OPEN ACCESS Journal of Entomology

ISSN 1812-5670 DOI: 10.3923/je.2020.

Research Article First Report of Psyttalia lounsburyi, a New Larval Parasitoid of the Mediterranean Fruit Ceratitis capitata in Moroccan Argane Forest

1Hallouti Ayoub, 3Zahidi Abdelaziz, 2Lamharchi El Hassane, 1Boubaker Hassan, 1El Mousadik Abdelhamid and 2Ait Benaoumar Abdellah

1Laboratory of Microbial Biotechnology and Plant Protection, Faculty of Sciences, Ibn Zohr University, B.P 8106, Agadir, Morocco 2Laboratory of Biotechnology and Valorization of Natural Resources, Faculty of Sciences, Ibn Zohr University, B.P 8106, Agadir, Morocco 3Faculty of Applied Science, Ait Melloul Campus, Ibn Zohr University, Agadir, Morocco

Abstract Background and Objective: Ceratitis capitata is considered as one of the most economically destructive pest. Several alternatives have been used but efficacy remains limited. The objective consists in the search for other natural enemies such as parasitoids and evaluate parasitism rate of larval stages from infested fruits by medfly collected in argane forest. Materials and Methods: About 400 fly larvae per sample and per locality were isolated and placed in breeding boxes at optimal temperature until the emergence of adults. Number of Ceratitis adults and parasitism rate have been observed. Morphological and molecular identification using COI-F/COI-R primers of collected parasitoids were also occurred. Results: Morphological and molecular analyses confirmed the occurrence of a new haplotype of the larval parasitoid belonging to Psyttalia genus in argane forest. In addition, phylogenetic analyses revealed that these specimens were 92% (E-value = 2e-85) identical to Psyttalia lounsburyi. The presence of this parasitoid was noted in all studied localities with a variable parasitism rate which reached a value of 21.5% in the localities where agricultural activities are present. Conclusion: It can be concluded that this natural parasitoid could be native to argane forest and will be used as biological control agent against Ceratitis capitata and Bactrocera oleae in Morocco. Also in Mediterranean region which constitute a handicap to the extension of biological agriculture of fruit trees.

Key words: Psyttalia lounsburyi, Ceratitis capitata, parasitoid, phylogenetic, biological control, argane forest

Received: Accepted: Published:

Citation: Hallouti Ayoub, Zahidi Abdelaziz, Lamharchi El Hassane, Boubaker Hassan, El Mousadik Abdelhamid and Ait Benaoumar Abdellah, 2020. First report of Psyttalia lounsburyi, a new larval parasitoid of the mediterranean fruit fly Ceratitis capitata in Moroccan Argane forest. J. Entomol., CC: CC-CC.

Corresponding Author: Zahidi Abdelaziz, Laboratory of Biotechnology and Valorization of Natural Resources, Faculty of Sciences, Ibn Zohr University, B.P 8106, Agadir, Morocco

Copyright: © 2020 Hallouti Ayoub et al. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

Competing Interest: The authors have declared that no competing interest exists.

Data Availability: All relevant data are within the paper and its supporting information files. J. Entomol., 2020

INTRODUCTION the most successful examples of using natural parasitoids to control fruit including C. capitata was in Hawaii by The Mediterranean fruit fly (Ceratitis capitata introducing over 20 parasitoids including one egg-larval Wiedemann) also called the medfly is a fruit fly of the parasitoid, Fopius arisanus (Sonan) and seven larval family. Despite of being native to tropical Africa parasitoids: Psyttalia xetheri, F. Vandenbosch, and especially West Africa, this pest is continuously expand its Diachasmimorpha tryoni, Diachasmimorpha longicaudata, geographic distribution globally and now occurs on five Procecidochares incise, Opius Artun and Tetrastichus continents1. Attacking over 300 host plant species, this fruit fly givardianus (Silvestri)12,14,17-20. is considered one of the most destructive pests of fruits Psyttalia lounsburyi (Silvestri) is a larval parasitoid of production in the world especially in the Mediterranean Braconidae family used against Bactrocera oleae. It can also region and Morocco2-4. The damages of the medfly are caused parasitize Ceratitis capitata larvae as reported by several by the laying of the female in the fruit and the larval studies11,21,22. This parasitoid discovered for the first time in development inside fruits. Because larvae of this pest live East Africa (Kenya), is known by its endoparasitism of inside the fruit, control is exceedingly difficult and expensive. B. oleae23. The female of P. lounsburyi attacks the first larval Consequently, the medfly is responsible of extensive stages of its host24,25 . In addition, larvae located near to the economic and crop losses5,6. Besides causing large damages surface of infested fruits are more susceptible to this ’s to production, producer countries lose their international attacks and therefore these larvae are easily infected than markets due to quarantine regulation against this pest in those protected by fruits tissues13,26,27. Currently, several several importing countries. Accordingly, the success of fruit researches are interested in this type of parasitism and its industries in the Mediterranean region and in Morocco application in biological control especially in USA and France depends heavily on the medfly management5,7. against B. oleae24,25,28. Host suitability and host range studies Moroccan moderate climate coupled to intensive are demonstrated that B. oleae is the specific host of monoculture of fruits and wide Argania spinosa forests P. lounsburyi24,28, however, other studies showed the (wild host plant of medfly) form a favourable environment possibility of attacking other pest such as C. capitata26. In fact, with uninterrupted supply of host fruits for the medfly to this parasitoid attacks successfully larvae of C. capitata under multiply and breed3,8. Under such circumstances, the laboratory conditions and could be easily multiplied on this development of extensive control programmes becomes a fly26. In this context and with the aim of widening the range of priority. In the current time the control of the medfly mostly natural enemies against ceratitis, this work has been based on using cover sprays of insecticides with the undertaken. The objective consists in the search for other carbamate, organophosphate and synthetic pyrethroid when natural enemies such as parasitoids and evaluate parasitism fruits becomes susceptible to oviposition in order to prevent rate of larval stages from infested fruits by medfly collected in direct attacks of fruits5. In addition, spot sprays of protein baits argane forest. coupled to orchard sanitation and fruit wrapping aimed at reducing the medfly population. This chemical compounds MATERIALS AND METHODS when misused can induce ecological and toxicological problems5,9,10. In order to reduce the negative effects of The study was carried out at the Faculty of Sciences chemical control, it is necessary to develop other alternative Agadir, from March, 2018-February, 2019 in Biotechnologies strategies for managing populations of this pest. and Valorization of Natural Resources and Microbial The use of biological control agents such as parasites, Biotechnologies and Plant Protection. Molecular analyzes parasitoids and predators could be a promising and (DNA extraction and sequencing) were carried out at the sustainable alternative to chemical control by pesticides. In Technical Support Units for Scientific Research at the National addition to being safe, economical and permanent solution, Center for Scientific and Technical Research (CNRST) Rabat natural enemies are proving effective in reducing medfly Morocco. This study is in continuity until today. populations in many regions to economic tolerance thresholds11. Furthermore, a major biological control effort Insect collection and identification: During research work on against the medfly was carried out using egg-larval parasitoids entomopathogenic fungi against C. capitata isolated from belonging to Psyttalia and Fopius genera such as argane (wild host plant of medfly) soil (thesis data), infested Fopius arisanus, Fopius concolor and Fopius ceratitovorus fruits with this fly were also collected in Souss region. and Psyttalia xetheri (Braconidae)11-16. In this context, one of Following this sampling, which occurred in May 2018, it was

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Table 1: Collect localities geographical references Kit (ExoSAP-IT) to remove primers and nucleotides (dNTPS) not Sampling sites Latitude and longitude used during the PCR reaction. All sequencing reactions were Amskroud forest 30E27'22.7"N 9E23'09.8"W prepared using a Big Dye Terminator v3.1 according to the Bousehab site 30E29'22.6"N 9E13'11.8"W Tamaayt site 30E24'28.6"N 9E20'51.7"W manufacturer’s recommendations. Admin forest 30E20'17.0"N 9E28'13.8"W The obtained purified products were prepared for Ihchach site 30 17'10.8"N 9 30'31.8"W E E sequencing in the forward and reverse directions using 2 µL (3.5 pmol µLG1) of COI primers, BigDye Terminator v3.1 noted the presence in addition to fruit fly in stars several cycle sequencing kit’s (1 µL), Sequencing buffer (5x, 3 µL), DNA specimens (adults) of Hymenoptera. In addition, several matrix (1.5 µL) and Milli-Q water (2.5 µL). The products were infested larvae and pupae of C. capitata were also observed. sequenced using an Applied Biosystem ABI 3130xl sequencer In order to study this new parasitoid, several collects of (Genetic Analyzer, 16 capillaries, Applied Biosystem). infested fruit by the host insect (C. capitata) were done The obtained sequencing results were submitted to a between May-June, 2018 in different localities in Argania homology test using BLAST search function from the spinosa Forest (Table 1). Infested fruits thus collected were NCBI GenBank website. To confirm BLAST results, a placed at room temperature in laboratory to recover pest Maximum-Likelihood phylogenetic analysis was applied to larvae. About 400 larvae were isolated from each sample per compare the specimen’s generated DNA sequences with locality and placed in boxes until the emergence of adults. other species form the Opiinae subfamily which are During this study, the parasitism percentage and the number morphologically near to our insect using the Neighbor-Joining of the insect’s adults were calculated for each sampling site. method. These species are: Diachasmimorpha longicaudata Some parasitoid were conserved in 90% alcohol and stored at (GU097655.1), Phaenocarpa sp. (KJ412475.1), -4EC. Collected specimens were identified according to Pseudognaptodon sp. (KJ412473.1), Asobara japonica Wharton description keys11,21,22,29,30-32. Other specimens were (JF430429.1), Cotesia autumnatae voucher (KC414765.1), sent to Technical Support Unit for scientific research at the Cotesia griffini (AF102704.1), Glyptapanteles indiensis National Center for Scientific and Technical Research (CNRST) (AF102713.1), Cotesia salebrosa voucher (KC414773.1), in Rabat for molecular analysis. Habrobracon hebetor (MG744442.1), Fopius arisanus Molecular analysis: In order to confirm morphological (KC581417.1), Psyttalia lounsburyi (DQ983810.1) and Psyttalia concolor TN0227 (EU761025.1). The DNA sequences of these identification, total genomic DNA was extracted from insect’s species were collected from the NCBI Genbank website then adults according to kit protocol (BIOLINE) following standard compared to our P. lounsburyi (UIZ FSA-H1) COI gene protocol for tissues. Concentration and purity of DNA sequence using MEGA6 software31. of sample are measured by a 8000-Nanodrop-photometer. Primers used for PCR amplification in this study were COI-F Data analysis: Analysis of Variances (ANOVA) with one factor (5'GTCACTTGCCGGCATTTGGATTATTT-3') as a forward and COI-R (5'TGATTTTTT GTCACCCA GAAGTGTTA-3') as a reverse. (sampling site factor) was adopted. The Least Significant The PCR amplification of the mitochondrial Cytochrome Difference test (LSD " = 5%) of equality of means was used to Oxidase I (COI) gene was performed in a total of 25 µL reaction compare differences between means for the three variables: volume with the following concentrations: PCR buffer (5x), Number of Psyttalia sp. adults, percentage of parasitism and percentage of natural mortality. All statistical analyzes were MgCl2 (2 mM), dNTP (0.2 mM of each), primer (COI-F 10 µM and COI-R 10 µM (1 µL of each)), Taq polymerase (0.2 µL) performed using the statistical version 6 software. (MyTaq DNA polymerase kit, Bioline) insect DNA (150 ng). The conditions for PCR were the following: Initial RESULTS denaturation (95EC, 1 min) followed by 35 cycles of denaturation (95EC, 15 sec), primer annealing (60EC, 20 sec) Morphological identification: Several morphological criteria and DNA extension (72EC, 15 sec). The reaction was held characterize the parasitoid collected (Fig. 1). Size of individuals at 4EC. was between 2.3-3.4 mm (female adult) and 1.9-2.9 mm The size of the amplified COI-region was determined (male adult), with orange to brown color. The ovipositor is using a 7 µL aliquot of the PCR product electrophoresed on brown and 1.8±0.1 times longer than mesosoma. Wings are 1.5% agarose gel. The ladder used was 100 bp. A volume of hyaline containing brown veins, with a large stigma about 5 µL of the amplified products were purified using purification 2.9 times longer than wide, with rarising slightly in stigma

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(a) (b)

1 mm

(c) (d)

1 mm 1 mm

(e) (f )

1 mm

Fig. 1(a-f): Morphological characteristics of parasitoid, (a) Female lateral view, (b) Male lateral view, (c) Female up view (d) Female antennas (e) Male antennas and (f) Head midpoint; 2RS weakly sinuate; 3RSa 2.5 times longer than r; seemingly bare (no bristles), large, 1.5±0.3 times longer than 3RSb ending slightly in wing tip (Fig. 2). Antennas with brown wide, the temples are very receding in dorsal view. The color are 3.0-3.1 times longer than the mesosoma and maxillary palps are longer than the height of the head. constituted by 31-37 segments in adult females and between Mesosoma 1.3±0.05 times longer than high and 30-35 for adult males. The head of this insect is characterized 1.65±0.05 times longer than broad. In the field, during by variable dimensions, it is 1.55-1.75 (average = 1.65±0.07) the month of May, 2018 as well as in 2019, around 5 pm, a times wider than long. In addition, it was 1.25-1.35 times remarkable activity of the parasitoid was noted on some broader than mesoscutum, with face distinctly punctate infested fruits by Ceratitis. This activity is observed at tree throughout and containing an ocellar triangle with brown level and even on the fruit on the ground as illustrated colour. Eyes are brown to black and they are very large and in Fig. 3.

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Fig. 2: Observed Psyttalia sp. wings venation under binocular loupe (Gx40)

(a) (b)

(c) (d)

(e) (f )

Fig. 3(a-f): Activity of larvae parasitoid Psyttalia lounsburyi noted on some infested fruits by Ceratitis capitata in the field during 2018 -2019

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0.72 0.08 Phaenocarpa sp. KJ412475.1 0.66 0.11 Glyptapanteles indiensis: AF102713.1 0.15 0.64 Cotesia griffini : AF102704.1 0.79 0.05 Habrobracon hebetor: MG744442.1 0.55 Fopius arisanus: KC581417.1 0.71 Diachasmimorpha longicau data: GU097655.1 0.05 0.02 0.27 Cotesia autumnatae voucher: KC414765.1 0.01 0.56 Cotesia salebrosa voucher: KC414773.1 0.81 0.03 Psyttalia lounsburyi haplotype 1: DQ983810.1 0.66 Psyttalia lounsburyi UIZFSA-H1 0.63 0.26 Asobara japonica: JF430429.1 0.69 0.05 Pseudognaptodon sp.: KJ412473.1 0.74 0.03 Psyttalia concolor TN0227: EU761025.1

Fig. 4: Evolutionary relationships of taxa. The evolutionary history was inferred using the Neighbor-Joining method33. The optimal tree with the sum of branch length = 9.29029587 is shown (Next to the branches). The evolutionary distances were computed using the Jukes-Cantor method and are in the units of the number of base substitutions per site. The analysis involved 13 nucleotide sequences34. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 563 positions in the final dataset. Evolutionary analyses were conducted in MEGA631

influenced significantly the three parameters studied (Table 2). Indeed, variable parasitism rate was detected in C. capitata larvae obtained from all the studied sampling sites (Fig. 4). The highest parasitism (%) was observed in “Ihchach site” with 21.5% followed by “Amskroud site” with 5.5% then Admin forest with 5% of parasitism by the Psyttalia sp. In addition, these highest percentages were observed in localities near to the agricultural area. Natural mortality was also observed and could be explained by the effect of temperature, conditions inside boxes or even by the parasitism.

Molecular analysis: The PCR showed positive amplification results of the COI-F/COI-R primers with the insect’s extracted Fig. 5: PCR amplification results of insect DNA with COI-F DNA (Fig. 5). The analysis of the obtained DNA sequences primer and NAV-F primer, the positive result was clearly observed with the COI-F primer using BLAST search revealed that these specimens were 92% (E-value = 2e-85) identical to the Psyttalia lounsburyi The identification results revealed that this species (Family Braconidae, subfamily Opiinae) haplotypes 1, 2 and 3 belongs to the genus Psyttalia (Opius) from the Braconidae Cytochrome Oxidase Subunit I (COI) genes with the family, subfamily Opiinae know by their larval parasitism of corresponding Genbank accession DQ983810.1, DQ983811.1 other such as fruit flies. and DQ983812.1, respectively. The phylogenetic tree obtained from comparison of the specimen’s generated DNA sequences Species occurrence and distribution: Adults of this with other species from the Opiinae subfamily demonstrated Psyttalia sp. were observed in all boxes of different samples the relationship with specimens (Psyttalia lounsburyi collected in the studied localities. The sampling site factor UIZFSA-H1) and Psyttalia lounsburyi species (Fig. 6).

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9 (a) 8 a a 7 6 5 4 b b 3 c

Natural mortality (%) mortality Natural 2 1 0

50 (b) 45 a 40 35 30

yttalia 25 s

P 20 15 b b 10 d c

Number of Number 5 sp. adults 0

25 (c) a 20

15

10

Parasitism (%) Parasitism b b 5 c bc

0 Bousehab site Amsekroud forest Tamaayt site Admin forest Ihchach site Sampling sites

Fig. 6(a-c): Observation of (a) Percentage of natural mortality, (b) Number of Psyttalia sp. adults and (c) Percentage of parasitism in the 5 sampling sites of the argane forest, Homogeneous groups (a-d) obtained by means comparison method Least Significant Difference (LSD) at an alpha significance level of 5%

Table 2: Analysis of variance of 3 characters observed at the 5 sampling sites (Bousehab, Amskroud forest, Tamaayt, Admin forest and Ihchach) Mean square ------Source of variations Degree of freedom Natural mortality (%) Number of Psyttalia sp. adults Parasitism (%) Sampling site factor 4 14.72** 463.6** 115.90** Error 9 6.62 470.5 117.63 **Significant at 0.05

DISCUSSION areas where the pest is more abundant. Indeed, the parasitoid population levels are directly affected by the presence of the Several adults of larval parasitoid were observed among host insect. Which parasitic activity was also noticed in the individuals emerged of the fruit fly in all boxes. These forest mainly at the level of trees bearing argane fruits. individuals are from samples collected in the localities Monitoring of population dynamics of the fruit fly and its studied in the Souss region of Morocco. This parasitoid was natural enemy in the field seems to be a necessity in order to morphologically and molecularly identified using a universal recommend methods of controlling this pest in the future. primer pair COI-F/COI-R. Sampling of the host larvae Regarding the identification of this parasitoid, (C. capitata) from several localities proved the attack of this morphological criteria are identical to fruit fly’s egg-larval pest by the parasitoid in all the studied samples with parasitoid belonging to Psyttalia (Opius) genus, family parasitism rates ranged from 3.5-21.5%. The parasitoid Braconidae, subfamily Opiinae. Starting with C. capitata occurrence seems to be higher in localities near to agricultural larvae and pupae in boxes and obtaining as results

7 J. Entomol., 2020 parasitoid individuals after adult emergence confirms the the study37 didn’t use DNA sequencing for identification endoparasitism of medfly larvae. In this type of parasitism, and therefore, never mentioned the occurrence of any females lay their eggs inside larvae of their host. It attacks P. lounsburyi species in Morocco. No further studies were first-instar larvae of the fruit fly14,16,22. Indeed, a major biological found in this context which confirms that the first report of control effort against fruit flies was carried out using several this larval parasitoid in Morocco and in North Africa. Psyttalia egg larval parasitoids from of the Opiinae subfamily11,12,16,19. lounsburyi, the subject of this study, is a larval parasitoid One of the most successful examples of using natural probably native of Kenya, southern and central Africa11,21. This parasitoids to control fruit flies including C. capitata and insect has often been introduced from Africa into many Bactrocera oleae was in Hawaii by introducing about agricultural areas around the world to control olive fruit fly 20 parasitoids including one egg-larval parasitoid, Fopius Bactrocera oleae. The most successful biological control arisanu (Sonan) and seven larval parasitoids: Fopius programs using this hymenoptera were against B. oleae in vandenbosch, Diachasmimorpha tryoni, Diachasmimorpha California, USA24,25 and France28. Comparing to other longicaudata, Psyttalia xetheri, Procecidochares incise, Psyttalia parasitoids which are not selective for their hosts, 12,17-20 Psyttalia artun and Tetrastichus givardianus (Silvestri) . P. lounsburyi has been reported for been specific for However, those parasitoids seem not to be specific to the B. oleae11,22,30 and C. capitata24,26. The parasitism rates of medfly and could attack non-target and beneficial insects. In C. capitata observed on a small sample of 400 larvae are very such situations, the choice of specific parasitoids is essential in promising. Mass collect of argane infested fruits with order to succeed this biological control method and therefore C. capitata could be a solution to have more parasitoid and control pest populations. Recently, many works were carried therefore mass produce it under controlled conditions. This out to test Fopius ceratitivorus (Wharton) in fields as a new parasitoid could be a good candidate as biological agent specific parasitoid to medfly13,30,35. These studies are clearly against C. capitata and B. oleae if it’s mass produced with demonstrated that this parasitoid attacks successfully eggs flooding releases following the cycle of its host in the fields in and larvae of C. capitata with the advantage of being specific Morocco and in the Mediterranean region. Its different to this pest, it appears unlikely to attack non-target morphology suggests that this species could be a new Tephritidae in Hawaii14,15. haplotype of P. lounsburyi and probably native to Morocco. The DNA sequencing using COI-F/COI-R primers revealed 92% of similarity to P. lounsburyi species COI gene sequence. CONCLUSION This result was confirmed by the Maximum-Likelihood phylogenetic analysis. Morphological criteria of our species To minimize the harmful impact of pests on citrus such as body color are different with description of production, using parasitoid as this new haplotype of P. lounsburyi species22 and P. lounsburyi (Silvestri) which are P. lounsburyi would be an effective alternative for controlling known by a dark body color with a black pigmentation on the C. capitata populations. Monitoring of population dynamics mesosoma (thorax) and metasoma21,23 . The observed species was orange with no black spots, this color is similar to Psyttalia of the fruit fly and its natural enemy in the field seems to be a concolor. Several researches showed that variation in color has necessity in order to recommend methods of controlling been observed in P. lounsburyi during his introduction from medfly pest in the future. Africa to Hawaii in a great biological control program21,36. Approximately 50 species of Psyttalia genus are described, all SIGNIFICANCE STATEMENT are native of South and Central Africa21,22,30. Due to the morphological similarity of Psyttalia species researchers This study is the first report of a new haplotype of confronted major difficulty in establishing their taxonomic Psyttalia lounsburyi as larvae parasitoid of medfly which is identities based on morphology criteria only22,32. Thus, various considered as one of the most economically destructive pest names have frequently been treated as synonyms or as of wide range of fruits and vegetables. It will be a very separate species or confused with other species26,29. The DNA promising alternative to reduce the use of pesticides and sequencing data in the present study using BLAST and ensure phytosanitary protection, respectful of environment phylogenetic tree showed that our specimens are genetically and human health. This study will also allow researchers to near to P. lounsburyi but not to Psyttalia concolor or Fopius focus their research on optimizing the conditions of mass arisanus. Psyttalia concolor has been reported in Morocco in production in order to recommend strategies to control a study of the specific wealth of natural enemies of C. capitata medfly populations by inundative or augmentive releases associated to citrus orchards37 from 2001-2011. However, depending on the availability of host under field conditions.

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ACKNOWLEDGMENTS 10. Quesada-Moraga, E., A. Ruiz-Garcia and C. Santiago-Alvarez, 2006. Laboratory evaluation of entomopathogenic fungi We gratefully acknowledge the Moroccan Ministry of Beauveria bassiana and Metarhizium anisopliae against Scientific Research, the presidency of Ibn Zohr University for puparia and adults of Ceratitis capitata (Diptera: Tephritidae). financial support. Our thanks to administration, technical J. Econ. Entomol., 99: 1955-1966. service of CNRST (National Center for Scientific and Technical 11. Wharton, R.A., 1989. Biological Control of Fruit-Infesting Tephritidae. In: Fruit Flies of Economic Importance 87, Research) for their technical assistance and molecular Cavalloro, C.V. (Ed.)., CRC Press, New York, pp: 323-332. analyzes. We thank the anonymous reviewers and office 12. Wang, X.G. and R.H. Messing, 2003. Intra-and interspecific journal which provided helpful comments that greatly competition by Fopius arisanus and Diachasmimorpha tryoni improved the manuscript. (Hymenoptera: Braconidae), parasitoids of tephritid fruit flies. Biol. Control, 27: 251-259. REFERENCES 13. Bokonon-Ganta, A.H., M.M. Ramadan and R.H. Messing, 2007. Reproductive biology of Fopius ceratitivorus (Hymenoptera: 1. Gasperi, G., M. Bonizzoni, L.M. Gomulski, V. Murelli, C. Torti, Braconidae), an egg-larval parasitoid of the Mediterranean A.R. Malacrida and C.R. Guglielmino, 2002. Genetic fruit fly, Ceratitis capitata (Diptera: Tephritidae). Biol. Control, differentiation, gene flow and the origin of infestations of the 41: 361-367. medfly, Ceratitis capitata. Genetica, 116: 125-135. 14. Bokonon-Ganta, A.H., M.M. Ramadan, X.G. Wanga and 2. Rachid, E. and M. Ahmed, 2018. Current status and future R.H. Messing, 2005. Biological performance and potential of prospects of Ceratitis capitata Wiedemann (Diptera: Fopius ceratitivorus (Hymenoptera: Braconidae), an Tephritidae) control in Morocco. J. Entomol., 15: 47-55. egg-larval parasitoid of tephritid fruit flies newly imported to 3. Alaoui, A., A. Imoulan, Z. El Alaoui-Talibi and A. El Meziane, Hawaii. Biol. Control, 33: 238-247. 2010. Genetic structure of Mediterranean fruit fly 15. Wang, X.G., A.H. Bokonon Ganta, M.M. Ramadan and (Ceratitis capitata) populations from Moroccan endemic R.H. Messing, 2004. Egg-larval opiine parasitoids forest of Argania spinosa. Int. J. Agric. Biol., 12: 291-298. (Hym., Braconidae) of tephritid fruit fly pests do not attack the 4. Morales, P., M. Cermeli, F. Godoy and B. Salas, 2004. A list of flowerhead feeder dubautiae (Dipt., Tephritidae). Mediterranean fruit fly Ceratitis capitata Wiedemann J. Applied Entomol., 128: 716-722. (Diptera: Tephritidae) host plants based on the records of 16. Benelli, G., G. Gennari and A. Canale, 2013. Host INIA-CENIAP museum of insects of agricultural interest. discrimination ability in the tephritid parasitoid Psyttalia Entomotropica, 19: 51-54. concolor (Hymenoptera: Braconidae). J. Pest Sci., 86: 245-251. 5. Bernard, J.L., 2014. Face aux ravageurs, les solutions de lutte 17. Bess, H.A., R. van den Bosch and F.H. Haramoto, 1961. Fruit fly directe. Phytoma-La Défense Des Végétaux, 675: 9-14. parasites and their activities in Hawaii. Proc. Hawaiian 6. FAO. and IAEA., 1995. Economic evaluation of damage caused Entomol. Soc., 17: 367-378. by and methods of control of the Mediterranean fruit fly in 18. Vargas, R.I., W.A. Walsh and T. Nishida, 1995. Colonization of the Maghreb: An analysis covering three control options, newly planted coffee fields: Dominance of Mediterranean including the sterile insect technique. Report of an Expert fruit fly over oriental fruit fly (Diptera: Tephritidae). Group Organized by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. J. Econ. Entomol., 88: 620-627. 7. Allwood, A.J., 1997. Control Strategies for Fruit Flies 19. Wong, T.T. and M.M. Ramadan, 1987. Parasitization of the (Family Tephritidae) in the South Pacific. In: Management of Mediterranean and oriental fruit flies (Diptera: Tephritidae) in Fruit Flies in the Pacific, Allwood, A.J. and R.AJ. Drew (Eds.)., the Kula area of Maui, Hawaii. J. Econ. Entomol., 80: 77-80. Brown Prior and Anderson, Melbourne, pp: 171-178. 20. Fischer, M., 1971. Zwei von Uganda nach Hawaii 8. Sacantanis, K.B., 1957. La forêt d’arganier: Le plus grand foyer importierte Opius-Arten (Hymenoptera, Braconidae). de Ceratitis capitata connu au monde. Bull. Lab. Entomol. Anzeiger Schädlingskunde Pflanzenschutz, 44: 10-12. Agric., 15: 1-53. 21. Wharton, R.A. and F.E. Gilstrap, 1983. Key to and status of 9. Dimbi, S., N.K. Maniania, S.A. Lux, S. Ekesi and J.K. Mueke, opiine braconid (Hymenoptera) parasitoids used in biological 2003. Pathogenicity of Metarhizium anisopliae (Metsch.) control of Ceratitis and Dacus sl (Diptera: Tephritidae). Sorokin and Beauveria bassiana (Balsamo) Vuillemin, to Ann. Entomol. Soc. Am., 76: 721-742. three adult fruit fly species: Ceratitis capitata (Weidemann), 22. Wharton, R.A. and P.M. Marsh, 1978. New world opiinae C. rosa var. fasciventris Karsch and C. cosyra (Walker) (Hymenoptera: Braconidae) parasitic on tephritidae (Diptera). (Diptera: Tephritidae). Mycopathology, 156: 375-382. J. Washington Acad. Sci., 68: 147-167.

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23. Silvestri, F., 1914. Report of an expedition to Africa in search 30. Wharton, R.A., 1999. A review of the old world genus of the natural enemies of fruit flies (Trypaneidae): fopius wharton (Hymenoptera: Braconidae: Opiinae), with With descriptions, observations and biological notes (No. 3). description of two new species reared from fruit-infesting Board of Agriculture and Forestry, Division of Entomology. Tephritidae (Diptera). J. Hymenoptera Res., 8: 48-64. 24. Daane, K.M., K.R. Sime, X.G. Wang, H. Nadel and M.W. Johnson 31. Tamura, K., G. Stecher, D. Peterson, A. Filipski and S. Kumar, et al., 2008. Psyttalia lounsburyi (Hymenoptera: Braconidae), 2013. MEGA6: Molecular evolutionary genetics analysis potential biological control agent for the olive fruit fly in version 6.0. Mol. Biol. Evol., 30: 2725-2729. California. Biol. Control, 44: 79-89. 32. Wharton, R.A., 1987. Changes in nomenclature and 25. Wang, X.G., M.W. Johnson, V.Y. Yokoyama, C.H. Pickett and classification of some opiine Braconidae (Hymenoptera). K.M. Daane, 2011. Comparative evaluation of two olive Proc. Entomol. Soc. Washington, 89: 61-73. fruit fly parasitoids under varying abiotic conditions. 33. Saitou, N. and M. Nei, 1987. The neighbor-joining method: BioControl, 56: 283-293. A new method for reconstructing phylogenetic trees. 26. Billah, M.K., S. Kimani-Njogu, W.A. Overholt, R.A. Wharton, Mol. Biol. Evol., 4: 406-425. D.D. Wilson and M.A. Cobblah, 2005. The effect of host larvae 34. Jukes, T.H. and C.R. Cantor, 1969. Evolution of Protein on three Psyttalia species (Hymenoptera: Braconidae), Molecules. In: Mammalian Protein Metabolism, Munro, H.N. parasitoids of fruit-infesting flies (Diptera: Tephritidae). (Ed.). Academic Press, New York, pp: 21-132. Int. J. Trop. Insect Sci., 25: 168-175. 35. Lopez, M., J. Sivinski, P. Rendon, T. Holler and K. Bloem et al., 27. Giunti, G., G. Benelli, R.H. Messing and A. Canale, 2016. 2003. Colonization of Fopius ceratitivorus, a newly discovered Early adult learning affects host preferences in the tephritid African egg-pupal parasitoid (Hymenoptera: Braconidae) of parasitoid Psyttalia concolor (Hymenoptera: Braconidae). Ceratitis capitata (Diptera: Tephritidae). Florida Entomol., J. Pest Sci., 89: 529-537. 86: 53-60. 28. Groussier Bout, G., M. Thaon, A. Auguste Maros, N. Treuvey, 36. Clausen, C.P., D.W. Clancy and Q.C. Chock, 1965. Biological B. Frank, C. Gratraud and J. Malausa, 2009. Introduction control of the oriental fruit fly (Dacus dorsalis Hendel) and en France d’un nouvel auxiliaire, Psyttalia lounsburyi. other fruit flies in Hawaii. Technical Bulletin No. 1322. Le Nouvel Olivier, 72: 3-7. Agricultural Research Service. US Department of Agriculture, 29. Kimani-Njogu, S.W., M.K. Trostle, R.A. Wharton, J.B. Woolley USA. and A. Raspi, 2001. Biosystematics of the Psyttalia concolor 37. Smaili, M.C., M. Abbassi, J.A. Boutaleb and A. Blenzar, 2013. species complex (Hymenoptera: Braconidae: Opiinae): The Richesse spécifique des ennemis naturels associés aux identity of populations attacking Ceratitis capitata (Diptera: vergers d’agrumes au Maroc: Intérêt et implication pour la Tephritidae) in coffee in Kenya. Biol. Control, 20: 167-174. lutte biologique. EPPO Bull., 43: 155-166.

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