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Complementary sex determination (CSD) is a widespread sex determination mechanism in haplodiploid Hymenoptera. Under CSD, sex is determined by the allelic state of one or multiple CSD loci. Heterozygosity at one or more loci leads to female development, whereas hemizygosity of haploid eggs and homozygosity of diploid eggs results in male development. Sexual (arrhenotokous) reproduction normally yields haploid male and diploid female offspring. Under asexual reproduction (thelytoky), diploidized unfertilized eggs develop into females. Thelytoky is often induced by bacterial endosymbionts that achieve egg diploidization by gamete duplication. As gamete duplication leads to complete homozygosity, endosymbiont-induced thelytokous reproduction is presumed to be incompatible with CSD, which relies on heterozygosity for female development. Previously, we excluded CSD in four Asobara (Braconidae) species and proposed a two-step mechanism for Wolbachia-induced thelytoky in Asobara japonica. Here, we conclusively reject CSD in two cynipid wasp species, Leptopilina heterotoma and Leptopilina clavipes. We further show that thelytoky in L. clavipes depends on Wolbachia titer but that diploidization and feminization steps cannot be separated, unlike in A. japonica. We discuss what these results reveal about the sex determination mechanism of L. clavipes and the presumed incompatibility between CSD and endosymbiont-induced thelytoky in the Hymenoptera.

Horizontal transfer in Drosophila has been inferred for several families of transposable elements. Specifically, the retroelement roo has been suggested to have been horizontally transferred between the species D. melanogaster, D. simulans, D. sechellia and D. yakuba. The inferences were based on the observation that divergence between transposable elements in different species was lower than the divergence found in typical nuclear genes and in the incongruence of phylogenies of the species and their TEs. Here, we address the question of the possible horizontal transfer of roo between species of the Drosophila genus by studying the presence absence of a duplication of 99 bp in the 5'UTR of the transposon, as well as comparing the sequences of the paralogous and orthologous duplicated repeats within and between species. First, the repeats were only found in five species of the melanogaster subgroup. Second, the date of occurrence of the duplication event originating the repeats was posterior to the split of the subgroup. The duplication date suggests an origin previous to the split of D. simulans and D. sechellia and close to the divergence of D. melanogaster from the D. simulans complex. These data point to horizontal transfer to the afrotropical species D. yakuba and D. erecta from one of the cosmopolitan species D. melanogaster or D. simulans. We propose that the parasitoid wasp Leptopilina could have been the vector of horizontal transfer after the observation that a sequence of 845 bp with high homology to a fragment of roo was isolated from this wasp.

A major hypothesis for the evolution of chemical signals is that pheromones arise from non-communicative precursor compounds. However, data supporting this hypothesis are rare, primarily because the original functions of the antecedent compounds often have been lost. A notable exception, however, is the parasitoid wasp speciesLeptopilina heterotoma, whose compound (−)-iridomyrmecin is used as a defensive secretion, a cue for females to avoid competition with con- and hetero-specific females, and as the primary component of the females’ sex pheromone. To better understand the evolution of sex pheromones from defensive compounds, we examined the chemical ecology ofL. pacifica, the sister species ofL. heterotoma. Here, we show thatL. pacificaalso produces a defensive secretion containing a species-specific mixture of mostly iridoid compounds. However, the composition of the secretion is more complex than inL. heterotoma, and iridomyrmecin is only a minor component. Moreover, in contrast toL. heterotoma, conspecific female competitors were not avoided by female subjects, and a role of the iridoids in the female sex pheromone ofL. pacificacan be excluded, as only the females’ cuticular hydrocarbons (CHCs) resulted in the elicitation of courtship by males. Although closely related, the two sister species show substantial differences in the use of the defensive secretion for communicative purposes. Variation in pheromone usage in this genus still presents a conundrum, highlighting the need for additional studies to understand the selective forces shaping the evolution of pheromone composition.

When a parasite attacks an insect, the outcome is commonly modulated by the presence of defensive heritable symbionts residing within the insect host. Previous studies noted markedly different strengths of Spiroplasma-mediated fly survival following attack by the same strain of wasp. One difference between the two studies was the strain of Spiroplasma used. We therefore performed a common garden laboratory experiment to assess whether Spiroplasma-mediated protection depends upon the strain of Spiroplasma. We perform this analysis using the two strains of male-killing Spiroplasma used previously, and examined response to challenge by two strains of Leptopilina boulardi and two strains of Leptopilina heterotoma wasp. We found no evidence Spiroplasma strain affected fly survival following wasp attack. In contrast, analysis of the overall level of protection, including the fecundity of survivors of wasp attack, did indicate the two Spiroplasma strains tested varied in protective efficiency against three of the four wasp strains tested. These data highlight the sensitivity of symbiont-mediated protection phenotypes to laboratory conditions, and the importance of common garden comparison. Our results also indicate that Spiroplasma strains can vary in protective capacity in Drosophila, but these differences may exist in the relative performance of survivors of wasp attack, rather than in survival of attack per se.

As generalist parasitoid wasps, Leptopilina heterotoma are highly successful on many species of fruit flies of the genus Drosophila. The parasitoids produce specialized multi-strategy extracellular vesicle (EV)-like structures in their venom. Proteomic analysis identified several immunity-associated proteins, including the knottin peptide, LhKNOT, containing the structurally conserved inhibitor cysteine knot (ICK) fold, which is present in proteins from diverse taxa. Our structural and docking analysis of LhKNOT’s 36-residue core knottin fold revealed that in addition to the knottin motif itself, it also possesses a Cation–Polar–Cation (CPC) clip. The CPC clip motif is thought to facilitate antimicrobial activity in heparin-binding proteins. Surprisingly, a majority of ICKs tested also possess the CPC clip motif, including 75 bona fide plant and arthropod knottin proteins that share high sequence and/or structural similarity with LhKNOT. Like LhKNOT and these other 75 knottin proteins, even the Drosophila Drosomycin antifungal peptide, a canonical target gene of the fly’s Toll-NF-kappa B immune pathway, contains this CPC clip motif. Together, our results suggest a possible defensive function for the parasitoid LhKNOT. The prevalence of the CPC clip motif, intrinsic to the cysteine knot within the knottin proteins examined here, suggests that the resultant 3D topology is important for their biochemical functions. The CPC clip is likely a highly conserved structural motif found in many diverse proteins with reported heparin binding capacity, including amyloid proteins. Knottins are targets for therapeutic drug development, and insights into their structure–function relationships will advance novel drug design.

International audience; The Drosophila endoparasitoid wasps Leptopilina boulardi and L. heterotoma (Hymenoptera: Cynipidae) are proovigenic species, i.e., females contain their lifetime number of mature eggs at emergence. They are therefore able to immediately parasitize many hosts when present. In response to parasitoid oviposition, the larval host D. melanogaster can mount an immune response, encapsulation, that can destroy the parasitoid eggs. This response is counteracted by the venom the wasp injects during oviposition. Here, we estimated the amount of venom injected into a D. melanogaster host larva using immunodetection of venom proteins and we attempted to correlate this amount with the number of eggs a female can lay on successive days. The venom reservoir of L. boulardi contains enough venom for at least 100 ovipositions while that of L. heterotoma contains venom for about 16 ovipositions. While a female L. boulardi may have enough venom for three days of parasitism when 20 or 40 larval hosts were presented each day, L. heterotoma certainly needs to synthesize new venom to parasitize the number of hosts offered. Interestingly, parasitism stopped (L. boulardi), egg protection (L. heterotoma) and egg hatching decreased (both species) after three days of parasitism. Thus, although venom does not appear to be a limiting factor for parasitism, our data suggest that it may have less effectiveness on the egg protection and on egg/host development after high repetitive egg laying.

Drosophila suzukii (Matsumura, 1931) (Diptera: Drosophilidae) is an invasive pest species of various fruit crops in the USA and Europe. Although D. suzukii has been recently reported in strawberry in Erzurum and other newly invaded areas in Turkey (e.g., Ankara, Bolu, Çanakkale and Düzce), there is only limited information on its indigenous parasitoids. In this study, four hymenopteran parasitoids, the larval parasitoids of Leptopilina boulardi (Barbotin, Carton & Kelner-Pillault, 1979), Leptopilina heterotoma (Thomson, 1862) (Figitidae) and the pupal parasitoids of Pachycrepoideus vindemmiae (Rondani, 1875) (Pteromalidae) and Trichopria drosophilae (Perkins, 1910) (Diapriidae), were collected from frugivorous drosophilid species. Leptopilina boulardi and T. drosophilae were found for the first time in Turkey. Leptopilina heterotoma and P. vindemmiae were the most common parasitoid species, reared from field-collected fruit samples in this study. The laboratory assays revealed that both pupal parasitoids developed from D. suzukii pupae, but the association of L. heterotoma and L. boulardi with D. suzukii is yet to be confirmed. The PCR amplification of the cytochrome c oxidase subunit I loci of mtDNA of the representative four parasitoid samples produced different lengths of DNA fragments, ranging from 633 bp to 658 bp. BLASTn queries based on the COI of the parasitoid samples showed that the sequences were 99-100% identical to those of the corresponding species in the GenBank database., Drosophila suzukii (Matsumura, 1931) (Diptera: Drosophilidae), Avrupa ve Amerika’da çeşitli meyvelerde zarara neden olan istilacı bir türdür. Drosophila suzukii, son zamanlarda Türkiye’de Erzurum’da çilekte rapor edilmesine ve o zamandan beri yeni alanları (Ankara, Bolu, Çanakkale ve Düzce vs.) istila etmesine rağmen, onun yerli parazitoitleri çok az bilinmektedir. Bu çalışmada, dört hymenopter parazitoit, larval parazitoitler Leptopilina boulardi (Barbotin, Carton & Kelner-Pillault, 1979), Leptopilina heterotoma (Thomson, 1862) (Figitidae) ve pupal parazitoitler Pachycrepoideus vindemmiae (Rondani, 1875) (Pteromalidae) ve Trichopria drosophilae (Perkins, 1910) (Diapriidae) türleri, meyvelerde bulunan drosophilid türlerinden elde edilmiştir. Leptopilina boulardi ve T. drosophilae türlerinin Türkiye’de ilk defa varlığı belirlenmiştir. Leptopilina heterotoma ve P. vindemmiae, toplanan örneklerden en fazla bulunan türler olmuştur. Laboratuvar denemeleri iki pupa parazitoitinin D. suzukii pupalarından geliştiğini belirlenmesine rağmen, L. heterotoma ve L. boulardi’nin D. suzukii’den geliştiklerini hala doğrulanamamıştır. Dört parazitoit türü temsil eden örneklerin mitokondrial DNA’sının cytochrome c oxidase subunit I lokusunun PCR ile çoğaltılması sonucu 633 bp ile 658 bp arasında DNA segmentlerini ürettiği belirlenmiştir. Bu ürünlerin BLASTn analizi sonucu GenBank’daki referans bireylerin sekanslarıyla %99-100 benzerliğe sahip olduğu tespit edilmiştir.

Predicting the outcome of interspecific interactions is important in designing biological control programs that involve multiple species introductions. Three solitary larval endoparasitoids, Asobara japonica Belokobylskij (Hymenoptera: Braconidae), Ganaspis brasiliensis Ihering and Leptopilina japonica Novkovic & Kimura (both Hymenoptera: Figitidae) from Asia are potential agents for classical biological control of the invasive Drosophila suzukii Matsumura (Diptera: Drosophilidae) in North America. We compared the developmental rates of early immature stages of the three parasitoids and then evaluated their potential interactions, including elimination of competitors in intrinsic competition, interspecific discrimination of previously parasitized hosts, and the outcomes of interspecific competition at three host densities (5, 15 or 30 host larvae). On average, L. japonica eggs hatched first, followed by A. japonica and then G. brasiliensis. All three species exhibited interspecific competition after egg hatch through physical combat or in some cases through physiological suppression, with L. japonica out-competing the other two parasitoid species in multi-parasitized hosts. Ganaspis brasiliensis adults discriminated strongly against hosts previously parasitized by L. japonica, and A. japonica adults discriminated against hosts previously parasitized by L. japonica, whereas L. japonica only discriminated against hosts parasitized by G. brasiliensis. Regardless of host density, the observed parasitism when both A. japonica and L. japonica were present was lower than expected, a likely consequence of interspecific competition, but the combined impacts on host suppression by L. japonica and G. brasiliensis were additive, likely due to strong interspecific discrimination by G. brasiliensis.

Deciphering the processes driving the evolution of the diverse pheromone-mediated chemical communication system of insects is a fascinating and challenging task. Understanding how pheromones have arisen has been supported by studies with the model organism Leptopilina heterotoma, a parasitoid wasp whose defensive compound (-)-iridomyrmecin also evolved as a component of the female sex pheromone and as a cue to avoid competition with other females during host search. To understand how compounds can evolve from being non-communicative to having a communicative function and to shed light on the evolution of the multi-functional use of iridomyrmecin in the genus Leptopilina, the chemical communication of two additional species, L. ryukyuensis and L. japonica, was studied. We demonstrate that in both species a species-specific mixture of iridoids is produced and emitted by wasps upon being attacked, consistent with their putative role as defensive compounds. In L. ryukyuensis these iridoids are also used by females to avoid host patches already exploited by other conspecific females. However, females of L. japonica do not avoid the odor of conspecific females during host search. We also show that the sex pheromone of female L. ryukyuensis consists of cuticular hydrocarbons (CHCs), as males showed strong courtship behavior (wing fanning) towards these compounds, but not towards the iridoid compounds. In contrast, males of L. japonica prefer their females' iridoids but CHCs also elicit some courtship behavior. The use of iridoid compounds as defensive allomones seems to be common in the genus Leptopilina, while their communicative functions appear to have evolved in a species-specific manner.

Leptopilina syphax (Hymenoptera: Figitidae) is a newly recorded species of parasitic wasp, and it attacks the larval stage of Drosophilidae, mainly the Drosophila species. Few works have been done in the basic study of L. syphax, including the data of mitochondrial genome. In this study, the complete mitochondrial genome of L. syphax (GeneBank accession number: MT649407) was sequenced using Illumina HiSeq X Ten system. The mitochondrial genome is 15,882bp long and comprises 13 protein-coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes. Meanwhile, 26 genes are in majority strand, and the remaining 11 genes are in minority strand. The overall base composition is 41.7% for A, 6.0% for G, 13.6% for C, and 38.7% for T, with an A + T content of 80.4%, respectively. We also performed a phylogenetic analysis with other known mitochondrial genomes of some parasitic wasps. The results show that L. syphax is closely related to L. boulardi, which is another Drosophila parasitoid.

Asobara japonica (Hymenoptera: Braconidae), Ganaspis brasiliensis and Leptopilina japonica (Hymenoptera: Figitidae) are Asian larval parasitoids of spotted wing drosophila, Drosophila suzukii (Diptera: Drosophilidae). This study evaluated these parasitoids’ capacity to attack and develop from 24 non-target drosophilid species. Results showed that all three parasitoids were able to parasitize host larvae of multiple non-target species in artificial diet; A. japonica developed from 19 tested host species, regardless of the phylogenetic position of the host species, L. japonica developed from 11 tested species; and G. brasiliensis developed from only four of the exposed species. Success rate of parasitism (i.e., the probability that an adult wasp successfully emerged from a parasitized host) by the two figitid parasitoids was low in hosts other than the three species in the melanogaster group (D. melanogaster, D. simulans, and D. suzukii). The failure of the figitids to develop in most of the tested host species appears to correspond with more frequent encapsulation of the parasitoids by the hosts. The results indicate that G. brasiliensis is the most host specific to D. suzukii, L. japonica attacks mainly species in the melanogaster group and A. japonica is a generalist, at least physiologically. Overall, the developmental time of the parasitoids increased with the host’s developmental time. The body size of female A. japonica (as a model species) was positively related to host size, and mature egg load of female wasps increased with female body size. We discuss the use of these parasitoids for classical biological control of D. suzukii.

Intraspecific competition is a major force in mediating population dynamics, fuelling adaptation, and potentially leading to evolutionary diversification. Among the evolutionary arms races between parasites, one of the most fundamental and intriguing behavioural adaptations and counter-adaptations are superparasitism and superparasitism avoidance. However, the underlying mechanisms and ecological contexts of these phenomena remain underexplored. Here, we apply the Drosophila parasite Leptopilina boulardi as a study system and find that this solitary endoparasitic wasp provokes a host escape response for superparasitism avoidance. We combine multi-omics and in vivo functional studies to characterize a small set of RhoGAP domain-containing genes that mediate the parasite’s manipulation of host escape behaviour by inducing reactive oxygen species in the host central nervous system. We further uncover an evolutionary scenario in which neofunctionalization and specialization gave rise to the novel role of RhoGAP domain in avoiding superparasitism, with an ancestral origin prior to the divergence between Leptopilina specialist and generalist species. Our study suggests that superparasitism avoidance is adaptive for a parasite and adds to our understanding of how the molecular manipulation of host behaviour has evolved in this system., Evolutionary arms races can drive adaptations in hosts and parasites as well as among competing parasites. A combination of multi-omics and functional tests identifies a set of genes that allow a parasitic wasp to minimize intraspecific competition by inducing hosts to escape before more wasps can parasitize them.

Chemical communication via pheromones is considered the oldest and most widespread form of communication in nature. However, the way that the enormous diversity of species-specific pheromones evolved is still of debate. One possible process driving pheromone evolution is the mate-finding and dispersal behavior, as long-distance mate-finding requires highly volatile compounds. In contrast, less volatile compounds might be sufficient attractants in species that search for mates within proximity. In the parasitoid wasp genus Leptopilina, the composition of species-specific sex pheromones ranges from highly volatile iridoid compounds through combinations of iridoids with low volatile cuticular hydrocarbons (CHCs) to only CHCs. To study the selective forces shaping the composition of sex pheromones in Leptopilina, we examined the dispersal behavior, i.e., the proportion of male and female wasps dispersing after emergence, in four species with known sex pheromone compositions. If males and females disperse immediately, long-range mate attraction might become necessary, favoring volatile iridoids over CHCs. If mating occurs directly on the host patch, short-range mate attraction by low volatile CHCs might suffice. Our analyses have revealed that the dispersal behavior of Leptopilina males and females after emergence does indeed differ between species with differently volatile sex pheromones. Specifically, males of species with iridoid sex pheromones start to disperse immediately before their females’ emergence, whereas males of species with CHC sex pheromones delay dispersal until their conspecific females emerge. While the differences in female dispersal behavior turned out to be species-specific, differences in male dispersal correlated with the volatility of female-produced sex pheromones of each species. This study significantly contributes to our understanding of the evolution of sex pheromones by differences in dispersal behavior.

Leptopilina lasallei Buffington and Guerrieri sp. nov. (Figures 1 – 4) Diagnosis Female. Setal band complete at base of metasoma (Figures 1b and Figure 2e); dorsal surface of scutellum anteriorly striate, posteriorly foveate (Figure 2f); posteroventral corner of metapleuron glabrous (Figure 2e); antenna with 10 flagellomeres (Figure 2c); petiole as long as wide (Figure 2e). Male similar to female except setal band incomplete at base of metasoma (Figure 3a); antenna with 13 flagellomeres (Figure 3a), F1 the same size as F2, not distinctly excavated laterally (Figure 3b). See Table 2 for a summary of characters. Description Female. Holotype length 1.4 mm. General. Body overall very smooth, glabrous, lacking sculpture except on scutellum (Figures 1 and 3). Head, mesosoma, metasoma dark brown, wings hyaline, legs honey yellow. Head. In anterior view, ovate (Figure 2a). Head glabrous with very sparse setae scattered on face, clypeus and mandibles. Lateral margin of occiput smooth. Ratio of length of gena (I, Figure 2a) to length of compound eye (II, Figure 2b) 1 to 3. Gena smooth. Lateral margin of occiput evenly rounded, not well defined. Occiput smooth. Ratio of maximum diameter of a lateral ocellus (III, Figure 2b) to shortest distance between lateral ocelli (IV, Figure 2b) 1:2. Posterior margin of anterior ocellus clearly separated from anterior margins of posterior ocelli. Ratio of vertical distance between inner margin of antennal foramen and ventral margin of clypeus (V, FIgure 2a) to vertical distance between anterior ocellus and antennal rim (VI, Figure 2a) 1:4. Median keel absent. Vertical carina adjacent to ventral margin of antennal socket present, minute. Facial sculpture absent, surface smooth. Facial impression absent, face flat. Antennal scrobe absent. Anterior tentorial pits small (VII, Figure 2a). Vertical delineations on lower face absent. Ventral clypeal margin laterally, close to anterior mandibular articulation, straight. Ventral clypeal margin medially with 6 setae. Clypeus smooth with gently curved ventral margin. Malar space adjacent to anterior articulation of mandible evenly rounded, smooth. Malar sulcus (VIII, Figure 2a) present, simple. Ratio of distance between compound eye and posterior mandibular articulation to distance between posterior ocellus and compound eye 1:1. Compound eyes, in dorsal view, not distinctly protruding from the surface of the head (Figure 2). Short, sparse setae on eyes (Figure 2). Orbital furrows absent. Lateral frontal carina of face absent. Dorsal and posterior aspects of vertex smooth. Hair punctures on lateral aspect of vertex absent. Posterior surface of head deeply impressed around postocciput. Labial-maxillary complex. Apical segment of maxillary palp with pubescence, consisting of 1 long erect seta. Apical seta on apical segment of maxillary palp longer than twice length of second longest apical seta. Maxillary palp composed of 4 segments. Last 2 segments of maxillary palp (in normal repose) straight. Apical segment of maxillary palp 2Χ longer than preceding segment. Antenna. Terminal flagellomere with 3 basiconic sensillae. Basiconic sensillae present on F5 – F9. Articulation between flagellomeres in antenna moniliform, segments distinctly separated by narrow neck-like articulation. Antenna composed of 10 flagellomeres (Figure 2c); F1 2.5Χ longer than F2. Flagellomeres cylindrical, distinctly widened towards apex, clavate. Placoidal sensilla present on F5 – F10 (Figure 2c). Mesosoma. Macrosculpture on lateral surface of pronotum absent dorsally and laterally (Figure 1). Anteroventral inflection of pronotum narrow. Pubescence on lateral surface of pronotum present in pronotal trough. Anterior flange of pronotal plate distinctly protruding anteriorly, transversely strigate (Figure 2). Ridges extending posteriorly from lateral margin of pronotal plate distinct but short, not extending to the dorsal margin of pronotum. Lateral pronotal carina absent. Crest of pronotal plate absent. Dorsal margin of pronotal plate (in anterior view) spatulate. Submedian pronotal depressions open laterally, deep (Figure 2). Lateral margin of pronotal plate defined all the way to the dorsal margin of the pronotum. Width of pronotal plate narrow, not nearly as wide as head. Mesoscutal surface convex, evenly curved (Figure 1). Sculpture on mesoscutum absent, entire surface smooth, shiny, with sparse long hairs. Notauli absent. Median mesoscutal carina, anterior admedial lines and median mesoscutal impression all absent. Parascutal carina nearly straight. Mesopleuron entirely smooth (Figure 1). Subpleuron entirely smooth, glabrous. Lower pleuron entirely smooth, glabrous. Epicnemial carina absent. Lateroventral mesopleural carina present, marking abrupt change in slope of mesopectus. Mesopleural triangle absent. Subalar pit present, located under subalar area, not easily observed. Speculum absent. Mesopleural carina present, complete, composed of one complete, uninterrupted carina. Anterior end of mesopleural carina inserting above notch in anterior margin of mesopleuron. Dorsal surface of scutellum distinctly striate on anterior 2/3, posterior 1/3 foveate (Figure 2). Circumscutellar carina present, complete, delimiting dorsal and ventral halves of scutellum. Posterior margin of axillula marked by distinct ledge, axillula distinctly impressed. Lateroventral margin of scutellum posterior to axillula smooth. Dorso-posterior part of scutellum rounded. Transverse median carina on scutellar plate absent. Scutellar plate, in dorsal view, exposing more than half of scutellum. Scutellar fovea present, 2, distinctly margined posteriorly, smooth on bottom. Longitudinal scutellar carinae absent. Single longitudinal carina separating scutellar foveae present, short, ending at posterior margin of foveae. Posterolateral margin of scutellum rounded. Lateral bar smooth, narrow. Metapleural-propodeal complex. Posterior impression of metepimeron absent (Figures 2 and 2e). Metapectal cavity anterodorsal to metacoxal base present, well defined. Anterior margin of metapectal-propodeal complex meeting mesopleuron at same level at point corresponding to anterior end of metapleural carina. Posteroventral corner of metapleuron (in lateral view) not extended posteriorly, glabrous. Anterior impression of metepimeron absent. Posterior margin of metepimeron distinct, separating metepimeron from propodeum. Subalar area broadened anteriorly, narrowed posteriorly. Prespiracular process present, blunt, lobe-like, rough. Dorsellum absent. Anterior impression of metepisternum present. Pubescence consisting of few hairs on posterior part of metepisternum, dense hair on propodeum. Propodeal spurs absent. Lateral propodeal carinae present, not reaching scutellum, lyre-shaped, stout. Ventral end of lateral propodeal carina reaching nucha, carinae separated from each other. Inter-propodeal carina space lightly setose, underlying surface smooth. Petiolar rim of uniform width along entire circumference. Petiolar foramen removed from metacoxae, directed posteriorly. Horizontal carina running anteriorly from lateral propodeal carina present medially, effaced laterally. Calyptra, in lateral view, rounded; in posterior view, elongate. Propodeum neck-like, drawn out posteriorly. Legs. Pubescence posterolaterally on metacoxa, present, small, rounded, with adjacent sparse pubescence (Figure 1). Microsculpture on hind coxa absent. Longitudinal ridge on the posterior surface of metatibia absent. Metafemoral tooth present, elongate, with adjacent serrate ridge posteriorly. Ratio of first metatarsal segment to remaining 4 segments 2.1:1. Wings. Wing vein M absent (Figure 3). Pubescence of fore wing present, long, dense on most of surface. Apical margin of fore wing rounded; Rs+M of fore wing defined but nebulous at point of origin from basal vein at posterior third; mesal end of Rs+M vein situated closer to anterior margin of wing, directed towards middle of basalis; vein R1 forming marginal cell completely; basal abscissa of R1 as broad as adjacent wing veins. Colouration of wing absent, entire wing hyaline. Marginal cell of fore wing membranous, similar to other wing cells. Areolet absent. Hair fringe along apical margin of fore wing present, of medium length. Metasoma. Petiole about as long as wide (Figures 2). Surface of petiole longitudinally costate, ventral keel absent. Posterior part of petiole not abruptly widened. Ventral and lateral parts of petiolar rim broad. Setal band at base of tergum 3 present, uninterrupted dorsally and ventrally (Figures 1 and 2). Tergum 3 indistinct, fused with syntergum. Posterior margin of tergum 4 evenly rounded. Sternum 3 encompassed by syntergum. Sculpture on metasomal terga absent (Figure 1). Syntergum present with terga 3 to 5 fused, ventral margin rounded. Peglike setae on T6 – T7 absent. Postero-ventral cavities of female metasoma T7 present, glabrous save for few, long setae. Female postero-ventral margin of T6 – T7 straight, parallel. Terebrum and hypopygium (in lateral view) straight, pointing posteriorly. Ovipositor clip present. Male: Similar to female except for antenna with 13 flagellomeres (Figure 2) with unmodified F2 (Figure 2), absence of setal band at base of tergum 3 (Figure 2). Metasoma, posteriorly, directly ventrally, somewhat truncate. Variation. Body size ranges from 1.2 to 1.5 mm. Overall body colouration varies slightly from dark brown to nearly black; dorsal surface of scutellum can range from slightly striate to deeply striate, foveate at posterior end of scutellum; very faint setal tracks present on the mesoscutum of some specimens, absent in others. Etymology. Named in honour of the late Dr John La Salle. John ’ s dedication to Hymenoptera research, and biodiversity research in general, will be greatly missed. We hope this honorific helps to keep his memory alive for years to come. Material examined. Holotype:. ♀, CHINA, Kunming, Xiao He Research Farm, Pan Long District 25.176593°N, 102.794697°E, 2209 m above sea level (asl), 20 July 2015, from banana trap, EGWY124 (Wang Yan) USNMENT00896641; left fore and hind wings mounted on slide USNMENT01525760. Paratypes: 1♀, CHINA, Kunming, Xiao He Research Farm, Pan Long District 25.176593°N, 102.794697°E, 2209 m asl, October 2014, from banana trap, EGWY36 (Wang Yan) USNMENT00896646; 1♀, CHINA, Kunming, Xiao He Research Farm, Pan Long District 25.176593°N, 102.794697°E, 2209 m asl, October 2014, from banana trap, EGWY41 (Wang Yan) USNMENT00896639; 1♀, CHINA, Kunming, Xiao He Research Farm, Pan Long District 25.176593°N, 102.794697°E, 2209 m asl, 20 May 2014, from banana trap, EGWY87 (Wang Yan) USNMENT00896644; 1♀, CHINA, Kunming, Xiao He Research Farm, Pan Long District 25.176593°N, 102.794697°E, 2209 m asl, 20 July 2015, from banana trap, EGWY103 (Wang Yan) SEM USNMENT01525765; 1♀, CHINA, Kunming, Xiao He Research Farm, Pan Long District 25.176593°N, 102.794697°E, 2209 m asl, 20 July 2015, from banana trap, EGWY135 (Wang Yan) USNMENT00896648; 1♀, CHINA, Kunming, Xiao He Research Farm, Pan Long District 25.176593°N, 102.794697°E, 2209 m asl, 20 July 2015, from banana trap, EGWY137 (Wang Yan) USNMENT00896633; 1♀, CHINA, Kunming, Xiao He Research Farm, Pan Long District 25.176593°N, 102.794697°E, 2209 m asl, 20 July 2015, from banana trap, EGWY129, DS095 (Wang Yan) USNMENT01525764; 1♂, CHINA, Kunming, Xiao He Research Farm, Pan Long District 25.176593°N, 102.794697°E, 2209 m asl, 20 July 2015, from banana trap, EG15CZ6R BT-26, DSZ102 (Wang Yan) USNMENT01525761; 1♀, CHINA, Kunming, Xiao He Research Farm, Pan Long District 25.176593°N, 102.794697°E, 2209 m asl, 20 July 2015, from banana trap, EG15CZ6R-BT, DS194 (Wang Yan) USNMENT01525762; 1♀, CHINA, Kunming, Xiao He Research Farm, Pan Long District 25.176593°N, 102.794697° E, 2209 m asl, 20 May 2015, from banana trap, EGWY119 (Wang Yan) USNMENT00896626; 1♀, CHINA, Kunming, Xiao He Research Farm, Pan Long District 25.1178°N, 102.4842°E, 23 June 2015, from banana trap, EG15CZ6RBT-19 (E. Guerrieri, M. Giorgini, K. Hoelmer) USNMENT00896625; 1♀, CHINA, Kunming, Xiao He Research Farm, Pan Long District 25.112378 °N, 102.481742°E, 23 June 2015, from banana trap, EG15CZ6RBT-27 (Wang Yan) USNMENT00896628; 1♀, CHINA, Kunming, Xiao He Research Farm, Pan Long District 25.112378°N, 102.481742°E, 23 June 2015, from banana trap, EGCZ6 RBTR19-4 (Wang Yan) USNMENT00896627; 1♀, CHINA, Kunming, Kunming Botanical Gardens, Pan Long District, 25.145348°N, 102.741543°E, 1958 m asl, 12 July 2015, from blackberry fruits, EG15CZ5-6 (Wang Yan) USNMENT00896638; 1♂, CHINA, Kunming, Kunming Botanical Gardens, Pan Long District, 25.145348°N, 102.741543°E, 1958 m asl, 12 July 2015, from blackberry fruits, EG15CZ5-23, DSZ 184 (Wang Yan) USNMENT01525763; 1♀, CHINA, Kunming, Kunming Botanical Gardens, Pan Long District, 25.145348°N, 102.741543°E, 1958 m asl, 12 July 2015, from blackberry fruits, EG15CZ5-32 (Wang Yan) USNMENT00896620; 1♀, CHINA, Kunming, Kunming Botanical Gardens, Pan Long District, 25.145348°N, 102.741543°E, 1958 m asl, 12 July 2015, from blackberry fruits, EG15CZ5-34 (Wang Yan) USNMENT00896649; 1♀, CHINA, Kunming, Cheng Jiang County, Long Jie Zuo Suo Cun, 24.711991°N, 102.870912° E, 2053 m asl, 24 July 2013, from banana trap, EGCZ1 BTR2-2 (E. Guerrieri, M. Giorgini, K. Hoelmer) USNMENT00896624; 1♀, CHINA, Kunming, Cheng Jiang County, Long Jie Zuo Suo Cun, 24.711991°N, 102.870912°E, 2053 m asl, 24 July 2013, from banana trap, EGCZ1 BTR2-3 (E. Guerrieri, M. Giorgini, K. Hoelmer) USNMENT00896647; 1♀, CHINA, Kunming, Cheng Jiang County, Fu Xian Lake, 24.506364°N, 102.860508°E, 1759 m asl, 24 July 2013, from banana trap, EGCZ2 BTR4-2 (E. Guerrieri, M. Giorgini, K. Hoelmer) USNMENT00896635; 4♀, 6♂, CHINA, Yunnan Prov., Fumin, 25.1475°N, 102.5289°E, ex D. suzukii or D. pulchrella on Myrica rubra, 7 February 2016, Jinping Zhang (CABI2) USNMENT01525940-USNMENT01525949; 1♀, 3♂, CHINA, Yunnan Prov., Kunming, West Mountain, 25.1475°N, 102.5289°E, ex D. suzukii on Myrica rubra, 7.II.2016, Fang Huan/Wu Hao (CABI3) USNMENT01525936-USNMENT01525939. Biology. Reared from Drosophila suzukii or D. pulchrella on Myrica rubra (CABI specimens). Other specimens were collected in the wild with banana-baited traps from May to October, with the majority in July (USNMENT00896649 emerged from blackberries). Banana traps were set up in wild vegetation in natural reserves (Kunming, Cheng Jiang County, Long Jie Zuo Suo Cun), in natural habitats surrounding orchards (Kunming, Xiao He Research Farm, Pan Long District), in blueberry crops (Kunming, Cheng Jiang County, Fu Xian Lake), and in a botanical garden (Kunming Botanical Gardens, Pan Long District). Comments. Leptopilina lasallei possesses some unusual character states for members of Leptopilina. Using van Noort et al. (2015), male L. lasallei runs to Leptopilina; a female specimen may also run to Leptopilina after some hesitation, but an often relied upon character, the ‘ broken ’ or ‘ interrupted ’ metasoma hairy ring character state, is not present. Instead, the hairy ring is complete in the females of L. lasallei, and this may add some confusion to diagnosing this taxon. In fact, this dimorphism is stronger than in most Eucoilinae, and interestingly, the pattern is inverted compared to other Leptopilina where the hairy ring of the female is more reduced than that of the male. This switch in dimorphism begs for clarifying observations of the behaviour of live specimens and the physiology of this trait. Leptopilina is one of the better studied genera of Eucoilinae, with laboratory strains genetically and behaviourally studied, and a relatively large number of species that have been described or redescribed in modern times. Nevertheless, a large portion of worldwide diversity is still unaccounted for. Leptopilina often gets confused with other small drosophilid parasitoids such as Ganaspis (Ganaspini) and Hexacola (Ganaspini) but is easily recognised by the characters that reveal its belonging in Eucoilini rather than Ganaspini: F 2 in male antennae equally or more modified (curved/excavated/elongated) than F1, glabrous and more or less oblique posteroventral corner of metapleuron. Furthermore, Leptopilina are characterised by a well-developed petiolar rim. The setal bands (‘ hairy ring ’) of the base of the metasoma are often reduced to varying extents. In a few species, female flagellomere number is reduced to 10. Most but not all species have a high, convex scutellum. These additional characters commonly occur among the genera of Ganaspini but distinguish Leptopilina from most of its closer relatives within the Eucoilini. Within Leptopilina, two species that L. lasallei can be confused with are L. decemflagella and L. tsushimaensis, as females in both of these species have 10 flagellomeres in their antennae (Lue et al., 2016). However, L. decemflagella and L. tsushimaensis females both have incomplete hair rings at the base of their metasoma. The striate dorsal surface of the scutellum in L. lasallei is shared with L. freyae and L. boulardi (Allemand et al., 2002); again, the latter two have a metasomal hairy ring in the female which is more or less strongly reduced (incomplete or even absent); male L. lasallei have the F1 and F2 of the antennae equal in size, whereas in in L. freyae and L. boulardi, F2 is clearly longer than F1. Another useful character for distinguishing L. lasallei from L. freyae and L. boulardi, and from L. orientalis which is similar too, is the shape of the propodeal carinae: in L. lasallei, these are heavily sclerotised and thick, overall lyre-shaped; in the other species of Leptopilina that are overall similar looking, the propodeal carinae are finer, less massive and parallel sided.

Drosophila suzukii (Matsumura, Diptera: Drosophilidae) is a key pest of sweet cherry and small fruits worldwide. Biological control remains unutilized in the framework of D. suzukii management. Nonetheless, natural enemies may play an important role in regulating this pest. We report for the first time the presence of Leptopilina japonica Novković and Kimura (Hymenoptera: Figitidae) in Europe. Two specimens emerged from ripened fruits and one was collected after direct observation on a cherry tree in June 2019. They showed the distinctive morphological traits already described and shared more than 99% sequence similarity with specimens of L. japonica collected in Asia. This first finding was confirmed by a wider survey carried out in 2020, L. japonica emerged from cherry fruit samples collected in five other sites across the Trentino region, suggesting that L. japonica has already colonized a wide area. Detection of this Asian species is relevant to the future direction in managing D. suzukii, both in Europe and North America. In fact, L. japonica showed similarity with Ganaspis brasiliensis (Ihering) (Hymenoptera: Figitidae), the most promising candidate for the classical biological control, in terms of developmental time, egg maturation, host age preference and lifetime fecundity.

In the search for native Asian parasitoids of Drosophila suzukii, the notorious spotted-wing Drosophila (SWD), an odd new species of Eucoilinae was discovered. Leptopilina lasalleisp. nov. is herein described and diagnosed relative to other eucoilines associated with drosophilid hosts. Morphologically, L. lasallei is somewhat aberrant within Leptopilina; phylogenetically, L. lasallei is sister group to the core Leptopilina. In the process of investigating L. lasallei, a de novo molecular phylogeny of Leptopilina was generated and is included here. The integrated approach used for the characterisation of L. lasallei, and the resulting phylogeny of Leptopilina, produced data useful to select parasitoid species for SWD biological control.http://www.zoobank.org/urn:lsid:zoobank.org:act:402D504A-4616-4524-85D7-1C13A6276F06http://www.zoobank.org/urn:lsid:zoobank.org:act:402D504A-4616-4524-85D7-1C13A6276F06.

We report the presence of two Asian species of larval parasitoids of spotted wing Drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), in northwestern North America. Leptopilina japonica Novkovic & Kimura and Ganaspis brasiliensis (Ihering) (Hymenoptera: Figitidae) were found foraging near and emerging from fruits infested by D. suzukii at several locations across coastal British Columbia, Canada in the summer and fall of 2019. While G. brasiliensis was found in British Columbia for the first time in 2019, re-inspection of previously collected specimens suggests that L. japonica has been present since at least 2016. Additionally, we found a species of Asobara associated with D. suzukii in British Columbia that is possibly Asobara rufescens (Förster) (known only from the Palearctic Region) based on COI DNA barcode data. These findings add to the list of cases documenting adventive establishment of candidate classical biological control agents outside of their native ranges. The findings also illustrate the need for revisiting species concepts within Asobara, as well as host and geographic distribution data due to cryptic and/or misidentified species.

Some species of parasitic wasps have domesticated viral machineries to deliver immunosuppressive factors to their hosts. Up to now, all described cases fall into the Ichneumonoidea superfamily, which only represents around 10% of hymenoptera diversity, raising the question of whether such domestication occurred outside this clade. Furthermore, the biology of the ancestral donor viruses is completely unknown. Since the 1980s, we know that Drosophila parasitoids belonging to the Leptopilina genus, which diverged from the Ichneumonoidea superfamily 225 Ma, do produce immunosuppressive virus-like structure in their reproductive apparatus. However, the viral origin of these structures has been the subject of debate. In this article, we provide genomic and experimental evidence that those structures do derive from an ancestral virus endogenization event. Interestingly, its close relatives induce a behavior manipulation in present-day wasps. Thus, we conclude that virus domestication is more prevalent than previously thought and that behavior manipulation may have been instrumental in the birth of such associations.

Following the global invasion of Drosophila suzukii (spotted-wing drosophila or SWD), nearly 100 studies have explored biological control of this pest. In 2019, a review summarized 75+ papers covering 57 species of SWD parasitoids, predators, competitors, and pathogens and identified the most promising ones. This review provides an update with recent studies. Since parasitoids are promising natural enemies that can be host-specific and self-disperse, this chapter focuses on SWD parasitoids in its invaded and native ranges, and prospects for classical biological control. To date, six species have been confirmed to attack SWD in the invaded regions including three widely studied generalist pupal parasitoids, Pachycrepoideus vindemiae, Trichopria drosophilae, and T. anastrephae. No locally occurring larval drosophila parasitoids can develop from SWD. In contrast, foreign explorations in China, Japan, and South Korea have revealed 19 species of SWD larval parasitoids. Asobara japonica, Ganaspis brasiliensis, and Leptopilina japonica spp. japonica have been evaluated. Ganaspis brasiliensis is a complex of cryptic species/strains with varying host specificity, some which also occur in regions outside of Asia, but one East Asian strain was found to be the most host-specific to SWD and is currently being petitioned for introduction into North America and Europe.