. Agric. ESAB, Beja. DELRIO, G. (1985) - Biotecnical methods forolive pest control. In Cavalloro, R. & Crovetti, A (Eds.), Integrated Pest Control in Olive-Groves - Proc. of the CECIFAOIIOBC International Joint Meeting, Pisa 3-6 April 1984: 394-410. DELRIO, G., PROTA, R. & USCIDDA, C. (1983) - Prove di comparazione di sostanze attrattive de! Dacus oleae Gmelin. Atti XIICongr. Naz. /ta/. Entomol.,Roma 1980: 361-368. DELRIO, G. ( 1989) - Biotechnical methods for the fiuit flycontrol. In CAV ALLORO, R. (Ed.), Fruitflies of economic importance 87 - Proc. of the CECIIOBCInternational Symposium, Rome, 7-10 April 1987: 359-372. EVARISTO, F. 1980 - Calibragem de armadilhas para avaliai,lio da popula9ao da Dacus oleae (Gmelin). I Congresso Portugues de Fitiatria e Fitofarmacologia, Lisboa, 15-19 Dezembro, 5: 165-173. HANIOTAKIS, G. & VASSILIOU-WAITE, A. (1987) - Effect of combining food and sex attractants on the capture of Dacus oleae flies.Entomologia Hel/enica, 5: 27-33. PAT ANITA, M. I. (1995) - Estudo sabre a mosca da azeitona- Bactrocera oleae (Gme/in) ea trafa da oliveira - Prays o/eae Bernard na regiiio de Moura numa perspectiva de protecfiio integrada. Dissert. Cur. Mest. Prot. Int. UTL, ISA, Lisboa, 2 l 8pp. PAT ANITA, M. I. & MEXIA, A (1997) - Utiliza9ao de diferentes tipos de armadilhas na monitorizayao da mosca da azeitona. II Congresso Iberoamericano e III Congresso Iberico de Ciencias Horticolas, 11 - 15 Mari,o, 1997, Vilamoura, Portugal. Actasde Horticultura, 15: 124 - 131. RAMOS, P., JONES, 0. & HOWSE, P. (1983) - The present status of the olive fiuitfly (Dacus o/eae Gruel.) in Granada, Spain and techniques formonitor ing its populations. In CAV ALLORO, R. (Ed.), Fruit flies of economic importance- Proc. of the CECIIOBC International Symposium, Athens, Greece, November 1982: 38-40. - 121 -

RAMOS, P , RAMOS, J. & JONES, 0. ( 1989) - Population monitoring and trap catch/infestation correlations in Dacus oleae from studies carried out over three seasons in an olive grove near Granada, Spain. In CAV ALLORO, R. (Ed.), Fruit fliesof economic importance 87 - Proc. of the CEC!OBC InternationalSymposium, Rome, 7-10 April 1987: 427-432. SOBREIRO, J. (1989) - Firts results on the use of chromotropic traps to control Dacus oleae (GmeL) In CAYALLORO, R. (Ed ), Fruit flies of economic importance 87 - Proc. of the CEC..JOBCInternational Symposium, Rome, 7-10 April 1987: 413-417. SOBREIRO, J. (1990) - Estudo da sensibilidade de diferentes cultivares de oliveira a ataques de mosca da azeitona Dacus o/eae (Gmelin). I Congresso lberico de Ciencias Horticolas. Lisboa, 18 - 22 Junho1990. Actasde Horticu/tura, 6: 265 - 270. - 122 -

UTILIZATION OF LOW COST MEDIA FOR REARING THE MEDITERRANEAN FRUIT FLY

A.G. Manoukas & E.N. Zografou National Center for Scientific Research "Demokritos" Athens 153 10. Greece

Summary Several experiments were conducted to evaluate fifteen media with low cost ingredients for larval growth and development of a Mediterranean fruit fly genetic sexing strain. It was found that wheat flour or corn starch and dried local brewer's yeast replaced successfully sugar and imported brewer's yeast respectively. Sugar beet seed byproducts with molasses could replace satisfactorily sugar and wheat bran of control. It was also found that a commercially available formula could be a very low cost alternative in rearing medfly. It was calculated that media containing this formula in various proportions reduced the cost by more than five times and one of them sustained results, in most parameters, equivalent or better than the control for at least three generations.

1 - Introduction Among the prime requirements for mass rearing of insects is the formulation of high efficiency, practical and low-cost media. Furthermore it is of importance that such media are produced, guaranteed and quality controlled by well established feed manufacturers. The Mediterranean fruit fly, Ceratitis capitata (Dipt., Tephritidae), WIED, has been artificially reared and routinely used for the application of the sterile insect technique throughout the world. The most expensive ingredient used in the larval medium was brewer's yeast followed by sugar and wheat bran. This is particularly true for countries (i.e. Greece) which import brewer's yeast. Several investigators utilized locally available products in place of brewer's yeast, sugar or wheat bran, in order to reduce cost or increase efficiency (Monro, 1968; Cavalloro and Girolami, 1969; Nade� 1970; Tanaka et a� 1970; Hafez and Shoukry, 1972; Peleg and Rhod, 1970; Chan et a� 1990; Economopoulos et a� 1990; Canato and Zucoloto, 1993; Zucoloto, 1993). To our knowledge no one tested complete diets produced by well established feed manufacturers for larval rearing of this insect. Reported here are results of one such diet together with results of fourteen other media which were found most suitable for rearing the Mediterranean fruit fly and which reduced cost substantially under local conditions. The complete diet was used for three generations.

2 - Materials and Method Table I presents the composition of the control and experimental media. Sugar, molasses and S.Beet Bypr. (Sugar beet seed byproducts) were from EBZ, Thessaloniki; Brewer's yeast from Amstel Co., Athens and Schwechat,Vienna; wheat flour and wheat bran was from Mills of Ag. Georgiou, Athens; corn starch from Yotis Co., Athens and Formula KASO from VIOZOKAT, Katerini. Formula KA 50, has a shelf life of six months and contained media of cereals and their byproducts, soybean mea� alfalfa mea� sunflower mea� certain salts, amino acids and vitamins. The proximate analysis of the control (C) and Formula KA SO (1) medium is given in Table II. Table I. Composition of control (C) and of experimental media, containing KA 50 and other local ingredients

Number of diet Ingredients % C 1 2 3 4 s 6 7 8 9 10 11 12 13 14 15 Water 54.5 64.S 64.5 70.7 60.6 55.0 55.l 59.6 55.l 55.8 54.6 55.9 53.3 43.6 41.8 43.6 Sugar 17.5 9.1 - - - 9.1 18.2 13.7 13.7 21.6 17.5 - - 14.0 - - Molasses - - 9.1 - 14.5 ------13.4 14.0 Wheat flower ------17.9 - - - - Corn Starch ------17.l - - - Yeast,Schwechat 8.8 ------9.0 8.6 - - 7.0 Yeast, Amstel ------8.8 - - 7.0 6.7 - Sodium benzoate 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.4 0.5 0.5 0.5 0.4 0.4 0.4 Citric acid 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.5 0.6 0.6 0.6 0.5 0.5 0.5 Wheat bran 17.9 ------17.9 16 19.9 - - - S. Beet Bypr. ------34.5 37.2 34.5 Formula KA50 - 25.4 25.4 28.2 23.8 35.0 25.l 25.l 29.6 21.6 ------I\.) c,., I

Table II. Proximate analysis of Media

Component,% Control {C)1 KA SO (1)2 Moisture 57.4 57.3 Proteins (NX6.25) 8.0 6.7 Lipids 0.9 0.8 Ash 1.7 3.5 Nitrogen Free Extracts3 32.0 31.7

On the basis of the analysis of its ingredients On the analysis given by the manufacturer Taken as a difference from 100 - 124-

A blender (BRAUN, model KM 321) and a mixer (BRAUN, model KM 321) was used for mixing of the ingredients and for preparation of media to desired texture, as follows: Approximately three quarters of total tap water was added to the blender and then sugar or molasses and brewer's yeast were added and blended for approximately three minutes. Then benzoic acid was blended for 2-3 minutes and pH was recorded. Finally citric acid was added and blended for an additional 3-4 minutes. The amount of citric acid was adjusted to give final pH of 4.0-4.5. The liquid diet was transferred to the mixer, the remaining of the water was used to rinse the blender and it was added to the mixer. Then wheat bran or S. Beet Bypr. was added gradually under continuous mixing until a uniform and presumably best "texture" for the insect was obtained.Formulae in pellets were soaked in water containing sugar or molasses, sodium benzoate and citric acid for the required time prior to adding in the mixer with the appropriate amount of water. Approximately 60 g of each test medium were placed in each (replicate) plastic container with covers (KRIS-PAN Co, Greece), of 7.5 cm low diameter, 9.5 cm top diameter and of 3.5 cm height, and three replicates per each (treatment) ingredient or formula were used. Twenty-five eggs per gram were used in all media. Eggs were obtained from flies of white pupa strain (genetic sexing strain GSWP-1-62) maintained under standard conditions (25± Z' C, 12:12 photoperiod and environmental humidity). Eggs were placed directly on the surface of the medium, except 100 eggs/replicate which were placed on a filter paper on the medium for recording hatchability of eggs. All replicates were kept in an incubator under 25±1° C, 12:12 photoperiod and environmental relative humidity. They were inspected regularly during the larval developmental period and prior to starting of pupation were uncovered to let larvae to pupate into an appropriate dry substrate (wood scrap). Pupal weight was recorded at least 6 days after pupation was completed. The results were compared with those of a routinely used larval medium (Nadel, 1970; Hendrichs et airy 1983; Hooper, 1987; Calkins et al, 1996), which was included as control Statistical procedures were those of Steele and Torrie (1%0).

3 - Results Table III presents the results of media C, 1, 2 and 3 for three generations

Table m. Performance of the Mediterranean fruit fly sexed strain (white pupa) larvae grown in the control and the experimental media for three generations1.

Number of Generation Hatchability Pupae/g Weight Adults% mediun:. � diet mg/pupa on pupae C 79.7 10.3· 8.5° 94.3 bc l F45 827 1s.o· 8.l 95.2 2 77;0 16.8bc 7.3b %.0 3 80.0 15.0b 5.5• 94.5 C 78.6 7.3• 8.8b 97.8b b b b l F46 77.3 16.0 7.7 92.2 b 2 85.0 16.8b 5.9" 95.3 b 3 84.3 16.8 4.9" 11.2· C 81.3 13.2" 8.3d 91.8 1 F47 80.0 17.lb 7.4° 91.8 2 81.7 15.4b 6.7b - 3 76.3 15.3b 5.3• - 1 Mean numbers of the same column followed by the same letter or no letter don't differ significantly at the probability level of 0.05, compared to each control (C). - 125 -

It is clear that, formula KA50 with sugar or molasses (medium I and 2) gave results equivalent to the control for hatchability and adult emergence and higher number of pupae/g diet compared to the control Pupal weight was much higher than 7.0 mg for medium l, which is considered suitable for the sterile male technique (Calkins et al., 1996) and equivalent to the control for F45 and F46. Medium 2 gave pupal weight lower than 7.0 during the F46 and F47 generation. Medium 3 (without sugar) was unsatisfactory. The lower pupal weight of medium 1 in the last generation (F47) suggests that this medium should be used only for release purposes and not for breeding, unless it is further improved. Table IV presents the results of media C and 4 to 15.

Table IV. Performance of Mediterranean fruit fly sexed strain (white pupa) larvae grown in 1 the control and the experimental media (Table I) .

Number HatchabHity Pupae/g Weight Adults% on of media % diet mg/pupa pupae 79.7 10.3• 8.5 94.3 C b 4 83.7 14.9 7.7 96.8 b b C 76.3 9.3• 8.8 91.7 s 77.0 16.2b 7.2" 34.5" ° 87.0 12.2" 8.5 92.0' C bc b 87.0 5.Q" 7.7•b 84.S c 6 1 bc b b 7 84.6 18.5 7_7• 80.3 83.7 21.0' 7.4· 67.5" 8 b 86.7 15.7abc 7.2· 78.8 9 b bc b 10 84.0 14.6" 8.3 81.5 b 73.0b 11.7 8.8 94.2 C b b 11 81.7 10.0 8.6 92.2 12 57.7" 10.2 8.1 73.8" b 81.0 7.8· 9.4 95.8 C b b 13 88.7 9.9 8.1• 98.3 88.0 9 l b 7.6" 98.0 14 . b b 15 91.7 9.8 8.2" 98.3 1 Mean numbers of the same column followed by the same letter or no letter don't differ significantly at the probability level of 0.05, compared to each control (C).

All experimental media containing formula KA50 (4 to 9) gave satisfactory results for all parameters recorded, with the exception of medium 5 which gave very low adult emergence. In most cases KA 50 combined with other ingredients gave larval survival (number of pupae/g diet) significantly higher than the control. It was also found that Amstel dried brewer's yeast (medium 10) when it replaced Schweschat yeast and wheat flour (medium 11) or corn starch (medium 12) when they replaced sugar, gave very satisfactory results. The same was true when Amstel brewer's yeastwas combined with sugar beet seed byproducts (medium 13) and with sugar beet seed byproducts and molasses (medium 14). Finally sugar beet seed byproducts with molasses (medium 15) could replace satisfactorily sugar and wheat bran of control. - 126 -

Table V presents a comparison of performance and cost of the medium KA 50 compared to Control

Table V. Comparison of Larval Media for the Mediterranean fruit fly larvae.

Media Parameter recorded Control' Low cost1 Control2 Eggs / g medium 25 25 25 Eggs hatchability, % 80 83-84 70-80 Pupae / g medium 10 15-18 10-14 Pupal weight, mg 8.5 7.7-8.1 8.0-9.5 Adult emergence, % 94 95-97 85-100 US$/ Kg medium 1.0 0.2 0.8-0.9 US$ / million pupae 100 12 75

1• Control and low cost (medium 1) used in this work. 2 • Similar to control used in a pilot mass rearing facility ..

It should be emphasized that the formula containing medium 1 is easier to mix and of very low cost. The cost in US Dollars per Kg medium is about 0.2 and the cost of million pupae 12 for medium l compared to 1.0 and 100.0 respectively for the control

4 - Discussion Over all consideration of the data, suggested that formula KA 50 could be a very low cost alternative in rearing Medfly. Further more, low cost local ingredients such as yeast, molasses, wheat flour, corn starch and S. Beet Bypr., could replace more expensive ingredients, alone or in combination with KA 50. It was calculated that experimental media containing KA 50 could .reduce the cost of larval medium by more than five times. For most of these diets pupal weight is somewhat lower than that of the control but well within satisfactory levels. The lower weight partly is due to the fact that larval survival is considerably higher than the control. All media and ingredients should be monitored for certain quality parameters (Manoukas,'1991). Also the data of proximate analysis (Table II) suggested that medium KA 50 could be improved by decreasing salt content and increasing protein.

Acknowledgmen'i:s We would like to acknowledge the European Union and the Greek Secretariat for Research and Development, for financing this work and the Athenian Brewing (AMSTEL), EBZ, VIOZOKAT and VITEMA Industries Greece, for the supply and quality control of ingredients and formulae. The contribution of Dr. C. lsrailides (National Institute for Agricultural Research) for drying brewer's yeast, is greatly appreciated.

References CALKINS, C.O� ASHLEY, T.R. and CHAMBERS, D.L. (1996) - Implementation of Technical and Managerial Systems for Quality Control in Mediterranean Fruit Fly (Ceratitis capitata) Sterile Release Programs. In: Fruit Fly Pests, B.A. McPheron and G.J. Steck (Eds.). St. Lucie Press, pp. 399-404. CANATO, C.M. and ZUCOLOTO, F.S. (1993) - Diet Selection by Ceratitis capitata Larvae (Diptera: Tephritidae): Influence of the Rearing Diet and Genetic Factors. J. Insect Physiol 39(11): 981-985. CAV ALLORO, R. and V. GIROLAMI. (1969) - Miglioramenti nell'allevamento in massa di Ceratitis capitata Wiedemann (Diptera, Trypetidae) Redia51: 315-327. CHAN, H. T. Jr., HANSEN, J.D. and TAM., S.YT. (1990) - Larval diets from different protein sources for Mediterranean fruit flies (Diptera:Tephritidae). J. Econ. Entomol 83(5): - 127 -

1954-1958. ECONOMOPOULOS, A.P, AL-TAWEEL, A.A. and BRUZZONE, N.D. (1990) - Larval diet with a starter phase for mass-rearing Ceratitis capitata: substitution and refinement in the use of yeasts and sugars. Entomol exp. appl SS: 239-246. HAFEZ, M. and SHOUKRY, A. (1972) - Effect of irradiation on adult fecundity and longevity of the Mediterranean fruit fly Ceratitis capitata Wied., in Egypt (Diptera:Tephritidae) Z Angew. Entomol 2: 59-66. HENDRICHS, J, G. ORTIZ, P. LIEDO and A. SCHWARZ (1983) - Siz years of successful med fly program in Mexico and Guatemala, pp. 353-365. In Fruit Flies of Economic Importance, Proceedings of an International Symposium organized by CEC/IOBC, November 1982, Athens A.A. Balkema, Rotterdam. HOOPER, G.H.S. (1987) - Application of quality control procedures to large scale rearing of the Mediterranean fruit fly. Ent. exp. appl 4<4: 161-167. MANOUKAS, A.G. (1991) - Dietary control in insects. The case of the olive fruit fly, Dacus oleae (GMEL.). Workshop proc. Int. Org. Biol. Control In Quality Control of Mass Reared Arthropods. F. Bigler, Edit pp. 174-182 MONRO, J. (1968)- Improvement in mass rearing the Mediterranean fruit fly Ceratitis capitata Wied. pp. 91-104. in Radiation, Radioisotopes and Raring Methods in the Control of Insect Pests. Proceedings of a Panel Organized by the Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture. NADEL, DJ. (1970) - Current mass-rearing techniques for the Mediterranean Fruit Fly pp. 13- 19. In Sterile Male Technique for Control of Fruit Flies. Proceedings of a Panel Organized by the Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture. PELEG, B.A. and RHODE, R.H. (1970) - New larval medium and improved pupal recovery method for the Mediterranean fruit fly in Costa Rica. J. Econ. Entomol 63: 1319-1321. STEELE, R.J. and TORRIE, J.H. (1960) - Principles of statistics Procedures. MacHil� London. TANAKA, N., OKAMOTO, R. and CHAMBERS, D.L. (1970) - Methods of mass-rearing the Mediterranean fruit fly currently used by the U.S. Departement of Agriculture. pp. 19- 23. In Sterile Male Technique for Control of Fruit Flies. Proceedings of a Panel Organized by the Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture. ZUCOLOTO. F.S. (1993) - Adaptation of a Ceratitis capitata population (Diptera, Tephritidae) to an animal protein-based diet. Entomol exp. appl 67: 119-127. - 128 -

MONITORIZA(:AO DA MOSCA-DA-AZEITONA (BACTROCERA OLEA£)NA REGIAO DE CASTELO BRANCO, PORTUGAL

J. Coutinho, M. Sequeira* & C. Veiga Escola SuperiorAgraria de Castelo Branco, Qt" Sr Mercules, P6000-Castelo Branco. Portugal Direc9ao Regional Agricultura Beira Interior, R. Amato Lusitano, L3 - P6000-Castelo Branco. Portugal*

Summary

Monitoring of the olive-fly (Bactrocera oleae) in the region of Castelo Branco, Portugal

The catches of the olive-fly Bactrocera oleae (Gmelin) in the region of Castelo Branco, Portugal, between 1995 and 1996 are analysed in the present work. The traps used were the followings: vertical yellow sticky traps with feromone lure; vertical yellow sticky traps without feromone lure; dome traps with ammonium salts and jackson traps with protein hydrolysate. The risk estimate and the economic threshold in "galega" variety are analysed. The most efficient catches were those using vertical yellow sticky traps with feromone lures. There were significant differences among the different types of trap. The jackson trap did not show any attractive power over B. oleae.

Resumo

Sao analisadas as capturas de mosca-da-azeitona, Bactrocera oleae (Gmelin), na regiao de Castelo Branco, Portugal, nos anos de 1995 e de 1996, em armadilhas cromotr6picas amarelas corn feromona e sem feromona, em armadilhas do tipo mcphail corn soluyao de sulfato de am6nio e em armadilhas tipo jackson corn hidrolisado de proteinas. Sao discutidos a estimativa do risco e os niveis econ6micos de ataque, na variedade galega. A armadilha cromotr6pica amarela cornferomona foi a mais eficiente na captura dos adultos de B. oleae, havendo diferenvas significativas entre todos os tipos de armadilhas. A armadilha tipo jackson nao revelou qualquer poder atractivo sobre B. oleae.

1. Introdu�ao

Este trabalho tern por objectivo conhecer a evoluyao das populavoes de mosca-da­ azeitona Bactrocera oleae (Gmelin) num olival da variedade galega, para apoiar a tomada de decisao quanto ao combate a mosca-da-azeitona, tendo em conta os niveis econ6rnicos de ataque definidos para a praga. Pretendeu-se assim ter um melhor conhecimento do comportamento da especie, de modo a poder-se implementar a protecvao integrada nos olivais da regiao de Castelo Branco.

2. Material e Metodos

0 ensaio decorreu nos anos de 1995 e de 1996 num olival da cultivar galega na Quinta da Sra de Mercules, propriedade da Escola Superior Agniria de Castelo Branco, corn aproximadamente 3 5 anos de idade, denorninado"olival da casa do pastor" - 129-

0 compasso e de 1 Orn x 1 Orn. Nao foram realizados tratamentos fitossanitarios. A colheita foi realizada em 15 de Dezembro em 1995 e em 12 de Novembro em 1996. Para a determina9ao das curvas de voo de Bactrocera oleae (Gmelin) foram utilizados tres tipos de armadilhas em cada ano, colocadas aleatoriamente segundo um esquema de casualiza9ao completa, corn quatro repeti95es. A distancia minima entre os locais de coloca9ao das armadilhas foi de 40 metros. Em 1995 foram usadas: armadilhas cromotr6picas amarelas quadradas de 20 cm de !ado, corn cola dos dois lados, corn uma capsula de 20 mg de feromona ("Agrisense - Biological Control Systems"), mudada de 4 em 4 semanas; armadilhas cromotr6picas amarelas quadradas de 20 cm de !ado, corn cola dos dois lados, sem feromonas ("Agrisense") e garrafas mosqueiras do tipo macphail ("Agrisense"), corn uma solw;:ao de sulfato de am6nio a 2 % (Agrisense, s/d). Em 1996 a armadilha cromotr6pica amarela sem feromona foi substituida por uma armadilha do tipo jackson corn atractivo de hidrolisado de proteinas ("Endomosyl, Hoechst"), impregnado semanalmente em duas bandas de cartao absorvente colocadas nas faces intemas laterais da armadilha (Pereira & Carvalho, 1993). As armadilhas cromotr6picas foram colocadas no !ado sudeste da copa das arvores, a uma altura media de 1.8 metros e tangencialmente a copa. As garrafas mosqueiras e as armadilhas tipo jackson foram colocadas no interior da copa das arvores, a mesma altura das outras armadilhas. Em 1995 as armadilhas foram colocadas em 7 de Julho e retiradas no dia 14 de Dezembro. Em 1996 as armadilhas foram colocadas em 16 de Julho e retiradas em 22 de Janeiro de 1997. Em 1995 foi colhida semanalmente uma amostra aleat6ria de 25 azeitonas por arvore, em 8 arvores, prefazendo 200 frutos por amostra semanal. As azeitonas foram colhidas a diferentes alturas em varias orientai;oes da copa. Ap6s a rnistura da amostra de 200 frutos foram retirados 100, sobre os quais incidiram as observai;oes dos estadios de desenvolvimento da mosca-da-azeitona. Em 1996 a amostra colhida foi de 100 a 400 fiutos em 20 arvores, colhendo-se inicialmente 5 azeitonas por arvore (Sobreiro, 1993) Da observai;ao das azeitonas registaram-se as picadas sem evolui;ao; preseni;a de larva viva; preseni;ade larva morta; preseni;ade pupa e galeria abandonada.

3. Resultados

No Quadro 3.1 e no Quadro 3.2 apresentam-se os valores totais e os valores medios das capturas nas armadilhas, respectivamente em 1995 e em 1996.

Quadro 3.1 - Capturas totais de adultos de Bactrocera oleae (Gmelin), por armadilha, em Ires tipos de armadilhas, em 1995.

Armadilha Atractivo Total adultos Media por capturados armadilha* Garrafa mosqueira sulfato de am6nio 1425 356.3 b 2% Cromotr6pica sem cor 472 118.0 C feromona

Cromotr6pica corn cor e feromona 4040 1010.0a feromona sexual

*As medias seguidas de letra diferente sao significativamente diferentes para p:s;0.05 pelo teste LSD - 130 -

Quadro 3.2 - Capturas totais de adultos de Bactrocera o/eae (Gmelin), por armadilha, em tres tipos de armadilhas, em 1996.

Armadilha Atractivo Total adultos Media por capturados armadilha* Garrafa mosqueira sulfato de am6nio 956 239a 2% Jackson hidrolisado de 0 0 proteina feromona sexual 2540 635b Cromotr6pica corn feromona *As medias seguidas de letra diferente sao significativamente diferentes para ps0.05 pelo teste LSD

No Quadro 3.3 e na Figura 3.1 e no Quadro 3.4 e na Figura 3.2 apresentam-se os valores medios das capturas nas armadilhas, respectivamente em 1995 e em 1996.

Quadro 3.3 - Numero medio semanal de adultos de Bactrocera o/eae (Gmelin). por armadilha, capturados em tres tipos de armadilhas, em 1995.

. -- ·� '-=•- ··-· -· , .. Datas Garrafas Cromotr6picas sem Cromotr6picas corn·... ·- -s·oma das mosqueiras feromona feromona medias*

13 / 07 1.75 1.50 24.75 28.00 def 20 I 07 4.25 1.50 16.00 21.75 def 27107 1.50 3.25 8.50 13.25 ef 03 / 08 1.25 0.25 10.00 11.50 f 10 / 08 0.75 0 11.50 12.25 ef 17 / 08 0 0.75 2.50 3.25 f 24 /08 0 0.25 6.00 6.25 f 31 / 08 0.50 0.25 3.00 3.75 f 07 ! 09 0.25 0.25 25.50 26.00 def 14 / 09 4.00 2.25 64.00 70.25 cde 21 / 09 4.00 3.25 54.25 61.50 cde 28 / 09 13.00 3.50 70.00 86.50 cd 05 / 10 15.00 3.00 79.00 97.00 cd 12 / 10 33.75 8.50 131.00 173.25 b 19 / 10 92.75 13.75 124.50 231.00 a 26 / 10 108.00 29.25 118.75 256.00 a 02 / 11 52.75 26.50 159.25 238.50 a 09 I 11 22.25 14.50 80.50 117.25 C 16 / 11 0 1.25 4.50 5.75 f 23 / 11 0.50 4.25 15.75 20.50 ef 30 /11 0.25 1.00 0.50 1.75 f 07 / 12 0.25 0 0.25 0.50 f .. -·, �-· .,,. . --- -· ··,- - ,,... .. ,. ... *As medias seguidas de letra diferente sao significativamente diferentes para ps0.05 pelo teste LSD - 131

Quadro 3.4 - Numero media semanal de adultos de Bactrocera o/eae (Gmelin). por armadilha, capturados em tres tipos de armadilhas, em 1996.

-· Datas Garrafas Jackson corn Cromotropicas corn Soma das mosqueiras hidrolisado de feromona medias protElfnas

22107 0.75 0 3.25 4.00 29 / 07 0.25 0 2.75 3.00 05 / 08 0.25 0 0.75 1.00 12 / 08 0.50 0 1.25 1.75 19 / 08 0.25 0 3.25 3.75 26 /08 0.25 0 0.75 1.00 02 / 09 0.25 0 6.25 6.75 09 / 09 3.25 0 24.50 27.75 16 / 09 5.25 0 39.50 44.75 22 /09 11.25 0 90.00 101.25 29 / 09 22.50 0 65.50 88.00 07110 99.50 0 150.00 249.50 13 / 10 77.75 0 68.00 145.75 20 / 10 3.50 0 58.75 62.25 28 / 10 9.50 0 77.25 86.75 04 / 11 3.00 0 24.50 27.50 11 / 11 0.75 0 7.00 7.75 19 / 11 0 0 0 0 27 / 11 0 0 3.75 3.75 05 / 12 0 0 2,75 2.75 11 / 12 0 0 0.75 0.75 17 / 12 0.25 0 2.00 2.25 23 / 12 0 0 1.50 1.50 31 / 12 0 0 1.00 1.00 06 / 01 0 0 0 0

Nos Quadros 3.5 e 3.6 apresentam-se os diferentes periodos em que podemos dividir as captura de adultos de B. oleae, respectivamente em 1995 e em 1996.

Quadro 3.5 - Numero media de individuos de Bactrocera o/eae (Gmelin) capturados semanalmente por armadilha, durante periodos em que a resposta as armadilhas varia devido a factores ambientais ou factores biologicos, em 1995 .

.. . . _.,,...,· ... ---- . - -, -� Periodo Garrafa mosqueira Cromotropica sem Cromotropica corn feromona feromona

I (13/07/95 - 7/09/95) 1.14 0.94 11.97

II (7/09/95 - 5/10/95) 9.00 3.00 78.81

Ill (5/10/95- 9/11/95) 61.90 18.50 203.20

IV (9/11/95 - 7 /12/95) 0.20 1.63 5.25 ·-- . --·J - 132 -

Quadro 3.6 - Numero media de indivfduos de Bactrocera oleae (Gmelin) capturados semanalmente por armadilha, durante perfodos em que a resposta as armadilhas varia devido a factores ambientais ou factores biol6gicos, em 1996.

Perfodo Garrafa mosqueira Jackson Cromotr6pica corn feromona

I (22/07 /96 - 2/09/96) 0.35 2.62

II (2/09/96 - 16/09/96) 4.25 32.01

Ill (16/09/96 - 4/11/96) 32.43 76.25

IV (4/09/96 - 31/12/96) 0.12 2.34

Nos Quadros 3.7 e 3.8 apresentam-se as percentagens de fiutos atacados por B. oleae agrupados por diferentes criterios, relativamente a 1995 ea 1996, respectivamente.

Quadro 3.7 - Percentagem de azeitonas atacadas por Bactrocera o/eae (Gmelin), agrupadas por diferentes criterios, referentes a 1995 .

�� •• - M • .. •••-• • ...... -· --; ...... -·------· - ... -.,.-- .-,.�,,.---- �--·"· ., ..•.. ····" ·' ' ..,.-· ---··- ·� �- . Data Picada Larva Larva Pupa Galeria Frutos Frutos sem morta viva (4) abandonada atacados atacados evoluyao (2) (3) (5) corn totais (1) · evoklyao (1+2+3+4+5) larvar (2+3+4+5)

13 / 07 0 0 0 0 0 0 0 20 I 07 0 0 0 0 0 0 0 27107 0 0 0 0 0 0 0 03 / 08 0 0 0 0 0 0 0 10 / 08 0 0 0 0 0 0 0 17 / 08 0 0 0 0 0 0 0 24 / 08 0 0 0 0 0 0 0 31 / 08 3 0 0 0 0 0 3 07 / 09 5 0 0 0 0 0 5 14 / 09 5 0 0 0 0 0 5 21 / 09 10 0 0 0 0 0 10 28/ 09 8 0 0 0 0 0 8 05 / 10 5 0 1 0 0 1 6 12 / 10 0 0 1 1 3 5 5 19 / 10 4 1 2 2 4 9 13 26 / 10 4 0 0 0 5 5 9 02 / 11 0 0 4 2 8 14 14 09 / 11 0 0 4 5 16 25 25 16 / 11 0 4 13 2 8 27 27 23 / 11 0 1 7 2 16 26 26 30 / 11 0 0 13 1 7 21 21 07 / 12 5 1 26 1 14 41 47 ...---�------.. ,,...... _...... - 133 -

Quadro 3.8 - Percentagem de azeitonas atacadas por Bactrocera o/eae (Gmelin), agrupadas por diferentes criterios, referentes a 1996.

Data Picada Larva Larva Pupa Galeria Frutos Frutos sem morta viva (4) abandonada atacados atacados evolui;:ao (2) (3) (5) corn totais (1) evolui;:ao (1+2+3+4+5) larvar (2+3+4+5)

22 I 07 1 0 0 0 0 0 1 31 / 07 1 0 0 0 0 0 1 05 I 08 3 0 0 0 0 0 3 12 / 08 5 0 0 0 0 0 5 19 / 08 4 0 0 0 0 0 4 26 I 08 6 0 0 0 0 0 6 02 I 09 5 0 0 0 0 0 5 09 I 09 4 0 0 0 0 0 4 16 / 09 * * * 22 I 09 3 0 1 0 0 1 4 29 I 09 7 0 11 1 7 19 26 07 I 10 5 0 15 0 12 27 32 13 / 10 2 2 20 1 7 30 32 20 I 10 0 2 32 2 22 58 58 27 I 10 0 1 32 4 28 65 65 05 I 11 0 1 18 5 37 61 61 • Nao foram feitas observagoes.

Capturas medias de B. oleae em tres tipos de armadilhas e percentagem de frutos atacados � Sra. de Mercules - E.S.A. - C. Branco 1995 160 45

140 40

�Armad. McPhail 35 120 ---0--Armad. Cromotr6pica S/ Feromona -ltr--Armad.Cromotr6pica C/ Feromona 30 100 --M-- % de Frutos Atacados Cl Evolw;::ao 25 j so 20 60 15 * 40 10

20 5

Oatas de observafSo

Fig. 3.1 - Curvas de voo de Bactrocera oleae (Gmelin) utilizando tres tipos de armadilhas ea evolui;:ao da percentagem de frutos atacados corn evolui;:ao larvar. Quinta da Senhora de Mercules - E.S.A., Castelo Branco, 1995. - 134 -

Capturas medias de B. oleae em dois tipos de armadilhas e percentagem de frutos atacadoscl evolu�ao � Sra. de Mercules - E.SA. - C. Branco 1996 160 70 140 60 .� 120 50 100 40 80 30 i 60 20 40

20 10 "'

0 0 3 3 g, "'0 8. 0 ;; ;; ;; ;; -s -s i'i i'i i'i � :I � � :I ..,. . .,. g> 'i' "' "' � 9 'r 'i' N "' � d, N :!. 0l -:1. "' "' 3. 8 0

Fig. 3.2 - Curvas de voo de Bactrocera oleae (Gmelin) utilizando tres tipos de armadilhas e a evolU!;:ao da percentagem de frutos atacados corn evolu9ao larvar. Quinta da Senhora de Mercules - E.S.A., Castelo Branco, 1996.

Na Figura 3.3 apresentam-se os valores medios das temperaturas e das precipita�oes no ano de 1995, durante o periodo em que decorreu o ensaio.

T( °C) - Precipita,;ao R(mm) 40,0 -+-Med.Tem p. max. 90,0 -+-Med.Tem p. mn 35,0 80.0

70,0 30,0

60,0 25,0 50,0 20,0 40,0 15.0 30,0

10,0 20,0

5,0 + 10,0

0,0 +"oa..,--+--+--+--+--+--+- l 0,0 JL > > ,; ,; ,; :'i :5 :5 0 0 0 0 0 ;j ;j ;j a; '5 '5 '5 '5 0 0 0 0 0 7 0) "' "' Cl) Cl) 0 z .....

Fig. 3.3 - Termopluviograma referente ao periodo de capturas de 8. o/eae. Quinta da Senhora de Mercules - E.S.A., Castelo Branco, 1995. - 135 -

4. Discussiio

Dos quatro tipos de armadilhas usadas para a monitorizac;:ao de B. oleae, a armadilha cromotr6pica corn feromona foi a mais eficiente quanta ao mimero de individuos capturados, pennitindo a detecc;:ao mais cedo e um melhor acompanhamento dos niveis populacionais. As diferenc;:as de capturas nos quatro tipos de armadilhas sao sempre significativas estatisticamente (Quadros 3 .1 e 3 .2). A armadilha tipo jackson corn hidrolisado de proteinas nao revelou qualquer poder atractivo sobre B. oleae, pois nao houve qualquer captura. Estas armadilhas nao mostraram qualquer interesse para a monitorizac;:ao de B. oleae, nas condic;:5es em que decorreu o ensaio. Estas armadilhas, a terem niveis de captura semelhantes as garrafas-mosqueiras, poderiam substitui-las, corn vantagem, visto serem mais praticas. Analisando as capturas nas armadilhas ao longo do tempo podemos distinguir 4 periodos, correspondendo a niveis populacionais bem diferenciados, como podemos observar nos Quadros 3.3, 3.4, 3.5 e 3.6 e nas Figuras 3.1 e 3.2, relativamente aos anos de 1995 e de 1996. No I periodo, compreendido entre 13/07 e 7/09 de 1995 e entre 22/07 e 16/09 de 1996, verificamos que o nivel de capturas e reduzido, em qualquer dos tipos de armadilhas utilizadas. Este periodo corresponde a eclosao dos adultos provenientes dos estados hibernantes. As altas temperaturas (Fig.3.3) e a baixa humidade relativa do ar provocam uma paragem da actividade dos adultos, embora ja haja frutos corn as caracteristicas necessarias as posturas. No II periodo, compreendido entre 07/09 e 5/10 de 1995 e entre 2/09 e 16/09 de 1996, verificamos um aumento de actividade devido ao abaixamento das temperaturas e ao aumento da humidade relativa e de alguma precipitac;:ao (Fig. 3.3). Tambem este periodo correspondera a adultos provenientes dos estados hibernantes. No III periodo, compreendido entre 5/10 e 9/11 de 1995 e entre 16/9 e 4/11 de 1996, verificamos uma intensa actividade da especie corn capturas de adultos elevadas em todos os tipos de armadilhas, o que indica uma elevada actividade tanto de machos como de femeas. Este periodo correspondera a eclosao dos adultos da 1• gerac;:ao. Neste periodo verifica-se tambem o grande aumento dos frutos atacados como podemos verificar nas Figuras. 3 .1 e 3 .2. A este periodo correspondem tambem temperaturas e humidades relativas 6ptimas para o desenvolvimento da especie. No IV periodo, compreendido entre 9/11 e 7/12 de 1995 e entre 4/11 e 31/12 de 1996, verificamos uma acentuada baixa de actividade dos adultos devido provavelmente ao facto de ter terminado a eclosao dos adultos da 1• gerac;:ao e devido as baixas temperaturas que se comec;:aram a registar neste periodo, tendo deixado tambem de se fazer notar o poder atractivo da feromona sexual devido as temperaturas crepusculares terem baixado para valores inferiores a l 6°C. 0 nivel econ6mico de ataque (NEA) da azeitona para azeite adoptado em Portugal e de 10% de fiutos corn larva viva (Sobreiro ,1993). Verificamos, no entanto, que em 1995 e em 1996, na variedade galega, o NEA e atingido ja muito perto da colheita, dificultando a escolha dos insecticidas a utilizar. Verificamos tambem que quando se atinge o NEA, nesta variedade, a percentagem de fiutos atacados e ja bastante elevada (Quadros 3.7 e 3.8) o que nos leva a questionar a oportunidade do tratamento e a utilizac;:ao deste NEA nesta variedade. 0 ano de 1996 foi um ano de contra-safra, cornproduc;:5es estimadas da ordem de 4- 5Kg/ arvore. Nestas situac;:5es de baixas produc;:oes pensamos que o NEA devera integrar o nivel de produc;:ao, ta! como utilizado em Espanha (Bueno, 1984). Neste caso - 136 -

(produ96es inferiores a 15Kg/ arvore) o NEA seria atingido quando 20% das azeitonas apresentassem larva viva. 0 NEA foi atingido em 1995 (10% de frutos corn larva viva) em 16/11, correspondendo a uma percentagem de frutos atacados cornevolu9ao larvar de 27%. Em 1996 o NEA, considerando l 0% de frutos corn larva viva, foi atingido em 29/9, correspodendo a 19% de frutos atacados corn evolu9ao larvar. Considerando 20% de frutos corn larva viva, o NEA foi atingido em 13/10, correspodendo a 30% de frutos atacados cornevolu9ao larvar.

Referencias bibliograficas

AGRISENSE (s/d). Fniitflies. Integratedpest management. Agrisense, Mid Glamorgan.

AGRISENSE (s/d). Insect monitoring and lures in fn,it crops. Agrisense,Mid Glamorgan.

BUENO, A. M. (1984). Strategy for the integrated control of Spanish olive trees. Technical recommendations forintegrated control programmes. In: CAV ALLORO, R. & CROVETTI, A. (Eds.). Proceedings of the CECIFAOIIOBC InternationalJoint Meeting. Pisa 3-6 April, 1994: 470-480.

PEREIRA, R. & CARVALHO, J. Passos (1993).Estudo da degradai,ao de proteina hidrolisada como atractivo da mosca do mediterraneo (Ceratitis capitata). Actas 1 ° Congres. de Citricultura, 20-22 Jan. 1993, Silves: 387-392.

SEQUEIRA, M. (1977). Influencia da mosca da azeitona Bactrocera oleae (Gmelin) na qualidade do azeite. Relat. Fim Curso Eng. Prod. Oleos Alimentares (CESE). Escola Superior Agraria. Castelo Branco.

° SOBREIRO, J. (1993). Guia para a protec9iiofitossanitaria da oliveira. Guias e catalogos n 4. CentroNacional de Proteq:ao a Produi,ao Agricola, Lisboa.

VEIGA, C. (1977). A mosca daazeifona na r.egiiio de Castelo Branco. Relat. Fim Curso Eng. Prod. Agricola (Relat. Preliminar). Escola Superior Agraria. Castelo Branco. - 137 -

CAPTURE DES FEMELLES DE LA MOUCHE MEDITERRANEENNE DES FRUITS, CERATJTISCAPITATA, DANS LES AGRUMES: EVALUATION DE NOUVEAUX PIEGES ET APPATS.

A. BAKRI & H. HADIS

Universite Cadi Ayyad, Faculte des Sciences-Semlalia, Departement de Biologie, Unite de Controle Biologique des Insectes B.P: S/15., Marrakech. MAROC

Resume:

La Mouche Mediterraneenne des fruits, Ceratitis capitata est un ravageur tres redoutable pour l'agrurniculture marocaine. Dans le but de reduire les effets nefastes de la Jutte chimique, une nouvelle methode de Jutte biotechnologique est testee en utilisant differents types de combinaisons piege - attractif. L'essai a ete conduit pendant 8 semaines dans un verger de clementinier localise dans la region du Souss ( sud-ouest marocain). Ce verger est situe au voisinage de la foret d'arganier, un foyer important de la Ceratite.

Cinq types d'associations piege - attractif ont ete testes: trois nouveaux pieges sees, DTFA2, DTFA4 et CC sont comparees a deux pieges standards: Jackson (Trimedlure) et Mc-Phail (Hydrolysat de proteine): Les pieges sees, DTFA2, DTFA4 et CC sont appates de deux nouveaux leurres synthetiques, ammonium acetate et putrescine recemment developpes par Heath et al. (1994). Le piege CC simple et economique est de notre propre conception.

Les resultats obtenus ont montre que Jes pieges de Jackson capturent toujours un plus grand nombre de Ceratite suivis par Jes pieges de Mc-Phail, puis les pieges sees DTFA2, CC et DTFA4. Mais, Jes pieges de Jackson capturent presque exclusivement que des males alors que Jes pieges sees et Jes pieges de Mc Phail capturent aussi bien Jes males que Jes femelles de la Ceratite, et avec une forte proportion des femelles par rapport a celle des males. De plus, la performance des pieges de Mc Phail est plus importantes que celles des pieges sees. Dans tous Jes cas, Jes captures ont ete tres influencees par Jes variations climatiques. Une correlation positive a ete enregistree entre Jes captures et Jes temperatures et negatives entre Jes captures et I' humidite relative minimale.

L'eventualite de !'utilisation de ces nouveaux pieges dans Jes programmes d'eradication de la mouche mediterraneenne des fruits au moyen de la technique d'insectes steriles est discutee ici. - 138 -

1-INTRODUCTION

Au Maroc, la principale methode utilisee pour la Jutte contre la Ceratite est basee sur les pulverisations d'appats et Jes traitements de couvertures par les insecticides a grande echelle sur Jes cultures commerciales destinees a !'exportation et au marche interieur. Cette technique donne de meilleurs resultats lorsqu'elle est generalisee. Toutefois, elle est couteuse et presente des incidences nefastessur les ennemis naturels de ravageurs comme Jes Aleurodes, Jes Cochenilles, Jes Pucerons et Jes Acariens. Le piegeage, au moyen de pieges a attractifs, serait un outil tres essentiel pour la surveillance et la detection de la population. Estimation de la variation du niveau de la population d'insectes permettrai de rationaliser les interventions avec insecticide et done reduire le cout de la production. Aussi, le !ache des predateurs serait faitau moment opportun quand la population est elevee. II y' a eu une evolution dans la techniques de piegeage de la ceratite. Trimedlure (Beroza et al., 1961), parapheromone synthetique, est le meilleur appat et utilise en association avec des pieges englues de type Jackson (Harris et al., 1971) pour la surveillance et la detection de la Ceratite. Cependant, trimedlure est males-specifique et done ne convient pas pour Jes programme de Jutte par les males steriles. Les hydrolysats de proteines en association avec des pieges Mc Phail (Mc Phail, 1939) capture aussi bien les males que Jes femelles de la Ceratite mais son mode d'emploi est delicat et son efficacite interfere avec Jes conditions climatiques humides. Des etudes recentes (Heath et al., 1995) ont pu developper de nouveaux systemes de pieges sees et d'attractifs alimentaires synthetiques ( acetate d'ammonium et putrescine), pour la detection des populations femellesde la Ceratite. Ces produits ont ete isoles a partir des volatils liberes par Jes appats proteiques. Ce travail a pour objectif d'obtenir des donnees sur ces nouveaux pieges sees et les attractifs alimentaires synthetiques d'une part, et de faire la comparaison avec les pieges Jackson appates au Trimedlure et les pieges Mc Phail appates a l'hydrolysat de proteine.

2-MATER!EL ET METHODES

L'essai a ete conduit du 17/11/1995 au 12/1/1996 dans la region du Souss (Sud-est du Maroc), particulierement dans un verger d'agrume (Variete: Clementine) localise dans la ferme experimentale de l'lnstitut Agronomique et Veterinaire Hassan II d'Agadir. La parcelle est d'une superficie d'un hectare. les arbres sont plantes en lignes (17) et en colonnes (23). les fruitsont deja ete recoltes un mois auparavant. Ainsi le verger a ete abandonne et Jes traitements chimiques sont arretes. Le protocole experimental consiste a comparer cinq systemes d'associations piege­ attractif. Piege Jackson (JT).C'est un piege en papier blanc cire de formetriangulaire (9 cm x 9 cm x 12.5 cm). A son interieur, une plaque engluee de couleur blanchatre. L'appat utilise est une pastille de Trimedlure(TML)(Agrisens) placee dans un mini-panier en plastique suspendu au milieu de la partie horizontale d'un filmetallique servant aussi a accrocher le piege sur l'arbre. Piege sec phase 2. C'est un cylindre (15.5 cm de hauteur et 9 cm de diametre) construit a partir d'un film d'acetate de couleur vert fluorescent. Le haut et le bas de ce cylindre sont fermespar deux boites de petri en plastique. Sur chacune de ces boites est colle une petite plaque(4 x 2.5 cm) qui incorpore un insecticide a base de methomyle. Au milieu de la partie laterale du piege sont perfores trois trous (2.5 cm de diametre) equidistant et qui permettent - 139 - l'entree des insectes. Un long fil metallique permet a la fois de maintenir et fixer les boites sur le cylindre et de suspendre le piege sur l'arbre. Piege sec phase 4. 11 est similaire au precedent, cependant, certaines modifications ont y ete apportes: La coloration est verte foncee, la base est laissee ouverte et une plaque jaune (12.5 x 7.5 cm) engluee des deux faces est suspendue verticalement au milieu du piege. Un long fil metallique permet a la fois de maintenir la plaque verticale et de suspendre le piege a l'arbre. Piege sec a conception local. Le piege est prepare a partir de bouteilles cylindriques et jaunes d'huile de table "Huilor" d'une capacite d'un litre (8 cm de diametre, 20 cm long). Ces bouteilles ont ete prealablement lavees avec un detergent puissant et sechees pour eliminer toutes contaminations ou odeurs. Le systeme de capture adopte pour ce piege est identique a celui du piege precedent. La base de la bouteille a ete supprimee et 3 trous de 2.5 cm de diametre chacun ont ete perfores a travers la partie mediane de la paroi de la bouteille. Un fil metallique suspend la plaque jaune engluee verticalement a l'interieur du piege. Chacun de ces pieges sees a ete appate de deux leurres synthetiques pour les femelles decrit par Heath et al. (1995): Leurre 1- L'acetate d'ammonium formule dans un petit sachet en Cellophane muni d'une fine membrane qui permet !'evaporation controlee de cet attractif (Consep Inc., Bend, OR). Leurre 2- Le putrescine d'evaporation(l,4-diaminobutane) formule dans un papier d'aluminium nitrate muni d'un petit orifice au centre est couvert d'une membrane (Epsky et al. 1995). Ces deux attractifs sont colles separement sur la face interne du piege. Piege Mc Phail (International Pheromone, Ltd. ). C'est la version en plastique qui est utilisee durant cette etude. Ce piege est constitue de deux parties qui peuvent se tier entre elle par emboitement. La moitie superieure est formee d'un plastique transparent en forme conique et la moitie inferieure est formee d'un plastique jaune invagine au milieu avec un trou d' entre des insectes. Dans cette moitie inferieure est versee l'attractif alimentaire qui consiste en 300 ml d'une solution d'attractif proteique: Nulure (9%), Soduim borate ou Borax (3%) et l'eau (88%). Les insectes tues par inondation dans la solution proteique sont recueillis par tamisage. Protocole experimental. Dans la parcelle d'etude, les pieges ont ete distribues en blocs aleatoires complets et suivant un carre latin. 5 blocs (lignes) ont ete etablis avec 5 pieges (1 JT, 1 DTFA2, lDTFA4, 1 CC et 1 IPMT) par bloc; soit au total 25 pieges par site. Les pieges sont suspendus 1 a 2 m au dessus du sol, dans la partie Sud-est de l'arbre. Les pieges a interieur d'un meme bloc et entre deux blocs successifs sont distants de deux arbres (30 a 50 m); ils sont places dans l'arbre dans un espace relativement ouvert ne touchant pas le feuillage. Dans Jes pieges Jackson, une meme pastille de Trimedlure est utilisee toutes les deux semaines. Dans les pieges sees, Jes deux leurres synthetiques sont changes tous les mois. Dans les pieges Mc Phail, la solution proteique est changee toute Jes semaines. L'ancienne solution est recyclee au milieu de la semaine par addition du volume d'eau evaporee. Le test a ete effectue pendant 8 semaines et le prelevement des donnes est realise tous les 3-4 jours. Pour chaque piege, les ceratites captures ont ete triees par sexe puis comptees. Le nombre des insectes autres que la ceratite a ete aussi determine. Au laboratoire, des echantillons de 25 femelles ou moins sont selectionnes au hasard a partir de chaque type de piege a appat alimentaire. Ces femelles sont ensuite dissequees sous la loupe binoculaire pour determiner l'etat de maturite des ovaires. Sur une population de laboratoire, les femelles immatures et ne manifestant pas de comportement d'accouplement presentent des ovaires et des conduits genitaux non developpes avec des ceufs de tres petites tailles et de teintes rosatres. Alors que les femelles matures montrant des tentatives de ponte presentent des ovaires bourres d ' oeuf de forme allongees et de teintes blanche eclatante. - 140 -

- Analyses statistiques. Les resultats obtenus ont ete soumis a une analyse de variance. La comparaison multiple des moyennes a ete realisee a !'aide du test HSD de Tuckey.

3- RESULTATS/ DISCUSSION

L'analyse des donnees climatiques (FIG. 1) de la station d'etude montre que Jes conditions les plus favorable a l'activite de la Ceratite sont remplies pendant la premiere semaine puis deviennent defavorable a la deuxieme et la troisieme semaine et commencent a se retablir pendant les deux dernieres semaines de l'essai. Les captures atteignent en moyenne 40 mouches par semaine/ piege (19,6 mouches/ prelevement) et 62% du total des mouches capturees etaient des males contre 38% de femelles.L'analyse de variance des donnees montre qu'il y' a une distribution homogene de la population dans la parcelle et l'effet du bloc etait non significatif (p>0.05). Cependant, Jes captures subissent des variations significatives( p<0.05) au fil des semaines. En effet, les captures sont fortement influencees par les variations de temperatures , d'hwnidite et de pluies. Une correlation positive existe entre Jes captures et la temperature d'une part et negative entre Jes captures et l'humidite relative minimale et la pluviometrie d'autre part (Tableau 1). Ainsi, en cas de condition climatique favorable et de niveau de population eleve, Jes captures dans les pieges sees (particulierement de DTFA2) sont statistiquement comparable a celles des pieges standards les plus performants JT et IPMT. Dans ce cas aussi, les pieges IPMT capturent mieux que les pieges Jackson (Fig.2). D'autant plus, dans chacun des pieges testes, les differences de captures entre les males et les femelles sont significatives. Hentz (1990) avait rapporte que ces deux types de pi�ges standards fonctionnent mieux lorsque le climat est chaud et sec. En revanche, quand le niveau de la population est modere, le piege Jackson reste toujours en tete et capturent significativement plus que Jes autres pieges. Mais la performance des pieges sees est significativement similaire a celle des pieges Mc Phail. Les pieges Jackson a Trimedlure capturent un pourcentage plus eleve ( 51% ) de la population totale, suivi dans l'ordre par Jes pieges IPMT (23,4 %), DTFA2 (11.6%), CC (6,9%) et DTFA4 (6,5%). Les trois versions des pieges sees a attractifs synthetiques presentent a peu pres la meme efficacite. La version DTFA2 est la plus prometteuse car son efficacite est statistiquement comparable au piege Mc Phail. Mais, Jes pieges Jackson capturent presque exclusivement que des males (99,37%) contre 0,63% de femelles et presque specifique pour la ceratite (99,5 %) contre 0,5 % pour les insectes non-cibles. Au contraire, les pieges Mc Phail et les trois pieges sees capturent aussi bien les males que les femelles de la Ceratite avec une tendance d'attirer d'avantage des femelles. Les pourcentages de femelles capturees par rapport a ceux des males sont 77%, 82%, 83,33% et 74,4% respectivement pour les pieges DTFA2, DTFA4, CC et IPMT. D'autre part, ces pieges a appat alimentaire synthetique attrapent mains si non autant d'insectes non-cibles que les pieges IPMT (Table 2): DTFA2 (11,2 %), IPMT (17,6 %), DTFA4 (23,3 %) et CC (24,5 %). La dissection des ovaires montre aussi que les 3 pieges a attractif alimentaire synthetique et IPMT capturent d'avantage de femelles immatures (Table3) mais le difference n'est pas significative (p>0,05). D'apres Heath et al. (1995) la variation des doses d'acetate d'ammoniwn et de putrescine determine le type de femelles capturees par les pieges. Les essais effectuees en parallele avec les notres dans differents pays a climat tempere tel que l'Espagne, la Grece, la Turquie ou tropical-subtropical (Guatemala, Argentine, Costa Rica, Mexico et Honduras ont tous montres que les pourcentages des captures des femelles par le piege DTFA2 sont plus eleves par rapport a ceux des males (Ros 1995; Katsoyannos et al. - 141

1995: ZUmreoglu 1995: Heath et Epsky 1995; Jeronimo et al. 1995; Vatuone 1995; Camacho 1995: Paxtian et al. 1995; Sponagel 1995). II est aussi interessant de signaler que J'efficacite de ces pieges est directement liee au niveau de Ja population qui a son tour depend des conditions climatiques ambiantes. A la lumiere de ces resultats, ii parait necessaire de citer Jes utilites des ces pieges sees a attractifs pour femelles a grande echelle dans Jes programmes de lutte contre la Ceratite au moyen de la technique d'insecte sterile (TIS). Les pieges JT, en capturant des effectifs eleves de Ja Ceratite particulierement des males contribuent a une amelioration de la TIS a condition d'etre utilise avant le declenchement du programme de Ja TIS. Cependant, Jes pieges destines a capturer Jes femelles presentent une particularite importante et plus d'avantage pour la TIS. Premierement, la reduction de la population des femelles sauvages diminuera les degats dues aux piqfires et pontes sur Jes fruits. Deuxiemement, contribuer a augmenter la probabilite de rencontre males steriles - femelles sauvages. Troisiemement, permet d'estimer le niveau de Ja population femelle fertile par rapport a celle sterile. Par ailleurs, dans Jes programmes de la TIS, Jes pieges Mc Phail, bien qu'ils capturent des proportions importantes des femelles par rapport a celle des males, ils paraissent non commode. Les resultats obtenus avec les pieges sees appates d'attractifs synthetiques a base alimentaire ouvre une meilleur perspectives devant Ieurs utilisation en conjonction avec la TIS. Les nouveaux attractifs alimentaires particulierement en combinaison avec Ja phase 2 des pieges sees presentent un potentiel d'attraction comparable a celui de la solution proteique utilisee dans les pieges Mc Phail.. Au niveau pratique, ils sont plus facilesa manipuler que les pieges Mc Phail. Les attractifs ant une longue remanence. Le cofit bas et Ja facilite de manipulation de ces pieges les rend plus pratique que Jes pieges Mc Phail. Par ailleurs, les deux versions americaines DTFA2, DTFA4 presentent la meme performance d'attraction pour la Ceratite que la version marocaine de notre propre conception (CC). Cette demiere construite uniquement a partir de bouteilles jaunes d'huile de table est la mains chere de toutes Jes autres que les agriculteurs peuvent fabriquereux meme.

REFERENCES

Beroza M., Green N., Gertler S. !., Steiner L. JF.,& Miyashita D. H. (1961). New attractants forthe Mediterranean fruit fly. J. Agric. Food Chem. 9, 361-365. Camacho V.H. (1995). Comparative evaluation of Jackson and dry traps for medfliesin support of the Sterile Insect Technique. First F AO/IAEA Research Co-Ordination Meeting. Division of Nuclear Techniques in Food and Agriculture. Co-Ordinated Research Programme on' Development of Female Medfly Attractant Systems for Trapping and Sterility Assessment'. Antigua, Guatemala, 22-26 May 1995. Epsky, N.D., R.R. Heath, A. Guzman and W.L. Meyer (1995). Visual cue and chemical cue interactions in a dry trap with food-based synthetic attractant for Ceratitis capitata and Anastrepha ludens (Diptera: Tephritidae). Environ. Entomol. 24:1387 - 1395. Heath R. B., Epsky N. D., Guzman A., Dubben B. D., Manukian A., & Meyer W. L. (1995). Development of a dry plastic insert trap with food-based synthetic attractant forthe mediterranean and mexican fruit flies (Diptera: Tephritidae). J. Econ. Entomol. 87: I 008- 1013. Heath R. & Epsky N.D. (1995). Recent improvements in adry trap with synthetic food-based attractant for female medfly attractant systems. First F AO/IAEA Research Co-Ordination Meeting. Division of Nuclear Techniques in Food and Agriculture. Co-Ordinated Research - 142 -

Programme on' Development of Female MedflyAttractant Systems for Trapping and Sterility Assessment'. Antigua, Guatemala, 22-26 May 1995. Hentz F. (1990). Standarization ofMedflytrapping for use in Sterile Insect Technique programmes. Report of a Research Co-ordination Meeting , Joint F AO/IAEA Division of Nuclear Technique in Food and Agriculture. Casablanca, Morocco, 5 - 9 November 1990. Jeronimo F., Rendon P., Villatoro C. (1995). Medfly female attractant-trapping studies. First FAO/IAEA Research Co-OrdinationMeeting. Division of Nuclear Techniques in Food an,d Agriculture. Co-Ordinated Research Programme on' Development of Female Medfly Attractant Systems forTrapping and Sterility Assessment'. Antigua, Guatemala, 22-26 May 1995. Katsoyannos B.I., Kouloussis N.A. & Papadopoulos N.T. (1995). Medfly female attractant studies in support of the Sterile Insect Technique: Experiment conducted in Chios, Greece, in! 994. First FAO/IAEA Research Co-OrdinationMeeting. Division of Nuclear Techniques in Food and Agriculture. Co-Ordinated Research Programme on' Development of Female Medfly Attractant Systems for Trapping and Sterility Assessment'. Antigua, Guatemala. 22- 26 May 1995. Paxtian J., Orozco D., Rull G., & Lie(l.oP. (1995). Evaluation of aMedly female attractant in a trapping system in the Soconusco region of Chiapas, Mexico. First F AO/IAEA Research Co-Ordination Meeting. Division of Nuclear Techniques in Food and Agriculture. Co­ Ordinated Research Programme on' Development of Female Medfly Attractant Systems for Trapping and Sterility Assessment'. Antigua, Guatemala, 22-26 May 1995. Ros J.P. (1995). Medfly female attractant studies in Spain. First FAO/IAEA Research Co­ Ordination Meeting. Division of Nuclear Techniques in Food and Agriculture. Co-Ordinated Research Programme on' Development of Female Medfly Attractant Systems forTrapping and Sterility Assessment'. Antigua, Guatemala, 22-26 May 1995. Sponagel K. (1995). Field experiments on female medfly trapping with two trap systems in Honduras. First FAO/IAEA Research Co-Ordination Meeting. Division of Nuclear Techniquesin Food and Agriculture. Co-Ordinated Research Programme on' Development of Female Medfly AttractantSystems for Trapping and Sterility Assessment'. Antigua, Guatemala, 22-26 May 1995. Vattuone E. M. (1995). New attractant trial and trap forthe capture of females of Certaines capitata Wied. First FAO/IAEA Research Co-Ordination Meeting. Division of Nuclear Techniques in Food and Agriculture. Co-Ordinated Research Programme on' Development of FemaleMedfly Attractant Systems forTrapping and Sterility Assessment'. Antigua, Guatemala, 22-26 May 1995. Ziimreoglu A. (1995). Investigation on the efficiency of various medflyfemale trapping combinations in the western partof turkey in support of the Sterile Insect Technique. First FAO/IAEA Research Co-Ordination Meeting. Division of Nuclear Techniques in Food and Agriculture. Co-Ordinated Research Programme on' Development of Female Medfly Attractant Systems for Trapping and Sterility Assessment'. Antigua, Guatemala, 22-26May 1995. - 143 -

Fig. : Correlation entre les facteurs climatiques et les captures de la ceratite dans le verger de clementine (Agadir, automne 95)

c::::::JPopula1mn T0max _.,_T'm,n _.__IIRm in ----Pluic

S J S2 S3 S4 S5 S6 S7 S8 semames

FlG.2: Evolution des captures do la �ratite pules differentspiegas testes dans le verger de Ch\mentlna. ------140 , a, 120 �• -' / j I ------1 C> ,a, I 'ii .:, 100 C a, E a, > •GI :qi... 60 a. I C: 40 C: a, >, Q :!!; ' JT ·- IPMT '--�-� ·- DTFA2 S1 __. ., Pieges - -- .. - DTFA4 S -��- 2S...... 3 54 -- cc S5 S6 - S7 S8 Semaines • 144 •

TABLEAU 1: LES COEFFICIENTS DE CORRELATION ENTRELES CAPTURES DE LA CERATITE DANS LE VERGER DE CLEMENTINE ET LES PARAMETRES CLIMATIQUES.

captures precipitation RH min Tmin Tmax

-0.59 -0,6 0.44 0.58 Captures p= P= 0, 11 p= 0, 1 4 p=0,26 P= 0, 1 2

p� probabilite

TABLEAU 2: Pourcentages des captures des insectes non cibles par les differents pieges testes dans la parcelle du Clementinier.

JT DTFA2 DTFA4 cc IPMT

Ceratites 99,5 88,8 76,7 75,5 82,4

Insectes non cibles 0,5 11,2 23,3 24,5 17,6

Totale 100 100 100 100 100

TABLEAU 3: POURCENT AGE DE FEMELLES lMMATURES ET MATURES CAPTUREES PAR LES DIFFERENTS PIEGES A ATTRACTIFSALIMENT AIRES DANS LE VERGER DE CLEMENTINE.

...... DTFA2 DTFA4 cc IPMT % Femelles immatures 64 52 55 68

% Femelles matures 36 48 45 32

Totale 100 100 100 100 - 145 -

CAPTURAS DE ADULTOS DE BACTROCERA OLEAE (GMEL.) MEDIANTE ARMADILHAS CROMOTROPICASE FERORMONICAS PARA A PREVISAO DA INFESTA(AO

C. Puce: <*>, A. F. Spanedda <*>, S. Speranza <*>, F. Lipizzi <.. >

(*) Dipartimento di Protezione delle Piante, Universita della Tuscia, Viterbo - Italy (**) Via Revoltella, 35 Roma - Italy

Resumo

Os Autores apresentam os resultados duma experiencia efectuada em 1988, 1989 e 1994 num olival situado no Alto Lazio (Italia Central). 0 ensaio incluiu a monitoragem dos adultos, a observayao da infestai;:ao e dos dados climaticos. A monitoragem dos adultos foi realizada atraves da coloca<;:ii.o de armadilhas adesivas do tipo Cromo-Trap (amarelas) e do tipo Trap-Test activadas e nao corn ferormona sexual, produzida por duas diferentes firmas. A finalidade da prova experimental foi a de testar, por todos os tipos de armadilhas e pelos dois sexos, a aplicai;:ao dum modelo de previsao da infestai;:ao, antecedentemente ensaiado em varios ambientes de cultivo das oliveiras, utilizando s6mente armadilhas cromotr6picas amarelas (Cromo­ Trap) sem ferormona sexual. Os resultados obtidos evidenciaram que: - o numero medio de femeas capturadas corn todosos tipos de armadilhas (menos que a Trap-Test sem ferormona) deu resultados satisfat6rios quanta a aplicayao do modelo de previsao da gravidade de infestai;:ao - s6mente o numero media de machos capturados mediamente corn a armadilha cromotr6pica amarela sem ferormonadeu resultados concordes corn o modelo de previsao.

Summary

CAPTURES OF BACTROCERA OLEAE (GMEL.) ADULTS BY CHROMOTROPIC AND PHEROMONES TRAPS FOR THE INFESTATION FORECAST

The Authors show the results of an experiment made in 1988, 1889 and 1994 in an olive-grove in Alto Lazio (Central Italy). The trial included the monitoring of adults, the observation on the infestation and climaticdata. The monitoring of the adults was carried out by setting of adhesive traps of Chroma-Trap (yellow) and Trap-Test type with and without female sexual pheromone, produced by two different companies. The aim of the trial was to test, forall types of traps and for the different sexes, the applicability of a forecasting model of the infestation, previously tested in various olive-grove environments using yellow Chromo-Trap without sexual pheromone. The results underlined that: - the average number of females captured by all types of traps (except Trap-Test without sexual pheromone) gave satisfactory response as regards the application of infestation forecastingmodel - only the average number of males captured by yellow Chroma-Trap without sexual pheromone gave results according to the application of infestation forecasting model.

I - lntrodm;ao

Na Italia Central a B. olae cumpre a I" gera9ao no periodo Julho-Agosto e a 2• gera9ao no periodo Setembro-Outubro (PUCCI e FORCINA, I 982). Em linha geral, a gera9ao estiva, isto e • 146 - a primeira, e de modesta entidade e nii.o provoca prejuizos que justifiquem seu controle. Isto devido a dois motivos: 1) - as drupas sii.oreceptivas so no fim da fasede endurecimento do caror;:o, que nas nossas zonas e para a cultivar objecto do presente estudo, verifica-se dentro da primeira decada do mes de Agosto (cfr. LOI et al., 1982); 2) - as ovideposir;:oes que interessam as drupas receptivas no mes de Agosto sii.o sensiveis as altas temperaturas, as quais determinam uma elevada mortalidade (PUCCI et al., 1982) 0 controle das popular;:oesde B. oleae pode ser actuado racionalmente por meio de metodologias que pressupooem a observar;:ii.o da diniimica dos voos dos adultos, utilizando diferentes tipos de armadilhas, juntamente corn o conhecimento do andamento da infestar;:ii.o detectada nas drupas. Atraves de tais elementos e possivel programar as intervenr;:oes corn tratamentos insecticidas, quer dirigidos contra os adultos quer contra os estados preimaginais contidos nos frutos. 0 requisito fundamental para a actuar;:iio das supracitadas metodologias de controlo e o estudo da relar;:ii.o entre capturas e infestar;ii.o em ligar;ii.o corn os factores bi6ticos e abi6ticos de contenr;ii.o das popular;oes (cfr. RICCI et al., 1979; RICCI e BALLATORI, 1981; PUCCI e FORCINA, 1981; ECONOMOPOULOS et al., 1982; CROVETTI et al., 1982). Mais recentemente foi afinada uma analise estatistica a fim de se achar a relar;:ii.o entre capturas e infestar;:ii.o nas zonas de cultivo das oliveiras do Alto Lazio (Italia Central). 0 estudo da relar;ii.o foi sintetizado num valor (Z) que representa a gravidade da infestar;:ii.o (PUCCI et al., 1990), cujo calculo e dado pelo seguinte modelo matematico: Z = 0,039 (Fm- 9,7)- 0,136 (Tm - 22,1) Dito modelo baseia-se no levantamento do m'.unero medio de remeas (Fm) capturadas corn armadilhas adesivas cromotr6picas de cor amarela e na temperatura media (Tm) registada na semana da captura. Na elaborar;:iio do modelo previsional estabeleceu-se tambem um nivel de controle (Z = 0, 10), alem do qua! a intensidade da infestar;:iiotera um crescimento ta! que justifica a aplicar;:ii.o de um tratamento insecticida (PUCCI e PAPARATTI, 1994). 0 tratamento eventual pode ser adulticida, se for efectuado dentro de dois dias ap6s o superamento do nivel, ou larvicida se foraplicado niiomais tarde de dez dias. Sucessivas aplicar;:oes teem demonstrado a validez do modelo tambem por cultivar e zonas de oliveiras diferentes(PUCCI e CASTORO, 1996). Emrecentes publicar;:oes VIGGIANI e MONDILLO (1996), VIGGIANI et al. (1997), afirmam que o uso das armadilhas cromotr6picas adesivas de cor amarela activadas cornferormona sexual de B. oleae regista um numero consideravel de hymen6pteros parasit6ides, determinando um grave prejuizo ecol6gico a ponto tal que os mencionados Autores niioaconselham seu uso. Com base na esposir;:ii.o anterior,as motivar;:oesdo presente trabalho foram as de: - utilizar armadilhas selectivas, como as do tipo Trap-test que sem duvida resultam ter um menor impacto sobre a entomofaunautil - verificar a aplicar;:ii.o do modelo de previsiio acima mencionado, corn base nas capturas, tanto dos machos como das remeas, observadas semanalmente nas armadilhas ensaiadas.

2 - Materiais e metodos

Na base das considerar;:oesreportadas na parte introdutiva, as provas experimentais tiveram 1ruc10 em correspondencia da fasefinal de endurecimento do cameo e se prolongaram ate o periodo da colheita dos frutospendentes. 0 olival em que se efectuou a prova abrange uma superficie de cerca 7 ha e antes do inicio do ensaio em 1988 niio tinha sido tratado quimicamente por um periodo de dez anos. 0 terreno e situado na vila de Canino (Viterbo) na zona do Alto Lazio na Italia Central e e constituido por plantas homogeneas de 40 anos de idade pertencentes a mesma cultivar para a produr;:ii.ode azeite chamada "Canino". 0 compasso e feito a quadrado 6x6 metros. 0 campo experimental foi - 147 - subdividido em 4 parcelas (repetidas); em cada parcela foram colocadas as armadilhas (tipo) a serem ensaiadas. A pesquisa articulou-se nas seguintes fases: 1. Captura das moscas adultas e contagem distinta por sexo 2. Levantamento dos valores da temperatura 3. Recolha de amostras de drupas e analise da infestacao 4. Aplicacao do modelo de previsao da gravidade da infestacao

Capturas das moscas adultas Por cada ano em que foi realizado o ensaio (1988,1989 e 1994) e em cada uma das quatro parcelas experimentais foram colocadas as seguintes armadilhas: - l Trap-test (TTl) activada cornferormona sexual produzida pela firma AGRIMONT - 1 Trap-test (TT2) activada corn ferormona sexual produzida pela firma SIAPA - 1Cromo-trap (CTl) activada cornferormona sexual produzida pela firmaAGRIMONT - l Cromo-trap (CT) sem feronnona sexual Para. alem das armadilhas acima mencionadas, em 1989 e 1994 foiincluida: - 1 Cromo-trap (CT2) activada corn ferormonasexual produzida pela firma SIAPA Por fun, em 1994 foitambern ensaiada: - l Trap-test (TT) sem ferormonasexual As armadilhas foram colocadas a uma distancia entre elas nao inferior aos 30 metros, espostas no !ado da oliveira orientado a sul e tangentes a zona mediana da copa. Com periodicidade semanal foi contado, distintamente por planta, por arrnadilha e por sexo, o numero de adultos capturados. Apos a contagem eles foram arrancados das armadilhas. As capsulas ferormonicas foram substituidas cada 30 dias.

Levantamento da temperatura Para a relevacao dos valores diarios de temperatura foi utilizado um termografo. Os valores minimos e maximos de cada dia da semana da captura foramregistados e elaborados de maneira a obter o valor da temperatura media semanal.

Recolha de amostras de drupas e analise dainfesta9iio Nas mesmas plantas em que se encontravam as armadilhas foram colhidas, sempre corn periodicidade semanal, amostras de 20 drupas/planta, na parte mediana da copa e de maneira casual. As amostras foram transferidas para o laboratorio, a fim de se averiguar o nivel de infestacao activa, distinta nas seguintes categorias: (i) - ovos e larvas de primeira idade (ii) - larvas de segunda e terceira idade (iii) - pupas (nesta categoria foram tambem incluidos puparios e galerias, enquanto abandonados pelas larvas da ultima idade)

Aplica9iiodo mode/ade previsiioda infesta9lio Por cada tipo de armadilha e pelos dois sexos, procedeu-se semanalmente ao calculo do valor Z, estreitamente relacionado corn a gravidade da infestacao, aplicando a seguinte expressao maternatica: Z = 0,039 (Fm ou Mm - 9,7)- 0,186 (Tm - 22,1) onde: Z = valor explicativo da gravidade da infestacao (inclusive! o prejuizo devido a frutos caidos) Fm = numero medio/armadilha de femeas capturadas semanalmente - 148 -

120

e �

14 outubro novembro

1989 120

julho

novembro

1994

120

.. 100 .. .. ,0 :.!! .. � 20

julho TT setembro 3 10 17 outubro novembro

Fig. 1. Comparai;iiodas capturas medias de femeas entre as diferentes armadilhas - 149-

450

400

� 350

300 E� 250

� 200 CT1 150 E� ,:: 100 50

agosto

outubro 14 novembro

1989 450

400

� 350

300 � 250 � 200 150 CT1 e 100 ,:: 50

ju\ho 26 2 9 16 outubro novembro

199

450 � 400 350 � 300 � 250 � 200

1,0 E� 100

julho 10 17 outubro novembro

Fig.2. Compara!;ao das capturas medias de machos entre as diferentes armadilhas - 150 -

100 95 o pupas + puparios e galerias abandonados 90 1988 85 DL2+L3 80 75 •ovos+ L1 70 65 60 55 ii 80 ,! .!: 45 � 40 35 30 25 20 15 10

26 15 22 29 12 19 26 10 17 24 31 14 Julho agosto setembro outubro novembro

100 95 CJpupas + pupirios e galerias abandonados 90 1989 85 DL2+ L3 80 75 1:1ovos•L1 J 70 c______0 65 .. 60 55 80 .!:j 45 � 40 35 30 25 20 15 10

21 28 11 18 25 15 21 28 12 19 26 2 9 16

Julho agosto setembro outubro novembro

100 D pupas + pup.\rlos e galerlas abandonados ..90 lllL2+L3 1994 ..80 75 l!lovos+ L1 70 65 60 55 80 ,! .!: 45 � 40 35 30 25 20 15 10 5 0 28 11 18 25 15 22 29 13 20 27 10 17 Julho agosto setembro outubro novembro

Fig. 3. Percentagem da intensidade de infestac;ao geral no cam po experimental - 151 -

Mm = numero medio/armadilha de machos capturados semanalmente Tm = temperatura media da semana da captura

3 - Resultados

Anitlise da dinamica da populai;:i'ioadulta Os graficos das Figs. I e 2 mostram a flutuav1i.o da populav1i.o adulta. Em termos de intensidade das capturas notam-se as diferenvas entre machos e remeas, entre os diferentes tipos de armadilhas e entre os tres anos de observav1i.o. Com base nos resultados podem-se fazer as seguintes observavoes de caracter geral: - a presenva mais abundante de B. oleae manifestou-se em 1988, enquanto que a densidade populacional nos anos 1989 e 1994 foi bem menor. 0 que indica que as respectivas infestav5es deverao reflectir a mesma ordem de grandeza. - as armadilhas Cromo-Trap activadas corn ferormona sexual capturaram uma quatidade maior de machos. Achamos que isso tenha sido causado pela presenva contemporanea de dois factores atrativos, a cor amarela e a ferormonasexual femenina - as armadilhas. Trap-test activadas corn ferormona sexuais atrativas para os machos, tambem capturaram remeas. Este facto verificou-se ja a partir do primeiro ano da prova e repetiu-se no segundo. Sendo un fen6meno nao esperado, em 1994 foi ensaiada a Trap-Test sem feronnona a fim de verificar se as capturas de remeas fossem casuais, isto e determinadas pelo tipo de armadilha, ou pelo contrario se existisse um certo poder atrativo exercitado pela presenva dos machos capturados. Os dados relevados orientam-nos para a segunda hip6tese.

Anitlise da intensidade de infestai;:i'io As percentagens da intensidade da infestav1i.o sao ilustrados nos graficos da Fig.3. Como era de esperar, pelo andamento das capturas, o 1988 foi o ano em que se verificou uma elevada infestav1i.o. Comevando do nivel de cerca 8-10% ja na semana de 12 a 19 de Setembro, a infestav1i.o atingiu sucessivamente os maximos valores, pr6ximos a 100%, na epoca da colheita. Em 1989 a infestav1i.o manifestou-se s6 a partir da penultima semana de Outubro e alcanvou cerca de 35% na primeira semana de Novembro. Em 1994 o inicio de um ataque evidente aos frutos verificou-sena semana de 13 a 20 de Outubro, atingindo o seu ponto maximo em meados de Novembro corn valores de infestav1i.o de cerca 28 %.

Verificai;:i'io da. aplicai;:i'io do mode lo de previsi'io da infestai;:i'io

A comparav1i.o do andamento dos valores Z teve o prop6sito de verificar, por cada tipo de armadilha e pelos dois sexos, a capacidade de determinar o alcance e o superamento do nivel de controle (Z = 0, 10), a fimde se aplicar o eventual tratamento adulticida ou larvicida. (Figs 4 e 5). Assumindo como referimento o andamento dos valores Z, calculados na base das capturas de remeas cornarmadilhas cromotr6picas amarelas sem ferormona, a analise dos resultados leva-nos as seguintes observavoes: 1) - em todos os anos do ensaio, o numero de remeas capturadas corn todos os tipos de armadilhas, fomeceu indicav5es sobre o momento de alcance do nivel de controle, perfeitamente coincidentes corn aquelas do modelo de referimento, excepv1i.o feita para a Trap-Test sem ferormonasexual cujas capturas foram quase nulas durante todo o periodo da prova; 2) - no que diz-respeito aos machos, nota-se uma certa variabilidade nos valores Z. Duma maneira geral, o sinal de superamento do nivel de controle dado pelas armadilhas ferorm6nicas e adiantado em relav1i.o ao da armadilha de referimento. Essa antecipavao porem, nao resulta constante nos • 152 • ... • ---en 1988 5,5 --n2 --n1 4,5 - - eT (referimento)

3;5 N

2,5

> 2 1,5 nivel de controle Z = 0, 10

0,5 ...... \ :i

-0,6 -1 ·1,5 22 29 12 19 26 10 17 24 31 14 agosto setembro outubro novembro

6,5 ---en 1989 6,5 -er2 --n1 4,5 4 --n2 3,5 - • er (referimento) N

I!! 2,5 �> 2 1,5 nivel de controle Z = o, 10 0,6 \ -0,5 -1

28 11 18 25 15 22 29 12 19 26 16 jutho agosto setembro outubro novembro

6,5 ---en

6,5 --e-eT2 1994 --n1

4,5 --n2 4 -<>-TT 3,5 - • eT (referimento)

I!! 2,5 �> 2 1,5 nivel de controle Z = 0, 10

0,5 ···-·--·············· ...... \. ················

-0,5 -1 ·1,6 28 11 18 25 15 22 29 13 20 27 10 17 julho itgosto setembro outubro novembro

Fig. 4. Comparac;ao dos valores Z calculados na base das capturas de femeas - 153 -

------•·• ------<------� --+--CT1 1988 ---rr1 5,5 ---rr2 5 --+-CT 4, .. - referimento 3,5

N.. 5 ! , 2

5 1,

0,5

-0,S nivel de controle Z = O, 1 O ·1 -1,5 J______, 22 29 12 19 ,. 10 17 " agosto setembro outubro 31 novembro 14

6,5------+--CT1 1989 5 5 -o-CT2 , -rr1

4,6 ---n2 --+-CT 3,6 • - re.ferimento

� 2,6 ;;

1,5

5 0,

0

-0,S nivel de controle Z = 0,10 -1,5·1 L��--�-31:;�J..------21 211 11 18 25 15 22 29 12 13 2i julho agosto setembro outubro novembro 15 5 6, ,----;======;------=�-----, • -en 5,5 -o-CT2 -n1 5 4, ---n2 --+-CT 3,5 -o-TT N.. 5 - • referimento , i 2

1,5 nivel de controle Z = 0, 10

0,6 \ -0,6

-1

5 ·1, 28-'--- 4 ------25 ------'27 julho 11agosto 18 setembro15 22 outubro13 20 novernbro3 10 17 1 Fig. 5. Compara(;ao dos valores Z calculados na base das capturas de machos - 154 -

varios anos e pelos diferentes tipos de armadilha. A unica excep9iio diz-respeito a armadilha cromotr6pica sem ferormona sexual, a qua!, em virtude da igualdade entre capturas de machos e capturas de femeas( sex ratio = 1: I) no periodo antecedente ao superamento do nivel de controle, fomeceos mesmos valores Z da correspondente captura de femeas.

4. Conclusoes

A aplica9iio do modelo matematico para a previsiio da gravidade da infesta<,:iio utilizando os dados medios de captura somente das femeastern fomecido resultados univocos niioso, como era previsto, para as armadilhas Cromo-Trap, mas tambem em rela9iio as Trap-test activadas corn ferormona sexual. Inserindo no modelo o numero medio de individuos de sexo masculino o valor do nivel de controle e atingido corn uma certa antecedencia, a qua! porem niio resulta ser constante por cada tipo de armadilha e ao longo dos anos. Considerando que as Trap-Test teem um menor impacto sobre a entomofauna util, sobretudo nos primeiros meses do veriio (Junho-Julho) e que ao mesmo tempo possuem um custo de cerca 10 vezes superior ao das Cromo-Trap, resta por avaliar a efectiva incidencia das capturas dos Hymen6pteros parasitas comparada corn a densidade de popula9iio dos mesmos, no periodo em que siioefectivamente utilizadas as armadilhas, isto e de Agosto ate meados de Outubro.

Referencias

CROVETTI A., QUAGLIA F., LOI G., ROSSI E., MALFATTI P., CHESI F., CONTI B., BELCARI A., RASPI A., PAPARATTI B. (1982) - Influenza di temperatura e urnidita sullo sviluppo degli stadi preimmaginali di Dacus o/eae (Gmel.). Frustula entomologica 5:133-166.

ECONOMOPULOS A. P., HANIOTAKIS G. E., MICHELAKIS S., TSIROPOULOS G. J., ZERVAS G. A., TSITSIPISJ. A., MANOUKAS A. G., KIRITSAKIS A., KINIGAKIS P. (1982) - Population studies on the olive fi:uit fly, Dacus oleae (Gmel.) (Dipt., Tephritidae) in western Crete. Zeit. Ang. entomol. 93(5):436-476.

LOI G., BELCARI A., MALFATTI P. (1982) - Studi sull'applicazione di metodologie statistiche computerizzate in olivicoltura. Predisposizione di un piano sperimentale per la individuazione di soglie econorniche d'intervento contro ii Dacus oleae (Gmel.). Primo esame dei dati relativi al 1980. Frustula entomologica 5 (4):223-246.

PUCCI C., CASTORO V. (1996) - Applicazione di un modello statistico di previsione della gravita dell'infestazione di Bactrocera o/eae (Gmel.) (Diptera, Tephritidae) nell'ambiente olivicolo materano: esperienze condotte nel biennio 1994-95. Giomate Fitopatologiche 1:505-512.

PUCCI C., COPPI R., PAPARATTI B., DI CIACCIO A. (1990) - Relationship between infestation of olives and capturing of Dacus oleae (Gmel.) females(Diptera: Tephritidae) by means of chromotropic traps. Atti del Convegno "IOBC Fruit Fly Open Meeting, Working Group on Fruit Flies of Economic Importance", Sassari, 26-27 novembre 1990.

PUCCI C., FORCINA A (1982) - Dinamica di popolazione degli stadi preimmaginali de! Dacus oleae (Gmel.) nelle drupe. Frustula entomologica 4 (1982):3-43. - 155 -

PUCCI C., FORCINA A., SALMISTRARO D. (1982) - Incidenza della temperatura sulla mortalita degli stadi preimmaginali, sull'impupamento all'intemo delle drupe e sull'attivita dei parassiti de! Dacus oleae (Gmel.). Frustula entomologica 4: 143-155.

PUCCI C., PAPARATTI B. (1994) - Prospettive di controllo guidato della Bactrocera oleae (Gmel.) mediante l'applicazione di un nuovo modello statistico di previsione della gravita dell'infestazione. Atti del Convegno "Lotta Biologica ed Integrata per la difesa della colture agrarie e delle piante forestali", Ferrara 24-25 ottobre 1994. 209-211.

RICCI C., BALLATORI E. (1981)-Dinamica di popolazione degli adulti di Dacus oleae (Gmel.) Frustula entomologica 4 (17):45-75.

RICCI C., FORCINA A. (1981) - Dinamica di popolazione degli stadi preimmaginali del Dacus oleae (Gmel.) nelle drupe. Frustula entomologica 4:3-43.

RICCI C., PUCCI C., BALLATORI E., FORCINA A. (1979) - Alcuni aspetti della dinamica di popolazione di adulti di Dacus o/eae (Gmel.) e analisi della relazione tra infestazione e catture con cartelle cromotropiche. Notiz. Malattie Piante 100 (3• serie, n. 26):261-282.

VIGGIANI G., JESU R., GARONNA A. P. (1997) - Catture d'Imenotteri parassitoidi con trappole innescate a feromone sessuale per ii monitoraggio di Bactrocera oleae (Gmelin) (Diptera: Tephritidae). Boll. Lab. Ent. agr. Filippo Silvestri 53(1997):123-135.

VIGGIANI, G. & MONDILLO, A. (1996) - Catture d'Imenotteri con trappole gialle innescate a feromoni sessuali per ii monitoraggio di Bactrocera oleae (Gmelin). Boll. Lab. Ent. agr. Filippo Silvestri 51 (1994):83-99. - 156 -

UTILISATION DE LA PHEROMONE DE MARQUAGE DANS LA LUTTE CONTRE LA MOUCHE DES FRUITS, CERATITIS CAP/TATA. WIED, (DIPTERA, TEPHRITIDAE)

M.H. Dhouibi (1) et H. Fellah (1)

RESUME

Dans le ca dre de l'amenagement de la Jutte contre la mouche des fruits nous arnns eu recours a un produit nature! isole des femelles de la Ceratite: la pheromone de marquage (Host marking ph<:romone). Cette substance secretee par lcs femelles apres le depot de leurs oeufs, a pour role de repousser et d'eviter eventuellement une seconde ponte; d'ou son importance pour la protection des fruits sains. Pour une meilleurc et eventuelle utilisation de cette substance, nous l'avons essaye pour la Jutte contre la Ceratite sur Pecher, Vigne, Figuier et Mandarinier. Ces essais ont rnontre quc Jes concentrations utilisees perrnettcnt d'obtenir des taux d'infestation plus foiblesque le temoin et proches de ceux obtcnus par !'insecticide de reference. Les concentrations Jes plus elcvees ( 1500 et 2000 ppm), sont Jes rneilleures.

Mots clcs: Ccratite, pheromone de marquage, concentration, taux d'infestation

INTRODUCTION

La mouche mediterranccnne des fruits, Ceralitis capita/a wicd (Diptera, Tcphritidac) reprcscnte le ravageur le plus important des fruits dans notre pays. La luttc chimiquc dcmeurc a nos jours l'am1e la plus dcployec contre ce ravagcur avcc un seul stade cible, l'adulte (Bateman, 1972), lcs ocufs et lcs larvcs exigent des produits systcmiqucs a longue remanence et trcs nocifs pour la faunc. Yana et Stancic (1967) ant mis au point pour la prcmicre foisen Tunisic une mcthode de traitcmcnt aerien a tres bas volume (U.L.V) utilisant un melange a 20% d'insecticidc et 80% de solution protcique. La methode est encore utiljsce par le G.I.A.F. Sans nier ses grands meritcs, le controle chimique de la Ceratite s'accomode mal aux principes de la protection de l'cnvironnement et de la preservation des cquilibres naturels. C'est pourquoi, la recherche de mcthodes alternatives a cette lutte chimique s'impose d'autant p.lus qu'on a procede ces demieres annees a l'introduction,.dequelques entomophages dans les vergers d'agrumcs pour le controlc biologique de ccrtains ravageurs. C'est ainsi que fut introduit Cales noaki en 1991 pour le controle de I' A!eurodc floconneux Ale11rothri.ms jlocosus Mask. En 1992, on a introduit Eretmocerus debachi Rose et Rosen, pour le controle de Parabemisia m1,ricae (Dhouibi, 1992; Cherrniti et orullon,1992). Par ailleurs, le cortege parasitaire autochtone des cochenilles et des pucerons est assez riche (Boukhriss, 1994) et merite un peu plus d'attention; sans compter que certains fruits hotes n'ont qu'une courte periode de maturite (les figues par exemple) et ne peuvent tolerer des applications d'insecticides classiques sans risque de laisser des residus. Dans le cadre de cette recherchc de methodes alternatives a la lutte chimique, des etudes sur le comportement de ponte de la Ccratite ant permis de mettre en evidence une pheromone de marquage secretee au nivcau du tube digcstif et deposec par la femelle sur le fruitjuste apres la pontc, afind'ecarter

Laboratoire d'entomologic. Departemcnt de biologic appliquee. Institut National Agronomique de Tunisie - 157 -

lcs autrcs fcincllcs il la rccherchc d'un site de pontc, cc qui pcm,et d'cviter la surcharge d'un mcme fruit (Prokopy, !981) Ccttc pheromone une fois isolce, identifice et synthetisce pcut ctrc pulvcrisee sur Jes fruits hotes, et constitucr unc importante alternative de Jutte contrc lcs Tephritidae (Prokopy, 1972, 1976; Katsoyannos et Boller, 1976, 1980). La methode utilisant cette pheromone differcdes autres methodes non chimiques et de la technique des males sterilcs. Le produit agit ici commc une allomone vis it vis du ravageur, le rcpousscrait et peuvent etre utilise de la meme fa�on que Jes produits conventionnels et par Jes memes moyens, sur de grandes superficies (Boller, 1981). A cet effet, nous avons teste cette substance de marquage sur differentes cultures susceptiblcs d'etre attaquees par la Ceratite dans le soucis d'epargner la faune utile et d'eviter le probleme de residu dans Jes fruits. II convient alors de detcmiiner la dose optimale d'utilisation, c'cst ii dire !'equivalent de la quantite sceretee par la feriidleapres le depot des oeufs.

MATERIELET METHOD ES

L'expcrience a consiste it comparer l'effet de !'application de differentes doses de la pheromone de marquage sur differences cultures attaquees par la Ceratite, situces dans trois biotopes similaircs ii vocation arboricole, (Momag, Sidi .Thabet et Sidi Fredj). Des pieges Rebell ii glu, appatcs il la Trimedlure, ont ete implantcs dans lcs diffcrentssites d'etude pour le controle du niveau de la population.

Pheromone de marquage

Caracteristiques du produit La solution phcromonale a etc extraitc it partir de feccs de l::i. Ccratite originaire de la region mediterraneenne et conservcc dans le methanol au laboratoire de \Vadcnswill, Suisse. la substance a ete tcstee au laboratoire pour son aetivite vis ii vis de mouches des fruits provcnant de diffcrentes regions du monde. La solution mere (50 mg d'cxtrait de McOH aqueux par ml de McOH) a etc testee dans des essais aux laboratoires (souche de Kenya). La pheromone n'etant pas stable dans l'cau, lcs solutions it utiliscr ne doivent etre preparees qu'au moment de l'cmploi. Doses uli Ii sees A partir de la solution mere de 50 ppm c'est a dire 50 mg d'cxtrait par ml de methanol, nous avons l]tilise !ors du: lcr essai sur Pecher a Momag; CJ = l 00 ppm !'equivalent de 0, 1 mg d'extrait / ml de methanol. C2 500 ppm !'equivalent de 0,5 mg d'extrait / ml de methanol. = i C3 = 1000 ppm !'equivalent de l mg d'c:-..-trait/ ml dt methanol. cme 2 essai sur vigne a Sidi Thabet, CJ = 500 ppm !'equivalent de 0,5 mg d'extrait / ml de methanol. C2 = l OOO ppm !'equivalent de 1 mg d'extrait / ml de methanol. C3 = 1500 ppm !'equivalent de 1,5 mg d'cxtrait / ml de methanol. 3 eme cssai sur mandarinier a Momag, C 1 = 1 OOO ppm !'equivalent 1 mg d'e:-..-trait/ml de methanol C2 = 1500 ppm !'equivalent de 1,5 mg d'extrait / ml de methanol. C3 = 2000 ppm !'equivalent de 2 mg d'extrait / ml de methanol. - 158 -

4cme essai sur figuicr a Sidi Frcdj, C 1 = 1 OOO ppm !'equivalent I mg d'e:-.irait/mlde methanol C2 = 1500 ppm !'equivalent de 1,5 mg d'extrait I ml de methanol. C3 = 2000 ppm !'equivalent de 2 mg d'e�irait I ml de methanol. Les applications de la solution pheromonale ont ete faites a !'aide de pulverisateurs a main d'un demi litre de contenance et n'ont concerne que les fruits. Les differentes concentrations ont ete preparees au moment de l'emploi.

Insecticide de reference Les pyrethrino1dcs de synthcse sent consideres pamli les produits selectifs, leur efficacite vis a vis de la mouche des fruits � ete prouvee. C'est pourquoi avons nous choisi un pyrethrinoide, la deltamethrine a la concentration de 2,5 gran1mes de matiere active par hectolitre conune insecticide de reference pour cct essai. Pour la commodite de !'application, ii a cte recommande d'additionner un agent mouillant !ors du traitemcnt. A cet effet, nous avons utilise un detergent a raison de 0, l % pour tous lcs traitements Protoco/es experimentaux Essai de pecher Cet cssai a cte cffectue dans la region de Mornag sur Pecher de la variete "JHL", iigce de 8 ans avec une densite de plantationde 500 pieds/ha et en conduite libre. L'essai a porte sur 15 pieds rcpartis en 5 blocs, chaque bloc est destine a recevoir 5 traitements;. C [, C2, C3 (diffcrentes concentrations de la pheromone), C4 (insecticide de reference) et C5 (Tcmoin). Chaque concentration est appliquee sur unc moitie de l'arbre. Le traitcment a ete realise le 12 Aout 1994 aprcs avoir ramassc tous les fruits attaqucs c'est a dire ramcne le taux d'infcstationde 4,22% a zero pourcent. On a precede au comptage du nombre total de fruits dans chaque bloc. Le controle consiste ii ramasser les fruits attaques a l'interieur de chaque traitement et ce tous lcs 3 a 4 jours, le rapport du nombrc de fruits·attaquesa l'interieur du bloc par le nombre total de fruits donne le nouveau taux d'infestation. Essai sur Vigne Cet essai a ete realise dans la region de Sidi Thabet sur 20 ceps de vigne de la varictc Rich BaBa Sam, ages de 6 ans et conduits en pergola. Dans cet essai, 4 blocs renfennant chacun 5 pieds ont cte corn;us, ou chaque pied est destine a recevoir un seul traitemcnt. Ces traitements· sont C i: 500 ppm, C2:· WOO ppm et C3: 1500 ppm (differentes concentrations de la pheromone de marquage), C4 (insecticide de reference) et C5: (Temoin), le traitement a ete realise le 16 Septembrc 1994 quand le taux d'infestation etait de 6,12%. Nous avons procede a !'elimination de toutes les baies infcstees puis au comptage de la totalitc des baies pour chaque traitement Le contr6le tous les 3 a 4 jours se fait par le comptage des nouvelles baies attaquces. Essai sur Mandarinier Cct essai a ete realise dans la region de Momag sur 25 pieds de mandarinier de la variete "Wilkins" ages de 15 ·ans et en conduite lib re. Nous avons administre 5 types de traitement pour chaque bloc de cinq pieds, trois concentrations de pheromone (C [: 1000 ppm, C2: 1500 ppm et C3: 2000 ppm), une concentration d'insccticide: C.i et un Temoin: C5. - 159 -

L'installation de l'cssai a ctc faitc le 30 Decembre 1994 lorsquc le taux d'infcst:i.tion ct1it de 3,32'%. Les controles hebdomadaires consistaient it prclever un echantillon au hasard; pour chaque type de traitemcnt !'equivalent de 150 a 200 fruits. Essai sur Figuier Cet essai a ete realise dans la SMVDA d'El Khir sur une parcelle de 9 ha de figuicrs noirs. Les traitements ont touche 50 picds representant I O blocs et cc pour avoir le maximum de fruits milrs par echantillon. Les traitements sent formcs par trois concentrations de pheromone de marquage C I: I OOO ppm, C2: 1500 ppm et C3: 2000 ppm, une concentration de Deltametrine, C4 et le Temoin, C5: ils ont ete realises le 8 Aoilt 1995 et les controles sont hebdomadaires. L'infestation initiate etait de 0,68%. Evaluation des resultats Les echantillons ont ete collecter aussi bien sur les pieds traites que sur ceux 11011 traites pour le calcul du taux d'infestation. Le dcgrc d'efficacite du traitement, c'est a dire le pourccntagc de protection apporte par la pheromone de marquage ou !'insecticide de reference, est calcule a partir de la fomrnle d'Abott.

% d'cfficacite du traitement = (I _Taux d'infestation du traitcment ) X 100 Taux d'infestation du Tcmoin

RESULTATS ET DISCUSSION

Les rcsultats obtcnus a partir de ect cssai de la pheromone de rnarquage sur lcs difforcntcs plantcs hotes sont consigncs dans lcs tableaux ci-dessous. !Is reprcsentent !'evolution de !'infestation sur Peche (tableau. I), sur raisin (tableau 2), sur Mandarinier (tableau 3) et sur Figue (tableau 4). La prcmiere remarque qu'on peut faire est que !'evolution de !'infestation sur les diffcrcntcs cultures montrent que la pheromone de marquage a diffcrentes concentrations contribue a attenuer le taux d'infcstation.

Tableau 1 Evolution de !'infestation en fonction des traitements sur pccher J.h.l. Date 16/08/1994 19/08/1994 23/08/1994 Traitement nbrc fruits Fruits Fruits Fruits Total examines attaques attagues attaqucs Temoin 607 14 12 25 51 C1=100 ppm 505 10 8 12 30 C2=500 ppm 588 2 14 20 36 C3= 1000 ppm 436 5 4 13 22 Insecticide 370 9 11 16 36 - 160 -

Tableau 2 Evolution de !'infestation en fonction des traitements Sur Vigne Rich baba sam. Date 19/09/ l 994 23/09/ l 994 26/09/ l 994 Traitcment nbre fruits Fruits Fruits Fruits Total examines attagues attagucs attagues Tcrnoin 1338 52 31 9 92 C2=500 ppm 1539 54 36 10 100 C3= 1000 ppm 1469 48 44 9 101 C4= 1500 ppm 1758 68 32 12 112 Insecticide 1839 64 57 21 142

Tableau 3 Evolution de !'infestation en fonction des traitements sur Mandarinier de la varicte Wilkins. Date 6/12/1995 13/12/1995 20/12/1995 Trnitcment Fruits Fruits Fruits Fruits Fruits Fruits cxaminCs attagucs examines, attagucs examines atta� Ternoin 152 6 101 5 102 6 C3=1000 ppm 102 4 90 4 123 7 C-l= 1500 ppm 98 4 95 4 95 5 CS= 2000 ppm 112 4 115 5 112 6 Insecticide 89' 3 104 4 96 5

Tableau 4 Evolution de ['infestation en fonction des traitemcnts sur Figuicr. Date 15/08/1995 23/08/1995 30/08/1995 Traitcment Fruits Fruits Fruits Fruits Fruits Fruits examines attagucs examines attaqucs examines attagucs C3= 1000 ppm 124 2 125 6 84 6 C4= 1500 ppm 134 2 112 4 102 4 CS= 2000 ppm 143 "J 124 4 95 6 Tcmoin 102 2 148 6 105 7 Insccti cide 140 3 107 6 105 10

Aussi bien pour la premiere mcthode que pour la seconde, l'cchantillonage sc fait ;;n fonction du degrc d'infestation. Si pour lcs deux premiers essais, la collecte n'a conceme que Jes fmits attaques sur la totalite de ceux traites, pour les essais sur M;u1darinier et sur Figuier, nous avons pris des cchantillons au hasard pour !'estimation du niveau de l'attaque. Ces niveaux d'infestation seront lcs paran1i:tres de comparaison entre lcs differentstraitements. Pour Jes differents cssais on obtient, des taux d'infestations sur le temoin de 9, 72 pour le Pecher; 7,71 pour la Vigne; 5,88 pour le Mandarinier et 9,52% pour le Figuier. Des taux toujours superieurs aux autres taux obtenus pour les trois concentrations de la pheromone (100 ppm, 500 ppm. !OOO ppm, 1500 ppm et 2000 ppm) et pour l'msecticide (0,25% de M.A.deltanietrine) 11 faut remarquer aussi que !'evolution des infestations n'est pas correlce linc':iirement avec la concentration utilisce de la pheromone de marquage. Averill et Prokopy ( 1987) pe11se11t qu.: le taux de repulsion des hates traitcs diminue suite a !'exposition repctcc de la fcmclle au produit et aussi en consequence de la privation prolongec d'hotcs favorables a la ponte. - 161 -

En effct, on observe sur la varictc ''Jh.l" de pccher 6, 12 % de fruits attaqucs pour une concentration en pheromone de 1000 ppm; alors quc cc pourccntage est de 8,38% pour la concentration de 100 ppm. Sur la varictc "Rich baba sam" de la vignc, un taux de 6,49% a etc cnrcgistre suite a !'application de 1000 ppm de pheromone de marquagc. Cc taux s'cst elcve a 8,87% lorsque la concentration est passee a 1500 ppm. Un taux plus foible de 5,33% a cte obtenu sur Jes mandarines Wilkins lorsqu'on a utilise une concentration de 2000 ppm contre 5,61 % realise avec seulement I OOO ppm de pheromone. Cette meme concentration sur lcs figuesdonne un pourccntage de 7,04% contre seulement 6,35% de fruits attaques avee une concentration de 2000 ppm. Il apparait done, a premiere vue, que les traitements a la pheromont de marquagc, produit nature! utilise a differentes concentrations; pem1ettent d'obtenir des taux d'infestation inferieurs it cdtii du temoin et superieurs a celui obtenu par le traitcment insecticide. D'autre part, une amelioration des rcsultats a ctc enregistree pour lcs traitcrnents a fortes concentrations. Les resultats par especc seront rcpris et an:1lyscs dans ce qui suit.

Essai sur Pecher: Les traitcments sur pccher cffectues le 12 Aout 1994 sur 15 pieds ont pcrmis d'obtcnir les rcsultats suivants (voir fig28a et 28b ). On peut rcmarquer que !'evolution de !'infestation est correlee a !'evolution des captures de males. Le taux d'infcstation du depart ctant nu!, le controle du 16 Aout 1994 dorme des t:1ux d'infcstations foibles pour Jes concentrations de 100 ppm et de 500 ppm; 2,3% et 1,98% rcspectivcment alors que le tcrnoin prescnte 2,43% de fruits infcstcs. La concentration la plus forte C3=1000 ppm pcrmct l'obtcntion de 0,34% contrc I, 14% pour !'insecticide de reference. Ces taux d'infestation sont obtcnus par l'cxarncn de !'ensemble des fruits traitcs au scin de ch:1quc type de traitement. r-.1:ilcsipiCgciscmnine

� C:ipturcssur pCchcr

3

2

12/8/94 16/8 19/8 23/8 Date Figure 1 a : Evolution de la capture des males durant la periode d'expcrimentation sur Peches a Momag - 162 -

"O infc�1.,1ion 10 -.------_ ,.,c-,-----r / // � C1=100ppm .,,,.,.,,... . --+-- C2=500ppm ,, /' / -

16!8 Figure lb : Evolution de !'infestation des pcches "fH.L" scion lcs traite111e)1ts i Moriiag, 1994.

A partir du second controlc, lcs differences sont plus ncttes. On obticm des tau:-; de 4,27; 3,56 et 3,72% pour les trois concentrations Cl= 100 ppm; C2= 500 ppm et C3= !OOO ppm, c011trc 2,05 % pour !'insecticide. Le temoin prcsente 5,40% de fruits attaqucs. Ceci s'expliqucrait par l':u1grn,·11tation du niveau de la population adultc de la mouche des fruits au cours de cctte periode et dont b 111oycnne de c:ipture passe de 2,2 individus par pii:geet par scmaine ii 3,33 individus par picge et par s.:rnainc. Le troisicme controlc du 23 Aotit nous a permis de confirmcr l'import:rncc de cette nouvdlc substance puisquc lcs taux d'infcs:ation des trois concentrations 8,38; 5,94 et 6,12% pour Cl= 100 ppm; C2= 500; C3= 1000 ppm, sont infcricurs it cclui du temoin 9,72%. L'insecticidc rcstc de loin le plus efficacc avcc 5,04% de fruitsattaqucs (figure. lb). Le traitcmcnt statistique, prcnant en comptc lcs diffcrcnts controlcs commc d:1nt des repetitions, pcm1et de classer Jes concentrations scion !cur efficacite; on rcmarque alors quc !'insecticide est le meillcur traitement avcc unc difference significative avec la concentration C3= I 00 ppm· puis vicnt la C2=500 ppm et enfin la Cl=IOO ppm. Enfo1, le temoin sc situc en dcmier lieu avec le t:iux d'infcstation le plus elevc par rapport aux.autres traitemcnts �t significativcment diffcrcnts d'cux. Essai sur la vigne Les traitemcnts ont etc cffcctucs le 16 Scptcmbre 1994 :'i Sidi Thabct. L,;;s l:lllx d'infcstatio11 obtcnus lors du premier controlc, c'cst a dire le 19 Septcmhrc 1994, sont trcs prochcs lcs uns des autrcs. L'effet des traitcments n'a pas -pu etrc degage !ors de ce premier controlc. Cc n'cst qm: !ors du second c_ontrole, realise le 23 Septcmbrc 1994 qu'on a rclcve un taux d'infestation du tcllloin de 7,56'Yo corrcspondant au taux le plus cleve. Par centre, !'insecticide a donnc le taux le plus faihlc soit 6,37% de fruits infestes. Les taux correspondants aux trois concentrations de la pheromone (C2=:i00 ppm; C3= I OOO ppm et C4=1500 ppm) se situcnt cntrc les dCU)'.(respectivement 6.87: 6,49 et 6,87%). - 163-

f,.-1;'ilc!'.;'piCgc.'scmoinc 70

60

lO

40

30

20

JO I 2/3i94 16i8 19,8 Date Figure 2a: Evolution de la capture des miilcsclurant la pcriodc d'expcrimcntation ,ur vignc a Sidi Thabct. %infostation

-1:1-- C4•1500

-4-··- insecticide

� temoin

o-+------1.'f------r------,c------..,....---d:11,.'--1- 1619 I 9i9 2319 :?6,9 Figure 2b. :.Evolution de !'infestation du raisins scion lcs traitcmcnts a Sidi Th abet 1994.

Nous rcmarquons clans cc cas quc !'evolution de !'infestation suit ccllc des c:1pturcs des miilcs (figure 2a). La chute du nive:rn de la population adulte se traduit par unc diminution ck la pcntc de la courbe de !'evolution du taux d'infestation. Les traitcments statistiques ainsi que la comparaison par le test multiple du Duncan nc pem1cttcnt de deceler aucune difference significativc cntrc Jes differents traitemcnts. Ccci est en partie du a ['architecture complcxe des grappes de raisin recevant les produits de traitcment ainsi qu'c'l !cur repartition sur les ceps. En effct, la grappe a une forme tclle que seule une partic est cxposcc a !'application des traitements. Done, la Ceratite peut se develo"pper sur Jes baies ou Jes parties des baics n':want pas re�u la pheromone et donner ainsi des taux d'infestation proches du temoin. Il faudrait essayer de trouver une methode d'application de la pheromone !ors des traitements pour pouvoir realiser des rcsultats interessa.nts. De plus, ii fauttenir compte de l'effctde la particularitc du microclimat que fournitJa conduite en pergola. Mais, malgre ces contraintes et bien qu'ils nc soicnt pas significativement diffcrcnts, les resultats obtenus sur vigne sont en faye1:r des traitcment a la pheromone. Essai s11r JIIandarinier Cct essai a etc mis en place le 30 Dccembrc I 994, Jorsquc le taux d'infcstation ctait de 3,32%. II faut signaler que contrairement au deux cssais precedents, cet cssai -:st conduit cbns u11 climat froid et humide. - 164 -

Le premier cont role du 6 Janvier 1995, pcrmct de distingucr le 111aintic11t du faux d'i11fcstation· par !'insecticide, (3,37%), ainsi que l'cffct tri:s positif de la plus forte concentration C5 =20il0 ppm qui nous pcrmct d'obtenir 3,57% de fruits attaqucs. Le temoin et la concentration C.+ 011t donnc ,rcs taux similaircs respectivement 3,94% et 3,92% (figure 3b). Millos/piE!gelsemaine ,O -0--- C':11)tun.:s!-tll" m:1111!,1,ini..:i-

20

iO

30/12 6001 13,01 20 n I Figure 3a : Evolution des captures des miilcsdurant la pcriode cl'cxpcrimcntation sur mandarinier a Momag

% infcsliltion

3()112 6/01 �;1.111 Figure.3b : Evolution de !'infestation des mandarines scion Jes traitc111cnts sur :\'Llnclarinier.

Nous remarquons !ors du premier contriilcque pour la concentration ck 1500 pp111 nous obtcnons k taux d'infcstation le plus clcvc ceci scrait du i un-maavais fohantillo1mage !ors Ju .n,111r6Jc Les taux o�tenus !ors du second contriileconfinnc la cause accidentelle de cc rcsultat. Urn.: scrnainc apri:s, le 13 Janvier 1995, l'effct des trois concentrations est plus net (6, 19: 5,83 et 6,25% pour C I= 1 OOO ppm; C2=1500 ppm et C3=2000 ppm), contre 6,71% de fruits attaques pour l'insccticick t.:t 7,71% pour le temoin. De meme que pour le troisieme contriile du 20 Janvier, la concentration de I OOO ppm permet le taux le plus proche du taux procure par le teriloin. Alors que les concentrations C2 = 1500 ppm et C3=2000 ppm, realisent des taux qui se rapprochent plus de celui de !'insecticide (respectivem.:nt 5.37% et 5,33% contre 5,20%). Le traitement statistique et le test multiple du Duncan pem1ettent de confinlll:r !'existence de difference significative d'une part, entre les traitcmcnts a la pheromone de marqu��c t.:t le tcmoin, et d'autrc part, entre Jes traitemcnts a la pheromone et le traitement insecticide. En ce qui concemc lcs trois concentrations, elles sont elassccs par ordn:: dccroissant des concentrations c'est a dire rcspcctivcment C5=2000 ppm; C4=!500 ppm: C3= I OOO ppm presentant cntrc eux des differences significativc. - 165 -

Essai .mr lejiguier L'essai sur figucs a ctc cffcctuc au cours de l'cte 1995, lcs traitcmcnts sont cffcctucsle 8 A0tit, date ou le taux d'infcstation est de 0,68%. La figure 4b montre quc lcs figues prcsentent des taux d'infestation analogues pour Jes differents traitements apri:s une semaine de !cur application (voir tableau 4). Alors qu'au cours du second controle du 22 Aout 1995, Jes trois concentrations de la pheromone et de !'insecticide sc detachent du temoin (respectivement 4,8; 3,57 et 3,00% pour Cl; C2 et C3, 1,96 et 2,01%). La conccntr:ition C3 =2000 ppm realise meme un taux d'infestation inferieur a !'insecticide et qui se maintient legerement infcrieur ace dernier apres 3 semaines d'essais c'est a dire jusqu'au 29 Aout 1995. �f!les.'piCgcr,cmainc 10 � caprurn ,ur figuicr

1 dates 810St95 1 �,os :::::: 08 '.:9,()8 Figure 4a: Evolution des captures des miles durant la periode d'experimcntation sur figuier.a Sidi Frcj. •tu1f�t.1oon 10 CJ:a:1000;,pm

C4=iSC0ppm

C5=200Jppm

lnsect1c1do

remain

1S08 22/08 Figure 4b: Evolution de !'infestation des figues selon Jes traitements .

Les figues etant des fruits sensibles aux produits insecticides et vu Ieur periode de maturation trcs rapide nous constatons ici !'importance de \'utilisation de la pheromone de marquage pour ces fruits. Le test multiple de Duncan a pcrmis de montrer que pour Ies figues, les traitcments a \'insecticide et a Ja pheromone de marquage aux concentrations C4 =1500 et C5 =2000 ppm reaJisent Jes memes performances sans aucune difference significative et qu'elles sont hautement significatives par rapport au temoin.

Evaluation des resultats A partir de ce qui a precede, nous avons pu montrer !'importance des traitements a Ja pheromone de marquage par rapport au temoin et a !'insecticide. L'evaluation de l'cfficacitedes traitements par la formule d'Abott perrnetd'obtcnir le tableau 5. - 166 -

Tableau 5 Efficacitc des traitements scion la formule d'Abbott. Traitcments Pecher vigne Wilkins Fi!(Uicr C1= 100 ppm 13,78% C2= 500 ppm 38,88% 10,89% C3=1000 ppm 37,03% 15,82% -1,59% 26,05% C-1= 1500 ppm 10,89% 8,67% 27,9-1% Cs= 2000 ppm 9,35% 33,29% Ins= 2.5 g Ma/hi 48,1-1% 17.38% 11.56% 30.46%

Nous remarquons que les concentrations elevees de pheromon�s peuvent donner des niveaux d'efficacitetres proches de celui de !'insecticide. Les concentrations C2 et C3 permettent des efficacites respectives de 38,88% et 37,03 % contre 48, I 4% pour !'insecticide sur pecher, alors que sur figue l'efficacite de la concentration C5=2000 ppm est de 33,29% contre 30,46% uniquement pour !'insecticide. Alors que sur les deux autres cultures, l'efficacite des meilleures concentrations est rcspectivement de 15,82% pour la C3=IOO ppm pour la vigne et de 9,35% pour la C5=2000 ppm pour le Mandarinier.

CONCLUSION

Les resultats obtenus dans les precedents essais nous pem1ettent d'insister sur !'importance de la place que doit occuper dans l'avcnir la pheromone de marquage dans lcs programmes de lutte contre la mouche mediterrancene des fruits, puisque le traitcmcnt des fruits par cette substance lcur procure une protection appreciable. Ccpendant !'utilisation de cctte pheromone restc encore imprevisible et irrcproductible d'un hotc ;i._ l'autre et d'une localite a l'autrc (Boller, 1994).0n ma1trise mal !'application qui reste encore difficilc it ·· realiser ce qui se rcpercute sur les resultats obtenus qui sont souvcnt alcatoires. Aluja et Boller ( 1992a. I 992b) pensent qu'il est preferable de ne pas traiter le feuillagc en vue d'une meillcure rcponsc des mouches, ainsi qu'une rapide reaction au contact de la pheromone de marquage. Les differences peuvent cgalemcnt etre dues aux niveaux des populations adultes. Averill et �rokopy ( 1989) montrcnt que le taux de repulsion des hotes traites a la pheromone de marq.uage diminuc en consequence a la privation prolongce d'hotes favorablcs a la ponte. En cc qui conceme la remanence, le produit reste efficace pendant 10 a 15 jours au cours de la saison chaudc sur fruits d'eteetjusqu'a 20jours en hiver sur mandarinier.

BIBLIOGRAPHIE

ALUJA M., AND BOLLER E.F., 1992a - Host marking pheromone of Rhagoletis cerasi: Foraging behaYior in response to synthetic pheromonal isomers. J. Chem Ecol.18: 1299-1311. ALUJA M., AND BOLLER E.F., 1992b - Host marking pheromone of Rhagoletis cerasi: Field deployment of synthetic pheromone as a novel cherry frnit fly management strategy. Entomol E:xp. Appl. 65: 1-11-1-17. AVERILL A. L., AND PROKOPY R.J., 1987 - Residual actiYity of OYiposition-dcterring pheromone in Rhagolctis pomonclla fliesand female response to infested frnit. Journal of Chemical Ecology, 13 : 167 - 177. - 167 -

AVERILL A.L., AND PROKOPY R.J.. 1989 - Host marking pheromones. Clwptcr 3.5, In: A.S.Robinson & G.H.S.Hoopcr (Eds.). Fruit Flies: Their Biology, Natural Enemies and Control Vo! 3A: 207-219. Elsevier Press, Amsterdam. BATEMAN M.A., 1972 - The ecology of fruit flies. An.nualReview of Entomology, 17 : .\93-513. BOLLER E.F., 1931 - Oviposition-detcrring pheromone of the European cherry fruit fly: status of rcseach and potentiel application. In: E.R.Mitchell (Editor), Management of Insect Pest With Semiochemicals. Plenum Press, New York, pp: .\57-462. BOLLER E.F. 199-l - Host marking pheromone in fruit flies: state of the art. In J.Piedade-Guerreiro (editors). Fruit flies of economic importance. InternationalOpen Meeting. Lisbon, Portugal 14-16 October 1993. Bulletin OILB/SROP Vol.17 (6) 1994.

BOUKHRISS.S., 199-l - Etude du complexe aphidien present dans un verge d'agrumes.' Memoire de fin d'etudes du cycle de specialisation INAT. CHERMITI.B., ET ONILLON C., 1992 - A propos de la presence en Tunisie de deux nouvclles especes d'aleurodes nuisibles aux Agrumes.4 /eurothrixus jloccosus Maskell et Parabemisia 111yricae (Kuwana) (Homoptera, Aleurodidae). Fruits, Vol -17 (3), .\05--lll. DHOUIBI M.H., 1992 --Efficacite-decertains produits chimiques a l'egard des mouches blanches: Aleurothrixus jlocossus Mask. et Parabemisia myricae Knw. (Homoptcres ,Aleurodidae) et leur impact sur lcs auxiliares Cales noacki Howard et Encarcia transvena Timberlake (Hymenoptera:Aphclinidae). Mcd. Fac.Landbouww, Rijksuniv. Gent. 57/2b, .\93-503. GAHBICHE H., 1993 - Contribution :i l'Etude de la Bioccologie de la mouchc mediterrancennc des fruits Ceratitis capita/a Wiedemann 1829 (Diptcra : Tcphritidae) dans dcux biotopcs du Nord de la Tunisie. Memoire de fin d'etudes du cycle de specialisation de !'INAT. KATSOYANNOS B.l., AND BOLLER E.F.. 1976 - First field application of oviposition-deterring marking pheromone of Europc:m cherry fruit fly. Enviromcntal Entomology. 5: 151-152. KATSOY ANNOS B.!., AND BOLLER E.F., 1980 - Second field of the oviposition-deterring pheromone of the European cherry fruit fly, Rhagaletis cerasi L. (Diptcra : Tephritidac). Zeitschrift Fiir Angewandtc Entomologie. 89: 273-281. PROKOPY R.J., 1972 - Evidence for a marking pheromone deterring repeated oviposition in apple maggot flics:­ Environmcntal entomology, 1 :326-332. PROKOPY R.J., 1976 - Significance of fly marking of oviposition site (in Tephritidae) In:V.L. Delucchi (Ed). Studies in Biological Control. Cambridge University Press, Cambridge, pp. 23-27. PROKOPY R.J., 193 l - Oviposition-dcterring pheromone system of apple maggot flies. In. E.K. Mitchell (Ed), Management of Insect Pests with Semiochemicals. Plenum Press, New York, pp. -177--19-l. YANA A., ET STANCIC J., 1967 - Esais de lutte chimiquc contre Ceratitis capita/a wied en Tunisie. Rapport d'activites de !'!NRAT • 168 •

EFICACIA DE DOS INSECTI[:IDASEN APLICATION AEREA CONTRA LA MOSCA DEL MEDITERRANEO (CERATITISCAP/TATA (WIED.)EN CITRICOS. ALICANTE (ESPANA)

Llorens Clirnent Jose M. G ilabertA.rtigues J. Servicio de Sanidad y Certificaci6n Vegetal de la Conselleria de Agricultura. Pesca y Alirnentaci6n de La CornunidadValenciana. Profesor Manuel Sala 2. 03003 Alicante (Espana)

RESUMEN. Se plantea un ensayo para conocer la eficacia y persistencia de rnalation 50 en aplicaci6n cebo con proteina hidrolizada en tratarniento aereo frente a fention 50 que se utiliza durante rnas de 30 anos en los tratarnientos oficiales en la CornunidadValenciana.

Los resultados de la experiencia fueron cornparables. per lo que se decidi6 el carnbiode rnateriaactiva insecticida.

Se sigue la evoluci6n de capturas de aduttos a lo largo del tiernpo que dur6 la carnpana. en zonas donde se realiz6 tratarniento aereo y en zonas donde solo se realiz6 tratarnientocebo terrestre. observandose grandes diferencias en las capturas.

PALABRAS CL.A.VE. Ceratitis c.apitata fention. malation. aplicaci6n aerea. aplicaci6n cebo.

INTRODUCCION.

La rnosca de la fruta fue declarada plaga de especial incidencia agrfcola per Orden del Ministerio de Agricultura. el 26 de Junie de 1955.

Desde 1966. en la Comunidad Valenciana. se realizan. de forma continuada. tratamientos cebo en aplicaci6n aerea. para disminuir la poblaci6n. en casi toda el area citrfcola.

La zona citricolase dividi6 en polfgonos y retfculas.

Un poligono lo forma un area mas o menos uniforme y esta compuesto per retfculas.

Cada retfculaes un rectangulo que abarca una superficie de 1 km. de lado per la longitud del polfgono.

En cada reticula se colocan de una a tres baterlas de mosqueros. dependiendo de su longitud.

Cada bateria la componen tres mosqueros de cristal. tipo McFail. cargados con una disoluci6n de fosfato biam6nico al 4%. Yun mosquero de plastico tipo Nadel. con una capsula de trimedlure.

El numero de moscas capturadas en cada bateria. se cuenta una o dos veces per sernana. dependiendo del estado del cultivo. - 169 -

El indice de una rnosca por mosquero y dia sirve de referencia para iniciar los tratarnientos. es decir, dar la orden de vuelo a las aeronaves para que realicen la aplicaci6n cebo en un area deterrninada.

Desde el inicio de los tratarnientos. la aplicaci6n insecticida se rea.liza con una mezcla de fention 50 LE al 1.5% y proteina hidrolizada a 1.2 kg. %. aplicado a gota gruesa, con un gasto de 20 I/hay una superiicie tratada del 40% lo que equivale a un gasto real de 81/ha.

OBJETl\/0.

Al iniciar la exportaci6n de frutos cit1'icos a mercados como el de EE U U donde el Ltvl R para el fention es de 0,05 ppm, es decir, el limite de detecci6n. era necesario cambiar dicho insecticida por otro de similar eficacia, pero que no aportara residuos de productos fitosanitarios no autorizados.

Por tanto. se plante6 el cambio de fention 50 LE por rnalation50 a la misma dosis.

CARACTERISTICAS DE LA ZONA DEL ENSAYO

La zona elegida para el ensayo fue la Zona Norte de la provinc,a de Alicante. denominada IMarinaAltat con mas de 7.000 ha de citricos.

Las variedades cultivadas en esta zona son:

Mandarinas.- Marisol. orovaL okitsu y clementina de Nules.

Naranjas.- Navelina, Valencia late y salustiana.

PRODUCTOS.

Los productos a ensayar fueron:

lnsecticidas.- Fention 50 LE a 1.5 I en 100 I de agua Malation 50 LE a 1.5 I en 100 I de agua

Cebo.- Proteina r1idrolizada a 1.2 kg. por 100 I de agua

\/ehiculo: - Agua

DIS ENO DEL ENSAYO.

Se realiz6 tratamiento aereo en aplicaci6n cebo.

Se disenaron tres areas diferentes que abarcaban cuatro poligonos del tratamiento oficial

Cada uno de ellos. se d1vidi6 en dos bloques, mas o menos iguales. uno por cada insecticida. - 170 -

En cada bloque es establecieron cinco repeticiones, ccincidiendo con las retfculas disenadas para la carnpafiaofic1al.

En total se colocaron 97 controles, formados por tres mosqueros de cristaL con disoluci6n de fosfato biam6nico al 4% y un mosquero de plastico. tipo Nadel, con una capsula de trimedlure + DD\/P

Se realizaron dos conteos sernanales,

El indice obtenido represent6 el numero de moscas capturadas por mosquero y dia.

CARACTERISTICAS DE LAS AERONA\/ES

Para el ensayo se emplearon dos aerona:ves iguales, tipo PZL M-18 A (Dromader), con una capacidad de carga de 2.000 I de mezcla insecticida, trabaiando simultaneamente en cada una de las areas. El tipo de boquilla ut1lizado fue D-8 (8mm) con un total de 17 boquillas por aeronave.

CARACTERISTICAS DE LAAPLICACION.

El gasto de producto fue de 20 I por ha, realizandose el tratamiento por bandas, con un 40% de superficie tratada.

La altura media de vuelo fue de 15 m. La velocidad media de vuelo fue de 120 krnJhora

La presi6n de trabajo fue de 35-45 psi.

RESULTADOS.

Los resultados de las eficacias de los productos ensayados, expresados en n• de moscas por mosquero y dia, viene reflejado en el grafico l, que representa las capturas en los mosqueros de fosfato biam6nico, es decir, fundamentalmente capturas de hembras. - 171 -

Comparacion tratamientos contra Ceratitis capitata Wied. 6itMosqueros de� fosfato� biamonico� - ;iFl�- .

27-jul 10.ago 20-ago 27-ago 3-sep 10-sep 21-sep ::t ----�Fechas de . --�� conteo � . _ ;,'.

Grafico 1 .- Comparaci6n de capturas segun productos utilizados

Como se puede apreciar, los resuttados de las capturas antes y despues de la aplicaci6n de ambos insecticidas son comparables.

COMPARACION DE CAPTURAS DE MACHOS Y HEM BRAS

En el grafico 2 se comparan los \ndices de moscas/mosquero y d\a obtenidos en mosqueros de fosfato biam6nico (hembras). con los obtenidos en mosqueros de trimedlure (machos)

Comparacion tratamientos contra Ceratitis capitata Wied.

B

10..ago 20-ago 27-ago 3-up 10-sep 21-sep

Grafico 2. Comparaci6nde capturas de machos y hembras seg(m productos utilizados

Se puede apreciar que la evoluci6n de los \ndices en funci6n de los tratamientos es similar - 172 -

CAPTURAS YT RATAMIENTOS A LO LARGO DE LACAMPANA OFICIAL

.Aunque se tenia previsto una segunda parte del ensayo intercambiando las zonas de ensayo. es de cir, aplicar fention donde se habia aplicado malation y viceversa, los resultados fueron tan concluyentes que se decidi6 directamente el cambio a malation en las aplicaciones posteriores.

A primeros de septiembre. se produjeron en la zona. lluvias abundantes durante mas de una semana. o que redujo el incremento de indices de capturas de moscas .

.ANALISIS ESTADISTICO

Los datos de las capturas se analizaron comparando cada uno de los tratamientos en cada una de las aplicaciones.

Se realiz6 un AN OVA a nivel de significaci6n de 95%

TES!S DATOS SI# TRTAR /P TR.4VlN 2PTRA1AM i:._?P TRAT.4M EST.4DISTIC. 27-7A 15-8 20-8A21-9 21-9A 18-10 25-!0A 22-11 MALATION media 0.85 b 0.19 a 0,18 b 0;21 b hembras error standard 0.09 0.03 0.04 0,02 FENTION media 0.68 b 0.16 ab 0.21 b 0.21 b hembras error standard 0.05 0.02 0.05 0.04 MALATION media 1.33 a 0,13 ab 0.79 a 1.99 a machos error standard 0.17 0.02 0,17 0.24 FENTION media 1.20 a 0.10 b 0.97 a 1,76 a machos error standard 0.11 0.01 0,23 0.24

No existen diferencias entre tratamientos. pero si entre los distintos sexos o sisternas de captura.

DATOS DE CAPTURAS EN LAS CUA TRO ZONAS DE LA PROVINCIA DE ALICAITTE.

La zona norte o Marina Alta. donde se realiz6 loa aplicaci6n es una de las cuatro zonas citricolas en las que se divide la provincia de Alicante.

En una segunda zona al sur. denominada IVega Baja del Rio Segura. tambien se realizan tratarnientosaereos cebo. simliares a los descritos para la zona norte.

Existen dos zonas mas. Ila Marina Bajal y L'Alacanti-Carnp d'Elxl que por sus especiales caracteristicas de diserninaci6n de cultivos. turismo etc. no se realizan tratamientos aereos colectivos. sino que se reparte el producto insecticida y la proteina entre los citricultores para que realicen tratamiento cebo terrestre. - 173 -

En el grafico 3 se aprecia la evolucion de las capturas (en rnosqueros de fosfato biam6nico) de hernt,ras por mosquero y dia de las cuatro zonas yen el grafico 4 se hace lo mismo con los macl,os capturados en rnosquerosde trimedlure.

Tratamientos contra Ceratitis caipitafa Wied.

D

------10- 20- 27- 3- 10- 21- 5- --2- 22- Fechas de conteo Grafico 3. Capturas de hembras en las cuatro zonas citricolas

Tratamientos contR"a Ceratitis

27- 10- l- 10- 21- 5- 18- 2· 22- M ------= = Fechas de conteo

Grafico4. Capturas de machos en las cuatro zonas citricolas

Como se puede apreciar. los indices de las zonas donde no se realiza tratamiento aereo. son mucho mas elevados. correspond1endose tambien con un indice de daFios en fruto mas elevado.

DISCUSION.

Despues de este ensayo se concluye que la eficacia y persistencia del malation a la dosis de 1.5 I por 1 00 I de agua en mezcla con proteina hidrolizada a 1 .2 kg. por 1 00 I de - 174 - agua en aplicaci6n cebo aerea. es similar a la de fention a la misma dosis. mezcla y aplicaci6n.

Tai vez. la persistencia de ambos productos este condicionada por la presencia activa de gotas humedas sobre las hojas; al secarse dichas gotas. puede desaparecer el poder atrayente de la protefna y por tanto la efica.cia del insecticida.

En las zonas donde no se realizaron tratarnientos aereos y solo se dieron tratarnientos cebo en aplicaci6n terrestre por parte de los agricultores. la eficacia fue rnucho menor. el nivel de capturas mas elevado y esto tuvo como consecuencia un mayor dano en los frutos por Cerat1tisca,01tata

CONCLUSION

Es aconsejable el carnbio de fention por malation en el tratamiento que anualmente realiza la Administraci6n en determinadas areas citricolas de la Cornunidad Valenciana Session 3 GENETIC ASPECTS AND MICROORGANISMS OF FRUIT FLIES POPULATIONS

Chairman: R. Milani Secretaries: Salah Oukil Dolores Ochando - 175 -

FITNESS OF MITOCHONDRIAL DNA HAPLOTYPES IN CERATITIS CAP/TATA

A. Reyes & M.D. Ochando Departamento de Genetica. Facultad de Ciencias Biol6gicas. Universidad Complutense. Madrid 28040. Spain.

Sllmmary Two mitochondrial DNA variants of Ceratitis capitata (Wied.) have been placed under the same nuclear background in order to test the action of selection. Two fitness parameters have been measured in different kind of crosses: fecundity and egg-to-pupa developmental rate. The results show that for each kind of haplotype female no differences in these parameters can be observed when males carrying different mtDNAs have been crossed with. However, important differenceshave been detected between the two different haplotype females. Selection acting on those mitochondrial DNA haplotypes is considered to explain the observed differences, though nucleus-cytoplasmic interactions and paternal leakage cannot be absolutely neglected.

1 - Introduction Since the discovery of mitochondrial DNA (mtDNA) about 30 years ago, a large amount of data have been accumulated on the structure and function of this extranuclear genome. The special featuresof this genome have made it a very popular tool for population and evolutionary analysis. Thus, in the last years, many studies have focused on the analysis of the level of polymorphism in mitochondrial DNA in an ever-increasing number of species. In most of those surveys mitochondrial DNA variants were implicity considered to be neutral markers (Wilson et al., 1985; Avise et al., 1987; Moritz et al., 1987). However, it is known that some regions of the mitochondrial genome are more frequently subject to substitutions than others (Simon, 1991; Kaneko et al., 1993; Saccone & Sbisa, 1994; Rand et al., 1994). In other cases, mtDNA variants have been found to show a different phenotypic expression, mainly in plants and yeasts, and in some cases in mammalian cells and human diseases (Saccone & Sbisa, 1994). These characteristics would suggest the action of selection. In order to test selection at mitochondrial DNA level, two kinds of approaches have been used: competition of mtDNA variants in population cages, and sequence analysis of mitochondrial genes. Competition experiments have been limited to a few surveys on differentDrosophila species (MacRae & Anderson, 1988; Fos et al., 1990; Nigro & Prout, 1990; Nigro, 1994). The observed differencesbetween mtDNA variants have been explained in some cases as a consequence of incompatibility-causing microorganisms without selection (Nigro & Prout, 1990). In other cases, the exclusive influence of the nuclear background (Nigro, 1994) or the simultaneous action of selection on the mitochondrial DNA variants in addition to nuclear-cytoplasmic interactions (MacRae & Anderson, 1988; Fos et al., 1990) have been proposed. - 176 -

The analysis of the sequence of mitochondrial genes is based on the assumption that under a strictly neutral model, the ratio of synonymous and asynonymous nucleotide substitutions will be the same within and between species (McDonald & Kreitman, 1991) Using this approach, the existence of non neutral evolution has been reported in mice (Nachman et al., 1994), and differentDrosophila species (Kaneko et al., 1993; Rand et al., 1994; Akashi, 1995). The Mediterranean fruit fly (medfly), Ceratitis capitata is a polyphagous species from tropical and temperate regions that affects more than 250 agricultural crop species and varieties, both commercial and ornamental (Fimiani, 1989). The great importance of this pest is due to the high economic cost derived, on one hand, on the direct damage in the fruits and, on the other hand, on the indirect costs related to quarantines and control and erradiaction programs. Effective pest control programs can only be succesfully developed if we have previously a comprehensive knowledge of the different aspects of the behavior, physiology, genetics, etc. of the species under consideration. In this way, it is of special interest for integrated pest control programs to know the possible existence of genetic variants with differential fitness, as in the case of induced male sterility or genetic incompatibility. In this paper we examine two fitness parameters, fecundity and developmental rate, in two strains of Ceratitis capitata, characterized by different mitochondrial DNA haplotypes. The aim of this work was to determine whether the differences on those parameters could be detected in our strains and whether they could be correlated with the presence of differentmitochondrial genomes.

2 - Materials and Method Two differentstrains ofCeratitis capitata (Wied.) derived from a natural population from Atajate (Malaga, Spain) were used in the experiments. One of them belonging to the haplotype IV, characterized by the existence of three Hae III sites, while the other strain was representative of the haplotype VI,. which exhibited four Hae III sites (Figure 1). The designation of the haplotypes was according to Reyes (1995) and no other differences were detected between this two strains when 21 other restriction endonucleases were used for RFLP analysis (Asp 718, BamH I, Bel I, BstE II, Cjo I, Cla I, Dra I, EcoR I, EcoR V, Hind III, Hin/ I, Hpa I, Hpa II, Pst I, Pvu II, Rsa I, Sac I, Sal I, Sma I, Xba I and Xho I, unpublished data). The two mitochondrial genomes were placed under the same nuclear genome control in order to minimize the importance of the nuclear background (Figure 2). We firstobtained a nuclear donor strain by means of consanguineous crosses for more than forty generations in a laboratory strain. Then, repeated· backcrosses of females with haplotype IV or VI with males from the nuclear donor strain were carried out for 15 generations. Thus, the original nuclear background of those two strains were replaced by the nuclear genotype of the laboratory consanguineous strain. - 177 -

M I 2

kb 23.l - 9.4 - - 6.6 - - 4.3 - - -

2.3- 2.0- -

0.6 :.eml - - Figure 1. Representation of I% agarose gel showing the Hae m patterns of the two Ceratitis capitata mitochondrial DNA variants used in the present work. Lane I: mitochontlrial DNA type IV. Lane 2: mitochondrial DNA type VI. Phage lambda digested with Hind ill was used as molecular marker (M).

After those 15 generations, different kind of crosses were established. On one hand, females with males carrying both the same mitochondrial DNA haplotype, i.e., females IV with males IV and females VI with males VI. On the other hand, crosses between lines with different mitochondrial genome were established: females IV with males VI and females VI with males IV. In all kind of crosses two fitness parameters were estimated: fecundity and egg-to­ pupa developmental rate. Fecundity, considered as the number of eggs laid by a one day old female during a period of 30 consecutive days, was estimated in 30 couples per kind of cross. Those cases in which the female or the male died before the end of those 30 days were eliminated for the analysis, as well as those females that after 10 days did not lay eggs. Egg-to-pupa developmental rate was measured in 200 eggs per kind of cross.The eggs came from the day 10 after cross establishment. In order to avoid larval competition, each egg was allowed to develop in lgr of larval medium (N-propyl p-hydroxibenzoic acid, 1 gr; p-hydroxibenzoic acid metil ester, 1 gr; beer yeast extract, 3 5 gr; glucose, 70 gr; wheat bran, 140 gr; HCI, 3.5 ml; water, 300 ml). - 178 -

Naturalpq:mlatioo La\:xiratorypcpulatioo

Consanguineouscro,ses > 40 generations §i1otyµ:IV i §rlotyµ:VI i

'¥ t Males Females Females t

Backcros;esfer 15 generations

HaµotypeIV \\i1h Haµotype VI with lab nuclar genome lab mclar genome

Females Males Females Males l Female!V Female ,Vf Female VI FemaleVI MaleIV Male VI MaleIV MaleVI

Fecundity (XIcouples) Egg-to-iuradevelcpmental rate (200 eggs)

Figure 2. Schematic representation of the crosses carried out in the present study between populations of C. capitata.

The differences between crosses were statistically tested by means oft-Student test, both forfecundity and egg-to-pupa developmental rate. - 179 -

3 - Results A- Fecundity. Table 1 shows the overall values and standard errors forthe cumulative fecundity in each kind of crosses. As it can be observed, when different types of males are considered for each kind of female, no statistically significant differences were detected. However, the comparison between the total mean value of females IV (1111±372 eggs) and females VI (1453±359 eggs) make evident a statistically significant difference(t118 = 5.1243; p < 0.001). The means cumulative fecundity of each kind of crosses are presented in Figure 3. As it can be observed, there are two well defined types of curves, one corresponding to those cases in which females IV are involved, and the other in which females VI are present. One fact that can be noticed from this figure and Table I is that there are only slight differenceswhen differentmales are taken into account for each kind of female. The comparison between different types of females reveals that females IV start to lay eggs between the fourth and fifth day of observation, while females VI do it one day later. Nevertheless, the main difference between the. curves representing to females IV and VI is that the slope in the case of females VI is higher than that in the case of females IV, indicating a higher number of eggs per day in the former cases. This fact results in a higher overall productivity in femalesVI.

Table !. Means and standard errors for fecundity in four different types of crnsses between lineages of C capitata carrying different mitochondrial genomes. Mean va!ues per kind of female and male are also presented. Roman numbers Teffer to the mitochondrial haplotype according to Figure 1. T-Studer.t test significance is shown close to the values under consideration. (n.s. not significant; *** p<0.001). Fecundity

Female IV Male IV 1053±350 158 = 1.1663 n.s. Male VI 1169±420

Female VI Male IV 1498±403 158 = 0.8649 n.s. Male VI 1408±308

Female IV Mean value 1111±372 1118 = 5.1243 *** Female VI Mean value 1453±359

Males IV Mean value 1276±378 1118 = 0.1582 n.s. Males VI Mean value 1289±351 - 180-

Fecundity

1600

1400 --+- Female IV Male 1200 IV

--o- Female IV Male 1000 I VI ell ell 800 -k- Female VI Male IV 600 -1r- Female VI Male VI 400

200

0 "'1" r- N � ::; ::e °' N N"' N Days

Figure 3. Cumulative fecundity of different kind of crosses between lineages of Ceratitis capitata carrying two different mitochondrial genomes. Roman numbers reffer to the mitochondrial haplotype according to Figure 1.

B - Egg-to-pupa developmental rate. The quantification of the results corresponding to egg-to-pupa developmental rate are shown in Table 2. It can be seen that no statistically significant differences are detected when different types of males are considered for each kind of females. However, important differences are detected between both kind of females. The total mean value of offsprings for femalesIV (12.1850±1.3032) is statistically lower (1798 = 38.0608; p < 0.001) than that observed forfemales VI (15.6750±1.2903). The results based on the developmental rate of 200 eggs per kind of crosses are presented in Figure 4. As in the case of fecundity, the results of crosses in which females IV are present are different from those in which femalesVI were crossed. - 181 -

Table Il. Means and standard errors for egg-to-pupa developmental rate in the four different types of crosses between linezges of C. capitata carrying different mitochondrial genomes. Mean values per kind of female and male are also shown. Roman nl!mbers reffer to the mitochondrial genotype according to Figure I. T­ Student test significance is shown close to the values under consideration. (n.s. not sigr.ificant; *** p<0.001). Egg-to-pupa developmental rate

Female IV Male IV 12.0700±1.1584 t398 = 1.7734 n.s. Male VI 12.2950±1.3702

Female VI Male IV 15.6350±1.3642 t398 = 0.6226 n.s. Male VI 15.7150±1.2004

Female IV Mean value 12.1850±1.3032 1798 = 38.0608 *** Female VI Mean value 15.6750±1.2903

Males IV Mean value 13.8525±1.4238 t798 = 1.5555 n.s. Males VI Males VI 14.0050±1.3406

Progeny of females IV begin to reach the pupa stage after 10 days, with a peak the day 12. Within 14 days, the 90% of the eggs sample have reached the pupa stage. In the case of progeny of females VI we did not find pupal stage but after 13 days, with a double peak the days 15 and 16. To reach the 90% of eggs sample in pupal stage it was needed 18 days. In both cases, however, for each kind of female, no great differences were observed when the males presented different haplotypes.

4 - Discussion The first surveys carried out on mtDNA considered their variants were selectively neutral (Wilson et al., 1985; Avise et al., 1987; Moritz et al., 1987). Later on, the existence of selection have been determined in different species with diverse approaches (MacRae & Anderson, 1988; Fos et al., 1990; Kaneko et al., 1993; Nachman et al., 1994; Rand et al., 1994;Akash, 1995). Selection acting on mitochondrial DNA variants have been described in Drosophila (McRae & Anderson, 1998; Fos et al., 1990) by means of the changes detected in the frequencies of different haplotypes accross time in population cages. In other cases, as in human cells (King & Attardi, 1988), different mtDNA types appeared to have effects on · 182 •

Developmental rate

--+- Female IV I Male IV -o- Female IV Male VI -h- Female VI Male IV -f:r- Female VI Male VI

Days

Figure 4. Number of individuals of Ceratitis capitata at pupa! stage and days they need to reach this stage. The values are for the four different kinds of crosses between lineages with different mitochondrial genomes. Roman numbers reffer to the mitochondrial genotype according to Figure 1. fitness, as ones were drug-resistant while the others not. Moreover, it has been described how single nucleotide variations (in protein coding genes forNDl, ND2, ND4, ND5, ATP6, and t-RNAs for Lys, Thr, and Leu) can cause phenotypic variants generally associated to human diseases such as Leber' s hereditary optic neuropathy or mitochondrial encephalopathy, among others (Saccone & Sbisa, 1994). Thus, nowadays, there seem to be no doubt about the possibility that mitochondrial DNA variants could be under selection. With respect to Ceratitis capitata, indirect support for the existence of selective pressures acting on mitochondrial DNA can also be obtained from previous surveys (Sheppard et al., 1992; McPheron et al., 1994, Reyes, 1995), taken into account that the existence of geographical variation could imply that different haplotypes are being favoured and predominating in different areas (Nigro, 1994). However, until now, no direct support have been published providing information for the null hypothesis that Ceratitis capitata mitochondrial DNA evolve according to a model of molecular evolution that assumes selection. In our results, when analyze fecundity (Table 1, Figure 3) or egg-to-pupa developmental rate (Table 2, Figure 4) there are two important aspects to focus on: the lack - 183 -

of differences between males with different haplotypes and the sharp differences detected between females carrying different mitochondrial DNAs. The fact that the nuclear genome has been uniformized for both lineages make it possible to assume that the observed differences are due to the different mitochondrial genomes. In fact, if according to the methodology we have used, nuclear background is the same in all strains, and considering exclusively maternal inheritance of mitochondrial DNA, we would not expect to find differences when different males are crossed with the same type of female. Indeed, as it can be observed in Figures 3 and 4, only slight differences exist, that resulted to be not statistically significant(Tables 1 and 2). Under laboratory conditions, selection can be acting on these lineages, and thus we observe that females VI lay more eggs than females IV (Table 1, Figure 3), while at the level of egg-to-pupa developmental rate progeny of females IV develope faster than progeny of females VI (Table 2, Figure 4). Based on these data, we find that selection is acting differentially in each parameter, i.e., the haplotype with higher fecundity is that with slower developmental rate (female VI), while that with lower fecundity sho.ws a faster developmental rate (female IV). Counteracting selective advantages have been reported for nuclear genotypes in Drosophila (Marinkovic & Ayala, 1975 a,b ), but never, until now, for mitochondrial genotypes. Selection can be acting in different ways, but in all cases those individuals with higher total fitness will be selected. To increase fitness two different strategies can be addressed: one of them based on a high fecundity, what would be correlated with high fitness. The other possibility would be a fast developmental rate; in this case, even though the number of descendents could be lower, if they develope faster the likely of reaching the adult stage increase, as they can profit the availability of food resources. In future studies, it will be interesting to see the evolution of those two lineages when they are placed together in competition experiments. Taking all the above considerations, we can assume that selection must be the main process acting on our mtDNA variants. However, we should also take into account other processes such as nucleus-cytoplasmic interactions, incomplete replacement of nuclear background and paternal leaking. Most respiratory chain complexes subunits are encoded by both, mitochondrial and nuclear genes and, subsequently, nuclear-cytoplasmic interactions must be also of great importance. In fact, the differences observed in our results are due to different fitness of the mtDNA haplotypes in a specific nuclear background. Thus, the differences detected at mitochondrial level would be increased by the interaction of different mitochondrial subunits with the nuclear ones. In this way, Nigro (1994) has found that nuclear effects can act in different directions in each cytoplasm, suggesting that different haplotypes under the same nuclear background can show significantdifferences, even in opposite directions. In the case of human cells (King & Attardi, 1988) the relationship between nuclear and mitochondrial genomes plays a crucial role in determining the growth rate of different classes of cell lines. This nuclear-cytoplasmic interactions would be of higher importance in the case of an incomplete replacement of the nuclear background. The fact that we do not observe differences for males carrying different mitochondrial DNAs in any case (Tables l and 2), suggests that complete replacement has taken place, or at least no significant differences among nuclear genomes may exist in our case. - 184 -

Paternal leakage has been estimated to range from 0.017 to 0.035% of the whole mtDNA detected in the zygote of Drosophila species (Matsuura, 1991). Thus, it is expected that a certain amount, however small, of paternal leakage could also exist in C. capitata. Nevertheless, we have found that in the medfly this is not an important phenomenon (unpublished data). In conclusion, we can say that the analysis of two fitness parameters, fecundity and egg-to-pupa developmental rate, in two different mtDNA variants have shown that the cytoplasmic genotype of the male does not affect significatively these parameters. However, the fitnesses are highly affected by the mitochondrial genotype of the female. The action of selection at mitochondrial level is invoked as the main responsible of the high differences observed in those two parameters, although nucleus-cytoplasmic interactions and paternal leakage cannot be absolutely neglected.

Acknowiedgements We would like to thank to Dr. F. Budia and Dr. E. Vifiuelas for providing us with the laboratory population and to B. Reyes for providing us with the natural infested fruits. This work was supported by grants from DGICYT PB89-0114 and PB93-1210.

References AKASH, H. 1995. Inferring weak selection from patterns of polymorphism and divergence at "silent" sites in Drosophila. Genetics 139: 1067-1076. AVISE, AC., ARNOLD, J., BALL, R.M., BERMINBHAM, E., LAMB, T., NEIGEL, J.E., REEB, C.A. & SAUNDERS, N.C. 1987. Intraspecific phylogeography: The mitochondrial DNA bridge between population genetics and systematics. Ann. Rev. Ecol. Sy-st. 18: 489-522. FIMIANI, P. 1989. Mediterranean region. In: Fruits Flies: Their Biology, Natural Enemies and Control. Vo! 3A. A.S. Robinson & G.H. Hooper (Eds). Elsevier, Amsterdam. pp. 37-50. FOS, M., DOMINGUEZ, M.A., LATORRE, A. & MOYA, A 1990. Mitochondrial DNA evolution in experimental populations of Drosophila subobscura. Proc. Natl. Acad. Sci. USA 87: 4198-4201. KANEKO, M., SATTA, Y., MATSUURA, E.T. & CHIGUSA, S.I. 1993. Evolution of ATPase 6 gene in Drosophila: Unuasually high level of polymorphism in D. melanogaster. Genet. Res. 61: 195-204. KING, M.P. & ATTARDI, G. 1988. Injection of mitochondria into human cells leads to rapid replacement of the endogenous mitochondrial DNA. Cell 52: 811-819. MACRAE, A.F. & ANDERSON, WW. 1988. Evidence for non-neutrality of mitochondrial DNA haplotypes in Drosophila pseudoobscura. Genetics 120:485-494. MATSUURA, E.T. 1991. Selective transmission of mitochondrial DNA in Drosophila. Jpn. J. Genet. 66: 683-700. MARINKOVIC, D., AYALA, F.J. 1975 a. Fitness of allozyme variants in Drosophila pseudoobscura. I. Selection at the Pgm-1 and Me-2 loci. Genetics 79:85-95. MARINKOVIC, D., AYALA, F.J. 1975 b. Fitness of allozyme variants in Drosophila pseudoobscura. II. Selection at the Est-5 , Odh, and Mdh-2 loci. Genet. Res. 24:137- 149. - 185 -

McDONALD, J.H. & KREITMAN, M. 1991 Adaptive protein evolution at the Adh locus in Drosophila. Nature 351: 652-654. McPHERON, B., GASPARlCH, G.E., HAN, H., STECK, G.J. & SHEPPARD, W.S. 1994. Mitochondrial DNA restriction map for the Mediterranean fruit fly, Ceratitis capitata. Biochem. Genet. 32: 25-33. MORITZ, C., DOWLING, T.E. & BROWN, W.M. 1987. Evolution of animal mitochondrial DNA: Relevance for biology and systematics. Ann. Rev. Ecol. Syst. 18: 269-292. NACHMAN, M.W., BOYER, S.N. & AQUADRO, C.F. 1994. Nonneutral evolution at the mitochondrial NADH dehydrogenase subunit 3 gene in mice. Proc. Natl. Acad Sci. USA 91: 6364-6368 NIGRO, L. 1994. Nuclear background affects frequency dynamics of mitochondrial DNA variants in Drosophila simulans. Heredity72: 582-586. NIGRO, L & PROUT, T. 1990. Is there selection on RFLP differences in mtDNA?. Genetics 125: 551-555. RAND, D.M., DORFSMAN, M. & KANN, L.M. 1994. Neutral and non-neutral evolution of Drosophila mitochondrial DNA. Genetics 138:741-756. REYES, A. 1995. Analisis de la variabilidad genetica en poblaciones espafiolas de Ceratitis capitata Wied. mediante la utilizaci6n de marcadores moleculares. Ph. D. Thesis. Universidad Complutense. Madrid. pp. 202. SACCONE, C. & SBISA, E. 1994. The evolution of the mitochondrial genome. In: Principles of Medical Biology. Vo! lB.E.E. Bittar (Ed). JAI Press Inc. Greenwich, Connecticut. pp. 39-72. SHEPPARD, W.S., STECK, G.J. & MCPHERON B. 1992. Geographical populations of the medfly may be differentiated by mitochondrial DNA variation. Experientia 48: 1010-1013. SIMON, C. 1991. Molecular systematics at the species boundary: exploiting conserved and variable regions of the mitochondrial genome of animals via direct sequencing from amplified DNA. In: Molecular Techniques in Taxonomy. G.M. Hewitt, A.W.B. Johnston & J.P.W. Young (Eds). Springer-Verlag, Berlin. pp. 33-71. WILSON, AC., CANN, R.L., CARR, S.M., GEORGE, M., GYLLESTEIN, U.B., HELM­ BYCHOWSKY, K.M., HIGUCHI, R.G., PALUMBI, R.S., PRAGER, E.M., SAGE, R.D. & STONECKING, M. 1985. Mitochondrial DNA and two perspectives on evolutionary genetics. Biol. J. Linn. Soc. 26: 375-400. · 186-

RECENT ADVANCES iN CERATITIS CAPITATA GENETICS. RESEARCH PROGRAMS DEVELOPED AT THE DEPARTMENT OF ANIMAL BIOLOGY OF THE UNIVERSITY OF PAVIA

R. Milani Dipartimentodi Biologia Animale, Universita di Pavia

Summary. The research activity on Ceratitis capitata developed at the Department of Animal Biology of the University of Pavia has focused on the mapping of the medfly genome, on the genetic aspects of the worldwide colonization process of Ceratitis capitata and on the contribution of the genetic approach to the solution of taxonomic problems. Multilocus enzyme electrophoresis data from 26 polymorphic loci (124 alleles) concurrently indicate that the geographical dispersal of the medfly, from its ancestral source area (East Africa), is associated with a great reduction in variability. Intraspecific differentiative processes affect mainly the two island populations, Reunion and Hawaii. The population genetic changes observed in the species range are consistent both with the chronology and with the historical circuitus course of the medfly colonization process. Sympatric samples of C. capitata, C. rosa and T coffeae showed marked between species differences in genetic variability, which seem related to their host­ dependence. Similar results were provided by assessment of genetic distances and cluster analysis. Genetically evidentiated phylogenetic affinities and classical taxonomy of the Tephritidae family share only limited areas of agreement. A complete Ccmar 1 element has been isolated and characterized.

I-Introduction My present communication refers only to the work done by my younger collegues at the University of Pavia. My colleagues regret their absence from the present meeting. I assume that the group of medfly research workers of Pavia is familiar to most participants at this Second International Open Meeting; however, I would like to acknowledge that everythings I am going to say now is based on their research and experimental activity: I am merely a reporter of their work, which goes far beyond my personal mastership of the modern technicalities. Their programs are aimed both at extending the knowledge of the medfly genome and at spotting promising tools for the transposition of the basic knowledges into applied research programs. A fine detailed knowledge of the genome is a prerequisite both for interpreting natural occurring phenomena and for providing clues and tools to programs of outstanding social and economical importance. - 187 -

2-Mapping the medfly genome. A paper published in 1994 by Rossler et al., lists 85 morphological mutants and 33 biochemical and functional loci. At present, the corresponding numbers are much higher, but reliable countings require a praeliminary accurate nomenclatorial pinpointing revision, in order to eliminate both synonimy for individual loci and identity of names assigned to different loci or alleles (Malacrida, personal information).Many of these loci have been mapped on the five autosomes, and provide the framework for a more complete representation of the genotype and of the genetic structure of this species. The interest of the Pavia team initially centered mainly on functional gene markers, because their suitability for genetic analysis has the additional bonus that these markers are possibly also potential targets for future manipulations. At present, 9 morphological mutants and 33 functional gene markers have been assigned to five of the six linkage groups expected in a species that has six autosomes. Each of the linkage groups carries structural genes coding for morphological traits, enzyme functions, sex ratio distorter factors and developmental proteins. The electrophoretic patterns usually allow the recognition both of alternative homozygous variants (allozymes) and of their heterozygous combination. Therefore, they provide a picture of both phenotype and genotype of the sample analysed. Electrophoresis is particularly suitable for studies which operate at the gene/locus levels, such as formal genetics, population structure, comparative genetics and taxonomy.

3-Genetic aspects oi the worldwide colonization process of C.capitata. Multilocus enzyme electrophoresis data from 26 polymorphic loci, involving 124 alleles, has allowed the evaluation of the genetic changes which occurred during the worldwide dispersal of C. capitata · from East Africa, its assumed ancestral source area, to continental and insular areas of the tropical and subtropical regions of the world. Out of the 26 available enzyme loci, only four provided monomorphic patterns (Aki, Adh2, alfa-Gpdh and Me); all other loci showed polymorphism. Altogether, 74 alleles were detected in 11 population samples (Baruffi et al., 1995). The average number of alleles per locus (A), the proportion of polymorphic loci (P) and the average number of heterozygous individuals (H) showed a general trend of decreasing genetic variation from the putative source area (Kenya) toward the periphery of the species range (Gasperi et al., 1991). The wild peripheral populations (Italy, Australia) and the five laboratory strains provided variability estimates in the same (low) ranges, apart from a laboratory strain (M84) established to mantain polymorphism at six loci, which mark three autosomes (dp and Got2: 3° chr; bw, Zw, Fh: 5th chr; ye2, Got I: 6th chr.). Multilocus enzyme electrophoresis (MLEE), based on allozymes, was, and still is, a powerful tool for assessing the genetic make-up of populations (Milani et al., 1989), for constructing genetic maps of the medfly (Malacrida et al., 1990), - 188 -

and for inferring taxonomic relationships between this species and several other tephritids (Malacrida et al., 1991). The genetic ·variability of allozymes shows spatial and temporal differentiation between geographical populations (Malacrida et al., 1995). As direct products of gene activities, allozymes testify very closely to the genes themselves; however, their information regards the activity, not the structure, of the coding genes.

4-The study of DNA variations. DNA variations can be studied directly by the applycation of molecular biology techniques. Genomic DNA, mitochondrial DNA, and DNA transposable elements have been tested for variability. Mitochondrial DNA variation has been used to discriminate between medfly populations (Frohlich et al. 1993; Mc Pheron et al., 1994). Techniques based on polymerase chain reaction (PCR) using random primers enable us to analyse genotypic DNA through the production of large amounts of (anonymous) gene markers. This technique of analysis, named Random Amplified Polymorphic DNA (RAPD) (Williams et al., 1990; Welsh & Mc Clelland, 1990) has been applied to C.capitata, and it has revealed a much larger amount of genetic variations tl1an has the one evidentiated by conventional :rvt:LEE; it has also produced a large amount of markers for genome mapping and for population genetics (Gasperi et al., 1993; Haymer & Mc Innis, 1994). Baruffi et al. (199 5) used single arbitrary prinlers to amplify, by polymerase chain reaction (PCR), genomic DNAs of individuals randomly sampled from those populations that had previously been analysed by :rvt:LEE. RAPD patterns of individual fliesshow about 10-20 main bands, ranging from50 to 2000 bp in size. The same patterns were obtained in independent experiments, showing the reproducibility of such patterns. The :rvt:LEEtechnique has the great advantage of (generally) providing distinct patterns for the alleles, even in their heterozygous condition. This property allows direct countings of the single alleles. The RAPD markers show dominance, and variations are revealed by the presence or the absence of a given band. Presence is evidence of nucleotide sequences homologous to the primer, which can either be in the homozygous or in the heterozygous condition, so actual allele countings are possible only for the absence/absence condition. Presence/presence and presence/absence provide the same band and their relative frequencies canonly be inferred. Baruffiet al. (1995) computed RAPD data "making the assumption that RAPD markers are in Hardy­ Weinberg equilibrium within and between loci". Specific analytical programs are now being developed to overcome the ambiguity introduced by dominance (Gasperi, personal information). The analysis of PCR amplified polymorphisms is based on the comparison of the banding patterns between pairs of individuals and on the calculation of the dissimilarity index d.. Index dis the ratio between the number of fragments which differ between the two individuals and the total number of fragments obtained from them. Details on the complete procedure and on the experimental data were - 189 - provided by Baruffi et al.(1995). It seems sufficient now to quote the results obtained by these Autors through amplification with four primers of DNAs of individual flies. The RAPD patterns of the samples of African populations show great variability both within and between populations, whereas those provided by the two peripheral populations (Italy and Australia) as well as those by the five laboratory ones, show a much reduced variability within populations and an overall similarity between peripheral and laboratory populations. The results obtained with the H parameter (Heterozygosity, MLEE) and the dissimilarity index are significantly correlated (r=0,67, P<5%). (Baruffi et al., 1995). The MLEE and RAPD methods evidentiate similar trends of the polymorphism variations during the expansion of tl1e species'areal; however, the DNA amplified fragments provided much higher mean values of poymorphism than did those detected by the analysis of enzyme variability (Baruffi et al., 1995). Such discrepancy may be due to the fact that MLEE deals with codifying loci, whereas RAPD involves the complete DNA complement.

5-Genetic relationships and taxonomy. The various alleles of single gene loci show qualitative and quantitative between-population differences, so the MLEE method allows us to evaluate the contribution of single gene loci to differentiation. The contribution of single loci to population differentiation are estimated by the fixation index (FSJ). Baruffi et al. (1995), published the FTS estimates in wild and laboratory populations of the medfly; their estimates were based on allelic frequencies at 22 polymorphic biochemical loci. The FTS values show that private alleles of two loci, namely Est] and Est2, mainly contribute to the differentiation of Hawaii and Reunion populations, whereas alleles of four loci ( Got2,, Hkl, Got 1 and Fh) are mainly involved in the differentiation of the multimarked M84 laboratory strain. The medfly belongs to a very large family (Tephritidae) of flies, for which, despite years of taxonomic efforts, no satisfactory classification and phylogeny exist. Malacrida et al.(1996) analysed allozyme data from 24 orthologous loci, in the attempt to elucidate the relationships between species of the related genera Ceratitis, Thirithrum, Capparimia, Bactrocera, Anastrepha and Rhagoletis. Sympatric samples of C. capitata, C. rosa and T. coffeae collected together in Kenya, one of their putative original areas, showed marked between-species differences in genetic variability, which seem related to their host-dependence. The polyphagous and cosmopolitan C. capitata is the most polymorphic, whereas T.coffeae, which is monophagous and endemic to Westem Africa, has the lowest level of genetic variability. C. rosa shows intermediate levels both of genetic variability and of geographical diffusion. The two monophagous species Capparimya savastanoi and Bactrocera oleae, rank, with the endemic T.coffeae, at the lowest level of genetic variability. - 190 -

Four different methods of cluster analysis concurrently indicated the presence of the same clusters of species (Malacrida et al., 1996). Concomitant results were provided by the parameters of genetic variability, by tl1e assessment of genetic distances and by cluster analysis. Areas of agreement exist between the electrophoretic trees and the relationships assessed in the Tephritidae subfamily on tl1e basis of morphological traits. However, some species-genus relationships concurrently indicated by electrophoretic trees do not match the generally accepted classifications of the Tephritidae. This non-concordance confinns that external morphology may poorly reflect genetic affinity. Electrophoretic data provide critical information for the reorganization of the taxonomy of the Tephritidae flies, particularly at the species-genus levels. The indication that the degree of genetic variability of a particular species has some relation to geographic distribution and to biological traits peculiar to the species opens the problem of the role of genetic variability in dispersion processes of the species (Malacrida et al., 1996).

6-The Ccmar 1 eiement. RAPD analysis has indicated in the medfly genome a redundance of highly polymorphic genetic material, which has only partial correspondence with the coding genes and/or loci evidentiated electrophoretically. Genomic DNA other than codifying genes can be explored through transposable elements. An amply distributed and well studied transposon is mariner. A mariner-like (transposable) element is inserted in the first intron of the alcohol dehydrogenasel (Adhl) gene. Very recently, Gomulski et al., (1997) reported the isolation and the characterization of a full-lengthmariner element, Ccmar 1, in the genome of the medfly. Comparingthe genome size with the length of the putativeintact element, these Authors found a copy number of about 500 homologous elements in the genomes of the flies from the six geographic population tested. Four Ccmar 1 clones were isolated. These clones "form a tight cluster on the periphery of the mellifera subfamily of mariner elements. The clones show no close relationships with any previous published medfly mariner element" (Gomulski, et al., 1997). Each of the four Ccmar 1 clones has a vast majority of unic mutations, indicating independent evolution over a long time. Allozymes, RAPD and single copy DNA studies have shown (Gomulski et al., 1997) that the genetic variability decreases by 2-9fold from the ancestral to the peripheral populations. In contrast, theCcmarinerl element is present in large similar numbers of copies in both ancestral and derived populations. The mariner-like elements, like all transposons, are subject to a progressive reduction in copy number, mainly due to stochastic loss, an evolution which requires a long time. The separation of the medfly-derived populations is too recent for the detection of stochastic loss (Gomulski et al.,1997). • 191 •

Ccmar 1 appears a suitable candidate for transformation of the medfly and for transgenical engineering aimed at the improuvement of the SIT means of medfly control. The genomic DNA of the three species of the Ceratininae subfamily which form a close phylogenetic cluster, namely Ceratitis capitata, C. rosa, Trirhythrum coffeae, contains very similar mariner elements, which are respectively Ccmar 1, Crmar 1, Tcmar 1. These elements have phylogenies and patterns of divergence which suggest that they might have been present in the ancestor lineage prior to the divergence of these three species. The related elements Ccmarl, Crmarl, Tcmarl differ dramatically in abundance in the three species; their copy numbers are 10, 500 and 5000 respectively in T. cojfeae, C. capitata and C. rosa, a difference which suggests that the acquisition of the element by T. coffeae is a much older event than that of the other two species (Torti et al., 1997).

References BARUFFI, L., DAMIANI, C.R., GUGLIELMINO, C., BANDI, C., MALACRIDA, A. R. and. GASPERI, G. (1995) - Polymorphism within and between populations of Ceratitis capitata : comparison between RAPD and multilocus enzyme electrophoresis data. Heredity,74: 425-437. FROHLICH, D. R., ROBINSON, A. S. AND WELLS, M. A. (1993). Mediterranean fruit fly, Ceratitis capitata (Wiedemann) mitochondrial DNA: genes and secondary structures for six t-RNAs. Insect Mo!. Biol., 1,165-169. GASPERI, G., MALACRIDA, A. R., GUGLIELMINO, C. R., BARUFFI, L., TORTI, C., MILANI, R., DAMIANI, G. AND BANDI, C. (1993) - Ceratitis capitata: suitable markers for population genetics and genome analysis. In: Management of Insect Pests: Nuclear and Related Molecular and Genetic Techniques, pp.251-256. International Atomic Energy Agency Press, Vienna, Austria. GOMULSKI, L. M., TORTI, C., MALACRIDA, A. R. AND GASPERI, G. (1997) - Ccmarl, a full lenght mariner element from the Mediterranean fruit fly, Ceratitis capitata. Insect Molecular Biol.,6: 1-13. HAYMER,D, S. AND McINNIS, D. 0. (1994) - Resolution of the populations of the Mediterranean fruitfly at the DNA level using random primers for the polymerase chain reaction. Genome, 37:244-248. MALACRIDA,A.R., GASPERI, G., BARUFFI, L. AND MILANI,R. (1990) - The contribution of formal genetic studies to the species characterization of Ceratitis capitata (Wied.). In: Genetic Sexing of the Mediterranean Fruit Fly, pp.85-89. International Atomic Energy Agency Press, Vienna, Austria. MALACRIDA, A. R., GUGLIELMINO, C. R., GASPERI, G., BARUFFI, L., VILLANI, P. C. AND MILANI, R. (1991) - Genetical approach to systematics and phylogeny of Trypetinae (Diptera, Tephritidae). Boll. Zoo!., 58: 355-361. MALACRIDA, A. R., GUGLIELMINO, C. R., D' ADAMO P., TORTI , C., MARINONI, F. AND GASPERI G. (1996) - Allozyme divergence and phylogenetic relationships among species of tephritid flies.Heredity, . 79: 592-602. McPHERON, B. A., GASPARICH, G. E., HAN, H. I., STECK, G. I. AND SHEPPARD, W. S. (1994). Mitochondrial DNA restriction map for the Mediterranean fruit fly, Ceratitis capitata. Biochem. Genet., 32: 25-33. · 192-

MILANI, R., GASPERI, G. AND MALACRIDA, A. R. (1989) • Biochemical genetics [ofCeratitis capitata J. In: Robinson, A. S. and Hooper, G.H.S. (Eds), Fruit Flies.Their Biology, Natural Enemies and Control, vol.3B, p.33-56. Elsevier, Amsterdam, The Neederlands. ROSSLER, Y., MALACRIDA, A. AND ZAPATER, M.C. (1994). Mutants, Chromosomes, and Genetic Maps in the Mediterranean Fruit Fly. CRC Press, Inc., 97- 112. TORTI, C., GOMULSKI, L. M., MALACRIDA, A.R., CAPY, P. AND GASPERI, G., (1997). Characterisation and evolution of mariner elements from closely related species of fruit flies (Diptera:Tephritidae). J Mo/. Evol.,accepted forpublication. WELSH, J. AND McCLELLAND, M. (1990). Fingerprinting genomes using PCR with arbitrary primers. Nucl. Acids Res., 18: 7213-7218. WILLIAMS, J. G. K., KUBELIK, A. R., LIVAK, K.J., RAFALSKI, J. A. AND TINGEY, S. V. (1990) - DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nucl. Acids Res., 18: 6531-6535 - 193 -

ACTION D'UNE DOSE SUBSTERILISANTE DES IRRADiATIONSAUX RA YONS GAMMA SUR LA DESCENDANCE DE CERATITJS CAP/TATA (WIED.) (DIPTERA: TRYPETIDAE)

S. OUKIL* et R. CAUSSE**

• Station Regionole de la Protection des Vegetaux d',.\lger BP 80 E! Harrach ,.\LGERIE. '* !NRA Station de zoologie site Agroparc Avignon FRANCE Resume: Une dose substerilisante de rayons gamma de 30 Gy determinee experimentalement, appliquee uniquement sur les males de c. capitata a chaque generation n 'induit qu'une sterilite residuelle tres foible chez ies descenc!ants. Ces descendants males reirradies a 30 et 90 Gy, presentent a certair.es generations une fertilite plus elevee que celle observee lors d'un premier traitemert a ces memes doses, pouvcnt faire penser a un phenomene de resistances aux irradictions. ;\lets c!es : Cerotitis copitata- Diptere- Trypetiaae- Rayons gamma- Doses substerilisanres.

- Introduction Ceratitis capitata Wiedemann 1824, denommee le plus souvent «Mouche mediterraneenne des fruits)) est l'un des plus redoutables ravageur des cultures fruitieres en Algerie. Signale pour la premiere fois aux environs d'Aiger en 1858, cet insecte a trouve dans les regions du littoral, et surtout dans les oasis, des conditions climatiques et une diversification des especes fruitieres favorables a son extension. II a pris aussi un grand developpement causant des degats evalues en 1990 par la F.A.O a 2,5 millions _de Dollars . Pour limiter les degats causes par ce ravageur sans modifier la biocenose assez complexe notamment des agrumes , les responsables algeriens ont envisage d'avoir recours a un moyen de luttebiotechnique dite «lutte autocide)> sous forme de lachers de males sterilises aux rayons gamma. Cette conception de lutte a suscite le plus vif interet et necessite en fait , des etudes de base dont l'une des plus importantes est celle qui conduit a obtenir des males, non seulement steriles mais egalement non affectes physiologiquement et dent les potentialites sexuelles ne sont pas diminues par !'agent de sterilisation. Dans le but d'acquerir Jes connaissances de base a la conception d'une methode de Juttepar !'utilisation des doses substeriles, nous avons aborde dans cette etude l'effet de ces doses sur la descendance et Ja mise en evidence d'un eventuel phenomene de resistance chez C. capitata aux radiations ionisantes.

2 - Materiels et Methodes

2. ·1 Protocole experimental Les insectes utilises pour nos experimentations sont ceux de Ja souche du Jaboratoire, maintenus en elevage isole de fa9on a maltriser la descendance des parents irradies. Les insectes sont traites au stade pupes agees de 7 jours, soit 24 heures avant l'eclosion. Des leur emergence, les adultes sont maintenus pendant quelques minutes a une temperature de 11° C, afin de les immobiliser temporairement et de favoriser le - 194 - sexage au moyen d'un aspirateur a bouche. En effet, seuls les males irradies sont utilises et mis en presence d'un meme nombre de femelles normales (issues d'un autre lot de pupes) du meme �ge et non irradiees. Les essais de fertilite sont conduits dans des cages cylindriques construites en altuglass transparent de 20 cm de diametre x 25 cm de hauteur. Dans l'une des faces planes est amenagee une porte grillagee a glissiere, tandis que l'autre face comporte une ouverture circulaire de 6 cm de diametre destinee a recevoir le dispositif de ponte. Celui-ci est constitue par une timbale en matiere plastique de couleur rouge orange, prealablement percee de petits trous d'epingle. Les essais sent effectues a 25° C avec une humidite relative de 40 a 60% et sous eclairage photoperiodique L.D: 12.12.

2.2 L'irradiateur Nos echantillons ont ete irradies a la station de Zoologie qui est equipee d'un irradiateur 60 co de 1200 cu�ies Le dispositif d'irradiation est forme d'une cellule, ou 6 barreaux de cobalt 60 disposes en couronne permettent de traiter un volume de 2 litres. A l'interieur, se deplace verticalement un piston en plomb contenant la chambre d'irradiation qui permet d'introduire et de retirer les echantillons. Les rnouches sont irradiees sous forme de pupes dans des tubes en Pyrex de 1 cm de diametre et 5 cm de hauteur, places sur un support en couronne a l'interieur de la chambre d'lrradiation .

3 - Resultats

3.1 Action des doses substerilisantes sur la descendance Le traitement des individus par des doses sterilisantes provoque souvent la diminution de leur potentialite sexuelle. Cela conduit a la necessite d'accroTtre la quantite d'insectes a lacher et a !'augmentation du prix de revient en raison de l'eievage. C'est la raison pour laquelle on a tendance a s'orienter vers !'utilisation des doses substerilisantes, qui presentent certains avantages par rapporta la sterilisation complete.

3.1.1 Determination de la dose substerilisante Dans un premier temps, nous avons compare 5 doses substerilisantes d'irradiation: 10,20,25,30 et 40 Gy. Pour chacune d'elles, nous introduisons dans des cages cylindriques 20 couples d'insectes formes de 20 males issus de pupes irradiees avec 20 femelles normales, tandis qu'une cage contenant 20 males et 20 femelles non traites sert de temoin. Les oeufs sont preleves quotidiennement et des lots de 100 par cage sont places ensuite avec un peu d'eau dans des boTtes de petri. Apres incubation pendant 20 jours, les larves ecloses sont denombrees du 3eme au 5eme jour. les resultats portant sur le taux de fertilite des oeufs obtenus pour chaque dose sont exprimes dans le tableau I ci­ dessous.

Doses en Gy Nb de couples % moyen de fertilite Ecarts-type 10 20 65 1,05 20 20 51 1,05 25 20 45 0,97 30 20 30 1.23 40 20 19 1,05

Au vu des ces resultats, notre choix s'est porte sur la valeur 30 Gy. En effet nous devons avoir une dose relativement elevee de fac;:on a assurer l'efficacite de la methode, mais - 195 - pas trop de maniere a disposer d'une population descendante suffisamment elevee, permettantla poursuite des experimentations. Dans ce cas notre nouvelle souche est constituee de males irradies a 30 Gy croises avec des femelles normales. Ensuite, a chaque generation nous avons accouple les descendants males et femelles de cette population ainsi obtenue, avec leurs partenaires normaux de la soucha d'elavaga soit M. F1 X F. normales F. F1 X M. normaux De plus nous irradions nouveaux ces males F1 a 30Gy et a 90Gy et nous les croisons avec des femelles normales soit · M. F1 Irr 30 Gy X F. normales M. F1 Irr 90 Gy X F normales

A chaque generation et pour tous ces croisements, nous avons constitue des lots de 20 males et 20 femelles sur lesquels nous avons etudie la mortalite des adultes, le taux de fertilite et le sex-ratio. Notons que le sex-ratio est exprime selon la formule

Nb de males/ (Nb de ma!es + Nb de Femelles)

Le m�me suivi a ete effectue pour deux lots temoins, a savoir . M. normaux X F. normales M. Irr 90 Gy X F. normales

Ce travail a ete effectue pour toutes les generations jusqu'a !a 8eme. A partir de c::tte derniere generation, les descendants males ont ete toujours irradies a 30 Gy et seul le sex-ratio a ete etudie sur les generations 9,10,11,12,14,15,16,21 et 23. Pour les tests de mortalite et !'estimation du taux de fertilite. ils n'ont ete effectues que ponctuellement sur la 13,17,18,19,20 et la 22eme generation.

3.1.2 Effet sur la mortalite

a) Mortalite des maies Au vu de l'histogramme de la (figure I) illustrant les mortalites cumulees des differents croisements, on remarque que le taux de mortalite des males descendant de parents males irradies est plus faible que celui des males temoins. En effet, cette mortalite varie de O a 15% pour les males issus de parents males irradies a 30 Gy. Elle est de 5 a 10% chez ces memes males irradies a leur tour a 30 Gy a chaque generation et elle se situe entre 10 et 30% chez les temoins. De plus au sein de la population temoin, on peut remarquer que la mortalite des males est presque toujours superieure a celle des femelles. Les tests statistiques nous montrent une difference significative au seuil de 0,001 du test T, entre la mortalite des males irradies et celle des males normaux, ainsi qu'entre celles des males normaux et des femelles normales.

b) Mortalitedes femelles En ce qui concerne les individus femelles descendants de parents males issus eux memes de parents (M. Irr a 30 Gy a chaque generation), le taux de mortalite est inferieur chez ces femelles par rapport a celui des femelles normales. On observe une legere diminution de la mortalite chez les femelles issues de parents males substeriles au fil des generations. mais la difference n'est pas significative au seuil de 0,01 du test T. - 196-

Fig l Mortalites des imagos de la F 1 a la F22 en fonction des generations.

30

25

-�.,. ::. 15 3

10

5 Croiscm::n ts A B 8 C C Tcmoins 1 l 1 1 D D1 Temoins 2

§ r.·!ales � Femelles

-Males descendants de males Irr a 30 Gy (A) X Femelles normales (Al) -Males nonnaul((Il) X Femellcs desccndantes de miles Irr a 30 Gy (Il 1) -Males descendants de males ·rrr 30a Gy a chaque generation puis Irr a h:ur tour :I 30 Gy (C) X Femellernonnales (C t) -Temoin. l: Males nonnaux X femelles nonnales -Males descendants de males substcriles et Irra leur tour a 90 Gy a chaque generation (D) X Fcmelles nonnaks (D 1J -Tcmoin 2: Males Irr a 90 Gy X Femelles normales - 197 - 3.1.3 Effetsur le taux de fertilite

a) Taux de fertilitedes males Si l'on considere le taux de fertilite des descendants males issus des parents males irradies a la dose substerile, on remarque que ces derniers presentent une sterilite residuelle plus importante aux generations 1, 2 et 3 comparee a celle des autres generations (figure II). Les insectes de la premiere generation sont relativement beaucoup plus fertiles (environ 2 fois et demie plus) que leurs parents males irradies a la dose substerilisante de 30 Gy . lls presentent tout de meme une legere sterilite residuelle, mais celle-ci est trop faible pour que l'on puisse envisager des lachers de tels insectes dans un programme de lutte autocide. Par la suite, cette fertilite augmente legerement a partirde la F4 avec un taux de 86%, qui se stabilise jusqu' a la F20 et a la F22 on retrouve pratiquement la meme valeur que pour la F3 avec 83%. Par contre, si l'on compare les taux de fertilite de ces memes descendants males soumis a une nouvelle irradiation de 30 Gy, au vu de la figure 111, on note tout d'abord une sterilite residuelle maximale de la F1 a la F3. Ceci pourrait etre du a la sterilite residuelle qui s'est ajoutee a celle provoquee par de nouvelles irradiations gamma. Ensuite apparaft une diminution progressive a la F4 avec une valeur de 35% de fertilite. Cette valeur depasse celle des parents. ce qui pourrait laisser supposer qu'il n'y a plus de sterilite residuelle a partir de ce moment. A la F5, on note une tres legere remontee de cette sterilite, puis une stabilisation avec legere variation autour d'une valeur insignifiante de 1 a 2%. Le calcul statistique nous montre une difference significative par rapport aux parents au seuil de 0,01 du test T, uniquement pour la generation 4.

b) Taux de fertilite des femelles Les femelles descendantes des males substeriles presentent une fertilite aussi importante que celle enregistree chez les males. Sur la figure II. on constate en F1 un taux plus faible (70,58%) que celui des males (78,20%). Ceci peut s'expliquer par le fait que les femelles necessitent des doses d'irradiation plus faible que les males ( Feron 1969, Hafez et Shoukry 1972). Mais au fur et a mesure des generations et pour la majorite d'entre elles ce taux a tendance a etre egal avec celui des males.

3.1.4 Effetssur le sex-ratio En ce qui concerne le sex-ratio, le calcul a ete fait sur 8 lots de 100 pupes, a savoir 4 pour la descendance des males lrradies a 30 Gy et 4 pour les temoins a chaque generation. On constate au vu de la figure IV une nette modification a l'avantage des males dans la descendance des croisements (M. Irr 30 Gy X F. normales). Ainsi, on enregistre de la F1 a F8 un sex-ratio qui varie de 0,53 a 0,58. A partir de la F8. on voit une stabilisation du sex-ratio qui se situe entre 0,52 et 0,54, c'est a dire toujours superieur a 0,50 impliquant un nombre plus eleve de males. Les temoins (M. normaux X F. normales) qui presentent un sex-ratio plus variable mais se situant neanmoins en dessous de 0,5, se caracterisent par une population a dominante femelle. L'analyse statistique nous montre une difference significative entre ces deux populations a chaque generation.

3.2 Etude de la resistance aux irradiations ionisantes Pour la mise en evidence d'un eventuel phenomene de resistance aux irradiations des males (descendants de parents M. irradies aux doses substerilisantes de 30 Gy) nous avons soumis ces memes individus, a des doses de 90 Gy de la 1 ere a la 22eme generation. A chaque generation, nous mettons 20 femelles d'elevage non traites en presence de ces males. En comparaison, nous avons suivi aussi la fertilite des males d'elevage irradies a 90 Gy en tant que temoins. Nous observons sur la figure V que les - 198-

Fig II Fertilite des males et des femelles descendants de males d'elevage irradies a 30 Gy pendant 22 generations.

100

so

20

GCnCrJtior.s 3 5 6 7 8 I 3 I 7 I 8 1 9 20 22

I L' ·') Temoins ( miles nonnaux X femelles nonnaks)

� Fenilitedes miles descendants des males substcriles

[222jFenilite des femelles descendants des miles substcrile;5

Fig Ill : Evolution du taux de fertilite des maies descendants de parents males d'elevage irradies a 30 Gy et reirradies a leur tour a 30 Gy, en fonction des generations de la F 1 a la F22 .

-�·.r.

Gene rJri ons PJ.renrs 2 3 "' S 6 7 8 I 3 I 7 I 8 I 9 20 ,

� Fenilire des remains

� Fertilire des rniilcs irradies a 30Gy - 199 -

Fig IV: Evolution du sex-ratio en fonction de;;, gbn61·2,tio,,s de ic·

Croisement des Maks irro.dies � chaqut g�neiation ---G---- a 30Gy X Femclle normaks

Tcmoins ('k\Jes norrnau, X Femellcs nom1aks)

Fig V : Evolution du taux de fertilite des descendants de parents rnales substeriies apres irradiation a 90 Gy. en fonction des generations.

; )

-�

I} I:; ? 5 6 7 8 I fj

� TCmoins : Miles LIT a 90 Gy X Fcmcl!cs nom1:dcs

� Miles d::sccndantsde pa.rents males subs1C.riks X rcmcllcs norm:ilcs - 200 - males de la F1 a la F22 (issus successivement de parents M. substeriles) irradies a 90 Gy presentent dans !'ensemble des generations une tres faible fertilite, qui varie neanmoins entre 0,55 et 2,75% en moyenne selon les generations, comparees a celles des temoins qui se situent entre 0,20 et 0,80%. Bien que ces valeurs soient faibles, la comparaison avec les temoins au vu des analyses statistiques montre une difference significative au seuil de 0,01 du test T, pour toutes les generations a !'exception de la generation 1. Cette derniere a un taux de fertilite de 0,55% contre 0,65% chez les temoins. Par contre, nous constatons que certaines generations presentent des taux de fertilite superieurs, a savoir les generations 4,13,17 et 18 avec successivement 12,70%, 4,20%, 6,40% et 7% de fertilite figure V. L'analyse statistique pour les 4 generations nous montre des ecarts significatifs au seuil de 0,001 du test T comparees a leurs temoins (M. Irr 90 Gy X F. normales). Ce resultat peut-il faire penser a un debut de phenomene de resistance, provoque par les doses substerilisantes de 30 Gy appliquees a toute les generation. A la generation 4 on enregistre un taux de fertilite moyen de 35%. II s'agit du seul cas qui differe significativement par rapport au taux de fertilite des parents (M. Irr a 30 Gy X F. normales). Concernant les mortalites de ces males substeriles et irradies a 90 Gy, les resultats font apparaitre un ecart significatif par rapport aux temoins. Ainsi, chez ces memes males on enregistre une mortalite inferieure (6,78%) par rapport a celle des males d'elevage irradies a 90 Gy (11,07%). (Figure I).

4 - Discussion

Apres avoir determine experimentalement·une dose substerilisante de 30 Gy sur les males de C. capitata, nous avons etudie la mortalite, le taux de fertilite des descendants et le sex-ratio sur plusieurs generations successive, en soumettant a chacune d'entre elles les males seuls a cette meme dose d'inadiation. La mortalite suivie pendant 30 jours pour chaque type de croisement fluctue en fonction des ces derniers et des generations, mais dans I 'ensemble nous remarquons que les males descendants de parents males substeriles, y compris lorsqu'ils sont irradies a nouveau soit a 30 Gy ou 90 Gy, presentent une mortalite significativenient plus faible que les temoins (0 a 15% au lieu de 10 a 20%). Seuls les males irradies pour la premiere fois a 90 Gy ainsi que les femelles descendantes de parents males substeriles ne presentent pas de differences de mortaiite. En ce qui concerne cette mortalite en relation avec les irradiations, Enfield et al. (1981) estiment que !'augmentation de la longevite des individus irradies est peut etre le resultat de la selection de genes, ayant un rapport direct avec la resistance aux irradiations ou le resultats de la selection de genes qui augmentent la longevite. Ces auteurs ont realise une selection genetique sur un coleoptere Antonomusgrandis pour !'evaluation du niveau de mortalite apres irradiation. Dans cette experience dix generations ont ete selectionnees sur lesquelles un accroissement de la longevite a ete constate au fur et a mesure des generations. Au niveau de la fertilite,on remarque que celle-ci se maintient au cours des generations a un niveau relativement eleve (variant de 70% a 90%) chez les descendants des deux sexes issus de parents males substeriles, meme si l'on continue a irradier partiellement les males a la meme dose. Ainsi, contrairement aux nombreux lepidopteres tels que: Trichop/usia ni (North et Holt 1968), Heliothis virescens (Pros hold et Barte Ii 1970) ou Laspeyresia pomonella (Bulyginskaya et Velcheva, 1977) on ne retrouve pas chez C. capitata cet effet des doses substerilisantes donnant lieu a une descendance a sterilite notable, ce qui interdit done toute utilisation de ces individus a des fins de lachers. Lorsque les males des differents generations sont eux memes irradies a nouveau a 30 Gy, ils presentent une fertilite assez faible en F1, F2 et F3 par rapport a leurs parents males. Celle-ci augmente significativement par la suite a la F4, pouvant faire penser a - 201 - un phenomene de resistance, mais a la F5 on remarque a nouveau une baisse de cette fertilitequi se stabilise par la suite a une valeur identique a celles des parents jusqu'a la F22. Quand ces memes mi'.\les sont soumis a une dose de sterilisation complete, les taux de fertilite restent tres faibles et pratiquement identiques a ceux observes generalement dans ce type d'experimentation, mais neanmoins significatifs compares a !eurs temoins, Ces resultats et plus particulierement en F4, F13, F17, et F18 ou apparaissent des taux de fertilite significativement plus eleves, pourraient permettre de dire que malgra la variabilite de ces donnees, une amorce de ce phenomena de resistance aux irradiations n'est peut �tre pas a ecarter. En ce qui concerne le sex-ratio, on remarque chez les individus descendants des males irradies, un taux qui evolue de 0,52 a 0,58 au cours des differentes generations, ce qui denote une legere majorite des males. Au contraire chez les individus normaux, ce sont les femelles qui sont majoritaires par rapport aux males avec un sex-ratio qui varie de 0,42 a 0,49. Ces observations ont ete faites plusieurs fois, essentieilement chez !es lepidopteres tels que : Heliothis verescens par Proshold et Sarteli (1970), Laspeyresia pomonellapar Charmilot et al. (1973). Les resistances aux insecticides chez !es insectes est a present bien connue et certains mecanismes qui interviennent dans ce phenomene ont ete analyses. Par ailleuis certains auteurs ont commence a mettre en evidence une resistance aux radiations ionisantes. C'est ainsi que Terzian et Stahler (1966), en exer9ant une pression de selection au moyen de radiations gamma sur une souche d'elevage de moustique Aedes aegypti par exposition des oeufs a une dose de 20 Gy, montrent sur les dix premieres generations une resistance aux effets de ces rayonnements et une augmentation significative de la duree de vie de -ces moustiques comparee aux temoins. Toujours sur Aedes aegypti et par exposition a 25 Gy pendant 90 generations, Stahler en 1971 met en evidence ce phenomene de resistance sur la fertilite qui atteint 58,30 en moyenne de la 64eme a la 90eme generation, comparee aux temoins qui ne depassent pas 11 de fertilite moyenne. De plus cette pression de selection se traduit chez ces individus par une mortalite ne depassant par 30% par rapport a celle de 93% enregistre chez les temoins. Enfield et al. (1983) en suivant experimentalement sur 20 generations l'accroissement de la duree de vie d'un coleoptere Antonomus grandis soumis a !'exposition des radiations ionisantes, trouvent sur les 12 premieres generations une diminution de la mortalite de 35% a 90% durant 14 Jours. A partir de la 17eme generation les resultats montrent qu'il n'y a pas de difference de mortalite avec les temoins . Par ailleurs a !'issue de cette etude, ils en deduisent que !'augmentation de la duree de vie a aussi un effet positif sur la fertilite et rapportent que des experimentations anterieures sur d'autres insectes, suggerent une probable variabilite genetique pour la resistance aux irradiations. Falconer (1960) dans une etude bibliographique, indique que l'accroissement de la resistance a partir d'une selection cesse habituellement en 25 generations, tandis que Dudley, en 1977; et Yoo (1980) montrent que !'augmentation de la resistance a partir d'une selection peut continuer jusqu'a 50 voire 100 generations. Par ailleurs, Hill (1982) pretend qu'a partir d'une pression de selection aux irradiations, ajoute au fait de travailler sur des populations tres importantes tout en augmentant le nombre de generations, est souvent a l'origine de !'apparition de phenomenes de resistance de nature differente (biologique ou physiologique) lies entre eux. Eiche (1973) en utilisant les techniques de substitution des chromosomes dans le cas de Drosophila mefanogaster, a montre que les genes qui interviennent dans les phenomenes de resistance aux irradiations sont disperses dans les differents chromosomes, rendant le caractere polygenique nature!. Cela corrobore le travail anterieur de Parsons et al. (1969) qui ont demontre que les genes polymorphes sont associes aux effetscumules. aussi bien sur le 2eme que sur le 3eme chromosome chez - 202 - Drosophila. Les analyses des auteurs Ogaki et Nakashima-ianaka (1966) ont confirme l'hypothese selon laquelle un tres grand nombre de genes pour la resistance aux irradiations seraient localises a !'extreme bout du chromosome 3.

REFERENCES

BULYKINSKA YA M.A., VELCHEVA N.V. ( 1977) - The effet of the complete steniisation of imago of Laspeyresia pomonella L. (Lepidoptera, Tortricidae) on the reproductive functions of their offspring. Entomol. Obzr .• 56 (3) : 475. CHARMILOT P.T., FOSSATI A., STAHL J. (1973) - Production de males steriles du carpocapse des pommes (Laspeyresia pomonella L.) descendant de parents substeriles et examen de leur competitivite en vue de la Jutte autocide. Rech.agrom. Suisse. 12: 181. DUDLEY J.W. {1977] - 76 generations of selection for oil and protein percentage in maize. !n : pollak E; Kimpthorne 0., Bailey T.B. (eds) Proc. lnt. Conf. Quant. Genet. Iowa State University Press. Ames: 459. EICHE A. (1973] - Localization of genes controlling radioresistance in Drosophila melanogaster. Hereditas '75 29. ENFIELD F.D.. NORTH D.1., ERICKSON R. (198 l J - Response to selection for resistance to gamma irradiation in the cotton boll weevil. Ann. Entomol. Soc. Am., 74 422. ENFIELD. F.D.. NORTH D.L ERICKSON R .. ROTERING L.. ( 1983) - A selection response plateau for radiation resistance in the cotton boll weevil. Theor Appl. Genet. 65 277. FALCONER D.S. (1960) - Introduction to quantitative genetics. The Ronald. Press. Campany. New york : 35 p FERON M. (1969] - Etude sur la mouche mediaterraneenne des fruits en Tunisie. in . FAO IAEA Insect ecology and the sterile-ma! technique. Proc. of a panel on insect ecology as related to control of noxious insects by the sterile-male technique. Vienna, 7- 11 august 1967. IAEA, Vienne : 102 p. HAFEZ M., SHOUKRY A. (1972) - Eft'et of irradiation on adult fecundity and longevity of the Mediterranean fruit fly Ceratitis capitata Wied. (Diptera: Tephrytidae) in Egypt. Z. Angew; entomo.l., 72 59. HILL W.G. (1982) - Rates of change in quantitative traits from fixation of new mutants. Proc. Nat. Acad. Sci. Usa., 72 142. NORTH D.T., HOLT. G.G. (1968) - Genetic and cytologic basis of radiation induced sterility in the adult male cabbage looper, Trichoplusia ni In Isotopes and radiation in entomology. Proc Symposium FAO/IAEA, Vienna, 4-8 dee. 1967. IAEA, Vienne · 391. OGAKI M., NAKASHIMA-TANAKA E. (1966) - Inheritance cf radioresistance in Drosophila Mutat. Res., 3 : 438. PARSONS P.A., MACBEAN 1.T.. LEE B.T.O. (1969) - Polymorphism in natural populations for genes controlling radioresistance in Drosophila. Genetics, 61 : 211. PROSHOLD F.J.. BARTELi J.A. (1970) - Inherited sterility in progeny irradiated male tobccco budworms : effets on reproduction, developemental time, and sex-ratio. J. Ecom. Entomol.. 63 280. STAHLER N. (1971 J - Changes in survival and tolerance levels in a radiation-resistance strain of Aedes aegypti (Diptera Culicidae) during 90 generations. Ann. Entomol. Soc. Am., 64 643. TERZIAN LA. STAHLER N. (1966) - A selected strain of Aedes aegypti resistant to gamma radiations. Radiat. Res., 28 643. YOO B.M. (1980] - Long term selection for a quantitative character in large replicated populations of Drosophila melanogaster. Genet. Res., 35 : 1 - 203

ETUDEDE LA VARIABILITE GENETIQUECHEZ 6 POPULATIONS DE CERATITIS CAP/TATA DANS DIFFERENTES ZONES GEOGRAPHIQUES DU BASSIN MEDITERRANEEN S. OUKIL*, R. BUES**, R. CAUSSE** et J.F. TOUBON** * Station rcgionale de la protection des vegetaux d'Alger BP 80El Harrach ALGERlE ** !NRA Station de zoologie site Agroparc Avignon FRANCE Resume \'elude de la variabilitc gcnctique et des 11ux gcniques, par clectrophorese enzymatiquc de 22 loci, sur 6 populations de C. capitata prelevees dans Jes zones geographiques du Bassin Mediterraneen; nous a perrnisre confirrnerle faible polymorphisme deja observe par d'autres chercheurs dans ces memes regions. La comparaison des frequences allcliques realisee au locus Pgm, fail apparaHre une asscz forte heterozygotie entre Jes 6 populations et malgre le faible polymorphisme enregistre sur ces dcrnieres, nous avons pu deduire que Jes echantillons recoltes a Pcrpignan et a Montpellier ont pour origine probable la population d'Afrique du Nord, tandis que celui d'Avignon ser.iit plutot originaire de Corse ou d'Espagne. l - INTRODUCTION

L'objectif de cette etude est, d'une part de comparer la structure genetique des populations de ceratitiscapitata dans le sud de la France (ou cette espece ne peut hivemer et ou on observe assez regulierement des degats sur pomme, poire et kaki) avec celle d'autres populations des pays du Bassin Mediterraneen, avec lesquels existent d'importants echanges commerciaux et d'autre part d'etendre a d'autres zones geographiques les travaux realises en Afrique et en Amerique. 2- MATERIEL .ET M.ETHODES.

2.1 - OlUGINE DES POPULATIONS

Les 6 echantillons de C. capitata ex.amines sont issus de pupes prelevees sur fruits contamines. Trois echantillons preleves en France dans les regions de Perpignan, Montpellier et Avignon, une souche d'Algerie (Boufarik), une souche de Corse (San Giuliano) et une souche d'Espagne (Gerone). Dans cette etude chaque echantillon est considere comme une sous­ population appartenant a une population totale. La souche de reference est maintenue en elevage de masse depuis plus de 120 generations sur alimentation artificielle.

2.2 - ANALYSE ENZYMATIQUE

le genotype des individus a ete etabli pour 22 loci. 21 ont ete analyses sur gel d'amidon: Pgm, Hbdh, ldh, Aat+, Aat-, Pgi, Adh+, Adhl-, Adh2-,Akl, Hkl, Hk2, Mdh, Me, Gpdh, Ldh, Pgd, Tox, Lap 1, Lap2, Aco. Seule l'esterase a ete analysee sur gel d'acrylamide. a) Electrophorese sur gel d'amidon

Elle est realisee selon la technique de Pasteur et al. ( 1987) a la concentration de 13% (15 g de saccharose et 39 g d'amidon), avec un tampon Tris citrate pH 6,3 selon Unruh ( 1990)soit : Tris 0,29 g pour 330 ml, acide citrique 0,19 g pour 300 ml, avec un pH de 6,7 pour le gel et 6,3 pour la cuve. Chaque individu est broye dans dans 25 µI de tampon (Tris 14,5 mM, acide borique 0,9 mM, EDTA 0,1 rnM, bleu de bromophenol I mM). La migration dure 6 heures pour une tension reglee a 150 volts. Les revelations sont conduites selon Jes procedes soit de Pasteur et al. ( 1987)soit de Hillis et Moritz ( 1990). - 204-

b) Electrophorese sur gel de polyact"ylamide Elle est realisee sur gel d'acrylamide en plaque (T 24,6%, C 2,4%) pour le locus esterase, avec un tampon Tris-acide borique et un pH 8,4. Les adultes de C. capitata sont broyes entiers individuellement dans 80 µI de tampon pH 6,9 (Tris 14,53 mM, acide borique 0,85 mM, EDTA 0, 12 mM, bleu de bromophol 50 mM). La migration s'effectue sous une tension constante de 250 volts pendant 16 heures. La revelation est obtenue avec de !'alpha naphtyl acetate et la col oration avec du fastgarnett.

2.3 - ANALYSE DES RESULTATS Suivant Jes auteurs, la limite du polymorphisme pour un locusdonne est situee pour une frequence de !'allele majoritaire soit a 99 ou 95 % (Pasteur, 1985). Nous avons utilise la limite de 99%; si la frequencede !'allele majoritaireest superieure a 99%,le locus est considere monomorphe. Les resultats du test de conformitedes structures genotypiques avec celles donnees sous l'hypothese du respect de la Joi de Hardy Weinberg, consistent a comparer les frequences des genotypes observes et celles attendues pour chaque locus dans chacune des solis­ populations. La presence de faibles effectifs attendus (inferieurs a 5) interdit quasi­ systematiquement !'utilisation du X2 pour tester l'homogeneite des frequencesalleliques. Nous avons dans ce cas utilise le test exact de Fishe�, a l'aide du Jogiciel Biosys 1. - L'appreciation des equilibres de la loi de Hardy Weinberg est egalement obtenue grace aux indices definis par Wright (1943; 1951). On distingue deux indices de fixation pour chaque locus: -Fit: Mesure les deviations de la population totale. -Fis: Mesure Jes deviations dans les sous-populations. Les valeurs des indices de fixation varient entre -1 et +l. Une valeur negative traduit un exces d'heterozygotes, tandis qu'une valeur positive traduit un defaut d'heterozygotes. Les F statistiques et les distances genetiques (Nei 1978) ont ete effectues a !'aide de Biosysl, Swofford et Selander (1989). Les frequences alleliques des loci polymorphes, sont comparees a !'aide du test G ( Scherrer, 1984 ). L'etude de Ja differenciation genetique des sous-populations est abordee par le calcul de l'indice Fst.Wright en 1951 a defini Fst comme indice mesurant le degre de differenciationgenetique entre sous-populations.Les valeurs de cetindice sont calculeespar le logiciel Biosys-1, selon la formulationde N ei (1977) et par Diploid (Wier, 1990). Ce demier logiciel nous a permis d'utiliser la procedure de Wier et Cockerham (1984) pour calculer Fst (rebaptise-

Nm= (I/ Fst- 1)/4 3 - RESULTATS

Les frequences alleliques pour chacun des loci polymorphes sont indiquees sur le tableau I; est egalement presente le nombre d'heterozygotes observes et attendus. Seu! le locus Pgm de la sous-population de Montpellier presente un nombre d'heterozygotes significativement superieur. (test des probabilites exactes).

Tableau 1: Frequences alieliques des differents loci polymorphes

Loci Perpignan Boufarik Montpellier Avignon Gerone San Elevage Guiliano Pgm (N) 24 96 48 48 24 23 24 A 0,000 0,000 0,000 0,000 0,000 0,000 0,000 B 0,917 0,906 0,833 0969 1,000 0,976 0,875 C 0,083 0,094 0,167 0,031 0,()00 0,021 0,125 Obser. 4 16 16 3 I 1 6 Allen. 3,745 16,398 13,474 * 2,937 I l 5,362 H bdh (N) 24 96 36 35 23 24 36 A 0,000 0,005 0,000 0,000 0,000 0,000 0,000 B 1,000 0,990 1,000 1,000 1,000 1,000 1,000 C 0,000 0,005 0,000 0,000 O,

Sur 22 loci analyses, 5 seulement sont polymorphes (Pgm, Hbdh, Idh, Aat+ et Pgi) et presentent pour une ou plusieurs populations, une frequence de !'allele le plus representatif inferieur ou egale a 99%, ceci pour un nombre moyen d'individus analyses par locus jamais • 206 - inferieur a 20 (tableau II). Seule la population d'Espagne est monomorphe pour tous les loci. Toutefois, la souche d'elevage reste polymorphe pour 2 loci malgre un grand nombre de generations.

Tableau II : V ariabilite genetique des 22 loci pour les differentes sous-populations

Populations Moyennes des Nb moyen d'alleles pourcentagesdes heterozygoties echantillons par par locus loci polymorphes moyennnes locus (3) (3) (2) (]) obsef\ees (3) Pemignan "7,8 (",3) 1,1 (0,1) 9,1 13,6 0,018 (0,010) Boufarik 79,8 (7,1) 1,3 (0,1) 4,5 13,6 0,011 (0,008) Montpellicr 30,0 (1,9) 1,1 (0,1) 4,5 13,6 0,018 (0,015) Avignon 27,9 (3,9) 1,1 (0,l) 0,0 9,1 0,004 (0,003) Gcronc 22,0 (1,8) 1,0 (0,0) 0,0 0,0 0,000 (0,000) San Guiliano 20,6 (1,7) 1,1 (0,1) 0,0 13,6 0,006 (0,003) Elcvagc 24,0 (2,2) 1,1 (0,1) 9,1 9,1 0,019 (0,013) (]) Allele le plus frequent a 95'7': (2) Allele le plus frequent a 99%. (3) Ecarl type de la moyenne L'heterozygotie moyenne observee est faible avec un taux moyen pour les sous­ populations de 0,0095. La comparaison des frequences observeesavec celles donnees par la loi de Hardy Weinberg montre que sur les 18 loci analyses seule la population de Montpellier fait appara1tre un exces d'heterozygotes au locus Pgm Dans le tableau III sont indiques les differents F statistiques de Wright pour les 6 sous-populations. Les valeurs du Fit, bien que toutes negatives n'indiquent qu'un tres faible exces d'heterozygotie, car tres voisines de 0. L'indice du Fis est egalement negatif pour toutes les sous-populations, mais tres voisin de 0, excepte pour la Pgm ou l'exces d'heterozygotes est sensible comme l'indique ci­ dessus !'analyse de Hardy Weinberg. Les valeurs du Fst, qui mesurent le degre de differenciation genetique entre les sous-populations, varient de 0,003pour le locus ldh a 0,051 pour Pgm avec une moyenne de 0,037.

Tableau III: Differents F statistiques de Wright des loci polymorphes

Loci Fis Fit Fst Pgm -0,098 -0,042 0,051 Hbdh -0,008 -0,001 0,007 Idh - 0,004 - 0,001 0,003 Aa+ -0,048 -0,024 0,0"3 p g i -0,031 -0,014 0,016 Movenne -0,075 - 0,033 0,037 La differenciation genetique entre sous-populations peut egalement s'apprecier a !'aide du calcul des distances genetiques qui varient de O a 0,00048, ce qui confirme la tres faible variabilite genetique entre les populations. Le calcul des flux geniques (Nm) entre les sous-populations d'apres Wright (1951)

Nm = ( 1/0,038) - I = 6,32 4 montre que quelle que soit !'importance de la population locale 6 a 7 individus en moyenne peuvent theoriquement s'echanger avec d'autres sous-populations. - 207

Les resultats des calculs du Fst selon Weir et Cockerham (appele e par ces auteurs en 1984) sont indiques sur le tableau IV.

Tableau IV : Resultats des Fst selon la methode Weir et Cockerham et flux genique (Nm) entre sous-populations differemmentregroupees.

Fst selon Weir Ecart-type Intervalle de Nm Sous-populations et Cockerham confiance a 95 % (1984) Perpignan Boufarik Montpellier 0,0250 0,0167 -0,0048 II 0,034 9,75 Avignon Gerone San Guiliano Perpignan Montpellier 0,0391 0,0223 0,0018 // 0,0547 6,147 Avignon Boufarik Gerone 0,0182 0,0241 - 0,0104 II 0,030 13,486 San Guiliano

Dans le tableau IV , differents regroupementsde sous-populations.ont ete. realises. La difference entre les 2 techniques de calcul est sensible, 0,038 pour la methode de Wright, 0,025 pour celle de W�fr et Cockerkam pour !'ensemble des 6 sous-populations. L'adjonction de la souche d'elevage ne modifie pas sensiblement Jes valeurs (Fst = 0,0229). L'observation des frequences alleliques du tableau I, fait apparaltre deux groupes de populations differenciees par les pourcentages d'alleles rares : d'une part les sous-populations de Perpignan, Montpellier et Avignon, d'autre part celles de Boufarik,Gerone et San Guiliano. Le calcul montre que les valeurs de Fst sont respectivementde 0,0391 et 0,0182 ce qui conduit a un flux genique inferieur pour les sous-populations de France(Nm= 6,1) par rapport a celles de Boufarik, de Gerone et de San Guiliano (Nm = 13,4). Le Nm calcule pour toutes Jes populations selon e est de 9,75, c'est a dire peu different de celui calcule par Fst Biosys 1 qui est de 6,32. Malgre le faible polymorphisme observe Jes frequences d'alleles entre sous­ populations ont ete comparees pour Jes deux loci Jes plus polymorphes (Pgrn et Aat) a !'aide du test G (Tableau V) Tableau V: Comparaison des frequence alleliques a l'interieur des differents groupes des sous-populations (test G)

Sous-populations Pgm Aat Perpignan, Boufarik,Montpellier, Avignon, Gerone, San Guiliano 23,24* * * 11,66 et la souche d'elevage Perpignan,Boufarik,Montpellier, A vignon, Gerone et San Guiliano 22,13* * * 8,38 Perpignan, Montpellier et 10,75* * 5,32 Avignon Boufarik, Gerone et San- 10,39* * 1,68 Guiliano • • S1gruf1cat1ve au seml de I% * * * Significative au seuil de 1 % - 208 -

Pour le locus Pgrn la valeur du X2 est de 23,24 avec 2 alleles et 7 echantillons, soit 6 ddl; ii est significatif au seuil de l % . Par contre le locus Aat de 11,66 pour 6 ddl n'est pas significatif. La valeur du X2 varie peu et reste hautement significativelorsque l'on ne considere que les 6 sous-populations. La souche d'elevage est peu differente de !'ensemble des sous-populations. Par ailleurs, Jes deux derniers groupes consideres dans le tableau IV, en !'occurrence les 3 sous­ populations recoltees en France (Perpignan, Montpellier et Avignon) et celles de Boufarik, Gerone et san Guiliano, montrent une valeur du X2 sensiblement egale et significativeau seuil de I%, uniquement pour le locus Pgm. Par ailleurs, la comparaison par le test G montre que la souche d'elevage est significativement different� au seuil de 5% de la sous-population geographiquement tres voisine, recoltee dans l'Ile de la Barthelasse (region d'Avignon); elle est de plus significativement differente des sous-populations de Gerone et de San Guiliano. Par contre e\le ne se differencie pas de celle de Montpellier, ce qui est surprenant si !'on considere la derive genetique possible apres plus de 120 generations d'elevage. 4 - DISCUSS!ON

L'electrophorese enzymatique de 22 loci realisee sur 6 sous-populations prelevees dans 4 zones geographiques du Bassin Mediterraneen: Algerie, Corse, Espagne et France, a permis de degager Jes resultats suivants: - Un faible taux de polymorphisme enzymatique, 5 loci seulement sont polymorphes au seuil 99% pour !'allele le plus frequent. - Les analyses montrent que les echantillons sont en conformite avec les postulats de la loi de Hardy Weinberg ( excepte pour le locusPgrn de l'echantillon de Montpellier). - L'heterozygotie observee sur !'ensemble des 22 loci analyses varie de O a 0,019 (moyenne = 0,010) et le nombre moyen d'alleles par locus de 1,1. - Le calcul du Fst moyen varie selon la methode utilisee de 0,025 (Weir et Cockerham, 1984) a 0,037 (Wright, 1951). Ce demier mode de calcul etant celui le plus couramment utilise dans la litterature. - Les distances genetiques calculees suivant Nei (1978) entre sous-populations varient de O a 0,00048(moyenne = 0,0007) - Le flux genique calcule suivant la methode de W!"ight (1951) a partir du Fst est de .6,37. - La comparaison des frequences alleliques selon 'letestGrealise au· locus Pgm fait apparaJtre une assez forteheterozygotie entre Jes 6 sous-populations. Plusieurs auteurs ont etudie le polymorphisme enzymatique de cette espece dans differentesregions du monde. Morgaae et al. en 1981 au Bresil, sur 4echantillons issusde 2 localites differentes et 2 plantes hotes (peches et oranges) observent au seuil de 99% un taux de polymorphisme de 4 loci sur 13 analyses (30,70%), soit legerement superieur a celui que nous observons (22,7%). D'apres ces auteurs le faible taux observe serait la consequence d'une introduction relativement recente de l'espece au Bresil. Les travaux les plus importants dans le Bassin Mediterraneen ont ete realises par une equipe italienne. Gasperi et al. (1991) ont compare la differenciation genetique des populations originaires des Iles mediterraneenne (Sardaigne et Procida) avec, d'une part une population de l'ile de la Reunion et d'autre part d'une population du Kenya. La comparaison realisee a !'aide du critere des distances genetiques (D = 0,011) ne revele qu'une tres faible differenceentre Jes 1les de la Sardaigne et de Procida par contre, la distance genetique observee entre les populations mediterraneenneset celles du Kenya est nettement superieure ( D = 0,026 - 0,028) et c'est avec 1'11e de la Reunion que celle-ci est la plus grande (0,101 - 0,102). La difference entre l'tle de la Reunion et le Kenya est egalement elevee (D = 0, 107). Rappelons qu'elle etait de moins de 0,0004 entre nos 6 sous-populations. Le Fst moyen calcule par ces auteurs pour les 4 populations est de 0, 123, valeur elevee due a !'introduction des populations de l'ile de la Reunion et du Kenya. Les resultats de ces auteurs montrent une variabilite - 209 nettement plus e!evee dans les populations preleves en zones tropicales et avancent l'hypothese que l'espece pourrait etre originaire de cette zone geographique. Gasperi et al. avaient montre dans une etude anterieure ( 1987) que la population de Li bye etait moins variable que celles de Sardaigne et de l'ile de Procida. La comparaison entre sous-populations libyennes et celles etudiees dans ce travail montre un fort degre d'homogeneite des principaux criteres (Heterozygotie, % de loci polymorphes et nombre moyen d'alleles par locus). KourH et al. (1992) considerent les populations d'Amerique et du Bassin Mediterraneen comme etant introduites a partir d'une population originelle situee au Kenya. Huetteli et al. ( 1980) travaillant sur des populations d'Afrique du Sud, d'Israel, des iles Hawaii et du Costa-Rica constatent que la population d'Afrique du Sud est la plus variable, mais ces auteurs ne calculent pas d'indice du degre d'isolement. La comparaison des flux geniques calcules par Jes differents auteurs est delicate, compte tenu du faible polymorphisme ou de l'isolement spatio-temporel des differentes populations. La synthese des resultats fait cependant apparaitre un flux genique suffisamment eleve pour expliquer la relative homogeneite de la plupart des populations, exceptees celles de l'ile de la Reunion, du Kenya et d'Afrique du Sud. D'apres Hagen et al. (1981), c'est seulement depuis 1842 que Jes premieres populations de C. capitata ont ete observees en Espagne, sensiblement a la meme periode qu'en Algerie 1858 d'apres Balacilowsky ( 1932). Gasperi et al. 1991 formulentl'hypothese que l'espece serait originaire du Kenya, dans laquelle se retrouvent Jes alleles detectes dans Jes autres populations notamment celles du Bassin Mediterraneen. Cette hypothese est compatible avec un effetfondateur (Wallace 1974). Une fraction seulement du pool genique de l'espece colonise une autre zone geographique. Sur cette nouvelle population s'exerce de nouvelles pressions de selection,. (une derivegenetique) qui conduisent a une differenciation entre sous-populations dans une meme aire geographique. Si l'on considere Jes echantillons recoltes dans le sud de la France ou l'espece ne peut pas hivemer et compte tenu de la similitude des alleles observes, on peut faire l'hypothese que les echantillons re.coltes a Perpignan et a Montpellier ont pour origine probable la population d'Afrique du Nord. La propagationde C. capitata aurait pu se faireaccidentellement par les ports de Sete ou de Port-Vendres. Par contre la population de la region d'Avignon aurait plutot comme origine la Corse ou l'Espagne et un port du sud-Est aurait pu etre a l'origine des contaminations. II est done vraisemblable que la structure genetique des echantillons recoltes dans le sud de la France varie d'une annee a l'autre selon l'origine des adultes introduits. Les resultats obtenus dans cette etude confirment le faible polymorphisme deja observe par d'autres auteurs. Toutefois, la technique de l'electrophorese enzymatique ne permet de revel er qu'une partie de la variabilite totale des populations. D'autres techniques directement appliquees a l'A.D.N. (R.F.L.P., R.A.P.D.) sont aujourd'hui disponibles et ont fait l'objet d'essais preliminaires sur C. Capitata par Haymer et Mcinnis en 1994. Neanmoins, le facteur limitant que nous avons rencontre demeure l'echantillonnage des populations dans des aires geographiques eloignees.

References BALACHOWSKY A. (1932) - La mouche des fruits (CeratitiscapitataWeid) Ministere de !'agriculture Service de la defense des vegetaux France : 7 p. GASPERI G., GUGLIELM!NO C.R., MALACllUDA A.R., MILANI R. (1991) - Genetic variability and gene flow in geographical population of Ceratitis capitata(Wied.) (medfly) Heredity, 67: 347. GASPERI G., MALACR!DA R., MILANI R.(1987) - Protein variability and population genetics of Ceratitis capitata In : Economopoulos, A P.( ed), Fruit flies. Elsevier, Amesterdam, 149. HAGEN S.K., ALLEN W.W., TASSAN L.R. (1981) - Mediteranean fruit fly. The worst may be yet to come. CaHf. Agric. (march-april) : 5. HAYMER D.S., McINNIS D.O. (1994) - Resolution of populations of the Mediterranean fruit fly at the DNA level using random primers forthe polymerase chain reaction. Genome, 37 : 244. - 210 -

HILl.iS D.M., MORITZ C. (1990) - Molecular systematics. Sinauer Associates, Sunderland, 588 p. HUETTEL M.D., FUERST P.A., MARUYAMA T., CHAKRABORTY R. ( 1980) - Genetic effects of multiple population bottlenecks in the mediterranean fruit fly Ceratitiscapitata. Genet��s, 94 : 47. KOURTI A., LOUKAS M., SOURDJ!S J. (1992) - Dispersion pattern of the medfly from its geographic centre of origin and genetic relationships of the medfly with two close relatives. Entomol. Exp. Appt, 63 : 63. MORGANE J.S., DE SOUZA M.L., DE CONT! E., CYTRYNOWiCZ M. (1981) - Allozyme variability in an introduced fruit fly pest CeraJitis capita/a (Wiedemann), 1824, (Diptera-Tephritidea). Rev. Brasil., Genet., 4 : 183. NEI M. (1977) - F-statistics and analysis of gene diversity in subdivided populations. Ann. Human. Gen. 41 : 225. NEI M. (1978) - Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics, 89 : 583. PASTEUR G. (1985) - Les parametres statistiques communement utilises dans !'exploitation des resultats de l'electrophorese des proteines et leur avenir en systematique. In: GOYFFON M., D'HONDT J.1.., E!ectropno:.-ese et taxonomie. §odet

Chairman: A M. Wakid Secretaries: Afellah Mohamed Dhouibi M. Habib - 211

MADEIRA-MEO, SIT PROGRAM AGAINST MEDFLY IN MADEIRA, PORTUGAL

1 1 1 1 R. Pereira , A. Barbosa', A. Brazao , N. Silva , J. Pacheco', A. Rodrigues , J. Caldeira', 2 3 L. Dantas1, J.P. Carvalho and D. Lindquist •

l-Direc9iio de Servi9os de Investiga9iioAgricola, Estrada Eng' Abel Vieira, 9135 Camacha, Madeira, Portugal 2-Esta9ao Agron6mica Nacional, Quinta do Marques, 2780 Oeiras, Portugal 3-Insect control consultant, Friedlg. 25/2, I I 90 Vienna, Austria

Summary

The Mediterranean fruit fly, Ceratitis capitata (Weid.), is a major pest of fruit in the Portuguese Autonomous.Region.of Madeira (Vieira, 1952). The medfly was reported to attack more than 40 species of fruit. It was reported as a pest primarily below 400m but occurred up to 700m on the south coast. The Sterile Insect Technique (SIT) is a genetic method of insect control. Huge numbers (frequently more than 50 million/week) of the target-insect are reared in specially designed factories (Lindquist et al., in press). These insects are sterilised with gamma radiation and released by aircraft into the target area. Mating between the factory reared sterile males and fertile wild females produce no progeny. Thus if sufficient sterile males are introduced into the target area on a continuos basis their is a very high probability that fertile wild females will mate with sterile males. Under these conditions the birth rate of the target species is greatly reduced and will rapidly reach zero if no fertileinsects are brought into the target area. To use the SIT effectively against the medfly in Madeira requires an area-wide approach. This is different from the usual field by field approach in that the entire pest population in the target area is attacked rather than only the insects in the individual farmer's field. Thus for the Madeira-Med Program, the entire medfly population on the island is the target.

!-Introduction

Madeira is located 980 km W-SW from mainland Portugal and has a population of approximately 255,000. The island is volcanic with very little level land suitable for large agricultural production. Approximately 47% of the land area is above 700m. Thus the area likely to require medfly control is about half of the island. Agricultural production is in small scale, frequently part time and mostly terraced because of the volcanic nature of the land. Grapes for wine and bananas are the predominate fruit crops. Neither are primary medflyhosts. Citrus and tropical fruits are not produced in large quantities and are generally not high quality. This is to a large extent because intensive medfly attack has prevented the establishment of citrus and tropical fruit production. Medflies are present year round below 300m, resulting in the necessity of continuos control measures, usually - 212 insecticide bait sprays. Current annual losses from the medfly in Madeira are estimated at US$ 3 million. In 1992 the agricultural officials of Madeira applied for an European Union (EU) grant to eliminate the medfly from Madeira using the Sterile Insect Technique (SIT). After extensive discussions the project was changed from eradication to control and approved in late 1993 with EU support of about US $ 4 million over a 7 year period. Subsequently the Madeira officials applied for and received a technical assistance project from the International Atomic Energy Agency (IAEA). The IAEA support is primarily for training, technical advise and consultant services. The shift from eradication to control using the SIT is significant as it represents the first planned use of the SIT for medfly control. Eradication has been the objective of all other medfly SIT programs. The Madeira-Med Program was composed by field activities, mass production, quality control, flyhandling and releases and public relations.

2-FielrlActivities

Ecological data on medfly population distribution, numbers, host preference and overwintering are being collected (Pereira et al., in press a). Surveys of medfly adults and larvae, host species and phenology, .temperature, winter temperatures .and medfly history all will be used to guide the control program. All of these data will be used to guide the program, specifically the sterile fly releases.

2.1-Climatic Conditions

Madeira is compose by two populated islands at 32° N in latitude and 17° W in 2 longitude. Porto Santo is small (about 50km ) with topographic and temperature conditions favourable to medfly. However, poor soil and little precipitation (380mm a year) do not permit an abundance of host fruits. The maximum elevation is 517m. The major Island (Madeira) is 740km2, has variable climatic conditions depending on altitude and north or south side of the island. The north coast is cooler than .the south (sea level in the north corresponds to 300m elevation in the south). According to the limits proposed by Bodenheimer (1951) for temperature and relative humidity, the ecoclimatograms of Funchal (58m), Camacha (680m) and Arieiro (1610m), were calculated (Fig. 1). Conditions in Funchal (capital city) are very favourable for medfly throughout the year. Eight generations per year of C. capitata are produced in the Funchal area (Vieira, 1952). Camacha, at 680m above sea level, is marginally favourable for the medfly during the winter. The medfly does not exist at Arieiro (1610 m) because of harsh winter conditions. - 213 -

T °C 40 impossible zone

non-favourable zone 35 favourable zone

30 optimal one

25

20

15

10 m-S-Funchal-58m -""l,- Camacha-680m --,t,-Arieiro-1610m 5

0 30 40 5.0 HR% 60 70 80 90 100

Fig. 1-Ecoclimatograms for Ceratitis capitata at various altitudes in Madeira island.

2.2-Adult Survey

In late 1994 we start a study to determine relative medfly populations at different altitudes. Two replicates each at four elevations (0-200m; 200-400m; 400-600m and 600-800m) both on the north and south coasts plus Porto Santo are included in the study. Each replicate consists of 20 Jackson traps, 10 baited with trimedlure and I O with protein hydrolysate (Pereira & Carvalho, 1994; Pereira & Carvalho, 1996). The traps are placed in the most prevalent hosts at the selected location and remain in these host throughout the study. The trap catches are counted bi-weekly. The protein bait is replaced each visit and the trimedlure every two months. We observe the decrease of the fly catch with the increase in altitude. In general medfly populations are higher in the south than in the north because of warmer weather and a wider variety of hosts in the south. In the winter months, the populations are high up to 400m above sea level in the south and up to 200m above sea level in the north. In March and April the pest exists only below 400m in the north and 600m in the south. Starting in May the pest exists in all altitudes under study. In Porto Santo island the population is constant for the seven first months of the year. The medfly population increased in August because of ripening figs, the most important host on Porto Santo. - 214 -

2.3-Larval Survey

In late 1995 we start to evaluate the level of infestation in different hosts at different altitudes. The fruitsare helded in the laboratory forthe larvae mature and leave the fruit. Larvae are recorded as number of larvae per fruit and number of larvae per kg of fruit. In 1996 we done 2,412 fruit samples, totalling about 660kg of fruits. A total of 30,702 larvae were recovered. In 1997, until July we do 2,088 fruit samples, about 726kg of fruits are collected. A total of 78 349 larvae were recovered. The medfly was reported to attack more than 40 species of fruit in Madeira Islands (Pereira et al., in press b; Vieira, 1952).

3-Mass Production

The objective of the medfly mass production unit is produce high quality sterile male medflies for the Madeira-Med medfly control program. This involves medfly mass production, process control of the rearing ingredients and sterilisation of the medflies that will be released. The IAEA has developed medfly strains (genetic sexing strains) which permit the release of sterile males only (Hendrichs et al., 1995). Data to date demonstrate that the release of males essentially doubles the efficacy of the sterile male medfly. Thus the number of sterile males required when using a genetic sexing strain is half the number of sterile males required when releasing equal numbers of sterile males and females. Aerial release costs are reduced with the genetic sexing strain. If a genetic sexing strain based on killing females in the egg or neonate larval stage is used, rearing costs are reduced (Kerremans & Franz, 1994). The Madeira-Med rearing factory is self contained with a biologically secure entrance and exit to prevent the escape of fertile medflies. It is anticipated the factory will operate 7 days a week (Barbosa et al., in press). The plan for production was presented in Table 1. Afterthe mass produced, medfly male pupae are sterilised whit 145Gy (14.5krad) in a Cobalt 60 source. This factory is designed for a weekly production of 50 million medflies. All the mass rearing operation we present in Fig. 2. It is anticipated that Madeira-Med will be able to supply other programs, either research or action, with limited numbers of sterile medflies. Mainland Portugal, Azores Islands, Spain, Italy, Morocco or Middle East are possibilities.

Table 1- Mass rearing perspectives for late 1997 and 1998. Production Year Months (N° flies/week) Colony Releases (only males) 1997 Sep. - Dec. 5 millions 20 millions

1998 Jan. - Dec. 8 millions 32 millions - 215

PRODUCTION

ADULTS � EGGING

SECOING

LARYAL !NI11A TION

P.l

I=: CLEAN TRAY · LARYAL MATURATION STORAGE IANOII' IRRADIATION t -< 1RAY LARVAL WASJIING ' ..COLLECTION PRE-IRRADIATION ,:.:: DIET' t DISPOSAL PUPATION PUPAL ' MATIJRATION PUPAL , SEPARATION

Fig. 2-Mass rearing operations - 216

4-Quality Control

Quality control is to insure that high quality sterile male medflies are released. This includes quality control of the medfly strain, diet ingredients, rearing conditions, sterile flies at the factory door and sterile flies released. Field cage test to measure the compatibility of the mass produced genetic sexing strains with wild Madeira medflieshave been doing and the strains arecompatible. Other component is the evaluation of the competitiveness between the strain actually under rearing "Vienna60" and the wild flies from Madeira. The last May, 1997 in Vienna, was discussed the tests necessary to do at the moment on the final product. It was decided that the necessary tests are: weigh of the pupae; sex ratio; emergence and fliers; longevity without food and water and competitiveness of sterile males with wild population in field cages.

5-Releases

After the production the pupae are transferred to the packaging, emergence and handling facility were they are prepared to emerge. After emergence they are prepared for aerial releases. After transfer to the airport, they are loaded on to an aircraft which disperse the sterile males on pre-determined flight patterns, altitudes and frequency. Sterile flies are released twice per week over all parts of the island receiving SIT treatments. In the Table 2 we present the area of treatment, release density and total males released by week supported by the ecological data (adult survey, larval survey and abiotic factors).

Table 2-Area and release density of sterile males per hectare per week in the Autonomous Region of Madeira. N° of sterile Coast and area of releases Area Release density males to release (ha) (N° males/ha) (x 103) 0-400m, North and South Coast 17 500 1 500 26,250

400-600m, South Coast 7 900 500 3, 950

Porto Santo 3300 500 1,650

Total 28 700 31,850

The type of aircraft (twin engine) and released system (chilled adult releases) are plan to start in November 1997. During March, 1997 we start the ground releases in paper bags, on three pilot areas. The Fig. 3 show the number of males released between week 11 to 38 of 1997. - 217 -

flies•1000 8500 8000 7500 r 7000 6500 6000 I 5500 . 5000 I\ I 4500 I \ 4000 I \ I 3500 I .. 3000 \ 2500 \ I 2000 \ 1500 \ I 1000 � I \ - 500 /�/ '-- / ...... ,... 0 -- 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 Week

Fig. 3-Number. of sterile males released in the pilot areas between 17 March to 21 September, 1997 (week 11 to week 38).

6-Public Relations

The medfly has been present on Madeira for a long time and thus most citizens do not consider it a problem. The objective of Public Relations activities for Madeira-Med activities is to insure public support for the project and must sensitise the Madeira people to the medfly problem and encourage their direct assistance in the project. Are in use video, radio, print and personal contacts. It is planned to use television too. The main work at this time are doing with high schools, including particle demonstrations of trapping and fruit sampling and a presentation in video and slides of the Madeira-Med Program.

7-Conclusions

The overall strategy of medfly control for Madeira is to apply sterile males in areas of the island where medfly overwintering occurs and the pest is thus active year round. If correctly conducted, the medfly will not move into areas where temperatures are too cold for year round reproduction. It is estimated that about half of the island will require treatment and of this, about half will require year round treatment. Confirmation of these assumptions must a wait collection of additional ecological data and sterility data from initial releases of sterile male medflies. Of particular importance in Madeira is the medfly population in urban areas. Madeira is densely populated. Most people live in individual houses with medfly hosts in their gardens. This posses problems with regard to reducing the medfly population - 218 - sufficiently low that sterile males can be used economically for medfly control. Special provisions thus must be made to reduce medfly populations in urban areas. This will require strong support from the urban human population.

Acknowierlgements

MADEIRA-MED is financially supported by the Government of Madeira, the European Union through POSEIMA (Programa de Op9oes Especificas para Fazer Face ao Afastamento e Insularidade da Madeira e A9ores) and REGIS II and the IAEA (International Atomic Energy Agency).

References

BARBOSA, A., CARVALHO, J. P., BRAZAO, A. & LINDQUIST, D.A. (in press). The Madeira medfly factory. 1st International Symposium on Biological Control in European Islands. 23-29 Set 1995 Ponta Delgada - A9ores. BODENHEIMER, F.S. (1951). Citrus entomology in the Midle East. Junk Ed., The Hague. HENDRICHS, J., FRANZ, G. & RENDON P. (1995). Increased effectiveness and applicability of the sterile insect technique through male-only releases for control of Mediterranean fruit flies during fruit seasons. J Appl. Ent. 119: 371-377. KERREMANS, P. & FRANZ, G. ( 1994). Cytogenetic analysis of chromosome 5 from the Mediterranean fruit fly, Ceratitis capitata. Chromosoma, 103: 142-146. LINDQUIST, D.A., CARVALHO, J.P., PEREIRA, R., BARBOSA, A.M. & BRAZAO, A. (in press). Area-wide control of the Mediterranean fruit fly, Ceratitis capitata, on Madeira with the sterile insect technique. ]Sf International Symposium on Biological Control in European Islands. 23-29 Set 1995 Ponta Delgada - A9ores. PEREIRA, R. & CARVALHO, J.P. (1994). Utiliza9ao em Portugal de sistemas de armadilhas na luta contra Ceratitis capitata (Wied.) em citrinos. Bulletin OILB, 17 (6): 139-148. PEREIRA, R. & CARVALHO, J.P. (1996). Trap utilisation on study of Mediterranean fruit fly populations at citrus groves in Portugal. in: McPheron & Steck (Ed.). Fruit fly pests, a world assessment of their biologyand management. 135-140. PEREIRA, R., CARVALHO, J.P., BRAZAO, A. & LINDQUIST, D. (in press a). Ecological data required for effective Mediterranean fruit fly, Ceratitis capitata, control using the sterile insect technique in Madeira. 1st International Symposium on Biological Control in European islands. 23-29 Set 1995 Ponta Delgada - A9ores. PEREIRA, R., BARBOSA, A., BRAZAO, A., CARVALHO, J.P. & LINDQUIST, D. (in press b ). Area-wide control of the Mediterranean fruit fly on Madeira with the sterile insect technique. VII] Congress of the international Societyof Citriculture, Sun City, South Africa, 12-17 May, 1996. VIEIRA, R. (1952). A mosca da Fruta (Ceratitis capitata, Wied.) na !Iha da Madeira. Gremio dos Exportadores de Frutas da Ilha da Madeira. - 219 -

CONTROL OF MEDITERRANEAN FRUIT FLY WITH VIENNA-43/44 TSL STERILE MALES IN TUNISIAN OASES 1 2 4 4 J.P. Cayol , E.J. Buycloc , F. Loussaier, M. Zaral, M. Boukhari & T. Arfaoui FAO/IAEA Agriculture and Biotechnology Laboratory, Agency's Laboratories, Entomology Unit, A-2444 Seibersdorf, AUSTRIA Consultant for the Insect and Pest Control Section, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, IAEA, Wagramerstrasse 5, P.O. Box 100, A-1400 Vienna, AUSTRIA 3 DGPA, Ministere de l'Agriculture, 30 avenue A Savary, 2000 Belvedere, Tunis, TUNISIA Commissariat Regional au Developpement Agricoie, 2200 Tozeur, TUNISIA

SUMMARY Recent developments of Medfly genetic sexing strains enable only males to be produced and released forarea-widacontrol usingJhe SterileInsect Technique (SIT). ln a project supported by the FAO Technical Co-operation Programme, Medfly pupae of a genetic sexing strain (Vienna-43/44 based on a temperature sensitive lethal (!m) mutation) were shipped from the IAEA mass-rearing facility of Seibersdorf (Austria) and sterile males released weekly from the ground in oases. of the. .Tozeur Governorate .from mid-.February until October 1994. Wild fly population data obtained through a network of Jackson and McPhail traps in two oases, Foum El Khanga and Mides where sterile males were released, were compared to those from the control oasis of Bir Kastilia. They show that, despite some reductions in sterile fly quality due to shipping conditions, .the sterile male releases .had a g_reat impact on the wild population inducing a seven to ten fold reduction in 1994 when compared to the data from Bir Kastilia (in October, 32.96 flies per trap per day in Bir Kastilia, compared to 3.44 in Foum El Khanga and 3.14 in Mides), thus confirming the efficiency of the tsl genetic sexing strain. However the pest was. not .eliminated. mainly due to failure to adhere strictly to the release calendar, and damage to pupae due to delayed air delivery. Comparison of trapping data collected in the same oases before (1993) and during (1994) the sterile male releases shows the suppression effect of the sterile males on the wild population. However, the wild population increased again during June-July 1995. Fruit infestation monitoring in Bir Kastilia revealed that the wild population builds up in May, mostly in plums and early figs, then in June-July in apricots and peaches, in September in figs and oranges and finally in pomegranates in October. ln the oases under SIT control, fruit infestation was delayed until August when larvae were detected on figs.

Key-words: Medfly, Ceratitis capitata, SIT, genetic sexing strain, Tunisia - 220- 1 - INTRODUCTION The Tunisian Government has participated with Algeria, Libya and Morocco in a regional programme for the control and eradication of the Mediterranean fruit fly (Medfly), Ceratitis capitata (Wied.) (Diptera: Tephritidae) in North Africa, using the Sterile Insect Technique (SIT). With the technical assistance of the International Atomic Energy Agency (IAEA), the first phase of this programme (1988-1993) called MAGHREBMED (!AEA,1995a) contributed to an increased knowledge of Medfly distribution and biology in the Maghreb Region, and to an economic evaluation of losses and control costs (IAEA, 1995b). A second phase initiated in January 1993 involved pilot trials aiming to: (i) assess the impact on isolated Medfly populations of the release of only sterile males and (ii) adapt SIT operations to local conditions and train national staff. This paper deals with the first objective. A suitable experimental release site had to satisfy the following criteria: sufficient isolation from surrounding agricultural land infested by the Medfly so as to (i) avoid re-infestation by immigration during and after SIT operations and (ii) minimise as far as possible quarantine operations; close to an international airport as sterilised Medfly pupae were to be imported from abroad; not larger than 12 square kilometres due to the limited availability of males. Among the areas proposed by the participating governments, one located in the Tozeur Governorate, south-western Tunisia, was selected as it adequately satisfied the above criteria. Initially planned for 36 months, the duration of the project had to be reduced to 24 months as only one donor was found. The FAO Technical Co-operation Programme allocated US$ 250,000 as from October 1992, whilst the Tunisian Ministry of Agriculture and the IAEA provided national and expatriate personnel, premises, transport, equipment and supplies, including sterilised Medfly pupae.

2- MATERIALS AND METHOD PROJECT AREA Nearly all of the 559,287 hectares of the Governorate are devoid of host plants of the Medfly, being either salt flats or dry steppe with few perennial grasses and scrubs. Crops are restricted to oases and to a few river (so-called "oueds") banks located in the Djebel El Negueb ··motmtain ·range forming the boundary with Algeria. All of the 26 oases in the Governorate are well isolated from each other and total about 8,000ha. These oases are groves composed of date palm Phoenix dactylifera L., the main fruit crop in the area. Tozeur, the Governorate capital city, has an international airportand daily flights to and from Tunis. Seven oases about 5"'0tTamerza, the effectiveness of two different genetic sexing strains has been reported (Cayol & Zara'i, submitted). In the plain oases of Chebika, Foum El Khanga and Bir Kastilia, the monthly means of daily maximum and minimum temperatures are over 35°C in July-August, and down to 7°C in January. In the other five mountain oases, the temperature is several degrees lower in winter, near 0°C in January, whilst summers are hotter than in the steppe. From the end of May to October, the high temperature permits a rapid turnover of Medfly generations. Relative humidity (RH) oscillates around 60% with a marked maximum in winter and an obvious - 221 minimum in summer (Le Houerou, 1989). In the palm groves, dense vegetation and irrigation modulate RH during summer. As water is in short supply and expensive, irrigated soil is intensively cultivated in the oases. Ancient palm groves present three vegetation levels, each one producing host fruit for the Medfly. At ground level, fodder crops and vegetables such as chilli peppers and tomatoes are grown, sometimes surrounded by hedges of box thorn, Lycium europaeum L. and clusters of prickly pear, Opuntia vu/garis Miller. At middle level there are fig, pome-and stonefruit, citrus and pomegranate, as well as buckthorn (Rhamnus a/atemus L. ssp. myrtifolia (Willk.), Rhamnus lycioides L.) and some jujube trees (Ziziphus ssp.). The date palm crowns compose the top level, a dense canopy which filters light and protects the lower vegetation from the dry and hot desert winds. Unfecundated dates of the predominant variety (Deglet Nour) reach the end of their development in November remaining on the palms for months and they may be infested by the Medfly (Buyckx, 1994a). Bir Kastilia is a typical ancient palm grove of the Tozeur Governorate and the ripening periods of its various host fruit are shown in table 1. Temperature, as well as the amount of fruit available for oviposition during each season, governs size and fluctuation of a Medfly population, a detailed floristic inventory was planned for each experimental oasis, but was undertaken only for Mides (Quilici, 1993) due to shortage of staff. In the two other oases only a qualitative estimation of the available medfly host plants was done. In general, there were more host-plants in Bir Kastilia than in Mides and still less in Foum El Khanga. Only one road enters the. selected area at Chebika oasis. coming from. Gafsa.or.Tozeur, passing through Tamerza and Ain El Karma oases, leading either to the mining area of Redeyef (Gafsa Governorate) or to the border post with Algeria. As Tunisian plant protection regulations cover quarantine control at international boundaries only, no internal quarantine post were established. With Algeria, efficient controls set up over the past decade to prevent the spread of Bayoud disease of date palm, prohibit the introduction of any plant material. No control or suppression measures, i.e. protein-insecticide bait sprays or fruit stripping weref: applied prior to sterile fly releases. STERILE MALE MEDFLIES In Foum El Khanga and Mides males from a genetic sexing strain based on a temperature sensitive lethal (ts/) mutation were released. In this strain, females are killed by heat treatment of eggs. Predominantly male pupae of GS strain Vienna-43/44 were mass-reared for the project by the Entomology Unit of the FAO/IAEA Agriculture and Biotechnology Laboratory in Seibersdorf, Austria. They were irradiated 48 hours before emergence (minimum absorbed dose: 90Gy), marked with fluorescent dye, packed in plastic bags (under hypoxia) and dispatched to Tunisia. From 10 February until 6 October 19941 , 16 to 32 litres of pupae were airfreighted in an insulated cardboard box weekly on Thursday afternoon from Vienna to Tunis and by a late evening local flight to Tozeur, limiting the duration of hypoxia to 12 hours. However during July, August and September the flight Tunis-Tozeur was postponed to the subsequent evening, entailing a 24 hour increase of hypoxia. To avoid excessive exposure to heat, cardboard boxes were placed in cold storage at 12°C at Tunis airport. In Tozeur, the pupae were distributed at a rate of 6,600 per 10 litre release paper bag (containing crumbled paper, a sheet of sugar coated paper plus two pieces of wet sponge). A sex ratio and a flight ability test (as described by Brazzelet al., 1986) were conducted for each batch received (Cayo! ,1 Zarai, submitted) in Tozeur, and in Seibersdorf before and after irradiation. Emerging flies were kept in closed bags in a temperature controlled room (24°C; 85% RH). Once a week from 14 February until 10 October, bags were manually placed (one release site each four hectare) and opened in the oases in the shade of host-trees. No releases were made on 17 February, 8 and 23 July as pupae arrived dead due to delayed air-

1 Two shipments of pupae were made on 13 and 20 January for training and checking procedures as per the project protocol. - 222 - delivery. Releases were completed between 05:00 and 07:00 AM. Several times "control" bags transported to the oases were returned to the laboratory for rapid quality control tests and results compared with the corresponding data collected before transportation of the bags to the oases. MEDFLY MONITORING Medfly numbers were monitored by trapping and fruit infestation assessment to follow the fluctuations of the wild population, the ratio of sterile males to wild flies as well as the dispersal of sterile males from the release site. One Jackson dry trap with trimedlure plug as attractant and one McPhail trap with liquid bait to catch female flies (Katsoyannos, 1994), were installed in March 1994 for every eight hectares along a grid established by Quilici (1993) for each of the experimental oases larger than eight hectares; all together six pairs at Foum El Khanga and Mides. However, as there was no time to lay out a grid for Bir Kastilia only two pairs of traps were placed there. Traps were checked weekly until 3 November 1994 and results expressed in flies/trap/day (ftd). After this date trapping was pursued by the Centre Regional de Developpement Agricole (CRDA) of Tozeur with Jackson traps only checked fortnightly until December 1995. A major but unavoidable disadvantage of trapping is that many released flies are caught which have then to be identified under ultra-violet light of a Nikon® fluorescent microscope (Enkerlin et al., 1996). As expected, very few sterile females were identified in McPhail traps, confirming the accuracy of 99.9% in sex separation at the egg stage, characteristic of a tsl GS strain. Fruit infestation studies monitored the level of the larval population in various fruits and indirectly the effectiveness of the released males for population suppression. From February to November 1994, samples of fruit at a suitable stage for oviposition were collected from host plants growing in each experimental oasis and in Bir Kastilia, weighed and placed separately in suitable plastic containers for larval emergence. Numbers of pupae obtained were then converted to pupae per kilogram of fruit (pkg).

3 - RESULTS BIR KASTILIA As this relatively large oasis presents a great diversity of cultivated and wild host plants besides date palm, host fruit is available for oviposition practically the whole year round (table 1). A fairly accurate picture of the annual fluctuations of the Medfly population can be drawn from the current trapping data (figure 2) and those obtained for 1992-1993 in other oases around Tozeur (Merhaben, 1993; Buyckx, 1994a; Cayol, 1994). During the relatively cool winter months (December to March), except for unfecundated dates, fruit is scarce with larvae developing slowly in the few available sour oranges and prickly pears and in whatever remaining fruits of jujube, buckthorn are suitable for oviposition. Trap catches are at their lowest until March, indicating the low level of the Medfly population and activity; they increase in April to a monthly mean of 6 ftd. Breeding accelerates in mid-spring as box thorn and early fruiting varieties of apricot, apple and plum become available for oviposition, and intensifies with the ripening of figs, pears and late fruiting apples and the rising temperature. Cayol (1996) showed that in Tozeur during the warm season Medflies have a bimodal pattern of activity, confirming the findings in Egypt of Hendrichs & Hendrichs (1990). Peaks in trap catches appear in June and September (monthly means of 34 and 36 ftd respectively) with a drop during July-August due to hot weather and less fruit available for breeding (Buyckx, 1994a; Cayol, 1994). The population begins to decrease in October (33 ftd) and collapses in the following two months. In November temperature is falling and most cultivated fruit has dried out or been collected, whilst date harvesting is at a peak. Fruit sampling initiated in March 1994 with unfecundated dates and citrus, the only fruit available, showed a slight infestation in the latter. The first plums and figs started ripening and were infested by the end of May, followed by apples in June and tomatoes in July. Thereafter figs and peaches became heavily infested, with peak infestations of 800 pkg at the end of July - 223 - and 811 pkg in mid-September for figs and a moderate drop to 353 pkg for peaches in mid­ August. Breeding in oranges started at the end of September (110 pkg) and in pomegranates in early October (18 pkg). Overall figs were the main infested crop. No Medfly infestation could be found in local varieties of lemon. Regarding unfecundated dates, all the 27 samples (in total 9.4 kg) collected in the seven experimental oases during March, April, May, early June, and in September were not infested, confirming the extremely low level of infestation of this crop (Buyckx, 1994a). MIDES Although Mides is relatively small, there are many trees under the date palms producing enough fruit to support a significant Medfly population. About 71 % are citrus, followed by fig (15%), pomegranate (6%), apricot (3%), peach (2,4%), apple (2%) and pear (0,3%) (Quilici, 1993). At the lowest level some red pepper, buckthorn and hedges of box thorn are found, whilst a few clumps of prickly pear and box thorn grow on the outskirts of the oasis. In preparation of the pilot trial, the CRDA started operating Maghrebmed dry traps (with trimedlure plug and DDVP dispenser) (Katsoyannos, 1994; IAEA, 1995a) in May 1993 until mid-December in four oases of the project area, including Mides, and in August in two others, Foum El Khanga and Dafria. Catches in Mides (figure 3) show a Medfly population increasing at the end of July 1993 to reach a high monthly- mean by October of 2.17 ftdand falling.abruptly in November-December. During releases in 1994, trap catches from April show the impact of sterile males on the wild population resulting in a high degree of suppression, which delayed its usual build up. Compared with the previous year, the mean monthly trap catch reached only 1.6 ftd in August 1994 (28.6 in 1993); 8.3 in September(56.3 in-1993), 4.J.ir:1 October.{217-3in 1993), 3.7 in November (80 in 1993) and 3.9 (5.1 ftd in 1993) in December. In 1995, no Medfly was caught from February to May. Zero catches in Jackson traps during winter months may not mean absence of Medfly but rather unattractiveness of the trap for a low Medfly population. In Guatemala; during periods of low population, Hentze (1990) found that white Jackson inserts did not catch any flies while yellow ones caught a few. In June 1995, the wild population built up slightly and reached a maximum of 21.92 ftd in December. Based on the trapping data, the population present in Mides in 1995 was three to seven fold higher than during-the male-only releases (1994), but four to sixteen fold lower than two years previously (1993). Fruit collection was initiated in March 1994. The only fruits available were prickly pears, sour oranges and unfecundated dates. Pupae were recovered from the first two but none from the dates. In April and May no larvae were found in unfecundated dates, prickly pears, buckthom and apricots. Only one slight infestation (16 pkg) of apricots was detected in mid June and one in figs (3 pkg) at the beginning of August. From the end of August until the end of September, infestation in peaches increased with a peak of 58 pkg in mid September. At this time the first oranges, still green, already contained larvae. Citrus fruit continued to be infested till the end of sampling (30 October 1994). FOUM EL KHANGA The middle level of Foum El Khanga oasis is well diversified with apricot being the most abundant, the other fruits being fig, orange, apple, peach, pomegranate, pear and quince. However, these trees are scattered among the date palms at a much lower density than in Mides, mainly due to the low number of citrus. At the ground level tomato and red pepper are grown. Trap catches are much lower than in Mides and Bir Kastilia, indicating a smaller Medfly population. Monthly means of 6.4, 6.25, 5.35 and 9.2 ftd were recorded for August, September, October and November 1993 respectively (figure 3). Trapping data during and after releases show the impact of sterile males on the wild population which remained at very low level from March through September 1994, with a monthly mean of around 0.5 ftd or less. However in October captures increased seven fold (3.6 ftd). After releases (figure 3), a reduction from December to January 1995 was followed by nearly zero captures from February to May. Then from May to December 1995, the population built-up again, reaching a maximum - 224 - nearly twice as high in November 1995 than in November 1993, before the sterile male releases. Sour oranges, the first sample collected in March 1994 yielded 10 pkg but thereafter until mid August no pupae were obtained from fruit samples. At the end of August figs became infested up to a maximum of 198 pkg in mid September. From the end of September until the end of October pomegranates became infested (18 pkg). Obviously the higher catch of adults during October correlates with the high larval infestation during the preceding month.

4 - DISCUSSION The comparison of trap catches and fruit infestation data from Mides and Foum El Khanga with those from Bir Kastilia show that sterile males suppressed to a great extent the Medfly population (figures 2 and �)- This confirms the appropriate behaviour and competitiveness of GS tsl strains as shown in a survivaldispersal study (Hendrichs et al., 1993) and in extensive green-house and field cage tests (Hendrichs et al., 1996). One of the advantages of male­ only release is due to the absence of sterile females (Hendrichs et al., 1994; 1995), and sterile males can mate only with wild females, they appear to disperse more in search of them than under normal SIT operations. This effect has been observed in a field pilot test in Hawaii (Mcinnis et al., 1993), in Israel (Nitzan et al., 1993), and in Greece with the Vienna-42 strain (Hendrichs et al., 1993). It also occurred in Tozeur where the numbers caught in traps (Cayol, 1994) indicated that sterile males dispersed well from the release sites. Medfly suppression lasted till July as shown by zero catches in June against 38.4 ftd in Bir Kastilia, and prevented the infestation of fruit, in particular of early season apricot varieties and figs. However, as from July the wild population started building up in both oases as shown by a monthly mean of 0.6 ftd. In October it reached 4.7 ftd in Mides and 3.6 ftd in Foum El Khanga, still respectively seven and nine fold less when compared to 31.8 ftd in the control. Until 21 April 1994, weekly requirements of pupae were calculated first at the rates of 3,450/ha for Mides and2,920/ha for Foum El Khanga. Quality control tests showed that only about one male from every five pupae was able to fly away in the field. Cayol & Zarai (submitted) attributed this reduction in emergence and flight ability partly to irradiation but mainly to duration of transport and procedure of transportation. This was already observed for shipments of a white pupae genetic sexing strain (T:Y(wp)30c) from Vienna to Italy by Nitzan et al. (1993) who found that emergence fluctuated greatly between deliveries, dropping oftento less than 30%, and suggested that air-transport and handling of boxes contributed most to pupal mortalityand poor quality of males. The quality of the flies at the release site is a major concern. Calkins & Ashley (1989) showed that, using a normal Medfly strain, a drop in the percentage of effective flies released, from a theoretical value of 100% to 17% induced a two-fold increase ofthe operational cost of a programme, and doubled the number of Medfly generations required to theoretically achieve eradication (from three to six generations, with a 100:1 sterile to fertile ratio). For this reason it was decided to double the number of pupae as from 28 April to 6,890/ha at Mides and 5,830/hiat Foum El Khanga. Based on the quality control data, about 584 and thereafter 1,166 sterile flying males should have been present per hectare in Foum El Khanga, and in Mides the figures are 690 and 1,380. However, some of them were caught in the traps and others captured by natural enemies, mainly wasps (Cayol, 1994); others may have been killed on 17 May by the dust storm followed by heavy rain and on 5 October by a violent rainstorm and flooding in the mountain oases. In the argan tree "forest" in south-western Morocco, Sacantanis (1957) observed that the hot, dry and dusty southerly windstorm (the "chergui") was the most destructive factor for the Medfly population. After a one or two-day storm, he found millions of dead insects accumulated in places sheltered from the wind, such as ditches. Hot weather reaching 40°C was also detrimental to the wild population and sterile flies as reflected by trapping. Predation is probably also an important cause of mortality of Medflies in the Tozeur oases. Baker & Van der Valk (1992) found numbers of released sterile - 225 - males declining rapidly and suggested that predators, including wasps, might account for this. In Greece, Hendrichs et al. (1993) observed a considerable number of predations on sterile males and found foraging yellowjacket wasps entering opened field cages and killing within a few hours all sterile male flies released therein. Hendrichs et al. (1994) confirmedthat foraging yellowjacket wasps were attracted by the pheromone-calling males aggregated in leks. Therefore, it may be assumed that there were less sterile males per hectare than indicated above, and in numbers too low to eliminate all wild flies. Immigration may originate from wild host plants on the outskirts of the oasis. Outside Mides, box thorn and prickly pear growing in inaccessible places such as canyons, were found infested (Cayol & Zarai", submitted). No sterile males were released nor bait spray applied there, so wild flies may have moved into the experimental area. Another possible source of infestation is the importation of infested fruit. In the Maghreb, the main cause of Medfly dispersal is transport of fruit produced in the coastal plains where yearly widespread infestations occur (Buyckx, 1994b). In Morocco, Sacantanis (1957) observed that trade and transport by motor vehicle were the main dispersion factors. As there was no quarantine control at two of the three points of entry into the experimental area, infested material may have been introduced and some wild flies may have originated from discarded rotting fruit. This happens mostly in the fruit and vegetable markets, however as people live outside of Mides and Foum El Khanga, no imported fruit is sold in the oases. Some fruit was imported for sale on the market of Tamerza oasis and for consumption at the Tamerza hotel. Tourist movement presented very little risk of introduction as it is limited to one-day tours of Tamerza and Mides canyon. Therefore the risk of introduction of immature stages, despite-the lack. oL quarantine, appears remote in comparison to re-infestation originating from the reservoir of wild host plants growing in the outskirts of the oases.

5 - CONCLUSION The main reasons for the cessation of the suppression of the wild population in July and its subsequent build up are the interruption of releases on 12 and 27 July, and a significant drop in quality of Vienna-44 pupae as from July. These failures, which occurred during the fifth month of releases jeopardised any possibility of eliminating the Medfly from the two oases. They happened during a period of increased breeding and rapid turnover of generations due to relatively high temperature and a large amount of ripening fruit. During this time, only 17 days for the development from egg to adult were required (Buyckx, 1994a). In spite of this and problems of predation and incomplete quarantine control, the Vienna 43/44 strain performed well, largely suppressing the wild population during four months and limiting fruit infestation considerably. These results confirm those obtained with the white pupae genetic sexing strain (T:Y(wp)30c) in 1986 in Procida Island, Italy, and in 1989 and 1990 in Gvulot, Israel (Cirio fil &. 1987; Nitz.anet al., 1993; Economopoulos et al., 1994). As natural factors drastically reduce the wild population at the end of December, the suppression period could have been moved backwards by four weeks, startingreleases earlier, in the beginning of January. It may also be concluded that the number of sterile males released during the first five months was sufficientfor suppression because the wild population was relatively low (less than 10 ftd). For higher wild population levels a preliminary reduction would have been indispensable as in Procida (fruit destruction) and in Gvulot (insecticide application). Even in the case of Mides and Foum El Khanga one may assume that such reduction could have increased the degree of suppression and even resulted in elimination of the pest. In arid and hyper-arid zones, an integrated approach appears the most suitable solution for SIT against the Medfly (Buyckx, 1994b). In November-December, collection and destruction of fallen fruit, culling, toxic bait application and intensive trapping of female flies would result in a very low residual population for elimination by releases of sterile males. Cultural control should be applied by the growers. However such an integrated programme could not be adopted as - 226 - there was no time during the preparatory period before releases to undertake a public information campaign in the oases nor time to organise cultural control and intensive trapping. Only after release operations had started, were the growers actually informed about the project, and as a result, they were not able to take the additional control measures necessary. Timely public information is an indispensable element of success in SIT programmes. Among the advantages of using GS strains in fruit fly SIT programmes outlined by Hendrichs et al. (1995) are the considerable savings not only in rearing, irradiation, packing, transport and release of sterile males only, but also in monitoring operations in the field by trapping wild females only and by fruit sampling, dispensing with the need to process male captures. Adoption of this procedure in the pilot trial would have avoided the elimination of a number of released sterile males. Another advantage is that Medfly males can be released at a more mature age whereby losses due to predation before they are sexually more effective are reduced. A further advantage of using GS strains is the increase in quality of sterile males obtained by irradiating 24h before emergence instead of 48h when pupae of both sexes have to be irradiated. In pilot trials aiming at eradication of Medfly, strict adherence to the release calendar is indispensable. Duration of hypoxia time and irradiation timing are key factors for quality of released males.

Acknowledgements This field research was carried out under IAEA Technical Co-operation Project RAF/5/013 and Project TUNl2256(E) "Control and Eradication of Medf/y" of the FAO Technical Co-operation Programme. The authors thank the Tunisian authorities for their support, in particular Mr. Hamza Mekki, Regional Commissar for Agricultural Development in the Tozeur Governorate, the Project Director, Mr. Hasnaoui Zaidi, Head of the Plant Protection Directorate, and the other members of the Tunisian team for their contributions in project execution. Special thanks go to Mr. T. Lakhdar for his great and unforgettable help. The assistance in implementing the project of Mr. B. Amouri, FAO Representative and his staffare gratefully acknowledged. We also thank Dr. J. Hendrichs, Dr. A.S. Robinson and Dr. P. Gomes for critically reviewing the draft of the manuscript.

References Baker P.S., Van der Valk H., 1992. Distribution and behaviour of sterile Mediterranean fruit flies in a host tree. J. Appl. Entomol., 114: 67-76. Brazzel J.R., Calkins C., Chambers D.L., Gates D.B., 1986. Required quality control tests, quality specifications, and shipping procedures for laboratory produced Mediterranean fruits flies for Sterile Insect Control programs. USDA manual, APHIS 81-51: 28p. Buyckx E.J., 1994a. Unfecundated dates, host of the Mediterranean Fruit Fly (Diptera: Tephritidae) in the oases of Tozeur, Tunisia. Proc. of IOBC/WPRS lnt. Open Meet. - Working group "Fruit flies of economic importance", Lisbon, Portugal, 14-16 oct. 1993. IOBCIWPRS Bull., 17(6): 25-37. Buyckx E.J., 1994b. Bioclimatic effects on the distribution of the Mediterranean fruit fly (Diptera: Tephritidae) in the Maghreb. In: C.O. Calkins, W. Klassen and P. Liedo (eds), Fruits fliesand the Sterile Insect Technique. Boca Raton, Florida, CRC Press, 139-164. Calkins C.O. & Ashley T.R., 1989. The impact of poor quality of mass-reared Mediterranean fruit flies on the sterile insect technique used for eradication. J. Appl. Ent., 108: 401-408. Cayo! J.P., 1994. Projet TCP/TUN/2256(E) "Controle et eradication de la ceratite": resultats, conclusions, recommandations. FAO, Rome, 17p. Cayol J.P., 1996. Box thorn, key early season host of the Mediterranean Fruit Fly. InternationalJournal of Pest Management, 42(4): 325-329. - 227 - Cayol J.P. & Zara"f M., submitted. Field releases of two genetic sexing strains of the Mediterranean fruit fly in two isolated oases of Tozeur Governorate, Tunisia. J. Appl. Entomol. Cirio U., Caparella M., Economopoulos A.P., 1987. Control of medfly (Ceratitis capitata, Wied.) by releasing a mass-reared genetic sexing strain. In: "Fruit flies: proceedings of the Second International Symposium", Colymbari, Crete. Elsevier Sc. Publ. Co. Inc., Amsterdam, 513-522. Economopoulos A.P., Nitzan Y., Rossler Y., 1994. Mediterranean fruit fly effective control by combination of initial population reduction followed by successive releases of sterilized genetic sexing males. Proc. of IOBC/WPRS lnt. Open Meet. - Working group "Fruit flies of economic importance", Lisbon, Portugal, 14-16 oct. 1993. IOBCIWPRS Bull., 17(6): 255- 263. Enkerlin W., Lopez L., Celedonio H., 1996. Increased accuracy in discrimination between captured wild unmarked and released dye-marked adults in fruit fly (Diptera: Tephritidae) sterile released programs. J. Econ. Entomol., 89(4): 946-949. Hendrichs J. & Hendrichs M.A., 1990. Mediterranean fruit fly (Diptera: Tephritidae) in Nature: location and diel pattern of feeding and other activities on fruiting and non-fruiting hosts and non-hosts. Ann. Ent. Soc. Am., 83: 632-641. Hendrichs J., Wornoayporn V., Katsoyannos B.I., Gaggl K., 1993. First field assessment of the dispersal and survival of mass reared sterile Mediterranean fruit fly males of an embryonal, temperature sensitive genetic sexing strain. In: IAEA, FAO (Eds). Management of insect pests: nuclear and related molecular and genetic techniques Proc. FAOIIAEA lnt. Symp., Vienna, Austria, 19-23 oct. 1992. IAEA, Vienna, 699p. Hendrichs. J., Katsoyannos B.I., Wornoayporn V., Hendrichs M.A., 1994. Odour-mediated foraging by yellowjacket wasps (Hymenoptera: Vespidae): predation on leks of pheromone­ calling Mediterranean fruit fly males (Diptera: Tephritidae). Oecologia, 99: 88-94. Hendrichs J., Franz G., Rendon P., 1995. Increased effectiveness and applicability of the sterile insect technique through male-only releases for control of Mediterranean fruit flies during fruiting seasons. J. Appl. Entomol.,.119: 371-377. Hendrichs J., Katsoyannos B., Gaggl K., Wornoayporn V., 1996. Competitive behavior of males of Meditteranean fruit fly, Ceratitis capitata, genetic sexing strain Vienna-42. In: McPheron B.A. & Steck G.J. (Eds). Fruit flypests: a world assessment of their biology and management. Delray Beach, Florida, St Lucie Press, 405-414. Hentze F., 1990. Improving Medfly capture efficacy in Jackson traps. In: Aluja M. & Liedo P. (Eds). Fruit flies: Biology and management. Springer-Verlag, New York, 231-233. IAEA, 1995a. Regional programme for the control and eradication of the Mediterranean fruit fly in North Africa (Maghreb). Terminalreport RU-6074, Vienna,####-. IAEA, 1995b. Economic evaluation of damage caused by, and methods of control of, the Mediterranean fruit fly in the Maghreb. IAEA-TECDOC-830, Vienna, 65p. Katsoyannos B.I., 1994. Evaluation of Mediterranean fruit-fly traps for use in sterile-insect­ technique programmes. J. Appl. Ent., 118: 442-452. Le Houerou H.N., 1989. Classification ecoclimatique des zones arides (s.l.) de l'Afrique du Nord. Ecologia Mediterranea, 1S(3). Mcinnis D.O., Tam S., Grace C., Miyashita D., 1994. Population suppression and sterility rates induced by variable sex ratio, sterile insect releases of Ceratitis capitata (Diptera: Tephritidae) in Hawaii. Ann. Entomol. Soc. Am., 87(2): 231-240. Merhaben J., 1992. Tunisie, DOC - Etat d'avancement des travaux. Report of the 5th Meeting of National Co-ordinators, MAGHREBMED Programme. IAEA, NCl5/92, appendix 4, Vienna. - 228 - Merhaben J., 1993. Tunisie, DOC - Etat d'avancement des travaux. Reportof the 6th Meeting of National Co-ordinators, MAGHREBMED Programme. IAEA, NC/6/93, appendix 4, Vienna. Nitzan Y., Rossler Y., Economopoulos A.P., 1993. Mediterranean fruit fly: Effective control by genetic sexing male only SIT releases during 1989-1990. In: Management of Insect Pests: Nuclear, and Related Molecular and Genetic Techniques. IAEA-SM-327, Vienna, Austria, 331-344. Quilici S., 1993. Rapport de mission en Tunisie du 24/10 au 09/11/1993. IAEA, Vienna, 21p. Sacantanis K.B., 1957. La foret d'arganier. Le plus grand foyer de Ceratitis capitata Wied. connu au monde. Boll. Lab. Ent. Agraria Portici, 15: 1-53. Jan Feb Mar Apr May Jun Jui Aug Sep Oct Nov Dec

HOST PLANT

Prickly pear Unfecundated date +++++++++++++++++++++ + ++ Citrus(**) xx-I xxxxxxxI x Box-thorn xxxxxxxx xxxxx Apricot X X X X X X X X X X X ? ? ? ? ? ? X X Plum X X X X Prune X X X X Peach X X X X

Apple X X X X X I'.) I'.) Fig X X X X X X X xlx (0 Pear X X X X X X Red pepper X X X X Grape X X X X X Persimmon X X Pomegranate xx 1 Tomato xlx X X XIXX

(+)= Prickly pears left on the plant can be found all year round (-)= If not harvested (?)= To be confirmed (°)= Depending on the date of plantation (**)= Excluding lemon in early spring (non hosl)

Table 1: Ripening periods ofMedfly host plants in the Bir Kastilia oasis ofTozeur Govemorate, Tunisia [after Merhaben (1992), Buyckx (1994a) and Cayol (1996)] TUNISIA ,./

.z//

N LEGEND <,) 0 border main road e!GAFSA] Governorate capital city "[Neftal city, village or oasis

15km �--,,c,d

Fig. 1: Location of the FAO pilot project TCP/TUN/2256(E) "Control and eradication ofMedfly" 50 40 -1

35

40 t . - I . \ 30 . . ', \ I. \ \ i;' 25 30 ... _ .... # .; . \ c. . c. E 2! i c::=::=::J Wild males 20 .i3 G>... M c. f! 1- c. m G> E l:_:_-__:_:_:_:__� ..E 20 � J\.) ,:, 15 <,)...... � -.. -..

10

10 n 5 * * * * o I I , I I I I I I I I I I I I 0 I M J J A D F M A s 0 N 1994

Fig. 2: Monthly means of wild male Medfly catches in Jackson traps, and of maximum (M) and minimum (m) temperatures in Bir Kastilia control oasis * no trapping data available 00 217.3 Sterilemale releases Mides oasis I ------.. 'Cl :t I 1: :;;� 60 Q, 0 . 30 0 Q, - � &) � i!! i 40 M_; , ...... � ., \' 20 i1i � 30 E E I- :E 20 �.. : .. m .. • I 10 � 10 *:* 0 0 M J J A s 0 J F M A M J J F M A M J J A s 0 N D 1993 1994 1995

18 50 Sterilemale releases N 16 Foum El Khanga oasis N >, 14 .J_ 40 'Cl.. I � 12 Q, 0 Q, M ...... , 30 !:.... 10 . ' 1:: ,. '... I i!! � ' ' a, 8 .. _.__ � Q, .• 20 [ 6 E ..E , � m .. 'Cl 4 ...... 10 � -· - 2 * * * * * *

al I 0 I ,I I I I I I I ii o M J J A s 0 N D J F M A M J J A •S 1:1 O N •1· D J F M A M J J A s 0 N D 1993

Fig. 3: Monthly means of wild male Medfly catches in Maghrebmed (Katsoyannos, 1994; IAEA, 1995a) traps (fromMay to December 1993) and in Jackson traps (fromMarch 1994 to December 1995) in Mides and Foum El Khanga oases, and monthly means of maximum (M) and minimum (m) temperatures during the sterile male release period * no trapping data available - 233- EFFECTS OF TWO MODERN PESTICIDE§: AZADIRACHTINAND TEBUFENOZIDE ON THE PARASITOID OP/USCONCOLOR (SZEPU:GETI)

M. Gonzalez & E. Viiiuela Protecci6n de Cultivos. E.T.S.I.Agr6nomos. E-28040-Madrid. Spain

Summary Effects of azadirachtin and tebufenozide, on the parasitoid of the olive fruit fly Opius concolor Szepl., has been investigated in laboratory. Adults of the wasp were exposed to both products via drinking water at the highest field recommended concentrations or via parasitization on treated Ceratitis capitata Wied. larvae with sublethal doses. Continuous feeding on azadirachtin-treated water during adult life span, gave a significant reduction on both the longevity and the beneficial capacity of the parasitoid measured as number of attacked hosts and size of progeny per female. Tebufenozide orally administered only caused a reduction on the longevity of 0. concolor. Azadiractin added to the larval C. capitata rearing medium, gave a reduction on adult emergence of the fly, while tebufenozide had no effect on this pest. When treated larvae of the fly were offered for parasitization to 0. concolor, no effect on the longevity was detected, but for both products a significant decrease on the number of emerged parasitoid adults was observed.

'i - lnt.rcduction One of the most serious pests of olive fruits all over the Mediterranean region, is the olive fly Bactrocera oleae (Gmsi.) (Diptera, Tephritidae) (Viggiani, 1986; Fimiani, 1989; Jimenez, 1989), which causes severe economic losses that may account for up to 60% of the total insect damage to the crop (Mazomenos et al., 1977). For the control of the fly, the endoparasitoid Opius concolor Szepl. (Hymenoptera, Braconidae), has been inundatively released in several countries of the area, since beginning of the sixties. However, as a satisfactory control of the fly, could not be achieved in most of the cases by means of this parasitoid (Kapatos, 1989), its use was relegated. Current control measures in Spain against B. oleae and many other pests of olive groves, still rely heavily on the use of pesticides (Jacas et al, 1992; Jacas & Viriuela, 1994.). At present however, several environmentally sound methods such as the joint use of natural enemies and pesticides that exhibit low mammalian toxicity and selectivity, are under investigation to achieve a more reasonable approach to the management of the pest complex. A prerequisite for the successful joint use of enemies and pesticides is to ascertain their compatibility by stablishing the pesticide side-effects on the natural enemies. In this study, the two modern pesticides investigated, azadirachtin and tebufenozide, belong to the class of insect growth regulators (IGR) and have been reported to be relatively safe to beneficial insects (Sipcam lnagra, 1996; AgrEvo, 1997). Azadirachtin is a naturally derived compound from the neem tree Azadirachta indica A. Juss, which main mode of action seems to be the inhibition of the ecdysone hormone. Tebufenozide is a nonsteroidal ecdysteroid agonist representative of a new class, the moulting accelerating compounds. - 234

Out of the many possible routes of pesticide uptake in nature by natural enemies (Croft, 1989), in this study we have investigated two on the parasitoid 0. concolor : the contamination via the drinking water and via parasitization on treated Ceratitis capitata Wied. larvae, their alternative host in laboratory.

2 - Materials and methods 2.1 - Chemicals The test chemicals were the commercial products Align® (Sipcam lnagra, Espana}, aqueous emulsifiable concentrate formulation with 32 g/1 of azadirachtin, and Mimic® (Rohm & Haas, Espana), flowable formulation with 240 g/1 of tebufenozide. Both products were diluted in distilled water prior the assays. 2.2 - Insects Wasps used in these tests came from a mass-reared stock maintained in our laboratory in the substitution host C. capitata as described by Jacas & Vinuela (1994b), Insects were reared at a temperature of 25 (± 2)°C, 75 (±5)% R.H. and 16:8 h light: dark photoperiod. Each experiment consisted of 4 replicates per dose level and for the control, and were performed under the same conditions as described for rearing the insects. Control groups were supplied with distilled water. 2.3 - Exposure via ingestion Newly emerged females of 0. concolor were exposed continuosly to the test products via treated drinking water, at the highest field recommended concentrations in Spain: 150 cc/hi of Align® ( equivalent to 48 mg/I of azadirachtin), and 60 cc/hi of Mimic® (equivalent to 144 mg/I of tebufenozide), following IOBC recommendations (Hassan, 1994). Groups of 15 insects were placed in plastic boxes ( 12 cm in diameter, 5 cm high) with a hole (5.5 cm in diameter) on the upper side for ventilation, covered by a gauze. Food consisting of a milled mixture of brewer's yeast and sugar (1 :4, w:w) was placed on the bottom of the cages. Insecticide solutions were offered in small glass vials (3 cm in diameter, 3.5 cm high), closed with Parafilm® and a piece of Spontex® wiper, and were fixed to the bottom of the cages with plasticine. Food, water (in controls) and insecticide solutions were replaced as needed. Daily mortality was monitored until the last insect died. To assess possible effects on reproduction, eight females per treatment and control, were individually isolated three days after the beginning of the experiment and provided with water for one day. During the following four days, 20 fully­ grown C. capitata larvae were offered daily to each female for parasitization. Parasitoid exposed hosts were kept until the emergence of adults following a standard procedure (Jacas & Vinuela, 1994b). Number of attacked hosts (difference between the number of offered hosts and the number of emerged flies) and progeny size per female was recorded. 2.4 - Exposure via treated host For these assays, fully-grown C. capitata larvae were treated with sublethal concentrations of every insecticide and offered to 0. concolor - 235- females for parasitization. According to previous assays, two doses of azadirachtin were chosen: 0.015 and _0.15 g a.i./kg diet. For tebufenozide two high doses were selected: 0.6 and 0.06 g a.i./kg diet, because the product posses a high potency and selectivity against lepidoptera (Smagghe et al., 1996). Treated larvae were obtained by placing groups of 50 neonate C. capitata larvae in non-ventilated rearing cages (50 g of artificial diet, 9 cm in diameter by 2 cm high) (Budia & Vinuela, 1996), where 5 ml of the insecticide solutions (or water for the controls), were added 6 days later. After 48 hours in contact with the insecticide, the fully grown larvae were offered to the parasitoids at the rate 6: 1 (host:parasitoid}, for 1 hour, as described by Jacas & Viriuela (1994b). The effects of insecticides on the host, were ascertained by studying the development of groups of 30 non-parasitized, treated and untreated fully grown C.capitata larvae. 2.5 - Stat!stical analysis One-way analysis of variance (ANOVA) and LSD multiple range test or Bonferroni test (if the F was non significant) were performed on the data to determine significant (P= 0.05) dose differences, using Statgraphic (Stsc, 1987). Percentages were transformed to arsine .../ x before analysis only if data were not normal (Milliken & Johnson, 1984).

3 - ResultG 3.1 - Exposure via ingestion Continuous feeding on treated water during adult life span, at the highest field recommended concentrations of azadirachtin and tebufenozide, caused a negative effect in adults of 0. concolor(Table 1 ).

Table 1: Effects of feeding azadirachtin and tebufenozide in water to adult Opius concolor during the lifespan, on the longevity and the beneficial capacity of the wasp.

Treatment Active Doses Longevity Beneficialcapacity in�redient m� a.i./ml {dal'.s} % Attacked hosts %Pro�enl'. Control 31.4±0.7" 87.5±4.s• 65.0±7.98 ® b Align Azadirachtin 48 4.9±0.1° 64.1±8.2 43.9±4.Sb Mimic® Tebufenozide 144 27.6±0.5b 91.3±3.18 70.0±5.4.

Within the same column, data followedby the same letter do not differ significantly (P = 0.05; LSD mean separation)

Azadirachtin was far more toxic for the parasitoid than tebufenozide, and caused a significant (P< 0.05) decrease not only in longevity but also in the beneficial capacity of the wasp. The most conspicuous effect of both insecticides was in longevity and a significant reduction was observed in every treatment. Adult mean longevity in azadirachtin-treated insects was only 4.9 days, while in tebufenozide-treated and control parasitoids, was 27.6 and 31.4 respectively. Cumulative mortality during the experiment was similar in controls and tebufenozide treated insects (Figure 1). - 236

Figure t: Cumulative mortality of Opius concolor adults fed azadirachtin and tebufenozide continuously from emergence during the life span.

Exposure via drinking water

so

60

40

20 .....Azadirachtin 48 mg/ml -e-Tebufenozide 144 mg/ml 0 ...... _...... 0 6 -s:t��------'======:'J10 16 20 25 30 36 40 46 Days

The beneficial capacity of O. conco!or was only modified after ingestion of azadirachtin. The percentage of attacked C. capitata hosts by 0. concolor female, decreased from 87.5 in controls to 64.1 in treated insects. The progeny size per female was also significanctly reduced a 32.5%. 3.2 - Exposure via treated hcst Table 2 shows the biological activity of azadirachtin and tebufenozide on the host-parasitoid complex when fully grown C.capitata larvae.reared in treated media were offeredto females of 0. concolor for parasitization.

Table 2: Effects of azadirachtin and tebufenozide on the complex Opius concolor - Ceratitis capitata, when treated C. capitata larvae were offered for parasitization to 0. concolor.

Non parasitized Parasitizedlarvae Active Doses larvae Treatment ingredient g/kgdiet % C. capitata % 0. concolor 0. concolor adults emer�ed· adults emer1:1ed· lon�evi!}'.{ d aist Control 94.0±2.2· 45.7±4.o· 33.2±2.0· Aligric, Azadirachtin 0.015 48.8±3.9b 27.3±4.0b 32.1±2.0·

., 0.15 0.0 0.0 Mimic Tebufenozide 0.6 92.s±o.s• 40.8±6.3ab 30.7±1.1· 6 91.4±2.38 26.5±8.4b 31.1±2.0· • Whitinthe samecolumn, data followedby thesame letter are significantlyequal (P = 0.05, LSD meanseparation) •• Data followed by the same letter do not differ significantly (P = 0.05, Bonferroni mean separation) - 237-

C. capitata larval development was very affected by the selected doses of azadirachtin. At a dose of 0.15 g/kg of diet, larvae were unable to reach the adult stage, and at a dose of 0.015 g/kg of diet, a 48.1 % reduction on adult emergence was scored. Contrary to these results, the selected doses of tebufenozide (0.6 and 6 g/kg of diet), had no significant effect on the fly. Neither azadirachtin nor tebufenozide modified the longevity of the parasitoids, but around a 40% reduction on the number of emerged adults was observed with the lowest dose of the first product, 0.015 g/kg of diet and the highest dose of the second one, 6 g/kg of diet. The lowest applied dose of tebufenozide, 0.6 g/kg of diet, did not have any effect on the parasitoid. Pattern of daily mortality and control and treated insects, was similar in every case {Figure 2).

Figu:re 2: Cumulative mortalityof Opius concolor adults emerged afterparasitization in Ceratitis capitata treated larvae with azadirachtin and tebufenozide.

Exposure via treated host

Cumulative mortallty(%) 100

80 ······························· .,:,.Control 80 ·· ..,__Azadlrachtln0.015 g/kg diet -e-Tebulenozlde0.8 g/kg diet 40 .. * Tebulenozlde 6 g/kg diet

20

0 ---II--IO'

4 - Discussion 4.1 - Longevity It has been generally reported in literature, a lack of effects on adult survival after adult treatment with IGR pesticides. In contrast with that, our results show that adult longevity of 0. conco/or decreased drastically after continuous ingestion of azadirachtin. Effects of neem on the survival of hymenopterans, seem to be contradictory however, and both a decrease and a prolongation of the life span have been reported in literature in different species {Schmutterer, 1995). When tebufenozide was supplied to adults, a slight reduction was observed, although more severe effects have been reported by Jacas et al. { 1995) in this insect, at considerably higher doses of this product {between 1OOO and 10000 mg/I). Contrary to these· results, 0. concolor adults that emerge from C. capitata treated pupae with the two studied insecticides, appeared - 238 morphologically normal and exhibited daily mortalities similar to those recorded in controls. 4.2 - Beneficia! capacity The beneficial capacity of 0. conco/or evaluated by means of the percentage of attacked hosts and progeny size per female, was only negatively affected after oral administration of azadirachtin during parasitoid adult life span. Contrary to these results, tebufenozide had no effect on the wasp. The naturally derived compound azadirachtin seems to be rather selective for beneficials {Sipcam lnagra, 1996), but small non statistically significant effects on reproduction have been detected in other Hymenoptera, such as a reduction in parasitized whiteflies for Encarsia formosa Gahan or a reduction in fecundity in Trichogramma minutum Riley (Ruiz et al., 1997). 4.3 - Adult emergence Azadirachtin has a visible effect on the host C. capitata when larvae were reared on the presence of the toxic. Adult emergence was totally inhibited at the highest dose studied, while at the lowest dose, a 48.1 % reduction was obtained. Drastic effects of this insecticide on the fruit fly, had already been reported by Stark et al. (1990), but when mature third instar larvae pupated in treated sand with ethanol dilutions of a highly purified and concentrate neem seed extract. As it could be expected according to the high selectivity of tebufenozide against lepidopterans (Smagghe et al., 1996), the insecticide had no effect on this fly at the studied doses, which were on the other hand, considerably high. Our results show that both pesticides had an effect on the parasitoid emergence, when they developped in treated larvae, even though the larvae seemed not to be affected and gave normal C. capitata emergence rates, as it was the case for tebufenozide. The decrease on 0. concolor emergence was very conspicuous for azadirachtin and for the highest dose of tebufenozide, while no effect was detected at the lowest dose of this last product. However, emerged 0. concolor adults seemed to be normal, and had longevities similars to those recorded in controls. 5 - Conclusion In conclusion our results indicate the compatibility of tebufenozide and 0. concolor, and the toxicity of azadirachtin in laboratory to this parasitoid both when ingested via drinking water or when the parasitoids emerge from treated C. capitata larvae. Further studies both in laboratory, semi-field and field, should investigate however other possible routes of contamination with these products, before establishing the compatibility or incompatibility of these pesticides in 1PM (integrated pest management) programmes with 0. concolor. Acknowledgements We acknowledge the kind of gift of azadirachtin from Sipcam lnagra and of tebufenozide from Rohm & Haas Espana. This research was partially subsidized by projects AGF94-0084 from the Spanish Ministry of Education and Culture and 06MI022/96 from the Community of Madrid to Dr. E. - 239 -

Vinuela. A Gonzalez is recipient of a pre-doctoral grant from the Community of Madrid.

References AgrEvo (1997) - Mimic®, l'insectticida diverse. Nota tecnica. 28 pp. i3UDIA, F. & VINUELA, E. (1996) - Effects of cyromazine on adult C.capitai"aon mortality and reproduction. J. Econ. Entomol. 89: 826-831. CROFT, B.A. ed. (1989) - Arthropod biological control agents and pesticides. John Wiley & Sons. New York. 723 pp. FIMIANI, P. (1989) - Mediterranean region. In: Fruit flies. Their biology, natural enemies and control. Vol. 3A, AS. Robinson & G. Hooper eds. Elsevier. The Netherlands. pp. 39-50. HASSAN, S.A. (1994) - Activities of the IOBC/WPRS working group "pesticides and beneficial organisms". /OBC Bull., 'i7: 1-5. JACAS, J.; GONzALEZ, M. & VINUELA, E. (1995) - Influence of the applicati.on method on the toxicity of the moulting accelerating compound tebufenozide on adults of the parasitic wasp Opius conco/or. Med. Fae. Landbouww. Univ. Gent 60/lb: 935-939. JACAS, J. & VINUELA, E. (1994.) - Side-effects of pesticides on Opius conco/or; a parasitoid of the olive·fly. /OBC Bull.,17: 143-146 ... JACAS, J. & VINUELA, E. (1994b) -Analysis of a method to study the side-effects of pesticides on the parasitic wasp Opius concolor, a parasitoid of the olive fly Bactrocera oleae. Bioc. Sci. Technol., 4: 147-154. JACAS, J.; VINUELA, E.; ADAN, A.; BUDIA, F.; DEL ESTAL, P. & MARCO, V. (1992) - Efectos secundarios de algunos plaguicidas utilizados en el olivar espanol sabre adultos de Opius conco/or, parasitoide de la mosca de laaceituna Bo/.San. Veg. P/agas, 18: 315-321. JIMENEZ, A (1989) - La utilizaci6n de Opius concolor en la lucha contra la mosca de la aceituna, Dacus oleae. Tesis Doctoral. UPM. Madrid. 289 pp. MAZOMENOS, B.E.; STEFANOU, D.; LANGLEY, P. & PANTAZI­ MAZOMENOS, A. (1997) - Effects of sugar-formulated triflumuron treated targets on reproduction in the olive fruit fly Bactrocera oleae. Bull. Entomo/. Res. 87: 169-172. MILLIKEN, G.A. & JOHNSON, D.E. (1984) - Analysis of messy data. Vol. I: designed experiments.Van Nostrand Reinhold, New York. RUIZ, A.; PRADES, J.; CANO, F.J. & ABRIL, E. (1997) - Align®, un bioinsecticida de origen vegetal, biodegradable, compatible con el media ambiente y los enemigos naturales. Actas II Congreso SEAE Pamplona 21- 27 setiembre 1996 (in press). SCHMUTTERER, H. (1995) - Side effects of beneficials and other ecologically important non-target organims. in: The neem tree. pp. 495-527. VCH. Weinheim. SIPCAM INAGRA (1996) -Align®. lnformaci6n tecnica. 16 pp. SMAGGHE, G.; VINUELA, E.; BUDIA, F. & DEGHEELE, D. (1996) - In viva and in vitro effects of the nonsteroidal ecdysteroid agonist tebufenozide on cuticle formation in Spodoptera exigua: an ultrastructural approach. Arch. Insect Biochem. Physiol. 32: 121-134. - 240 -

STARK, J.D.; VARGAS, R.I. & THALMAN, R.K. (1990) -Azadirachtin: effects on metamorphosis, longevity and reproduction of three tephritid fruit fly species. J. Econ. Entomol. 83: 2168-2174. STSC (1987) - Statgraphics user's guide, version 5.0. Graphic software system. STSC, Rockville, MD. VIGGIANI, G. (1986) - La protection phytosanitaire en oleiculture. In: Traite d'entomologie oleicole. pp. 339-347. International Olive Council. Spain. - 241 -

LIST OF PARTICIPANTS

ARGELIA - ALGERJE - ALGERIA Gaouar, Nassira Institut de Biologie Universite de Tlemcen BP358 - RP 13000 Tlemcen Oukil, Salah Station Regio:.alede la Protection des Vegetaux 12 - Avenue Hacene Badi EI-Harrach BRASIL - BRE:SIL- BRAZIL Vatter, Arthur Center for Nuclear Energy in Agriculture University of Sao Paulo Av. Centenario 303 P.O. Box 96- Piracicaba - SP - 13400-970

ESPANHA - ESPAGNE - SPAIN Climent, Jose M. L. Generalitat Valenciana Coselleria de Agricultura, Pesca y Alimentacion Seccion de Sanidade y Certificacionde Sanidad Vegetal Prof. Manuel Sala, 2 03003 Alicante Del Pino , A. !NIA - Dept. Protecci6n Vegetal Apartado Oficial461 13 Moncada - Valencia Montiel, Bueno Alfonso Delegacion Provincial de Agricultura Avenida de Madrid 25 E - 23071 JAEN Munis, Mariano Consejo Superior de Investigaciones Cientificas Centro de Ciencias Medioambientales Serrano 115 Dpdo -28006 - Madrid Ochando , M. Dolores Universidade Complutense de Madrid Facultad de Ciencias Biologicas Departamento de Genetica 28040 Madrid Ros, Pedro !NIA - Centro de lnvestigaci6n y Tecnologia Area de Protecci6n Vegeta[ Aptd 8111 - 28080 Madrid - 242

ESPANHA - ESPAGNE- SPAIN Vinuela, Elisa Sandual Proteccion de Cultivos ETSI Agronomos E - 28040 Madrid FRAN(:A - FRANCE Quilici, Serge Inst. de Recherches sur Fruits et Agrumes Station de Bassin Martin F-97455 Saint-Pierre Cedex - B.P. 18 - La Reunion

GRECIA - GRECE Manoukas, A.G. Institute of Biology N.R.C. "Demokritos" P.O. Box 60228 GR- 15310 Aghia Paraskevi

ITALIA - ITALIE Buyc!u, E. J. Insect and Pest Control Section Joint FAO/IAEA Division Viale dei Primati Sportivi, 86 - I - 00144 Roma Canale, Angelo Universita Degli Studi di Pisa Dipartimento di "Coltivazione e Difesa delle Specie Legnose" Sez: Entomologia Agraria Via delBorghetto 80 - 56124 Pisa Lipizzi, F. Dipartimento di Protezione delle Piante Universita Degli Studi della Tuscia Via S. Camillo de Lellis I - 01100 Viterbo Milani ,R. Dipt. Biologia Animate Universita di Pavia Piazza Botta 9 - I - 27 l 00 Pavia Pucci , Claudio Dipartimento di Protezione delle Piante Universita Degii Studi della Tuscia Via S. Camillo de Lellis I - 0 I J 00 V iterbo Raspi , Alfio Universita Degli Studi di Pisa Dipartimento di "Coltivazione e Difesa delle Specie l.egnose" Sez: Entomologia Agraria Via de! Borghetto 80 - 56124 Pisa • 243 •

!TA.LIA - ITALJE Spanedda, A.F. Dipartimento di Protezione delle Piante UniversitilDegli Studi della Tuscia Via S. Camillo de Lellis I- 01100 Viterbo

SPERANZA, S. Dipartimento di Protezione delle Piante Universita Degli Studi della Tuscia Via S. Camillo de Lellis I - 01100 Viterbo MARROCOS - MAROC - MOROCCO Afellah , Mohamed I N RA- Laboratoire de Zoologie B.P. 578 Meknes Bakri, Abdeljelil Universite Cadi Ayyad Faculte des Sciences Semlalia Unite de Controle Biologique des Insectes 40000 Marrakech PORTUGAL - PORTUGAL Alves, Catarina Instituto de Medicina Tropical Rua da Junqueira - 1300 - Lisboa Camboa, Filomena Martins DGPC Quinta do Marques 2780 Oeiras Cardoso, Miguel J. Viegas R. Escritor Aquilino Ribeiro n° 25, 2° esq. 7800 Beja Carneiro, Ana Paula D. G. P. C. Tapada da Ajuda - Edificio l 1300 Lisboa Carvalho, Ana Catarina R. Publia Hortensia deCastro 11, 2°D 1500 - Lisboa Costa, Ana Cristina R. Av. 24, 1087 - 2° 4500 Espinho Costa, Jorge P. Nuno DRAEDM - Esta9ao de Avisos Rua da Restaura9ao, 336 4050 Porto Coutinho, Jose P. Ribeiro Escola Superior Agraria de Castelo Branco Quinta da Sra de Mercules 6000 Castelo Branco - 244 -

PORTUGAL - PORTUGAL Frescata, Carlos BIOSANI, Lda Casal St0 Isidro, S_erra Louro P - 2950 Palmela Gon�alves, Mario Leal DGPC Tapada da Ajuda 1300 Lisboa Guardado, M. I. IICT -Centro de Zoologia Rua da Junqueira 14 -1300 Lisboa Lavadinho, A. M. P. DGPC Quinta do Marques 2780 Oeiras Martins, Ana Luzia F. G. P. R. Escritor Aquilino Ribeiro n° 25, 2° Esq. 7800 Beja Matias, Carlos Laborat6rio de Proteq:iiolntegrada EstayiioNacional de Fruticultura de Vieira Natividade Estrada de Leiria - Apa1t. 167 2461 -Alcoba,.aCodex Patanita, Isabel Cardoso Escola Superior Agraria de Beja Praceta Rainha D. Leonor 7800 Beja Pereira, Claudia R. do Carmo R. UniiioPiedense 86, 3° Dt0 2810 Laranjeiro Pereira, Rui Direcyiiode Servi1rosde fnvestigayiio Agricola Estrada Eng0 Abel Vieira 9135 Camacha- Madeira Piedade-Guerreiro, J. IICT -Centro de Zoologia Rua da Junqueira 14 -1300 Lisboa

TUNISIA - Tl/NISIE Habib, Dhouibi Mohamed lnstitut National Agronomique de Tunisie 43 Avenu Charles Nicolle I 082 -Tunis - Mahrajene - 245

INDEX OF AUTHORS

Afellah, M. Hurtrel, B. Alves, C. Koch, A. Arfaoui, T. Lindquist, D. Arlhur, V. Lipizzi, F. Artigncs, M. Loussaief, F. Bakri,A. Manoukas,A. lfarbosa. A. Mexia,A. Ben H,im::di. ! Milani, R. Boukhari, l\l. !Vluiiiz,M. Br:iziio,A. Naamani, K. Bues,R. Ochando, D. Buyck:<,E. Ouk.ii,S. Caldeira, J. Pacheco, J. Canale,A. Patanita, I. Cardoso,M. Pereira,R. Carvalho,P. Piedade-Gut'rreiro,.J. Causse,R. Pucci, C. Cayol,J. Putruele, M. Chemssedine, M. Quilici, S. Climent, .i.. Raspi,A. Coutinho,.!. Reyes, A. DaGtas, L. Rodrigues,A. DelPino,A. Ros,P. Uhouibi,M. Sequeira, M. Dias,M. Silva, G. Feilah, H. Silva,N. Felicioli,A. Smailici, C. Gaouar,N. Spanedda, A. Garrido,A. Speranza, S. Gomes da Silva, J. Toubon,J. Gonzalez, M. Veiga, C. Guardado, I. Viiiuela,E. Hadis, H. Zalom, F. Herbaut, C. Zarai,M. Hila!, A. Zografou,E.