JHR 80: 49-70 (2020) pee eS," JOURNAL OF: She wentenicetente doi: |0.3897/jhr.80.57024 RESEARCH ARTICLE ) Hymenopter a Q https://jhr.pensoft.net ‘The International Society of Hymenopterisss RESEARCH First report and integrated analysis of two native Trissolcus species utilizing Bagrada hilaris eggs in California Fatemeh Ganjisaffar', Elijah J. Talamas*, Marie Claude Bon?, Thomas M. Perring' | Department of Entomology, University of California, Riverside CA 92521, USA 2 Florida State Collection of Arthropods, Division of Plant Industry, Florida Department of Agriculture and Consumer Services, Gainesville, FL 32608, USA 3 USDA-ARS European Biological Control Laboratory, 810 Avenue du Campus Agropolis, 34980 Montferrier le Lez, France Corresponding author: Elijah J. Talamas (billy.jenkins@gmail.com) Academic editor: Petr Jansta | Received 28 July 2020 | Accepted 18 November 2020 | Published 29 December 2020 http://zoobank.org/6 DE3A894-CDFC-4D3C-946E-25E492C4C851 Citation: Ganjisaffar F, Talamas EJ, Bon MC, Perring TM (2020) First report and integrated analysis of two native Trissolcus species utilizing Bagrada hilaris eggs in California. Journal of Hymenoptera Research 80: 49-70. https://doi.org/10.3897/jhr.80.57024 Abstract Surveys with sentinel eggs of Bagrada hilaris (Hemiptera: Pentatomidae) in southern California retrieved two parasitoids that were not previously known to be associated with this stink bug, Trissolcus hullensis and T. utahensis (Hymenoptera: Scelionidae). Molecular and morphological analysis of these specimens is used to modify the concept of 7’ utahensis and assess the factors that contribute to intraspecific variation. We provide an updated couplet to separate 7’ utahensis from a morphologically similar species, 7’ cosmopeplae. Keywords Bagrada bug, egg parasitoids, painted bug, Scelionidae, sentinel eggs Introduction Bagrada hilaris (Burmeister) (Hemiptera: Pentatomidae) is native to Africa, Asia, and the Middle East (Howard 1907; Husain 1924). This stink bug first was reported in the United States in Los Angeles County, California, in 2008 (Arakelian 2008). By 2015, it had spread to 21 other counties in California, and six other states (Nevada, Arizona, Copyright Fatemeh Ganjisaffar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 50 Fatemeh Ganjisaffar et al. / Journal of Hymenoptera Research 80: 49-70 (2020) Utah, New Mexico, Texas, and Hawaii) (Palumbo and Natwick 2010; Palumbo et al. 2016; Bundy et al. 2012, Vitanza 2012; Perring et al. 2013; Reed et al. 2013; Matsunaga 2014). Bagrada hilaris also has been reported from six states of Mexico (Sanchez-Pefha 2014; Torres-Acosta and Sanchez-Peha 2016; Hernandez-Chavez et al. 2018) and Chile (Fatindez et al. 2016; Fatindez et al. 2017). Bagrada hilaris attacks various vegetable crops, weedy mustards, and several ornamental plants within the mustard family (Brassicaceae). In the United States, B. hilaris has been a serious pest of cole crops (Perring et al. 2013; Reed et al. 2013; Palumbo et al. 2016). The bugs are particularly damaging to young seedlings, but they also feed on leaves, stems, flowers, and seeds of older plants (Palumbo and Natwick 2010; Huang et al. 2014). Chemical applications have been the main approach used against this pest (Palum- bo 2015) and efforts to establish a biological control program were initiated in 2014. Three egg parasitoids of B. hilaris were collected in Pakistan (Mahmood et al. 2015) and brought to the United States to be evaluated as biological control candidates: Trissolcus hyalinipennis Rajmohana & Narendran (Rajmohana 2006), Gryon gonikopalense Sharma (Platygastroidea: Scelionidae) (Sharma 1982), and Ooencyrtus mirus Triapitsyn & Power (Chalcidoidea: Encyrtidae) (Triapitsyn et al. 2020). Laboratory evaluations of these parasitoids are still ongoing, and no release permits have been issued to date (Sforza et al. 2017; Martel et al. 2019; Power et al. 2020a, b). Meanwhile, monthly surveys us- ing B. hilaris sentinel eggs have been conducted in California since the fall of 2017 to identify potential native or introduced parasitoids. Through these surveys, two scelionid species, I’ hyalinipennis and Trissolcus basalis (Wollaston) (Ganjisaffar et al. 2018), and a new encyrtid species, Ovencyrtus lucidus Triapitsyn & Ganjisaffar (Triapitsyn et al. 2020), have been identified. This study reports the discovery and identification of two additional scelionid species that parasitized B. hilaris sentinel eggs in southern Califor- nia: Trissolcus hullensis (Harrington) and Trissolcus utahensis (Ashmead). Our analysis includes 7’ utahensis reared from sentinel eggs of Podisus maculiventris (Say) (Hemiptera: Pentatomidae) in British Columbia, Canada, to assess the limits of this species and determine its intraspecific variation using molecular and morphological data. Previous identification of the Canadian specimens brought attention to problems with the couplet that separates 7’ utahensis and Trissolcus cosmopeplae (Gahan) in the key to Nearctic Trissolcus of Talamas et al. (2015). This key has been used in annual workshops to iden- tify stink bug egg parasitoids throughout North America, during which this couplet has proven to be problematic as well. We here employ molecular phylogenetics to determine which characters are variable, and which are sufficiently stable to be used for identification in these species, and we provide an updated couplet for 7’ utahensis and T; cosmopeplae. Materials and methods Survey locations The Agricultural Operations of the University of California, Riverside, was the main site for the surveys. Fields that had been planted for various research were used for New records of Trissolcus parasitizing Bagrada hilaris 1 ¢ {ita er Sree Figure |. Survey locations are displayed in red dots. The black rectangle shows the Agricultural Opera- tions of the University of California, Riverside, where most of our surveys were conducted. Five Trissolcus hullensis were recovered from an alfalfa field (33.96508°N, 117.34084°W), one Trissolcus utahensis was recovered from a squash field with mustard weeds (33.96611°N, 117.34230°W), and eleven 7’ utahensis were recovered from roadside mustard weeds (33.99105°N, 117.33360°W). our sentinel egg deployments. A mixed vegetable field available from October 2017 to March 2018, an alfalfa field (Medicago sativa L.) available from October 2017 to Janu- ary 2019, and a squash field (Cucurbita moschata L., variety black futsu) available from January 2018 to September 2019 were used. The mixed vegetable field was selected par- ticularly because of its two rows of broccoli (Brassica oleracea L., variety Italica), a favora- ble host for B. hilaris. The alfalfa field hosted several stink bug species during spring and summer, serving as a potential source of stink bug egg parasitoids. The squash field was selected because B. hilaris adults were found on shortpod mustard weeds, Hirschfeldia incana (L.) Lagr.-Foss., within the field. Surveys also were conducted at other locations on the Agricultural Operations property where mustard weeds were found. In addition, various locations in the urban area with brassicaceous weeds surrounding the Univer- sity of California, Riverside, were surveyed (Fig. 1). Previous monthly samplings from January to December 2011 by Reed et al. (2013) had shown that the primary weeds supporting B. /ilaris in this area were London rocket (Sisymbrium irio L.), shepherd’s purse (Capsella bursa-pastoris (L.) Medik.), and shortpod mustard. Bagrada hilaris also were found occasionally on Russian thistle (Sa/sola australis R.Br.) and telegraph weed (Heterotheca grandiflora Nutt.) when they were in close proximity to senesced shortpod 52 Fatemeh Ganjisaffar et al. / Journal of Hymenoptera Research 80: 49-70 (2020) mustard. According to Reed et al. (2013), these weeds were available following fall and winter rains through June and the peak B. /ilaris abundance was during the spring on these host plants. We used the same locations for our sentinel egg deployments, and surveys were conducted monthly from 21 October 2017 through 27 September 2019. Sentinel egg card preparation and parasitoid recovery Bagrada hilaris eggs (< 24 hours old) were used for the surveys. Adult mating pairs of greenhouse-grown B. /ilaris (Reed et al. 2017) were maintained on organic broccoli florets in plastic containers (15 cm diameter x 6.5 cm height) with 2 screen openings for air circu- lation in an insectary room at 30 = 1 °C, 40-50% humidity and 14:10 (L:D) photoperi- od. White paper towels were cut in circles to fit the bottom of each container to provide a substrate for oviposition. Approximately 15 mating pairs were placed into each container. Bagrada hilaris eggs were collected daily and glued (Gorilla Super Glue Gel, The Gorilla Glue Co., Ohio, USA) on a 3 x 5 grid of squares on a weatherproof card so that each card contained 15 eggs (Ganjisaffar et al. 2018). For each location, a sentinel card was taped to the wire of a landscape flag, positioning it about 30 cm from the ground. Previous studies showed that eggs placed on cards on the soil surface were eaten by predators (Ganjisaffar et al. 2018). The number of cards used for each survey date varied from 10 to 20, depending on the availability of B. hilaris eggs. Cards were left in the field for 3-5 days (3.7 days on average) to avoid B. ilaris eggs from hatching in the field. According to Reed et al. (2017), B. hilaris eggs hatch after 5 days at temperatures approximating the warmest temperatures on the days of the field surveys. Once collected, the grid was cut and placed in glass vials plugged with cotton. The vials were maintained in the same insectary room that was used for the B. Ailaris colony and were examined for parasitism and wasp emergence. Emerged wasps were transferred to vials containing 95% ethanol for identification. Molecular analysis Genomic DNA was non-destructively isolated from the entire specimen using the Qia- gen DNeasy Blood and Tissue kit (Hilden, Germany) as described in Sabbatini Peverie- ri et al. (2018). A comprehensive list of all samples extracted with author and year, host, and locality data is given in Table 1 in Supporting Information. The barcode region of the mitochondrial Cytochrome Oxidase Subunit I (CO/) was amplified using the universal barcoding primer set LCO1490/HCO2198 (Folmer et al. 1994) (Table 1). The primer set LCO1490puc (Cruaud et al. 2009)/C1-N-2353 (Simon et al. 2006) was used in six samples to amplify a longer region (~850 bp) than the classical barcode region (~710 bp), providing more than enough coverage for the barcode that is not always obtained when using universal primer sets (Table 1). All PCRs were performed as described in Ganjisaffar et al. (2018), except the PCR conditions. The thermocycling conditions were as follows: 1 cycle of denaturation at 94 °C for 3 min, 35 cycles at 94 °C for 30 s, 52 °C (LCO1490/HCO2198) or 50 °C (LCO1490puc/C1-N-2353) for 30 s, 72 °C for 1 min with a final extension step of 10 min at 72 °C. All samples were 39 New records of Trissolcus parasitizing Bagrada hilaris T9ZF08.LW 092708. LW 6SZ708. LW 8SZP08. LW LSL¥08.LW 9SZF08. LIN SSLYO8.LW VSLFO8.LIN €SZP08. LW CSLPO8.LW TSZP08.LN 0SZ708. LW 67Z708. LW 8PZV08. LW LYLVO8.LIN 9VLF08.LN SPLYO8.LW VVLFO8.LN €VLV08. LW CYLVO8.LI T¥ZP08.LN OVZF08. LW 6€ZF08. LW 8EZ¥08. 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A COT consensus sequence was established for each specimen. All sequences generated from this study are deposited in GenBank, and all residual DNAs are archived at EBCL or FSCA (Table 1). Voucher specimens which have been reexamined following the molecular analysis are presently archived at FSCA (Table 1). All sequences were translated into amino acids to check for stop codons and frame shifts. All sequences obtained were compared with se- quences present in GenBank using the Basic Local Alignment Search Tool (http://www. ncbi.nlm.nih.gov/BLASTn). BOLD identification engine (Ratnasingham and Herbert 2007) was similarly datamined for barcodes of Trissolcus species and evaluated for barcode identification success. The 28 sequences obtained in this study were aligned with 28 bar- code sequences of Trissolcus retrieved from GenBank. ‘The final alignment of 56 sequences was performed using the default settings of CLUSTAL W (Thompson et al. 1994) as implemented in MEGA X (Kumar et al. 2018) and resulted in 576 characters with 174 parsimony informative sites. The phylogenetic relationships among specimens were re- constructed following a Bayesian analysis as implemented in MrBayes v. 3.2 (Ronquist et al. 2012). Searches were run for | million generations, in two independent runs, using default priors and the GTR+1+G substitution model that was selected using the Bayesian information criterion (BIC) in MEGA X. One sequence of Trissolcus thyantae Ashmead (GenBank MN615574.1) was specified as the outgroup based on the results of Talamas et al. (2019). Because a network approach is well adapted to infer intraspecific genealogi- cal relationships, a haplotype network was built for 7! utahensis using TCS 1.21 (Clement et al. 2000). As the resulting clades were not connected under 90% statistical parsimony limits, we reran the TCS analysis by fixing connection limits at 50 steps. To estimate the divergence within and between terminal taxa and clades, we calculated the uncorrected p-distance using MEGA X, since the generally used K2P distance (Kimura 1980) could be inappropriate when applied to closely related taxa (Srivathsan and Meier 2012). Morphology Terminology follows that of Talamas et al. (2017). Following non-destructive DNA ex- traction, six specimens used in the molecular analysis were photographed (1 7! hullensis and 7 1! utahensis) to document morphology of specimens from different haplogroups and those reared from different hosts. Images were produced with a Macropod imag- ing system. Image stacks were rendered with Helicon Focus and further processed in Adobe Photoshop CS6. Abbreviations and characters annotated in the figures aem = anteroventral extension of the metapleuron (Figs 6, 13) gc genal carina (Fig. 16) msct metascutellum (Figs 4—5) mshs_ mesoscutal humeral sulcus (Figs 6, 21) mtnm metanotum (Figs 4—5) New records of Trissolcus parasitizing Bagrada hilaris 55 mtpm metapostnotum (Figs 4-5) not notaulus (Figs 7, 20, 22) oc occipital carina (Figs 22, 25) ppm _ propodeum (Figs 4—5) Results Field surveys Our survey period of March 6-9, 2018, in the alfalfa field, yielded a sentinel card with 6 parasitized eggs from which 5 specimens of 7’ hullensis and 1 specimen of T. basalis wasps emerged. A month later during our April 6—9, 2018, survey in the roadside mustard weeds (33.99105N, 117.33360W), two sentinel cards were parasitized; one of them had 5, and the other one had 7 parasitized eggs, which yielded 11 7’ utahensis. One 7. utahensis wasp was recovered from a sentinel card that was deployed in the squash field with mustard weeds on October 12-16, 2018. Molecular analysis The phylogenetic analysis based on the CO/ barcode data revealed a relatively well- resolved and supported topology identifying six terminal taxa (Fig. 2). The deepest node corresponds to the split between 7’ utahensis and the other species, including TL. hullensis. The T: hullensis clade comprised two specimens reared from B. hilaris eggs and one specimen reared from frozen sentinel eggs of Halyomorpha halys (Stal) (Hemiptera: Pentatomidae) in Napa County, California. The 7’ utahensis cluster con- tains four main clades. The TCS analysis yielded four haplotype sub-networks, which were not connected under the 90% statistical parsimony limits, corresponding to these four clades of which clade 2 and clade 3 are each represented by a single haplotype, and clade 1 and clade 4 by three haplotypes each (Fig. 3). Clades 1, 3 and 4 contained specimens from British Columbia that were reared from P maculiventris eggs. Clade 1 contained specimens from Canada and southern California, reared from the eggs of P maculiventris and B. hilaris, respectively, indicating that they are conspecific. Inter- estingly, clade 2 included only Californian specimens reared from B. hilaris. Genetic distance between all clades ranged from 2.8% (clade 1/clade 2) to 9.9% (clade 1/clade 4) but remained lower than the range (from 10.6% to 14.3%) of interspecific distances obtained with the five other species in our analysis (Table 2). Trissolcus hullensis (Harrington) Remarks. The identification of Trissolcus hullensis is straightforward using the char- acters presented in Johnson (1985) and repeated in Talamas et al. (2015). Charac- ters of the posterior mesosoma are particularly useful for determining this species: In 56 Fatemeh Ganjisaffar et al. / Journal of Hymenoptera Research 80: 49-70 (2020) MN615641 MN615642 { MN615640 ; = MN615639 Trissolcus hyalinipennis MN615637 MN615638 MN615651 rE MN615649 : L-MN615653 Trissolcus colemani |_ MN615652 (East Asia) L_MN615648 LMN615650 FSCA 00091873, CA, ex. B. hilaris ; +] FSCA 00091874, CA, ex. B. hilaris Sai ok | FSCA 00090585, CA, ex.H. halys} S'S MN603797 MN603800 oe" MN603799 MN603798 MK906048 | Trissolcus semistriatus MN615636 MK906049 MN615634 MN615635 TSP398 TSP410 ee Trissolcus colemani (Europe, Middle East) TSP400 MK906051 TSP401 : ; ; Trissolcus FSCA 000094714, BC, ex. P. maculiventris utahensis FSCA 00033043, BC, ex. P. maculiventris FSCA 000094715, BC, ex. P. maculiventris Clade 1 _FSCA 00000302, BC, ex. P. maculiventris | Clade 2 Clade 3 FSCA 00033042, BC, ex. P. maculiventris FSCA 00094713, BC, ex. P. maculiventris ] FSCA 00094712, BC, ex. P. maculiventris FSCA 00033045, BC, ex. P. maculiventris FSCA 00033044, BC, ex. P. maculiventris Clade 4 FSCA 00094711, BC, ex. P. maculiventris FSCA 00033040, BC, ex. P. maculiventris — FSCA 00033041, BC, ex. P. maculiventris MN615574 Trissoicus thyantae 0.1 Figure 2. The Bayesian 50% majority rule consensus tree inferred from the 56 COJ sequences of the six Trissolcus species including 7! hullensis and T. utahensis. Only posterior probabilities >90% are indicated on the nodes. ‘The tree is rooted with the outgroup Trissolcus thyantae (GenBank MN615574). The scale bar corresponds to 0.1 estimated substitutions per site. TL. hullensis the propodeum and metanotum are directly adjacent between the meta- postnotum and metascutellum (Fig. 4), whereas in other species of Nearctic Trissolcus, the metapostnotum extends medially toward the metascutellum, and separates the pro- podeum from the metanotum (Fig. 5). New records of Trissolcus parasitizing Bagrada hilaris a7 Clade 4 FSCA 00094712 FSCA 00094711, 00033044—00033045 45 mutation steps © FSCA 00033040-00033041 Clade 2 FSCA 00091859, 00091872 8 mutation ih steps 34 mutation steps oN FSCA 00033042, 00094713 FSCA 00033239 Clade 3 FSCA 00000302 Clade 1 FSCA 00094714—000094715, 00033043 Figure 3. TCS CO/ haplotype network for the four clades of 7’ utahensis by fixing connection limits at 50 steps. Each haplotype is represented by a colored circle. Lines represent one mutational step between haplotypes, and dark circles represent unsampled haplotypes inferred from the data. Interrupted lines were used when haplotypes were separated by a long branch of more than 7 mutation steps. Material Examined. 5 females, FSCA 00091873—00091874, 00091886— 00091888 (deposited in FSCA) USA: CA: UC Riverside Agricultural Operations, 33.96508N, 117.34084W, alfalfa field, ex. Bagrada hilaris sentinel eggs deployed 6-9. III.2018, parasitoids emerged 23.III.2018, Coll. Ganjisaffar. Trissolcus utahensis (Ashmead) Remarks. ‘The concept of Trissolcus utahensis was most recently treated in a revisionary context by Johnson (1985). This treatment separated 7’ utahensis from T’ cosmopeplae 58 Fatemeh Ganjisaffar et al. / Journal of Hymenoptera Research 80: 49-70 (2020) Table 2. Genetic distances (p-distances %, 1,000 bootstrap replications) for the COJ barcode at the levels of intra-species, inter-species and clades of 7’ utahensis. Data are expressed as mean + S.E. T. hullensis T. utahensis T. utahensis T. utahensis Clade 3 T. utahensis Clade 4 T. colemani (Europe, Middle T. hyalinipennis B 3 $ St R2) S 8 3 3 S N N iS) T. colemani 4.2 + 0.6 (East Asia) 12.7 + 1.31] 14.1 + 1.4 T. colemani (Europe, Middle East) T. hullensis 12.5+ 1.3 T. utahensis 11.4+1.2 12.3 + 1.4 14.1£1.4] 13.14 1.4] 0.7+0.2 Clade 1 T. utahensis 11.6£1.2 | 10.8£1.3 | 14.14£1.4] 14.3+1.4] 12.8+1.3] 2.8+0.6 0 Clade 2 T. utahensis 126+1.3]11.8£1.3} 1641.5 | 145+1.4| 12.2+1.3) 7.78 + 1.1) 7.12+1.1 0 Clade 3 T. utahensis 11.941.2 |12.941.3 |) 126+1.3]14.74£1.5]11.9+1.3)9.9741.1] 8.65+41.1]9.6141.2| 0440.1 Clade 4 12.4+1.3) 13.1413 10.8+1.3) 142+1.4 based on the length of the anteroventral extension of the metapleuron, the absence of a genal carina, the shape of the gena in lateral view, and if notauli could be distinguished from the surface sculpture of the posterior mesoscutum. These characters were used again in the key to Nearctic Trissolcus by Talamas et al. (2015), with the addition of the form of the mesoscutal humeral sulcus, which was treated as variable within 7’ cosmopeplae. Talamas et al. (2015) also treated the anteroventral extension of the metapleuron as vari- able within 7’ cosmopeplae and emphasized the shape of the gena in lateral view to sepa- rate these species. This modification to the key sought to reconcile variability in the shape of the gena with other, seemingly variable characters. ‘The shape of the gena has proven to be one of the more difficult characters to interpret because there is not a discrete bound- ary between “narrow” and “bulging”. Because the variation in these characters does not correspond to clades in our phylogeny, we treat them as intraspecifically variable and the T. utahensis clade as a single species (Fig. 2). Based on the morphological analysis provid- ed below we propose the following replacement for couplet 14 in Talamas et al. (2015): 14 Anteroventral extension of the metapleuron long, extending to base of meso- coxa (Fig. 6); mesoscutal humeral sulcus comprised of cells (Figs 6—7).......... 2 SEP Fas ERT ROR ath 3 Fee eta Cato ch were T. cosmopeplae (Gahan) - Anteroventral extension of the metapleuron short, not approaching base of mesocoxa (Figs 12-19); mesoscutal humeral sulcus indicated by a smooth Furtowe( Pies? 20222) cuccd.c. cath vac deve ccaesatsndecPeceates veces T. utahensis (Ashmead) Sculpture of the dorsal frons. Figs 8—11 illustrate variation in the size of the smooth area directly below the preocellar pit, and the striation that radiates from the New records of Trissolcus parasitizing Bagrada hilaris 59 4 , eG Sts Figure 4-5. 4 Trissolcus hullensis (FSCA 00091886), head, mesosoma, metasoma, dorsolateral view 5 T utahensis (FSCA 00000302), head, mesosoma, metasoma, dorsolateral view. Scale bars in millimeters. antennal scrobe. Fig. 8 illustrates a specimen that emerged from a B. hilaris egg. As was found in 7’ basalis, specimens that developed in B. hilaris eggs have reduced sculpture relative to those that developed in other hosts (Ganjisaffar et al. 2018). The specimens in Figs 9, 11 were both reared from the eggs of P maculiventris in British Columbia, and have identical CO/ barcode sequences, yet the size of smooth area on the dorsal frons differs between them. The specimen in Fig. 11 is the largest (1.35 mm) among these, and the specimen in Fig. 8 is the smallest (0.93 mm). The specimens in Figs 9, 10 have the smooth area on the frons about equal in size and these specimens are also approximately equal in length (1.11 and 1.07 mm, respectively). These two specimens were retrieved in different haplogroups (clades 1 and 4), and we thus postulate that sculptural differences on the frons are size dependent. Variation on the gena. The shape of the gena varies between and within the four clades of 7’ utahensis. In clades 1 and 3, the specimens have a rather narrow gena, and 60 Fatemeh Ganjisaffar et al. / Journal of Hymenoptera Research 80: 49-70 (2020) 6 — Figures 6-7. Trissolcus cosmopeplae, holotype female (USNMENT00989096) 6 head and mesosoma, lateral view 7 head and mesosoma, dorsal view. Scale bars in millimeters. Figures 8-11. Zrissolcus utahensis, head, anterior view 8 DPI_FSCA00033239 (ex. B. hilaris) 9 FSCA 00033041 (ex. PR maculiventris) 10 FSCA 00000302 (ex. P maculiventris) 11 FSCA 00033040 (Ex. P maculiventris). Scale bars in millimeters. in clades 2 and 4 the gena is moderately to distinctly bulging in lateral view. Figs 12— 15, 24 and 27 illustrate this variation. ‘The degree to which the gena is bulging in lateral view does not appear to be host or size related. Specimens reared from B. hilaris eggs New records of Trissolcus parasitizing Bagrada hilaris 61 See Le ih > | aa A