D 4 10 22 Limited Distribution
WORKING MATERIAL
INCREASING THE EFFICIENCY OF LEPIDOPTERA SIT BY ENHANCED QUALITY CONTROL
Third Research Co-ordination Meeting within the FAO/IAEA Co- ordinated Research Programme held in Phoenix, USA from 12 to 16 September 2012
Reproduced by the IAEA Vienna, Austria 2013
NOTE The material in this document has been supplied by the authors and has not been edited by the IAEA. The views expressed remain the responsibility of the named authors and do not necessarily reflect those of the government(s) of the designating Member State(s), In particular, neither the IAEA nor any other organization or body sponsoring this meeting can be held responsible for any material reproduced in this document. 2
Table of Content
Introduction 5
The Coordinated Research Project 5
The Third RCM 6
Quality Management Workshop 6
Agenda 8
List of Participants 9
Working group Reports 14
Working Papers 46
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Introduction
Some species of Lepidoptera are among the most damaging pests of food and fibre crops around the world. Nineteen out of the 37 worst insect pest threats facing US agriculture are Lepidoptera (51%). Codling moth Cydia pomonella (L.), diamondback moth Plutella xylostella (L.), oriental fruit moth Grapholita molesta (Busck), the American cotton bollworm or corn earworm Helicoverpa zea (Boddie), and pink bollworm Pectinophora gossypiella (Saunders) are all examples of globally important moth pests that in most cases are being controlled with broad-spectrum and persistent insecticides. The extensive use of insecticides as the major control tactic is putting the farmer for some serious challenges as many Lepidoptera have developed resistance to the commonly used insecticides. Moreover, recently, several effective pesticides have been removed from the European market due to increased recognition of long-term human health and environmental harm caused by their wide-spread use. In addition, there is an increasing market pressure to reduce pesticide residues in food commodities. Finally, global increases in trade and travel have resulted in an increase in the rate of invasion of lepidopteran species, which threaten agricultural systems, markets, communities, and biodiversity on a worldwide basis.
Insect pests can be controlled or eradicated through a “birth control” method based on genetic manipulation, known as autocidal pest control, or the sterile insect technique (SIT). It involves the colonization and mass- rearing of the target pest species, sterilization of the insects through the use of gamma radiation, and their release into the field on a sustained basis and in sufficient numbers to achieve appropriate sterile to wild insect over-flooding ratios. The sterile insects need to find and mate with fertile, virgin insects, rendering the wild population infertile. Due to the absence of offspring, the natural pest population will decrease. The validity of this method has been demonstrated for many insect pests, including many moths, screwworm, tsetse and fruit flies.
A refinement of the SIT for Lepidoptera, known as inherited sterility or F1 sterility, involves lowering the irradiation dose and consequently increasing the competitiveness of the released parental insects. Moths treated with a lower sterilizing dose live longer, are stronger fliers, and mate more frequently than moths treated with higher radiation doses. When inherited sterility systems are implemented into moth SIT programs, great improvements in programme efficiency can be obtained.
There is broad international consensus that intervention campaigns against Lepidopteran pests should be based on the area-wide concept of integrated pest management (AW-IPM), and that the SIT can be considered as a key tactic for creating pest free areas or for pest management within IPM programmes.
The use of SIT as a control tactic has many advantages, including its species specificity, its environment friendliness, and its compatibility with the use of other area-wide control tactics such as mating disruption, biological control, cultural control methods and the use of bio-rational pesticides. There are several successful examples of the use of SIT against Lepidoptera including operational containment, suppression and eradication programmes: codling moth, pink bollworm, cactus moth Cactoblastis cactorum (Berg), painted apple moth Teia anartoides Walker, false codling moth Thaumatotibia leucotreta (Meyrick). In addition, there have been pilot projects to demonstrate feasibility in the field; e.g. gypsy moth Lymantra dispar (L.), tobacco budworm Heliothes virescens (F.), corn earworm, oriental fruit moth, carob moth Ectomyelois ceratoniae Zeller, and Asian corn borer Ostrinia furnacolis (Guenée). The Coordinated Research Project (CRP)
In the past years, it has become clear that expansion of the SIT to more key lepidopteran pests will require improvements that increase the quality control and management of mass-rearing, irradiation, shipping, release and field assessment activities. During a consultants meeting at the IAEA in Vienna from 3-7 December 2007 3 key areas of research were identified that could improve the quality management of Lepidoptera for use in SIT programmes: 5
• identifying and characterizing factors and variables that affect quality and field performance of released moths;
• developing and improving tools and methods to assess, predict and enhance the field performance of released moths based on insect quality;
• developing new and improved methods for enhancing rearing systems, facilitating the selection for performance and fitness traits that improve colony establishment, refurbishment and production, and the field performance of released moths.
The consultants considered a CRP as an excellent mechanism to deliver the required research.
The Third Research Coordination Meeting (RCM)
The 3 rd RCM of the RCP on “Increasing the efficiency of Lepidoptera SIT by enhanced quality control” was hosted by the USDA-APHIS-PPQ and held at the Fiesta Resort Conference Centre in Phoenix, Arizona, US (12-16 September 2012). Eighteen contract and agreement holders from 14 countries attended the meeting. In addition, invited speakers from USDA (Drs Bob Staten and Michelle Walters), Canada (Ms Cara McCurrach and Mr Scott Arthur), and South Africa (Mr Martin Wolfarther) gave presentations on the pink bollworm programme in the US, the SIR Codling Moth Programme in Canada, the SIT programme against codling moth in South Africa and on the use of strontium for marking sterile moths. The CRP also welcomed two new contract holders from Argentina and Chile. This third RCM was very well organised and the great efforts of the local counterpart Mr Greg Simmons are very much appreciated.
The first two days of the RCM were devoted to presentations of the participants. They presented the progress made with their research since the last RCM in Stellenbosch, South Africa. From the presentations, it was obvious that most participants had made good progress and had been able to carry out most of the activities planned during the second RCM in Stellenbosch, South Africa.
After reviewing the progress made so far, the participants discussed the work plans for the coming months. The details of the progress made so far and the work plans for the last phase of the CRP can be found in this working material document. The final RCM of this CRP is scheduled for 2-6 June 2014 and will be hosted by the Okanangan Kootenay Sterile Insect Release Progamme, in Kelowna, British Columbia, Canada.
There were no major changes made in the programme or objectives of the CRP.
Workshop on “Assessing quality management aspects of Lepidoptera mass produced for the Sterile Insect Technique in large operational settings”
Prior to the third RCM, a 2-day workshop was organised and hosted by the USDA-APHIS-PPQ, Arizona on “Assessing quality management aspects of Lepidoptera mass produced for the Sterile Insect Technique in large operational settings” (10-11 September 2012). As part of the workshop, the group viewed the USDA Pink Bollworm Facility (Phoenix Arizona) that produces the sterile insects for the eradication programme in Texas, New Mexico, Arizona, California and northern Mexico (Sonora, Chihuahua). Participants were able to view and discuss the various quality control management issues that are put in place in the facility to ensure the production of high quality sterile insects for release. The group also viewed the loading of the release planes and the quality control aspects related to the chilled adult release system. During the workshop, discussions were held with staff of the Arizona Cotton Research Board and with scientists who have developed transgenic pink bollworm strains. The workshop also included a visit to the University of 6
Arizona, where Dr Bruce Tabashnik gave a presentation on the use of sterile insects as a substitute to “refugia” to manage resistance in pink bollworm on Bt cotton. In addition, a demonstration was given of the Picarro Carbon Isotope Analyser that uses stable isotopes to distinguish wild from mass produced moths.
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AGENDA Third FAO/IAEA Research Co-ordination Meeting on ‘Increasing the Efficiency of Lepidoptera SIT by Enhanced Quality Control ’
12-16 September 2012
Fiesta Resort & Hotel
Phoenix, USA
Wednesday 12 September 2010
Opening Session
08:30 – 08.45 Official opening of the RCM by Eoin Davis , Director, Pink Bollworm Rearing Facility, USDA-APHIS-PPQ.
08.45 – 09.00 Greg Simmons / Marc Vreysen - Administrative details, objectives of the meeting and outline of the agenda.
SESSION 1: Chairperson: Greg Simmons 09.00 – 09.45 Invited speaker Bob Staten - Progress in eradication of the pink bollworm Pectinophora gossypiella (Lepidoptera: Gelechiidae) across the Southwestern cotton belt.
09.45 – 10.00 Discussion
10.00 – 10.30 COFFEE BREAK
10.30 – 11.00 Harari, A. - Inherited sterility and quality control of Lobesia botrana
11.00 – 11.30 Lobos, C., G. Alvaro, G., and Eduardo, M. - Domestication and mass-rearing of Lobesia botrana
11.30 – 12.00 Saour, G. - Inherited sterility and irradiated male's flight ability in the European Grapevine Moth (Lepidoptera: Tortricidae).
12.00 – 12.30 Carabajal, L.Z., Cagnotti, C.L., Viscarret, M.M., Ferrari, M.E., Lauría, J.P. and López, S.N. - Development of the sterile insect technique to control of the tomato moth Tuta absoluta (Lepidoptera: Gelechiidae
Discussion
12.30 – 14.00 LUNCH
SESSION 2: Chairperson: Des Conlong 14.00 – 14.30 Addison. M.F. - Enhanced production and quality control of mass-produced codling moth in South Africa
14.30 – 15.00 Carpenter, J., Hight, S., and Blomefield, T. - Quality control tests of lab-reared Cydia pomonella and Cactoblastis cactorum field performance: Comparison of laboratory and field bioassays
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15.00 – 15.30 Invited speaker Cara McCurrach - Current Status and progress made on codling moth control by Okanagan SIR program.
15.30 – 16.00 COFFEE BREAK
16.00 – 16.30 Invited speaker Wohlfarter, M., and Stenekamp D. - Codling moth SIT, from pilot research based to commercial mass-rearing: recapture the true indicator
16.30 – 17.00 Chen, M., Men, Q., Li, Y., Feng, J., and Zhang, Y. - Gene flow among Cydia pomonella populations in China: knowledge on the population genetics for the SIT
17.00 – 17.30 Pérez-Staples, D., Barradas-Juanz, N., and Díaz-Fleischer, F. - Modifications on the rearing method and larval diet of Hypsipylla grandella (Lepidoptera: Pyralidae)
17.30 – 18.00 Discussion
Thursday 13 September 2012
SESSION 3: Chairperson: R. K. Seth
08.30 – 09.00 Simmons, G. - Development of the Sterile Insect Technique for the Light Brown Apple Moth, light brown apple moth, Epiphyas postvittana (Lepidoptera: Tortricidae)
09.00 – 09.30 Conlong, D.E., Walton, A.J., Mudavanhu, P., and Potgieter, L. - Sterile Insect Technique in Southern African sugarcane pest management
09.30 – 10.00 Koekemoer, W. - Expansion of the SIT programme for FCM suppression in South Africa
10.00 – 10.30 COFFEE BREAK
10.30 – 11.00 Suckling DM, El-Sayed AM, Stringer, LD, Mas, F., Stanbury,M., Battisti, A. and Hardwick, S. - New tools for insect quality assurance and surveillance
11.00 – 11.30 Hood Nowotny, R., and Carpenter, J. – New isotopic methods for determining the origin of moths and who has mated who
11.30 – 12.00 Invited speaker Michelle Walters - Use of Strontium chloride as an additional marker for sterile pink bollworm
12.00 – 12.30 Discussion
12.30 – 14.00 LUNCH
SESSION 4: Chairperson: R. Hood Nowotny
14.00 – 14.30 Woods, B., Kaur, A., Lacey, I. and Soopaya, A.- The importance of spermatophore dissection in laboratory and field studies of fitness of irradiated light brown apple moth (LBAM) in Western Australia
14.30 – 15.00 Wee, S.L., and Park, K.C. - Electrophysiological and behavioural assays of cabbage- derived volatiles on diamondback moths
15.00 – 15.30 Seth, R. K., Zubeda and Mahtab Zarin - Sperm dynamics in correlation with mating behaviour of sub-sterilized male moths, Spodoptera litura (Fabr.) and their F1 progeny, as a crucial assessment of ‘Inherited Sterility’ technique for Lepidopteran pest suppression 9
15.30 – 16.00 COFFEE BREAK
16.00 – 16.30 Mei-ying Hu, M.Y., Weng, Q-F, Deng, Y.Y., Zhu, F.W., and Fu, H.H. - Stability on Inherited Sterility (IS) of Litchi Stem-end Borer (Conopomorpha sinensis Bradley) by irradiation and its evaluation of field cage trial.
16.30 – 17.00 Lebdi Grissa, K., and Chakroun, S. - Comparison between two radiation sources (Tunis and Vienna) for the biological parameters and the sterilization dose of the carob moth
17.00 – 18.00 Discussion
Friday 14 September 2012
09.00 – 12.00 Visit to CPHST lab – visit to see research on transgenic pink bollworm Pectinophora gossypiella for use in SIT.
12.00 – 14.00 LUNCH
14.00 – 17.00 Establishment of working groups – discussions of working groups etc.
Saturday 15 September 2012
08.30 – 10.00 Discussion in working groups on status of research, planned activities, recommendations, work plans
10.00 – 10.30 COFFEE BREAK
10.30 – 12.00 Discussion in working groups on status of research, planned activities, recommendations, work plans etc.
12.00 – 14.00 LUNCH
14.00 – 17.00 Discussion in working groups on status of research, planned activities, recommendations, work plans etc.
Sunday 16 September 2012
08.30 – 10.30 Preparation of report of each working group
10.30 – 11.00 COFFEE BREAK
11.00 – 12.30 Preparation of report of each working group
12.00 – 14.00 LUNCH
14.00 –16.00 Presentation of work plans of each working group, closure of the RCM
16.00 – 18.00 Departure airport
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LIST OF PARTICIPANTS
ARGENTINA Ms. Silvia LOPEZ (represented in the meeting by Ms Carabajal) Instituto National de Tecnologia Agropecuaria Nicolas Repeto de los Reseros 1668 Hurlinham email:[email protected] Buenos Aires
AUSTRIA Ms. Rebecca HOOD (in lieu of Wanek) Dept of Chemical Ecology and Ecosystem Research Tel: +43 4277 54251 Vienna Ecology Center Fax +43 4277 9542 Faculty of Life Sciences – University of Vienna Althanstrasse 14 1090 Vienna
CHINA Ms. Mei-Ying HU Tel: +86 20 852 80308 South China Agricultural University Fax: +86 20 852 80292 Department of Plant Protection e-mail: [email protected] 510642 Guangzhou
Mr Maohua CHEN College of Plant Protection; Northwest A&F University P.O. Box 133, 3 Taicheng Road, Yangling Xianyang 712100 Shaanxi China email: [email protected]
CHILE Mr. Carlos Roberto LOBOS AGUIRRE Programa Mosca del Mediterráneo (Programa MOSCAMED) Servicio Agrícola y Ganadero (SAG) Casilla 4088, Avenida Bulnes 140, 3r. piso Santiago Chile email: [email protected]
ISRAEL Ms. Ally HARARI Tel: +972-3-9683252 Ministry of Agriculture and Rural Development; Fax: +972-3-9665327 Agricultural Research Organization Volcani Centre email [email protected] Department of Entomology P.O. Box 6 50250 Bet Dagan
INDIA Mr. Rakesh SETH Tel: +91-11-27666564 Department of Zoology Fax: +92-11-27666564 University of Delhi email [email protected] New Delhi 110 007
MALAYSIA Ms. Suk Ling WEE Tel: +603 8921 5925 Universiti Kebangsaan Malaysia Fax: +603 8925 3357 School of Environmental & Natural Resources Sciences email: [email protected] Faculty of Science & Technology 43600 Bangi, Selangor Darul Ehsan
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MEXICO Ms Diana PEREZ STAPLES Universidad Veracruzana Apartado Postal 250 91090 Xalapa Mexico email: [email protected]
NEW ZEALAND Mr. David M. SUCKLING Tel: +64 3 977 7340 Horticultural and Food Institute of New Zealand Ltd. Fax: +64 21 2268 344 Mt. Albert Research Centre email: [email protected] 120 Mt. Albert Road Private Bag 92169 Auckland
38 Bryndwr Rd Christchurch New Zealand
SOUTH AFRICA Mr. Des CONLONG Tel: +27 (0) 31 508 7477 South African Sugar Research Institute (SASRI) Fax: +27 (0) 31 508 7597 Private Bag X02 email: [email protected] 4300 Mount Edgecombe
Mr. Matthew Friend ADDISON Tel: +27 (0) 21 882 8470/1 Deciduous Fruit Producers Trust Fax: +27 (0) 21 882 8996 Suit 275 email: [email protected] Postnet X5061 7599 Stellenbosch
Mr. Willem KOEKEMOERr XSIT Tel: 022 921 2993 P.O Box 422 Fax: 022 921 2993 7340 Citrusdal email:[email protected]
SYRIAN ARAB REPUBLIC Mr. George SAOUR Tel: +963 11 2132580 Atomic Energy Commission of Syria (AECS) Fax: +963 11 6112289 Department of Molecular Biology and Biotechnology email: [email protected] Kafar Sousah 17 Nissan Street Damascus
TUNISIA Ms. Kaouthar LEBDI-GRISSA Tel: +216 71 289 431 Institut National Agronomique de Tunisie Fax : +216 71 799 391 43, avenue Charles Nicole email : [email protected] 1082 Tunis
UNITED STATES OF AMERICA Mr. James CARPENTER Tel: +1 229 387 2348 Crop Protection and Management Research Unit Fax: +1 229 387 2321 Agricultural Research Service (ARS) email: [email protected] 2747 Davis Road Tifton, GA 31794
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Mr. Gregory SIMMONS CPHST Light Brown Apple Moth Coordinator Tel: 831-796-9605 Temporary Duty Location Cell: 602-708-7286 USDA-APHIS-PPQ- Fax: 831-632-0474 7697 Highway 1, Building 20 email: [email protected] Moss Landing CA 95039-9672
Observers
Martin WOHLFARTER ENTOMON TECHNOLOGIES (Pty) Ltd Tel/Fax: 27 (0) 21 882 9164 P.O. Box 12669, Die Boord, 7613, Mobile: 27 (0) 72 288 2060 South Africa email: [email protected]
Cara NELSON and Scott ARTHUR Okanagan Kootenay Sterile Insect Release Program Tel: (250) 469-6182 or (250) 808-0287 1450 KLO Road, Kelowna, BC V1W 3Z4 Email: [email protected]
Michelle WALTERS and Bob STATEN, USDA – APHIS Pink Bollworm Programme
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WORKING GROUP REPORTS
The 3 working groups discussed the achievements obtained so far and the plans for the coming 18 months.
Working Group 1: Factors and Variables that Affect Quality of Lepidopterans Used in SIT Programs.
Working Group 1. 2010-2012 Report of Progress
1. Biological attributes that may affect quality
Cactoblastids cactorum Planned activity 1. Laboratory and field bioassays developed previously will be used to evaluate the effect of pupal weight and moth size on quality of the cactus moth (Carpenter and Hight).
Experimental design and methods are under discussion and development.
Planned activity 2. Increasing radiation doses will be used to degrade insect quality and this quality will be assessed with previously developed laboratory and field bioassays (Carpenter and Hight).
Laboratory (mating and flight ability) bioassays and field (field cage and open field release) bioassays were conducted simultaneously to discern the ability of the different bioassays to predict quality and field performance of codling moths produced in a commercial mass-rearing facility. Moth quality was degraded by different levels of radiation during the sterilization procedure. Both the laboratory flight bioassay and the field cage bioassay successfully detected quality and performance differences that were relevant to moth performance in the field. However, the study data suggests that the field cage bioassay was a better predictor of the daily performance of males that had been released in the orchard than the laboratory flight bioassay. Conversely, data suggests that the controlled climatic conditions of the laboratory allowed the flight cylinder bioassay to be more sensitive in detecting daily fluctuations in the quality of moths caused by factors within the mass rearing facility. Therefore, both laboratory and field bioassays may be required to provide feedback on quality and performance of mass-reared moths in a SIT program.
Lobesia botrana
Planned activity 3. A suitable field site will be identified to carry out field bioassays using Lobesia botrana with different size classes from laboratory insect colony (Saour).
Field site has been identified and field bioassays have been initiated.
Planned activity 4. Laboratory and field bioassays will be conducted to assess the impact of size of Lobesia botrana on longevity, mating propensity, flight ability (in the laboratory) and trap captures in field cages setting and in orchards (in the field). Bioassays will be conducted at the same time, with same cohort of insects, and the same insect size classes. If a suitable field site cannot be identified, a field study will be attempted in a non-grape setting (Saour).
Laboratory bioassays have been initiated and are ongoing to examine the effect of different diets on moth quality. Parameters under investigation include longevity, flight ability and mating propensity.
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Planned activity 5. Identifying characteristics in male moths that may affect the size of the transferred spermatophores, i.e. male size, age, irradiation, and the number of times that a male mated. Sperm volume and apyrene: eupyrene ratio may also influence the male reproductive success, i.e. number of eggs oviposited/fertilized and female mating behavior. A correlation between sperm quality and male characteristic, e.g. male size, could be used to predict the quality of a male as a mating partner (Harari). Characteristics were identified in male moths that may affect male reproductive success after irradiation with 100 Gy: Compared to untreated males irradiation with 100 Gy (i) did not affect male or female emergence, (ii) did not affect male and female survival, and (iii) did not affect male flying propensity to baited traps in a field cage. Comparing reproductive success of untreated males with that of males treated with 100 Gy revealed: (i) irradiated males and females mated significantly less (~5%) than untreated adults, and (ii) fertilization success of irradiated males and females was significantly reduced. The effect of irradiation on sperm characteristics showed (i) no significant effect in sperm production between irradiated males in the pupae stage and untreated males, (ii) similar numbers of apyrene and eupyrene sperm were found in the spermatophores located in the female’s bursa copulatrix right after natural copulation. In summary – no deleterious effects were detected on the quality of males after being irradiated with 100 Gy. Generally, there was a correlation between male size and size of the spermatophores but no effect of spermatophore size on reproductive success was detected.
Spodoptera litura
Planned activity 6. Sperm descent in the reproductive tract of irradiated male Spodoptera and their F1 progeny will be correlated with mating success and reproductive competence (Seth).
The circadian rhythmic release of sperm during the photophase and scotophase was not affected by
irradiation in sub-sterilized parent male moths and their F 1 generation male moths. In the photophase and scotophase there was very little decrease in the quantity of sperm descending at the duplex level in P1 males irradiated with 130 Gy and in F 1 males that were descendants from parents irradiated with 100 and 130 Gy. In F 1 males the overall impact of irradiation on the quantity of sperm descending was higher than in the P1 generation. It also indicated that the effect of irradiation was more pronounced on eupyrene sperm descent than on apyrene sperm descent in P 1 and F 1 moths, although this was not statistically significant.
Planned activity 7. Sperm activation will be studied in irradiated male Spodoptera and their F 1 progeny and correlated with mating success and reproductive competence (Seth).
After standardization of ‘ in-vitro assay’ of sperm activation in adult Spodoptera litura moths the in-vitro sperm activation was studied in gamma irradiated male moths. The activity of sperm of irradiated male parents and their F 1 progeny was assessed in terms of (i) % sperm eliciting activity, and (ii) the degree (intensity) of activity in sperm. The impact of the irradiation treatment on percentage sperm activation in mated moths was apparent from a dose of 130 Gy and above, but the profile of sperm intensity of mated male moths treated with 100-130 Gy was not significantly affected as compared to the untreated males, except 60-90 min after incubation. The profile of the percentage of sperm showing activity after mating was
more or less similar in untreated and irradiated P1 moths, whereas there were slight differences in F1 moths as compared to P1 moths. Mated males showed an increase in percentage sperm activation and a decrease in
intensity of sperm activity in P 1 moths (0 Gy, 100 Gy, 130 Gy) as compared to virgin moths. Overall, the degree of sperm activity was not much influenced by the radiation treatment but there was slightly more impact when treated with 130 Gy in both generations of this lepidopteran moth. These findings suggest that sperm dynamics can definitely act as a one of the reliable tools to assess the viability of irradiated moths.
Planned activity 8. Sperm transfer from irradiated male Spodoptera and their F1 progeny to female up to bursa copulatrix and spermatheca will be correlated with mating success and fertility. Studies will be conducted to assess whether various characteristics of the sperm behavior (sperm activation and sperm
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transfer) can be used as a qualitative tool for released irradiated insects and their F1 progeny in the field (Seth).
Sperm transfer from irradiated male moths (P 1 and their untreated F1 descendants) was ascertained at the spermatheca level where sperm is retained after mating, and used for fertilizing the female gametes (eggs).
Untreated females mated to F 1 males stored less sperm in their spermatheca than those mated with P 1 treated moths, although this effect was not drastic, and it could be correlated with successful matings (i.e. those matings where sperm was transferred to the spermatheca) and the degree of sterility. For example, sperm
transfer in P1 and F 1 males could be correlated with sterility in F 1 (70-80%) as compared to sterility in the parental generation (50-55%).
Planned activity 9. The effect of age of female, irradiated male, and F1 male Spodoptera on their mating success and fertility will be assessed (Seth).
Male irradiation affected the mating success and remating propensity of female moths of different age, but at each particular gamma dose the age of the female had little effect of mating success. Female age has a larger impact on re-mating propensity of the females with irradiated male moths than that on its mating success. Furthermore, the fertility of females mated with F1 males was similar among crosses with 0-1 and 2-3 day old females, whereas there was a significant reduction in egg fertility of females aged 5-6 days that were mated with F1 males derived from 100 – 130 Gy treated males.
Planned activity 10. The effect of mating status of female Spodoptera on the reproductive performance of irradiated male and F1 male moth will be assessed (Seth).
The effect of mating status of female Spodoptera on the reproductive performance of irradiated male and F 1 male moth was assessed using sequential matings. This species exhibited strong second male sperm precedence.
Conopomorpha sinensis
Planned activity 11. Bioassays will be developed to compare quality of colony Conopomorpha sinensis reared on fresh fruit with insects collected from the wild in terms of flight ability, longevity, mating performance. These studies will be conducted using field cage settings (Hu).
Bioassays were carried out to compare the quality of colony Conopomorpha sinensis reared on fresh fruit collected from the wild in terms of flight ability, longevity, mating performance. No significant differences between wild moths and those reared were obtained.
Planned activity 12. A high quality artificial diet will be developed for the litchi stem-end borer Conopomorpha sinensis in terms of different protein sources, different sugars, removal of preservatives, optimize pH etc. (Hu).
Studies on developing an artificial diet for Conopomorpha sinensis have been initiated and are ongoing. Hypsipyla grandella Planned activity 13. A preliminary study will be carried out on the size of the spermatophore of Hypsipyla grandella to determine if it can be used to predict male quality. This study will test the hypothesis that larger males elaborate larger spermatophores; and that larger spermatophores reduce the likelihood that females will remate (or extend their refractory time). Data also will be collected to examine the following questions: Does the size of the male affect the number of times the male mates? Does the size of the spermatophore change as a function of mating frequency (Diaz)?
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Female Hypsipyla grandella were dissected and the spermatophore was identified. However, due to low sample sizes we were not able to statistically test differences in size of males, refractory period or mating frequency.
Planned activity 14. Determine mating propensity and mating competitiveness of Hypsipyla grandella in relation to male and female age (Diaz).
Due to small sample sizes available, these experiments were not carried out. All colony individuals were used to test artificial larval diets, rearing methods and density.
Planned activity 15. The effect of larval density of Hypsipyla grandella on growth rate, pupal and adult size will be assessed. In addition, the effect of adult size will be assessed in terms of flight ability, longevity, and mating. Adult Hypsipyla grandella flight ability will be assessed in the lab and compared with their flight ability in field cages using females as pheromone baits (Diaz).
The effect of increasing larval density was tested on growth rate, pupal size and longevity. However, due to the cannibalistic behaviour of H. grandella, unacceptable levels of mortality were observed whenever more than one individual was reared in the same medium. Adult flight ability has not been tested due to low colony numbers.
Tuta absoluta
Planned activity 16. Bioassays will be developed to compare biological quality of laboratory-reared Tuta absoluta on tomato plants with insects collected from the wild in terms of flight ability, longevity, mating performance. These tests will be carried out in field cages (Lebdi-Grissa).
This work was done in the laboratory. The longevity, mating performance, fecundity and fertility of wild adults reared on tomato plants were studied.
Planned activity 17. An artificial diet for Tuta absoluta will be developed and parameters assessed including growth rate, survival, adult longevity, pupal and adult size, fecundity and fertility (Lebdi- Grissa).
Evaluations of artificial diets have been initiated but so far rearing of Tuta absoluta has not been successful.
Planned activity 18. Laboratory and field cage release bioassays will be developed and used to evaluate the effect of moth ( Tuta absoluta ) size on flight ability, mating, and longevity; same size classes and insects from same rearing cohort will be used in bioassays; compare lab and field results (Lebdi-Grissa).
No activities to report.
Chilo sacchariphagus
Planned activity 19. Evaluations of the various diet formulations for Chilo sacchariphagus will be repeated in Mauritius (to be completed by MSc Student) (Conlong).
A visit of Dr Jim Carpenter to Mozambique in July 2011 to assess rearing attempts for Chilo sacchariphagus led to the development of a diet assessment protocol using established diets formulated for this insect from CIRAD in Reunion, MSIRI in Mauritius and a diet for C. partellus from ICIPE in Kenya. These diets were formulated and dispensed into multicell trays and sent to the rearing lab in Mozambique, and to the student at MSIRI, Mauritius. The first consignment of diet to Mozambique was lost in Mozamabican customs sheds, and the consignment to Mauritius had to be destroyed because of
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contamination with E. saccharina neonates. The second consignment of diets reached their destinations, but no pupae could be obtained because of gross contamination of diets. Moving the rearing of this insect to controlled conditions in a quarantine laboratory in Stellenbosch, South Africa is being investigated.
Eldana saccharina
Planned activity 20. The change in focus of Eldana saccharina rearing from “parasitoid host” to “sterile insect technique” has allowed the opportunity to modify the diet and to include less expensive ingredients and a more efficient production system. Therefore, new diets and protocols for Eldana rearing will be evaluated and resulting insect quality will be compared to insect quality with the current rearing system (Conlong).
A diet incorporating rice mixed with fusarium as an alternative to chickpea is being investigated and is showing promise as an alternative, more productive diet to the current one. In addition, trials are underway to mass rear Eldana in large trays used for medfly SIT rearing, going through a series of trials using trays of increasing size.
Other Progress. The effect of different doses of X-rays on emergence, morphology, fecundity and fertility of irradiated Tuta
absoluta males and females and their descendants (F 1) was studied. Irradiation had a detrimental effect on all the variables as doses increased. An X-ray dose equivalent to 200 Gy was proven to be able to sterilize females completely and males partially. These results were published in Journal of Pest Science (2012) (López).
The wild-type karyotype of T. absoluta was described. The analysis of mitotic metaphases from wing imaginal discs revealed that males and females of this species have 2n= 58 chromosomes. Most of the chromosomes are of small size, but two notoriously larger chromosomes were detected. Preparations from Malphigian tubes showed the presence of sex chromatin in females, indicating the existence of one W chromosome in the species (López).
The effect of different X-ray doses on spermatogenesis of T. absoluta was analyzed. In all the individuals a notable difference in size and morphology between eupyrene and apyrene sperm bundles was detected. Radiation significantly affected the proportion of eupyrene sperm bundles of normal morphology, being lower for the highest doses when compared to the control dose and the smaller dose. All the radiation doses generated an increase in the proportion of deformed eupyrene sperm bundles when compared to the control dose (López).
The T. absoluta morphology of the nuclei of the eupyrene spermatozoa transferred by irradiated males was described. An increase in the proportion of spermatozoa with deformed heads transferred by males irradiated with the highest doses was observed, which was correlated to a decrease in the proportion of normal spermatozoa (López).
2. Operational factors that may affect quality
Spodoptera
Planned activity 1. A collaborative relationship with governmental institutes will be developed to conduct field trapping studies. Substerilized male Spodoptera will be released and their performance measured as identified by trap captures (Seth).
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Studies have been initiated on the orientation of sub-sterilized males and F 1 males of sub-sterilized male parents towards pheromone traps (Fero-TTM procured from PCI) in field cages.
Cactoblastis cactorum
Planned activity 2. Studies to examine the influence of age, mating status, and cold storage on codling moth and cactus moth will be continued. Using the previously validated laboratory and field bioassays, additional trials to examine age, temperature ranges and handling procedures will be conducted (Carpenter and Hight).
Studies were initiated on codling moth to examine factors related to insectary rearing and handling protocols by conducting laboratory bioassays of moth quality at several points along the production-release chain. These studies were done in collaboration with Tom Blomefield and Martin Wohlfarter.
Planned activity 3. Flight performance of irradiated Cactoblastis cactorum from the two insectaries comparing flight cylinders and open field release/recapture will be continued (Carpenter and Hight).
Flight performance of irradiated Cactoblastis cactorum from a filter colony insectary and a production colony insectary was compared in flight cylinders and open field release/recapture trials. Results suggest that moth quality is reduced in the production rearing facility and indicate that additional studies are needed.
Hypsipyla grandella
Planned activity 4. Efforts will be made to develop better adult mating/oviposition cages for Hypsipyla grandella and a substrate that will facilitate egg collection (Diaz).
Three different oviposition media were tested. Promising results were obtained using frass smeared on paper towels. Fertile eggs were deposited on this material.
Planned activity 5. Egg production and egg fertility of adult Hypsipyla grandella will be assessed using various stocking rates and sex ratios (Diaz).
Experiments are still in progress.
Planned activity 6. Determine sterilizing irradiation dose for Hypsipyla grandella and its effect on adult flight ability in lab and field cage trials. (Diaz).
This activity has not been possible due to small sample sizes. Colony rearing first needs to be optimized to obtain adequate number of adults for irradiation tests.
Lobesia botrana
Planned activity 7. A full replicated study will be conducted with Lobesia botrana to confirm diet improvements (larval development time, pupal weight, fecundity, longevity, host suitability index (HSI)) (Saour).
Modified diets for Lobesia botrana were evaluated by examining larval development time, pupal weight, fecundity, longevity, and host suitability indices. Results from this study indicated that replacing maize oil with ginger root oils significantly improved the quality of reared moths (reduced developmental time, and increased moth weight, adult longevity and female fecundity)
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Planned activity 8. Evaluate flight ability of males irradiated at 150 and 400 Gy in laboratory and field cage studies, and open field releases (pistachio field if needed). Traps and pheromones are available (Saour).
Laboratory and field bioassays revealed that there were no significant differences in the flight performance of 150 Gy or unirradiated males.
Planned activity 9. A cage is being developed in which to conduct laboratory studies of Lobesia botrana . The cage would allow evaluations with irradiated and non-irradiated moths for flight ability, male response to calling females, male response to calling females with host plant, etc. (Saour).
A laboratory cage was developed to combine flight ability and male response to calling females. Bioassays using this laboratory cage revealed that there were no significant differences in the flight performance of 150 Gy or unirradiated males.
Planned activity 10. Study behaviours (flight, mating) of F1 Lobesia botrana sterile individuals in laboratory and field cages (using 1x1x1 m cage) (Saour).
No activities to report as yet.
Eldana - Chilo
Planned activity 11. A method will be developed from a multi cell tray rearing system to a larger open medfly system rearing tray for Eldana (Conlong).
A diet incorporating rice mixed with Fusarium as an alternative to chickpea is being investigated and is showing promise as an alternative, more productive diet to the current one. In addition, trials are underway to mass rear Eldana in large trays used for medfly SIT rearing, going through a series of trials using trays of increasing size.
Planned activity 12. Efforts will be made to further improve the rearing system for Chilo (Conlong).
The first consignment of diet to Mozambique was lost in Mozamabican customs sheds, and the consignment to Mauritius had to be destroyed because of contamination with E. saccharina neonates. The second consignment of diets reached their destinations, but no pupae could be obtained because of cross contamination of diets. Moving the rearing of this insect to controlled conditions in a quarantine laboratory in Stellenbosch, South Africa is being investigated.
Planned activity 13. Initiate mating competitiveness studies on the appropriate radiation doses in the lab and field ( Chilo ) (Conlong).
These studies are pending on the further development of an appropriate rearing system.
Conopomorpha sinensis
Planned activity 14. Optimal oviposition stocking ratios and optimal adult densities for maximum egg production of Conopomorpha sinensis will be investigated (Hu).
Optimal oviposition stocking ratios and optimal adult densities for maximum egg production of Conopomorpha sinensis were investigated. The stocking ratios is 1(male):2(female) and optimal adult densities is 30 female adults.
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Planned activity 15. Male Conopomorpha sinensis from irradiated pupae will be compared with normal males for their longevity, flight ability, and mating competiveness. Studies will be conducted in laboratory cages and studies will be initiated to develop field cage protocols (Hu).
The adults emerging from the pupae irradiated at 50, 100, 150, 200 and 250 Gy were crossed with untreated adults at the following combinations: ①-♂X♀; ②♂X-♀; ③-♂X-♀; ④♂X♀ （CK ）were carried out. Four combinations could be sterilized when parental pupae irradiated at 250 Gy. The combination of - ♂(P) by
♀(P) in F 1 could be sterilized when parental pupae were irradiated at 200 Gy.
Conopomorpha sinensis
Planned activity 16. Conduct study to determine the fate of 9% eggs that hatch from male Conopomorpha sinensis irradiated with 200 Gy (Hu).
Hatching rates of F 1 generation in male adults from pupae irradiated at 200 Gy was 12.97%. No adults emerged in this treatment.
Planned activity 17. Conduct studies to identify the formation of F1 sterile individuals (Hu).
The highest effects of irradiation were observed on F 1 generation in all treated combinations in 3 generations. When the parental pupae were irradiated at 150 Gy, the combination -♂ (P) x ♀ (P) could last three generations, and all were effective.
Tuta absoluta
Planned activity 18. Determine the optimum radiation dose for Tuta absoluta (Lebdi-Grissa).
No activities to report.
Planned activity 19. Longevity, mating competitiveness, and flight ability of wild and irradiated Tuta absoluta will be evaluated in laboratory and field bioassays (Lebdi-Grissa)
Laboratory trials have been initiated and results are pending.
Lobesia botrana
Planned activity 20. Studies that were initiated to examine sperm in spermatophores in the females’ bursa copulatrix immediately after mating with normal males and with males irradiated as pupae with 100 Gy 1-4 days after pupation will be completed, and expanded to include irradiation of males 5-8 days in pupation and adults. Studies on irradiation effects of Lobesia botrana will be completed using a dose of 100 Gy and the effects evaluated of treating pupae vs adults. Field studies will be conducted to evaluate fitness measures of irradiated Lobesia botrana pupae to compare with laboratory experiments, orienting to pheromones (synthetic and live calling females) performance will be tested in wind-tunnel and semi- field conditions. (Harari).
Studies on irradiation (100 Gy) effects of Lobesia botrana have been completed and field studies evaluated male flight ability (see above). Male orienting to pheromone source in a wind-tunnel was not performed due to problems in the newly built wind tunnel.
Published papers
Marti, O. G., Jr., Carpenter, J. E. 2009. Effect of irradiation on the incidence of mating in Cactoblastis cactorum . Florida Entomologist. 92: 159-160. 2009. 21
Blomefield, T., Carpenter, J. E., and Vreysen, M. J. B. 2011. Quality of Mass-Reared Codling Moth (Lepidoptera: Tortricidae) After Long-Distance Transportation: 1. Logistics of Shipping Procedures and Quality Parameters as Measured in the Laboratory. J. Econ. Entomol. 104(3): 814-822.
Carpenter, J. E., Blomefield, T., and Vreysen, M. J. B. 2012. A flight cylinder bioassay as a simple, effective quality control test for Cydia pomonella . Journal of Applied Entomology: doi: 10.1111/j.1439- 0418.2012.01711.x
Grasela, J. J., McIntosh, A. H., Ringbauer, Jr., J., Goodman, C. L. Carpenter, J. E., and Popham, H. J. R. 2012. Development of cell lines from the cactophagous insect: Cactoblastis cactorum (Lepidoptera: Pyralidae) and their susceptibility to three baculoviruses. In Vitro Cellular and Developmental Biology – Animals. 48: 293-300.
Carpenter, J. E. and S.D. Hight. Rearing the oligophagous Cactoblastis cactorum (Lepidoptera: Pyralidae) on meridic diets without host plant materials. Florida Entomologist. (in press)
Carpenter, J. E., Blomefield, T., and Hight, S. D. Comparison of laboratory and field bioassays to examine lab-reared Cydia pomonella (Lepidoptera: Tortricidae) quality and field performance. Journal of Applied Entomology: (submitted)
Conference presentations
Barradas-Juanz N., Diaz-Fleischer F., Pérez-Staples D. (2009). XV Simposio de Parasitología Forestal. Oaxaca, Mexico. "Variación temporal y espacial en la infestación de Hypsipylla grandella en el Vigía, Veracruz".
Pérez-Staples D. Barradas-Juanz N., Diaz-Fleischer F. (2009). XV Simposio de Parasitología Forestal. Oaxaca, Mexico. "Opitmización de la dieta larval de Hypsipylla grandella".
Vázquez Domínguez G., Pérez Staples D., Perroni-Ventura Y., Díaz-Fleischer F., Macías-Ordóñez R. (2010). III Congreso Mexicano de Ecología. Veracruz, Mexico. “Estequiometría de nitrógeno y fósforo en Hypsipyla grandella”.
Cagnotti, C. L., M. M. Viscarret, M. B. Riquelme, E. N. Botto, L. Z. Carabajal, D. F. Segura and S. N. López. 2012. Effects of X-rays on Tuta absoluta for use in inherited sterility programmes. J. Pest. Sci. Published on line DOI 10.1007/s10340-012-0455-9.
Lauría J. P., L. Z. Carabajal Paladino, C. Cagnotti, I. Muntaabski, J. L. Cladera and S. N. López. 2012. Caracterización de los euespermatozoides transferidos por machos irradiados de Tuta absoluta (Lepidoptera). XV Congreso Latinoamericano de Genética, XLI Congreso Argentino de Genética, XLV Congreso de la Sociedad de Genética de Chile y II Reunión Regional Sag-Litoral (abstract accepted).
Cagnotti C., M. Viscarret, M. Riquelme, E. Botto, L. Z. Carabajal Paladino and S. López. 2011. Estudios de radiobiología con rayos X en Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae): potencial para la aplicación de la Técnica del Insecto Estéril. Workshop: La polilla del tomate en la Argentina: estado actual del conocimiento y prospectiva para un manejo integrado de plagas. La Plata, Argentina.
Carabajal Paladino, L, E. Ferrari, J. P. Lauría, C. Cagnotti, M. Viscarret, D. Segura, J. Cladera and S. N. López. 2011. Genética y citogenética de Tuta absoluta. Workshop: La polilla del tomate en la Argentina: estado actual del conocimiento y prospectiva para un manejo integrado de plagas. La Plata, Argentina.
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Carabajal Paladino L. Z., C. Cagnotti, S. N. López, M. M. Viscarret, F. Marec and J. L. Cladera. 2011. Análisis citogenético clásico de Tuta absoluta (Lepidoptera, Gelechiidae). XL Congreso Argentino de Genética, III Simposio Latinoamericano de Citogenética y Evolución, I Jornadas Regionales SAG-NEA.
Ferrari M. E., C. Cagnotti, L. Z. Carabajal Paladino, J. L. Cladera, D. F. Segura, M. M. Viscarret and S. N. López. 2011. Impacto de la radiación en la espermatogénesis de Tuta absoluta (Lepidoptera: Gelechiidae). XL Congreso Argentino de Genética, III Simposio Latinoamericano de Citogenética y Evolución, I Jornadas Regionales SAG-NEA.
Cagnotti C. L., M. M. Viscarret, E. Botto and S. N. López. 2011. Estudios de radiobiología en Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae): Potencial para la integración de la Técnica del Insecto Estéril (TIE) y el control biológico. Seminario Internacional de Sanidad Agropecuaria (SISA).
Ferrari M. E. 2011. “Análisis del impacto de la radiación X sobre la espermatogénesis de la polilla del tomate Tuta absoluta”. Bachelor degree Thesis. Facultad de Ciencias Exactas, Químicas y Naturales, Universidad de Morón.
Cagnotti C. L., M. M. Viscarret, E. Botto and S. N. López. 2010. Estudios exploratorios de radiobiología con rayos X en Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) con miras a su aplicación en la Técnica del Insecto Estéril. XXXIII Congreso Argentino de Horticultura.
Thesis
Barradas-Juanz N. (2010). “Variación temporal y espacial en la infestación de Hypsipylla grandella en el Rancho “El Vigía”, Municipio de Actopan en Veracruz, México. BSc. Thesis. Facultad de Biología. Universidad Veracruzana. Xalapa, Veracruz, Mexico.
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Working Group 1. Activities planned for 2013-2014
2. Biological attributes that may affect quality
• Laboratory and field bioassays used to evaluate the effect of pupal weight and moth size on quality of the cactus moth will be continued (Carpenter and Hight).
• Studies to examine the ability of laboratory and field bioassays to predict the field performance of cactus moths will be continued (Carpenter and Hight).
• Conduct field release-recapture bioassays to examine quality differences of L. botrana males that have been reared on improved artificial diets (Saour).
• Laboratory flight ability bioassays will be conducted to examine the effects of age, mating status, and weight of L. botrana reared from the same diet (Saour).
• Conduct laboratory study to examine the effect of 150 Gy on female calling behaviour of L. botrana reared from the same diet (Saour).
• Elucidate the role of the apyrene sperm cells in female mating and remating behavior and of sperm competition between ejaculates of two males (irradiated and non-irradiated), using Lobesia botrana as a model (Harari) .
• Evaluate qualitative sperm transfer into spermatheca of irradiated male (P 1) and their F 1 progeny and correlate with mating competitiveness & degree of sterility induced. (Seth )
• Examine the orientation /attractiveness of irradiated male (P 1) & F 1 moths towards pheromone traps (Seth ).
• Biochemical investigations will be conducted to examine the radiation-induced alterations in
antioxidant status of P 1 and F 1 insects with respect to the glutathione system (GSH/GSSG), antioxidant enzyme SOD, glutathione reductase, catalase, and ascorbate peroxidase (Seth ).
• A molecular biology approach will be used to assess the effect of irradiation on the phenoloxidase cascade enzymes (pro-phenoloxidase-PPO & pro-phenoloxidase activating enzyme-PPAE) and
scavenging enzymes (SOD & CAT) in P 1 and F1 insects, by relative transcript levels of immune cascade genes ( ppo & ppae ) and oxidative stress genes ( sod & cat ) by Real-Time qRT-PCR] (Seth ).
• Laboratory trials will be carried out to investigate the efficacy of the sterile insect technology (SIT) for controlling litchi stem-end borer Conopomorpha sinensis Bradley (Hu ).
• The sexual competitiveness of sub-sterile male Conopomorpha sinensis adults will be evaluated using different mating ratios in laboratory and field cage trial (Hu ). • The efficacy of the sterile insect technology (SIT) for controlling litchi stem-end borer Conopomorpha sinensis Bradley will be investigated in the field (Hu ). • Conduct studies to determine the optimum radiation dose for Tuta absoluta SIT, and examine performance of irradiated moths in laboratory and field cage bioassays ( Lebdi-Grissa ).
• Evaluations of the various diet formulations for Chilo sacchariphagus will be repeated under quarantine in Stellenbosch (to be completed by post doc/contract) ( Conlong ).
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• The change in focus of Eldana saccharina rearing from “parasitoid host” to “sterile insect technique” has allowed the opportunity to modify the diet and to include less expensive ingredients and a more efficient production system. Therefore, new diets and protocols for Eldana rearing will be evaluated and resulting insect quality will be compared to insect quality with the current rearing system ( Conlong ). Continuing for 2012-2014.
• A method will be developed from a multi cell tray rearing system to a larger open medfly system rearing tray for Eldana (Conlong). Ongoing for 2012-2014.
• Efforts will be made to further improve the rearing system for Chilo (Conlong). Ongoing for 2012- 2014.
• Initiate mating competitiveness studies on the appropriate radiation doses in the lab and field ( Chilo ) (Conlong) . (Depends on successful initiation of a lab colony).
2. Operational factors that may affect quality
• Studies to examine the influence of age, mating status, and cold storage on codling moth and cactus moth will be continued. Using the previously validated laboratory and field bioassays, additional trials to examine age, temperature ranges and handling procedures will be conducted ( Carpenter and Hight ).
• Flight performance of irradiated Cactoblastis cactorum from the two insectaries comparing flight cylinders and open field release/recapture will be completed (Carpenter and Hight) .
• Quality control parameters will be obtained from Hypsipyla grandella reared on the new diet (egg hatch, larvae survival, pupal weight, pupal survival, adult size, fecundity). Flight ability of adults of this new diet will be tested under laboratory conditions ( Pérez-Staples ).
• A new diet that will be suitable for the first three instars of Hypsipyla grandella will be tested and compared to the use of fresh cedar leaves. Biological attributes of individuals reared on both methods will be quantified ( Pérez-Staples ).
• Experiments will be conducted to increase the rearing density of Hypsipyla grandella (Pérez- Staples ).
• Different sex ratios will be tested to determine which ratio has the highest probability of mating and oviposition ( Pérez-Staples ).
• A new oviposition medium will be tested using paper towels smeared with Hypsipyla grandella larval frass, and experiments will continue modifying the rearing cage so that only one side has mesh and is easily removable from the rest of the cage ( Pérez-Staples ).
• Determine the optimum radiation dose for Tuta absoluta (Lebdi-Grissa) .
• Longevity, mating competitiveness, and flight ability of wild and irradiated Tuta absoluta will be evaluated in laboratory and field bioassays (Lebdi-Grissa)
• Evaluate the effect of an X-ray dose equivalent to 200 Gy on the dispersion capability and mating competitiveness of male Tuta absoluta (López).
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• Evaluate the capability of irradiated individuals and their progeny to suppress a Tuta absoluta population under semi-controlled conditions. (López).
• Evaluate the acceptability and suitability for parasitoids and predators of the infertile eggs obtained as a result of irradiated individuals and inherited sterility. (López).
• Analyze the proportion of apyrene and apyrene sperm transferred by males irradiated at an X-ray dose equivalent to 200 Gy (López).
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Working Group 2: Tools and methods to assess field performance
The objective of this working group is to develop and improve tools and methods to assess, predict and enhance the field performance of released moths based on insect quality.
The following was achieved in 2010-2012 for each of the activities planned in the categories:
1. Theoretical Framework
Planned activity 1. Initiate an integrated framework for assessing population suppression using SIT, including modelling of population fitness parameters to estimate overflooding ratio, radiation dose, and related parameters (Kean, Suckling, Simmons, Carpenter, Woods).
Epiphyas postvittana (LBAM)
A model for determining the overflooding ratio was developed linked with other work. Main findings of general interest: the minimum overflooding ratio for light brown apple moth (LBAM) was estimated as 12:1; a limited effect from females in SIT was predicted. Important parameters that required estimation included:
• daily survival (or mean longevity) of adult males and females, wild and sterile
• relative performance of sterile males, in terms of finding mates
• relative performance of sterile females in terms of attracting males – are they any less attractive than wild ones?
• are females really sterile at a given dose, or do a few still produce viable eggs? If so, how fertile are they compared to wild females?
• mating competitiveness was assessed in field cages in Perth (Woods, McInnis) .
Kean, J. M., D. M. Suckling, L. D. Stringer, and B. Woods. 2011. Modeling the sterile insect technique for suppression of light brown apple moth (Lepidoptera: Tortricidae). Journal of Economic Entomology 104: 1462-1475. Eldana saccharina
An MSc (Operations Research) modelling SIT in this species was submitted for examination (Potgieter L. 2012. A mathematical model for the control of Eldana saccharina Walker via the sterile insect technique. Department of Logistics, Stellenbosch University). Very high marks were given by both examiners, and the work was upgraded to a PhD, due to be completed in 2014.
It describes two models formulated for the population growth of Eldana saccharina under the influence of partially sterile released insects. The first describes the population growth of and interaction between normal and sterile Eldana moths in a temporally variable, but spatially homeogenous environment. The model consists of a deterministic system of difference equations subject to strictly positive initial data. The second model decribes this growth and interaction under the influence of partially sterile insects which are released in a temporally variable and spatially heterogenous environment. This model consists of a discretized reaction-diffusion system with variable diffusion coefficients, subject to strictly positive initial data and zero-flux Neumann boundary conditions on a bonded spatial domain .
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Planned activity 2. Determine appropriate theoretical spatial scale(s) for using semi-field experiments to test field performance behaviours (such as dispersal and mate finding) using field cages and simplified field environments for small scale release experiments (Simmons, Suckling, Carpenter).
Epiphyas postvittana (LBAM)
A large scale operational field trial was conducted in California, and the data will enable estimates of the dispersal kernel (diffusion coefficient) for estimating release rate and frequency, retention (daily survivorship - emigration) (Simmons).
2. Trapping tools and studies
Planned activity 1. Develop and/or improve attractants and trapping systems for male and female moths (LBAM, cactus moth and DBM) that will enable assessment of field performance of released insects (Suckling, Wee, El-Sayed, Park, Woods, McInnis, Carpenter).
Epiphyas postvittana (LBAM)
Work continued on a female attractant but results have proved unreliable in this species so far (Suckling El- Sayed et al.).
Plutella xylostella (DBM)
There were a total of 12 bioactive peaks recorded by GC-EAD on the cabbage headspace against the diamondback moth’s (DBM) antennae. GC-MS analysis was conducted on cabbage headspace to determine the identity of these compounds and a tentative list was generated. Preliminary field trials using some of these tentatively identified compounds, either singly or in combinations caught some DBM, mostly males, but not in significant numbers (Wee, Park) .
Cactoblastis cactorum
New pheromone blends with additional compounds were identified and evaluated in field tests (Carpenter, Hight).
Chilo sacchariphagus
A commercial lure (used in mating disruption) was assessed for monitoring SIT. A mating disruption trial was commenced, and trap shutdown looked possible, but the collaborators did not change traps at the required dates. Lures are being used to determine moth cycles through the year. Will be completed in 2012 (Conlong).
Planned activity 2. Develop smart traps for monitoring moth behaviour in the field for CM, FCM and LBAM (Addison, Suckling, Simmons).
Cydia pomonella
Work is continuing on procuring a low cost GPS-enabled camera card for use in a delta traps (Addison ).
Epiphyas postvittana (LBAM)
A workshop on smart traps was organized in Phoenix and a discussion covered various aspects of the practical use of such traps, which varies between applications for surveillance, eradication and IPM. A 28 prototype web-enabled camera trap was developed with a New Zealand security company for fruit flies and moths (Suckling). A prototype iPhone trap streaming to the web was also developed (El-Sayed) .
3. Semi-field methods which appears to have potential as a research tool
Planned activity 1. Develop semi-field testing methods for LBAM, PBW, cactus moth and CM using simplified field environment systems (including field cages) to conduct small scale release experiments (Simmons, Tate, Carpenter, Hight, Suckling, Addison, Woods, McInnis).
Epiphyas postvittana (LBAM)
Field cages, transects and vineyard and urban grids were compared for key performance indicators such as longevity, recapture rates, flight distance and mating rate. A ventilated outdoor horticultural tunnel was built and preliminary testing was undertaken in New Zealand. (Simmons, Suckling, Woods).
Suckling, D. M., L. D. Stringer, V. J. Mitchell, T. E. Sullivan, G. S. Simmons, A. M. Barrington, and A. M. El-Sayed. 2011. Comparative fitness of irradiated light brown apple moths ( Lepidoptera : Tortricidae ) in a wind tunnel, hedgerow and vineyard. Journal of Economic Entomology 104: 1301-1308.
Cactoblastis cactorum
An array of 64 traps was tested for recapture of moths irradiated with different doses and released from a central release point in the field. Data were collected on recapture rate and distance from the release site (Carpenter, Hight) .
Cydia pomonella
An array of traps was tested in a ventilated outdoor horticultural tunnel for assessing thermal tolerance (Addison) .
Chidawanyika, F. and J. S. Terblanche. 2011. Rapid thermal responses and thermal tolerance in adult codling moth Cydia pomonella (Lepidoptera: Tortricidae). Journal of Insect Physiology 57: 108- 117.
Chidawanyika, F. and J. S. Terblanche. 2011. Costs and benefits of thermal acclimation for codling moth, Cydia pomonella (Lepidoptera: Tortricidae): Implications for pest control and the sterile insect release programme. Evolutionary Applications 4: 534-544.
Sørensen, J. G., M. F. Addison, and J. S. Terblanche. 2012. Mass-rearing of insects for pest management: Challenges, synergies and advances from evolutionary physiology. Crop Protection 38: 87-94.
Stotter, R. L., and J. S. Terblanche. 2009. Low-temperature tolerance of false codling moth Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) in South Africa. Journal of Thermal Biology 34: 320-325.
Planned activity 2. Carry out field cage tests on CM, LBAM, FCM, Chilo and Eldana to measure mating competitiveness and mating compatibility of insects under different a) irradiation doses and atmospheres and b) adult sex ratios c) flight or locomotion performance, d) ability of males to locate females and e) ability of laboratory-reared males to successfully mate with wild females, and f) different geographic origin. (Addison, Taret, Woods, Conlong and McInnis).
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Epiphyas postvittana (LBAM)
Cage testing on overflooding ratios was undertaken and data were collected to determine the effect of different overflooding ratios on population suppression (Woods) .
Eldana saccharina
Two chapters in a PhD thesis were sent in for examination in August 2012. (Mudavanhu, P. 2012, Performance of sterilized Eldana saccharina Walker (Lepidoptera: Pyralidae) adults in mating and cage trials: Further steps towards its control using the Sterile Insect Technique. Department of Conservation Ecology and Entomology, Stellenbosch).
Main findings of first chapter:
• Males exposed to 150 and 200 Gy called within an hour after darkness, wilds took 8 hrs
• Males exposed to 150 and 200 Gy mated with wild females 2 hrs before wild males did
• 150 Gy males remained in copula with wild for 2 hours, wild and 200 Gy for only 1.5 hrs
• On first night after emergence 150 and 200 Gy males mated with 3-6 wild females, while wild males mated with a max of 2; on second night, wild males mated with a max of 3 females, and the irradiated males a max of 2
• Under laboratory conditions, and without competition, males irradiated with 200 Gy (and 150 Gy) performed as well as, if not better than wild males at attracting and mating with wild females.
The second chapter showed that in general, despite differences in peak times of mating between the respective treatments, the data from both lab and field cages indicate that there is no evidence of any incompatibility between mass-reared, irradiated and wild E. saccharina strains. The results of the study thus provide the necessary evidence and confidence that the mass-reared E. saccharina strain currently produced at the SASRI insect rearing unit is suitable for use in SIT-based projects.
Mudavanhu. P., Conlong, D.E. and Addison P. 2011. Performance of sterilized Eldana saccharina Walker (Lepidoptera Pyralidae) in mating trials. Short Communication. Proceedings of the South African Sugar Technologists Association 84: 287 – 291.
Chilo sacchariphagus
A rearing protocol was developed together with Dr Jim Carpenter and sent to labs in Mozambique and Mauritius to provide insects for further research. A field visit was undertaken in Mozambique.
Cydia pomonella
There is no progress to report due to logistical difficulties and lack of insect supply (Addison).
4. Marking tools and studies
Planned activity 1. Develop reliable and cost-effective marking and monitoring systems (e.g. stable isotopes, staining systems, sperm morphology, genetic markers) for assessing mating status, fertility and F1 frequency (Hood-Nowotny, Woods, McInnis, Simmons, Suckling, Tate, Conlong)
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Epiphyas postvittana (LBAM), Thaumatotibia leucotrata, Eldana saccharina
Preliminary studies showed that natural abundance levels of isotopes can be used for marking insects and spermatophores of Epiphyas postvittana (New Zealand, Australia, USA), Eldana saccharina (South Africa) and Cactoblastis cactorum (USA), and diamondback moth (Malaysia) to determine mating success of sterile insects in the field, using tethered females (Hood-Nowotny).
Benedict, M. Q., R. C. Hood-Nowotny, P. I. Howell, and E. E. Wilkins. 2009. Methylparaben in Anopheles gambiae s.l. sugar meals increases longevity and malaria oocyst abundance but is not a preferred diet. Journal of insect physiology 55: 197-204.
Hood-Nowotny, R., L. Mayr, A. Islam, A. Robinson, and C. Caceres. 2009. Routine Isotope Marking for the Mediterranean Fruit Fly (Diptera: Tephritidae). Journal of Economic Entomology 102: 941-947.
Hood-Nowotny, R., M. Watzka, L. Mayr, S. Mekonnen, B. Kapitano, and A. Parker. 2011. Intrinsic and synthetic stable isotope marking of tsetse flies. Journal of Insect Science (Online) 11: 79-79.
Hood-Nowotny, R., R. Schwarzinger, C. Schwarzinger, S. Soliban, O. Madakacherry, M. Aigner, M. Watzka, and J. Gilles. 2012. An analysis of diet quality, how it controls fatty acid profiles, isotope signatures and stoichiometry in the malaria mosquito Anopheles arabiensis . PloS One In press .
ul Haq, I., L. Mayr, P. E. a. Teal, J. Hendrichs, A. S. Robinson, C. Stauffer, and R. Hood-Nowotny. 2010. Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon fly Bactrocera cucurbitae : effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast. Journal of Insect Physiology 56: 1807-15.
Cactoblastis cactorum
The histological method for identifying F 1 offspring of sterile males of Cactoblastis has been tested with insects mated in the field as part of an operational program (Carpenter, Hight).
Teia anartoides
A paper testing the histological method for identifying F1 males of painted apple moth was published (Wee/Suckling).
Wee, S.-L., D. M. Suckling, and A. M. Barrington. 2011. Feasibility study on cytological sperm bundle assessment of F1 progeny of irradiated male painted apple moth ( Teia anartoides Walker; Lepidoptera: Lymantriidae) for the sterile insect technique. Australian Journal of Entomology 50: 269-275.
Planned activity 2. Collect and analyse mass-reared and wild moths from the CRP participants to establish whether it is possible to distinguish populations using natural isotope markers (C3/C4). (Hood- Nowotny, CRP participants).
See above
Planned activity 3. Baseline data on isotope labelling of moths for sperm transfer studies will be collected to enable method development (Hood-Nowotny).
See above
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5. Laboratory tools to assess fitness or movement
Planned activity 1. Carry out tests to determine flight tube dimensions to effectively determine flightability of irradiated vs untreated LBAM and Spodoptera litura males and females (Woods, McInnis, Seth, Carpenter).
Epiphyas postvittana (LBAM)
Nothing further to report (Woods).
Planned activity 2. Develop wind tunnel, observational arenas and machine vision tools for assessment of key moth behavioural parameters that can reflect adult moth quality under controlled conditions (Suckling, El-Sayed, Conlong).
Epiphyas postvittana (LBAM)
Laboratory and field tests with LBAM were completed and a paper published (cited above).
Planned activity 3. Investigate thermal biology and locomotion in CM, Chilo and Eldana (e.g. using machine vision) to determine its significance in field performance (Addison, Terblanche, Conlong).
Cydia pomonella
A paper reporting results from arenas with controlled temperature was published (Addison, Chidawanyika) (cited above).
Eldana saccharina
Photo resolution on the thermal arena was not good enough to follow first instar Eldana larvae, and adults just did not move on the arena. Instead CT min and CT max comparisons were made between wild and lab reared adults, as well as adults exposed to 150, 200 and 250 Gy radiation doses. The wild moths had lower CT min and higher CT max values compared to the lab reared moths. As radiation dose increased the exposed moths became linearly less tolerant of colder and higher temperatures compared to the lab reared moths not exposed to radiation. (Mudavanhu, Conlong).
Planned activity 4. Develop methods to affect and measure quality in a uniformly quantifiable manner (radiation doses, moth age, and size) (Simmons, Carpenter, Tate).
Epiphyas postvittana (LBAM)
A system for monitoring adult output from cyclone collection of LBAM was tested to improve the quality and production of insects (Simmons) .
Cactoblastis cactorum
Recaptures of Cactoblastis were assessed in a release arena, see above (Carpenter, Hight) .
Planned activity 5. Marking tools and studies (Chen)
The geographical distribution of codling moth in China has been updated. Twelve C. pomonella populations were collected throughout the invested regions of China. Eight microsatellites were used to investigate the gene flow pattern and population genetic of these sampled populations. There was significant genetic 32
differentiation among populations. The Ili River valley populations have higher genetic diversity among northwestern populations, suggesting that the Ili region is most likely the origin of codling moth introduction into northwestern China. Populations were isolated from each other in the distribution region, suggesting SIT can be applied for control this invasive species in China.
Qiu, M., Chen, M., Zhang, Y. and Feng, J. 2012. Genetic structure and diversity of a newly invasive species, the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae) in China, Biological Invasions, DOI 10.1007/s10530-012-0299-5.
Qiu, M., Chen, M., Zhang, Y. and Feng, J. 2012. Amplifying stability and genetic diversity of microsatellite loci in codling moth Cydia pomonella (Lepidoperta: Tortricidae) populations in China, Acta Phytophylacica Sinica 39, 341-346. ( in Chinese )
Qiu, M. 2012. Genetic diversity and genetic strcture of codling moth populations in China revealed by micrasatellite makers. Ph.D. thesis of Northwest A&F University, Yangling, Shaanxi Provicne, China. (in Chinese )
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Working Group 2. Activities planned for 2013-14
1. Theoretical Framework
• Compare actual values and theoretical estimates of parameters required for SIT. This will involve testing the robustness of models with parameters from field and laboratory analysis for LBAM (Simmons) The Eldana model has been upgraded to a PhD project, and will incorporate spatial distribution. Due for completion 2014 (Conlong) .
• Determine appropriate theoretical spatial scale(s) for use in semi-field experiments to test field performance behaviours (such as dispersal and mate finding) using field cages and simplified field environments for small scale release experiments ( Simmons, Carpenter, Addison ).
2. Trapping tools and studies
• Attractants and trapping systems will be developed and/or improved for male and female moths (LBAM, cactus moth, Eldana , Chilo sacchariphagus, Lobesia botrana and Plutella xylostella ) that will enable assessment of field performance of released insects ( Suckling, El-Sayed, Park, Simmons, Wee, Carpenter, Conlong ).
• DBM behavioural bioassays to infested/non-infested plants; headspace and GCEAD; compounds with putative activity (RT) need to be identified; lab tests for single compounds/blends and field tests for single compounds/blends (Wee and Park ). • Continue chemical identification of antennal-active compounds in cabbage headspace. • Conduct choice-test with Y-olfactometer assays: (a) to confirm the attractiveness of DBM to odour of infested cabbage over healthy cabbage. (b) to evaluate the activity of the identified compounds against the DBM, either singly or in combinations. • Conduct field trial using several selected candidates of cabbage chemicals. • Develop non-pheromone-based monitoring tools in order to monitor efficacy of mating disruption and SIT (could include some of the following: Malaise, sticky pane traps, light traps, female traps, ultraviolet LEDs) ( Simmons, Addison, Suckling ).
• Egg sampling in the field will be developed as an independent indicator of SIT success, by isotopically determining the proportion of eggs laid by wild and sterile females ( Hood-Nowotny, Carpenter) .
• Develop and/or test smart traps for monitoring moth behaviour in the field for research or management of CM, FCM and LBAM ( Addison, Suckling, Simmons ). This could include IR LED traps, and image analysis of camera traps, with or without a telecommunications linkage.
3. Semi-field methods
• Develop semi-field testing methods for LBAM, FCM, cactus moth and CM using simplified field environment systems (including field cages) to conduct small scale release experiments ( Simmons, Carpenter, Hight, Addison ).
• Carry out field cage and pilot field tests on CM, LBAM, FCM, Chilo and Eldana to measure mating competitiveness or mating compatibility of insects under different parameters such as: a) irradiation 34
doses, b) adult sex ratios, c) flight or locomotion performance, d) ability of males to locate females, e) ability of laboratory-reared males to successfully mate with wild females, or f) different geographic origin ( Addison, Conlong, Hood-Nowotny ).
4. Marking tools and studies
• Reliable and cost-effective systems for assessing mating status, fertility or F 1 frequency (introgression of inherited sterility) will be developed. This may include stable isotopes, fluorescent powder marking systems, staining systems, sperm morphology and genetic markers (Hood-Nowotny, Woods, Simmons, Suckling, Chen, Walters).
• A protocol will be developed and tested for collecting samples from sticky traps, for use with the isotope identification method ( Hood-Nowotny, CRP participants).
• Mass-reared and wild moths will be collected from the CRP participants and analysed to establish whether it is possible to distinguish populations using natural isotope markers (C3/C4) ( Hood-Nowotny, CRP participants ).
• Baseline data on isotope labelling of moths for sperm transfer studies (including adult labelling) will be collected to enable method development ( Hood-Nowotny, Conlong ).
• At least one paper will be prepared ( Hood-Nowotny et al.).
5. Laboratory tools to assess fitness or movement
• Tests will be carried out to determine flight tube dimensions to effectively determine flight ability of irradiated vs untreated CM males and females ( Addison ).
• Molecular tools will be used to reveal gene flow patterns, genetic structures and dispersal distances of CM populations in China ( Chen ). This could include testing of private alleles/haplotypes between factory SIT and wild populations in order to determine population introgression at the F1 generation (Chen, Addison ). An attempt will be made to use colonies from several countries.
• Three mitochondrial genes will be used for further investigation of gene flow pattern among populations from the isolated oases of Hexi Corridor, the Ili Reiver valley and other northwestern regions.
• Phylogenic analysis for samples from northeastern and northwestern China, and from other nine countries will be done using the mitochondrial gene data.
• Preliminary research of lab-rearing for further irradiation treatment of northwestern and northeastern populations in China.
• Investigate the potential for a wing fanning assay for moths and characterise the relationship between wing fanning and arrival behaviour at a pheromone source (or female moths), in order to develop a generalised tool for quality assessment which can be compared with other quality measures, such as flight tubes ( Suckling, Simmons, El-Sayed, Conlong, Carpenter, Addison).
• Thermal biology and locomotion will be investigated in CM, Chilo and Eldana to determine its significance in field performance ( Addison, Terblanche, Conlong ).
• Investigate and assess the quality of sterile insect ( Cydia pomonella ) production and shipping methods for support of eradication programs against new outbreaks in remote locations or in other countries 35
without existing SIT infrastructure: for shipping eggs and pupae for post shipping rearing and irradiation at destination and compare to quality of irradiated adults for immediate release. ( Wohlfarter, Woods, Addison, Simmons)
• Develop and test protocols using stable isotope mass spectrometry and new carbon and deuterium combustion analysis machines (elemental cavity ring down systems) for improved identification of trapped insects in operational programmes. These will include European grapevine moth, pink bollworm, and light brown apple moth. Important factors needing investigation include: refining methods for sample collection and preparation; turn-around time to get results to programme; investigation of traps currently used to determine if glue removal methods can be developed or if programmes need to use new dry glue or bucket trapping systems (Hood-Nowotny, Simmons, Walters ).
• Investigate and develop new tools to improve: sterile insect monitoring, pest detection and surveillance, and development of new methods for determination of pest host status and provenance for improved response to pest invasions. The new tools to be investigated include: pyrolysis GCMS fatty acid finger
printing for host verification; endophytic bacteria for F 1 marking and parental egg identification; and deuterium analysis for validation of geographical origin ( Hood-Notowny, Simmons, Suckling, Walters ).
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Working Group 3: Quality Control Aspects in Relation to Rearing of Moths
The objective of this working group is to develop new and improved methods for enhancing rearing systems and to facilitate the selection of performance and fitness traits that improve colony establishment.
The following was achieved in 2010-2012 for each of the activities planned in the categories:
1) Developing new and improved methods for enhancing rearing systems.
Planned activity 1: To investigate use of alternative and/or optimize current collection systems for moth stages (pupal separation, pheromone separation/collection, light sources, UV, NIR, air systems – cyclone, plenum) (Simmons, Ploski, Addison, Conlong).
Cydia pomonella
Various collection methods were tested throughout a full production season looking at; 1) attraction to collection point, 2) collector type (plenum vs. cyclone), 3) collection air-speed and 4) cooling. Time between moth emergence and collection was analyzed and found critical for moth quality. Where the former time was too long male & female moths could find one another and effectively mate, which is undesirable prior to radiation. Currently a standard BLB light source is utilized for attractance, however a light tunnel has been developed allowing testing of various wavelengths, as emitted from LED light sources, which will be done during 2012-2014.
Eldana saccharina
A plenum-based collection system is currently being investigated and developed.
Epiphyas postvittana
An insect collection system was developed for modular rearing systems which includes new LED UV lights, high efficiency small scale dust collection systems, enhancements to cyclone collection equipment and development of a protocol for managing sex ratio from cyclone collections to improve production and quality of egging cage systems.
Planned activity 2: To investigate and develop enhanced methods for insect mass-rearing (post collection handling methods, enhancements for neonate establishment [phagostimulants, light, substrate enhancement], post irradiation treatment therapy, effects of different temperature, rearing regimes, diapause rearing, thermal conditioning,etc.) (Simmons, Conlong, Addison/Terblanche, Carpenter, Hight, Hahn, Stotter).
Cydia pomonella
Initial findings have been published (Chidawaniyka et al. 2010) – further research has attracted additional industry funding and will therefore continue.
Thaumatotibia leucotreta
With the expansion of FCM SIT into the Sunday river valley (Eastern Cape Province, South Africa), transportation of moths needed to be critically analysed to ensure minimal quality degradation before releases. A new cardboard box system was developed that reduced the handling steps, allowed larger volumes to be handled during radiation, reduced condensation damage to moths and reduced cooling volumes required due to more efficient spatial use. Parameters relating to container capacity during radiation as well as cassettes directly connectable to aerial release devices will be investigated during 2012-2014.
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Epiphyas postvittana
A biomedical packaging system for organ transplant shipping was adapted for shipping sterile insects from the production facility to remote release locations.
Planned activity 3: To investigate effects of nutrition and diet composition on insect quality and resistance to stress from irradiation, handling (reduced nutritional quantity diets, fillers agars, recycled agar, agar substitutes, fatty acids, role of ascorbic acids, ascorbic acid – rooibos tea as substitute, etc.) (Simmons).
Epiphyas postvittana
No progress to report.
Planned activity 4: To investigate diet preparation methods, storage and shipping methods effects on insect quality (pelletization, preservation and packaging, centralized production) (Simmons, Conlong, Addison).
Cydia pomonella
Non-nutritious fillers ( Pinus sp. wood shavings/-chips) have been added to mass rearing diet during the past production year, to comprise on average 2.84% of the diet. No production limiting effects have so far been observed, however moisture management has improved, with diet being dryer (desirable), whilst diet cost decreased by 0.3%.
Eldana saccharina
No progress to report research objective will be started the 4 th phase of the CRP in 2012-2014.
Pectinophora gossypiella
A freeze dried diet preservation system was developed for the pink bollworm diet used in mass-rearing. Preliminary data show that the diet can be reconstituted and produces insects of similar quality to fresh production diet. Many other rearing programs on other species will be evaluating this diet.
Planned activity 5: To develop and test alternative egg and diet treatment methods for disease control and enhancement of insect quality (heat, ozone, formaldehyde replacements, CHG, irradiation hormesis) (Simmons, Addison, Conlong, Seth).
Cydia pomonella
No progress to report, this research objective will be started in the 4 th phase of the CRP in 2012-2014.
Eldana saccharina
No progress to report, this research objective will be started the 4 th phase of the CRP in 2012-2014.
Epiphyas postvittana
A protocol for effective treatment of eggs was developed using chlorohexadine glutamate (CHG) resulted in improved eclosion in rearing trays and reduced pathogen incidence.
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2) Facilitating the selection for performance and fitness traits that improve colony establishment
Planned activity 1: To investigate effects of relaxed pressure rearing systems (filter rearing) on moth quality and possible use for mass-rearing; identify potential stresses in mass-reared colonies (Carpenter & Hight).
Flight performance of irradiated Cactoblastis cactorum from a filter colony insectary and a production colony insectary was compared in flight cylinders and open field release/recapture trials. Results suggest that moth quality is reduced in the production rearing facility and indicate that additional studies are needed.
ACTIVITIES PLANNED FOR 2013-2014
The following activities are planned:
• Continue to investigate effects of relaxed pressure rearing systems (filter rearing) on moth quality and possible use for mass-rearing; identify potential stresses in mass-reared colonies ( Carpenter, Hight ).
• To investigate collection methods of Cydia pomonella and Eldana saccharina with particular focus on attraction to a light source, by use of different wavelengths LED lights, and collection from such source by cyclonic- or plenum based collectors (Addison, Conlong, Simmons, Stenekamp, Wohlfarter).
• Develop and enhance rearing methods and test artificial diets for Tuta absoluta , Lobesia botrana , Hypsipyla grandella and Chilo sacchariphagus (Conlong, Garrido, Grissa, Perez-Staples, Saour, Simmons).
• Develop quality control measurement protocols, for Tuta absoluta , Lobesia botrana , Hypsipyla grandella and Chilo sacchariphagus (Conlong, Garrido, Grissa, Perez-Staples, Simmons).
• Investigate and develop cost efficient methods and compare quality aspects to larger scale rearing systems, for insect mass-rearing of Cydia pomonella, Eldana saccharina, Lobesia botrana and Chilo sacchariphagus, using a modular rearing design (Conlong, Garrido, Simmons, Wohlfarter).
• Develop methods for insect rearing and improved handling to enhance neonate establishment by modifying existing natural diets to develop and test meridic diets, eliminate handling steps in pre- and post irradiation, packaging and transport, effects of different thermal conditioning, phagostimulants (ginger oil), substrate enhancement for Cydia pomonella, Thaumatotibia leucotreta, Tuta absoluta , Lobesia botrana , Hypsipyla grandella and Chilo sacchariphagus (Conlong, Garrido, Grissa, Koekemoer, Perez-Staples, Saour, Simmons, Wohlfarter).
• To investigate effects on insect quality related to diet preparation-, storage- and shipping methods (peletization, preservation and packaging, centralized production, addition of non-nutritious fillers) for Cydia pomonella, Pectinophora gossypiella, Thaumatotibia leucotreta, Tuta absoluta , Lobesia botrana , Hypsipyla grandella and Chilo sacchariphagus (Addison, Conlong, Garrido, Wohlfarter, Walters, Simmons, Stenekamp).
• Develop and test alternative egg and diet treatment methods for disease control and enhancement of insect quality (heat, ozone, formaldehyde replacements, addition of anti-microbials and CHG (Addison, Stenekamp,Wohlfarter).
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The logical framework
Narrative Summary Objective Verifiable Means of Verification Important Indicators Assumptions
Overall Objective:
To provide the basis N/A N/A • The problems for improved efficacy caused by pest of lepidopteran (moth moths will pest) SIT through the remain of development of regional / systems that enable interregional assessment and relevance prediction of the field performance of sterile • SIT will insects remain an important component of area-wide integrated pest management campaigns against moth pests.
Specific Objectives:
To identify and N/A N/A characterise factors and variables that affect quality and field performance of released moths.
To develop and N/A N/A improve tools and methods to assess, predict and enhance the field performance of released moths based on insect quality.
To develop new and N/A N/A improved methods for enhancing rearing systems, to facilitate the selection for performance or fitness traits that improve 40
Narrative Summary Objective Verifiable Means of Verification Important Indicators Assumptions
colony establishment, refurbishment and production, as well as the field performance of released moths.
Expected Outputs (1)
Factors and variables One or more biological Biological and operational Appropriate biological that affect moth quality attributes and operational factors reported in oral or attributes and correlated with field factors identified and written form operational factors performance characterised. exist and can be measured.
Quality assessment protocols used by researchers and practitioners in ongoing projects
Expected Outputs (2)
New and improved One or more methods Accessible tools and New and improved tools and methods to developed for laboratory, methods for laboratory, methods and tools are measure moth quality semi-field and field semi-field and assessment agreed and accepted assessment that predict the by operational field performance of programme managers released moths based on insect quality
Expected Outputs (3)
Enhanced rearing One or more methods Methods enhancing Enhanced rearing systems that enable evaluated that enhance rearing systems have been systems are agreed and assessment and rearing systems based on reported in oral or written accepted by prediction of the field fitness traits, colony form operational performance of sterile establishment, programme managers insects refurbishment and Methods investigating production fitness traits have been reported in oral or written form
Expected Outputs (General)
Publications in peer- Papers drafted and Journal with published Data is adequate for reviewed journals and submitted papers publication final results published in a special volume
New and improved Recommendations Progress report Recommendations can general and specific available be agreed upon by quality measures for operational 41
Narrative Summary Objective Verifiable Means of Verification Important Indicators Assumptions
moth SIT programmes progr amme managers
New or refined Methods and protocols Progress report Methods and protocols methods and protocols available can be agreed upon by for improving moth operational quality in operational programme managers SIT programmes
CRP OUTPUTS LIST
Peer reviewed publications 1. Benedict, M. Q., R. C. Hood-Nowotny, P. I. Howell, and E. E. Wilkins. 2009. Methylparaben in Anopheles gambiae s.l. sugar meals increases longevity and malaria oocyst abundance but is not a preferred diet. Journal of insect physiology 55: 197-204.
2. Brockerhoff, E. B., D. M. Suckling, M. Kimberley, B. Richardson, G. Coker, S. Gous, J. L. Kerr, D. M. Cowan, D. R. Lance, T. Strand, and A. Zhang. 2012. Aerial application of pheromones for mating disruption of an invasive moth as a potential eradication tool. PLoS ONE 7(8): e43767.
3. Carpenter, J. E., O. G. Marti, S. L. Wee, and D. M. Suckling. 2009. Cytological attributes of sperm bundles unique to F 1 progeny of irradiated male Lepidoptera: relevance to sterile insect technique programs. Florida Entomologist 92: 80-86.
4. Chidawanyika, F., and J. S. Terblanche. 2011. Costs and benefits of thermal acclimation for codling moth, Cydia pomonella (Lepidoptera: Tortricidae): Implications for pest control and the sterile insect release programme. Evolutionary Applications 4: 534-544.
5. Chidawanyika, F., and J. S. Terblanche. 2011. Rapid thermal responses and thermal tolerance in adult codling moth Cydia pomonella (Lepidoptera: Tortricidae). Journal of Insect Physiology 57: 108-117.
6. El-Sayed, A. M., V. J. Mitchell, L. A. M. Manning, and D. M. Suckling. 2011. New sex pheromone blend for the lightbrown apple moth, Epiphyas postvittana . Journal of Chemical Ecology 37: 640-646.
7. Hood-Nowotny, R., L. Mayr, A. Islam, A. Robinson, and C. Caceres. 2009. Routine isotope marking for the Mediterranean Fruit Fly (Diptera: Tephritidae). Journal of Economic Entomology 102: 941-947.
8. Hood-Nowotny, R., M. Watzka, L. Mayr, S. Mekonnen, B. Kapitano, and A. Parker. 2011. Intrinsic and synthetic stable isotope marking of tsetse flies. Journal of insect science (Online) 11: 79-79.
9. Hood-Nowotny, R., R. Schwarzinger, C. Schwarzinger, S. Soliban, O. Madakacherry, M. Aigner, M. Watzka, and J. Gilles. 2012. An analysis of diet quality, how it controls fatty acid profiles, isotope signatures and stoichiometry in the malaria mosquito Anopheles arabiensis . PloS One In press.
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10. Jang, E. B., D. O. McInnis, R. Kurashima, B. Woods, and D. M. Suckling. 2012. Irradiation of adult light brown apple moth, Epiphyas postvittana (Lepidoptera: Tortricidae): egg sterility in parental and F1 generations. Journal of Economic Entomology 105: 54-61.
11. Kean, J. M., D. M. Suckling, L. D. Stringer, and B. Woods. 2011. Modeling the sterile insect technique for suppression of light brown apple moth (Lepidoptera: Tortricidae). Journal of Economic Entomology 104: 1462-1475.
12. Simmons, G. S., D. M. Suckling, J. E. Carpenter, M. F. Addison, V. A. Dyck, and M. J. B. Vreysen. 2010. Improved quality management to enhance the efficacy of the sterile insect technique for lepidopteran pests. Journal of Applied Entomology 134: 261-273.
13. Soopaya, R., L. D. Stringer, B. Woods, A. E. A. Stephens, R. C. Butler, I. Lacey, A. Kaur, and D. M. Suckling. 2011. Radiation biology and inherited sterility of light brown apple moth (Lepidoptera: Tortricidae): developing a sterile insect release program. Journal of Economic Entomology 104: 1999-2008.
14. Sørensen, J. G., M. F. Addison, and J. S. Terblanche. 2012. Mass-rearing of insects for pest management: Challenges, synergies and advances from evolutionary physiology. Crop Protection 38: 87-94.
15. Stotter, R. L., and J. S. Terblanche. 2009. Low-temperature tolerance of false codling moth Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) in South Africa. Journal of Thermal Biology 34: 320-325.
16. Suckling, D. M., and E. G. Brockerhoff. 2010. Invasion biology, ecology, and management of the light brown apple moth (Tortricidae). Annual Review of Entomology 55: 285-306.
17. Suckling, D. M., B. Woods, V. J. Mitchell, A. Twidle, I. Lacey, E. B. Jang, and A. R. Wallace. 2011. Mobile mating disruption of light-brown apple moths using pheromone-treated sterile Mediterranean fruit flies. Pest Management Science 67: 1004-1014.
18. Suckling, D. M., E. B. Brockerhoff, L. D. Stringer, R. C. Butler, D. M. Campbell, L. K. Mosser, and M. F. Cooperband. 2012. Communication disruption of Epiphyas postvittana (Lepidoptera: Tortricidae) using two formulations at four point source densities in vineyards. Journal of Economic Entomology in press.
19. Suckling, D. M., L. D. Stringer, V. J. Mitchell, T. E. Sullivan, G. S. Simmons, A. M. Barrington, and A. M. El-Sayed. 2011. Comparative fitness of irradiated ight brown apple moths (Lepidoptera : Tortricidae ) in a wind tunnel, hedgerow and vineyard. Journal of Economic Entomology 104: 1301-1308
20. Suckling, D. M., P. C. Tobin, D. G. McCullough, and D. A. Herms. 2012. Combining tactics to exploit Allee effects for eradication of alien insect populations. Journal of Economic Entomology 105: 1-13.
21. Suckling, D. M., T. E. S. Sullivan, L. D. Stringer, R. C. Butler, D. M. Campbell, A. M. Twidle, W. J. Allen, A. Mafra-Neto, and A. M. El-Sayed. 2012. Communication disruption of light brown apple moth ( Epiphyas postvittana ) using a four-component sex pheromone blend. Crop Protection in press .
22. Tooman, L. K., C. J. Rose, C. Carraher, D. M. Suckling, S. R. Paquette, L. A. Ledezma, T. M. Gilligan, M. Epstein, N. B. Barr, and R. D. Newcomb. 2011. Patterns of mitochondrial haplotype diversity in the invasive pest Epiphyas postvittana (Lepidoptera: Tortricidae) Journal of Economic Entomology 104: 920-932.
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23. ul Haq, I., L. Mayr, P. E. a. Teal, J. Hendrichs, A. S. Robinson, C. Stauffer, and R. Hood- Nowotny. 2010. Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon fly Bactrocera cucurbitae : effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast. Journal of Insect physiology 56: 1807-15.
24. Wee, S.-L., D. M. Suckling, and A. M. Barrington. 2011. Feasibility study on cytological sperm bundle assessment of F1 progeny of irradiated male painted apple moth ( Teia anartoides Walker; Lepidoptera: Lymantriidae) for the sterile insect technique. Australian Journal of Entomology 50: 269-275.
25. Qiu, M., Chen, M., Zhang, Y. and Feng, J. 2012. Genetic structure and diversity of a newly invasive species, the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae) in China, Biological Invasions, DOI 10.1007/s10530-012-0299-5.
26. Qiu, M., Chen, M., Zhang, Y. and Feng, J. 2012, Amplifying stability and genetic diversity of microsatellite loci in codling moth Cydia pomonella (Lepidoperta: Tortricidae) populations in China, Acta Phytophylacica Sinica 39, 341-346. ( in Chinese )
Conferences 1. Harraca, V., Conlong, D.E. and Rutherford, R.S. 2011. Eldana saccharina sex appeal: A future method to monitor and control this pest? Short Communication. Proceedings of the South African Sugar Technologists Association 84 : 271- 274.
2. Mudavanhu. P., Conlong, D.E. and Addison P. 2011. Performance of sterilized Eldana saccharina Walker (Lepidoptera Pyralidae) in mating trials. Short Communication. Proceedings of the South African Sugar Technologists Association 84: 287 – 291.
3. Rutherford, R.S. and Conlong, D.E. 2010. Combating sugarcane pests in South Africa: from researching biotic interactions to bio-intensive integrated pest management in the field. Proceedings of the International Society of Sugar Cane Technologists 27: 1-17.
4. Suckling, D. M. 2010. LBAM biology, range extension and control, pp. Abstract 1004, Entomological Society of America 58th Annual Meeting, San Diego, U.S.A.
5. Suckling, D. M., B. Woods, and E. B. Jang. 2010. Mobile mating disruption of light brown apple moth using sterile medflies, pp. 38, 84th Annual Western Orchard Pest And Disease Management Conference, Portland, Oregon.
6. Suckling, D. M., B. Woods, G. Baker, D. Williams, and A. M. El-Sayed. 2011. Widening the toolkit: developing biosecurity solutions from chemical ecology (Poster), pp. 68, CRC Plant Biosecurity - Science Exchange 2011, Barossa Valley, S.A.
7. Suckling, D. M., E. G. Brockerhoff, A. Roques, H. Jactel, A. M. El-Sayed, M. Branco, and V. Mastro. 2009. Developing pyramid lure combinations to improve surveillance efficiency, pp. 82-83, IUFRO International Forest Biosecurity Conference : Popular summaries, Rotorua.
8. Suckling, D. M., L. D. Stringer, and J. M. Kean. 2011. Integrated pest eradication: trends, tools and technologies for horticultural pests, 85th Annual Western Orchard Pest And Disease Management Conference, Portland, Oregon.
9. Walton A.J and Conlong, D.E. 2008. Use of oil soluble dyes to mark adult Eldana saccharina Walker (Lepidoptera: Pyralidae). Proc Sth Afr Sug Technol Ass 81: 298-309.
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10. Walton, A.J., Conlong, D.E. and Addison, M.F. 2011. Parental and F1 sterility of Eldana saccharina Walker (Lepidoptera: Pyralidae). Short Communication. Proceedings of the South African Sugar Technologists Association 84: 281 – 286.
11. Way, M.J., Conlong, D.E. and Rutherford, R.S. 2011. Biosecurity against invasive alien insect pests: a case study of C hilo sacchariphagus (Lepidoptera: Crambidae) in the southern African region. Proceedings of the South African Sugar Technologists Association 84: 84 – 91.
12. Woods, B., G. Baker, D. M. Suckling, and D. Williams. 2011. Achieving successful impact management of invasive plant pests using sterile insect release and mating disruption (Oral), pp. 76, CRC Plant Biosecurity - Science Exchange 2011, Barossa Valley, S.A.
MSc Thesis Walton, A. J. 2011. Radiation biology of Eldana saccharina Walker (Lepidoptera: Pyralidae). Unpublished MSc thesis. Department of Conservation Ecology and Entomology, Stellenbosch University. South Africa.
Potgieter, L. 2012 . A mathematical model for the control of Eldana saccharina Walker via the sterile insect technique. Unpublished MSc thesis. Department of Logistics, Stellenbosch University.
PhD Thesis Mudavanhu, P. 2012. Performance of sterilized Eldana saccharina Walker (Lepidoptera: Pyralidae) adults in mating and cage trials: Further steps towards its control using the Sterile Insect Technique. Unpublished PhD thesis. Department of Conservation Ecology and Entomology, Stellenbosch University. South Africa.
Qiu, M. 2012. Genetic diversity and genetic strcture of codling moth populations in China reveaing by micrasatellite makers. Ph.D. thesis of Northwest A&F University, Yangling, Shaanxi Province, China. ( in Chinese )
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WORKING PAPERS
Irradiation biology of the European grape vine moth, Lobesia botrana Ali Harari Ministry of Agriculture and Rural Development, Agricultural Research Organization Volcani Centre, 50250 Bet Dagan Inherited sterility using 100G We measured the effect of 100Gy on mating behavior and reproductive fitness. We first conducted a flying test, out of a 30 cm 3 cylinder, placed in a small cage. All irradiated and control males and females passed the flying test in the small cage, flew out of the cylinder and landed on the cage’s walls or floor. The second test was performed in a field cage (3m 3), examining flight ability of irradiated vs. control males to a pheromone source located in a trap 2m high, as compared with non-baited traps. In the control trap, only a few males were caught. A similar number of irradiated and non-irradiated males were caught in the pheromone trap. The third test was to confirm mating ability of sterile males and females. Control males and females readily mated, as did irradiated males and females. However, a larger number of irradiated females did not produce viable eggs (23%) (f1) as compared to control females (11%). The fourth test was to confirm inherited sterility of irradiated males and females. The number of eggs laid did not differ in irradiated individuals and control but fertilization ratio was different, with a lower number of fertilized eggs to irradiated individuals. Sex ratio among offspring was slightly male biased in irradiated individuals. Strong female biased sex ratio was observed in offspring of females that were irradiated as adult. F2- Male offspring of irradiated males had significantly lower percentage of fertilized eggs than males and females offspring of non-irradiated parents and of irradiated females. The calculated potential reproductive success of irradiated males and females, revealed that irradiated females may have low reproductive success through both their sons and daughters, whereas irradiated males will have higher reproductive success though their daughters than through their sons The effect of irradiation on spermatophore size As females tend to remate if the spermatophore is small and sperm amount is limited, we measured the effect of irradiation in various stages of pupation on sperm number in the testis and in the spermatophore. Irradiation during early stages has led to a decrease in number of sperm in the adult testis. Surprisingly, however, the amount of eupyrene sperm that was transferred to the female via the spermatophore was not significantly reduced. The number of both eupyrene and apyrene sperm did not affect the male reproductive success after one mating. Separating males from females in a laboratory population As a portion of the irradiated females in our study retained their reproductive ability, we tried to define a mechanisms that will allow a confident separation of males from females before radiation. We separated larvae before pupation, numbered them identified their RGB colors, and measured their length at the pupae stage. All individuals were sexed as pupae. We then looked for special characteristics typically for males that allow a separation of males from females in a mix population. Date of roaming and length were such characteristics but not RGBs. Collecting all pupae of a certain size and that were
46 roaming during the first and second day will obtain a high proportion of females. As this protocol may lead to selection for small body size, collecting all males that are 5.4 mm or less, will obtain 87% of all males but this requires the acceptance of a 30% percent error.
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Development of the sterile insect technique in Lobesia botrana (Denis & Schiffermüller): domestication and mass-breeding Álvaro Garrido, Carlos Lobos and Eduardo Marti Agriculture and Livestock Service - SAG, Chile. Abstract Lobesia botrana (European Grapevine Moth, EGVM) is an exotic pest recently discovered in Chile (2008) and if it expands and takes root in the country it will severely damage and affect grape production in the country. In Chile there are 180,000 hectares of vines, which are threatened by the insect. The current plague infested area includes large urban areas where actions are not possible with conventional pesticides control or to the mechanism of mating disruption. From detection of Lobesia botrana in Chile, the government has implemented an Official Control Program throughout the country in order to eradicate and contain the insect. In this context the results obtained to date have been successful in farms, but has been difficult to control in urban areas. An alternative control may be applied in conjunction with mating disruption or a biological control program is the use of the Sterile Insect Technique (SIT) for these populations of Lobesia botrana in urban areas, an area in which no published scientific references have been found in the world. As a first step in this process of implementation of SIT in Lobesia botrana , a methodology for the rearing of Lobesia botrana will be developed. As a first phase, it is proposed to investigate and evaluate different existing diets reported in the literature or that can be obtained from specialists in the field, as well as to understand and to manage the environmental conditions (temperature, light, humidity, etc..) for this species. Once these preliminarily parameters are assessed, it is proposed to start the first larger breeding attempts and see if the species has any problems. After the parameters of controlled breeding have been defined, a medium-scale colony will be established, which is expected to reproduce the conditions achieved in the laboratory and to provide sufficient biological material to evaluate the SIT in field trials. Specimens of Lobesia botrana obtained under these conditions must be of adequate quality and biological development, so as to be able to compete successfully with wild insects present in the field, as this ensures the effectiveness of this tool as a method of control. KEY WORDS Lobesia botrana , mass rearing, sterile insect technique, urban areas Introduction Agricultural and Livestock Service (SAG) is the National Plant Protection Organization (NPPO), responsible for animal and plant protection in Chile, which is under the authority of the Ministry of Agriculture of Chile. In this sense, the SAG has faced since 2008, a phytosanitary emergency due to the introduction in Chile of the European grapevine moth ( Lobesia botrana ) (Lepidoptera: Tortricidae), which led to the development of a national program to combat it / eradication (PCOLB, Official Control Program of Lobesia botrana). Therefore, the SAG is interested in developing the sterile insect technique for the Lobesia botrana , especially for the control of this moth in urban areas of the country. For SAG, a prerequisite for the development of SIT is to achieve insect mass rearing under controlled conditions, which will serve as a basis for further development of the technique. This is why it is essential in a first phase, getting a proper production of large numbers of insects, define the infrastructure requirements needed for breeding, dietary ingredients, defining procedures, calculate
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necessary staff, the required equipment, environmental conditions in which they work, etc.. Also it is required to get and set management protocols for each stage of the process, from preparation of the diet, through methods to add the plague to the diet, the collection of insects and their care in its various stages. In the first part, the main objective of the project is to define a technically feasible method for mass rearing of Lobesia botrana that serves to initiate the laboratory and field experiences in the development of the Sterile Insect Technique for this species. The specifics objectives are collect wild insects for the establishment of a Lobesia botrana colony, assess effect of various artificial diets on the productivity and quality of the produced insects, and assess effect of abiotic factors on productivity and competitiveness of the reared insects. Development of Lobesia botrana (EGVM) mass rearing and SIT Based on Experience Gained from Several Operational Programs for Moths Materials and Methods To define and make two or three different diet (codling moth diet, pink bollworm diet and chilean diet) for Lobesia botrana , To collect wild insect of different zone of Metropolitan region To evaluate different parameters of process quality (temperature, humidity, photoperiod, etc) To evaluate different biological parameters of production quality on egg, larvae, pupae and adult (weight, longevity, fertility)
Results No results yet, because the project is starting now (September, 2012)
References No references yet, because the project is starting now (September, 2012)
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Inherited sterility and irradiated male's flight ability in the European Grapevine Moth (Lepidoptera: Tortricidae) George Saour Syrian Atomic Energy Commission, P. O. Box 6091 Damascus, Syria Abstract The European grapevine moth, Lobesia botrana (Denis and Schiffermüller) (Lepidoptera: Tortricidae) is a major grapevine pest. Fecundity and fertility of the F1 progeny of males irradiated with 150 Gy inbred or crossed to irradiated and unirradiated moths were recorded. A significant reduction in fertility was observed when F1 males mated with either F1 or unirradiated females. According to the sterility index, F1 females mated with F1 males had greater sterility than that when F1 females were crossed to 150 Gy- irradiated males. Larval developmental time, moth weight, moth longevity, fecundity, fertility, and flight-ability were used as criteria to evaluate the quality of a modified larval diet supplemented with different vegetable oils. Evaluations of male L. botrana field performance either produced using different diets or 150 and 350 Gy-irradiated males vis a vis unirradiated males emerged from the same rearing diet were performed by using field release-recapture tests in grape orchard. Released males were recaptured using delta traps baited with synthetic pheromone. Results indicated that the dose of 150 Gy did not affect field performance of the released males. In contrast, the dispersion of 350 Gy-irradiated males was negatively affected by the radiation dose. Introduction The European grapevine moth, Lobesia botrana (Denis and Schiffermuller) (Lepidoptera: Tortricidae), is a polyphagous insect that develops on more than 200 plant species from various families. However, L. botrana is one of the most serious pests in Mediterranean and southern European area vineyards. More recently, L. botrana was inadvertently introduced in Japan and was reported from Chile, Argentina and the Napa Valley in California (USA). Several control tactics have been evaluated against L. botrana including the use of insecticides, biological control agents and mating disruption methods. However, in most vineyards L. botrana is still controlled using broad-spectrum insecticides. The use of autocidal control methods (i.e., sterile insect technique (SIT) and inherited sterility or F1 sterility in Lepidoptera) represent an environmentally and medically benign option that offer great potential for managing L. botrana. The potential of using inherited sterility technique against L. botrana can be enhanced by providing information on biological factors affecting the quality and competitiveness (flight ability and field dispersal) of irradiated male moths. Materials and methods Fertility and fecundity of F1 progeny of 150 Gy-irradiated males inbred or crossed to irradiated and unirradiated moths Newly emerged L. botrana males were irradiated with a dose of 150 Gy and crossed to virgin unirradiated females. The deposited eggs were collected and allowed to hatch and sterility and fecundity in the F1 generation were calculated. F1 neonates from 150 Gy-irradiated ♂ x unirradiated ♀ were placed on artificial diet pieces. Pupae were collected and all emerging F2 adults were either inbred or paired singly with 150 Gy-irradiated or unirradiated counterparts of the opposite sex (n = 45 pairs for each cross). Moths were allowed to mate and lay eggs. Eggs were collected and incubated. The sterility and fecundity in F2 generation were calculated. Longevity for the F1 and F2 pairs were recorded. Moreover, the percentage of sterility index was calculated using the formula of Toppozada et al.: % Sterility = [1-(Ft x Fet/Fc x Fec)] x 100
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where Ft = fecundity of treated females; Fet = fertility of treated females; Fc = control fecundity; and Fec = control fertility. The experiment was conducted three times for each cross with 15 pairs per replicate. Effect of larval diet compositions on L. botrana moth's quality and flight ability Lobesia botrana larvae were reared on a semi-artificial diet (as described in Thiery and Moreau, 2005), with the following composition: 150 ml water, 3 g agar, 9 g maize flour, 11 g wheat germ, 9 g yeast, 0.9 g ascorbic acid, 0.3 g benzoic acid, 0.3 ml maize oil, 0.3 g Nipagine, and 0.2 g Iprodione (fungicide), at 25 ±1 ºC, 60±10% RH with a photoperiod of light/dark 15:8 + 1 of dusk. Grape seed oil or ginger root oil – Zingiber officinale Roscoe - instead of maize oil with or without 3 g of casein powder were used as specific ingredients in order to produce high quality L. botrana moths. The suitability of the improved diet to L. botrana larvae was evaluated depending on the following criteria: 1) larvae developmental time; 2) percentage pupation; 3) moth weight after emergence; 4) pupae developmental time; 5) percentage of emerged adults; and 7) fecundity. Moreover, the impact of diet composition on L. botrana quality was calculated by using several nutritional indexes including: 1) host suitability index (HSI) = [(Mean no. of eggs laid per female/larval duration x consumption) x 100% survival]; 2) suitability index (SI) = (Mean of eggs laid per female/larval duration) x 100% survival; 3) food consumption index (FCI) = Weight of food consumed/ larval duration x moth weight; and 4) growth index (GI) = 100% moth survival / larval duration. Newly hatched L. botrana larvae (n = 200) were fed on artificial diets with different oil composition (5g diet/larva). The diet cubs were weighted before and after the feeding period to determine the consumption rate. The formed pupae were removed and kept until adult emergence. Newly emerged males and females were weighted and paired individually in transparent plastic Petri dishes (9 cm). The oviposited eggs (fecundity) were collected daily and counted. Females that did not oviposit more than 10 eggs were discarded in order to achieve homogeneity of variance. For diet consumption rate, aliquots were used to adjust errors due to natural weight loss of the diet according to Kogan formula: C = Ie-Fe (I0/F0) where, C = corrected weight, Ie and I0 = initial fresh weight for the experimental and the aliquot diets, Fe and F0 = final fresh weight for the experimental and the aliquot diets 48 hours after feeding. To assess the flight-ability of L. botrana adult males, an experimental apparatus was constructed. The apparatus consists of a transparent plexiglass box of 70 x 40 x 50 cm with two compartments separated by a plexiglass screen fixed in the middle of the box. Three openings (or vents) of 38 cm long x 2 cm wide were made onto the screen at three heights (15, 30, and 45 cm). Once the moths go through the opening, they will not be able to return back since the opening was narrowed to 0.5 cm by gluing two sloping pieces of cardboard onto the other side of the opening. Airflow was created inside the box by two small electric fans fixed on both the left and right side of the box. The speed of each fan was adjusted with an external voltage regulator device. Only adult males that were < 24 h old were used for the flight-ability test. Two-d-old virgin females were confined inside small plastic mesh cylindrical box containing a cotton wick soaked in 5% sucrose solution (food source). Water-soaked cotton wicks in Petri dishes (n = 6) affixed over the bottom of the box provided moisture to the apparatus. Moreover, the inner walls of the male's compartment were covered with a thin layer of white talc to prevent males from climbing. Approximately 75 males resulted from three different diets (25 for each diet) were cooled for 3 min. and then released in the 1st compartment, while 5-6 virgin females inside the plastic mesh box were placed in the 2nd compartment and the no. of males that flew and went through the opening, at determined level (15, 30 or 45 cm), to female compartment was recorded after 24, 48, 72 and 96 h. Fluorescent powder was used to mark and distinguish between the adult males.
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Effect of gamma radiation on L. botrana male flight ability Newly emerged adult males (<24 h old) were irradiated at two doses: 150 and 350 Gy (n = 25 males for each dose). At each examined dose, a group of newly emerged adult males (n =25) were taken as a control. Irradiated and control males were cooled for 3 min. and then released in the 1st compartment of the flight ability apparatus. The same procedure as described before was followed. Field release-recapture tests for L. botrana 150 and 350 Gy-irradiated and unirradiated males The trial was conducted in a 0.5 h vineyard located nearby Damascus. The vineyard was moderate density plantation (250 plants/ha). The orchard consisted of 6 rows separated by a 5-m alley. The vine trees (2 m height) were spaced 3.5 m apart and maintained on trellis system. Capture of L. botrana males were assessed using pheromone-baited traps (Russell IPM. UK). All traps were spaced throughout the vineyard and hung at 2 m high. Fluorescent powder was used to mark and distinguish
Table 1. Means (±SE) of egg hatch, number of eggs/female, moth longevity, and Sterility index when the European grapevine moth males were irradiated with 150 Gy and crossed to fertile counterparts
(P 1), and when F 1 adults resulting from irradiated male x unirradiated female were inbred or crossed to 150 Gy-irradiated and unirradiated moths.
Crosses Mean egg hatch Mean no. eggs/ Mean moth longevity (day) Sterility index (%) female ♂ ♀ (%)
Parental generation (P 1) Irradiated ♂ x Unirradiated ♀ 38.9 ± 1.6b 71.8 ± 4.0a 12.2 ± 0.4a 12.9 ± 0.4a 54.3 ـ Unirradiated ♂ x Unirradiated ♀ 81.9 ± 2.3a 74.7 ± 3.4a 13.0 ± 0.5a 12.8 ± 0.6a
F1-genration
F1 ♂ x Unirradiated ♀ 19.6 ± 2.5bc 42.1 ± 2.1c 10.2 ± 0.4b 12.3 ± 0.7a 86.9
F1 ♂ x F 1 ♀ 10.2 ± 1.1d 37.7 ± 2.1c 10.1 ± 0.4b 10.0 ± 0.4b 93.9
150 Gy-Irradiated ♂ x F 1 ♀ 15.1 ± 1.9cd 66.7 ± 2.7b 12.3 ± 0.4a 10.3 ± 0.4b 84.0
Unirradiated ♂ x F 1 ♀ 24.6 ± 2.9b 72.8 ± 2.4ab 12.6 ± 0.3a 10.2 ± 0.3b 71.6 ـ Unirradiated ♂ x Unirradiated ♀ 82.9 ± 1.8a 76.1 ± 1.7a 12.8 ± 0.3a 12.7 ± 0.4a
Means within a column for each generation followed by the same letter are not significantly different at P < 0.05 (Fisher PLSD).
% Sterility index = [1-(F t x Fe t/F c x Fe c)] x 100,
Ft = fecundity of irradiated females; Fe t = fertility of irradiated females; F c = control fecundity;
and Fe c = control fertility. Mean of 3 replicates for each cross, 15 moth pairs per replicate. between the 150, 350 Gy-irradiated and unirradiated males. The release of moths was performed 1 h before sunset underneath the vine canopy. Traps were checked daily for 4 days after moth releases. Higher trap capture were interpreted as indicative of higher moth quality. Four releases were performed with 150 L. botrana males per release. Results Fertility and fecundity of F1 progeny of 150 Gy-irradiated males inbred or crossed to irradiated and unirradiated moths Fecundity and percentage egg hatch of L. botrana parental generation was significantly affected by the dose of radiation used (150 Gy). However, adult longevity was not affected by the applied dose (Table 1). F1 females crossed to unirradiated males had residual fertility comparable to that obtained from 150 Gy-irradiated males mated with unirradiated females. A significant reduction in percent egg hatch with
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Table 2. Means of developmental stages, moth weight, fecundity, moth longevity and diet consumption of the European grapevine moth fed on artificial deit with different supplements of oil and casein (25 ± 1 °C, 15h light: 8h dark + 1h dusk).
Diet supplements Larval mortality Larvae develop- Pupal mortality Moth weight (mg) Pupae develop- No. of emerged No. eggs/female Moth longevity Diet consumption/ (%) mental time (day) (%) ♂ ♀ mental time (day) adults (fecundity) (day) larva (g)* Maize oil (control) 45a 22.9a 13.2a 8.5e 10.9e 7.2a 84d 96.4d 10.5e 0.56e (n = 90) (41.8%) Grape seed oil 42.5a 21.6ab 12.3a 8.9c 11.4c 7.0a 90cd 106.8c 11.3d 0.71d (n = 85) (45.2%) Ginger oil 33.5b 18.6c 10ab 9.8b 12.6b 7.2a 113b 123.1b 13.3b 0.98b (n = 67) (56.5%) Maize oil + casein 39.5a 21.2ab 11.3a 9.1bc 11.6bc 6.9a 98c 116.7bc 11.6d 0.8cd (n = 79) (49.2%) Grape seed oil + 35.5b 20.3b 10.8a 9.3bc 12.1bc 7.3a 107b 119.2b 12.5c 0.91bc casein (n = 71) (53.7%) Ginger oil + casein 26c 17.7c 8.5b 10.8a 13.9a 7.1a 131a 133.6a 14.1a 1.23a (n = 52) (65.5%)
Means in column followed by the same letter are not significantly different (P < 0.05, Fisher PLSD). * C= Ie-Fe (Io/Fo), C = corrected weight, Ie and Io = initial fresh weight for the experimental and the aliquot diets, Fe and Fo = final fresh weight for the experimental and the aliquot diets. The experiment was replicated 4 times, with 50 larvae per replicate
high sterility index was recorded when F1 males were crossed to their female siblings (F = 200.2; df = 4, 131; P < 0.0001). In F1-generation crosses, the fecundity of crosses F1 male x unirradiated female and F1 male x F1 females was significantly lower than that of the other crosses tested (F = 65.9; df = 4, 127; P < 0.0001). The mean longevity of F1 adults was significantly lower than their irradiated parents (e.g. for male moths F = 8.8; df = 4, 127; P < 0.0001). Effect of larval diet compositions on L. botrana moth's quality and flight ability Larval and pupal mortality, larval and pupal developmental time, moth weight, no. of emerged moths, fecundity, moth longevity, and diet consumption per larvae differed significantly between the artificial diets tested (e.g. for larval developmental time and female fecundity F = 12.6; df = 5, 232; P < 0.0001 and F = 12.1; df = 5, 263; P < 0.0001, respectively) (Table 2). In general, replacing maize oil by either grape seed or ginger root oils significantly improved the quality of the produced moths (i.e. shortened the developmental time, increase the moth weight, adult longevity and female fecundity). However, the highest values of moth quality were recorded when L. botrana larvae reared on casein + ginger oil diet and the lowest from larvae reared on maize oil diet. Nutritional indexes calculated for L. botrana varied significantly with the diet supplements (Table 3). Host suitability, food consumption and growth rates were highest on casein + ginger oil diet. This suggests that the last diet is a better food source for larval growth and development than the other diets
Table 3 . Mean food consumption index (± SE) of Lobesia botrana as affected by different diet supplements
Diet supplements Host suitability indexº Suitability index¹ Food consumption index² Growth index³
Maize oil (control) 3.3 ± 0.15d 1.9 ± 0.20de 2.3 ± 0.12e 1.7 ± 0.07e
Grape seed oil 3.2 ± 0.15d 2.3 ± 0.12ce 3.3 ± 0.11d 2.1 ± 0.10d
Ginger oil 3.9 ± 0.13ab 3.8 ± 0.16b 4.9 ± 0.08b 3.1 ± 0.08b
Maize oil + casein 3.5 ± 0.11cd 2.6 ± 0.10c 3.4 ± 0.13d 2.2 ± 0.07d
Grape seed oil + 3.6 ± 0.11bc 3.3 ± 0.14b 4.3 ± 0.10c 2.7 ± 0.09c casein Ginger oil + casein 4.3 ± 0.15a 5.4 ± 010a 5.8 ± 0.11a 3.9 ± 0.06a
Means in column followed by the same letter are not significantly different (P < 0.05, Fisher PLSD). º(Mean of eggs laid per female/larval duration x consumption) x 100% survival ¹ (Mean of eggs laid per female/larval duration) x 100% survival ² Weight of food consumed/ larval duration x moth weight ³ 100% moth survival / larval duration
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Table 4 . Mean percentages (± SE) of flight ability of Lobesia botrana male determined using calling virgin female for 4 days between male moths emerged from rearing diets with different supplements
Tested Type of diet Males flew up to calling female days after the initiation of the test Non-flyer groups supplements 1 2 3 4 moths
Maize oil (control) 19.2 ± 1.4b 38.4 ± 1.1b 16.2 ± 1.7a 8.6 ± 1.7a 17.8 ± 1.3a I Grape seed oil 23.6 ± 3.4ab 42.7 ± 1.5ab 12.7 ± 1.3ab 8.9 ± 1.1a 15.3 ± 1.3a Ginger oil 29.6 ± 4.6a 43.2 ± 1.9a 8.9 ± 2.0b 7.7 ± 2.6a 10.4 ± 0.9b Maize oil + casein 21.7 ± 1.6b 38.7 ± 1.6b 15.3 ± 1.2a 9.0 ± 0.8a 15.2 ± 1.3a II Grape seed oil + casein 23.9 ± 3.4b 42.5 ± 1.4ab 13.3 ± 1.6ab 7.0 ± 1.6a 13.2 ± 1.0a Ginger oil + casein 31.6 ± 3.7a 44.6 ± 0.9a 10.1 ± 1.9b 5.2 ± 1.6a 8.6 ± 0.5b
Means in column for each day and tested group followed by the same letter are not significantly different (P < 0.05, Fisher PLSD). Mean of 6 replicates, 75 Lobesia botrana males per replicate. tested. Of the six diets studied, maize oil was least suitable for L. botrana as shown by the lowest host suitability, food consumption and growth rates (Table 3). The mean percentage of males that successfully flew up was related to the food supplements added to the larval diets. Table 4 illustrates that there were significant differences in male's flight ability among the different diets. Irrespective of the 4th day following the start of the experiment, the mean percentage of flyer males of ginger oil diet was significantly higher than that of the maize oil diet either with or without the addition of casein. In contrast, the mean percentage of non-flyer males emerging from larvae that reared on ginger oil or casein + ginger oil diets was significantly lower than that emerged from the other diets tested (F = 11.7; df = 2, 15; P < 0.0009). Effect of gamma radiation on L. botrana male flight ability The number of L. botrana males that flew up and went through the opening to female's compartment was influenced by the applied radiation dose and number of days following the initiation of the test (Table 5). Regardless of the 3rd and 4th days following the release, the mean percentage of flyer moths differ significantly between 150 and 350 Gy-irradiated males (F = 47; df = 2, 15; P < 0.0001 and F = 33.1; df = 2, 15; P < 0.0001 for 1 and 2 days, respectively). The highest flight rate was recorded during the 2nd day after the start of the trial and no differences in the flight ability were found between 150 Gy- irradiated and unirradiated moths. The mean percentage of non-flyer moths of 350 Gy-irradiated males was significantly higher than that of 150 Gy-irradiated and unirradiated males (F = 67.2; df = 2, 15; P < 0.0001) (Table 5).
Table 5 . Mean percentages (± SE) of flight ability of Lobesia botrana male determined using calling virgin female for 4 days between 150, 350 Gy-irradiated and unirradiated male moths
Type of tested males Males flew up to calling female days after the initiation of the test Non-flyer 1 2 3 4 moths
150 Gy-irradiated males 21.0 ± 1.1c 42.7 ± 3.2a 14.2 ± 0.9a 7.3 ± 1.0a 14.8 ± 2.5b
350 Gy-irradiated males 11.5 ± 1.0b 19.0 ± 1.6b 16.0 ± 1.6a 6.5 ± 1.2a 47.0 ± 3.3a
Unirradiated males 26.5 ± 1.2a 45.0 ± 2.4a 12.3 ± 1.6a 7.1 ± 1.1a 9.1 ± 0.9b
Means in column for each day followed by the same letter are not significantly different (P < 0.05, Fisher PLSD). Mean of 6 replicates, 75 Lobesia botrana males per replicate.
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Table 6 . Effects of distance from the release site and time after release on the number of 150, 350 Gy-irradiated and unirradiated Lobesia botrana males recaptured in sex pheromone traps.
Time after Type of captured Number of moths captured Total moths Moth captured release (h) moths 5m 10m 15m 20m 25m 30m 35m 40m captured (%) 150 Gy-irradiated ♂ 8 5 6 9 3 0 1 0 30 5a 24 350 Gy-irradiated ♂ 0 0 0 1 0 0 0 0 1 0.2b Unirradiated ♂ 12 4 5 9 3 1 0 1 35 5.8a
150 Gy-irradiated ♂ 6 1 0 1 0 0 1 0 9 1.5a 48 350 Gy-irradiated ♂ 0 1 1 1 0 0 0 0 3 0.5b Unirradiated ♂ 7 0 1 0 0 1 1 0 11 1.8a 150 Gy-irradiated ♂ 2 2 1 2 0 2 2 0 13 2.2a 72 350 Gy-irradiated ♂ 0 0 0 1 1 0 0 0 2 0.3b Unirradiated ♂ 2 1 0 3 3 2 2 1 13 2.2a
150 Gy-irradiated ♂ 0 1 0 1 2 0 0 1 5 0.8ab 96 350 Gy-irradiated ♂ 0 0 0 1 0 0 0 0 1 0.2b Unirradiated ♂ 0 0 5 1 2 0 0 0 8 1.3a
Total - 37 15 19 30 14 6 7 3 131 - % Captured - 6.2 2.5 3.2 5.0 2.3 1.0 1.2 0.5 - 21.8
Percentages in column for each time after release followed by the same letter are not significantly different (P < 0.05, Analysis of proportion). 4 releases were performed, 150 Lobesia botrana males per release.
Field release-recapture tests for 150 and 350 Gy-irradiated and unirradiated male L. botrana The results showed that 131 marked 150, 350 Gy-irradiated and unirradiated male L. botrana were recaptured as a result of 4 releases and during 4 days after the initiation of the experiment (Table 6). The recapture rate was 21.8%. At a distance of 5 m from the release point only 150 Gy-irradiated and unirradiated males were recaptured. Three males (two unirradiated and one 150 Gy-irradiated) were trapped 40 m from the release point. The highest recapture rate occurred during the first day after release. Recapture percentages of 150 Gy-irradiated male moths did not significantly differ from that of unirradiated males. In contrast, the lowest recapture rate occurred within the 350 Gy-irradiated released males (7 recaptured from 200 released). These data suggest that the high doses of radiation negatively affected the ability of the male moths to fly and respond to the female sex pheromone.
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Development of the sterile insect technique to control the tomato moth Tuta absoluta (Lepidoptera: Gelechiidae) Silvia Noemí López Insectario de Investigaciones para Lucha Biológica - IMYZA-CNIA-INTA Castelar, Buenos Aires, Argentina Introduction Tuta absoluta (Meyrick 1917) (Lepidoptera: Gelechiidae) is an important tomato pest native to the Neotropics (García and Espul, 1982; Larraín, 1986). It also attacks potato, aubergine, cucumber, tobacco and other wild Solanaceae species (Mallea et al., 1972; Galarza, 1984; Notz, 1992; CIP, 1996). This pest causes crop losses that may reach 80 -100% (Lopez, 1991; Apablaza, 1992) due to the death of the plant by its attack and the attack of secondary pathogens, and by the rejection that attacked fruits have in national and international markets. This moth was only present in South American countries until 2006 when it was detected in Spain (Urbaneja et al., 2007). In 2008 it was found in Italy, France, Greece, Portugal, Algeria, Tunisia and Morocco (Potting et al., 2009). The next year T. absoluta attacks were reported in Crete (Roditakis et al., 2010), United Kingdom, Holland and Switzerland, but only in greenhouses (Potting et al., 2009). Finally, it has also invaded Cyprus, Germany (EPPO, 2010), Turkey (Kılıç, 2010), Bulgaria, Romania, Lithuania and other countries of the Middle East (Desneux et al., 2010). In order to control this pest, farmers from South America apply up to more than 36 insecticide applications within a single crop cycle (Suinaga et al., 2004). As a result, resistant populations to some of the recommended insecticides have been reported in Brazil (Guedes et al., 1994; Siqueira et al., 2000), Chile (Salazar and Araya, 1997) and Argentina (Lietti et al., 2005). Besides, this practice is against the current demands for a sustainable production with low contaminants levels. One of the pest control options that does not develop resistance and has also no significant negative impact on the environment is the Sterile Insect Technique (SIT). SIT programs have been successful against a number of Diptera pests (Bloem and Carpenter, 2001). In Lepidoptera species, being more resistant to radiation when compared to most other insects due to the holokinetic nature of its chromosomes (LaChance, 1967), full sterilization requires higher radiation doses which may affect mating ability and field performance (Bloem and Carpenter, 2001). As a consequence, Inherited Sterility (IS) or F1 Sterility is proposed as an alternative. In this case, completely sterile females and partially sterile males carrying chromosomes alterations are released in the field. These partially sterile males mate with wild fertile females and the deleterious effects induced by radiation are inherited and expressed for several generations, but mainly in the F1 generation (Carpenter et al., 2005). The use of IS as a component of population management programs for Lepidoptera has been studied by several researchers (Knipling, 1979; North and Holt, 1971; LaChance, 1985; Carpenter et al., 1987; Bloem and Carpenter, 2001). Field releases of partially sterile insects have demonstrated that some Lepidoptera species like the cabbage looper Trichoplusia ni (Hübner), the corn earworm Helicoverpa zea (Boddie), the gypsy moth Lymantria dispar (L.) and the codling moth Cydia pomonella (L.) could be controlled by this technique (North and Holt, 1969; Proverbs et al., 1978; Carpenter et al., 1987; Carpenter and Gross, 1993; Mastro, 1993; Bloem and Carpenter, 2001; Bloem et al., 1999). Regarding T. absoluta , IS may represent a suitable tool for managing pest populations either in the field or in greenhouses. However, little is known about its chromosomes, cytology and the effect that radiation may have on this species, to develop an IS program. Only Arthur (2004) analyzed the effect of gamma rays on different stages of this species in order to establish a quarantine treatment for packaged tomatoes.
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Another aspect that is taken into account in this research is the use of X-rays instead of gamma-rays to irradiate the individuals. Historically, sterilization has used gamma-radiation with a high radioactive material dependency. Nowadays, this type of irradiators have several difficulties to be purchased because the Nordion INC company (formerly MDS INC) stopped the production of self-shielded colbalt-60 irradiators and by the difficulties related to the transportation of Co sources (Mastrangelo et al., 2010; Mehta and Parker, 2011). On the contrary, the X-irradiators do not depend on a radioactive source and they are easier and safer to obtain than the cobalt-60 ones. The objectives of this research include: to analyze the impact of X-rays on emergence, morphology, fecundity, fertility and F1 sterility of irradiated males and females; to describe the wild-type karyotype of this species in order to have basic information to identify the chromosomes aberrations caused by X- rays; to study the impact of irradiation on the dichotomic spermatogenesis process, and to analyze the morphology of the nuclei of the eupyrene spermatozoa transferred by irradiated males. Materials and Methods Biological material Tuta absoluta specimen used in the studies were obtained from the laboratory colony in the Insectario de Investigaciones para Lucha Biológica, Instituto de Microbiología y Zoología Agrícola (INTA Castelar). Tomato moth was reared on tomato plants ( Solanum lycopersicum L.) in a controlled environment room at 22-30° C and 60-85% relative humidity. The voucher specimens were preserved in the IILB collections. Impact of radiation on biological parameters
Emergence and morphology Pupae 48 to 72 hours prior to emergence obtained from the rearing of T. absoluta were separated by sex according to the position of the genital opening and randomly assigned to the treatments. Groups of pupae were irradiated using a Constant Potential X-Ray System Mg 160 Philips at a dose rate of 0.679 Grays/second (Gy/s). The doses applied include: 0, 5208, 10416, 15625, 20833, 26041, 31250 and 36458 Roentgen, equivalent to 0, 50, 100, 150, 200, 250, 300, and 350 Gray (Gy) in gamma radiation. From now on, doses would be referred in Gy. The effect of X-radiation on both male and female emergence was evaluated considering the following variables: (1) complete adult emergence estimated as the proportion of adults that emerged completely/total irradiated pupae, and (2) proportion of deformed individuals estimated as the deformed individuals/total adults that emerged completely. Since the complete adult emergence is a binary response variable (success or failure), the proportion of complete adult emergence was analysed by a logistic regression with dose and sex as independent variables (Agresti, 1996; StatSoft, 2000). Male fertility and fecundity and inherited sterility Completely emerged males obtained from pupae exposed to increasing doses of X-radiation, were crossed with untreated females, establishing the parental generation (F0). For each dose level (0, 100, 150, 200, 250, and 300 Gy) couples were individually placed in a cylindrical acetate cage containing a tomato plant. The plant was renewed twice at 5-day intervals. The first two plants used were carefully inspected for the eggs laid by each female during the first 10 days of its lifespan. This procedure guaranteed that about 90 % of the fecundity is recorded, since after females are 10 days-old the number of eggs/female/day decays to zero (Pereyra and Sánchez, 2006). The third plant was kept until both adults were dead. Adult longevity was then recorded. Tomato plants with eggs were individually placed on humid tissue paper in rectangular plastic containers. After 7 days, F1 larvae were counted and placed on a new tomato plant in a cage of acetate until pupation. The pupae were counted and sexed. Fecundity (total eggs laid/female), longevity of
57 adults, fertility, number of F1 larvae/female, number of F1 pupae/female, and F1 sex ratio (number of males/total individuals) were recorded for all doses. The effect of radiation dose on fecundity, longevity and number of F1 larvae and pupae were analysed by a Negative Binomial regression for count data using the generalized linear model procedure in STATGRAPHICS Centurion XVI v16.1.17 (StatPoint Technologies, Inc. 1982–2011). Fertility was estimated as the proportion of emerged larvae from the recorded eggs (number of larvae/number of eggs). This resulted in an underestimation as the chorion of T. absoluta egg is very small and they broke and lost among the hairs of the leaves when larvae emerged. Since fertility and sex are binary response variables, the proportion of emerged larvae from the eggs and sex ratio were analysed by a logistic regression with dose as the independent variable. In order to analyze the inherited effects, emerging F1 adults were used for each dose level in two different types of crosses: MF1 x FU and MU x FF1 (where MF1 = male descendent from an irradiated male, MU = unirradiated male, FU = unirradiated female, and FF1 = female descendent from an irradiated male). F1 adult couples were held on a tomato plant in a cage as described above. F1 fecundity, longevity of adults, fertility, number of F2 larvae/female, number of F2 pupae/female, and F2 sex ratio (number of males/total individuals) were recorded for all crosses. The effect of dose radiation and cross type on the F1 fecundity and longevity and number of F2 larvae and pupae were analysed by a Negative Binomial regression for count data using the generalized linear model procedure in STATGRAPHICS Centurion XVI v16.1.17. Fertility and sex ratio were analysed by a logistic regression with dose and cross type as independent variables. Female fertility and fecundity Completely emerged adult females, obtained from pupae exposed to increasing doses of X-radiation 48– 72 h before emergence, were crossed with untreated males, establishing the parental generation (F0). The doses applied to the pupae were: 0, 150, 200 and 250 Gy. Adult couples were held on a tomato plant in a cage as described above. F0 fecundity, longevity of adults, F0 fertility, number of F1 larvae/female, and number of F1 pupae/female were recorded for all crosses. F0 Fecundity, longevity of adults, F0 fertility, number of F1 larvae/female, and number of F1 pupae/female were recorded for all crosses. Data of F0 fecundity and longevity were analysed by a Negative Binomial regression for count data (StatPoint Technologies, Inc. 1982–2011). Fertility was analysed by a logistic regression with dose as independent variable. Description of the karyotype The wild-type karyotype of this species was described using preparations made by spreadings from wing imaginal discs of third and fourth instar larvae according to Traut (1976) with slight modifications detailed in Bressa et al. (2009). After checking the preparations under a phase contrast microscope, the slides were dyed with the fluorochrome DAPI according to Poggio et al. (2011) for further analysis of chromosome number and morphology. In order to assess the presence of sex chromatin in this species, preparations from Malpighian tubes of wild-type third instar larvae were carried out. Malpighian tubes of male and female larvae were dissected in physiological solution for insects (Glaser, 1917 cited in Lockwood, 1961) and transferred into a drop of Carnoy’s fixative (absolute ethanol:chloroform:acetic acid, 6:3:1). After fixation, the structures were dyed with a drop (20 µl) of lacto-acetic orcein, and covered with a cover slide. Then, the preparations were observed under light field microscope and photographed. Impact of radiation on spermatogenesis Male pupae were treated with doses equivalent to 0, 100, 150, 200, 250 and 300 Gy. Immediately after emergence individuals were kept in 70 % ethanol in 1.5 ml microtubes until analysis. Preparations for spermatozoa bundles analysis were performed dissecting the testes of the fixed material in physiological
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solution for insects. The gonads were transferred into a drop of 60 % acetic acid to be dilacerated in order to liberate the sperm bundles. Afterwards, a drop (20 µl) of aceto-orcein was added to dye the cells. Then, the preparation was squashed under a cover slide, sealed and maintained in moist chamber for at least 24 hours before observation under microscope. The morphology (normal – abnormal) of the apyrene and eupyrene bundles, as well as their quantity were registered. The variables used for analysis included: (1) apyrene bundles/eupyrene bundles ratio, (2) normal eupyrene bundles/total sperm bundles, and (3) abnormal eupyrene bundles/total sperm bundles; and were analyzed by one-way ANOVA. The variable apyrene bundles/eupyrene bundles was transformed according to y=ln(x) to achieve homogeneity of variance. If significant differences among treatments were detected, LSD contrast was applied. Morphology of eupyrene spermatozoa transferred by irradiated males For analyzing the transference of euspermatozoa only doses equivalent to 0, 150, 200 and 250 Gy were applied, because higher doses proved to have a notorious negative impact on emergence, fertility and fecundity of males. Male pupae were irradiated as described above. If they completely emerged and showed normal morphology, immediately after emergence they were placed in independent glass tubes with one untreated female. Once a copula was detected, or after 7 days if copula could not be seen, both individuals were transferred alive into a 1.5 ml microtube and stored at -20° C until dissection. Spreading preparations from bursa copulatrix where performed. The structure was dissected in physiological solution for insects and then transferred into a slide with 15 µl of 60% acetic acid to release the sperm and fix the cells. Then, 35 µl of 5% Triton were added to the acetic acid to favor the separation of the cells. The whole volume was collected and transferred into a 0.2 ml microtube. In order to minimize the loss of tissue during transference, the slide was washed two times with 35 µl of 5% Triton, and this volume was also added to the microtube. Afterwards, the cell suspension was vigorously vortexed for 10 seconds to favor de separation of the cells, and then centrifuged 5 minutes at 14.000 rpm at room temperature. The supernatant was removed, and the cell pellet was resuspended in 15 µl of 60% acetic acid and transferred into a new slide. Then, a spreading preparation was performed by moving the drop to different positions on the slide on a hot plate at 55° C. These preparations were dehydrated in an ethanol series (70%, 80%, 100%, 30 seconds each) and stored at -20° C until use. For analysis, the sperm preparations were dehydrated and dyed with DAPI according to Poggio et al. (2011) and observed under an epifluorescent microscope. The number and morphology of the eupyrene spermatozoa were registered and analyzed. Results Impact of radiation on biological parameters Emergence and morphology The proportion of complete adult emergence decreased significantly with the increase of the X-radiation (Wald = 19.81; P<0.05; X2(1) = 20.64; P<0.05) (Fig. 1). For the same dose of radiation, the effect depended on the sex of the individual, with female emergence being higher than that of males (Wald = 5.52; P<0.05; X2(1) = 5.54; P<0.05). It was
also observed that with 350 Gy some Figure 1. Proportion of complete adult emergence (number of adults adults had deformities such as that completely emerged/total number of individuals) in the malformed wings and bent legs (Fig. 2). emergence study.
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Male fertility and fecundity and inherited sterility
Figure 2. (a) Incomplete emergence, (b) deformed wings, (c) deformed legs.
Fecundity of T. absoluta females mated with irradiated males decreased significantly with increasing doses of radiation (Wald = 1,590.17; P<0.05; X2(1) = 42.88; P<0.05). Due to the fact that a dose of 300 Gy affected the mating ability of treated males (females laid an extremely low number of eggs), this dose was not considered useful for IS and was not taken into account in the following analyses (Fig. 3a). Longevity of treated males was not affected by the application of radiation (Wald = 2.26; P>0.05; X2(1) = 0.51; P>0.05). The mean longevity was 17 ± 1 days. On the other hand, the dose affected significantly the fertility in the F0 generation (Wald = 101.84; P<0.05; X2(1) = 112.79; P<0.05) (Fig. 4). A significant reduction in the number of F1 larvae and pupae was observed when pupae of parental males were irradiated with increasing doses of X-rays (Wald = 388.31; P<0.05; X2(1) = 24.02; P<0.05 and Wald = 225.75; P<0.05; X2(1) = 20.54; P<0.05, respectively) (Fig. 3b, c). There were no significant differences in the F1 sex ratio among doses (Wald = 2.92; P>0.05; X2(1) = 2.96; P>0.05). The proportion of males was 0.66 ± 0.036. Since the number of pupae obtained from males irradiated with 250 Gy was very low (8.5 ± 2.99), and females did not emerge from the pupae, this dose was discarded to analyze the F2 progeny. Fecundity of F1 adults mated with untreated counterparts was affected by the dose of X-radiation applied to the F0 male (Wald = 911.80; P<0.05; X2(1) = 107.31; P<0.05) and the type of F1 mating (MF1 x FU or MU x FF1) (Wald = 141.10; P<0.05; X2(1) = 26.01; Figure 3 . (a) Parental fecundity (number of eggs/female), (b) number of F1 larvae, (c) number of F1 pupae when T. P<0.05) (Fig. 5a). F1 Fecundity decreased with absoluta males were irradiated as pupae and mated with the dose but daughters of irradiated males laid untreated females. F0= parental generation, F1= first more eggs than females mated with the sons of generation of descendants of irradiated males. irradiated males regardless of the dose applied.
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The dose was the only factor that affected significantly the fertility in the F1 generation (Wald = 217.70; P<0.05; X2(1) = 268.35; P<0.05), while the type of crossing did not have significant effects and was removed from the analysis (Wald = 1.19; P = 0.27; X2(1) = 1.14; P>0.05) (Fig. 6). There were no significant differences in F1 adult longevity among treatments (Wald = 2.46; P>0.05; X2(1) = 1.40; P>0.05 for dose and Wald = 3.53; P>0.05; X2(1) = 2.01; P>0.05 for type of crossing). The mean longevity was 14 ± 1 days. The total number of F2 larvae decreased Figure 4. Parental fertility (number of larvae/number of eggs) when T. absoluta males were irradiated as pupae and mated significantly when doses applied to F0 males with untreated females increased (Wald = 930.30; P<0.05; X2(1) = 210.29; P<0.05) and, according to F1 fecundity results, it was affected by the type of F1 crossing (MF1 x FU or MU x FF1) (Wald = 29.79; P<0.05; X2(1) = 4.12; P<0.05) (Fig. 5b). Moreover, a similar result was obtained for the total number of F2 pupae (Wald = 673.18; P<0.05; X2(1) = 116.77; P<0.05 for dose and Wald = 61.26; P<0.05; X2(1) = 5.55; P<0.05 for type of crossing) (Fig. 5c). Doses did not affect the F2 sex ratio (Wald = 1.08; P>0.05; X2(1) = 1.71; P>0.05) and there were no significant differences in this variable among types of crossing (Wald = 0.53; P>0.05; X2(1) = 0.62; P>0.05). The proportion of males was 0.49 ± 0.042. Female fertility and fecundity The fecundity of irradiated females was significantly affected by the X-radiation doses applied (Wald = 591.86; P<0.05; X2(1) = 465.49; P<0.05) (Fig. 7). Besides, the X- radiation affected significantly the fertility of the irradiated females (Wald = 73.28; P<0.05; X2(1) = 79.91; P<0.05) (Fig. 8). The eggs laid by females that were irradiated as pupae with 200 and 250 Gy did not hatch. The only F1 larvae that developed to pupae, and then to adults were the descendants from untreated females (Table 1). On the other hand, longevity of treated females was not affected by the doses of radiation applied to the pupae (Wald = 2.23; P>0.05; X2(1) = 2.52; P>0.05). The mean longevity was 12 ± 1 days.
. Figure 5. For both types of F1 crosses (F1 male x untreated female and untreated male x F1 female). (a) Fecundity of the F1 progeny (number of eggs), (b) number of F2 larvae, (c) number of F2 pupae.
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Figure 6 . Fertility of the F 1 progeny (number of larvae/number of eggs) when T. absoluta parental males were irradiated as pupae and mated with untreated females
Figure 7. Parental fecundity (mean number of eggs) when T. absoluta females were irradiated as pupae and mated with untreated males.
Figure 8. Parental fertility (number of larvae/number of eggs) when T. absoluta females were irradiated as pupae and mated with untreated males
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Table 1. Effects of X radiation on the parental generation (Irradiated female x Untreated male) and on their F1 progeny. I: irradiated; U: unirradiated.
Crosses Dose (Gy) Number of F larvae / female Number of F pupae/ female (F male x 1 1 (Replicates) 0 (Mean±SE) (Mean±SE) F0 female) 0 (8) U x U 57.63 ± 4.39 47.63 ± 4.78
150 (8) U x I 0.75 ± 0.49 0
200 (10) U x I 0 0
250 (3) U x I 0 0
Description of the karyotype
Figure 9 . Mitotic metaphases from wing imaginal discs. (a) Female, (b) male. Arrow= largest chromosomes. Bar= 10µm. Mitotic metaphases obtained from imaginal discs from T. absoluta males and females have 2n= 58 chromosomes (Fig. 9). Most of the chromosomes are of small size, with exception of two chromosomes that are notoriously larger. These kinds of preparations did not allow the identification of the sex chromosomes. Preparations from Malphigian tubes revealed the presence of sex chromatin in females, and its absence in males (Fig. 10).
a b
Figure 10. Cells from Malpighian tubes. (a) Female, (b) male. Arrow= sex chromatin.
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Impact of radiation on spermatogenesis The apyrene bundles have a fusiform shape and the nuclei and fragments of nuclei are located in the middle section. The length of these bundles is approximately 100 µm (Fig. 11). No altered morphology on this type of bundles was detected in irradiated individuals Two types of eupyrene bundles were observed, one with normal appearance and the other one with alterations in its structure (Fig. 12). The normal eupyrene bundles (Fig. 12a) are of elongated shape and its diameter was constant at all its length. Once mature, it acquired a wavy appearance. All the spermatozoa heads were organized together in one side of the bundle. The length of these Figure 11 . Apyrene sperm bundle. bundles is of approximately 400 - 450 µm. The abnormal eupyrene bundles (Fig. 12b) show the same general elongated morphology and length, but the nuclei are disorganized and nuclei fragments can be found in the group of tails. The apyrene bundles/eupyrene bundles ratio showed values ranging from 1.43 at 100 Gy to 1.79 at 300 Gy. Statistically significant differences among treatments were confirmed (F(5;48)=2.565, p=0.038). The dose equivalent to 300 Gy differed from all the doses except 250 Gy, but the latter did not differ from the control 0 Gy dose (Fig. 13). The normal eupyrene bundles/total sperm bundles ratio showed values ranging from 0.34 in the higher dose (300 Gy) to 0.41 in the untreated individuals (0 Gy) (Fig. 14). The analysis of variance detected statistically a b significant differences in the proportion of normal eupyrene Figure 12. (a) Normal eupyrene bundle, (b) abnormal eupyrene bundle. sperm produced by males Arrow= nuclei, arrow head= nuclei fragments. treated with different radiation doses (F(5;48)=4.741, p=0.001). The LSD contrast indicated that the proportion of normal eupyrene sperm bundles was significantly lower in the adults that were exposed to 200, 250 and 300 Gy when compared to the 0 Gy control dose. The proportion of abnormal eupyrene sperm bundles with respect to the total number of sperm bundles varied from 0.001 in non-irradiated individuals (0 Gy) to 0.029 in the highest dose applied (300 Gy) (Fig. 15). The ANOVA
showed statistically significant Figure 13. Apyrene bundles/eupyrene bundles ratio under different radiation differences among treatments treatments. (F(5;48)=30.89, p<0.05). The
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LSD contrast indicated that the proportion of abnormal eupyrene bundles produced by control males was significantly lower to that observed in irradiated individuals, and no differences were observed among irradiation treatments. Morphology of eupyrene spermatozoa transferred by irradiated males When the morphology of the euspermatozoa heads was
studied after its transfere to Figure 14. Normal eupyrene bundles/Total sperm bundles ratio under different untreated females, different radiation treatments. Different letters represent statistically significant differences. morphologies were detected. Even though this study is still in progress, it can be said that euspermatozoa with abnormal heads are observed in irradiated individuals preferentially. While normal euspermatozoa heads are slightly wavy (Fig. 16a) the abnormal euspermatozoa heads form a particular angle that is easily identified in spreading preparations (Fig. 16b). Discussion Impact of radiation on biological parameters
Emergence and morphology Selection of the most appropriate sub-sterilizing dose of radiation to induce IS in moths is a critical point. Thus, it should be high enough to avoid the release of fertile females but not extremely high, to maintain the mating competitiveness of the males (Bloem et al., 2003). Moreover, this dose should not affect normal adult emergence. The proportion of complete adult emergence of T. absoluta decreased with the increase of the X-radiation applied to parental pupa. Several authors have mentioned that Lepidoptera females are generally more sensitive to radiation than males (LaChance, 1985; Carpenter et al., 2005). However, we observed that for a same dose of radiation, the effect depended on the sex of the individual, and the emergence of female was higher than that of males. The results also suggest that a dose >350 Gy would affect not only the emergence of adults but also their normal morphology. Doses between 50 and 300 Gy Figure 15. Abnormal eupyrene bundles/total sperm bundles ratio under different reduced adult emergence radiation treatments. compared to the control but
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none of the moths showed deformities. This is consistent with other reports in other Lepidoptera species (Nguyen and Nguyen, 2001; Dhouibi and Abderahmane, 2001). On the other hand, our results do not agree with those of Arthur (2004) who studied the effect of gamma radiation applied on Figure 16. Eupyrene spermatozoa transferred to a non-irradiated female. (a) T. absoluta pupae and found normal head morphology, (b) abnormal head morphology. Arrow=peculiar angle that 300 Gy was the lethal formed in abnormal eupyrene spermatozoa. dose. The type of radiation and the way this affects the insect development can be a significant factor to explain the differences between these studies (Bakri et al., 2005). Male fertility and fecundity and inherited sterility Another key point for IS is that the sub-sterilizing dose of radiation should maintain mating ability of treated males (North and Holt, 1971; Carpenter et al., 2001; Bloem et al., 2003; Ayvaz et al., 2007). The results of fecundity indicated that mating ability of treated males was affected by the highest tested dose (300 Gy). North and Holt (1971) showed that when Trichoplusia ni (Hübner) wild-type females mated with an irradiated male, they often failed to lay a normal number of eggs. They suggested that this might be caused by inadequate sperm or accessory gland fluid transfer to the female during mating. On the other hand, radiation did not affect male mating ability in other moth species such as Cryptophlebia leucotreta (Meyrick) in the dose range of 100 – 350 Gy (Bloem et al., 2003), and Plutella xylostella (L.) and Cactoblastis cactorum Berg in the dose range of 100 – 500 Gy (Sutrisno et al., 1993; Carpenter et al., 2001). The fertility in the F0 generation was affected by the application of radiation. A decrease in the F0 fertility due to increasing doses of radiation was observed for untreated females that mated with males irradiated as mature pupae in different species of Lepidoptera (Carpenter et al., 1986; Henneberry and Clayton, 1988; Carpenter et al., 2001; Nguyen and Nguyen, 2001; Bloem et al., 2003; Ayvaz et al., 2007; Boshra, 2007). For example, Henneberry and Clayton (1988) showed a decrease in fertility in Pectinophora gossypiella (Saunders) when pupae were treated with 150 Gy (gamma rays) (43%) compared to the control dose (81%). This species is a gelechiid like T. absoluta . However, F0 fertility in T. absoluta was higher than that in P. gossypiella at 150 Gy. This could indicate that T. absoluta is more resistant to radiation than this species. Differences in the sensibility to radiation could be related to quantitative differences in total DNA content and to the developmental stage of the individuals when they are irradiated (Tobías, 1952; Ostergen et al., 1958; Sparrow et al., 1965; Mansour, 2010). On the other hand, the differences between T. absoluta and P. gossypiella could also be due to the type of radiation applied (X-rays or gamma-rays) since both have different energy levels and, as a consequence, affect the chromatin differently (Bakri et al., 2005). The number of F1 larvae and pupae was affected by increasing X-radiation doses. This result suggested that a smaller number of F1 larvae and adults from irradiated males was obtained with higher doses of radiation. One of the advantages of IS in Lepidoptera males is the distortion of the F1 sex ratio in favour of males (Proverbs, 1962; LaChance, 1985; Makee and Saour, 1997). Lepidoptera species show a typical WZ/ZZ (female/male) sex determination system (Suomalainen, 1969; Traut and Marec, 1997; Makee et al., 2008). Marec et al. (1999) suggested that the sex ratio distortion in the F1 generation in Lepidoptera is a result of recessive lethal mutations induced in the Z sex chromosomes of treated
66 parents that are responsible for the death of F1 females. However, in our study it was not observed this bias against females. We notice that Carpenter et al. (2001) (and references therein) also found a lack of F1 sex ratio distortion in C. cactorum . One of the most important attributes of the IS in Lepidoptera is that F1 male and female offspring are more sterile than the irradiated F0 parents (Proverbs, 1962; North, 1975; LaChance, 1985; Carpenter et al., 2005). The results of the present study showed that the F1 fertility was smaller than the F0 fertility. This result and the decrease in the number of larvae and pupae in the F1 and F2 generations with increasing X-radiation doses, suggest that the deleterious effects induced by X-radiation in F0 male were still present in F1 and F2 generations. Taking into account all the studied variables, we suggest that 200 – 250 Gy doses could be used to induce IS in T. absoluta males. Female fertility and fecundity The results of this experiment suggested that full sterility of T. absoluta females might be reached at 200 Gy when they are irradiated as pupae. Moreover, the larvae that descended from females irradiated with 150 Gy failed to reach pupae stage. This is consistent with other Lepidoptera species studied such as C. cactorum and E. kuehniella whose females became completely sterile at a dose of 200 Gy (Carpenter et al., 2001; Ayvaz et al., 2007). Concerning other gelechiid species, full female sterility in P. gossypiella and Phthorimaea operculella (Zeller) was achieved at 150 and 200 Gy, respectively (Henneberry and Clayton, 1988; Makee and Saour, 2003). Description of the karyotype The karyotype of lepidopteran individuals treated with radiation presents chromosomal aberrations (fragmentation and reordered fragments) responsible of the inherited sterility. In order to identify this kind of rearrangements in irradiated T. absoluta males and their descendants, it is indispensable to describe the wild-type karyotype of the species. Among the superfamily Gelechioidea, the chromosome numbers vary between n= 11 and n= 30, with a mode in n= 29 (Lukhtanov and Puplesiene, 1999). The chromosome number described for T. absoluta in the present work adjusts to this mode. Taking into account that the ancestral chromosome number proposed for the order Lepidoptera is n= 31 (Lukhtanov, 2000), it is evident that several chromosomal rearrangements have been involved in the karyotype evolution of Gelechioidea. The presence of two large chromosomes in the karyotype of T. absoluta makes this species a suitable model for further analysis in this topic. The presence of sex chromatin, cytological evidence of the presence of W chromosomes in the interphase nuclei of certain tissues, was observed in seven out of the 15 species of Gelechoidea studied up to now (Traut, 1999). The description of the presence of one body of sex chromatin in T. absoluta cells from Malpighian tubes, indicates that there is one W chromosome in the female complement. Impact of radiation on spermatogenesis The morphology of apyrene and eupyrene spermatozoa varies according to the lepidopteran species under study. They might be homomorphic or heteromorphic, although, in general, the apyrene sperm is shorter, thinner and with higher mithocondrial content than eupyrene sperm (Friedländer, 1997). After spermatogenesis, the spermatozoa form bundles that maintain the heteromorphism observed in the individual spermatozoon. The nuclei of the fertile sperm are grouped and organized (Carpenter et al., 2009) while the infertile sperm contains micronuclei in the tails that will eventually be eliminated (Lai- Fook, 1982a). In T. absoluta notoriously heteromorphic and discernable eupyrene and apyrene bundles were observed. The morphology described above for this species coincides to that proposed by Lai- Fook (1982a) and Friedländer (1997) for other lepidopterans, and is similar to that observed in Cydia
67 pomonella (L.), C. cactorum , Teia anartoides (Walker), Helicoverpa zea (Boddie) and Spodoptera frugiperda (Smith) (Carpenter et al., 2009). The length of the sperm in moths is very diverse, as the length of the eupyrene sperm varies between 110 and 12675 µm, and the length of the apyrene sperm between 106 and 883 µm (Morrow and Gage, 2000). In general, the length of the apyrene sperm is half or one third of that of the eupyrene sperm (Gage and Cook, 1994). The total length of the apyrene and eupyrene bundles of T. absoluta (approximately 200 µm for apyrene and 400-450 µm for eupyrene) are within the range described for Lepidoptera (Morrow and Gage, 2000) The apyrene:eupyrene ratio varies in the different lepidopteran species. While in some the percentage of apyrene sperm is of only 11%, in others it may reach the 99% (Gage and Cook, 1994; Cook and Wedell, 1996, 1999; Marec et al., 1996; Swallow and Wilkinson, 2002). The reported apyrene:eupyrene ratios include: 9.5:1 for Ephestia kuehniella Zeller (Koudelová and Cook, 2001), 7:3 in Bombyx mori (L.) (He et al., 1996; Kawamura and Sahara, 2002), 5:1 in Spodoptera litura (Fabricius) (Etman and Hooper, 1979), 9.6:1 for Manduca sexta (L.) (Silberglied et al., 1984), and 9:1 in Plodia interpunctella (Hübner) (Gage and Cook, 1994). In T. absoluta the apyrene:eupyrene ratio was 1.42:1, being smaller than that observed in other species. It is worth noticing that during this research, the only bundles that were quantified were those with distinctive morphology, meaning that the exclusion of those with dubious structure may cause a sub-estimation of this variable. The detection of eupyrene bundles of deformed morphology can be explained by the lesions that ionizing radiation may cause in the DNA. Even though many repairing mechanisms are triggered, these may be inadequate or insufficient. As a consequence, the cells may die or survive bearing mutations (Brown, 2008). When X-radiation is applied to insects, the most commonly observed effects include nuclear fragmentation, cellular necrosis and chromosomal breaks (Coggins, 1973). As the germinal cells are also sensitive to this type of treatment (Mathur, 1960; Mandl, 1964; Riemann, 1967), an insect may become sterile due to the damages caused to them (Adem et al., 1978; Watters, 1979; Dohino et al., 1994). In the particular case of lepidopteran species, it is well known that the euspermatogenesis is highly sensitive to genetic and experimental manipulation, while paraspermatogenesis is irregular and resistant to them (Friedländer, 1997). Thereby, eupyrene spermatozoa with nuclear rearrangements are expected as a result of irradiation, and have been described in several species (Carpenter et al., 2009). These kind of morphological rearrangements in individual spermatozoa, may lead to the observation of eupyrene sperm bundles of abnormal morphology. The low proportion of this kind of bundles observed in T. absoluta , may be due to the moment at which the specimens were irradiated. The most sensitive moment of euspermatogenesis occurs during the fourth larval instar, when meiosis takes place, afterwards, during the pupal and adult stages, only the production of spermatids continues (Lai-Fook, 1982b). According to the traditional protocol of irradiation applied in the laboratory, all T. absoluta males were irradiated at the pupal stage, meaning that the abnormal eupyrene bundles may have originated from random damage to the DNA of the spermatids that were already produced when the irradiation took place, or, from the damage to the DNA of the few euspermatogonia still present at irradiation, whose alteration was dragged during the whole process of euspermatogenesis. The generation of abnormal euspermatozoa affects the fertility and fecundity of their carriers. In previous reports of other species, a decrease in fertility is observed when the radiation dose to which males are exposed is increased (Carpenter et al., 1986; Henneberry and Clayton, 1988; Carpenter et al., 2001; Nguyen and Nguyen, 2001; Bloem et al., 2003; Ayvaz et al., 2007; Boshra, 2007). This fact was also described for T. absoluta (present report). A significant decrease in the fecundity of wild-type females when crossed by maled irradiated at 300 Gy was observed. A possible cause of this is the decrease in the number of normal eupyrene sperm and the increase in the production of abnormal eupyrene sperm, detected when making preparations from testes.
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The detection of abnormal eupyrene sperm bundles may also be useful for monitoring the release of irradiated individuals in the field. In pest management programs, one of the most important aspects includes monitoring before, during and after the treatment. In the case of SIT, it is essential to be able to differentiate the sterile individuals from the wild ones, in order to evaluate the performance of the former and the impact they have in the population (Vreysen, 2001). Several techniques have been developed to “label” the sterile insects, and so allow the study of their dispersion, abundance and survival (Schellhorn et al., 2004). The most widespread methodologies include the use of dyes and chemicals, as well as the use of genetic markers (Parker, 2005). In many SIT programs, the sterile pupae are dusted with fluorescence powders. In case of dipteran species, during emergence they inflate the ptilinum with hemolymph to beat against the puparium in order to break it (Gállego Berenguer, 2006). The fluorescence powder on the puparium is transferred to the ptilinum, and when this structure is transformed into scar tissue, the powder is incorporated and remains in the head during the whole insect lifespan (Hagler and Jackson, 2001). In case of lepidopteran species, the emergence from the puparium does not involve a ptilinum, but is caused by the secretion of chemical compounds that degrade the puparium. Due to this important difference, the fluorescence powders are not 100% effective, as it is only attached to the scales, but is not incorporated to any body structure, making it less persistent and susceptible of being eliminated together with the scales by action of wind, water and rubbing with other individuals. What is more, this kind of marking procedure has several drawbacks such as high cost, risks to human health, and it is prone to errors as the dust can be transferred from a sterile male to a wild type male (Hagler and Jackson, 2001). The detection of abnormal eupyrene sperm bundles in a statistically significant proportion in all the analyzed doses, and the extremely low prevalence in non-irradiated individuals (only one out of nine individuals showed one abnormal eupyrene sperm bundle), allows to postulate the making of squash preparations from testis as a complementary methodology to identify irradiated individuals in the field. This technique is relatively simple, does not require of sophisticated equipment and gives results almost immediately, so it could complement other traditional methods in small and medium scale projects. Morphology of eupyrene spermatozoa transferred by irradiated males This research is still in progress, but the data obtained up to now indicate that abnormal eupyrene spermatozoa are transferred during copula, and they may explain the decrease in fertility and fecundity observed in irradiated males. Conclusions The information obtained up to now indicates that: • Irradiation doses over 350 Gy are inadecuate for SIT in T. absoluta due to its impact on the morphology of the individuals. • An irradiation dose of 300 Gy alters mating ability of males, making it unsuitable for SIT programas. • A dose of 200 Gy is necessary to generate unfertile females. • The knowledge of the chromosome number of the species will allow the further detection of the chromosomal rearrangements responsible for inherited sterility in the tomato moth. • Doses over 300 Gy significantly alter the apyrene:eupyrene sperm ratio, which is known to be important for sperm motility and fecundity capacity after sperm transference during copula. • Doses over 200 Gy decrease the proportion of normal eupyrene bundles. • Doses over 50 Gy generate abnormal eupyrene bundles.
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• The analysis of the presence of abnormal eupyrene bundles may be used as complementary tool for monitoring the release of irradiated individuals. • Abnormal eupyrene spermatozoa are normally transferred to females during copula. In summary, the most appropriate strategy to control T. absoluta populations would be the release of completely sterile females and partially sterile males with an optimum dose of radiation of 200 Gy. To conclude, this study is the first one to analyze the effects of radiation on T. absoluta , providing a first step to address the viability of implementing IS as a control technique on this species.
NOTE: All the information presented in this report related to “Impact of radiation on biological parameters” was published in: Cagnotti CL, Viscarret MM, Riquelme MB, Botto EN, Carabajal LZ, Segura DF, López SN. 2012. “Effects of X-rays on Tuta absoluta for use in inherited sterility programmes”. J Pest Sci DOI 10.1007/s10340-012-0455-9
References Adem E, Watters FL, Uribe-Rendón R, De la piedad A. 1978. Comparison of Co 60 gamma radiation and accelerated electrons for suppressing emergence of Sitophilus spp. in stored maize. Journal of Stored Products Research 14: 135-142. Agresti A. 1996. An introduction to categorical data analysis. Wiley & Sons, Inc., University of Florida, USA. Apablaza J. 1992. La polilla del tomate y su manejo. Tattersal 79: 12–13. Arthur V. 2004. Use of gamma radiation to control three Lepidopteran pests in Brazil. In Proceedings: Irradiation as a phytosanitary treatment of food and agricultural commodities. Final research coordination meeting organized by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, 2002, Vienna, Austria. Ayvaz A, Albayrak S, Tunçbilek AS. 2007. Inherited sterility in Mediterranean flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae): effect of gamma radiation on insect fecundity, fertility and developmental period. Journal of Stored Products Research 43: 234–239. Bakri A, Mehta K, Lance DR. 2005. Sterilizing insects with ionizing radiation. In: Sterile insect technique. Principles and practice in area-wide integrated pest management. Dyck VA, Hendrichs J, Robinson AS (eds). Springer, Dordrecht. Bloem S, Bloem KA, Carpenter JE, Calkins CO. 1999. Inherited sterility in codling moth (Lepidoptera: Tortricidae): Effect of substerilizing doses ofradiation on insect fecundity, fertility and control. Annals of the Entomological Society of America. 92: 222-229. Bloem S, Carpenter JE, Hofmeyr JH. 2003. Radiation biology and inherited Sterility in false codling moth (Lepidoptera: Tortricidae). Journal of Economic Entomology 96: 1724–1731. Bloem S, Carpenter JE. 2001. Evaluation of population suppression by irradiated Lepidoptera and their progeny. Florida Entomologist 84: 165-171. Boshra SA. 2007. Effect of high-temperature pre irradiation on reproduction and mating competitiveness of male Sitotroga cerealella (Olivier) and their F1 progeny. Journal of Stored Products Research 43: 73-78.
70
Bressa M, Papeschi A, Vítková M, Kubíčková S, Fuková I, Pigozzi M, Marec F. 2009: Sex chromosome evolution in cotton stainers of the genus Dysdercus (Heteroptera: Pyrrhocoridae). Cytogenetic and Genome Research: 125: 292-305. Brown PA. 2008. Genomas. Médica Panamericana. Buenos Aires. Carpenter JE, Bloem S, Bloem KA. 2001. Inherited sterility in Cactoblastis cactorum (Lepidoptera: Pyralidae). Florida Entomologist 84: 537–542. Carpenter JE, Bloem S, Marec F. 2005. Inherited Sterility in Insects. In: Sterile Insect Technique: Principles and Practice in Area-Wide Integrated Pest Management. Dyck VA, Hendrichs J, Robinson AS (eds.). Springer. Dordrecht. Carpenter JE, Bloem S, Marec F. 2005. Inherited sterility in insects. In: Sterile Insect Technique Principles and Practice in Area-Wide Integrated Pest Management. Dyck VA, Hendrichs J, Robinson AS (eds.). Springer, Netherlands. Carpenter JE, Gross HR. 1993. Suppression of feral Helicoverpa zea (Lepidoptera: Noctuidae) populations following the infusion of inherited sterility from released substerile males. Environmental Entomology 22: 1084–1091. Carpenter JE, Marti OG, Wee SL, Suckling DM. 2009. Cytological attributes of sperm bundles unique to F1 progeny of irradiated male Lepidoptera: relevance to Sterile Insect Technique programs. Florida Entomologist 92 (1): 80-86. Carpenter JE, Young JR, Sparks AN, Cromroy HL, Chowdhury MA. 1987. Corn earworm (Lepidoptera: Noctuidae): effects of substerilizing doses of radiation and inherited sterility on reproduction. Journal of Economic Entomology 80: 483-489. Carpenter JE, Young JR, Sparks AN. 1986. Fall armyworm (Lepidoptera: Noctuidae): comparison of inherited deleterious effects in progeny from irradiated males and females. Journal of Economic Entomology 79: 47–49. CIP (International Potato Center). 1996. Insects. Major Potato Diseases, Insects, and Nematodes. Centro Internacional de la Papa. Lima. 87-89. Coggins PB. 1973. The effect of X-radiation on spermatogenesis and the fertility of Schistocerca gregaria (Forsk). Journal of Embryology & Experimental Morphology 30 (1): 163-177. Cook PA, Wedell N. 1996. Ejaculate dynamics in butterflies: a strategy for maximizing fertilization success? Proceedings of the Royal Society B 263: 1047-1051. Cook PA, Wedell N. 1999. Non-fertile sperm delay female remating. Nature 397: 486. Desneux N, Wajnberg E, Wyckhuys KAG, Burgio G, Arpaia S, Narvaez-Vasquez CA, Gonzalez- Cabrera J, Catalan Ruescas D, Tabone E, Frandon J, Pizzol J, Poncet C, Cabello T, Urbaneja A. 2010. Biological invasion of European tomato crops by Tuta absoluta: ecology, geographic expansion and prospects for biological control. Journal of Pest Science 83: 197–215. Dhouibi MH, Abderahmane CT. 2001. The effect of substerilizing doses of gamma radiation on the pupae of the carob moth Ectomyelois ceratoniae (Lepidoptera: Pyralidae). In: Proceedings: Evaluation of population, suppression by irradiated Lepidoptera and their progeny, FAO/IAEA Final Research Coordination Meeting, 1998. Penang, Malaysia. Dohino T, Tanabe K, Hayashi T. 1994. Comparison of Lethal Effects of Electron Beams and Gamma Rays on Eggs of Two spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). Research Bulletin of the plant Protection Service, Japan 30: 69-73.
71
EPPO (European and Mediterranean Plant Protection Organization). 2010. EPPO Reporting service. Pest & Diseases 1. Paris. 01-01-2010. Etman AAM, Hooper GHS. 1979. Developmental and reproductive biology of Spodoptera litura (Lepidopetra: Noctuidae). Journal of the Australian Entomological Society 18: 363-327. Friedländer M. 1997. Control of the eupyrene-apyrene sperm dimorphism in the Lepidoptera. Journal of Insect Physiology 43: 1085-1092. Gage MJG, Cook PA. 1994. Sperm size and numbers? Effects of nutritional stress upon eupyrene and apyrene sperm production strategies in the moth Plodia interpunctella (Lepidoptera, Pyralidae). Functional Ecology 8: 594-599. Galarza J. 1984. Laboratory assessment of some solanaceous plants as possible food plants of the tomato moth Scrobipalpula absoluta. IDIA 421/424: 30–32. Gállego Berenguer J. 2006. Manual de parasitología: morfología y biología de los parásitos de interés sanitario. Ediciones Universitat de Barcelona. Barcelona. Garcia MF, Espul JC. 1982. Bioecología de la polilla del tomate (Scrobipalpula absoluta) en Mendoza, República Argentina. Revista de Investigaciones Agropecuarias INTA 17(2): 135-145. Glaser RW. 1917: Ringer solutions and some notes on the physiological basis of their ionic composition. Comparative Physiology and Biochemistry 2: 241-289. Guedes RNC, Picanco M, Matioli A, Rocha DM. 1994. Efeito de insecticidas e sistemas de condução do tomateiro no controle de Scrobipalpuloides absoluta (Meyrick) (Lepidoptera: Gelechiidae). Anais da Sociedade Entomológica do Brasil 23: 321-325. Hagler JR, Jackson CG. 2001. Methods for marking insects: current techniques and future prospects. Annual Review of Entomology 46: 511-543. He L, Nakagaki M, Kajiura Z, Takei R. 1996. Influence of low temperature during eggincubation on spermatogenesis of the silkworm, Bombyx mori. Journal of Sericultural Science of Japan 65: 467-471. Henneberry TJ, Clayton TE. 1988. Effects of gamma radiation on pink bollworm (Lepidoptera: Gelechiidae) pupae: adult emergence, reproduction, mating, and longevity of emerged adults and their F1 progeny. Journal of Economic Etomology 81: 322-326. Kawamura N, Sahara K. 2002. In vitro cultivation of spermatocysts to mature sperm in the silkworm Bombyx mori. Development, Growth and Differentiation 44: 273-280. Kılıç T. 2010. First record of Tuta absoluta in Turkey. Phytoparasitica 38(3): 243-244. Knipling EF. 1979. The basic principles of insect population suppression and management, agricultural handbook, 512. USDA, Washington, DC. Koudelová J, Cook PA. 2001. Effect of gamma radiation and sex-linked recessive lethal mutations on sperm transfer in Ephestia kuehniella (lepidoptera: pyralidae). Florida Entomologist 84 (2): 172-182. La Chance LE. 1985. Genetic methods for the control of lepidopteran species: status and potential. USDA, ARS-28. LaChance LE. 1967. The induction of dominant lethal mutations in insects by ionizing radiation and chemicals – as related to the sterile-male technique of insect control. In: Genetics of Insect vectors of disease. Wright JW, Pal R (eds.). Elsevier, Amsterdam. LaChance LE. 1985. Genetic methods for the control of the lepidopteran species: status and potential. USDA, Washington, DC.
72
Lai-Fook J. 1982a. Structural comparison between eupyrene and apyrene spermiogenesis in Calpodes ethlius (Hesperiidae, Lepidoptera). Canadian Journal of Zoology 60(6): 1216-1230. Lai-Fook J. 1982b. Testicular development and spermatogenesis in Calpodes ethlius Stoll (Hesperiidae, Lepidoptera). Canadian Journal of Zoology 60(6): 1161-1171. Larraín P. 1986. Eficacia de insecticidas y frecuencia de aplicación basada en niveles poblacionales críticos de Scrobipalpula absoluta (Meyrick), en tomates. Agricultura Técnica 46: 329-333. Lietti MM., Botto E, Alsogaray RA. 2005. Insecticide resistance in Argentine populations of Tuta absoluta (Meyrick) (Lepidoptera: Gelechidae). Neotropical Entomology 34: 113-119. Lockwood APM. 1961: ‘Ringer’ solutions and some notes on the physiological basis of their ionic composition. Comparative Physiology and Biochemistry 2: 241–289. Lopez E. 1991. Polilla del tomate: Problema crítico para la rentabilidad del cultivo de verano. Empresa y Avance Agrícola 1: 6–7. Lukhtanov V, Puplesiene J. 1999. Polyploidy in bisexual Lepidoptera species (Insecta: Lepidoptera): old hypotheses and new data. Bonner Zoologische Beiträge 48(3-4): S313-328. Lukhtanov VA. 2000. Sex chromatin and sex chromosome systems in nonditrysian Lepidoptera (Insecta) Journal of Zoological Systematics and Evolutionary Research 27: 62-68. Makee H, Saour G. 1997. Inherited effects in F1 progeny of partially sterile male Pthorimaea operculella (Lepidoptera: Gelechiidae). Journal of Economic Entomology 90: 1097-1101. Makee H, Saour G. 2003. Noninherited sterility in irradiated Pthorimaea operculella females. Journal of Applied Entomology 127: 489-493. Makee H, Tafesh N, Marec F. 2008. Analysis of radiation-induced W chromosome aberrations in the codling moth, Cydia pomonella (L.), by Fluorescence in situ hybridization techniques. Journal of Pest Science 81: 143–151. Mallea AR, Macola GS, Garcia SJG, Bahamondes LA, Suarez JH. 1972. Nicotiana tabacum var. virginica, a new host of Scrobipalpula absoluta. Revista de la Facultad de Ciencias Agrarias, Universidad Nacional de Cuyo 18: 13–15. Mandl AM. 1964. The radiosensitivity of germ cells. Biological Reviews of the Cambridge Philosophical Society 39: 288-371. Mansour M. 2010. Effects of gamma radiation on the Mediterranean flour moth, Ephestia kuehniella, eggs and acceptability of irradiated eggs by Trichogramma cacoeciae females. Journal of Pest Science 83: 243–249. Marec F, Kollárová I, Pavelka J. 1999. Radiation- induced inherited sterility combined with a genetic sexing system in Ephestia kuehniella (Lepidoptera: Pyralidae). Annals of the Entomological Society of America 92: 250–259. Marec F, Pavelka J, Koudelová J, Kìivan V, Cook PA. 1996. Suppression of model populations of Ephestia kuehniella by releasing males balanced for two sex-linked recessive lethal mutations. In: Working Material, First FAO/IAEA Research Coordination Meeting, Evaluation of Population Suppression by Irradiated Lepidoptera and Their Progeny. Jakarta. Indonesia, 24-28 April 1995. International Atomic Energy Agency, Vienna. Mastrangelo T, Parker AG, Jessup A, Pereira R, Orozco-Dávila D, Islam A, Dammalage T, Walder JMM. 2010. A new generation of X ray irradiators for insect sterilization. Journal of Economic Entomology 103: 85–94.
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Mastro VC. 1993. Gypsy-moth F1 sterility program: current status. In: Proceedings, Symposium: Radiation induced F1 sterility in Lepidoptera for area wide control. FAO/IAEA, 9-13 September 1991, Arizona. Mathur RS. 1960. The effect of X-radiation on the spermatogenesis of Petrobius maritimus. Proceedings of the Royal Irish Academy, Section B 61: 275-281. Mehta K, Parker A. 2011. Characterization and dosimetry of a practical X-ray alternative to self- shielded gamma irradiators. Radiation Physics and Chemistry 80: 107–113. Morrow EH, Gage MJG. 2000. The evolution of sperm length in moths. Proceedings of the Royal Society of London Series B 267: 307–313. Nguyen TQH, Nguyen TTT. 2001. Radiation-induced F1 sterility in Plutella xylostella (Lepidoptera: Plutellidae): potential for population suppression in the field. Florida Entomologist 84: 199-208. North DT, Holt GG. 1969. Population suppression by transmition of inherited sterility to progeny of irradiated cabbage looper, Trichoplusia ni. Canadian Entomologist 101: 513-520. North DT, Holt GG. 1971. Inherited sterility and its use in population suppression of Lepidoptera. In: Proceedings of the symposium application of induced sterility for control of lepidoptera populations. FAO/IAEA, Viena, 1970. North DT. 1975. Inherited sterility in Lepidoptera. Annual Review of Entomology 20: 167-182. Notz AP. 1992. Distribution of eggs and larvae of Scrobipalpula absoluta in potato plants. Revista de la Facultad de Agronomía (Maracay) 18: 425–432. Ostergen G, Morris R, Wakoning T. 1958. A study in Hyacinthus on chromosome size and breakability by X-rays. Hereditas 44: 1-17. Parker AG. 2005. Mass-rearing for sterile insect release. In: Sterile Insect Technique. Principles and Practice in Area-Wide Integrated Pest Management. Dyck VA, Hendrichs J, Robinson AS (eds.). Springer. Dordrecht. Pereyra PC, Sánchez NE. 2006. Effect of two Solanaceous plants on developmental and population parameters of the tomato leaf miner, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae). Neotropical Entomology35: 671-676. Poggio MG, Bressa MJ, Papeschi AG. 2011. Male meiosis, heterochromatin characterization and chromosomal location of rDNA in Microtomus lunifer (Berg, 1900) (Hemiptera: Reduviidae: Hammacerinae). Comparative Cytogenetics 5(1): 1–22. Potting R, Jan van der Gaag D, Loomans A, van der Straten M, Anderson H, MacLeod A, Castrillón JMG, Cambra GV. 2009. Tuta absoluta, Tomato leaf miner moth or South American tomato moth. Ministry of Agriculture, Nature and Food Quality (LVN) Plant Protection Service of the Netherlands. Available online: http://www.minlnv.nl/cdlpub/servlet/CDLServlet?p_file_id=42402. Proverbs MD, Newton JR, Logan DM. 1978. Suppression of codling moth, Laspeyresia pomonella (Lepidoptera: Olethreutidae), by release of sterile and partially sterile moths. Canadian Entomology 110: 1095-1102. Proverbs MD. 1962. Progress on the use of induced sexual sterility for the control of the codling moth Carpocapsa pomonella (L.) (Lepidoptera: Olethreutidae). Proceedings of the Entomological Society of Ontario 92: 5-11. Riemann JG. 1967. A cytological study of radiation effects in testes of the screwworm fly, Cochliomyia hominivorax. Annals of the Entomological Society of America 60: 308-320.
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Roditakis E, Papachristos D, Roditakis NE. 2010. Current status of the tomato leafminer Tuta absoluta in Greece. OEPP/EPPO Bulletin 40: 163–166. Salazar ER, Araya JE. 1997. Detección de resistencia a insecticidas en la polilla del tomate. Simiente 67: 8-22. Schellhorn NA, Siekmann G, Paull C, Furness G, Baker G. 2004. The use of dyes to mark populations of beneficial insects in the field. International Journal of Pest Management 50: 153-159. Silberglied RE, Shepherd JG, Dickinson JL. 1984. Eunuchs: the role of apyrene sperms in Lepidoptera?. American Naturalist 123: 255-265. Siqueira de HA, Guedes RN, Picanço MC. 2000. Insecticide resistance in populations of Tuta absoluta (Lepidoptera:Gelechiidae). Agricultural and Forest Entomology 2: 147-153. Sparrow AH, Sparrow RC, Thompson KH, Schairer LA. 1965. The use of nuclear and chromosomal variables in determining and predicting radiosensitivities. Radiation Botany 5: 101-132. StatSoft. 2000. Statistica for Windows. Computer program manual. StatSoft Inc., Tulsa, USA. Suinaga FA, Dias Casali VW, Picanço M, Foster J. 2004. Genetic divergence among tomato leafminer populations based on AFLP analysis. Pesquisa Agropecuária Brasileira 39: 645-651. Suomalainen E. 1969. On the sex chromosome trivalent in some Lepidoptera females. Chromosoma 28: 298-308. Sutrisno S, Hoedaya MS, Sutardi D, Rahayu A. 1993. Radiation induced F1 sterility in diamondback moth, Plutella xylostella (L.), and tropical armyworm, Spodoptera lituria F. In: Proceedings of the symposium Radiation induced F1 sterility in Lepidoptera for area wide control. FAO/IAEA, 9–13 September 1991, Arizona. Swallow JG, Wilkinson GS. 2002. The long and short sperm polymorphism in insects. Biological Reviews 77:153–182. Tobías CA. 1952. The dependence of some biological effects of radiation on the rate of energy loss. In: Symposium on Radiobiology. J. Wiley & Son. NewYork. Traut W, Marec F. 1997. Sex chromosome differentiation in some species of Lepidoptera (Insecta). Chromosome Research 5: 283-29. Traut W. 1976: Pachytene mapping in the female silkworm Bombyx mori L. (Lepidoptera). Chromosoma 58: 275-284. Traut W. 1999. The evolution of sex chromosomes in insects: Differentiation of sex chromosomesin flies andmoths. European Journal of Entomology 96: 227-235. Urbaneja A, Vercher R, Navarro V, Garcıa Marı F, Porcuna JL. 2007. La polilla del tomate, Tuta absoluta. Phytoma-España 194: 16–23. Vreysen MJB. 2001. Principles of area-wide integrated tsetse fly control using the Sterile insect Technique. Médecine Tropicale 61: 397-411. Watters FL. 1979. Potential of accelerated electrons for insect control in stored grain. In: Proceedings: Second International Working Conference on Stored-Product Entomology. September 1978. Ibadan.
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Enhanced production and quality control of mass produced codling moth in South Africa M F Addison Department of Conservation Ecology and Entomology, Faculty of AgriSciences, Stellenbosch University, Matieland, South Africa Abstract The enhanced production and quality control of mass produced codling moth in South Africa is seen as critical in the development of a viable Sterile Insect Release (SIR) programme. The field performance of released moths under variable temperatures has been assessed. The phenotypic plasticity of codling moth has been demonstrated. Research on the morphological, physiological and genetic attributes of moths is being investigated. The application of thermal conditioning in large scale moth production is underway. The use of remote sensing equipment in the field has been investigated and suitable technologies identified.
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Comparison of laboratory and field bioassays of lab-reared Cydia pomonella (Lepidoptera: Tortricidae) quality and field performance J.E. Carpenter 1, T. Blomefield 2 and S.D. Hight 3 1. USDA-ARS, Crop Protection and Management Research Unit, Tifton, Georgia, USA, 2. Agricultural Research Council, Stellenbosch, South Africa, 3. USDA-ARS-CMAVE Center for Biological Control, FAMU, Tallahassee, Florida, USA Abstract Maximum production and fitness of insect species that are mass-reared for biological control programs such as the sterile insect technique (SIT) have benefitted from the employment of quality control and quality management. With a growing interest in the use of SIT as a tactic for the suppression/eradication of key lepidopteran pests, such as the codling moth, Cydia pomonella L. (Lepidoptera: Tortricidae), there is a parallel interest in inexpensive bioassays that can accurately detect differences in insect quality and monitor insect field performance. In this study we examined laboratory (mating and flight ability) bioassays and field (field cage and open field release) bioassays simultaneously to discern the ability of the different bioassays to predict quality and field performance of codling moths produced in a commercial mass-rearing facility. Moth quality was degraded by different levels of radiation during the sterilization procedure. Both the laboratory flight bioassay and the field cage bioassay successfully detected quality and performance differences that were relevant to moth performance in the field. However, the study data suggest that the field cage bioassay was a better predictor of the daily performance of males that had been released in the orchard than the laboratory flight bioassay. Conversely, data suggest that the controlled climatic conditions of the laboratory allowed the flight cylinder bioassay to be more sensitive in detecting daily fluctuations in the quality of moths caused by factors within the mass rearing facility. Therefore, both laboratory and field bioassays may be required to provide feedback on quality and performance of mass-reared moths in an SIT program. Key Words Quality control, sterile insect technique, flight ability, Cydia pomonella
Introduction Successful research, operational, and commercial programs which rely on mass-reared insects require reliable production and delivery of insects of high quality and performance (Calkins and Parker 2005). Accurate methods for monitoring potential quality degradation during each step of production, handling and release are crucial to insure quality control and success of these programmes (Huettel 1976; Singh and Ashby 1985; Calkins and Parker 2005). As there is a continued and growing interest in the use of the sterile insect technique (SIT) as a tactic for the suppression/eradication of key lepidopteran pests, such as the codling moth, Cydia pomonella L. (Lepidoptera: Tortricidae) (Addison and Henrico 2005; Bloem et al. 2005; Carpenter et al. 2005; Simmons et al. 2010; Vreysen et al. 2010), the establishment of simple and inexpensive bioassays that can detect differences in insect quality and monitor field performance is essential. Moth mobility, flight propensity and flight ability are elemental to the execution of more complex behaviours and abilities such as mating and sperm transfer, response of males to calling females or pheromone traps, dispersal, adult longevity, and infusion of sterility in the wild population (Calkins and Parker 2005; Vreysen 2005; Simmons et al. 2010). Therefore, bioassays for mobility, flight propensity and flight ability should be fundamental to monitoring the performance of sterile moths released in the field (Carpenter et al. 2012). Actographs (Bloem et al. 2006a, 2006b; Keil et al. 2001; Gu et al. 2006),
77 flight mills (Huettel 1976; Schumacher et al. 1997), and flight cylinders (Carpenter et al. 2012) have been used successfully to examine factors that affect moth mobility, flight propensity and flight ability. Other bioassays such as wind tunnels (Suckling et al. 2007), and release/recapture of marked adults (Butt et al. 1970; Bloem et al. 1998, 2004) have examined moth ability to perceive and respond to semiochemicals that induce flight. However, many of these bioassays are comparatively complicated requiring advanced expertise, specialized equipment, have a low throughput which limits sample size, and have limited portability between the field and laboratory. Also, the results from laboratory bioassays are seldom linked directly to corresponding behaviour or performance in the field. Additional studies are needed to evaluate the ability of simple laboratory bioassays to predict the quality and performance of codling moths released in the field. Therefore, we examined laboratory and field bioassays simultaneously to discern the ability of the different bioassays to predict quality and field performance of codling moths produced in a commercial mass-rearing facility. For this study conducted in November 2010, codling moth quality was degraded by exposing moths to several different doses of radiation (Bloem et al. 2004, Carpenter et al. 2010). Materials and Methods Test Insects and Handling . Codling moths were obtained from the Entomon Technologies (Pty) Ltd mass-rearing insectary in Stellenbosch, South Africa. This codling moth colony was initially established in 2002 from infested thinning apples collected from commercial and organic orchards in the Elgin apple production area. Wild codling moths collected from conventional orchards in Elgin were introduced into the laboratory colony during 2007, 2008 and 2009. The colony has been reared on an adaptation of the Guennelon et al. (1981) diet according to the methods of Stenekamp (2011). In the mass-rearing facility, moths emerge from open trays of diet and are attracted to a black light where they are collected by a wind trap and deposited into a cold room (2-6oC) similar to the methods of Dyck (2010). Male moths used in the bioassays were obtained from Entomon after they had been collected and placed in cold storage. Female moths used in the bioassays were collected immediately after they emerged from diet trays to ensure that they were virgin. Moth irradiation was conducted using a panoramic Cobalt60 point source surrounded by a turntable that was 1 m in diameter. Treatment samples were placed on one or more of eight smaller turntables, each 200 mm in diameter and situated equidistant on the periphery of the main turntable. The smaller turntables counter rotated to enable 360° treatment of the treatment samples. The Cobalt60 source delivered a dose rate of 2.38 -2.41 Gy/min during the time of the trials. The operational dose of radiation for the Entomon SIT program (Addison and Henrico 2005) is 150 Gy. Radiation dose treatments for all bioassays in this study
were 0, 150, 300, and Figure 1. Temperatures ( oC, median ± maximum-minimum) recorded at sunset, sunset 450 Gy. Male moths + 1 h, and sunset + 2 h ( 18:00, 19:00 and 20:00), the time of day that Cydia pomonella from each radiation would be initiating and engaged in pheromone-mediated flight. Temperatures were treatment were recorded during November 2010 at the University of Stellenbosch research farm distinguished by lightly during the period bioassays were being conducted.
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dusting with colored fluorescent powder. Bioassays . Laboratory bioassays were conducted at the Agricultural Research Council, Infruitec- Nietvoorbij, Plant Protection laboratories in Stellenbosch, South Africa, in environmentally controlled rooms [27°C ± 0.5°C, 18:06 (L:D) photoperiod, 70-75 % RH]. Field bioassays were conducted at the University of Stellenbosch research farm. Laboratory bioassays measured the ability/propensity of moths to mate while in small cages and the ability/propensity of male moths to fly from a flight cylinder. Field bioassays measured the ability of male moths released in a field cage and released in an apple orchard to engage in pheromone-mediated flight and locate a pheromone source (i.e. to be captured in a pheromone-baited trap). Temperatures were recorded at sunset, sunset + 1 h, and sunset + 2 h ( 18:00, 19:00 and 20:00), the time of day that codling moths initiated and engaged in pheromone- mediated flight (Ref?) (Fig. 1). Each day that releases were made in the field cage and apple orchard, laboratory bioassays were conducted using moths from the same cohort that were released in the field cage and the apple orchard. Releases were made on November 12, 15, 16, 17, and 22 for a total of five releases. Laboratory bioassays were conducted on the same dates as field bioassays, and also on November 19. Mating Cage . Mating ability/propensity was examined by placing 10 virgin females and 10 males in plastic mesh cages (30cm × 30cm × 30cm) for 24 h. All females were dissected and mating was determined by the presence of a spermatophore in the bursa copulatrix (Ferro and Akre 1975). For each test date, two cages (replications) were set up for each of the four radiation treatments (0, 150, 300, and 450 Gy). Flight Cylinders . Flight cylinders (Carpenter et al. 2012) were produced by cutting PVC irrigation pipe (color = dark gray; diameter = 16 cm) into heights of 16 cm. Cylinders were placed on tables in a sealed room and the inside surface of the cylinders was coated with talc to ensure that the moths used flight to escape the cylinders. Chilled (2°C) male moths (n=10) from each radiation treatment were placed inside a single flight cylinder, the cylinder determined randomly. Moths remaining in each cylinder after a period of 20 h were counted and removed from the cylinders. For each test date, there were four replications, each replicate on a separate table. Each replication had four cylinders, one cylinder for each of the four radiation treatments (0, 150, 300, and 450 Gy). Open Field Release/Recapture . Each release of male moths was made at a central point in an apple orchard block (60m × 100m) in the University of Stellenbosch research farm. Twelve Delta traps baited with codling moth pheromone lures were positioned in the orchard block. Six of the traps were placed in a circle around the release point, each trap 8 – 9 m from the release. Two traps were placed on the same row as the release, with each trap 22.5 m from the release in opposite directions. Four traps were placed 38 m from the release, with one trap near each corner of the orchard block. The number and type of male moths captured in each trap were recorded on November 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, and 26. Field Cage . A circular screen field cage (3m diameter × 2m height) was erected within an open area in an apple orchard on the University of Stellenbosch research farm. The cage was 60m from the edge of the “release” orchard block. A pheromone baited sticky trap was positioned in the center of the cage at a height of 2m. For each release made in the orchard, 20 male moths from each radiation treatment were placed together in the cage. The trap in the cage was checked according to the same schedule as the traps in the apple orchard. The number of males captured from each treatment was recorded. Moths that were not captured were left in the cage to be congruent with the moths that were not captured in the apple orchard. Data Analysis . Data from the mating cage study were analyzed using multi-factor analysis of variance (PROC GLM) (SAS Institute 1989) with the mean number of female moths that had mated as the dependant variable and date, replication and radiation exposure as sources of variation. The data from
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the flight cylinder study were analyzed using multi-factor analysis of variance (PROC GLM) (SAS Institute 1989) with the mean number of moths that flew from the cylinders as the dependant variable and date, replication and radiation dose as sources of variation. Data from the field cage study and the orchard study were analyzed using multi-factor analysis of variance (PROC GLM) (SAS Institute 1989) with the mean number of moths captured as the dependant variable and date and radiation dose as sources of variation.
The number of males captured from Figure 2. The mean number of Cydia pomonella male moths of the each radiation treatment was expressed initial 10 male moths that flew from the flight cylinders on each day as a percentage of the total moths the bioassay was conducted. Means with the same lower case letter captured each day and analysed as a are not significantly different ( P = 0.05). Vertical bars denote 0.95 dependant variable (PROC GLM) (SAS confidence intervals. Institute 1989), and we compared the rank (PROC RANK) (SAS Institute 1989) of the number of males captured each day from each treatment. Data from the orchard study also were analyzed using multi-factor analysis of variance (PROC GLM) (SAS Institute 1989) with the mean distance recaptured from the release site as the dependant variable and date and radiation dose as sources of variation. For each analysis that indicated a significant effect due to radiation dose, the relationship between dose and the dependant variable was examined with regression analysis (PROC GLM) (SAS Institute 1989). All interactions were included in the statistical models to test the null hypotheses of independent effects of the different sources of variation. For each analysis that indicated a significant effect due to day, means were separated by the Tukey-Kramer statistic at P = 0.05 (SAS Institute 1989). 7.5 Results
7.0 The mean (± S.D.) number of females (9.04 ± 0.9) (n=10) 6.5 mating in the mating cage study
6.0 was not affected by the day or the radiation dose, and there 5.5 was no interaction between day and radiation dose. Analysis of 5.0 data from the flight cylinder 4.5 bioassay revealed a significant main effect due to day and 4.0
Mean Moths that Flew from Cylinders from Flew that Moths Mean radiation dose, however, no
3.5 interaction between day and 0 150 300 450 radiation dose was detected. Radiation Dose (Gy) Significantly (F = 7.025; d.f. = Figure 3. Effect of radiation dose administered to Cydia pomonella adults on 5, 90; P = 0.00001) (Fig. 2) the mean number of male moths of the initial 10 male moths that flew from the more moths flew from the flight flight cylinders. Vertical bars denote 0.95 confidence intervals . cylinders when the bioassays
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were conducted on November 15 than when bioassays were conducted on November 12, 17 and 19, and significantly fewer moths flew from the flight cylinders when the bioassays were conducted on November 12 than when bioassays were conducted on November 15, 16 and 22. The number of male moths that flew from the flight cylinders was significantly reduced as the dose of radiation increased (Fig. 3) (y = 5.983 – 0.3098x; F =
9.977; d.f. = 3, 92; P = 0.00213). Figure 4 . The mean number of Cydia pomonella male moths captured each The mean number of male moths day in a pheromone trap located in the center of a field cage. Eighty male moths (20 moths for each of four radiation treatments) were released in the recaptured in the field cage varied field cage on November 12, 15, 16, 17, and 22. Means with the same lower significantly by day (F = 10.707; case letter are not significantly different ( P = 0.05). Vertical bars denote d.f. = 9, 30; P <0.0001) (Fig. 4), 0.95 confidence intervals . with the highest number of males recaptured on November 17, 18, and 25. Therefore, we compared the number of males captured from each radiation treatment as a percentage of the total moths captured each day, and we compared the rank of the number of males captured each day from each treatment. Radiation dose had a significant effect on the rank of the number of males captured each day (X2 = 8.401; d.f. = 3; P = 0.0384). There also was a significant relationship between the percentage of the total moths captured each day and the dose of radiation (y = 37.24 – 0.447x; F = 9.505; d.f. = 1, 38; P = 0.00381) (Fig. 5). Generally, males that were exposed to lower doses of radiation were more likely to be recaptured. No interaction between day and radiation dose was detected. Results from the orchard bioassay were similar to the results from the field cage bioassay. The mean number of male moths recaptured in the orchard varied significantly by day (F = 26.393; d.f. = 8, 27; P <0.0001) (Fig. 6), with the highest number of males recaptured on November 17 and 18, followed by November 15, 16 and 25. Therefore, we compared the number of males captured from each radiation treatment as a percentage of the total moths captured each day, and we compared the rank of the number of males captured each day from each treatment. Radiation dose had a significant effect on the Figure 5. Field cage bioassay demonstrating the effect of rank of the number of males captured each radiation dose administered to Cydia pomonella adults on the day (X2 = 8.359; d.f. = 3; P = 0.0391). There mean percentage of the total number of moths recaptured in a also was a significant relationship between pheromone trap each day. Eighty male moths (20 moths for the percentage of the total moths captured each of four radiation treatments) were released in the field cage each day and the dose of radiation (y = 30.07 on November 12, 15, 16, 17, and 22. Vertical bars denote 0.95 – 0.456x; F = 8.919; d.f. = 1, 34; P = 0.0052) confidence intervals.
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(Fig. 7). As with the field cage, males that were exposed to lower doses of radiation were more likely to be recaptured. The mean distance males were recaptured from the release site varied significantly by day (F = 3.065; d.f. = 8, 26; P = 0.01435), however, this dispersal was not influenced by the dose of radiation male moths received before release in the orchard. No interaction between day and radiation dose was detected for the number of males recaptured or the distance males were captured from the release site. Figure 6 . The mean number of Cydia pomonella male moths Discussion from four radiation treatments recaptured each day in pheromone traps located in an apple orchard. A total of 1115, 680, 1040, The benefit of quality control and quality 1122, and 1233 moths were released in the apple orchard on management to maximizing production November 12, 15, 16, 17, and 22, respectively. Means with the and fitness of insect species that are same lower case letter are not significantly different ( P = 0.05). mass-reared for release in area-wide Vertical bars denote 0.95 confidence intervals. autocidal control programs has been a topic of discussion for decades (Boller et al. 1981; Burt 2002; Calkins and Parker 2005). An integral element of this discussion has been the need for effective quality assurance tests for monitoring and providing feedback on the performance of insects during each step of production, handling and release to insure quality control and success of these programs (Huettel 1976; Singh and Ashby 1985; Calkins and Parker 2005). Initially, most assessment of sterile male quality was conducted within the mass-rearing facility, but more attention has recently been given to routine assessments of the performance and competitiveness of released sterile insects in the field (Vreysen 2005; Simmons et al. 2010). The use of quality assessments and standards has advanced in parallel with the extensive use of the SIT to control/eradicate fruit flies and the development of international trade in 40 sterile fruit flies (FAO/IAEA/USDA 35 2003).
30 Interest to include the SIT as a tactic for the control/eradication of key lepidopteran 25 pests such as the codling moth has been
20 increasing (Addison and Henrico 2005; Bloem et al. 2005; Carpenter et al. 2005; 15 Simmons et al. 2010; Vreysen et al. 2010).
10 The codling moth has become established Mean Percentage of Daily Capture Daily Percentage Mean of throughout most of the apple producing 5 0 150 300 450 areas of the world, however, there is a low Radiation Dose (Gy) level of genetic diversity among codling moth populations from different Figure 7. Effect of radiation dose administered to Cydia geographical origins (Pashley and Bush pomonella adults on the mean percentage of the total number of 1979, Buès and Toubon 1992, Buès et al. moths recaptured each day in pheromone traps located in an apple 1995, Timm et al. 2006) and no mating orchard. A total of 1115, 680, 1040, 1122, and 1233 moths were released in the apple orchard on November 12, 15, 16, 17, and 22, barriers have been detected between respectively. Vertical bars denote 0.95 confidence intervals . codling moths collected from five
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different continents (Robinson and Proverbs 1973; Bloem et al. 2010; Taret et al. 2010). The ability to use commercial freight routes for inter-continental transport of moths and pupae (Blomefield et al. 2011) underscores both the opportunity for international trade of sterile codling moths and the need for the development of international quality control standards. Monitoring the quality of mass-reared codling moths for use in a SIT program is essential to evaluate how well the insect performs in both the rearing facility (for insect production) and the field (for mating with the wild insects). Bioassays to monitor moth performance in the rearing facility typically include life history parameters such as pupal weight, developmental time, survival rate of the different developmental stages, fecundity, fertility, percentage mating and longevity. Bioassays to monitor moth performance in the field are more difficult and expensive to conduct (Stewart 1984) but may include mating ability and sperm transfer, response of males to calling females or pheromone traps, mobility, flight ability, dispersal, adult longevity, and infusion of sterility in the wild population (Calkins and Parker 2005; Vreysen 2005; Simmons et al. 2010). Each of these parameters measures different components of the field competitiveness of irradiated insects and is comprised of both behaviour and ability traits. Bioassays using flight cylinders are inexpensive, portable, and low-tech, they provide rapid feedback of data under controlled conditions, and they have been used successfully in assessing fitness and establishing standards for Tephritid flies (FAO/IAEA/USDA 2003). Carpenter et al. (2012) initiated the development of a flight ability test for codling moths to test the hypothesis that this simple bioassay could be effectively used to detect differences in moth quality. In these studies the flight cylinder bioassay was successful in detecting treatment differences in codling moth quality that had been identified previously by more complex laboratory bioassays and field trials. Therefore, they suggested that the flight cylinder bioassay might be a useful and effective quality assurance test for monitoring and providing feedback on the performance of codling moths during the production, handling and release phases of control programs that include an SIT component. For this study, we selected two laboratory bioassays that evaluated separate behaviours and abilities, and we selected two field bioassays that evaluated similar behaviours and abilities, but on different spatial scales. In the mating cage bioassay, males were evaluated for their ability to perceive the female sex pheromone, their ability/propensity to locate the calling female, and their ability to copulate with the female and elaborate a spermatophore. Because the size of the cage was small, male moths could be successful without taking flight. In the flight cylinder bioassay, males were evaluated for their ability/propensity to fly from the cylinder. Because only male moths were in the bioassay room, flight from the cylinders was not influenced by the ability of males to perceive the sex pheromone or the propensity of the males to respond to the pheromone. Both the field cage bioassay and the release/recapture bioassay in the orchard evaluated the ability of males to perceive the sex pheromone, ability/propensity to initiate directed flight, and the ability to engage in pheromone-mediated directed flight to locate the source of pheromone. Major differences between the two field bioassays were the spatial scale, the greater risk of moth predation in the orchard, attraction to wild females, and potential for movement of released moths out of the trapping zone in the orchard. Bioassays involving males released in laboratory flight cylinders, in field cages and in an orchard each detected the quality degradation caused by the radiation exposure, however, the mating cage bioassay did not detect quality differences. Also, no quality or performance differences were detected by comparing the mean distance males were recaptured from the release site in the orchard. These data suggest that the radiation treatment degraded the ability/propensity of the male moths to fly as opposed to the ability to detect and respond to the female sex pheromone. The daily capture of males in the orchard was significantly correlated with the daily capture of males in the field cage (r = 0.7550; n=11; P = 0.007), however, the number of males that flew from the flight cylinders was not correlated with the number of males captured in the field cage or the orchard. When the temperatures at sunset and early evening were low (< 20oC) (Fig. 1), the capture of males in the orchard (Fig. 6) and the field cage (Fig.
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4) also was low. It is likely that the influence of meteorological factors contributed to the field cage bioassay being a better predictor of the daily performance of males that had been released in the orchard than the indoor flight cylinder bioassay. Conversely, it is likely that the controlled climatic conditions of the laboratory allows the flight cylinder bioassay to be more sensitive in detecting daily fluctuations in the quality of moths caused by factors within the mass rearing facility. Therefore, both laboratory and field bioassays are required to provide feedback on quality and performance of mass-reared moths in a SIT program. In this study where quality degradation was caused by increasing doses of radiation, the flight cylinder and field cage bioassays successfully detected quality and performance differences that were relevant to moth performance in the field. Acknowledgements We would like to thank ARC Infruitec-Nietvoorbij for providing laboratory and cold room facilities, Entomon Technologies (Pty) Ltd for providing the codling moths, the University of Stellenbosch for providing the orchards, and Muriel Knipe and Niël DuPlessis for invaluable technical assistance. Thanks to the Deciduous Fruit Producers Trust, ARC Infruitec-Nietvoorbij and the International Atomic Energy Agency, Vienna, for providing funds to support the research. References Addison MF, and Henrico D, 2005. A pilot sterile insect release programme to suppress codling moth in South African apple and pear orchards, pp. 141-142. In: Book of Extended Synopses. FAO/IAEA International Conference on Area-Wide Control of Insect Pests: Integrating the Sterile Insect and Related Nuclear and Other Techniques, 9-13 May 2005, Vienna, Austria. IAEA-CN-131/166P. IAEA, Vienna, Austria, 141. Bloem KA, Bloem S, Carpenter J, 2005. Impact of moth suppression/eradication programmes using the sterile insect technique or inherited sterility, pp. 677-700. In: Dyck, V.A., J. Hendrichs, and A.S. Robinson (eds.), Sterile insect technique. Principles and practice in area-wide integrated pest management. Springer, Dordrecht, The Netherlands. Bloem S, Bloem KA, Knight AL, 1998. Assessing the quality of mass-reared codling moth (Lepidoptera: Tortricidae) by using field release-recapture tests. J. Econ. Entomol. 91, 1122–1130. Bloem S, Carpenter JE, Bloem KA, Tomlin L, Taggart S, 2004. Effect of rearing strategy and gamma radiation on field competitiveness of mass-reared codling moths (Lepidoptera: Tortricidae). J. Econ. Entomol. 97, 1891–1898. Bloem S, Carpenter JE, Dorn S, 2006a. Mobility of mass-reared diapaused and nondiapaused Cydia pomonella (Lepidoptera: Tortricidae): Effect of mating status and treatment with gamma radiation. J. Econ. Entomol. 99, 699–706. Bloem S, Carpenter JE, Dorn S, 2006b. Mobility of mass-reared diapaused and nondiapaused Cydia pomonella (Lepidoptera: Tortricidae): Effect of different constant temperatures and lengths of cold storage. J. Econ. Entomol. 99, 707-713. Bloem S, Carpenter JE, Blomefield TL, Harrison C, 2010. Compatibility of codling moths Cydia pomonella (Linnaeus) (Lepidoptera : Tortricidae) from South Africa with codling moths shipped from Canada. J. Appl. Entomol. 134, 201-206 Blomefield TL, Carpenter JE, Vreysen MJB, 2011. Quality of mass-reared codling moth (Lepidoptera: Tortricidae) after long-distance transportation 1. Logistics of shipping procedures and quality parameters as measured in the laboratory. J. Econ. Entomol. 104, 814-822.
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Boller EF, Katsoyannos BF, Remund U, Chambers DL, 1981. Measuring, monitoring and improving the quality of mass-reared Mediterranean fruit flies, Ceratitis capitata Wied. 1. The RAPID quality control system for early warning. J. Appl. Entomol. 92, 67–83. Buès R, Toubon JF, 1992. Polymorphisme enzymatique dans différentes populations de Cydia pomonella L. (Lep. Tortricidae). Acta Oecol. 13, 583–591. Buès R, Toubon JF, Poitout HS, 1995. Variabilité éco physiologique et enzymatique de Cydia pomonella L. en fonction de l’origine géographique et de la plante hôte. Agronomie 15, 221–231. Burt M, 2002. TQM (Total Quality Management): Managing quality, not just controlling or measuring it, pp. 29-31. In N.C. Leppla, K.A. Bloem & R.F. Luck, eds. Proc. Quality Control for Mass-Reared Arthropods, 8th and 9th Workshops of the IOBC Working Group on Quality Control of Mass-Reared Arthropods (http://biocontrol.ifas.edu/amrqc/IOBCproceedings/amrqcbook.htm). Butt BA, Hathaway DO, White LD, Howell JF, 1970. Field releases of codling moths sterilized by tepa or by gamma irradiation, 1964–67. J. Econ. Entomol. 63, 912–915. Calkins CO, Parker AG, 2005. Sterile insect quality, pp. 269-296. In: Dyck, V.A., J. Hendrichs, and A.S. Robinson (eds.), Sterile insect technique. Principles and practice in area-wide integrated pest management. Springer, Dordrecht, The Netherlands. Carpenter JE, Bloem S, Marec F, 2005. Inherited sterility in insects, pp. 115-146. In: Dyck, V.A., J. Hendrichs, and A.S. Robinson (eds.), Sterile insect technique. Principles and practice in area-wide integrated pest management. Springer, Dordrecht, The Netherlands. Carpenter JE, Bloem S, Taggart S, 2010. Effect of rearing strategy and gamma radiation on the fecundity & fertility of mass-reared codling moth. J. of Appl. Entomol. 134, 221-226. Carpenter JE, Blomefield TL, Vreysen MJB, 2012. A flight cylinder bioassay as a simple, effective quality control test for Cydia pomonella. J. Appl. Entomol. doi: 10.1111/j.1439-0418.2012.01711.x Dyck VA, 2010. Rearing codling moth for the sterile insect technique. FAO Plant Production and Protection Paper 199. Food and Agriculture Organization of the United Nations, Rome, Italy. (FAO/IAEA/USDA) Food and Agriculture Organization of the United Nations/International Atomic Energy Agency/United States Department of Agriculture. 2003. FAO/IAEA/USDA manual for product quality control and shipping procedures for sterile mass-reared tephritid fruit flies. Version 5. IAEA, Vienna, Austria. http://www.iaea.org/programmes/nafa/d4/index.html. Ferro DN, Akre RD, 1975. Reproductive morphology and mechanics of mating of the codling moth, Laspeyresia pomonella. Ann. Entomol. Soc. Amer. 68, 417-424. Gu H, Hughes J, Dorn S, 2006. Trade-off between mobility and fitness in Cydia pomonella L (Lepidoptera: Tortricidae). Ecol. Entomol. 31, 68–74. Guennelon G, Audemard H, Fremond J, El Idrissi Ammari MA. 1981. Progrès réalisés dans l’élevage permanent du Carpocapse (Laspeyresia pomonella L.) sur milieu artificel. Agronomie 1, 59-64. Huettel MD, 1976. Monitoring the quality of mass-reared insects: a biological and behavioural perspective. Environ. Entomol. 5, 807-814. Keil S, Gu H, Dorn S, 2001. Response of Cydia pomonella to selection on mobility: laboratory evaluation and field verification. Ecol. Entomol. 26, 495–501. Pashley DP, Bush GL, 1979. The use of allozymes in studying insect movement with special reference to the codling moth, Laspeyresia pomonella (L.) (Olethreutidae), pp. 333-341. In: Rabb, R.L., and G.G.
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Kennedy (eds.), Movement of highly mobile insects: concepts and methodology in research. North Carolina State University Press, Raleigh, NC, USA. Robinson AS, Proverbs MD, 1973. Hybridization between geographical races of the codling moth (Lepidoptera: Olethreutidae). Can. Ent. 105, 289–290. SAS Institute. 1989. SAS user’s guide. SAS Institute, Cary, NC. Schumacher PA, Weber DC, Hagger C, Dorn S, 1997. Heritability of flight distance for Cydia pomonella. Entomol. Exp. Appl. 85, 169-175. Simmons G, Carpenter JE, Suckling DM, Addison M, Dyck VA, Vreysen MJB, 2010. Improved quality management to enhance the efficacy of the sterile insect technique for lepidopteran pests. J. Appl. Entomol. 134, 261-273. Singh P, Ashby MD, 1985. Insect rearing management, pp. 185–215. In: P. Singh and R.F. Moore (eds.), Handbook of insect rearing. Vol. I. Elsevier, Amsterdam, The Netherlands. Stenekamp D, 2011. Optimization of a mass-rearing system to produce codling moth, Cydia pomonella, for a Sterile Insect Release programme in South Africa. pp. 125. PhD Thesis, University of Stellenbosch, South Africa. Stewart FD, 1984. Mass rearing the pink bollworm, Pectinophora gossypiella, pp. 176–187. In E.G. King and N.C. Leppla (eds.), Advances and challenges in insect rearing. Agricultural Research Service, United States Department of Agriculture, New Orleans, LA, USA. Suckling DM, Barrington AM, Chhagan A, Stephens AEA, Burnip GM, Charles JG, Wee SL, 2007. Eradication of the Australian painted apple moth Teia anartoides in New Zealand: trapping, inherited sterility and male competitiveness, pp. 603-615. In: Dyck, V.A., J. Hendrichs, and A.S. Robinson (eds.), Sterile insect technique. Principles and practice in area-wide integrated pest management. Springer, Dordrecht, The Netherlands. Taret G, Sevilla M, Wornoayporn V, Islam A, Ahmad S, Cáceres C, Robinson AS, Vreysen MJB. 2010. Mating compatibility among populations of codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) from different geographic origins. J. Appl. Entomol. 134, 207-215. Timm AE, Geertsema H, Warnich J, 2006. Gene flow among Cydia pomonella (Lepidoptera: Tortricidae) geographic and host populations in South Africa. J. Econ. Entomol. 99, 341–348. Vreysen MJB, 2005. Monitoring sterile and wild insects in area-wide integrated pest management programmes, pp. 325-361. In: Dyck, V.A., J. Hendrichs, and A.S. Robinson (eds.), Sterile insect technique. Principles and practice in area-wide integrated pest management. Springer, Dordrecht, The Netherlands. Vreysen MJB, Carpenter JE, Marec F, 2010. Improvement of codling moth Cydia pomonella (Lepidoptera: Tortricidae) SIT to facilitate expansion of field application. J. Appl. Entomol. 134, 165- 181.
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Gene flow among Cydia pomonella host and geographic populations in China: knowledge on the population genetics for the SIT Maohua Chen College of Plant Protection, Northwest A&F University, Taicheng Road, Yangling District, Shaanxi Province, China Abstract The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae) is one of the most serious pest insects in apple orchards. This pest was firstly reported in China in the 1950s and was considered one of the most serious invasive pests in fruit orchards. It spread rapidly from the original site in Xinjiang to other northwestern regions, and has also invaded into some northeastern regions since 2006. With its rapid invasion rate, most apple growing regions of China is being threatened. As yet there has been no research into the genetic structure and genetic diversity of codling moth populations in China. In this study, we collected C. pomonella populations throughout the invested regions of China. Microsatellites were used to investigate the genetic structure and diversity of these sampled populations. It was observed that there was significant genetic differentiation among populations. The Ili River valley populations have higher genetic diversity among northwestern populations, suggesting that the Ili region is most likely the origin of codling moth introduction into northwestern China. Populations are isolated from each other in the distribution region, suggesting SIT can be applied for control this invasive species. Mating compatibility should be tested when SIT is used to control this pest in China Keywords: SIT; Cydia pomonella ; genetic differentiation; gene flow Introduction The codling moth Cydia pomonella (L.) (Lepidoptera: Tortricidae) is the key pest of pome fruits in temperate areas worldwide (Barnes 1991). It was first reported in the country in 1957 (Zhang 1957), and expanded to the whole Xinjiang province, most part of Gansu province, some neighboring regions of Gansu province. If codling moth passed the natural barrier i.e. the high mountains named as Tsinling Mountains which traverses China from East to West, it will invade from the northwest to the other areas, and the apple production of the whole country would be affected seriously by this pest. Actually, codling moth was reported in the northeast of China in 2006, although this frigid region is not an important fruit growing region. Cydia pomonella typically completes two generations on apple per year in the infested regions of China (Zhang 1957, Qin et al. 2006, Bahatiguli 2009). In the last 30 years, increasingly severe damage caused by this species has been reported (Qin et al. 2006, Bahatiguli 2009). Since the area of infestation expanded rapidly, this resulted in the presently unknown patterns of gene flow, which is important for establishing an area wide control strategy (Dorn et al. 1999). Apple is a major fruit in China, the apple production account for about 30% percent of the total fruit production every year in China. China is one of the most important apple growing regions in the world. In 2008, the apple plantation area of China was 1 992 200 ha, accounted for 41.09% of the world’s total apple plantation area, and the apple output was 29 846 600 ton, accounted for 42.88% of the world’s total apple output (Wang et al. 2010). Although C. pomonella is an object of both internal and external quarantine in China, it is still invading to another northwestern province (Shaanxi province), which is a main fruit growing region in the country, as a direct result of the accidental introduction by travelers and commercial transport (Shi et al. 2008). It is known that C. pomonella lacks a mechanism to avoid temperatures lethal to progeny development and has a remarkable ability to adapt to a wide range of climatic conditions (Notter-hausmann & Dorn 2010). The control of codling moth relies on the use of broad spectrum insecticides including organophosphate, carbamates and pyrethroids in the country (Qin et al. 2006, Bahatiguli 2009), which are known to select
87 for resistance to several insecticide groups (Knight et al. 1994, Sauphanor et al. 1998, Dunley & Welter 2000, Fuentes-Contreras et al. 2007, Reyes et al. 2009). As food safety is becoming a major concern for consumers, the use of environment-friendly technologies such as the sterile insect technique (SIT) will be increasingly required for the integrated pest management of C. pomonella (Vreysen et al. 2010). Development and optimization of pest management practices requires the understanding of ecological strategies and evolutionary patterns that characterize successful biological invasions (Torriani et al. 2010). The SIT is highly species-specific and non-polluting, and the target is the reproductive system of sexually reproducing pests (Klassen 2005, Vreysen et al. 2010). It can be integrated with a number of other techniques, and has been successfully implemented as an effective and environment-friendly technique to control C. pomonella in British Columbia, Canada (Klassen 2005, Bloem et al. 2007, Vreysen et al. 2010). Studies on gene flow pattern of insect populations can provide sound baseline information to develop efficient insect pest-management strategies such as SIT (Klassen 2005, Krafsur 2005, Timm et al. 2006, Chen & Dorn 2010, Vreysen et al. 2010). Gene flow studies can reveal the distance over which members of a given species typically disperse, estimate the degree of genetic isolation of target populations from each other, and from untargeted populations, determine whether a sub-population or sibling species exist, find out whether the pest was introduced several times or just once to a newly infestation region, and establish the origin of outbreaks or reintroductions (Krafsur 2005, Chen & Dorn 2010). In practice, if the immigration of gravid adult female moths into the treated agro-ecosystem could be kept to a minimum, the SIT would be more effective. As moths released in SIT programs should be capable of mating with females from different geographic regions, it is important to know to which degree an insect species migrates between different field populations (Krafsur 2005, Chen & Dorn 2010). On the other hand, estimating rates of gene flow among populations can provide an estimate of the rate of invasion or reinvasion of eradicated areas from unchallenged populations (Krafsur 2005). As gene flow patterns of field populations may be affected by a variety of factors, including dispersal ability, dietary specialization, habitat persistence, spatial structure of habitat within the landscape, anthropogenic effects such as the insect control programme used and geographical distance (Peterson & Denno 1998, Bohonak 1999, Krafsur 2005, Chen & Dorn 2010), the codling moth populations showed different gene flow patterns in different countries (Timm et al. 2006, Franck et al. 2007, Fuentes- Contreras et al. 2008, Thaler et al. 2008, Chen & Dorn 2010). Thus, different countries should use different tactics when implementing SIT to control C. pomonella (Timm et al. 2006, Chen & Dorn 2010). It is not known which patterns of gene flow exist within C. pomonella populations in China. Approach and Methodology Sampling A total of 339 C. pomonella individuals were collected from 12 apple orchards in the C. pomonella distribution regions of China from 2008 to 2010. In Xinjiang, infested fruit was collected from each fruit tree per orchard and one second or third instar larva per fruit was used. In the newly invaded Gansu and Heilongjiang, more than 30 pheromone traps were used in each apple orchard (>5 ha), and only one moth from each trap was used. All the sample locations are illustrated in Fig. 1. For making comparisons with populations in China, samples collected by pheromone traps in Germany (samples from Karlsruhe in July, 2008) and Switzerland (samples from Chur in June, 2009) were included in the analysis. All the samples were stored at -20 ℃ prior to genetic analyses.
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Figure 1. (A) Sample locations of the total 12 populations in China; (B) Sample locations of two populations in northeastern Heilongjiang Province; (C) Sample locations of 10 populations in the Northwestern Xinjiang and Gansu Province
DNA extraction and microsatellite genotyping Genomic DNA was extracted from the thorax and leg of each individual sample using DNeasy Tissue Kit (QIAGEN, Hilden, Germany). Extraction was performed according to the bench protocol for animal tissues. Before extraction, xylene was used to dissolve the glue covering specimens taken from pheromone traps. Eight loci (Cp1.62, Cp2.39, Cp2.P, Cp3.56, Cp3.169, Cp3.K, Cp4.56 and Cp4.129) were selected for the present study, based on their characterization in the literature as highly polymorphic (Franck et al. 2005). Each locus was amplified using fluorescently-labeled primers (FAM) (Schuelke 2000). Polymerase chain reactions (PCR) were performed using a S1000 Thermal Cycler (BIO-RAD, Hercules, CA, USA) in a total volume of 25µl, containing 1×PCR amplification buffer (Takara, Dalian, China), 2mM MgCl2, 0.2mM of each dNTP (Takara, Dalian, China), 0.2µM of each forward primer, 0.8µM of each reverse primer, 0.8µM M13 primer, 1.0 U Taq polymerase (Takara, Dalian, China), and 1µl genomic DNA (10-30ng/µl). PCR amplification was employed with denaturation at 95 ℃ for 10 min, followed by 30 amplification cycles consisting of 95 ℃ for 30s, 45s at the primer-specific annealing temperature, 72 ℃ for 45s, then 8 cycles consisting of 95 ℃ for 30s, 53 ℃ for 45s and 72 ℃ for 45s, and a final step at 72 ℃ for 10 min.
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To examine the length and genotype of the amplified PCR products, an ABI3730XL automated DNA sequencer (Applied Biosystems, Foster City, CA, USA) and GENESCAN version 4.0 (Applied Biosystems, Foster City, CA, USA) were used. Primers selections, amplification of microsatellite loci and genotype scoring Genomic DNA was extracted from 25mg of larval material using the DNeasy Tissue Kit (QIAGEN. Based on the primary test of the microsatellites described previously by Zhou et al. (2005) and Franck et al. (2005), nine microsatellite loci will be selected this study, and each sample will be genotyped at the selected loci. The PCR will be carried out with PTC-200 (PTC200) DNA Engine Thermal Cycler (Bio- Rad Laboratories, Inc. USA). The microsatellite alleles will be separated in Takara company (Takara, Xi’ an China). Micro-Checker version 2.2.3 (Van Oosterhout et al. 2004) was used to check the data for null alleles. MICROSATELLITE ANALYSER (MSA) version 3.15 (Dieringer and Schlötterer 2003), were used to calculate number of alleles, and the observed and expected heterozygosity (Ho and He). GENEPOP version 4.0.1 (Rousset 2008) was used to estimate the Hardy-Weinberg equilibrium (HWE), HWE-P and genotypic linkage disequilibrium. Analysis of molecular variance (AMOVA) was performed using ARLEQUIN version 3.0 (Excoffier et al. 2005), along with calculating the inbreeding coefficients (FIS), the pair fixation indices (FST) and their significance with 10000 permutations (Weir and Cockerham 1984). The significances of fixation indices were corrected for multiple comparisons by the Bonferroni method (Rice 1989) using SPSS version 19.0 (SPSS Inc. Chicago). For AMOVA, samples were arbitrarily grouped according to the collection regions: (i) Xinjiang Province; (ii) Gansu Province; (iii) Heilongjiang Province; and (iv) the other countries. Results Microsatellite markers and Genetic diversity
Table 1. Population statistics for Cydia pomonella investigated using eight microsatellite loci
Population code N NA FIS Ho He HWE-P TPM SMM
Zha 25 5.3 0.484 0.201 0.416 0.006 0.99023 1.00000
Jiu 25 4.3 0.298 0.272 0.434 0.118 0.90234 0.98633
Dun 33 5.6 0.400 0.220 0.460 0.006 0.97266 0.99414
Kum 33 6.0 0.257 0.298 0.495 0.004 0.99609 0.99609
25 5.5 0.330 0.232 0.395 0.020 0.90234 0.98047
Kor 11 4.9 0.097 0.290 0.475 0.268 0.96094 0.98828
Uru 26 8.6 0.486 0.262 0.552 0.125 0.98633 0.99609
Jin 21 6.9 0.267 0.321 0.519 0.211 0.99414 0.99805
Ili 27 9.6 0.354 0.356 0.616 0.064 0.98633 0.99805
Kuy 26 8.4 0.275 0.387 0.537 0.054 0.99805 1.00000
Mud 24 10.6 0.295 0.517 0.821 0.084 0.62891 0.99609
Don 24 12.6 0.293 0.482 0.791 0.160 0.98047 0.98047
Ger 15 8.6 0.259 0.550 0.770 0.192 0.52734 0.98047
Swi 24 10.4 0.304 0.502 0.801 0.105 0.52734 0.99023
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The table includes population code, sample size (N), mean number of alleles per locus (NA), multilocus estimate of inbreeding coefficient (FIS), observed heterozygosity (Ho), excepted heterozygosity (He), P- value for Hardy-Weinberg equilibrium (HWE) analysis, and P-value for heterozygote excess using two phase model (TPM) and stepwise mutation model (SMM). Significant departures from Hardy-Weinberg equilibrium are given in bold, P<0.05. NA, FIS, Ho, He and HWE-P are all indicated by mean values over eight loci. Probability values for heterozygote excess were determined using one-tailed Wilcoxon tests, and P-values less than 0.05 were considered significant We genotyped 339 individuals for eight microsatellite loci. All of the eight selected loci were polymorphic and the number of alleles per locus ranged from four to 16. The frequency of null alleles ranged from 0.010 to 0.203 that is typical for lepidopteran (Dakin and Avise 2004; Meglécz et al. 2004). Twelve samples with two non-amplified loci were omitted. The mean number of alleles per locus ranged from 4.3 to 12.6 (Table 1). Two populations from the Heilongjiang Province in northeastern China had the largest number of alleles (Don, 12.6; Mud, 10.6). Of populations from northwestern China, the Ili population showed the highest value of mean number of alleles (9.6), followed by Urumqi population (8.6) and Kuytun population (8.4). In comparison with the Chinese codling moth populations, the German and Swiss population also had relatively high value of 8.6 and 10.4 respectively. The mean value of observed heterozygosity was between 0.201 and 0.550, as compared to the expected heterozygosity of between 0.395 and 0.821. The two populations from the Heilongjiang Province had the largest number of observed heterozygosity (Mud, 0.517; Don, 0.482), followed by populations from the north of Xinjiang Province (Kuy, 0.387; Ili, 0.356; Jin, 0.321). The German and Swiss population also showed relatively high value of observed heterozygosity (Ger, 0.550; Swi, 0.502). Four of the 14 populations revealed significant departures from HWE, together with high mean values of FIS ranging from 0.257 to 0.486, indicating the existence of heterozygote deficiencies (Table 1.)
Table 2. Analysis of molecular variance of populations from Xinjiang, Gansu, Heilongjiang and other countries Source of variation d.f. Sum of Variance Percentage of P-value Fixation squares components variation indices
Among groups 3 155.756 0.23546Va 9.64 P<0.0001 FCT =0.09644 Among populations 10 165.522 0.30390Vb 12.45 P<0.0001 FSC =0.13776 within groups
Within populations 666 1266.820 1.90213Vc 77.91 P<0.001 FST =0.22091 AMOVA results (Table 2) indicated that 9.64% of overall variation was explained by geographic locations, revealing significant variances existed among populations from different geographic locations. The majority of variance (77.91%) was found within populations. The values of pairwise FST ranged from 0.040 to 0.337 (Table 3). Sixty-eight comparisons out of ninety-one showed high or very high genetic differentiations. The pairwise FST values among Ili, Jin and Kuy populations that are from north of Xinjiang Province were less than 0.10, while other pairwise FST values were above 0.10 for Xinjiang populations. The pairwise FST values between the populations from northeast (Heilongjiang Province) and northwest (Xinjiang Province and Gansu Province) of China ranged from 0.157 to 0.324, indicating high or very high genetic differentiation (Wright 1978).
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Table 3 Pairwise population differentiation estimates (F ST ) averaged eight loci between all populations of C. pomonella (below the diagonal) and P-values (above diagonal) Zha Jiu Dun Uru Kum Kas Jin Kor Ili Kuy Mud Don Ger Swi Zha ** ** ** ** ** ** ** ** ** ** ** ** ** Jiu 0.182 ** ** ** ** ** ** ** ** ** ** ** ** Dun 0.100 0.195 ** ** ** ** ** ** ** ** ** ** ** Uru 0.116 0.155 0.146 ** ** ** ** ** ** ** ** ** ** Ku 0.152 0.121 0.142 0.173 ** ** ** ** ** ** ** ** ** m Kas 0.144 0.272 0.236 0.205 0.166 ** ** ** ** ** ** ** ** Jin 0.244 0.252 0.274 0.209 0.133 0.246 ** ** ** ** ** ** ** Kor 0.096 0.337 0.306 0.223 0.323 0.332 0.278 ** ** ** ** ** ** Ili 0.154 0.185 0.120 0.124 0.110 0.105 0.083 0.203 ** ** ** ** ** Kuy 0.196 0.105 0.199 0.159 0.112 0.210 0.098 0.277 0.080 ** ** ** ** Mu 0.292 0.303 0.294 0.194 0.316 0.324 0.244 0.231 0.187 0.217 ** ** ** d Don 0.277 0.264 0.288 0.204 0.268 0.301 0.196 0.241 0.157 0.161 0.112 ** ** Ger 0.264 0.298 0.282 0.154 0.300 0.301 0.214 0.220 0.158 0.198 0.060 0.088 * Swi 0.266 0.264 0.266 0.159 0.281 0.289 0.222 0.223 0.157 0.176 0.040 0.110 0.043 First conclusions There was significant genetic differentiation among populations. The Northeastern populations have higher genetic diversity than northeastern populations. The Ili River valley populations have higher genetic diversity among northwestern populations, suggesting that the Ili region is most likely the origin of codling moth introduction into northwestern China. Populations are isolated from each other in the distribution region, suggesting SIT can be applied for control this invasive species. Mating compatibility should be tested when SIT is used to control this pest in China Future work To use three mitochondrial genes for further gene flow and population genetic research. References Bahatiguli (2009). The damage and control of Cydia pomonella in Xinjiang Province. Protection Forest Science and Technology 4, 118–120. (in Chinese) Barnes, M.M. (1991) Codling moth occurrence, host race formation and damage. pp. 313–327 in van der Geest, L.P.S. & Evenhuis, H.H. (Eds) Tortricid pests: their biology, natural enemies and control, 9 July, 1991, Elsevier, Amsterdam, Netherlands. Bloem, S., McCluskey, A., Fugger, R., Arthur, S., Wood, S. & Carpenter, J. (2007) Suppression of the codling moth Cydia pomonella in British Columbia, Canada, using an area-wide integrated approach with an SIT component. pp. 591–601 in Vreysen, M.J.B., Robinson, A.S. & Hendrichs, J. (Eds) Area- wide control of insect pests. From research to field implementation, Springer, the Netherlands. Bohonak, A.J. (1999) Dispersal, gene flow, and population structure. Quarterly Review of Biology, 74, 21–45. Chen, M.H. & Dorn, S. (2010) Microsatellites reveal genetic differentiation among populations in an insect species with high genetic variability in dispersal, the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). Bulletin of Entomological Research 100, 75–81.
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Dakin, E.E., Avise, J.C. (2004) Microsatellite null alleles in parentage analysis. Heredity 93, 504–509. Dorn, S., Schumacher, P., Abivardi, C. & Meyhöfer, R. (1999) Global and regional pest insects and their antagonists in orchards: spatial dynamics. Agriculture Ecosystems and Environment 73, 111–118. Dunley, J.E. & Welter, S.C. (2000) Correlated insecticide cross-resistance in azinphosmethyl resistant codling moth (Lepidoptera: Tortricidae). Journal of Economic Entomology 93, 955–962. Excoffier, L., Laval, G. & Schneider, S. (2005) Arlequin ver. 3.0: An integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online 1, 47–50. Franck, P., Guérin, F., Loiseau, A. & Sauphanor, B. (2005) Isolation and characterization of microsatellite loci in the codling moth Cydia pomonella (Lepidoptera: Tortricidae). Molecular Ecology Notes 5, 99–102. Franck, P., Reyes, M., Olivares, J. & Sauphanor, B. (2007) Genetic architecture in codling moth populations: comparison between microsatellite and insecticide resistance markers. Molecular Ecology 16, 3554–3564. Fuentes-Contreras, E., Reyes, M., Barros, W. & Sauphanor, B. (2007) Evaluation of azinphosmethyl resistance and activity of detoxifying enzymes in codling moth (Lepidoptera: Tortricidae) from central Chile. Journal of Economic Entomology 100, 551–556. Fuentes-Contreras, E., Espinoza, J.L., Lavandero, B. & Bamírez, C.C. (2008) Population genetic structure of codling moth (Lepidoptera: Tortricidae) from apple orchards in central Chile. Journal of Economic Entomology 101, 190–198. Klassen, W. (2005) Area-wide integrated pest management and the sterile insect technique. pp. 39–68 in Dyck, V.A., Hendrichs, J. & Robinson, A.S. (Eds) Sterile insect technique. Principles and practice in area-wide integrated pest management, Springer, Dordrecht, the Netherlands. Knight, A.L., Brunner, J.F. & Alston, D. (1994) Survey of azinphosmethyl resistance in codling moth (Lepidoptera: Tortricidae) in Washington and Utah. Journal of Economic Entomology 87, 285–292. Krafsur, E.S. (2005) Role of population genetics in the sterile insect technique. pp.389–406 in Dyck, V.A., Hendrichs, J. & Robinson, A.S. (Eds) Sterile insect technique. Principles and practice in area- wide integrated pest management, Springer, Dordrecht, Netherlands. Meglécz, E,, Petenian, F,, Danchin, E,, Coeur, D’Acier, A,, Rasplus, J.Y,, Faure, E. (2004) High similarity between flanking regions of different microsatellites detected within each of two species of Lepidoptera: Parnassius apollo and Euphydryas aurinia. Molecular Ecology 13, 1693–1700. Miller, M.P. (1997) Tools for population genetic analyses v 1.3. Download from http://www.marksgeneticsoftware.net/. Nei, M. (1972) Genetic distance between populations. American Naturalist, 106, 283–292. Notter-hausmann, C. & Dorn S. (2010) Relationship between behavior and physiology in an invasive pest species: oviposition site selection and temperature-dependent development of the oriental fruit moth (lepidoptera: tortricidae). Environmental Entomology 39, 561–569. Peterson, M.A. & Denno, R.F. (1998) The influence of dispersal and diet breadth on patterns of genetic isolation by distance in phytophagous insects. American Naturalist 152, 428–446. Qin, X.H., Ma, D.C., Zhang, Y., Li, G.H. & Wang, P. (2006) The damage and development of Cydia pomonella in the northwest of China. Plant Quarantine 2, 95–96. (in Chinese)
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Reyes, M., Franck, P., Olivares,J., Margaritopoulos, J., Knight, A. & Sauphanor, B. (2009) Worldwide variability of insecticide resistance mechanisms in the codling moth, Cydia pomonella L. (Lepidoptera: Tortricidae). Bulletin of Entomological Research 99, 359–369. Rice, W,R, (1989) Analyzing tables of statistical tests. Evolution 43, 223–225 Rousset, F. (2008) GENEPOP’007: a complete re-implementation of the GENEPOP software for Windows and Linux. Molecular Ecology Resources 8, 103–106. Shi, H.L., Wang, P.X., Li, P.F. & Ren Y.C. (2008) Risk analysis for Cydia Pomonella invasion to Shaanxi Province. Shaanxi Forest Science and Tcchnology 4, 76–80. (in Chinese) Schuelke, M., (2000) An economic method for the fluorescent labeling of PCR fragments. Natural Biotechnology 18, 233–234. Thaler, R., Brandstätter, A., Meraner, A., Chabicovski, M., Parson, W., Zelger, R., Dalla, Via J. & Dallinger, R. (2008) Molecular phylogeny and population structure of the codling moth (Cydia pomonella) in Central Europe: II. AFLP analysis reflects human-aided local adaptation of a global pest species. Molecular Phylogenetics and Evolution 48, 838–849. Timm, A.E., Geertsema, H. & Warnich, L. (2006) Gene flow among Cydia pomonella (Lepidoptera: Tortricidae) geographic and host populations in South Africa. Journal Economic Entomology 99, 341– 348. Torriani, M.V.G., Mazzi D., Hein S. & Dorn S. (2010) Structured populations of the oriental fruit moth in an agricultural ecosystem. Molecular Ecology, 2010, in press. Vreysen, M.J.B., Carpenter, J.E. & Marec, F. (2010) Improvement of the sterile insect technique for codling moth Cydia pomonella (Linnaeus) (Lepidoptera Tortricidae) tofacilitate expansion of field application. Journal of Applied Entomololgy134, 165–181. Wang, J.Z., Xue X.M. & Lu, C. (2010), Shandong Agriculture Sciences6, 117-119. (in Chinese) Weir, B.S., Cockerham, C.C. (1984) Estimating F-statistics for the analysis of population-structure. Evolution 38, 1358–1370 Wright, S, (1978) Evolution and Genetics of Populations. University of Chicago, Chicago Zhang, X.Z. (1957) The first record of Cydia pomonella in China. Acta Entomologica Sinica 4, 467– 472. (in Chinese) Zhou, Y.H., Gu, H. & Dorn, S. (2005) Isolation of microsatellite loci in the codling moth Cydia pomonella (Lepidoptera: Tortricidae). Molecular Ecology Notes 5, 226–227.
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Modifications on the rearing method and larval diet of Hypsipylla grandella (Lepidoptera: Pyralidae) Diana Pérez-Staples, Nancy Barradas-Juanz, Francisco Díaz-Fleischer INBIOTECA, Universidad Veracruzana, Av. de las Culturas Veracruzanas 101, Col. E. Zapata, Xalapa, Veracruz, Mexico Hypsipylla grandella (Lepidoptera: Pyralidae) is one of the worst forestry pests in tropical America, severely limiting the commercial value of cedar and mahogany plantations. One method for controlling this pest without the use of toxic chemicals or controversial transgenics is the sterile insect technique (SIT). For this technique to be employed, it is necessary to find a relatively inexpensive artificial diet that can be used for mass-rearing, as well as modify the rearing method for laboratory conditions. We present preliminary data on a comparison between a previously reported artificial diet (“O diet”) for H. grandella and two less expensive diets tailored towards mass-rearing. We also report certain modifications on the rearing method so a full cycle of this species can develop under laboratory conditions. Adults of both diets successfully mated and laid eggs in laboratory conditions, which to our knowledge have not been previously achieved for this species. We also tested larval rearing methods and conclude that the use of Petri dishes is more efficient. As larvae will need to be placed together for mass-rearing, we tested larval growth at different densities. Preliminary experiments demonstrate that rearing larvae together may not be possible due to cannibalism. Results from one of the modified diets are promising, as it is less expensive than the original diet and adequate for mass-rearing. Further research is needed to reduce egg to larvae mortality, improve growth rate, and find adequate oviposition mediums. Keywords: Lepidoptera, SIT, diet, cedar, mahogany shoot borer, mass-rearing
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Sterile insect technique in southern African sugarcane pest management D.E Conlong 1,2 , A.J. Walton 1, P. Mudavanhu 2, and L. Potgieter 3 1. South African Sugarcane Research Institute, Mount Edgecombe, South Africa; 2. Department of Conservation Ecology and Entomology, Stellenbosch University, Matieland, South Africa; 3. Department of Logistics, Stellenbosch University, Matieland, 7600. South Africa) Abstract Stemborers are constraints to world sugarcane production. Adopting only one, or a few control measures has worked in certain countries, but in others stemborers remain problematic and are a biosecurity risk. A modern approach to integrated pest management (IPM) includes control measures based on holistic agro-ecosystem interactions. Such an approach is under development for incorporation into an area- wide (AW)-IPM system. Included here is Sterile Insect Technique (SIT). Eldana saccharina (Lepidoptera: Pyralidae), severely limits sugarcane production in South Africa. This paper reviews laboratory research indicating that E. saccharina is susceptible to F1 male sterility, and argues that SIT could be a viable control option. Calco Red N1700, fed to E. saccharina larvae in a diet coloured adult fat bodies red, with no ill-effects on development and reproductive biology and provided a means of distinguishing them from field adults. Radiation biology studies showed that F1 male sterility was attained by exposing “fathers” to 200 Gy. Minimal emergence from F1 progeny of males exposed to 150 Gy makes this radiation dose an alternative. Males irradiated at 150 and 200 Gy commenced calling at the same time as untreated laboratory reared males, and at least six hours before field males. Similarly, irradiated males commenced mating with field females within 30 minutes of calling initiation, and much earlier than wild males. They mated with three to five times more wild females than wild males, and remained in copula with wild females for an hour longer than wild males. In choice experiments irradiated males were as competitive at attracting and mating with wild females as wild males were. Based on these laboratory and field cage studies, SIT, using F1 sterility, can provide a further tool to be developed as part of an AW-IPM against E. saccharina , and considered against other sugarcane stemborers which can be laboratory reared.
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Expansion of the SIT programme for false codling moth suppression in South Africa. Wilhelm Koekemoer X-Sterile Insect Technique, Citrusdal, South Africa
Abstract Sterile Insect Technique (SIT) has been applied commercially as a means for suppressing false codling moth (FCM) in citrus orchards in the Citrusdal area of South Africa since November 2007. The area initially covered was ± 1600 hectares. Due to the success of the release programme, the area has increased to ± 6100 hectares by June 2012 of which 4500 hectares are in the Citrusdal area, Western Cape Province, and another 1600 in the Sundays River Valley, Eastern Cape Province. By September 2012, it is expected that the total release area will be approximately 8500 Ha, split equally between the two provinces. To cope with this increase, several changes have had to be made with regard to production of the FCM as well as the distribution of them. In the past, quad bikes were used almost exclusively to distribute irradiated FCM in citrus orchards. Due to the increase in area to be covered, an alternative method was sought to release irradiated FCM. In the past season, gyrocopters were used to release the insects by air. Release by gyrocopter was found to be more efficient during preliminary trials. A transportation system was developed to enable transportation of irradiated FCM without detrimentally affecting the quality of the released product. Various modifications had to be made to the rearing facility to cope with the necessary increase in production. The FCM larvae are currently reared in glass bottles. Due to problems associated with this, a new method of rearing has been sought and is in the testing stage. Currently, 20 million FCM are irradiated each week for releases. This will be increased to approximately 26 million FCM per week by September 2012.
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New technologies for quality assessment in Lepidoptera Suckling DM 1, El-Sayed AM 1, Stringer, LD 1, Mas, F. 1 , Stanbury, M. 1 , Battisti, A. 2 and S. Hardwick 3 1. The New Zealand Institute For Plant & Food Research Limited, Christchurch, New Zealand, .2 University of Padua, Legnaro, Italy, 3. AgResearch, Lincoln, New Zealand Abstract Irradiation affects flight performance so attention to insect quality is an essential part of ensuring the successful use of the sterile insect technique. New technology is being developed to measure wing fanning using a laser viobrometer and a web-enabled high resolution camera trap to help improve assessment of field performance of irradiated insects. Measurement of bouts of wing fanning, which is reduced according to earlier results after irradiation, can now be measured using the laser vibrometer, which indicated wing fanning at 266 Hz in Epiphyas postvittana stimulated with pheromone. Successful efforts by the research team to convince a New Zealand company to develop a high resolution camera trap with different configurations (including a delta trap for moths and Lynfield trap for fruit flies) mean that insect counts can be made remotely in real time. The trap will be demonstrated during the workshop. A second camera trap was also developed from a commercially available iPhone (Generation 3), which can stream directly to the inetrnet. Background We expect that flight track analysis and other techniques will be highly efficient in detecting any deficiency in the male flight behavior that might be associated with quality of mass-reared and irradiated males. If successful, the technique could be standardized and used as quality assurance tool in Figure 1 . Paired laser vibrometers for measurement of any Sterile Insect Technique (SIT) program. The wing fanning using noise subtraction from the background (built in-house by Dr AM El-Sayed). availability of such tools can significantly enhance the efficacy of SIT programs. Wing fanning has been further investigated as a measure of quality, since Suckling et al. (2011) reported a correlation with flight arrival, and wing fanning alone is easier to assess. Results obtained A laser vibrometer (Fig. 1) has been developed and wing fanning recorded from irradiated and untreated light brown apple moths. Vibrational sound produced during wing fanning of male LBAM exposed to sex pheromone was recorded by a prototype laser vibrometer (built in-house but also commercially available). The frequency of wing fanning was assessed by Fast-Fourier Transform (FFT) as 266 Hz (Fig. 2). This hi-tech equipment has potential to be used in the assessment of insect quality (in general), and in the next year further experiments will examine other species. This equipment has considerable potential to reveal valuable insights as well as to provide pragmatic solutions for assessing insect quality.
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Figure 2 . Sample recordings of wing fanning in male lightbrown apple moth. The frequency is estimated by Fast Fourier Transform at 266 Hz.
Camera Trap Two remotely powered camera traps were progressed for the CRP. The first project involves a New Zealand security company, with existing web-enabled low resolution cameras, active in 12 countries
Figure 3. Prototype traps demonstrated in Phoenix at the Cooperdinated Research Meeting Workshop .
99 with a global SIM card. A prototype web-enabled camera trap (Biocam Self-Reporting), with much higher resolution (up to 8 MP) has been developed and tested in New Zealand and Italy, with configurations for various pests and traps (Fig. 3). An 8MP image is capable of enabling resolution of small insects (Fig. 4), although images of higher resolution and frequency require more power. The company already provides data warehousing for images in the security field and proposes a similar business model for scaling up in biosecurity. The second camera trap was developed from an earlier generation iPhone (Fig. 5), which can upload streamed video data to the Facebook website. Conclusions drawn so far While the wind tunnel system looks promising for measuring insect quality and a paper comparing the attributes of the wind tunnel and field releases has been published following the agenda of the CRP, more work is still needed to develop the quality measures needed to progress this area. An automated system of single moth releases into behavioural assays would be desirable to advance automation of moth quality assessment.
Figure 4 . Bioeye Lynfield camera trap from Mi5 (Auckland), with Drosphila suzukii. Images are taken at 5 MP.
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Figure 5. A prototype camera trap for moths, developed by Dr Ashraf El-Sayed from an iPhone (3rd generation).
The laser vibrometer was successful at measuring wing fanning and the spouse ear design has potential for wider use in measuring insect quality and could prove to be amenable to automation. A web-enabled camera trap has also been developed by a company which may offer these globally in the near future, once the New Zealand researchers have demonstrated their reliability, long battery life and other attributes (http://www.mi5.co.nz/security). Acknowledgements This project is also supported by related projects with Better Border Biosecurity (www.b3nz.org), the Cooperative Research Centre for Plant Biosecurity, and the USDA APHIS. Related publications related to the Technical Contract 2011-12 Brockerhoff EB, Suckling DM, Kimberley M, Richardson B, Coker G, Gous S, Kerr JL, Cowan DM, Lance DR, Strand T, Zhang A 2012. Biological invasion management by aerial application of pheromones for mating disruption. Public Library of Science One in press. Barclay HJ, Matlock R, Gilchrist S, Suckling DM, Reyes J, Enkerlin WR, Vreysen MJB 2011. A conceptual model for assessing the minimum size area for an area-wide integrated pest management program. International Journal of Agronomy Article ID 409328: 12. El-Sayed AM, Mitchell VJ, Manning LAM, Suckling DM 2011. New sex pheromone blend for the lightbrown apple moth, Epiphyas postvittana. Journal of Chemical Ecology 37: 640-646. Jang EB, McInnis DO, Kurashima R, Woods B, Suckling DM 2012. Irradiation of adult light brown apple moth, Epiphyas postvittana (Lepidoptera: Tortricidae): egg sterility in parental and F1 generations. Journal of Economic Entomology 105: 54-61. Kean JM, Suckling DM, Stringer LD, Woods B 2011. Modeling the sterile insect technique for suppression of light brown apple moth (Lepidoptera: Tortricidae). Journal of Economic Entomology 104: 1462-1475.
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Soopaya R, Stringer LD, Woods B, Stephens AEA, Butler RC, Lacey I, Kaur A, Suckling DM 2011. Radiation biology and inherited sterility of light brown apple moth (Lepidoptera: Tortricidae): developing a sterile insect release program. Journal of Economic Entomology 104: 1999-2008. Suckling DM, Tobin PC, McCullough DG, Herms DA 2012. Combining tactics to exploit Allee effects for eradication of alien insect populations. Journal of Economic Entomology 105: 1-13. Suckling DM, Woods B, Mitchell VJ, Twidle A, Lacey I, Jang EB, Wallace AR 2011. Mobile mating disruption of light-brown apple moths using pheromone-treated sterile Mediterranean fruit flies. Pest Management Science 67: 1004-1014. Suckling DM, Stringer LD, Mitchell VJ, Sullivan TE, Simmons GS, Barrington AM, El-Sayed AM. 2011. Comparative fitness of irradiated ight brown apple moths (Lepidoptera: Tortricidae) in a wind tunnel, hedgerow and vineyard. Journal of Economic Entomology 104: 1301-1308. Tooman, L. K., C. J. Rose, C. Carraher, D. M. Suckling, S. R. Paquette, L. A. Ledezma, T. M. Gilligan, M. Epstein, N. B. Barr, and R. D. Newcomb. 2011. Patterns of mitochondrial haplotype diversity in the invasive pest Epiphyas postvittana (Lepidoptera: Tortricidae) Journal of Economic Entomology 104: 920-932. Wee S-L, Suckling DM, Barrington AM 2011. Feasibility study on cytological sperm bundle assessment of F1 progeny of irradiated male painted apple moth (Teia anartoides Walker; Lepidoptera: Lymantriidae) for the sterile insect technique. Australian Journal of Entomology 50: 269-275.
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Stable isotopes in Lepidoptera research Rebecca Hood-Nowotny and Wolfgang Wanek Dept of Chemical Ecology and Ecosystem Research, Vienna Ecology Center, Faculty of Life Sciences – University of Vienna, Vienna Background There is currently a lack of adequate tools and methods to link insect quality to field performance of sterile insects (Dyck 2005). Mating success of sterile insects and subsequent population suppression is the ultimate quality measure of operational performance in an SIT programme. Due to the problems of trapping live female Lepidoptera, assessing sterile mating events using conventional egg hatch data is near impossible. Stable isotopes offer a number of possibilities to study both the biology and ecology of moths and the implication of quality control in a mass rearing context. The advantages of stable isotopes pose no safety threat, and can be used in ecosystem studies in complex environments. Stable isotopes are non- radioactive and omnipresent in the environment, for example 13C with its one extra neutron constitutes one percent of all carbon atoms. Using stable isotope tools it is possible to trace the distribution and movement of populations across the landscape, to estimate the population sizes and to determine who is inseminating (wild, sterile or GM) the native female population; all factors critical in any insect control programme but essential in sterile insect technique or genetic based eradication strategies. It has been shown throughout the course of the CRP that natural differences in the isotopic signatures of moths reared on mass-rearing diets compared to those of wild moths reared on native species are excellent markers for establishing the source of individuals in a population. These Isotopic methods provide us with a useful tools in improving the quality control of lepidopteran species as they offer opportunities to field test factory quality parameters without the confounding factors such as predation biases due to dye marking etc. In particular the value of having an independent complimentary mass rearing marker has been highlighted in the course of the CRP. It has been stressed that these stable isotope markers are ideal as they do not degrade and are easily incorporated into operational programmes. Therefore collaborations have been established with operational programmes to further refine and develop these methods to function appropriately and effectively within programme setting. Isotopic techniques have been developed to establish the paternal identity of wild female spermataphores, be they sterile mass reared male or non-sterile wild. These techniques have been successfully tested in the laboratory and are currently being developed for field use on trapped moths. These techniques really allow us to answer the question, are mass reared specimens as competitive in the field as the wild types and are they competitive enough? This is a valuable tool as it really allows us to start linking production quality with field performance. Tasks for following 18 months Continue to support CRP members through the provision of isotopic analysis for pilot marking experiments. Complete the publication of moth identification using stable isotopes. Further refine and develop isotopic methods for determining paternal status of field caught females as the ultimate measure of SIT factory quality for a range of moth species. Investigate the role and cost of nuptial gifts and how they influence mating success in relation to SIT strategies.
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Investigate and develop a novel-tool box to improve the quality of sterile insect monitoring and provenance of pest out breaks. Pyrolysis GCMS fatty acid finger printing for host verification. Endophytic bacteria for F1 marking and parental egg identification. Deuterium analysis for validation of geographical origin. Initiate and test protocols for the use of isotope ratio analysis using elemental cavity ring down systems for improved identification insects which are response triggers, and integrate the technology into operational programmes. Initiate and test protocols for the use of isotope ratio analysis using elemental cavity ring down systems for improved identification insects which are response triggers, and integrate the technology into operational programmes.
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The importance of spermatophore dissection in laboratory and field studies of fitness of irradiated light brown apple moth (LBAM) in Western Australia Bill Woods, Ian Lacey, Alven Soopaya and Amandip Kaur Department of Agriculture and Food Western Australia Abstract The Australian Tortricid light brown apple moth (LBAM) is a pest in Australia, New Zealand and now California. Trials in Western Australia tested irradiation doses from 150-300Gy to determine the appropriate irradiation dose for SIT. Numerous spermatophore dissections were carried out during these trials and evidence of multiple mating was found at all doses tested. LBAM irradiated at 300Gy were tested in field cages for competitiveness with wild adults. Moths were released at dusk and females collected the following morning and egg hatch recorded with subsequent spermatophore dissection. Once again multiple mating was evident in some moths. In the summer of 2011 15 N vs air sterile LBAM were released in 3 -5 0 5 10 15 20 25 plots each of 2 ha in urban Perth as -18
Lab SA colony part of a trial testing integration of -20 SIT with SPLAT pheromone Lab Wild colony -22 treatment. Movement of sterile -24 moths was measured with 16 Wild Swan Valley pheromone traps and 5 live female -26 Wild Dalkeith traps per plot. Live virgin females -28 CVPDB vs Apple X wild were placed in the traps on Monday 13 -30 Wild glass house Perth and removed and dissected on -32 Friday for evidence of mating.
-34 Spermatophores dissected were sent
-36 to Vienna for isotope analysis. Once again evidence of multiple mating was present even if only one male moth was found in the traps. The importance of multiple mating to the effectiveness of SIT of LBAM will be discussed Figure 1 . Results of isotope analysis of LBAM adults from laboratory and field in Western Australia Introduction As part of trials on the irradiation biology of light brown apple moth (LBAM) dissection of females for spermatophores to determine if they had mated was carried out. During these dissections it was observed that females often remated. Remating by wild females is not necessarily detrimental to SIT but if there is a differential rate of mating by females first mated to steriles or if there is sperm selection this can impact on SIT effectiveness. Therefore the effect of irradiation on remating was studied in field cage competiveness trials and in field trials investigating sterile moth release in urban Perth. Additionally spermataphores were sent to Vienna for isotopic analysis to test if this technique could be used to separate laboratory females that had mated with wild or sterile moths. Previous work as part of this project had shown a good separation in isotope values between wild and laboratory reared moths
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Methods Spermatophore dissection Spermatophore dissection was carried out to confirm mating as part of irradiation dose trials, field cage competiveness trials and mark release recapture trials. Moths were collected live if possible and held in a freezer before dissection in 70% alcohol under a binocular microscope. Moths that died before dissection and were dried out required soaking in a detergent solution overnight to soften them before dissection. For spermataphores to be dissected for isotope analysis dissection was carried out with saline, not alcohol as it interferes with the 13C signature. Small circles of quartz filter paper were punched out using an office hole punch and the spermataphore placed onto it with a fine brush. The samples were then loaded into small foil cups and dried at 50 °C. After drying they were folded into a small ball and placed into a 96 well micro titre plate. Dissected spermatophores from the mark release recapture trial were forwarded to Rebecca Hood- Nowotny in Vienna for isotope analysis. Irradiation biology The radiation biology of two geographically isolated LBAM populations was studied in Perth. Pupae 2 days before emergence, pharate adults fully developed but not emerged and one-four day old adults and were exposed 250 and 300 Gy in two trials. To determine fertility female moths were paired individually with 3 untreated male moths that were removed after 8 days. Spermataphore dissection was used to confirm mating status. Field cage trials Moths were irradiated at 300Gy amd at dusk released into nylon tents with an artificial Ficus tree. Mating pairs were captured into plastic vials and the following morning females placed individually into plastic containers for oviposition. After eight days females were removed and dissected for presence of spermatophores, an indication of successful mating. Some cages had ♂ only releases and in some cages mating was not observed and mating pairs captured but ♀ moths collected in the morning and egged. Mating status was then determined by spermatophore dissection and egg hatch. Parallel control and sterility matings were run in the laboratory and used to calculate C value in the Fried test. In these cage trials 25 males and females were placed together in a small cage and removed after 4 days. Mating competitiveness is expressed in terms of Relative Sterility Index (RSI) or Fried competitiveness value(C). SPLATTM trial Organic SPLATTM HD was applied in late September 2011 manually using 50 ml syringes to deliver dollops of 1 ml on front verge trees, fences, and power poles a least 1 m above ground level. As far as possible a uniform distribution of dollops and a dose rate of 500 mg/ha were achieved in each SPLATTM -treated plot. Six plots (1 to 6) were treated with SPLATTM. The remaining 3 plots (7-9) were controls. Each plot was approximately 2 ha. The LBAM population was monitored using 9 traps per plot baited with 3 mg LBAM sex pheromone lures, checked at weekly intervals. In week 6 and 7 1000 male moths irradiated at 250 Gy and dyed with fluorescent powder were released into three of the SPLATTM plots. Following the release no dyed moths were recaptured in the monitoring traps, probably because the pheromone disruption effect was still being very effective. Therefore to test moth survival and movement, dyed sterile moths were instead released in control plots in weeks 8 to 11 on 23 Nov, 30 Nov and 7 Dec. From the end September to mid-December 6 virgin female mating cages were placed per plot (Fig 2). The trap design was a Delta trap with an inner fly mesh screen tube with invaginated ends which
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Figure 2. Checking a virgin female mating cage allowed entry of male moths but not escape of females. A moist dental wick was provided in each trap to ensure survival of moths. Virgin females were put out on Monday and removed on Friday. Each female was then dissected and successful mating with a wild male confirmed by the presence of one or more spermataphore. On most but not all occasions, male moths remained in the mating cage. These moths were observed under fluorescent microscopy for presence of dye indicating they were irradiated. Results and discussion Irradiation biology trials In laboratory trials female moths irradiated at 250 and 300Gy mated up to three times. Remating was observed in both irradiated and unirradiated moths. The stage at which irradiation was carried out did not appear to influence the average number of mating. There is no obvious explanation for the large number of remating of control moths in trial 2.
Table 1. Average number of matings determined by spermatophore dissection Irradiated pupae pharate moths males pupae
250Gy 1.5 1.2 1.5 control 1.6 1.2 1.4 pharate (+1d) (+2d) (+3d) (+4d) pupae moths moths moths moths
300Gy 1.6 1.4 1.7 1.8 1.7 control 2.5 2.2 1.5 2.2 1.8 Field cage trials Results indicated acceptable competitiveness and that male-only release may improve competitiveness. Estimating competitiveness by egg hatch and not by capturing mating pairs appeared to give a more realistic estimation of Fried C values but there was no difference in RSI between the techniques.
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Table 2 . Competitiveness values for male only and bi-sex release in field cage trials Treatment RSI ±SE Fried C ±SE
Bi -sex 0.4±0.1 0.9±0.4
Male only 0.8±0.1 1.9±1.3
Male only 0.7±0.0 2.4±0.4
Male only(not captured) 0.7±0.0 0.7±0.1
As part of the competitiveness trials control and sterility matings were carried out in the laboratory in small cages where female moths had the opportunity to mate with different partners over a number of days. From the results (Table 3) it can be seen in this situation multiple mating often occurred with up to 3 spermataphores per female. However in field cages where female only had the opportunity to mate over one night although multiple mating did occur but it was less frequent. We can hypothesise that if the females had remained in the field cages for a number of days the incident of multiple mating may have been higher.
Table 3. percentage of mated moths in competitiveness trials with one, two or three spermatophores Sperma tophores 1 2 3 Lab N ♂X N♀ 50% 50% Lab I ♂X N♀ 53% 41% 6% Field cage 89% 11%
SPLAT trials Average percentage trap shut down in the SPLATTM plots peaked at 98% on week 2 remained above 90% until week 12 week before showing a downward trend. No sterile moths were caught in the traps in the 3 SPLATTM treated plots for the duration of release. In the control plots a very low level of recapture and poor dispersal were observed. Dyed sterile moths were released on plot 7 and some moved to the adjacent plot 8. Most moths that were captured were within 10 metres of the release point and a few (2 moths) at a maximum distance of 35 m. In the three control plots, the average percentage of females in the virgin female traps that were mated were 40 % in plot 7, 45% in plot 8 and 25% in plot 9 with spermatophores per virgin female averaging 1.1 in all three plots. Therefore in the field only limited multiple mating occurred with most moths only mating one and a few twice. Attempts to us isotopic analysis to determine whether these matings were from sterile moths gave inconclusive results (Table 4). From previous results the 12C/13C ratio for laboratory reared moths varied between 20-22 and for wild moths from Dalkeith 25-28 (Fig 1). Therefore, if we assume all spermataphore with a signature below 24 are from sterile matings and excluding the results of Nov 4 which is probably anomalous, all results with a lower value could be the result of a mating with a sterile male. Sterile males were released in control plots from Nov 23. The female from plot 7 cage 4 on Nov 25 when dissected contained 2 spermatophores both which gave values below 24. As there was also a dead dyed sterile moth in the cage it is highly likely that this result is from mating with a sterile moth.
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Table 4. Results of isotopic analysis of spermataphores. Matings highlighted in bold are at level expected for sterile males reared on cane sugar. Result for Nov 4 is probably faulty as indicated by C/N ratio. First sterile release was on Nov 23 Ampl Ampl Plot-female Date 28 44 d 15N/14N d 13C/12C Amt% N Amt% C C:N ratio 7-1 Nov 4 631 655 10.528 -25.643 14.03 74.28 5.295789 7-2 Nov 4 41 63 4.433 -26.523 0.97 10.14 10.46863 7-5 Nov 4 104 115 5.793 -25.130 2.40 15.84 6.598843 8-1 Nov 4 14 34 1.671 -21.117 0.37 6.99 19.0583 8-1 Nov 4 435 339 12.750 -24.672 9.60 39.57 4.122067 9-3 Nov 4 430 302 10.805 -24.936 9.53 35.61 3.737896 8-5 Nov 11 137 133 5.579 -26.932 3.08 17.69 5.747998 8-1 Nov 11 660 502 9.376 -26.615 14.55 57.03 3.920613 8-1 Nov 11 84 93 4.064 -27.372 1.91 13.32 6.965784 8-4 Nov 11 395 285 5.416 -28.624 8.75 33.58 3.836285 7-4 Nov 11 662 466 7.695 -27.246 14.57 53.06 3.642694 9-1 Nov 11 792 568 7.275 -26.520 17.38 63.94 3.678298 9-4 Nov 11 513 425 7.894 -25.207 11.42 48.89 4.28118 7-1 Nov 18 551 434 7.152 -25.434 12.30 49.97 4.064484 8-4 Nov 18 444 418 8.730 -28.852 10.02 48.58 4.848816 8-5 Nov 18 604 445 6.567 -23.834 13.59 51.57 3.795825 8-3 Nov 18 51 62 5.309 -24.415 1.21 10.08 8.32589 9-2 Nov 18 509 351 13.138 -26.621 11.50 41.53 3.61165 9-4 Nov 18 538 378 10.943 -23.292 12.19 44.50 3.651724 9-5 Nov 18 65 80 5.363 -26.560 1.56 12.07 7.749707 9-5 Nov 25 50 110 5.251 -26.848 1.22 15.42 12.60501 7-4 Nov 25 579 478 10.048 -22.770 13.23 56.23 4.250214 7-4 Nov 25 488 417 9.954 -23.437 11.10 49.04 4.41925 8-1 Nov 25 71 97 5.011 -23.755 1.69 14.03 8.275578 7-1 Dec 2 91 206 4.858 -25.803 2.19 26.20 11.98722 7-1 Dec 2 565 484 7.373 -25.325 12.93 56.96 4.406694 7-2 Dec 2 696 588 9.537 -26.505 15.86 68.74 4.333595 8-2 Dec 2 610 507 9.142 -22.336 13.91 59.38 4.26779 8-3 Dec 2 359 311 10.316 -27.465 8.24 37.70 4.577916 9-5 Dec 2 313 244 8.325 -22.998 7.23 30.22 4.179372 8-4 Dec 2 626 485 14.974 -24.789 14.29 57.04 3.991981 7-4 Dec 9 556 416 5.743 -24.453 12.80 49.96 3.903229 8-1 Dec 9 645 454 11.020 -24.577 14.77 53.83 3.645216 8-1 Dec 9 139 216 5.369 -25.357 3.27 27.04 8.274532 9-5 Dec 9 655 586 5.145 -22.751 15.10 69.12 4.578177
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Although we did not catch sterile moths in pheromone traps in plot 9 results indicate that sterile males may have reached the area and mated. However these results may be also because there is a C4 host in the area which the LBAM are feeding on or the spermataphore sample is not clean enough. Future work will involve sending matched mated female leg when we supply the spermataphores and noting whether it is a clean sample or not. In the laboratory when exposed to multiple males LBAM females have the potential to mate many times. However, it appears that in the field only a small percentage will mate more than once. The impact of multiple matings on effectiveness of SIT is yet to be determined we need to have a reliable method to discriminate matings from sterile or wild males. The current isotope method needs refinement.
References Soopaya R, Stringer LD, Woods B, Stephens AEA, Butler RC, Lacey I, Kaur A, Suckling DM 2011. Radiation biology and inherited sterility of light brown apple moth (Lepidoptera: Tortricidae): developing a sterile insect release program. Journal of Economic Entomology 104: 1999-2008. Acknowledgements We thank Delyse Campbell of Plant & Food NZ and Latif Salehi from SARDI for helping to apply SPLAT. Max Suckling provided input on the design the SPLAT trials in Perth and Don McInnis helped set up the LBAM mating competiveness trials. Daniel Murphy from UWA supplied filter paper and foil cups for isotope dissection and Rebecca Hood-Nowotny from the University of Vienna carried out isotopic analysis of spermataphores. The project was supported by funding from the Cooperative Research Centre for National Plant Biosecurity in Australia.
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Electrophysiologal and behavioural assays of cabbage-derived volatiles on diamondback moth, Plutella xylostella Suk Ling Wee 1 and Kye-Chung Park 2 1. School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Malaysia, 2. The New Zealand Institute for Plant and Food Research Limited, Lincoln, New Zealand Abstract Cabbage (Brassica oleracea L. Subsp. capitata ) was consistently reported as the most preferred crucifer host for diamondback moth (DBM), Plutella xylostella (L.). Volatile emission from cabbage plants was sampled and tested on the excised antennae of DBM moths using gas chromatographic- electroantennogram (GC-EAD). A total of 12 antennal-active peaks were detected in infested cabbage which also included the 9 common peaks in non-infested cabbage plants. GC-MS analysis has tentatively identified these antennal-active peaks as dimethyltrisulfide, methyldihydrothiophenone, hexyl acetate/hexenyl acetate, ethyldimethyl benzene/acetylthiophenone, 2-phenyl-2-propanol/ethyl heptanoate, 2-phenylethyl alcohol, 4-ethyl-6-hepten-3-one, menthone/ tetramethyl benzene, 2- nonenal/octyl acetate, 2,6-nonadienal, geranyl acetone/carvyl acetate, caryophyllene alcohol/geranyl butyrate and need further confirmation. Preliminary field trials, using some of these components, either singly or in combinations, were conducted to determine if these compounds are behaviourally active. Results showed that a few chemicals caught a low number of male moths and one female moth. Problems encountered in the field trials was highlighted and discussed. Introduction Diamondback moth (DBM), Plutella xylostella (Plutellidae) is a notorious brassica specialist and is widely distributed in tropical, subtropical and temperate regions. Amongst all brassica hosts, cabbage (Brassica oleracea L. subsp. capitata ) is the most preferred host of DBM (Reddy & Guerrero 2000, Reddy et al. 2004). Insect antennae are the most sensitive detector of the volatiles that play a significant role in mate- and host-finding and recognition. Volatile and semi-volatile compounds such as green leaf volatiles (GLV) and allyl isothiocyanates from cabbage plant extract were shown to be attractive to DBM, with the former acting as long range attractant and the latter as arrestant leading to feeding and oviposition activities, respectively (Hillyer & Thorsteinson 1969, Reddy & Guerrero 2000, Renwick 2002). It was also reported that the larval-injured cabbage plants were more attractive to female DBM than the healthy plants (Reddy & Guerrero 2000, Reddy et al. 2004). Here, we demonstrate the positive antennal responses from male and female moths to the headspace extract from both healthy and larva-infested cabbage plants using gas chromatography (GC)- electroantennogram (EAD). There were 12 tentatively identified compounds which were used in preliminary field trials to determine the bio-activity of these compounds. Materials and Methods Headspace samplings were conducted for healthy and DBM larval-infested cabbage plants in plastic pots using the dynamic headspace method in an air entrainment system. Volatiles released from the intact plants were actively adsorbed onto a Tenax cartridge (GR 35/60, 100 mg) for 4-6 hours. The Tenax cartridges were extracted with solvent hexane (1 ml) and the eluate was concentrated to approximately 100 µl under a gentle argon stream. Aliquots of 2 µl and 1 µl were subjected to GC-EAD and GC-MS analysis, respectively.
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The electrophysiological response of DBM to cabbage headspace volatiles was analyzed using a Varian 3800 gas chromatograph with a splitless injector, coupled to an EAD Recording Unit (Syntech Research and Equipment, Hilversum, Netherlands). Samples were run on a ZB-5 (Phenomenex) capillary column (30 m x 0.25 mm ID) with a 1:1 split outlet between the flame ionisation detector and the electroantennogram. Nitrogen was used as the carrier gas at a flow rate of 1 ml/min. The gas chromatograph oven temperature was programmed from 80°C (1 min hold) to 240 °C at 10°C/min and held for 15 min. An excised antenna was held between two glass electrodes containing Ringer’s solution with 10% polyvinylpyrrolidone (MW 360,000). Each glass electrode held a length of 1 mm silver wire that electrically connected the preparation to the preamplifier of a recording unit. The temperature of the transfer line from the gas chromatograph to exit port was maintained at 200°C and the antennal preparation maintained in a filtered and humidified 400 ml/min air stream. Female antennae were subjected to GC-EAD analysis and screening of both healthy and infested plants to determine if there is any difference in antennal activity. Male antennae were also tested against the infested cabbage plant volatiles and compared to the antennal response of the females. Based on the results of the GC-MS analysis, nine tentatively identified compounds were tested in a preliminary field trials using single as well as combination compounds. An ogranic farm with brassica plots was selected for the field trial. The brassica plants planted were chinese broccoli, B. oleracea var. alboglabra , B. juncea group, pok choy ( B. rapa var. chinesis , B. rapa var. narinosa , and other brassicas. A summary of the single compounds and their combinations are listed in Tables 1 and 2.
Table 1. List of the tentatively identified compounds elicited positive response in the GC-EAD recordings of diamondback moth. Single compound was used in the field trials.
Compounds A B D E F H J
Hexyl acetate 10 0
Z3 -Hexenyl acetate 10 0
Ethyl heptanoate 10 0
2-Phenylethanol 10 0
Nonyl aldehyde 10 0
(Nonanal)
Geranyl acetone 10 0
Hexane 0
Red delta traps with cotton wick as dispensers were used for the trapping. Traps were positioned approximately 10-20cm above the canopy of the brassica. Traps were randomly placed in blocks of brassicas with 8-10 m apart. Lures were changed every 4-7 days. Five to ten replications were used for each of the treatments tested. Figure 1 shows the site and placement of traps in the organic farm.
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Table 2 . Combination of chemical compounds from the tentatively identified compounds elicited positive response in the GC- EAD recordings of diamondback moth used in the field trials.
Compounds A B C D E F G H I J
Hexyl acetate 0 10 10 10 10 10 10 10 10 0
Z3-Hexenyl acetate 10 0 10 10 10 10 10 10 10 0
3-Phenyl-1-propanol 10 10 0 10 10 10 10 10 10 0
Ethyl heptanoate 10 10 10 0 10 10 10 10 10 0
2-Phenylethanol 10 10 10 10 0 10 10 10 10 0
Nonyl aldehyde (nonanal) 10 10 10 10 10 0 10 10 10 0
Octyl butyrate 10 10 10 10 10 10 0 10 10 0
Geranyl acetone 10 10 10 10 10 10 10 0 10 0
Geranyl acetate 10 10 10 10 10 10 10 10 0 0
Results There were a total of 12 bioactive peaks recorded by GC-EAD on the cabbage headspace against the moths’ antennae. The pattern, amplitude and the number of EAG response peaks were consistent in all GC-EAD recordings from male and females (Figures 2a to 2c). There were no major differences in the EAD active peaks between males and females except a small peak #7 detected in the healthy cabbage (Figure 2a). However, there were three small EAD-active peaks (#6, 7 and 8) only present in the
Figure 1. Placement of traps containing cabbage-derived volatiles dispensed on cotton wicks in red delta traps in organic brassica plants
113 headspace samples of larvae-infested cabbage (Figure 2b & 2c). A summary of the retention time by which the compound was eluted in the GC-EAD under the temperature programme concomitant with the antennal response and thier tentative identification is given in Table 3. GC-MS analysis has tentatively identified these antennal-active peaks as dimethyltrisulfide, methyldihydrothiophenone, hexyl acetate/hexenyl acetate, ethyldimethyl
Figure 2 . Gas chromatograph (GC)-electroantennogram (EAD) recordings of diamondb ack moth to the headspace extract of cabbage plant, a preferred brassica host: (a) female-healthy cabbage, (b) female-larval infested cabbage, and (c) male - larval infested cabbage. Numbers on EAD peaks corresponded to the retention time (RT, mins) of chemi cal compounds eluted from the GC system under the programmed temperature of from 80°C (1 min hold) to 240°C at 10°C/min and held for 15 min. RT: peak 1 (4.22), peak 2 (4.43), peak 3 (4.49), peak 4 (5.08), peak 5 (5.10), peak 6 (5.27), peak 7 (5.31), pea k 8 (5.35), peak 9 (5.50), peak 10(5.55),peak 11 (7.31), and peak 12 (8.16). benzene/acetylthiophenone, 2-phenyl-2-propanol/ethyl heptanoate, 2-phenylethyl alcohol, 4-ethyl-6- hepten-3-one, menthone/ tetramethyl benzene, 2-nonenal/octyl acetate, 2,6-nonadienal, geranyl acetone/carvyl acetate, caryophyllene alcohol/geranyl butyrate and need further confirmation.
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Table 3. Tentative identification of EAD-active compounds present in the headspace collections of healthy or DBM larvae infested cabbage plants using GC-MS. EAD peak # RT Kovats RI Compounds identified
1 4.23 969 Dimethyltrisulfide
2 4.43 999 Methyldihydrothiophenone
3 4.49 1007 Hexyl acetate, hexenyl acetate
4 5.08 1089 Ethyldimethyl benzene, acetylthiophenone
5 5.10 1092 2-Phenyl-2-propanol, ethyl heptanoate
6 5.27 1115 2-Phenylethyl alcohol
7 5.31 1120 4-Ethyl-6-hepten-3-one
8 5.35 1126 Menthone, tetramethyl benzene
9 5.50 1147 2-nonenal, octyl acetate
10 5.55 1154 2,6-Nonadienal
11 7.31 1415 Geranyl acetone, carvyl acetate
12 8.16 1554 Caryophyllene alcohol, geranyl butyrate
Preliminary field trial results showed that these tentatively identified compounds, either singly or in combinations caught some DBM, mostly males, but not in significant numbers that could enable statistical analysis (Tables 4 & 5).
Table 4. Summary of diamondback moth trap catches from single chemical compound-baited delta trap.
Trap # Chemicals N Males Females Total
A Hexyl acetate 10 0 0 0
B Z3-Hexenyl acetate 10 0 0 0
D Ethyl heptanoate 10 0 1 1
E 2-Phenylethanol 10 0 0 0
F Nonyl aldehyde 10 0 0 0
H Geranyl acetone 10 0 0 1
J Hexane 10 0 0 0
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Table 5. Summary of diamondback moth trap catches from combination of multiple compound-baited delta trap. For details of mixture, refer to Table 3.
Chemical Mix N Males Females Total
A 10 1 0 1
B 10 1 0 1
C 10 0 0 0
D 10 1 0 1
E 10 0 0 0
F 10 3 0 3
G 5 1 0 1
H 5 1 0 1
I 5 0 0 0
J 10 0 0 0
Discussion The most challenging part of this research is the chemical identification based on the GC-EAD and GC- MS analyses. Plant headspace often yielded more than 30 GC peaks than insect pheromone profiles, and with less separation between retention times. Because the excised antennae having a shorter life span, the programmed temperature setting for GC-EAD was much faster than those in GC-MS and has resulted in a more ‘compressed’ GC profile in terms of the base peaks with respective retention times than in a normal GC-MS profile for chemical separation. This had some effects on the GC-EAD profiles whereby the shorter total run time also meant that the retention time will be different from those retention time obtained from the GC-MS. This has posed a challenge in accurately match GC peaks with GC-MS peaks, especially the minor peaks. On the other hand, using live and whole body moths would enhance the life span of the recordings but the baselines obtained was ‘noisier’ than the excised antennae as the live moth would struggle from time to time during the recording period. Therefore, our attention has been focused on chemical identification since then rather than to keep repeating the GC- EAD recordings. The positive but poor catches using the tentative identified chemical compounds means that further refining work needs to be done to confirm the chemical identity as well as the formulation/mixture of the chemicals. There were also some twenty brassica volatiles in the literature which are related to the chemical identified at this stage. These chemicals will be included in the early screening for attractive compounds in the next 18 months.
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The cotton wick was not a suitable dispenser in Malaysia as the chemical evoprated within 2-3 days. Polyethylene (PE) bag with felt, and PE vials may be a better substitude replacing cotton wick in the future field trials. Work Plan for the Next 18 Months 1. Continue and finalise chemical identification of antennal-active compounds in the cabbage headspace. 2. Conduct choice-test with Y-olfactometer assays: (a) to confirm the attractiveness of DBM to odour of infested cabbage over healthy cabbage. (b) to evaluate the activity of the identified compounds as well as other brassica volatiles in the literature against the DBM, either singly or in combinations. 3. Conduct field trial using several selected candidates of cabbage chemicals (from 2 (b)). References Hillyer, R.J. & Thorsteinson, A.J. 1969. The influence of the host plant or males on ovarian development or oviposition in the diamondback moth, Plutella maculipennis (Curt.). Can. J. Zool. 47: 805-816. Reddy, G.V.P. & Guerrero, A. 2000. Behavioral responses of the diamondback moth, Plutella xylostella, to green leaf volatiles of Brassica oleracea subsp. capitata. J. Agric. Food Chem. 48: 6025- 6029. Reddy, G.V.P., Tabone, E. & Smith, M.T. 2004. Mediation of host selection and oviposition behavior in the diamondback moth Plutella xylostella and its predator Chrysoperla carnea by chemical cues from cole crops. Biol. Control 29: 270-277. Renwick, J.A. 2002. The chemical world of crucivores: lures, treats and traps. Entomol. Exp. Appl. 104: 35-42.
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Sperm dynamics in correlation with mating behaviour of sub-sterilized male moths, Spodoptera litura (Fabr.) and their F1 progeny, as a crucial assessment of ‘Inherited Sterility’ technique for Lepidopteran pest suppression. R.K. Seth, Zubeda and Mahtab Zarin Department of Zoology, University of Delhi, Delhi, India,
Description of research carried out Spodoptera litura (Fabr.) is a serious polyphagous pest of Indian tropics (Lefroy 1908, Moussa et al. 1960, Chari and Patel 1983, Higuchi et al. 1994) whose reproductive capacity and migration ability over long distances has made it an economically important pest of many agricultural crops, with a wide geographic range throughout Asia. The development of resistance to chemical insecticides is one of the driving forces for change in approaches of insect pest management. At present, the increasing number of resistant insects is outstripping the development of new insecticides, and furthermore, the strict pre- requisite demand of environmentally safe chemicals has reduced the chances of finding new and effective insecticides. In view of increasing environmental hazards from the use of chemical pesticides and development of insecticide-resistance in this pest (Ramakrishnan et al. 1984; Armes et al. 1997), there is a serious need towards developing of environmentally sound alternative measures to control this pest. F1 sterility, as a parabiological (radio-genetic) control measure, has been proposed for the suppression of S. litura (Seth and Sehgal 1993; Seth and Sharma 2001), instead of using the Sterile Insect Technique (SIT) wherein the high (100%) sterilizing dose impairs the mating competitiveness of the moths. A range of 100-130Gy was suggested to be employed in order to implement F1 sterility for the suppression of S. litura populations, because under the influence of this range of doses of ionizing radiation, the mating viability and sperm behavior of the pest would not be affected significantly. In the present study the main focus was to identify some crucial bio-characteristics of the irradiated insects, and correlate such physiological and biochemical characteristics with adult behavioural competitiveness of sub-sterilized moths and their progeny. This would help us understand the irradiation mediated intrinsic disturbance or changes and degree of manifestation of somatic damage that could be correlated with reproductive competence of irradiated insects and their progeny. Materials and Methods A fresh set-up was established to exercise quasi-mass rearing of Spodoptera litura on semi-synthetic diet (Seth and Sharma 2001) at 27±1°C, 75±5% relative humidity and 12 hr Light:12 hr Dark regimen. Sperm descent from testes and its rhythmic pattern : Circadian rhythm of sperm descent from testes down in to the reproductive tract in radiation induced substerilized moths was assessed as per the method described by Seth et al. (2002). For determining the descent of sperm from the testes into the male reproductive tract, various regions of the male tract, viz. upper vas deferens (UVD), seminal vesicle (SV), and the ductus ejaculatorius duplex (Duplex) were dissected out in Belar’s saline (Flint and Kressin, 1969). These regions of the male tract, along with their contents, were carefully separated out on a microscope slide under the compound microscope (Olympus BX-60). For the assessment of eupyrene sperm (bundles), 50µl Lacto aceto orcein (LAO) was added to each of the reproductive tract portions, and 5µl aliquot was taken for each reading with mean of five such aliquot observations constituting each replicate for each tract portion. Since, apyrene sperm are not in bundles when they descend from the testes (Friedländer et al. 2005), their relative density was estimated in the various parts of the reproductive tract down the testes up to Duplex ejaculatory duct. For the assessment of apyrene sperm, the sample of ingredients (sperm + secretions) from each reproductive tract portion was diluted
118 in Belar’s saline (500µl for UVD and 500µl for SV and 1500µl for duplex). A 2µl aliquot was taken for each observation of counting apyrene dissociated (individual) sperm, with five aliquot observations constituting each replicate for a specific tract portion. The numbers of eupyrene bundles and individual (dissociated) apyrene sperm were quantified in different parts of the reproductive tract at two particular times of the day, at 10–11 AM (during photophase) and at 10–11 PM (during scotophase) in order to ascertain the sperm descent profile and confirm their descent rhythm in this lepidopteran pest under the influence of gamma irradiation. This is the first attempt to quantitfy number-wise, the dissociated (loose) apyrene sperm in different parts of the reproductive tract in respect of methodology unlike in the earlier report (Seth et al.2002) where apyrene density was determined scale-wise. Sperm decent and its rhythmic pattern were studied in irradiated substerilized male moths and their F1 progeny Standardization of ‘in-vitro assay’ of sperm activation in adult moth, Spodoptera litura and study the sperm activation in irradiated (substerilized) moths and their F1 progeny : An in-vitro assay was designed to study the apyrene sperm activation, in which sperm from the ‘Duplex’ region was mixed with secretions from the prostatic part (as activator). For the preparation of sperm, the virgin adult male moths (2-3day old after emergence) was dissected in Belar’s saline, and the contents of the ductus ejaculatorius duplex were mixed with the 0.3 M HEPES-KOH buffer(50µl/region) containing 20mgBSA/ml at pH 7.0, and incubated at room temperature (ca. 25°C). For the preparation of the activator, the prostatic part from virgin male moths was dissected, and its secretions were added to the ammonium bicarbonate-acetic acid buffer (40µl/region) at pH 7.0 and centrifuged at 6000 rpm at 4°C for 10min. The supernatant was incubated at room temperature. For the sperm activation assay, the sperm and activator were mixed thoroughly and incubated at room temperature. The observations on sperm activation in 5µl aliquots of this incubation mixture were conducted at 5min interval under phase contrast microscope. Sperm mixed in double distilled water (instead of activator) formed a ‘control’. After standardizing the in-vitro assay, the sperm activation was ascertained in terms of two parameters, (i) % sperm activation, and (ii) intensity of sperm activity. For studying % sperm activation, each replicate at one particular time point constituted the mean of 10 readings (10 different visual fields on the microscopic slide having 5µl aliquot of incubated mixture of sperm and activator) from each insect. It was replicated 25 times. For assessing intensity of sperm activity, each replicate at one particular time point constituted the mean of 10 readings (of individual sperm from different visual fields) on the microscopic slide having 5µl aliquot of incubated mixture of sperm and activator from each insect, and each data comprised of two phases (i) computing the number undulations per sec, (ii) transforming the number of undulations into scale from 0-4, viz. nil undulation – ‘0’, 1-5 undulations/sec-‘1’(+), 6-10 undulations/sec –‘2’(++), >10 undulations/sec – ‘3’(+++), > 15 undulations/sec – ‘4’(++++). Each regimen observation was replicated 25 times. The sperm activation was studied in irradiated moths treated with 100Gy-400Gy (sub-sterilizing, sterilizing and higher sublethal gamma doses) doses. Further, sperm activation was compared in moths treated with substerilizing doses (100Gy and 130Gy) and their F1 progeny moths in relation to control. Sperm activation profile was also studied in mated irradiated male moths and their F1 progeny and compared with virgin response. Sperm activation would act as a tool to assess the viability of irradiated moths and their F1 male moths. Effect of age of female moth on its receptivity towards irradiated male and its F1 male progeny : The effect of age of female moth was ascertained on its receptivity with respect to irradiated male and its F1 male progeny. It was assessed in terms of mating behaviour (percent mating success and percent remating propensity) of treated male moths and translated in terms of sperm transfer and resulting viability (fertility) of the eggs laid.
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Sperm transfer to spermatheca of female mated with irradiated male(P1) and F1 moths : Sperm transfer from irradiated male moths (P1) and their ensuing F1 progeny male was assessed in the spermatheca of untreated females mated with these irradiated male moths (P1 and F1) under phase contrast and fluorescence(using DAPI) microscopy. Attractiveness of sub-sterilized males and F1 males of sub-sterilized male parents towards pheromone traps in the field cages : The orientation of sub-sterilized males and F1 males of sub-sterilized male parents were assessed towards pheromone traps in the field cages (~ 9ft x 6ft x 7ft ht). Initially, a cohort (22-30) of 0-1 day old untreated male moths (unmarked and marked with fluorescent paint), were released and evaluated for their attractiveness towards pheromone traps that were obtained from PCI (Pest Control India Pvt Ltd). This pheromone trap (Fero-TTM), consisted of a funnel base, a canopy and a transparent sleeve. The pheromone trap had a pheromone lure (Spodo-lure) that was fixed to the canopy, which was later placed over the funnel. Further, 0-1 day old irradiated male moths, and F1 male moths (derived irradiated male parent) were released and evaluated for their attractiveness towards pheromone traps (Fero-TTM). Irradiation of insects : In the irradiation facility of the Institute of Nuclear Medicine and Allied Sciences (INMAS), Ministry of Defence, Delhi, S. litura was irradiated in a Cobalt-60 source at the dose rate of 1.20-1.76 kGy/hr for the present investigations. A sublethal dose range of 100-130Gy was used to irradiate male moths (0-1-day-old) to produce partially sterilized males that could be released to mate with normal females and produce F1 sterile progeny. These sub-sterilized male moths and their F1 sterile progeny (from irradiated male parents) were used for the present experimental study. Statistics: Data were usually obtained in replicates of 12-25 for sperm descent, sperm activation and mating behavioural experiments. Any variation in number of replicates for some experiments was specified appropriately in the text. The data was subjected to analysis of variance (ANOVA). Percentage data were transformed using arcsine√x before ANOVA. Means were separated at the 5% significance level by least significant difference (LSD) test (Snedecor & Cochran 1989). Results and discussion Sperm behaviour in Lepidopteran pests is quite complex because of two main reasons, (i) two types of sperm-(apyrene and eupyrene), and (ii) multiple mating. Therefore, it is very important to understand sperm dynamics in response to sub-sterilizing gamma doses used in operational ‘F1 sterility’ programmes, which has been proposed especially for lepidopteran pests owing to their extreme radio- resistance and inheritance capacity of enhanced degree of sterility with respect to viability of sub- sterilized parent moths. Effect of gamma irradaition on sperm descent and its rhythm in sub-sterilized moths and their F1 progeny There appeared to be a regular pattern or circadian rhythm of sperm descent from testes down the reproductive tract through UVD, SV up to Duplex ejaculatorius ductus (Duplex) as has been reported earlier (Seth et al 2002). In this moth, observations over a 24 hr period the relative density of eupyrene sperm and apyrene sperm was assessed at 10-11 AM during the photophase and at 10-11 PM during scotophase and to further identify the circadian rhythm of sperm descent. Eupyrene spermatozoa were present in the male tract only in the form of bundles, whereas apyrene spermatozoa were present within the tract only as individual sperm. Thus the disaggregation of the apyrene bundles occured as apyrene sperm descended from the testes. Neither eupyrene nor apyrene spermatozoa were motile anywhere in the male tract. Movement of spermatozoa within the male tract must therefore be due to movements of the tract itself. Photophase: In the photophase, more eupyrene sperm bundles were observed in the SV (51-59 bundles) in comparison to the sperm bundles in UVD (11-13 sperm bundles) indicating that the SV is used as a
120 storage organ in the photophase during the passage of sperm from testes down to Duplex, i.e. ~ 183 sperm bundles in 0-1 day old, ~ 327 sperm bundles in 1-2 day old, and ~ 455 sperm bundles in 2-3 day old virgin male moths were observed. Similarly, markedly more numbers of apyrene sperm were observed in SV in comparison to the sperm number in UVD, which reflected that the SV was a (temporarily) storage organ in the photophase during the passage of sperm from testes down to Duplex which showed about 134,479 sperm in 0-1 day old, about 271,622 sperm in 1-2 day old, and about 342,135 sperm in 2-3 day old virgin moths. A similar pattern of alternation of sperm (eupyrene and aprene sperm) was observed in UVD and SV in 1-2 day old and 2-3 day old male moths. A regular daily increase in eupyrene and apyrene sperm in the Duplex was observed in these moths, indicating the accumulation of both types of sperm in a circadian fashion. There was no significant difference among eupyrene sperm observed in the SV of differently aged moths, and the same was true for apyrene sperm within the SV. Likewise, the density of apyrene and eupyrene sperm was similar in UVD (although contrastingly less than that in SV) for each age group of moths. Under the influence of irradiation, there was a dose dependent but statistically insignificant variation (reduction) observed in eupyrene sperm and apyrene sperm accumulation in UVD for a regimen of particular aged moths. The same was true for the SV region in which there was no significant variation in number of sperm (apyrene, eupyrene) within the regimen of a particular age group due to irradiation. A dose dependent reduction was noticed in the eupyrene sperm and apyrene sperm accumulation in the Duplex day-wise with a little (significant) impact only at 130 Gy. Scotophase : Further, during the scotophase significantly more number of eupyrene sperm bundles in UVD (78-81 bundles) were observed in comparison to the sperm bundles in SV (14-19 bundles) that reflected that UVD is the first part to receive and store sperm in scotophase uring the passage of sperm descent from testes down to Duplex which showed ~ 306 sperm bundles in 0-1 day old, ~ 453 sperm bundles in 1-2 day old, ~ 616 sperm bundles in 2-3 day old virgin untreated (normal) male moths. Similarly, markedly more number of apyrene sperm were observed in the UVD (56,000-60,000 individual, dissociated sperm) in comparison to the sperm number in SV (9,000-13,000) which was an indication that sperm descent was restricted to UVD in the scotophase during the passage of sperm descent from testes down the tract to Duplex which showed about 217,327 sperm in 0-1 day old, 365,394 sperm in 1-2 day old, and 459,681 sperm in 2-3 day old virgin untreated male moths. A similar pattern of alternation of eupyrene and aprene sperm was observed in the UVD and the SV of 0-1 day old, 1-2 day old and 2-3 day old male moths. A regular daily increase in eupyrene and apyrene sperm was observed in the Duplex of these moths, indicating the accumulation of both types of sperm in a circadian fashion. There was no significant difference among eupyrene sperm observed in SV of differently aged moths, and the same was true for apyrene sperm. Likewise, the density of apyrene and eupyrene sperm was similar in the UVD (although contrastingly more than that in the SV) for each age group of moths. Under the influence of irradiation, there was an apparent dose dependent reduction in eupyrene and apyrene sperm accumulation in the UVD for a particular aged moth. Further, there was no significant variation witnessed for the SV region at 100Gy with respect to both types of sperm, whereas apyrene sperm showed a little increase (more accumulation) in SV at 130Gy after 24 hrs of irradiation treatment (i.e. in 1-2 and 2-3 day old moths) which might be due to a slightly slower passage of sperm. A dose dependent reduction was noticed in the daily eupyrene and apyrene sperm accumulation in the Duplex with a little impact only at 130 Gy, that was not markedly different from control (Tables 1a,1b). This rhythmic release of sperm was unaffected when moths were irradiated which could exert only a little influence on quantitative sperm descent at 130Gy in male(P1) moths. This sperm behaviour could be correlated with reproductive viability of treated moths. Circadian rhythmic release of sperm with respect to photophase and scotophase was not influenced by giving sub-sterilizing irradiation doses to parent male moths, nor was their any influence in the F1
121 generation male moths. In photophase and scotophase a small quantitative decrease in sperm descent at the Duplex level was observed in P1 male at 130Gy and in F1 males at 100 and 130Gy; In the Duplex the decrease in sperm decent was logical (due to relatively less sperm production under irradiation stress) as this region retained the sperm until mating was initiated. The little accumulation of sperm during decent in the UVD during photophase and in SV during scotophase at 130Gy in P1 and F1 moths indicated that sperm descent might be slightly slower in gamma irradiated individuals. A circadian rhythm of sperm descent from testes down the reproductive tract through the UVD, the SV up to the Duplex region was witnessed by assessing the relative density of euprene sperm and apyrene sperm at 10-11 AM during the photophase and at 10-11PM during scotophase. There was more eupyrene and apyrene sperm accumulation in UVD in contrast to sperm density in scotophase, and alternate situation of more sperm density in SV in comparison to that in UVD in photophase. This reflected a descent rhythm of sperm from testes to UVD in scotophase followed by a passage of sperm through the SV in photophase. In general, sperm was seen in the UVD in a high proportion of insects at night, but the proportion containing sperm in the UVD during the day was low. The reverse pattern was seen in the case of the SV, in which sperm were commonly seen during the day, but not at night. This rhythmic alternation between day and night occurred for both eupyrene and apyrene spermatozoa. In a high proportion of the insects examined, both kinds of spermatozoa were plentiful in the UVD during the scotophase, whereas few insects had either type of sperm in the UVD during the day. Where sperm was present, the numbers of both eupyrene and apyrene sperm in the UVD were significantly greater at night than in the day (P<0.01) for days 0–1, 1–2 and 2–3 after adult eclosion. It indicated that for each type of sperm, release from the testis into the UVD apparently occurs mostly, or possibly exclusively, at night. Spermatozoa were found only rarely in the lower vas deferens and this region of the male tract was therefore not examined systematically at all sampling times. The rarity of spermatozoa in this region implied that their passage through it was rapid. Sperm numbers in the duplex increased progressively with increase in age of the adult moth. The larger numbers of spermatozoa found here made it difficult to discern a clear rhythm of sperm movement into the duplex, but since loss of sperm from the SV occurred during the photophase, it was assumed that entry into the duplex also occurred principally in the light, and the process of transfer to the duplex from the SV had been completed by early in the scotophase. The eupyrene sperm was released from the testes into the UVD during the scotophase, and further descent of sperm from the UVD to the SV occurred during a restricted period at the end of the scotophase (around the time of the light–dark transition). The transfer of spermatozoa from the SV to the duplex occurred around mid-photophase (Seth et al. 2002). Sperm descent from testes down to UVD, SV followed by its passage upto Duplex, where the sperm accumulation would occur until mating initiated, was found to pursue a daily rhythmic fashion, and this rhythmic release of sperm was unaffected by radiation which could have only a little influence at 130Gy in male moths with respect to number of sperm being moved within the tract but not in rhythmic passage of sperm. This sperm behaviour could be correlated with reproductive viability of treated moths. A vast array of periodicity in function or activities is manifested by virtually every organism (Rusak and Zucker 1979). For many organisms, the most important interval measured by internal clocks is 24h cycle of light and dark. Rhythms that coincide with this cycle are called 'circadian rhythms' derived from Latin circa meaning about, and dies meaning day (Saunders 1977). It was noticed in S. litura that sperm movement occured rhythmically with the alternation of light and dark (Seth et al. 2002). Giebultowicz et al. (1996) found that there was a daily rhythm of myogenic contractility of the male reproductive tract in L. dispar which occurred at the time of expected sperm movement along the tract. It was noted in the present study on S. litura , during dissections that the wall of the male tract of S. litura was contractile but such contractions were not investigated. Giebultowicz et al. (1997) have
122 shown that in Lymantria dispar there is an epithelial barrier to the exit of sperm from the testis into the UVD. Sperm must penetrate this barrier by moving between epithelial cells and are evidently only able to do so during a brief period in the early part of the scotophase. Bebas et al. (2001) have recently shown that there is a diurnal rhythm of penetration of epithelial barrier of the testes by sperm in S. littoralis . Similar might be the situation in case of S. litura since the sperm are not motile in the male tract, movement of sperm along the reproductive tract is presumably effected by movement of the wall of the tract. Decrease in apyrene sperm (2-13% in photophase; ~2-17% in scotophase) and eupyrene sperm (~5-13% in photophase &~6-14% in scotophase) numbers in P1 moths due to 100-130Gy was noticed, whereas, decrease in apyrene sperm (6-24% in photophase; 5-30% in scotophase) and eupyrene sperm( ~8-24% in photophase & in scotophase) in F1 moths due to 100-130Gy was observed (Tables 1a,1b). Impact of irradiation was more obvious in the F1 progeny than Parent (P1) generation with respect to quantitative sperm descent. The effect of irradiation was slightly more pronounced on eupyrene sperm descent than on apyrene sperm descent in irradiated moths. UVD & SV acted as temporary storage receptacles during passage of sperm. In certain instances, an apparent increase in eupyrene sperm accumulation in UVD of F1 moths in photophase, and little increase in (apyrene) sperm accumulation in SV of F1 moths in scotophase, was noticed, that might be probably due to slightly delayed speed of sperm movement during descent. Overall the impact of irradiation on quantitative sperm descent was more pronounced in the F1 than in the P1 generation. It also indicated that the effect of irradiation was slightly more obvious on eupyrene sperm descent than on apyrene sperm descent while considering the observations in P1 and F1 moths, although this trend at many points was not statistically apparent. Effect of gamma irradiation on sperm activation in substerilized moths and their F1 progeny After standardization of ‘in-vitro assay’ of sperm activation in adult Spodoptera litura , the in-vitro sperm activation was studied in gamma irradiated male moths. The activity of sperm of irradiated male parents and their F1 progeny was assessed of apyrene sperm in two modes, (i) % sperm eliciting activity, (ii) the degree(intensity) of activity in sperm (Tables 2-5) Proportion of sperm eliciting activity The per cent sperm activation was observed at different time points after the mixture of sperm and activator (in vitro) from 0 min up to 225 min after incubation (Table 2a). In the control, as per in-vitro sperm bioassay, the sperm activity was observed up from 5 min onwards to 225 min after sperm- activator incubation. 55% or more sperm showed activity from 15 min up to 105 min, with more than 75% sperm being active at 25 min to 90 min, followed by a sharp decline in proportion of active sperm up to 225 min. At 100-130Gy, there was a gradational effect on the proportion of sperm showing activity, with ~6-20 % reduction in active sperm proportion, during the high activity period from 25min to 90min after incubation (i.e., peak range of sperm population being active in control). After 90 min. incubating the sperm of irradiated male parents(100Gy, 130Gy) did not show any significant variation in activity with respect to control. Therefore the pattern of sperm activity profile was similar in control and irradiated moths, with a little impact of irradiation stress during peak activity period (25-90min after in-vitro sperm-activator incubation). Further, the sperm activity was affected in a dose dependant manner with evident impact at 200-250Gy (high sterilizing doses). Sperm activity was drastically affected at 300-400Gy that were higher than sterilizing dose but still under sub-lethal gamma radiation. The sperm activity was also studied in F1 progeny of substerilized moths(100Gy, 130Gy) and compared with the repsonse of parent generation. Sperm activity was affected by irradiation up to 90 min (5-34% impact in Parent generation, and 6-36% impact in F1 generation), but the response of sperm activity
123 amongst parent and F1 generation due to irradiation was not significantly different among each other, although slight dose dependant response was apparent in parent as well as F1 generation(Table 3a) Effect of irradiation on sperm activation in mated moths: Investigations at gamma doses higher than 250Gy were not conducted for sperm activity as matings were not exercised in male moths exposed to higher doses (300Gy, 400Gy). Irradiation impact on per cent sperm activation of mated moths was apparent from 130Gy onwards, but sperm intensity profile of mated male moths at 100-130Gy was not significantly affected as compared to the control, except at 60-90 min after incubation. The profile of per cent sperm showing activity after mating was more or less similar to the control in P1 moths; whereas a slight effect was apparent in F1 moths in comparison to P1 moths. Mated males showed increase in sperm activation in P1 moths (0Gy, 100Gy, 130Gy) with respect to virgin moths unlike at higher doses. Further, the mated males F1 (like P1 male moths) showed an increase in % sperm activation with respect to virgin moths during the initial phase (30 min duration) at 0Gy, 100Gy, 130Gy. Relative response of mated irradiated moths was similar to mated control (mated normal moths). The sperm activation in F1 was slightly less pronounced than in P1 moths (Table 4a,5a). Intensity of sperm activity The activity of apyrene sperm of irradiated male parents assessed in second mode, i.e., the degree (intensity) of activity in sperm, was also important in order to correlate the proportion of sperm eliciting activity with the degree of activity in sperm, and obtain an overall activity profile of apyrene sperm. The degree of sperm activity was observed at different time points after the incubation of sperm with activator (in vitro) from 0 min up to 225 min. (Table 2b). In the control, from 1 min onwards, the sperm activity was observed up to 225 min after in-vitro sperm-activator incubation, with steep rise in activityfrom 1 min onwards up to 75 min., and a high degree of sperm activity (with 3-3.52 scale) was observed from 15 min up to 90 min, which was quite in consonance with % sperm active data that indicated more than 75% sperm being active at 25 min to 90 min, followed by gradual decline in sperm activity (proportion wise and intensity wise) up to 225 min. At 100-130Gy, there was a gradational effect on proportion of sperm eliciting activity, showing ~4-24 % reduction in sperm intensity, from 60 min up to 180 min in comparison to the proportion of sperm showing activity being affected(~6-20 % reduction) from 25min to 90min, that corresponded to peak range of sperm population being active. After 180 min of in-vitro incubation, the sperm of irradiated male parents(100Gy, 130Gy) did not show any significant variation in degree of activity. The pattern of intensity was similar in irradiated moths in comparison to control, with a little impact of irradiation stress during the period from 60 min up to 180 min, after in-vitro sperm incubation. Therefore, the intensity of sperm activation profile at 100Gy and 130Gy was quite similar to the control. Intensity of sperm activation showed almost similar peak responses (>3 scale/degree) from 15min to 90min at 100Gy and control (0Gy) whereas little impact was apparent at 130Gy with peak response from 15min to 60min. Further evident irradiation effect was observed at 200Gy onwards with decrease in activity and restriction of activity duration wise in a dose dependant manner. The intensity of sperm activity was also studied in F1 progeny of substerilized moths (100Gy, 130Gy) with respect to activity of parent generation, and control. Intensity wise, sperm activity was slightly affected in the beginning, but apparently no drastic effect was evident in the parent generation and F1 generation, except at the peak period (60-105min), when the activity shown by P1 (130Gy) and F1 generation(100Gy,130Gy) was little reduced. But the response of sperm activity amongst parent and F1 generation due to irradiation was not significantly different, although slight dose dependant response was apparent (not statistically significant) in parent as well as F1 generation (Table 3b)
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Effect of irradiation on degree of sperm activity in mated moths The sperm intensity profile of mated male moths at 200Gy (and higher radiation doses) was significantly reduced as comnpared to control males (mated male), with diminishing activity than at lower doses and control. The sperm activity (intensity wise) profile after mating was more or less similar for control P1 moths and F1 moths (except at 60-90min in F1 moths where activity was little influenced). Mated males showed decrease in sperm activity intensity in P1 moths (0Gy, 100Gy, 130Gy) with as compared to respect to virgin moths unlike at higher doses. Further, mated P1 males and F1 males (100Gy, 130Gy) showed decrease in intensity of sperm activity as compared to virgin moths during initial 60 min phase at 100Gy, 130Gy. However, little increase in intensity of sperm activity was observed during later phases of activation due to irradiation stress in both generations (P1 and F1) (Table 4b,5b). The two sperm types in Lepidopteran males result from two distinct modes of spermatogenesis (Friedlander 1997). The loose apyrene sperm and bundles of eupyrene sperm remain unchanged and immotile in the storage organs (seminal vesicles or duplex) of the male lepidopterans until they are transferred to the female bursa copulatrix during mating (Katsuno 1977, Kasuga and Osanai 1984, Koudelova and Cook 2001). Both types of spermatozoa are transferred to the female but only the eupyrene ones leave the spermatheca and fertilize the eggs (Holt and North 1970, Friedlander and Gitay 1972). Both types of spermatozoa lack intrinsic motility during their migration throughout the male genital tract, only becoming motile within the spermatophore (Shepherd 1974). A number of secretions produced by male genital tract have been reported to be involved in the induction of sperm motility (Omura 1938, Shepherd 1974, 1975). According to Shepherd (1974), when the sperm are ejaculated during copulation they are mixed with secretions of the males’ common duct, which renders them vigorously motile. It has been suggested that the acquisition of motility by either eupyrene or apyrene spermatozoa is regulated by different mechanisms (Thibout 1980, Osanai et al. 1987, Takemura et al. 1999, Friedlander et al. 2001). In S. litura the sperm activity was in the form of simple waves of shorter wavelength, and spiral coiling like motions. The wave was found to originate at one end (generally a partially fixed end) of the sperm and pass anteriorly. Such a finding in S. litura is in consonance with those of Davis (1965) in Cimex lectularius . The intensely active spermatozoa of S. litura were observed moving forward, however, the distance covered by them was not observed. During movement one end of spermatozoa looked like glued at one end and the other end moving vigorously in clockwise as well as anti-clockwise manner, and slightly moving ahead. Such a movement in the spermatozoa was also reported by Richards (1963) in case of American cockroach Periplanata americana . Tschudi-Rein and Benz (1990) reported that motility in apyrene spermatozoa is characterized by a much shorter wavelength, a higher frequency (higher than the eupyrene ones) and an almost spiral like coiling motion, whereas the eupyrene spermatozoa showed a simple wave of low frequency with a wavelength almost as long as the flagellum. The two types of spermatozoa are also activated at very different times. Apyrene spermatozoa are activated during copulation while descending the ductus ejaculatorius simplex and remain highly motile in the spermatophore with in the female. In S. litura the active sperm showed about 200-700 undulations per minute after mixing it with the secretions of prostatic part. The degree of sperm intensity varied with progress in time maintaining an almost plateau of peak activity from 15 min up to 90 min followed by gradual decline in sperm activity (proportion wise and intensity wise) up to 225 min. The effect of radiation was also studied on the frequency of undulations per unit time in the active sperm of P1 and F1 male moths. Hughes and Davey (1969) also studied the tail beat frequency in the active spermatozoa of P. americana . They reported that the tail beat frequency varied with time and active spermatozoa from seminal vesicle of P. americana showed as much as 508 tail beats per minute. The occurrence of sperm activation in S. litura by secretions from the prostatic part (simplex/common
125 duct) is in consonance with many other findings of various investigators: Pieris brassicae (Tschudi- Rein and Benz 1990), Cydia pomonella (Ferro and Akre 1975, Tschudi-Rein 1988), Bombyx mori (Omura 1938, Takemura et al. 1999), Trichoplusia ni (Hubner) (Holt and North 1970), Acrolepiopsis assectella (Zeller) (Thibout 1977) and Spodoptera litura (F.) (Etman and Hooper 1979). In all the above cases the apyrene sperm was either activated while passing through the simplex or a few minutes after entering the spermatophore. In many lepidopterans it has been shown that accessory secretions have a selective effect in activating the sperms and only apyrene sperms were activated. These highly motile sperms were reported to be used in the transport of non-motile eupyrene sperms to the spermatheca (Iriki 1941, Holt and North 1970). The eupyrene sperm were reported motile only after their transfer to spermatheca (Holt and North 1970, Thibout 1971). Kasuga et al. (1987), Osanai (1992), Osanai and Isono (1997) have reported that endopeptidase of serine protease type is important for acquisition of sperm motility. Thus, spermatophore formed in the females’ bursa copulatrix, is the site of sperm maturation as well as a reactor, and the apyrene sperm stir its viscous contents to promote the dissociation of eupyrene bundles. Effect of age of female moth on its receptivity towards irradiated male and its F1 male progeny Male irradiation affected the mating success and remating propensity with female moths of different age groups, but at each particular gamma dose, age of female had little effect of mating success, although effect witnessed on the remating propensity within each regimen was predominant reduction due to female age (Table 6). The mating success of irradiated males with untreated 0-1 day old female was influenced as compared to control, with significant effect (i.e. reduction) in case of F1 progeny of 130Gy treated male parent, whereas mating success of irradiated male with untreated 2-3 day old female was not influenced compared to control. Further the percent mating success of irradiated male with untreated 5-6 day old female was again influenced in comparison with the control, with significant effect in case of F1 male progeny of 130Gy male parent. Hence, the female age could reinforce or further influence the impact of male irradiation on the mating success of F1 male derived from treated male parent with normal (untreated) female (of different age groups). The remating propensity of 0-1 day old females with irradiated males was increased compared to the control, with significant effect in case of F1 male progeny of irradiated (100Gy, 130Gy) male parent, whereas the remating propensity of 2-3 day old female with irradiated male or F1 male was not affected as compared to the control, except in case of F1 male progeny of irradiated 130Gy male parent, where the remating success of female was reduced significantly (by 35.9.%) as compared to control). Further, it may be noticed that the remating propensity of 5-6 day old females with irradiated males was decreased (by 40.8-41.9 % with respect to control), during its mating with F1 male progeny of both 100Gy and 130Gy irradiated male parent. (Table 6). The impact of female age on its re-mating propensity with irradiated male moths was more pronounced than that on its mating success. Female mating success with irradiated male and its F1 progeny got reduced with ageing of female; whereas the remating propensity of female showed an increasing trend in case of 0-1 day old female, followed by decreasing pattern with female ageing, having more impact in crosses with F1 males in a dose dependant manner, where mating frequency was less than 1.5 that would lead to monoandrous situation as indicated by Arnqvist et al. (2000). The fertility (% egg hatch) within one regimen under treated parental cross was not significantly different due to difference in female age. Further, the fertility under regimen of F1 male mating is similar among crosses with 0-1 and 2-3 day old females, whereas there was significant reduction noticed in the egg fertility examined in the crosses of 5-6 day old females with F1 male, derived from 100Gy treated male as well as 130Gy treated male (Table 6).
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Effect of irradiation on sperm transfer to spermatheca of female mated with Irradiated male (P1) and F1 moths Sperm transfer from irradiated male moths (P1 & F1) was assessed at the spermatheca level where the sperm was retained, and used for fertilization with female gametes(eggs) before oviposition (Table 7). About 60,926 sperm (eupyrene+apyrene) were counted in control insects (spermatheca of normal female mated with untreated male). There was a dose dependant reduction in sperm transferred to spermatheca (5.2% and 24.3% reduction by P1 male at 100Gy and 130Gy respectively; 29.3% and 34.2% reduction by F1 male at 100Gy & 130Gy, respectively). The females mated to F1 males received less sperm in the spermatheca than those mated with P1, although this effect was not drastic, and it could be correlated with successful matings (that transferred sperm upto spermatheca) and the degree of sterility. For instance, 82-86% of the matings with P1 males and F1 males resulted in sperm transfer up to spermathecae, and the percent response of such successful matings was not significantly different among parent and F1 generations, except in case of F1 male at 130Gy(~75.6%) with little evident effect. The sperm transfer by P1 and F1 could be correlated with sterility being caused in F1 generation (70- 80%) in comparison to sterility in parent generation(50-55%). Further, studies on the qualitative sperm transfer (eupyrene and apyrene) to mated female moths up to spermatheca in response to matings with sub-sterilized male moths and their F1 male are in progress, and this is being quantified at the spermatheca level for the first time. Attractiveness of sub-sterilized males and F1 males of substerilized male parents towards pheromone traps in the field cages. The orientation of sub-sterilized males and F1 males of sub-sterilized male parents is being assessed towards pheromone traps in the field cages. In the initial phase, the proportion of released moths and timing of evaluation was standardized to undertake this behavioural investigation that would indicate the fitness of irradiated male and their F1 progeny towards wild females in the field for mating behaviour. The male moths generally were greatly attracted towards the pheromone lure and got trapped in the pheromone traps (Fero-TTM) positioned in the field cages. Initial experiment was performed to ascertain the attraction of N- ♂ unmarked and N- ♂ marked (Fluorescent yellow) towards pheromone trap in field simulated cage. About 66.4% insects got trapped when N- ♂ unmarked and N- ♂ marked were released, with trap ratio as almost 1:1 among these groups. In the following experiment, the orientational behaviour of N-♂ (unmarked) and F1 ♂ (marked) was assessed. About 63% insects were trapped when N- ♂ unmarked and F1(100Gy) ♂ marked were released, with trap ratio as 1:0.88 within these regimens. Experiments are in progress and the degree of attractiveness of irradiated and F1 male moths (derived treated male parents) will be compared with P1 moths and untreated moths (Control) (Table 8). References Armes, N. J., Wightman, J. A., Jadhav, D. R . and Ranga Rao, G. V. 1997. Status of insecticide resistance in Spodoptera litura in Andhra Pradesh, India. Pestic. Sci. 50: 240-248. Arnqvist G, Edvardsson M, Friberg U, Nilsson T (2000) Sexual conflict promotes speciation in insects. Proc Natl Acad Sci USA 97:10460–10464 Bebas, P., Cymborowski, B. and Giebultowicz, J.M. 2001. Circadian rhythm of sperm release in males of the cotton leafworm, Spodoptera littoralis: in vivo and in vitro studies. J. Insect Physiol. 47: 859-866. Chari, M. S. and Patel, N. G. 1983. Cotton leafworm Spodoptera litura (Fabr.): its biology and integrated control measures. Cotton Development 13: 7-8. Davis, N.T. 1965. Studies of the reproductive physiology of Cimicidae (Hemiptera)-II. Artificial insemination and the function of the seminal fluid. J. Insect Physiol. 11: 355-366.
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Etman, A.A.M. and Hooper, G.H.S. 1979. Sperm precedence of the last mating in Spodoptera litura. Ann. Entomol. Soc. Amer. 72: 119-120. Giebultowicz, J.M., Blackburn, M.B., Thomas-Laemont, P.A., Weyda, F. and Raina, A.K. 1996. Daily rhythm in myogenic contractions of vas deferens associated with sperm release cycle in a moth. Journal of Comparative Physiology A 178, 629–636. Giebultowicz, J.M., Weyda, F., Erbe, E.F., and Wergin, W.P. 1997. Circadian rhythm of sperm release in the gypsy moth, Lymantria dispar: ultrastructural study of transepithelial penetration of sperm bundles. J. Insect Physiol. 43, 1133–1147. Ferro, D.N. and Akre, R.D. 1975. Reproductive morphology and mechanics of mating of the codling moth, Laspeyresia pomonella. Ann. Entomol. Soc. Amer. 68 (3): 417-424. Flint, H.M. and Kressin, E.L. 1969. Transfer of sperm by irradiated Heliothis virescens (Lepidoptera: Noctuidae) and relationship to fecundity. Canadian Entomologist 101, 500–507. Friedlander, M. 1997. Control of the eupyrene-apyrene sperm dimorphism in Lepidoptera. J. Insect Physiol. 43(12): 1085-1092. Friedlander, M. and Gitay, H. 1972. The fate of the normal-anucleated spermatozoa in inseminated females of the silkworm Bombyx mori. J. Morph. 138: 121-130. Friedlander, M., Jeshtadi, A. and Reynolds, S.E. 2001. The structural mechanism of trypsin induced intrinsic motility in Manduca sexta spermatozoa in vitro. J. Insect Physiol. 47: 245-255. Friedländer, M., Seth, R.K. and Reynolds, S.E. 2005. Eupyrene and apyrene sperm: dichotomous spermatogenesis in Lepidoptera. Advances in Insect Physiology, 32: 206-308. Higuchi, H., Yamamoto, H., and Suzuki, Y. 1994. Analysis of damage to soyabeans infested by the common cutworm Spodoptera litura Fabricius (Lepidoptera:Noctuidae). II. Estimation of Leaf Area Damged by Young Larvae Using Spectral Reflectivity’, Japanese Journal of Applied Entomology and Zoology, 38, 297-300. Holt, G.G. and North, D.T. 1970. Effects of gamma-irradiation on the mechanism of sperm transfer in Trichoplusia ni. J. Insect. Physiol. 2211-2222. Hughes, M. and Davey, K.G. 1969. The activity of spermatozoa of Periplanata. J. Insect Physiol. 15: 1607-1616. Iriki, S. 1941. On the function of apyrene spermatozoa in the silkworm (in Japanese). Zool. Mag. 53: 123-124. Kasuga, H. and Osanai, M. 1984. Migration mode of two types of spermatozoa of Bombyx mori from testis to receptaculum seminis. Zool. Sci. 1: 942. Kasuga, H., Aigaki, T. and Osanai, M. 1987. System for supply of free arginine in the spermatophore of Bombyx mori: arginine – liberating activities of the contents of male reproductive glands. Insect Biochem. 17: 317-322. Katsuno, S. 1977. Studies of eupyrene and apyrene spermatozoa in the silkworm, Bombyx mori. L. (Lepidoptera: Bombycidae). Appl. Entomol. Zool.12: 142-153. Koudelova, J. and Cook, P.A. 2001. Effect of gamma radiation and sex-linked recessive lethal mutations on sperm transfer in Ephestia kuehniella (Lepidoptera: Pyralidae). Florida Entomol. 84: 172-182. LaChance, L.E., Richard, R.D. and Ruud, R.L. 1977. Movement of eupyrene sperm bundles from the testis and storage in the ductus ejaculatoris duplex of the male pink bollworm: effects of age, strain, irradiation, and light. Ann. Ent. Soc. Amer. 70, 647–651.
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Lefroy, H. M. 1908. The tobacco caterpillar, Prodenia littoralis Mem. Dept. Agric. India Entomol. Ser. 2: 79-93. Moussa, M. A., Zaher, M. A. and Kotby, F. 1960. Abundance of cotton leafworm, Prodenia litura (F.) in relation to host plants. I. Host plants and their effect on biology. Bull. Soc. Entomol. Egypte 44: 241- 251. Omura, S. 1938. Studies of the reproductive system of the male of Bombyx mori. II. Post-testicular organs and post-testicular behaviour of the spermatozoa. J. Fac. Agr. Hokkaido Imperial Univ. 40: 129- 170. Osanai. M. 1992. Arginine degradation cascade, a novel energy yielding system for spermatozoa of the silkworm, Bombyx mori (Bombycidae). Frontiers and New Horizons in amino acid Research 15: 295- 302 Osanai, M. and Isono, M. 1997. Dissociation of eupyrene sperm bundle by acids, specially succinate accumulated in the spermatophore of the silk moth, Bombyx mori. Invert. Reprod. Dev. 31: 99-108. Osanai, M., Aigaki, T. and Kasuga, H. 1987. Energy metabolism in the spermatophore of the silkworm, Bombyx mori associated with accumulation of alanine derived from arginine. Insect Biochem. 17: 71- 75 Ramakrishnan, N., Saxena, V. S. and Dhingra, S. 1984. Insecticide resistance in the population of Spodoptera litura (Fb.) in Andhra Pradesh. Pesticides 18: 23-27. Richards, A. G. (1963). The rate of sperm locomotion in the cockroach as a function of temperature. J. Insect Physiol. 9: 545-549. Rusak, B. and Zucker, I. 1979. Neural regulation of circadian rhythms. Physiol. Rev. 59: 449-526. Saunders, D.S. (1977). An introduction to biological rhythms. Glasgow, Scotland: Blackie. pp. 170. Seth, R.K. and Sharma, Vandana P. 2001. Inherited sterility by substerilizing radiation in Spodoptera litura (Lepidoptera: Noctuidae): Bioefficacy and potential for pest suppression. Florida Entomologist, 84 (2): 183-193. Seth, R.K., Rao, D.K. and Reynolds, S.E. 2002. Movement of spermatozoa in the reproductive tract of adult male Spodoptera litura: daily rhythm of sperm descent and the effect of light regime on male reproduction. J. Insect Physiol. 48: 119-131. Seth, R.K. and Sehgal, S.S. 1993. Partial sterilizing radiation doses-effects on F-l progeny of Spodoptera litura(Fabr.) : growth, bioenergetics and reproductive competence. In: Proc. International IAEA/FAO Symp. on Management of Insect Pests : Nuclear and Related Molecular and Genetic Techniques, I.A.E.A., Vienna (Oct. 19-23, 1992) : 427-440. Shepherd, J.G. (1974). Sperm activation in saturniid moths: some aspects of the mechanism of activation. J. Insect Physiol. 20: 2321-2328. Shepherd, J.G. (1975). A polypeptide sperm activator from male saturniid moths. J. Insect Physiol. 21: 9-22. Snedecor, G.W., and Cochran, W.G. 1989. Statistical Methods, (8th ed), Ames, IA: Iowa State University Press. Takemura, Y., Kanda, T. and Horie, Y. 1999. Artificial insemination using trypsin –treated sperm in the silkworm, Bombyx mori. J. Insect Physiol. 45: 471-477. Thibout, E. 1971. Description de l’appareil genital male et formation du spermatophore chez Acrolepia assectella (Lepidoptera: Plutellidae). C.R. Hebd. Seances Acad. Sci. Ser. D 237: 2546-2549.
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Thibout, E. 1977. La migration spermatique chez Acrolepiopsis (Acrolepia) assectella Zell. (Lepidoptera: Plutellidae): role de la motilite des spermatozoides et dela musculature de l’appareil genitale femalle. Ann. Entomol. Soc. Fr. (N.S.) 13: 381-389. Thibout, E. 1980. Evolution and role of apyrene sperm cells in lepidopterans: their activation and denaturation in the leak moth, Acrolepiopsis assectella (Hypnomeutoidea). In : Advances in Invertebrate Reproduction. Edited by Clark, W.H. and Adams, T.S. Elsevier Science, Amsterdam. 231-242 Tschudi-Rein, K. 1988. Aspects of the reproductive biology of Pieris brassicae (Lepidoptera: Pieridae) with special reference to eupyrene and apyrene spermatozoa. Doctoral thesis. Swiss Federal Institute of Technology, Zurich, Switzerland. Tschudi-rein, K. and Benz, G. 1990. Mechanism of sperm transfer in female Pieris brassicae (Lepidoptera: Pieridae). Ann. Entomol. Soc. Amer. 83(6): 1158-1164.
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Table 1a . Eupyrene and apyrene sperm descent from testes in the reproductive tract of gamma irradiated male Spodoptera litura 1 and its F1 progeny during photophase Gamma Age of Apyrene Sperm Eupyrene sperm dose to male (Number of dissociated sperm) (Number of sperm bundles) male (Gy) moth /Moth (days) UVD SV Duplex UVD SV Duplex generation 0Gy/P 1 0-1d old 10,385a 42,335a 1,34,479a 11.91a 54.62ab 183.2a male ±519 ±2369 ±5966 ±0.98 ±2.4 ±9.2 1-2d old 9,606a 49,021a 2,71,622b 13.67a 59.1a 327.1b ±814 ±2763 ±13688 ±1.64 ±3.6 ±21.6 2-3d old 10,622a 43,220a 3,42,135c 11.9a 51.2b 455.6c ±732 ±2498 ±17106 ±1.9 ±2.9 ±30.9 100Gy/ 0-1d old 9,908a 41,185a 1,45,743a 10.6a 55.3a 173.0a P1 male ±503 ±2,346 ±7082 ±0.86 ±3.0 ±11.0 1-2d old 10,440a 42,278a 2,58,258b 14.1a 58.4a 307.6b ±702 ±3027 ±14075 ±2.2 ±2.9 ±16.5 2-3d old 6,626b 39,538a 3,43,149c 8.7a 52.3a 433.9c ±426 ±2250 ±19220 ±1.2 ±2.6 ±22.2 130Gy/ 0-1d old 9,842a 44,758a 1,32,693a 11.2ab 50.2a 159.4a P1 male ±596 ±4344 ±9154 ±1.9 ±2.6 ±9.3 1-2d old 10,499a 39,515a 2,53,146b 8.4a 53.2a 284.4b ±647 ±1975 ±14425 ±0.95 ±2.9 ±13.3 2-3d old 7,128b 35,829a 2,98,006c 12.9b 53.6a 399.1c ±815 ±1824 ±22685 ±1.1 ±3.1 ±22.7 100Gy/ 0-1d old 7,890ab 45,892a 1,26,962a 13.3ab 48.1a 168.2a F1 male ±631 ±4923 ±6887 ±1.3 ±3.9 ±9.9 1-2d old 9,662a 40,350a 2,26,698b 16.5a 49.1a 289.1b ±763 ±2421 ±15642 ±1.4 ±4.4 ±19.9 2-3d old 6,990b 38,361a 3,25350c 11.4b 57.2a 388.6c ±575 ±3108 ±26028 ±0.67 ±4.3 ±30.6 130Gy/F 1 0-1d old 8,780ab 40,647a 1,26,056a 10.8a 44.5ab 154.5a male ±698 ±2033 ±9697 ±1.1 ±3.5 ±8.8 1-2d old 11,282a 43,907a 2,06,128b 13.3a 39.1a 264.2b ±979 ±3029 ±15017 ±0.92 ±2.9 ±23.9 2-3d old 8,413b 38,603a 2,74,108c 12.4a 48.2b 346.3c ±671 ±2663 ±25421 ±0.88 ±4.2 ±27.3
1 Virgin male moth (P1 male/F1 male) was examined in different age groups; For the assessment of apyrene sperm, the sample of apyrene sperm from each reproductive tract portion was diluted in Belar’s saline (500µl for UVD and SV each; and 1500µl for duplex), with 2µl aliquot for each observation of counting apyrene dissociated (individual) sperm, and mean of five such aliquot observations constituting each replicate; For the assessment of eupyrene sperm (bundles), 50µl Lacto aceto orcein(LAO) was added to each of the reproductive tract portions; 5µl aliquot was taken for each reading with mean of 5 such aliquot observations constituting each replicate for each tract portion.
Means ± SE followed by same letter in a column within each regimen of a particular gamma dose among different age groups are not significantly different at P<0.05 level (ANOVA followed by LSD post-test); n=25
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Table 1b. Eupyrene and apyrene sperm descent from testes in the reproductive tract of gamma irradiated male Spodoptera litura 1 and its F1 progeny during scotophase Gamma Age of Apyrene sperm Eupyrene sperm dose to male male (Number of dissociated sperm) (Number of sperm bundles) (Gy) moth /Moth (days) UVD SV Duplex UVD SV Duplex generation 0Gy/P 1 male 0-1d old 56,537a 9353a 2,17,327a 80.3a 14.01a 306.3a ±2844 ±508 ±12231 ±4.1 ±0.79 ±17.3 1-2d old 59,613a 12,758b 3,65,394b 78.1a 16.37ab 453.1b ±3048 ±676 ±18269 ±4.3 ±0.92 ±30.2 2-3d old 60,272a 9,846a 4,59,681c 80.2a 19.24b 616.3c ±3789 ±576 ±22419 ±4.8 ±1.3 ±28.4 100Gy/ 0-1d old 54,334a 11,224a 1,94,387a 74.3a 13.3a 278.6a P1 male ±3142 ±662 ±9719 ±4.22 ±1.2 ±15.6 1-2d old 51,088a 9,411b 3,26,516b 71.6a 12.8a 405.6b ±2860 ±564 ±18092 ±4.1 ±1.1 ±27.2 2-3d old 48,784a 12,821a 4,52,949c 75.6a 15.3a 576.7c ±2536 ±641 ±20336 ±5.2 ±0.9 ±33.2 130Gy/ 0-1d old 52,723a 9,309a 1,81,350a 68.3a 16.5a 263.5a P1 male ±2636 ±522 ±10155 ±3.4 ±0.92 ±14.19 1-2d old 49,827a 10,890a 3,20,580b 71.9a 12.4b 411.6b ±2790 ±544 ±18799 ±3.45 ±0.81 ±22.7 2-3d old 50,493a 14,029b 4,04,059c 70.8a 14.08ab 536.5c ±2524 ±786 ±22,915 ±4.49 ±0.88 ±31.3 100Gy/ 0-1d old 56,996a 13680a 1,43,852a 76.0a 10.25a 245a F1 male ±2849 ±1472 ±8055 ±4.43 ±0.9 ±15.5 1-2d old 45,736b 10,082b 2,82,956b 66.7a 14.9b 416.1b ±2561 ±596 ±14149 ±3.78 ±0.86 ±25.8 2-3d old 46,089b 12,141ab 4,39,285c 69.2a 12.7ab 550.7c ±2581 ±607 ±24,599 ±3.8 ±1.2 ±28.8 130Gy/F 1 0-1d old 51,885a 11,876ab 1,53,327a 68.3a 15.8a 243.9a male ±2224 ±690 ±8586 ±3.4 ±0.9 ±24.7 1-2d old 44,780a 10,288a 2,89,829b 71.9a 18.29a 386.7b ±2409 ±522 ±16238 ±4.28 ±1.1 ±21.2 2-3d old 46,899a 12,235b 3,92,244c 67.9a 15.1a 476.5c ±2626 ±685 ±21965 ±3.21 ±0.88 ±28.6
1 Virgin male moth (P1 male/F1 male) was examined in different age groups; For the assessment of apyrene sperm, the sample of apyrene sperm from each reproductive tract portion was diluted in Belar’s saline (500µl for UVD and SV each; and 1500µl for duplex), with 2µl aliquot for each observation of counting apyrene dissociated (individual) sperm, and mean of five such aliquot observations constituting each replicate; For the assessment of eupyrene sperm (bundles), 50µl Lacto aceto orcein(LAO) was added to each of the reproductive tract portions; 5µl aliquot was taken for each reading with mean of 5 such aliquot observations constituting each replicate for each tract portion.
Means ± SE followed by same letter in a column within each regimen of a particular gamma dose among different age groups are not significantly different at P<0.05 level (ANOVA followed by LSD post-test); n=25
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Table 2a. Sperm 1 activation in gamma irradiated 2-3 day old virgin male Spodoptera litura adults, assessed by in-vitro bioassay Gamma Per cent sperm showing activity Dose to Time interval (min.) after incubation of sperm with activator male moth (Gy) 0min 5min 10min 15min 20min 25min 30min 45min 60min 75min 90min 105min 135min 165min 195min 225min 0Gy No 22.58a 42.29a 55.48a 69.32a 76.12a 90.81a 89.71a 85.94a 83.73a 78.74a 57.49a 25.22a 14.58a 6.97a 3.02a activity ±1.88 ±3.83 ±3.59 ±4.05 ±2.73 ±1.19 ±2.39 ±3.13 ±2.28 ±2.97 ±5.82 ±4.69 ±3.51 ±1.06 ±0.94
100Gy - 12.49b 17.50b 36.42b 49.25b 65.87b 81.02b 83.66ab 81.49a 74.01a 67.18b 51.97a 22.01ab 13.52a 4.68ab 1.78ab ±1.08 ±3.11 ±5.39 ±4.36 ±2.88 ±1.86 ±1.79 ±2.12 ±3.93 ±5.04 ±3.65 ±1.4 ±1.14 ±0.59 ±0.37
130Gy - 11.31b 17.06b 36.61b 49.89b 61.81b 73.13c 78.38b 78.91a 73.78a 62.62b 48.62a 23.44ab 11.08a 3.93bc 1.40a ±1.68 ±2.51 ±2.91 ±3.21 ±2.33 ±1.54 ±2.03 ±2.71 ±2.15 ±3.46 ±3.10 ±2.24 ±1.53 ±0.86 ±0.36
200Gy - 13.63b 17.55b 39.18b 47.47bc 50.48c 61.39d 60.08c 58.48b 40.72b 38.42c 26.36b 13.59b 7.48ab 4.21b 0b ±3.75 ±3.94 ±3.25 ±2.71 ±2.57 ±2.35 ±2.35 ±2.53 ±2.17 ±2.69 ±2.42 ±1.94 ±1.99 ±1.70
250Gy - 10.6b 10.78bc 35.10b 41.7c 46.4cd 57.9d 48.30d 40.7c 39.70b 28.5d 22.3b 15.6b 5.68b 1.96c 0b ±2.71 ±4.13 ±3.07 ±2.35 ±5.18 ±4.35 ±2.12 ±4.09 ±4.03 ±3.28 ±3.71 ±3.07 ±3.64 ±0.64
300Gy - 2.9c 3.6c 16.12c 29.6d 41.6d 31.8e 24.30e 22..9d 16.7c 15.5e 11.6c 6.7c 6.84b 0d - ±0.72 ±2.55 ±2.26 ±3.32 ±4.30 ±4.92 ±4.39 ±3.78 ±4.39 ±4.13 ±3.74 ±2.22 ±1.54
400Gy - 0d 4.61c 10.55c 17.4e 15.1e 25.5e 21.67e 20.13d 11.01c 5.10f 1.72d 0d 0c - - ±0.65 ±2.36 ±2.81 ±2.36 ±4.76 ±4.19 ±3.03 ±3.75 ±1.03 ±0.53
Means ± SE followed by same letter in a column are not significantly different at P<0.05 level (ANOVA followed by LSD post-test); percentage data were arcsine transformed before ANOVA, but data in table are back transformations; 1 Apyrene sperm from 2-3 days virgin male adults; Each replicate at one particular time point constituted mean of 10 readings (10 different visual fields on the microscopic slide having 5µl aliquot of mixture of sperm and activator) from each insect. n=25
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Table 2b. Intensity of sperm activation in gamma irradiated 2-3 day old virgin male Spodoptera litura assessed by in-vitro bioassay Dose Degree of sperm activity to male Time interval (min.) after incubation of sperm with activator moth (Gy) 1 2 3 5 10 15 20 25 30 45 60 75 90 105 135 165 180 195 225 min min Min Min min min min min min Min min min min min Min min Min min min 0Gy 1.08a 1.52a 2.03a 2.59a 2.86a 3.06a 3.22a 3.4a 3.45a 3.48a 3.44a 3.52a 3.36a 2.84a 2.11a 1.79a 1.43a 1.03a 0.58a ±0.15 ±0.13 ±0.17 ±0.13 ±0.11 ±0.09 ±0.10 ±0.07 ±0.06 ±0.07 ±0.05 0.09 ±0.12 ±0.11 ±0.12 ±0.18 ±0.13 ±0.13 ±0.05
100Gy 0.58b 1.55a 2.14a 2.59a 2.74a 2.96a 3.21a 3.39a 3.42a 3.33a 3.28ab 3.03b 3.19ab 2.34b 1.98ab 1.63ab 1.23ab 0.96a 0.51a ±0.09 ±0.20 ±0.10 ±0.07 ±0.09 ±0.08 ±0.09 ±0.03 ±0.04 ±0.04 ±0.05 ±0.09 ±0.12 ±0.10 ±0.09 ±0.07 ±0.08 ±0.15 ±0.12
130Gy 0.63b 1.75a 2.15a 2.51a 2.88a 3.19a 3.24a 3.32a 3.43a 3.45a 3.09b 2.92b 2.54bc 2.30b 1.86b 1.50a 1.21ab 0.95a 0.48ab ±0.05 ±0.23 ±0.09 ±0.06 ±0.17 ±0.19 ±0.09 ±0.16 ±0.14 ±0.04 ±0.07 ±0.09 ±0.09 ±0.11 ±0.11 ±0.10 ±0.10 ±0.11 ±0.13
200Gy 0.49b 1.02b 1.48b 1.83b 2.10b 2.2b 2.40ab 2.60b 2.77b 2.82a 2.64b 2.44bc 2.18c 2.07bc 1.94ab 1.43b 1.13bc 0.43b 0b ±0.12 ±0.08 ±0.10 ±0.23 ±0.18 ±0.20 ±0.43 ±0.21 ±0.08 ±0.32 ±0.36 ±0.42 ±0.37 ±0.12 ±0.26 ±0.28 ±0.25 ±0.20
250Gy 0.41b 1.03b 1.41b 1.80b 1.70bc 1.60bc 1.80b 1.96bc 1.99c 2.01b 1.85bc 1.66c 1.59c 1.51c 1.29b 1.18b 0.69c 0c - ±0.09 ±0.07 ±0.19 ±0.19 ±0.30 ±0.29 ±0.21 ±0.32 ±0.25 ±0.36 ±0.40 ±0.30 ±0.27 ±0.33 ±0.31 ±0.22 ±0.26 0.28b± 0.02Ch eck 300Gy 0c 0.52c 0.91c 1.52b 1.70bc 1.80bc 1.80b 2.12bc 1.90cd 1.60bc 1.40cd 0.90d 0.80d 0.50d 0c 0c 0d 0c - ±0.17 ±0.11 ±0.09 ±0.10 ±0.12 ±0.10 ±0.14 ±0.10 ±0.16 ±0.15 ±0.09 ±0.04 ±0.02 0.56c± 0.59c± 0.02Ch 0.04 eck!!! Check! !! 400Gy - 0d 0.47d 0.70c 1.40c 1.50c 1.70b 1.40c 1.12d 0.95c 0.63d 0.42e 0.29e 0.11e 0c - - - - ±0.15 ±0.12 ±0.12 ±0.26 ±0.20 ±0.22 ±0.26 ±0.25 ±0.23 ±0.12 ±0.02 ±0.06
Means ± SE followed by same letter in a column are not significantly different at P<0.05 level (ANOVA followed by LSD post-test); 1 Apyrene sperm from 2-3 days virgin male adults; Each replicate at one particular time point constituted mean of 10 readings (of individual sperm from different visual fields) on the microscopic slide having 5µl aliquot of mixture of sperm and activator from each insect; Each data comprises of two phases (i)computing the number undulations per sec, (ii) transforming the number of undulations into scale from 0-4, viz. nil undulation – ‘0’, 1-5 undulations/sec-‘1’(+), 6-10 undulations/sec –‘2’(++), >10 undulations/sec – ‘3’(+++), > 15 undulations/sec – ‘4’(++++); n=25
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Table 3a . Sperm 1 activation in gamma irradiated sub-sterilized male Spodoptera litura and its F1 progeny assessed by in-vitro bioassay Gamma Per cent sperm showing activity Dose to Time interval (min.) after incubation of sperm with activator male moth 0min 5min 10min 15min 20min 25min 30min 45min 60min 75min 90min 105min 135min 165min 195min 225min (Gy) 0Gy No 22.58a 42.29a 55.48a 69.32a 76.12a 90.81a 89.71a 85.94a 83.73a 78.74a 57.49a 25.22a 14.58a 6.97a 3.02a activity ±1.88 ±3.83 ±3.59 ±4.05 ±2.73 ±1.19 ±2.39 ±3.13 ±2.28 ±2.97 ±5.82 ±4.69 ±3.51 ±2.06 ±0.94
100Gy - 12.49b 17.50b 36.42b 49.25b 65.87bc 81.02b 83.66ab 81.49ab 74.01ab 67.18b 51.97a 22.01a 13.52a 4.68a 1.78ab (P 1 ±1.08 ±3.11 ±5.39 ±4.36 ±2.88 ±1.86 ±1.79 ±2.12 ±3.93 ±5.04 ±3.65 ±1.4 ±1.14 ±0.59 ±0.37 moth) 130Gy - 11.31b 17.06b 36.61b 49.89b 61.81c 73.13c 78.38b 78.91bc 73.78ab 62.62b 48.62a 23.44a 11.08a 3.93a 1.40ab (P 1 ±1.68 ±2.51 ±2.91 ±3.21 ±2.33 ±1.54 ±2.03 ±2.71 ±2.15 ±3.46 ±3.10 ±2.24 ±1.53 ±0.86 ±0.36 moth) 100Gy - 10.32b 14.64b 38.15b 52.52b 71.27ab 83.65b 78.14b 72.96bc 70.09b 61.82b 51.29a 23.09a 12.3a 4.89a 0.58b (F 1 ±1.74 ±1.15 ±2.4 ±3.79 ±2.75 ±2.01 ±2.56 ±2.72 ±3.11 ±3.71 ±5.08 ±2.26 ±1.57 ±0.76 ±0.39 moth) 130Gy - 9.64b 12.77b 35.19b 52.23b 65.92bc 78.33bc 79.94b 69.99c 65.19b 58.70b 50.09a 21.04a 13.49a 5.11a 0.78b (F 1 ±1.80 ±1.05 ±1.66 ±2.61 ±1.70 ±1.77 ±3.51 ±5.03 ±5.91 ±5.93 ±5.01 ±2.16 ±1.18 ±0.47 ±0.08 moth)
Means ± SE followed by same letter in a column are not significantly different at P<0.05 level (ANOVA followed by LSD post-test); percentage data were arcsine transformed before ANOVA, but data in table are back transformations; 1 Apyrene sperm from 2-3 days virgin male adults; Each replicate at one particular time point constituted mean of 10 readings (10 different visual fields on the microscopic slide having 5µl aliquot of mixture of sperm and activator) from each insect. n=25
135
Table 3b. Intensity of sperm activation in gamma irradiated sub-sterilized male Spodoptera litura and its F1 progeny assessed by in-vitro bioassay. Dose Degree of sperm activity to male Time interval (min.) after incubation of sperm with activator moth (Gy) 1 2 3 5 10 15 20 25 30 45 60 75 90 105 135 165 180 195 225 min Min Min min min min min min min Min min min min min Min min Min min min 0Gy 1.08a 1.52a 2.03a 2.59a 2.86a 3.06a 3.22a 3.4a 3.45a 3.48a 3.44a 3.52a 3.36a 2.84a 2.11a 1.79a 1.43a 1.03a 0.58a ±0.15 ±0.23 ±0.17 ±0.13 ±0.11 ±0.09 ±0.10 ±0.07 ±0.06 ±0.07 ±0.05 0.09 ±0.12 ±0.11 ±0.12 ±0.18 ±0.13 ±0.13 ±0.05
100Gy 0.58b 1.55a 2.14a 2.59a 2.74a 2.96a 3.21a 3.39a 3.42a 3.33a 3.28ab 3.03b 3.19ab 2.34b 1.98a 1.63a 1.23ab 0.96a 0.51a (P 1 ±0.09 ±0.20 ±0.10 ±0.07 ±0.09 ±0.08 ±0.09 ±0.03 ±0.04 ±0.04 ±0.05 ±0.09 ±0.12 ±0.10 ±0.09 ±0.07 ±0.08 ±0.15 ±0.02 moth)
130Gy 0.63b 1.75a 2.15a 2.51a 2.88a 3.19a 3.24a 3.32a 3.43a 3.45a 3.09b 2.92bc 2.54b 2.30b 1.86a 1.50a 1.21ab 0.95a 0.48ab (P 1 ±0.05 ±0.13 ±0.09 ±0.06 ±0.17 ±0.19 ±0.09 ±0.16 ±0.14 ±0.04 ±0.07 ±0.09 ±0.09 ±0.11 ±0.11 ±0.10 ±0.10 ±0.11 ±0.03 moth)
100Gy 0.56b 1.57a 1.94a 2.32ab 2.59a 2.78ab 3.04a 3.15a 3.28a 3.28a 3.08b 2.78bc 2.52b 2.32b 1.86a 1.57a 1.18ab 0.93a 0.27b (F 1 ±0.06 ±0.17 ±0.56 ±0.08 ±0.09 ±0.26 ±0.28 ±0.09 ±0.24 ±0.19 ±0.20 ±0.17 ±0.29 ±0.15 ±0.13 ±0.12 ±0.16 ±0.10 ±0.11 moth)
130Gy 0.48b 1.48a 1.79a 2.12b 2.61a 2.78b 2.93a 3.14a 3.23a 3.18a 2.96b 2.69c 2.42b 2.03b 1.78a 1.53a 1.10b 0.84a 0.20b (F 1 ±0.05 ±0.15 ±0.08 ±0.06 ±0.17 ±0.09 ±0.31 ±0.28 ±0.26 ±0.26 ±0.18 ±0.11 ±0.07 ±0.17 ±0.11 ±0.10 ±0.08 ±0.09 ±0.01 moth)
Means ± SE followed by same letter in a column are not significantly different at P<0.05 level (ANOVA followed by LSD post-test); 1 Apyrene sperm from 2-3 days virgin male adults; Each replicate at one particular time point constituted mean of 10 readings (of individual sperm from different visual fields) on the microscopic slide having 5µl aliquot of mixture of sperm and activator from each insect; Each data comprises of two phases (i)computing the number undulations per sec, (ii) transforming the number of undulations into scale from 0-4, viz. nil undulation – ‘0’, 1-5 undulations/sec-‘1’(+), 6-10 undulations/sec –‘2’(++), >10 undulations/sec – ‘3’(+++), > 15 undulations/sec – ‘4’(++++); n=25
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Table 4a. Percent sperm activation in gamma irradiated mated male Spodoptera litura assessed by in-vitro bioassay. Gamma Per cent sperm showing activity Dose to male moth Time interval (min.) after incubation of sperm with activator (Gy) 0min 5min 10min 15min 20min 25min 30min 45min 60min 75min 90min 105min 135min 165min 195min 225min 0Gy No 36.75a 67.2a 74.6a 78.6a 85.7a 93.33a 87.47a 79.14a 72.1a 62.8a 57.06a 35.70a 19.13a 7.69a 1.28a activity ±4.69 ±4.29 ±3.49 ±4.41 ±3.05 ±2.01 ±3.79 ±2.01 ±1.68 ±2.39 ±2.41 ±2.65 ±2.34 ±1.25 ±0.28
100Gy - 29.2ab 57.7ab 63.43b 68.7a 74.8a 81.57b 75.73a 76.0ab 63.45b 58.0a 47.8b 26.60a 19.37a 3.95b 0b ±2.63 ±3.53 ±4.79 ±4.73 ±4.44 ±1.23 ±4.64 ±2.13 ±2.02 ±2.86 ±2.54 ±2.82 ±1.81 ±0.89
130Gy - 22.6b 49.7b 56.6b 70.7a 79.9a 80.55b 78.59a 71.17b 65.57b 56.80a 44.62b 28.92a 20.38a 3.12b 0b ±4.25 ±2.39 ±4.46 ±3.61 ±4.95 ±3.18 ±4.83 ±2.47 ±2.19 ±2.78 ±2.71 ±3.47 ±3.14 ±0.94
200Gy - 21.27b 26.22c 38.43c 39.09b 47.91b 60.74c 52.31b 47.72c 45.72c 32.77b 24.81c 18.16b 9.92b 0.58c 0b ±2.56 ±2.88 ±2.43 ±2.85 ±1.90 ±1.75 ±2.48 ±2.13 ±2.99 ±1.70 ±1.31 ±1.61 ±1.13 ±0.28
250Gy - 13.45c 18.54c 31.25c 38.61b 43.55b 54.12c 53.28b 48.84c 38.85c 27.50b 18.60c 9.33c 5.08c 0d - ±2.71 ±3.21 ±2.77 ±2.12 ±2.14 ±2..92 ±3.18 ±2.89 ±3.03 ±3.28 ±1.81 ±1.83 ±1.31
Means ± SE followed by same letter in a column are not significantly different at P<0.05 level (ANOVA followed by LSD post-test); percentage data were arcsine transformed before ANOVA, but data in table are back transformations; 1 Apyrene sperm from 2-3 days virgin male adults; Each replicate at one particular time point constituted mean of 10 readings (10 different visual fields on the microscopic slide having 5µl aliquot of mixture of sperm and activator) from each insect. n=25
137
Table 4b. Intensity of sperm activation in gamma irradiated mated male moth Spodoptera litura assessed by in-vitro bioassay. Dose Degree of sperm activity to male moth Time interval (min.) after incubation of sperm with activator (Gy) 1 2 3 5 10 15 20 25 30 45 60 75 90 105 135 165 180 195 225 min Min Min Min min min min min min Min min min min min Min Min Min min min 0Gy 1.02a 1.55a 1.89a 2.16a 2.48a 2.76a 3.11a 3.29a 3.35a 3.30a 3.18ab 3.03a 2.71ab 2.34a 1.97ab 1.83a 1.16a 0.70a 0.17a ±0.17 ±0.08 ±0.30 ±0.18 ±0.27 ±0.30 ±0.24 ±0.24 ±0.04 ±0.09 ±0.07 ±0.09 ±0.04 ±0.52 ±0.08 ±0.08 ±0.07 ±0.12 ±.09
100Gy 0.60a 1.69a 2.0a 2.23a 2.56a 2.88a 3.08a 3.23a 3.20a 3.30a 3.21a 3.14a 2.94a 2.72a 2.49a 2.11a 1.38a 0.82a 0.20a ±0.19 ±0.05 ±0.16 ±0.20 ±0.15 ±0.07 ±0.14 ±0.14 ±0.13 ±0.13 ±0.14 ±0.05 ±0.05 ±0.44 ±0.14 ±0.13 ±0.08 ±0.09 ±0.08
130Gy 0.58a 1.55a 2.05a 2.26a 2.54a 2.80a 3.04a 3.25a 3.20a 3.05a 2.89b 2.70b 2.52b 2.20a 2.03ab 1.68ab 1.28a 0.63a 0b ±0.21 ±0.17 0.09 ±0.06 ±0.06 ±0.07 ±0.08 ±0.09 ±0.14 ±0.06 ±0.07 ±0.09 ±0.06 ±0.06 ±0.05 ±0.08 ±0.11 ±0.13
200Gy 0b 0.73b 1.39b 1.90b 2.1b 2.23b 2.50b 2.75b 2.90ab 2.86ab 2.72b 2.48c 2.26bc 2.10 1.92b 1.52b 0.91b 0.25b 0b ±0.19 ±0.09 ±0.14 ±0.06 ±0.06 ±0.04 ±0.14 ±0.12 ±0.09 ±0.06 ±0.04 ±0.13 ±0.10 ±0.08 ±0.07 ±0.11 ±0.10
250Gy - 0.43b 1.32b 1.65b 1.97b 2.14b 2.33b 2.55b 2.66b 2.61b 2.40c 2.19d 2.06c 1.63 1.41c 0.99c 0.20c 0c - ±0.13 ±0.04 ±0.06 ±0.04 ±0.13 ±0.10 ±0.09 ±0.14 ±0.10 ±0.12 ±0.09 ±0.12 ±0.15 ±0.11 ±0.15 ±0.11
Means ± SE followed by same letter in a column are not significantly different at P<0.05 level (ANOVA followed by LSD post-test); 1 Apyrene sperm from 2-3 days virgin male adults; Each replicate at one particular time point constituted mean of 10 readings (of individual sperm from different visual fields) on the microscopic slide having 5µl aliquot of mixture of sperm and activator from each insect; Each data comprises of two phases (i)computing the number undulations per sec, (ii) transforming the number of undulations into scale from 0-4, viz. nil undulation – ‘0’, 1-5 undulations/sec-‘1’(+), 6-10 undulations/sec –‘2’(++), >10 undulations/sec – ‘3’(+++), > 15 undulations/sec – ‘4’(++++); n=25
138
Table 5a. Sperm 1 activation in gamma irradiated sub-sterilized male Spodoptera litura and its F1 progeny assessed by in-vitro bioassay. Dose to Per cent sperm showing activity male Time interval (min.) after incubation of sperm with activator moth (Gy) 0min 5min 10min 15min 20min 25min 30min 45min 60min 75min 90min 105min 135min 165min 195min 225min 0Gy No 36.75a 67.2a 74.6a 78.6a 85.7a 93.33a 87.47a 79.14a 72.1a 62.8a 57.06a 35.7a 19.13a 7.69a 1.28a activity ±4.69 ±4.29 ±3.49 ±4.41 ±3.05 ±2.01 ±3.79 ±2.01 ±1.68 ±2.39 ±2.41 ±2.65 ±2.34 ±1.25 ±0.28
100Gy - 29.2ab 57.7ab 63.43b 68.7a 74.3ab 81.57b 75.73ab 76.0ab 63.45b 58.0ab 47.8b 27.6a 19.37a 3.95b 0b (P 1 ±2.63 ±3.53 ±4.79 ±4.73 ±1.44 ±1.23 ±1.64 ±2.13 ±2.02 ±2.86 ±2.54 ±2.62 ±1.81 ±0.89 moth)
130Gy - 22.6bc 49.7b 56.6bc 70.7a 79.9ab 80.55bc 78.59ab 71.17b 65.57b 56.80ab 44.62b 28.92a 20.38a 3.12b 0b (P 1 ±4.25 ±2.39 ±4.46 ±3.61 ±4.95 ±3.18 ±4.83 ±2.47 ±2.19 ±2.78 ±2.71 ±3.47 ±3.14 ±0.94 moth)
100Gy - 12.62c 39.08c 50.6c 64.1ab 73.0b 75.88bc 69.38b 63.05bc 55.79c 53.59b 43.63b 18.73b 8.49b 2.25b 0b (F 1 ±5.27 ±3.52 ±3.16 ±2.83 ±2.85 ±2.99 ±2.30 ±3.10 ±2.29 ±3.83 ±2.96 ±2.39 ±2.75 ±0.12 moth)
130Gy - 12.61c 17.04d 32.5d 59.6b 74.47ab 72.05c 69.88b 55.49c 41.99d 25.71c 23.19c 14.30b 9.44b 2.08b 0b (F 1 ±4.30 ±5.35 ±4.70 ±3.76 ±4.29 ±2.59 ±2.16 ±3.61 ±3.15 ±3.26 ±1.63 ±1.61 ±1.04 ±0.80 moth)
Means ± SE followed by same letter in a column are not significantly different at P<0.05 level (ANOVA followed by LSD post-test); percentage data were arcsine transformed before ANOVA, but data in table are back transformations; 1 Apyrene sperm from 2-3 days virgin male adults; Each replicate at one particular time point constituted mean of 10 readings (10 different visual fields on the microscopic slide having 5µl aliquot of mixture of sperm and activator) from each insect. n=25
139
Table 5b. Intensity of sperm activation in gamma irradiated sub-sterilized male Spodoptera litura and its F1 progeny assessed by in-vitro bioassay. Dose Degree of sperm activity to male Time interval (min.) after incubation of sperm with activator moth (Gy) 1 2 3 5 10 15 20 25 30 45 60 75 90 105 135 165 180 195 225 min Min Min min min min min min min Min min min min min min Min Min Min min 0Gy 1.02a 1.55a 1.89a 2.16a 2.48a 2.76a 3.11a 3.29a 3.35a 3.30a 3.18ab 3.03a 2.71ab 2.34a 1.97a 1.83a 1.16a 0.70a 0.17a ±0.17 ±0.08 ±0.30 ±0.18 ±0.27 ±0.30 ±0.24 ±0.24 ±0.04 ±0.09 ±0.07 ±0.03 ±0.04 ±0.52 ±0.08 ±0.08 ±0.07 ±0.12 ±.09
100Gy 0.6a 1.69a 2.0a 2.23a 2.56a 2.88a 3.08a 3.23a 3.20a 3.30a 3.21a 3.14a 2.94a 2.72a 2.49a 2.11a 1.38a 0.82a 0.20a (P 1 ±0.19 ±0.05 ±0.16 ±0.20 ±0.15 ±0.07 ±0.14 ±0.14 ±0.13 ±0.13 ±0.14 ±0.05 ±0.05 ±0.44 ±0.14 ±0.13 ±0.08 ±0.09 ±0.08 moth) 130Gy 0.58a 1.55a 2.05a 2.26a 2.54a 2.80a 3.04a 3.25a 3.20a 3.05a 2.89b 2.70ab 2.52b 2.20a 2.03a 1.68ab 1.28a 0.63a 0b (P 1 ±0.21 ±0.17 0.09 ±0.06 ±0.06 ±0.07 ±0.08 ±0.09 ±0.14 ±0.06 ±0.07 ±0.09 ±0.06 ±0.06 ±0.05 ±0.08 ±0.11 ±0.13 moth) 100Gy 0.70a 1.63a 1.90a 2.1a 2.35a 2.51a 2.95a 3.16a 3.11a 3.10a 2.75b 2.65b 2.42b 2.24a 2.07a 1.75ab 1.29a 0.59a 0b (F 1 ±0.21 ±0.09 ±0.06 ±0.04 ±0.08 ±0.06 ±0.05 ±0.09 ±0.05 ±0.06 ±0.16 ±0.07 ±0.07 ±0.07 ±0.14 ±0.09 ±0.16 ±0.15 moth) 130Gy 0.45a 1.34a 1.80a 2.08a 2.28a 2.50a 2.70a 2.95a 3.14a 3.07a 3.02b 2.77ab 2.55b 2.33a 2.01a 1.59b 1.01a 0.39a 0b (F 1 ±0.20 ±0.19 ±0.07 ±0.05 ±0.06 ±0.08 ±0.07 ±0.17 ±0.09 ±0.13 ±0.04 ±0.05 ±0.05 ±0.07 ±0.08 ±0.09 ±0.16 ±0.12 moth)
Means ± SE followed by same letter in a column are not significantly different at P<0.05 level (ANOVA followed by LSD post-test); 1 Apyrene sperm from 2-3 days virgin male adults; Each replicate at one particular time point constituted mean of 10 readings (of individual sperm from different visual fields) on the microscopic slide having 5µl aliquot of mixture of sperm and activator from each insect; Each data comprises of two phases (i)computing the number undulations per sec, (ii) transforming the number of undulations into scale from 0-4, viz. nil undulation – ‘0’, 1-5 undulations/sec-‘1’(+), 6-10 undulations/sec –‘2’(++), >10 undulations/sec – ‘3’(+++), > 15 undulations/sec – ‘4’(++++); n=25
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Table 6 . Effect of age of normal (unirradiated) female adults on receptivity and mating propensity with gamma irradiated males and their F1 progeny male Spodoptera litura Dose administered to Nature of cross# Fertility %Mating % male parent success Remating* (Gy)
0Gy 0-1d T♂ x 0-1d N♀ 97.2a 88.1a 55.6a ±2.9 ±4.4 ±2.9 0-1d T♂ x 2-3d N♀ 95.7a 79.2a 56.5a ±1.5 ±4.2 ±2.8 0-1d T♂ x 5-6d N♀ 87.4a 80.1a 48.1b ±3.9 ±3.8 ±2.4 100Gy 0-1d T♂ x 0-1d N♀ 49.1a 85.2a 60.5a ±1.2 ±4.2 ±3.1 0-1d T♂ x 2-3d N♀ 48.3a 76.5ab 61.4a ±1.4 ±3.8 ±3.4 0-1d T♂ x 5-6d N♀ 44.3a 74.4b 41.3b ±1.9 ±3.7 ±1.9 130Gy 0-1d T♂ x 0-1d N♀ 42.8a 79.9a 60.8a ±0.6 ±3.9 ±4.2 0-1d T♂ x 2-3d N♀ 40.9a 74.3ab 52.2ab ±1.5 ±3.6 ±2.6 0-1d T♂ x 5-6d N♀ 40.3a 67.7b 45.8b ±0.9 ±3.3 ±2.2 100Gy 0-1d F-1♂ x 0-1d N♀ 35.3a 81.3a 64.6a ±0.9 ±4.1 ±3.2 0-1d F-1♂ x 2-3d N♀ 32.9ab 72.5a 57.5a ±1.4 ±5.6 ±2.8 0-1d F-1♂ x 5-6d N♀ 31.6b 71.5a 32.3b ±1.3 ±3.9 ±1.6 130Gy 0-1d F-1♂ x 0-1d N♀ 27.5a 75.9a 64.3a ±1.1 ±3.9 ±3.0 0-1d F-1♂ x 2-3d N♀ 26.4a 72.9a 35.6b ±0.6 ±3.6 ±1.6 0-1d F-1♂ x 5-6d N♀ 19.9b 67.9a 32.9b ±1.5 ±3.5 ±1.9
Means ± SE followed by same letter within a regimen in a column are not significantly different at P<0.05 level (ANOVA followed by LSD post-test); percentage data were arcsine transformed before ANOVA, but data in table are back transformations; n=12; # T♂: irradiated male parent moth; F-1♂: F-1 male progeny of irradiated male parent male; Evaluation of freshly emerged male moths and female moths of different age groups (16♂ vs 8♀) constituted each replicate for assessing mating success and remating propensity; * % rematings in females out of total matings that were recorded by presence of number spermatophores within bursa copulatrix. n=12-15
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Table 7 . Effect of gamma irradiation on the sperm transfer at spermatheca in correlation with mating success and fertility
Gamma dose to parent % mating success that Number sperm 1 Fertility(% egg hatch) male/ Nature of cross resulted in sperm transferred in transfer up to spermatheca of spermatheca
0Gy (Control) 96.4a 60,926a 91.4a (N♂ x N♀) ±2.8 ± 4203 ±3.1
100Gy 85.7b 57,716a 45.4b
(P 1♂ x N♀) ±2.9 ± 3232 ±2.7
130Gy 78.5bc 46,077b 41.2b
(P 1♂ x N♀) ±3.1 ± 2103 ±1.9
100Gy 82.1bc 43,042bc 26.7c
(F 1♂ x N♀) ±4.1 ± 2449 ±1.4
130Gy 75.6c 40,065c 20.9d
(F 1♂ x N♀) ±3.1 ± 1987 ±1.8
1 including both Eupyrene and Apyrene sperm; Means ± SE followed by same letter in a column within each regimen of a particular gamma dose among different age groups are not significantly different at P<0.05 level (ANOVA followed by LSD post-test); n=5 for % mating success resulting in to sperm transfer upto spermatheca, where a group of 12-15 pairs constituted each replicate; n=25 for sperm transfer in mated females; n=10 for fertility; For statistical analysis, the percentage data were transformed using arcsine √x but data in table are back transformations.
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Table 8 . Effect of gamma irradiation on orientation behavior of male Spodoptera litura towards pheromone lure (Fero-TTM) Nature of released % Total % Unmarked male % Marked # male moths insects trapped moths in pheromone male moths in trap pheromone trap Unmarked (0Gy ) 66.48 50.03a 49.96a vs. ±2.48 ±3.70 ±3.7 marked (0Gy) (50-73.3%) male moths
Unmarked (0Gy) 63.02 54.7a 48.6a vs. ±2.14 ±2.7 ±2.7 marked F 1-(100Gy) (54.1-70.8%) male moths
#Male adults were marked on thorax with florescent yellow colour; A group of 22-30 insect having 50% marked moths were released in (9ft x 6ft x 7ft) cage and number of males captured with in pheromone trap were observed after 24hr, n=10. For statistical analysis, the percentage data were transformed using arcsine √x but data in table are back transformations. Means ± SE followed by same letter in a row between marked and unmarked insects trapped are not significantly different at P<0.05 level (Student’s t-test)
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Inherited sterility (IS) stability of Litchi stem-end borer ( Conopomorpha sinensis Bradley) and field cage trials to evaluate moth quality Mei-ying Hu, Qun-fang Weng, Yue-ye Deng, Hao-hao Fu Department of Plant Protection, South China Agricultural University, Guangzhou, 510642 China
Abstract In the current study, the inherited sterility (IS) stability of litchi stem-end borer ( Conopomorpha sinensis Bradley) was confirmed and the efficiency of its control was evaluated in field cage trials. The parental generation emerged from pupae that were irradiated at doses of 0, 50, 100, 150 ，200 and 250Gy, respectively, were mated as the following combinations: ①-♂X♀(-♂: Male adults emerging from irradiated pupae, ♀: Untreated female adults), ②♂X-♀(♂: Untreated male adults, -♀: Female adults emerging from irradiated pupae), ③-♂X-♀, ④♂X♀ （CK ）, their F1 generation were then mated with untreated adults to get F2 generation, whose progenies were then mated with untreated adults to get F3 generation. The results showed that when the parental generation was irradiated at 200Gy, there were only 41% emerged from the combination of ①-♂X♀, while this value was 0% in the combination of ②♂X-♀ and ③-♂X-♀. The emergence rate of F1 generation who emerged from combination ①- ♂X♀ and crossed with untreated ones was only 9.37% when irradiated at 250Gy. The hatching rates, pupation rates, emergence rates and the longevity of parental generation decreased with the dosage increased. However, there were no significant differences in longevity between the parental generation and its offspring when irradiated with 150Gy, hence, 150Gy was selected as the sterile dosage for sterilizing litchi stem-end borer which would be released in the field. The irradiation effectiveness in F1 generation was the highest of all the tested generations, and the effectiveness sustained as the generation increased. The sex ratios showed that there were more male than female offspring no matter in which combination, and the rates would increase with the generation increasing. Field cage trial showed that releasing male adults irradiated with 150Gy at the rate of 10:1:1 (Treated male adults: Untreated male adults: Untreated female adults) could effectively control a natural population. Key words: Conopomorpha sinensis Bradley; inherited sterility stability ；field cage trial Introduction Litchi has been grown for over 3500 years and it belongs to the Sapindaceae family that is indigenous to the subtropics of southern China (Wiki 2011). Litchi fruits are rich in sugar, vitamin C, phosphorus, calcium and a spot of protein, fat and abundant mineral elements. Litchi is one of the most famous fruits in South China, especially in Guangdong Province, and is highly nutritious which makes it “the treasure of fruits” (Kong 2010). Litchi is of high economic value. It’s important for the food industry and is also one of the most competitive rare fruits in China (Yang 2006). Currently, litchi is mainly cultivated in the Provinces of Guangdong, Hainan, Guangxi, Sichuan, Fujian, Yunnan, Taiwan, etc. in China, and also in other countries such as Vietnam, Laos, Cambodia, Malaysia, Thailand, Burma, India, Sri Lanka, Indonesia, Puerto Rico, the Philippines, Mauritius, Malaysia, South Africa, Cuba, Honduras, Panama, Brazil, Trinidad, Florida of America, Hawaii, Guam and Queensland of Australia (Lu et al. 2004). In 2007, the planting area in China reached 588,000 hectares which produced a total yield of 1.65 million ton of litchi, accounting for 72.5% and 61.1% of the world planting area and production, respectively (Liu et al. 2009). However, the current export volume of fresh litchi fruit is not restricted and the major markets are still domestic. An important reason for restricting litchi export is the issue of persticde residues (Fang,
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2007). By using chemical pesticides to control Conopomorpha sinensis (Bradley) is difficult because it is a kind of borer pest. On the other side, most of the chemicals are forbidden during the process of producing organic litchi, which make it more difficult to control the pest (Zeng et al. 2007). The “3R” (Resistance, Resurgence and Residue) and “3E”(Energy, Environment and Economy) problems caused by immoderately abusing chemical pesticide have become more and more serious, resulting in ecological imbalance and environmental destruction. Meanwhile, pesticide abusing causes the problems of pesticide residues. Radiation sterility technology applied to control pests has advantages compared with traditional pest control methods. It is useful because of high specificity, effecting one pest of the whole biocoenosis and wouldn’t seem to be harmful to humans and animals and natural enemies, with no pollution for agricultural products and environmentally friendly as well as lasting effects, etc. It has broad prospects for exploitation (Peng 1994). In our previous work, the sterile dosage for the litchi stem-end borer had been studied. The quality and the sexual competitiveness of the litchi stem-end borer treated by sub-sterile dose were evaluated before releasing, and the sterile effects were evaluated by setting different mating ratios to obtain the optimal release ratio in this experiment. Materials and methods Source of insect and materials Source of insect The litchi stem-end borers were collected from litchi orchards of Guangdong Academy of Agricultural Sciences, and Zengcheng city ，Guangdong Province and Haikou city, Hainan Province, China by picking up the dropped fruit, collecting the pupae from leaves and deciduous leaves. Fruit materials Fresh litchi fruit and litchi kernel were used in this study. Radiation Source Cobalt 60 (designed by Nordion Company in Canada) was used for irradiation, located in the Guangzhou Furui High-Energy Technology Co., LTD., in Guangzhou. It was used as the source of gamma radiation. The dose rate was 3.2Gy/min. Effects of irradiation on parental generation of litchi stem-end borer Mature pupae with uniformity in size were irradiated with 0, 50, 100, 150, 200, and 250Gy. The adults developing from these irradiated pupae were then crossed with untreated adults using the following combinations: ①-♂X♀(-♂: Male emerging from irradiated pupae, ♀: Untreated Female), ②♂X-♀(♂: Untreated male, -♀: Female emerging from irradiated pupae), ③-♂X-♀, ④♂X♀ （CK). Each treatment was replicated three times. Each combination contained 30 pairs and was placed in cages (50cm × 40cm × 50cm). The cages were placed in an incubator set at 75±5% RH, 26±1 ℃ and 12:12(L: D) photoperiod, Adults were fed on 5 ％ litchi honey solution. The fresh litchi wrapped with tissue paper was placed in the cages and changed every 48 h. Their hatching rates, pupation rates, emergence rates as well as sex ratio and longevity of each generation were recorded. Effects of irradiation on progenies of litchi stem-end bore in different combinations The male and female adults of F1 generations, obtained from the 4 groups of parental generation combinations were crossed with untreated male and female adults, respectively. Then their male and female adults of F2 generation were crossed with untreated male and female adults, respectively. And it was the same as their F3 generations. Each combination contained 30 pairs and was placed in cages
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(50cm × 40cm × 50cm), each treatment was replicated three times. The cages were placed in an incubator, set at 75±5% RH, 26±1 ℃ and 12:12(L: D) photoperiod. Adults were fed on 5 ％ litchi honey solution. The fresh litchi wrapped with tissue paper was placed in the cages and changed every 48 h. Their hatching rates, pupation rates, emergence rates as well as sex ratio and longevity of each generation were recorded, respectively. Evaluation of sexual competitiveness of sub-sterile male adults by setting different mating ratios in laboratory Male adults emerging from pupae irradiated at 250Gy and emerged within 12 h were selected, and mated as the following combinations: treated male adults (TM): untreated female adults (UF) : untreated male adults (UM) with ratios of 1:1:1,3:1:1,5:1:1, 7:1:1 and 10:1:1, respectively. The number of UF was 30. Each combination was placed in cages (50cm × 40cm × 50cm). The cages were placed in the incubator at 75±5% RH, 26±1 ℃ and 12:12(L: D) photoperiod. Adults were fed on 5 ％ litchi honey solution. The fresh litchi wrapped with tissue paper was placed in the cages and changed every 48 h. The emergence rate and the number of laid eggs of each treatment were recorded, respectively. The laboratory evaluation of sexual competitiveness (C) of sub-sterile male adults was calculated using the following formula: H − E S C = a e ÷ Ee − H s N
Ha = egg hatching rate in mating between normal males and normal females ( Ha =85.33%)
Ee = egg hatching rate in competitive mating
Hs = egg hatching rate in mating between sterile males and normal females ( Hs =13.54%) S/N = ratio of sterile males and normal males Field cage triala Different kinds of litchi trees in litchi orchard of South China Agricultural University were selected and covered by nylon-screen cage (10.0 m × 3.0 m × 10.0 m), each cage contained 3 trees. 1000 sub-sterile male adults developed from the pupae irradiated at 150Gy and emerged within 12 h were released to each nylon-screen cage together as the combination of TM × UF× UM, whose ratio was 10:1:1. This experiment lasted for 5 months, and the number of population in field cages was recorded during this period and the control efficiency were evaluated. Results Effects of gamma irradiation on parental generation of litchi stem-end borer The results (Table 1,2,3) showed that the hatching rates, pupae rates, emergence rates all decreased as the dosage increased, and the highest effect on the F1 generation was observed. The pupae rate was 0% in the combination of -♂(P) by ♀(P) when irradiated at 250Gy, while this value was 0 in the combination of ♂(P) by -♀(P). There were no eggs hatching in the combination of -♂(P) by -♀(P) when irradiated at 200Gy. There were no significant differences in the longevity of their progenies of combinations -♂(P) by ♀(P) and ♂(P) by -♀(P) irradiated at a dose lower than 150Gy, compared to combination ♂(P) by ♀(P)(CK). However, there was a significant difference in the longevity of progenies when comparing combination -♂(P) by -♀(P) with combination ♂(P) by ♀(P)(CK) when the both the parental adults were treated at 50Gy at their pupae stage. There was more male than female offspring when irradiated at different dosages, and the sex ratio of F1 (Male adults: Female adults) increased as the dosage increased. The ratio was higher in the combination of ♂(P) by -♀(P) than in - ♂(P) by ♀(P), and it was the highest in the combination of -♂(P) by -♀(P) when the pupae of their parental generation were irradiated at dosages lower than 150Gy, which indicated that the irradiation
146 effect on female litchi stem-end borer was higher in female than male. There were no significant differences in longevity between the CK and combination -♂(P) by ♀(P) and ♂(P) by -♀(P) when the treated dosages were lower than 150Gy, but there was a significant difference between CK and combination -♂(P) by -♀(P), whose longevity of progenies was only 5.08 days when irradiated at 150Gy, while this value was longer in CK , reaching 16.4 days.
Table 1. Effects of irradiation on parental generation of various combinations
Dosage(Gy) ♀(P)
Hatching rates Pupa tion rates Emergenc e rates Sex ratio of Longevity Combination (%) (%) (%) F1(%) (days) 0 ♂(P) 85.03±0.45a 93.82±0.21 a 93.13±0.07 a 1.0:1 16.40±0.20a 50 -♂(P) 76.92±1.28b 80.68±0.71b 82.45±0.52b 1.3:1 15.80±0.08a 100 -♂(P) 62.45±0.65c 73.04±0.16c 75.08±1.02bc 2.5:1 15.10±0.30ab 150 -♂(P) 54.21±1.05d 59.70±0.48d 63.09±0.77c 3.9:1 14.30±0.09ab 200 -♂(P) 33.99±0.26e 34.13±0.69e 41.00±0.6d 5.1:1 13.30±0.30b c
250 -♂(P) 13.54±0.97f 0 — — 11.60±0.4c
Note: Mean values followed by the same letters are not significantly different at P = 0.05 level according to Duncan’s multiple range test (DMRT). Values are the mean of three replicates. “—“ means no observed data.
Table 2. Effects of irradiation on parental generation of various combinations
Dosage(Gy) ♂(p)
Hatching rates Pupa tion rates Emergenc e Sex ratio Longevity Combination (%) (%) rates(%) (%) (days) 0 ♀(P) 85.03±0.45a 93.82±0.21 a 93.13±0.07 a 1.0:1 16.40± 0.20a 50 -♀(P) 73.59±0.12b 77.32±0.46b 74.77±0.34b 1.4:1 15.30±0.11a 100 -♀(P) 60.92±0.05c 70.59±0.28bc 68.02±0.91bc 2.9:1 14.70±0.23ab 150 -♀(P) 48.77±0.38d 55.10±0.83d 52.30±0.41c 4.2:1 13.58±0.04 ab 200 -♀(P) 28 .05 ±0. 17 e 0 — — 11.60 ±0. 32 b 250 -♀(P) 10 .46 ±0. 34 f 0 — — 8.45 ±0. 68c
Note: Mean values followed by the same letters are not significantly different at P = 0.05 level according to Duncan’s multiple range test (DMRT). Values are the mean of three replicates. “—“ means no observed data.
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Table 3. Effects of irradiation on parental generation of various combinations Dosage(Gy) -♂
Longevity Hatching rates Pupation rates Emergence Sex ratio (days) Combination (%) (%) rates (%) (%) 0 ♂ X ♀ 85.03± 0.45a 93.82 ±0.21 a 93.13± 0.07 a 1.0:1 16.4 0±0.2 0a 50 -♀(P) 64.19 ±0.47b 61 .68 ±0. 15b 44.57 ±0.41b 2.1:1 9.30 ±0. 71b 100 -♀(P) 50.92 ±0.05c 50 .42 ±0.31c 32 .70 ±0.25c 5.3:1 8.02 ±0. 63bc 150 -♀(P) 38.77 ±0.38d 35 .30± 0.19d 15.91 ±0. 04d 7.0:1 7.18 ±0. 02c 200 -♀(P) 0 — — — — 250 -♀(P) 0 — — — —
Note: Mean values followed by the same letters are not significantly different at P = 0.05 level according to Duncan’s multiple range test (DMRT). Values are the mean of three replicates. “—“ means no observed data .
Effects of irradiation on progenies of litchi stem-end borer in different combinations. The results (Table 4, 5, 6) showed that the irradiated effects on the hatching rates, pupation rates, emergence rates and longevity were all higher than that were in the parental generation. The highest irradiation effects were observed on the F1 generation of all the parental generation, and these effects sustained as the generation increased. However, the irradiation effects of F1 generation were higher in the parental combination ♂ x -♀ (P) and -♂ x -♀ (P) when compared to the combination of -♂(P) x ♀, which suggested that the irradiation effects on female litchi stem-end borer were higher than male ones. When the parental male adults were irradiated at 150Gy before its emergence then crossed with untreated female adults, the hatching rates of combinations F1♂ x ♀ and ♂ x F1♀ were 40.98% and 36.72%, respectively. While they were 22.08% and 18.44% in F1 generation of ♂ x -♀(P), respectively; and 15.68% and 11.15% when both their male and female parental generations were irradiated at this dosage, and they were 12.97% and 0 when the male parental generation were irradiated at 200Gy in this combination. The pupation rates were 0 in the F1 generation of combinations ♂ x -♀(P) and -♂(P) x - ♀(P) when the parental generations were irradiated at 150Gy before emergence. However, there was still 10.1% pupated from larvae in combination F1♂ x ♀ when their parental male adults were irradiated at 200Gy (-♂(P) x♀) while it was 0 in combination ♂ x F1♀, which suggested that the sterility effect on female progenies were higher than it on male progenies, and combination -♂(P) x ♀could be applied in the field cage release trial. The emergence rates in the F1 generation of combination F1♂ x ♀ and ♂ x F1♀ were 51.79% and 46.81%, respectively, whose parental generation were treated at 150Gy before emergence in combination -♂(P) x ♀, while this value was 0 in F1♂ x ♀ when the parental male adults were irradiated at 200Gy before its emergence, and was 0 in F1 generation of combination -♂(P) x - ♀(P) which had been both irradiated at 100Gy before their emergence. There were no significant differences in hatching rates between in three combination styles of parental generation irradiated at 50Gy and the CK in F1 generation of combination -♂(P) x ♀ . No matter in which combination of parental generation, there was more male than female offspring in the F1, F2, F3 generations, but the trend would decrease as the generations increased. That is, the ratio of male: female would decease as the generation increased. There were no significant differences in longevity between CK and F1 progenies of combination -♂(P) x ♀ whose male adults were irradiated at 100Gy before its emergence, but there was a significant difference in combination ♂ x -♀(P) when the female adults were irradiated at the same dosage before its emergence. The longevity was only 7.25 and 6.48 days in the
148 combinations F1♂ x ♀ and ♂ x F1♀, respectively, when both of their male and female parental generations were irradiated at 50Gy before their emergence (-♂(P) x -♀(P)). The longevity was 10.25 and 10.06 days in the combinations F1♂ x ♀ and ♂ x F1♀, respectively, when their male parental generations were irradiated at 150Gy before emergence crossed with untreated female adults (-♂(P) x ♀), but they were only 9.91 and 9.04 days when whose female parental generation were irradiated at 150Gy then crossed with untreated male adults(♂ x -♀(P)), much less than CK which were 15.69 and 15.43 days, respectively. The longevity could be prolonged as the generation increased.
Table 4. Effects of irradiation on progenies generation of various combinations
Dosage(Gy) -♂(P)x ♀
Hatching rates Pupation rates Emergence Sex ratio Longevity Combination (%) (%) rates (%) (%) (days)
93.42± 0.62a 87.15±0.45 a F1♂ x ♀ 79.67±0.06a 1:1.1 15.69±0.23a 0(CK) 86.39±0.23 a ♂ x F 1♀ 79.64±0.15a 92.03±0.81a 1.2:1 15.43±0.02a
F1♂ x ♀ 70.47±1.02a 69.13±0.54b 72.65±0.02 a 2.4:1 14.27±0.61a 50 ♂ x F 1♀ 68 .19±0.90a 66 .68±0.33b 71.01±0.78 a 2.2:1 13.85±0.07a b
F1♂ x ♀ 51 .04±0.83ab 58.77±0.6c 63 .18±0.81 ab 5.3:1 13.04±1.02ab 100 ♂ x F 1♀ 48 .15±0. 36ab 55.54±0.04c 59 .55±0.01 ab 5.1:1 12.80±0.53ab
F1♂ x ♀ 40 .98±0. 06b 49.02±0.93d 51 .79±0.23 b 8.0:1 10.25±0.39b 150 ♂ x F 1♀ 36 .72±0. 48b 44.30±1.07d 46 .81±0.04 b 8.1:1 10.06±0.90b
F1♂ x ♀ 12 .97±0.01c 10.10±0.77d 0 — 9.45 ±0. 40b c 200 ♂ x F 1♀ 0 — — — 8.96 ±0. 37 bc
F2♂ x ♀ 81.43±0.58a 94.12±0.83a 88.85±0.01 a 1.2:1 15.75±0.03a 0(CK) ♂ x F 2♀ 80.75±0.14a 92.45±0.34a 87.54±0.15 a 1.1:1 15.60±0.39a
F2♂ x ♀ 76.16±0.68ab 70.07±0.94b 76 .42±0.65 a 2.2:1 15.01±0.76a 50 ♂ x F 2♀ 73.89±0.02ab 69 .32±0.41b 73 .77±0.18 a 2.1:1 14.79±0.15a
F2♂ x ♀ 53 .04±0.83b 63.03±0.61c 67.49±0.23 ab 5.1:1 13.79±0.08ab 100 ♂ x F 2♀ 50 .01±0. 49b 59 .72±0.34c 64.18±0.90 ab 5.0:1 13.45±0.36ab
F2♂ x ♀ 42 .34±0. 72bc 54 .15±0.03d 56 .01±0.45 b 7.9:1 11.97±0.40ab 150 ♂ x F 2♀ 38 .93±0. 13bc 49 .63±0.12d 49 .81±0.04 b 7.8:1 10.93±0.07ab
F3♂ x ♀ 81.97±0.23a 95.47±0.61a 89.17±0.58 a 1.1:1 16.13±0.64a 0(CK) ♂ x F 3♀ 81.03±0.89a 93.83±0.03a 88.89±0.04 a 1.1:1 15.79±0.03a
F3♂ x ♀ 78.53±0.41ab 73 .50±0.42b 80 .02±0.19 a 2.1:1 15.47±0.16a 50 ♂ x F 3♀ 76.97±0.13ab 72.89±0.04b 78 .54±0.32a 2.1:1 15.01±0.85a
F3♂ x ♀ 57 .68±0.23ab 65 .12±0.74c 72 .06±0.78 ab 5.0:1 14.66±0.10ab 100 ♂ x F 3♀ 53 .42±0. 66ab 62.54±0.27c 68 .83±0.09 ab 4.9:1 14.47±0.28ab
F3♂ x ♀ 48.78±0. 47b 59 .68±0.14d 58 .15±0.89 b 6.9:1 13.05±0.52ab 150 ♂ x F 3♀ 43 .69±0. 32b 54.07±0.64d 51 .04±0.79 b 6.8:1 11.58±0.43ab
Note: Mean values followed by the same letters are not significantly different at P = 0.05 level according to Duncan’s multiple range test (DMRT). Values are the mean of three replicates. “—“ means no observed data.
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It could be concluded that 150Gy could be used as the large scale release treated dosage of male adults before their emergence, and the sterility of litchi stem-end borer could be steadily inherited at least in three generations when the parental male adults were irradiated lower than 150Gy before their emergency.
Table 5. Effects of irradiation on progenies generation of combination ♂(P) by -♀(P)
♂ x -♀(P) Dosage(Gy) Combination Hatching rates Pupation rates Emergence Sex ratio Longevity (%) (%) rates (%) (%) (days) 93.42±0.62a 87.15±0.45a F1♂ x ♀ 79.67±0.06a 1:1.1 15.69±0.23a 0(CK) 86.39±0.23a ♂ x F 1♀ 79.64±0.15a 92.03±0.81a 1.2:1 15.43±0.02a
F1♂ x ♀ 61.06±1.12b 59 .63±0.42b 59.06±0.15 b 2.4:1 13.56±0.31ab 50 ♂ x F 1♀ 58.84±0.04b 56 .18±0.50b 57.83±0.11 b 2.3:1 12.97±0.10ab
F1♂ x ♀ 44.53±0.13 c 48.08±0.91c 41.56±0.41 c 5.2:1 11.49±0.65b 100 ♂ x F 1♀ 40 .71±0. 21 c 45.43±0.12c 37 .18±0.67 c 5.4:1 10.83±0.04b
F1♂ x ♀ 22 .08±0. 45 d 0 — — 9.91±0.18b 150 ♂ x F 1♀ 18 .44±0. 18 d 0 — — 9.04±0.85b
F2♂ x ♀ 81.43±0.58a 94.12±0.83a 88.85±0.01 a 1.2:1 15.75±0.03a 0(CK) ♂ x F 2♀ 80.75±0.14a 92.45±0.34a 87.54±0.15 a 1.1:1 15.60±0.39a
F2♂ x ♀ 64 .17±0.48b 63 .80±0.03b 64 .12±0.84 b 2.2:1 14.27±0.08ab 50 ♂ x F 2♀ 61 .01±0.56b 61.45±0.19b 61 .65±0.03b 2.1:1 13.80±0.02ab
F2♂ x ♀ 49 .83±0.01 c 53.00±0.22c 48 .21±0.68 c 5.1:1 12.69±0.03b 100 ♂ x F 2♀ 43 .51±0. 18 c 51.86±0.04c 44 .95±0.01 c 5.0:1 12.01±0.85b
F3♂ x ♀ 81.97±0.23a 95.47±0.61a 89.17±0.58 a 1.1:1 16.13±0.64a 0(CK) ♂ x F 3♀ 81.03±0.89a 93.83±0.03a 88.89±0.04 a 1.1:1 15.79±0.03a
F3♂ x ♀ 69 .01±0.06ab 68 .19±1.02b 70 .45±0.63 ab 2.1:1 15.01±0.48ab 50 ♂ x F 3♀ 65 .38±0.67ab 66.70±0.23b 65 .01±0.49 ab 2.1:1 14.95±0.10ab
F3♂ x ♀ 53 .57±0.12b 59 .15±0.68c 54.38±0.45b 5.0:1 13.45±0.72b 100 ♂ x F 3♀ 46 .01±0. 89b 54.01±0.47c 47 .04±0.68b 4.9:1 13.40±0.81b
Note: Mean values followed by the same letters are not significantly different at P = 0.05 level according to Duncan’s multiple range test (DMRT). Values are the mean of three replicates. “—“ means no observed data.
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Table 6. Effects of irradiation on progenies generation of combination -♂(P) by -♀(P)
-♂(P) x -♀(P) Dosage(Gy) Combination Hatching rates Pupation rates Emergence Sex ratio Longevity (%) (%) rates (%) (%) (days) 93.42±0.62a 87.15±0.45 a F1♂ x ♀ 79.67±0.06a 1:1.1 15.69±0.23a 0(CK) 86.39±0.23 a ♂ x F 1♀ 79.64±0.15a 92.03±0.81a 1.2:1 15.43±0.02a
F1♂ x ♀ 53.79±0.04b 46.06±0.58 b 24.07±0.90 b 4.7:1 7.25±0.51 ab 50 ♂ x F 1♀ 50.15±0.68b 44.45±0.12 b 20 .12±0.64 b 4.4:1 6.48±0.72 ab
F1♂ x ♀ 35.86±0.12c 38.25±0.89c 0 — 6.04 ±0. 15 bc 100 ♂ x F 1♀ 33.72±0.34c 36.75±0.22c 0 — 5.95 ±0. 03 bc
F2♂ x ♀ 81.43±0.58a 94.12±0.83a 88.85±0.01 a 1.2:1 15.75±0.03a 0(CK) ♂ x F 2♀ 80.75±0.14a 92.45±0.34a 87.54±0.15 a 1.1:1 15.60±0.39a
F2♂ x ♀ 58 .49±0.16b 51 .69±0.12 b 29 .69±0.02 b 4.6:1 8.01±0.49b 50 ♂ x F 2♀ 54 .58±0.11b 48 .18±0.63 b 24 .94±0.13 b 4.4:1 7.96±0.06b
F2♂ x ♀ — — — — — 100 ♂ x F 2♀ — — — — —
F3♂ x ♀ 81.97±0.23a 95.47±0.61a 89.17±0.58 a 1.1:1 16.13±0.64a 0(CK) ♂ x F 3♀ 81.03±0.89a 93.83±0.03a 88.89±0.04 a 1.1:1 15.79±0.03a
F3♂ x ♀ 65.78±0.19b 79.48±0.31 b 57.39±0.69 b 4.6:1 8.91±0.07b 50 ♂ x F 3♀ 64.21±0.58b 77.64±0.18b 55.53±0.40 b 4.5:1 8.72±0.40b
Note: Mean values followed by the same letters are not significantly different at P = 0.05 level according to Duncan’s multiple range test (DMRT). Values are the mean of three replicates. “—“ means no observed data.
Laboratory evaluation of sexual competitiveness of sub-sterile male adults by setting different mating ratios The results (Table 7) showed that under the varying numbers of normal male and irradiated adults and female adults, the sexual competitiveness increased as the sterile:untreated male ratio increased, while the hatching rate reduced as the sterile:untreated male ratio increased. There were significant differences in them as compared to the CK. When the ratios were 7:1:1 and 10:1:1, the number of egg laid by per female reduced as the hybridization proportion increased. When the ratio was 10:1:1, the hatching rate reached the lowest, which was 23.30% and could be selected as the optimal release ratio to applied to the field trial.
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Table 7. Laboratory evaluation of sexual competitiveness of sub-sterile male adults by setting different mating ratios Mating Numb er of Number of eggs laid Hatching rate Sexual competitiveness ratio adults by per female (%) index(C)
1:1:1 30:30:30 64.21±2.74b 60.53±1.94a 0.53±0.07a
3:1:1 90:30:30 70.62±3.68ab 40.28±3.00b 0.59±0.11a
5:1:1 150:30:30 77.83±3.89a 31.10±1.08c 0.62±0.05a
7:1:1 210:30:30 76.33±3.00ab 27.59±1.35cd 0.60±0.07a
10:1:1 300:30:30 75.14±5.07ab 23.30±0.74d 0.64±0.05a
Note: Mean values followed by the same letters are not significantly different at P = 0.05 level according to Duncan’s multiple range test (DMRT). Values are the mean of three replicates.
Field trial The results (Table 8) showed that the eggs laid by per female, hatching rate and the sexual competitiveness decreased slightly compared to the laboratory evaluation (Table 7). With the sterile:untreated male ratio of 10:1 the mean value sexual competitiveness was 0.47, lower than 0.5, which indicated that the sub-sterile male adults could compete the wild ones well. The value of sexual competitive index normally fluctuates between 0 and 1 (Ito et al. 1982; Iwahashi et al. 1983). The value of 1 indicates an equivalent level of competitiveness between the two types of males, while values close to zero indicate superior competitiveness of the wild male (Fried 1971). The results (Table 4) also showed that sexual competitiveness index increased as the ratio of UF: TM: UM increased, and the sexual competitiveness indexes were between 0.5333 and 0.6211 and showed no significant differences. Discussion In the present study, the inherited sterility (IS) stability of litchi stem-end borer was confirmed and the control efficiency was evaluated through semi-field cage trials. The results showed that increasing radiation doses caused reduced hatching ability, pupation, emergence in the parental generation, and these kinds of effects lasted for three generations, which implied that the inherited sterility (IS) could be inherited. The resulting F1 progeny were more sterile than the irradiated parent moths, and the males were more competitive than completely sterile males as a result of receiving a lower dose of radiation (Seth and Sharma, 2001). The results of this study showed that the irradiated effects on the F1 generation were greater than that on the parental generation, and these kinds of effects could sustain as the extension of generations. The results also showed that the progenies of combination -♂(P) by ♀were much competitive than other combinations, including that the effects on the parental male adults were less than the female adults. A further important component of mating behavior for the SIT is remating by wild females. In this study, the control efficiency was also evaluated through the field cage trial. As is known that, in the SIT, released sterile males mating with wild females produce the only sterilizing effect. If sterile females are also released, they can have a minor positive effect by distracting wild fertile males and acting as a “sperm sink”. However, the simultaneous release of both sexes is usually less economical, and also less
152 effective, than the release of only males, since there may be a tendency towards assortative mating (Robinson et. al. 1999). So irradiated male adults were released to mate with wild females as evaluation of control efficiency. In the current study, the value of the sexual competitiveness index of sub-sterile male adults by setting different mating ratios in laboratory was between 0.5333 and 0.6211, showing no significant differences as compared to the control, and the mean value of sexual competitiveness of the sub-sterile males in the field was 0.4756, suggesting that the sub-sterile males could successfully compete with wild litchi stem-end borers. Releasing male adults irradiated at 150Gy with the rate of 10:1:1 (Treated male adults: Untreated male adults: Untreated female adults) could effectively control the natural population.
Table 8. An evaluation of sexual competitiveness of released sterile males in field cages Number of Sexual Hatching rate Cage No. eggs laid by competitiveness (%) per female index (C)
1 27.07 25.32 0.51
2 49.43 27.57 0.41
3 42.33 26.00 0.48
mean±SE 39.61±6.60 26.30±0.67 0.47±0.03
Above all, it can conclude that 150Gy was the optimum sterile dosage for sterilizing litchi stem-end borer which would be released in the field, and the natural population could be effectively controlled when release at the ratio of treated male adults: untreated male adults: untreated female adults 10:1:1. References FANG J., LI Y.P., LIANG W. H., et al. 2007. Standard analysis of china litchi pesticide residue Journal of World Agriculture, 3:46-49. KONG F.L., 2010. Study on isolation, purification, structure and antioxidant activity of polysaccharides from pulp tissue of litchi (Chinensis sonn.). South China University of Technology, 6-28. LIU Y., ZHOU C.F., WAN Z., et al. 2009. 2008 annual Guangdong litchi and longan industry development present situation analysis. Journal of Guangdong Agriculture Scienc, 2:110-112. LU M.Y., GUO W, PAN J.C., et al. 2004. Production trade analysis of world litchi and longan. Journal of China tropical agriculture, 1:28-29. PENG Z.H., WANG S.W., CUI Y.S., 1994. Study on Behaviours of Aprione Germaric Imago and Radiation Sterility of Its Male lmago. Journal of Anhui Agricultural University, 50-53. Robinson, A. S., G. Franz, and K. Fisher. 1999. Genetic sexing strains in the medfly, Ceratitis capitata: development, mass rearing and field application. Trends in Entomology 2:81-104. Seth, R.K., Sharma, V.P., 2001. Inherited sterility by substerilizing radiation in Spodoptera litura (Lepidoptera: Noctuidae): bio-efficacy and potential for pest suppression. Florida Entomologist 84, 183–193. V. A. Dyck, J. Hendrichs and A. S. Robinson. Sterile Insect Technique Principles and Practice in Area- Wide Integrated Pest Management. [M]. The Netherlands: Springer. 2005. 278-280. WIKI. 2011. http://en.wikipedia.org/wiki/Lychee
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YANG B., 2006. Study on Preparation of Bio-active Compounds from Litchi (Litchi Chinensis Sonn.) Pericarp Tissues and Their Biological Activities. South China University of technology, 12-13 ZENG Z.A., LIANG G.W., LIU W.H., et al. 2007. The new record of selecting effective species of egg parasitoids of Conopomorpha sinensis Bradley (Lepidoptera: Graillariidae).Natural Enemies of Insects 29:6-11.
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Comparison between two radiation sources (Tunis and Vienna) for biological parameters and sterilization dose of carob moth, Ectomyelois ceratoniae (Lepidoptera, Pyralidae) K. Grissa, S. Chakroun, M. Skander and W. Hached INAT, Laboratoire d’Entomologie-Acarologie, Tunis The carob moth, Ectomyelois ceratoniae , is a major insect pest of dates, pomegranate, oranges and several other host plants in Tunisia. Larvae are polyphagous and attack both stored products and field crops in the Mediterranean basin and countries in the Near East region. This pest causes great economic losses and yearly infestation rates range from 20% in dates and oranges to 80% in pomegranate in Tunisia. The life cycle of the carob moth includes the adults (male and female with a wingspan of 16 to 22 cm), white eggs (0,5-0,8mm) when laid and pink eggs if fertilized, five instars (Table 1) and chrysalis. The development cycle of the carob moth depends on hosts and temperatures. At 25°C, the cycle is about 43,5 days in dates and 41,6 days in artificial diet (Fig. 1).
Table 1. Length in (mm) of the 5 larvae instars L1 mm L2 mm L3 mm L4 mm L5 mm 1,3-2,2 2-3,1 3,4-5,7 7,1-12,3 12,2-14,5
In Tunisia, the area of palm dates is about 6 000 Ha, the production is estimated at 162 000 T, the quantity of exported dates is 68 000 T which corresponds to 15% of the agricultural export. The pomegranate orchards are grown in arid regions (center and south) due to their adaptability to drought, poor soil and salinity of irrigation water (up to 4 g/l). The production of pomegranate is about 70 to 90 tons, whose export as biological product is improved every year to some countries as Libya, France, Gulf countries. The area of Navel oranges in Tunisia is equivalent to 3.650 ha which product about 41.600T (13.5% of the total production of citrus fruits) used essentially to local consumption (only some is export).
Figure 1. Development cycle of carob moth on dates and artificial diet at 25°C
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Bio-ecology, Symptoms and Damages of the carob moth on palm dates, pomegranates and Citrus The activity of the carob moth on palm dates occurs from September to December when the fruit is receptive. We observe 3 to 4 generations. The female lays eggs on fruit and the instar L1 mobile, enter by the perianth. The whole cycle takes place in the fruit, also the nymph instar and adult emerges from the fruit. Diapause occurs in dried or dropped fruits or in the soil. As symptoms, the larva spins and covers the inlet hole from the inside by the silk, the damage is due to the presence in date of caterpillar, droppings, silk which prohibits the consumption of the fruit. The activity of the carob moth on pomegranates takes place from May to August which generates 3 to 4 generations in the field. The female lays eggs in the calyx of pomegranate, the L1 instar enters in the fruit and at the end of its cycle, old larva leaves the fruit and pupate in the soil. Adult emerges from the soil. The Diapause occurs in dried or dropped fruits, under the bark, in the soil. The symptoms result from the fact that the larva spins and covers the calyx from the inside by the silk. As damage, the fruit rots and falls prematurely. Activity on Washington Navel occurs from July to November when the fruit is receptive, which generate 3 to 4 generations. The female lays eggs in the umbilicus, the L1 instar enters in the fruit and consumes the inside. At the end of its cycle, old larva leaves the fruit and pupate in the soil and the adult emerges from the soil. Diapause occurs under the bark, in dropped fruit or in the soil. The symptoms are silk and excrements in the umbilicus and as damages, the larva causes the fruit necrosis. Orange attacked before maturity changes its color (maturity false color) and fall to the ground. Many control methods have been used to keep populations below economic threshold levels. Controlling the carob moth with insecticides is not efficient because larvae feed and develop inside the fruit, where they are protected. Also, the harmful effect of insecticides on the environment and the risk of developing insecticide resistance restrict the use of this control method. Establishment of the sub-sterilizing dose for an SIT program Two experiments in Tunis (CNSTN and INAT) and Vienna (IAEA) were conducted to identify the sub- sterilizing dose for the carob moth. Five doses (100, 150, 200, 250 and 300 Gy) were tested. The parameters studied were fecundity, fertility, hatching eggs and adults emergence for 3 generations, parents, F1 and F2. The results shows no signifcant %Fecundity Vienne Tunis difference between the 100 parameters studied in Tunis 71 - 57-82% 47 -78 % 33 -70 % or those obtained at Vienne. ns ns ns The figures 2 to 5 confirm ns 75 * theses results. ns ns ns ns * ns ns Graph4. Adults emergences 50 of parents for four doses of radiation and the control 25
0 FixMi FixMi FixMi FixMi FixMn FnxMi FixMn FnxMi FixMn FnxMi FixMn FnxMi 0GY 150GY 200GY 250GY 300GY
Figure 2. Fecundity of parents for four doses of radiation and the control
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%Fertility Vienn 100 67- e ns 75 20- 16-42% ns 13- 50 ns 4-27% * ns ns ns ns ns 25 ns ns ns *
0 FixMi FixMi FixMi FixMi FixMn FnxMi FixMn FnxMi FixMn FnxMi FixMn FnxMi 0GY 150GY 200GY 250GY 300GY
Figure 3 . Fertiliity of parents for four doses of radiation and the control
F1 offspring Until 200 Gy, we still obtain a low emergence rate of adults for the F1 offspring. For the F2 offspring, we did not observe hatching eggs at 100, 150 and 200 Gy for Vienna experiment and for Tunis experiment, we obtained a low rate of larvae development. As a conclusion of this work, 250 Gy is the radiation dose needed to sterilize females and sub-sterilize males of carob moth (Figure 6).
%Hatching eggs Vienne Tunis 100
75 47-52% ns 50
10-15% 3-9% 0-5% 0-4% 25 ns ns ns ns ns ns ns ns ns ns ns ns 0
FixMi FixMi FixMi FixMi
FixMn FnxMi FixMn FnxMi FixMn FnxMi FixMn FnxMi 0GY 150GY 200GY 250GY 300GY
Figure 4. Hatching eggs of parents for four doses of radiation and the control
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%Adults Vienne Tunis emergence
100
75 42%
50 ns
4-10% 2-6% 25 0-4% 0-2% ns ns ns ns 0
FixMi FixMi FixMi FixMi FixMn FnxMi FixMn FnxMi FixMn FnxMi FixMn FnxMi 0GY 150GY 200GY 250GY 300GY
Figure 5. Adults emergences of parents for four doses of radiation and the control
%Adults emergence 100 Fi X Mn 80
60
40
20
0 0Gy 100Gy 150Gy 200Gy 250Gy 300Gy
Figure 6. Adult F1 emergence
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IPM control against the tomato leaf miner, Tuta absoluta , in Tunisia K. Grissa, M. Skander, R. Ben Jebara, A. Aissa and A. Cherif INAT, Laboratoire d’Entomologie-Acarologie, Tunis Establishement of a diet to the rearing of the tomato leaf miner An artificial diet composed by: 60% wheat bran, 12% sucrose, 12% glycerin, 10% distilled water, 2.3% yeast, 2% salt mixture, 0.67% vitamin C, 0.67% Aureomycine, 0.13% methyl parabene et 0.3% lysine; was tested to rear the tomato leaf miner but unfortunately we do not obtain good result. So, the experiment is in progress.
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