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Simple Summary: Insect hemolymph transports, exchanges, and eliminates soluble compounds from the hemocoel. These processes play a critical role in many physiological processes, including development and immunity. Since matrix metalloproteases (MMPs) are the major circulating gelatinases in the blood of various species, including mammals, we used gel zymography to analyze gelatinase and caseinase activities in Drosophila larval hemolymph under normal and pathological conditions. Our investigations demonstrate that the major gelatinases and caseinases in Drosophila larval hemolymph are not MMPs but serine proteases (SPs). We identified more than 60 SPs in these proteolytic active bands. While a role in immunity has been suggested for some of these circulating SPs, the physiological functions of most of them remain to be elucidated. After separation on gel zymography, Drosophila melanogaster hemolymph displays gelatinase and caseinase bands of varying sizes, ranging from over 140 to 25 kDa. Qualitative and quantitative variations in these bands were observed during larval development and between different D. melanogaster strains and Drosophila species. The activities of these Drosophila hemolymph gelatinase and caseinase were strongly inhibited by serine protease inhibitors, but not by EDTA. Mass spectrometry identified over 60 serine proteases (SPs) in gel bands corresponding to the major D. melanogaster gelatinases and caseinases, but no matrix metalloproteinases (MMPs) were found. The most abundant proteases were tequila and members of the Jonah and trypsin families. However, the gelatinase bands did not show any change in the tequila null mutant. Additionally, no clear changes could be observed in D. melanogaster gel bands 24 h after injection of bacterial lipopolysaccharides (LPS) or after oviposition by Leptopilina boulardi endoparasitoid wasps. It can be concluded that the primary gelatinases and caseinases in Drosophila larval hemolymph are serine proteases (SPs) rather than matrix metalloproteinases (MMPs). Furthermore, the gelatinase pattern remains relatively stable even after short-term exposure to pathogenic challenges. [ABSTRACT FROM AUTHOR]

Legumes can meet their nitrogen requirements through root nodule symbiosis, which could also trigger plant systemic resistance against pests. The pea aphid Acyrthosiphon pisum, a legume pest, can harbour different facultative symbionts (FS) influencing various traits of their hosts. It is therefore worth determining if and how the symbionts of the plant and the aphid modulate their interaction. We used different pea aphid lines without FS or with a single one (Hamiltonella defensa, Regiella insecticola, Serratia symbiotica) to infest Medicago truncatula plants inoculated with Sinorhizobium meliloti (symbiotic nitrogen fixation, SNF) or supplemented with nitrate (non-inoculated, NI). The growth of SNF and NI plants was reduced by aphid infestation, while aphid weight (but not survival) was lowered on SNF compared to NI plants. Aphids strongly affected the plant nitrogen fixation depending on their symbiotic status, suggesting indirect relationships between aphid- and plant-associated microbes. Finally, all aphid lines triggered expression of Pathogenesis-Related Protein 1 (PR1) and Proteinase Inhibitor (PI), respective markers for salicylic and jasmonic pathways, in SNF plants, compared to only PR1 in NI plants. We demonstrate that the plant symbiotic status influences plant–aphid interactions while that of the aphid can modulate the amplitude of the plant's defence response. [ABSTRACT FROM AUTHOR]

Legumes can meet their nitrogen requirements through root nodule symbiosis, which could also trigger plant systemic resistance against pests. The pea aphid Acyrthosiphon pisum, a legume pest, can harbour different facultative symbionts (FS) influencing various traits of their hosts. It is therefore worth determining if and how the symbionts of the plant and the aphid modulate their interaction. We used different pea aphid lines without FS or with a single one (Hamiltonella defensa, Regiella insecticola, Serratia symbiotica) to infest Medicago truncatula plants inoculated with Sinorhizobium meliloti (symbiotic nitrogen fixation, SNF) or supplemented with nitrate (non-inoculated, NI). The growth of SNF and NI plants was reduced by aphid infestation, while aphid weight (but not survival) was lowered on SNF compared to NI plants. Aphids strongly affected the plant nitrogen fixation depending on their symbiotic status, suggesting indirect relationships between aphid- and plant-associated microbes. Finally, all aphid lines triggered expression of Pathogenesis-Related Protein 1 (PR1) and Proteinase Inhibitor (PI), respective markers for salicylic and jasmonic pathways, in SNF plants, compared to only PR1 in NI plants. We demonstrate that the plant symbiotic status influences plant-aphid interactions while that of the aphid can modulate the amplitude of the plant's defence response. [ABSTRACT FROM AUTHOR]

Previous data showed that the parasitoid wasp Aphelinus asychis was more successful on Sitobion avenae clones artificially infected with different strains of Regiella insecticola compared to the same clones lacking facultative symbionts. To test whether this resulted from a specific interaction between the aphid and symbiont genotype, we used new clonal lines from a different genotype of S. avenae (Linyi) infected with the same R. insecticola strains. The parasitism rate of A. asychis was higher on the two Linyi lines infected with R. insecticola than on the uninfected control line, while the emergence rate was lower due to higher mortality during development. However, a different wasp species, Aphidius gifuensis, showed no difference in the parasitism rate and emergence rate between the three Linyi lines, suggesting that the increase in parasitism rate could be wasp species-dependent. The mortality of A. asychis during development in the presence of R. insecticola may be linked to (i) a direct toxic or metabolic effect since the weight of the emerged wasps was also reduced and (ii) a general effect on the aphid fitness since the survival and fecundity of infected aphids having survived A. asychis attack were clearly reduced compared to the control, while those surviving A. gifuensis attack had only a slight increase in their survival rate. Our data therefore enriched the panel of phenotypic effects that R. insecticola could contribute to the aphid and emphasized the potential implications of symbionts on biological pest control. [ABSTRACT FROM AUTHOR]

Highlights: [•] Biological control of Bactrocera oleae needs a reliable estimation of parasitoid efficiency. [•] We present a specific PCR tool to compare parasitism rates of Psyttalia species. [•] The PCR method allows early and sensitive detection of Psyttalia eggs. [•] The PCR method is more reliable than traditional ones (rearing and dissection). [ABSTRACT FROM AUTHOR]

Simple Summary: Global warming affects most species and their interaction s. Insects are ectotherms, meaning their body temperature is affected by the ambient temperature. This is particularly important for koinobiont parasitoids, insects that keep their host insect alive during development of their eggs and larvae, the host eventually being consumed before adult parasitoids emerge. Temperature changes could therefore affect parasitoids directly and/or indirectly through their impact on the host. Here, we tested the effect of temperature on the parasitic success of two parasitoid lines on two host species, and on each partner independently, to determine whether the host immune response and/or the parasitoid venom proteins, injected with the egg to counteract the host immune response, were affected. The host's immune defense consists of forming a capsule surrounding the parasitoid egg. In half of the interactions tested, the parasitic success increased with temperature. For one, the increase appeared to result solely from an increased capacity of the parasitoid to escape from a capsule, while for the second, it also appeared to involve a decrease in host encapsulation capacity. Finally, we observed a strong change in venom composition depending on the rearing temperature which may partially explain the change in parasitic success. Temperature is particularly important for ectotherms, including endoparasitoid wasps that develop inside another ectotherm host. In this study, we tested the impact of three temperatures (20 °C, 25 °C and 30 °C) on the host–parasitoid immune interaction using two Drosophila host species (Drosophila melanogaster and D. yakuba) and two parasitoid lines of Leptopilina boulardi. Drosophila's immune defense against parasitoids consists of the formation of a melanized capsule surrounding the parasitoid egg. To counteract this response, Leptopilina parasitoids rely on the injection of venom during oviposition. Here, we tested the effect of temperature on parasitic success and host encapsulation capacity in response to a parasitoid egg or other foreign body. Increased temperature either promoted or did not affect the parasitic success, depending on the parasitoid–host pairs considered. The mechanisms behind the higher success seemed to vary depending on whether the temperature primarily affected the host immune response or also affected the parasitoid counter-immune response. Next, we tested the effect of parasitoid rearing temperature on its success and venom composition. Venom composition varied strongly with temperature for both parasitoid lines, partially consistent with a change in their parasitic success. Overall, temperature may have a significant impact on the host–parasitoid immune interaction. [ABSTRACT FROM AUTHOR]

Meteorus pulchricornis (Ichneumonoidea, Braconidae) is an endoparasitoid wasp of lepidopteran caterpillars. Its parasitic success relies on vesicles (named M. pulchricornis Virus-Like Particles or MpVLPs) that are synthesized in the venom gland and injected into the parasitoid host along with the venom during oviposition. In order to define the content and understand the biogenesis of these atypical vesicles, we performed a transcriptome analysis of the venom gland and a proteomic analysis of the venom and purified MpVLPs. About half of the MpVLPs and soluble venom proteins identified were unknown and no similarity with any known viral sequence was found. However, MpVLPs contained a large number of proteins labelled as metalloproteinases while the most abundant protein family in the soluble venom was that of proteins containing the Domain of Unknown Function DUF-4803. The high number of these proteins identified suggests that a large expansion of these two protein families occurred in M. pulchricornis. Therefore, although the exact mechanism of MpVLPs formation remains to be elucidated, these vesicles appear to be "metalloproteinase bombs" that may have several physiological roles in the host including modifying the functions of its immune cells. The role of DUF4803 proteins, also present in the venom of other braconids, remains to be clarified. [ABSTRACT FROM AUTHOR]

For freshwater fish the motile period of sperm is extremely brief, even after a dilution in isotonic media. This result is in contrast to most other animals (ranging from invertebrates to mammals), in which sperm are generally motile for at least several hours. We have analyzed the reasons for the brevity of this movement by studying the relationships between the metabolism of trout sperm and the activation of their motility upon dilution. Sperm motility was not initiated when the dilution medium contained an elevated concentration of potassium (20-40 mM), but dilution in an isotonic solution of sodium chloride triggered an immediate activation of motility, and sperm swam vigorously. Motility of sperm decreased rapidly and 15 s after dilution sperm were moving slowly in small circles. Sperm became abruptly immotile at 20-30 s and flagella straightened. When millimolar concentrations of Ca2+ were also present in the dilution medium, movement did not not stop abruptly, flagella kept beating and stopped only after 1-2 min. When sperm remained immotile they retained a high concentration of ATP. The activation of motility induced a rapid decrease of ATP. In the absence of calcium, and after the cessation of motility, ATP increased slowly back to its original concentration. In the presence of millimolar concentrations of calcium the concentration of ATP decreased to a very low level and remained low thereafter. The progressive decrease of the flagellar beat frequency, that had been observed during the period of trout sperm movement, might be related to the rapid exhaustion of intraflagellar ATP. Motility could be reinduced in sperm that had recovered high concentrations of ATP, demonstrating the functional integrity of the motile apparatus even after flagellar arrest. In conclusion we suggest that the maximum duration of trout sperm motility, at most 2 min (as a consequence of a depletion of ATP during the movement), is due... [ABSTRACT FROM AUTHOR]

Symbiotic microbes have evolved to become an important source of phenotypic plasticity in host insects. In aphids, facultative endosymbionts have been found capable of influencing host phenotypes, such as against natural enemies and host fitness. However, the related evidence has been mainly gathered from a few model organisms, such as the pea aphid. Here, we explored the roles of a common facultative symbiont of the genus Rickettsia in the grain aphid Sitobion avenae. We first observed that Rickettsia-infected aphids (either natural or artificial status) benefit of a higher population increase rate (nearly 1.23-fold) than the Rickettsia-free aphids from the same genetic background. While Rickettsia presence did not confer resistance against the aphid parasitoid Aphidius gifuensis, emerged wasps from Rickettsia-infected aphids had a significantly reduced weight and their sex ratio was biased toward more female wasps. These results suggest that the presence of the symbiont ameliorates the fitness of its aphid host, but impacts the growth of parasitoid offspring and the parasitoid sex-ratio in a way that could be detrimental for the aphid. This demonstrates that the presence of a facultative symbiont may lead to both positive and negative trade-offs, which may ultimately not affect the fitness of the host population as they are compensatory. [ABSTRACT FROM AUTHOR]

Two different species of solitary parasitoid wasps have been used as biological control agents for the Asian citrus psyllid Diaphorina citri: the endoparasitoid Diaphorencyrtus aligarhensis and the ectoparasitoid Tamarixia radiata. Since little information was available on the role of the venom of these two parasitoid species in their reproductive success, we set up experiments to compare the effect of natural parasitism, natural envenomation (which was possible for T. radiata) and microinjections of D. aligarhensis and T. radiata venoms on the development and behavior of D. citri nymphs. We found that natural or artificial envenomation by T. radiata rapidly inhibited the crawling behavior of D. citri nymphs and subsequently induced a high nymph mortality rate, mimicking the natural effect of parasitization. In contrast, microinjection of D. aligarhensis venom did not affect nymphal crawling and had no clear effect on nymphal development apart from a slight delay in the days following injection, which may be consistent with the need to keep the host alive during parasitoid larval development. These results demonstrated the differential effects of venom from two types of parasitoid species sharing the same host, improving our understanding of the evolution of Hymenoptera parasitism and highlighting the use of parasitoid's venom as a potential source of bioinsecticidal molecules. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis kurstaki (Btk) is among the most used microbial agent worldwide to control lepidopteran pests in organic and non-organic crops. The extensive use of this bioinsecticide and its environmental accumulation may become a major issue for integrated pest management (IPM) programs that include Btk and non-target species (parasitoids and predators) if side effects on these organisms and their associated food-webs occur. Here, chronic effects of Btk bioinsecticide were explored in two Drosophila larval endoparasitoids, Leptopilina heterotoma and L. boulardi (Hymenoptera: Figitidae), indirectly on their development in the exposed host and directly on their adult longevity. Emergence rates of D. melanogaster “Canton-S” and “Sefra” host strains fed Btk bioinsecticide throughout larval development decreased at the highest concentration (10 times the recommendations), and no significant effect was observed on the parasitisation behaviour of female parasitoids toward Btk-fed larval hosts. Parasitoids’ developmental time in Btk-exposed hosts remained unchanged while emergence rate decreased as a function of bioinsecticide concentration, host fly strain and parasitoid species. In adult parasitoids Btk-fed, the longevity of L. heterotoma males and L. boulardi females were reduced even at recommended spraying concentrations. Our study reports that the Btk bioinsecticide may affect non-target parasitoids through chronic indirect host exposure and directly through food poisoning. In this context, since closely related parasitoid species may respond differently to bioinsecticide exposure, species-specific studies must be conducted when IPM strategies combine these natural enemies with the Btk bioinsecticide. [ABSTRACT FROM AUTHOR]

Abstract: The final stages of sperm differentiation occur outside the gonad, in the epididymal tubule. These last maturation steps, essential to the quality of spermatozoa, are not under the genomic control of the germ cells. A series of sequential interactions with the epididymal fluid, mostly specific proteins present in the lumen of different regions, are believed to induce the final steps of sperm maturation. In order to provide the luminal changes required for this maturation to occur, the epithelium may resort to two basic mechanisms: absorption and secretion. Far from being a uniform channel, the epididymal duct is a canal with highly specialized regional differentiation of its epithelial ultrastructure and its secretory and absorptive functions. This review focuses on the ultractructural characteristic of the epithelial cells, their specific secretory activity according to the epididymal regions and their eventual role in sperm maturation of the boar. The chronology of the changes that occur in and on the sperm and in the surrounding environment are described. Relationships between the highly regionalized epididymal activities, sperm characteristics linked to their survival and fertility potential are also presented in this review. [Copyright &y& Elsevier]

The pea aphid Acyrthosiphon pisum hosts different facultative symbionts (FS) which provide it with various benefits, such as tolerance to heat or protection against natural enemies (e.g., fungi, parasitoid wasps). Here, we investigated whether and how the presence of certain FS could affect phenoloxidase (PO) activity, a key component of insect innate immunity, under normal and stressed conditions. For this, we used clones of A. pisum of different genetic backgrounds (LL01, YR2 and T3‐8V1) lacking FS or harboring one or two (Regiella insecticola, Hamiltonella defensa, Serratia symbiotica + Rickettsiella viridis). Gene expression and proteomics analyses of the aphid hemolymph indicated that the two A. pisum POs, PPO1 and PPO2, are expressed and translated into proteins. The level of PPO genes expression as well as the amount of PPO proteins and phenoloxidase activity in the hemolymph depended on both the aphid genotype and FS species. In particular, H. defensa and R. insecticola, but not S. symbiotica + R. viridis, caused a sharp decrease in PO activity by interfering with both transcription and translation. The microinjection of different types of stressors (yeast, Escherichia coli, latex beads) in the YR2 lines hosting different symbionts affected the survival rate of aphids and, in most cases, also decreased the expression of PPO genes after 24 h. The amount and activity of PPO proteins varied according to the type of FS and stressor, without clear corresponding changes in gene expression. These data demonstrate that the presence of certain FS influences an important component of pea aphid immunity. [ABSTRACT FROM AUTHOR]

Background: Parasitoid wasps have fascinating life cycles and play an important role in trophic networks, yet little is known about their genome content and function. Parasitoids that infect aphids are an important group with the potential for biological control. Their success depends on adapting to develop inside aphids and overcoming both host aphid defenses and their protective endosymbionts. Results: We present the de novo genome assemblies, detailed annotation, and comparative analysis of two closely related parasitoid wasps that target pest aphids: Aphidius ervi and Lysiphlebus fabarum (Hymenoptera: Braconidae: Aphidiinae). The genomes are small (139 and 141 Mbp) and the most AT-rich reported thus far for any arthropod (GC content: 25.8 and 23.8%). This nucleotide bias is accompanied by skewed codon usage and is stronger in genes with adult-biased expression. AT-richness may be the consequence of reduced genome size, a near absence of DNA methylation, and energy efficiency. We identify missing desaturase genes, whose absence may underlie mimicry in the cuticular hydrocarbon profile of L. fabarum. We highlight key gene groups including those underlying venom composition, chemosensory perception, and sex determination, as well as potential losses in immune pathway genes. Conclusions: These findings are of fundamental interest for insect evolution and biological control applications. They provide a strong foundation for further functional studies into coevolution between parasitoids and their hosts. Both genomes are available at https://bipaa.genouest.org. [ABSTRACT FROM AUTHOR]

Host range in parasitoids could be described by the preference–performance hypothesis (PPH) where preference is defined as host acceptance and performance is defined as the sum of all species on which parasitoid offspring can complete their life cycle. The PPH predicts that highly suitable hosts will be preferred by ovipositing females. However, generalist parasitoids may not conform to this hypothesis if they attack a large range of hosts of varying suitability. Under laboratory conditions, we tested the PPH relationship of three aphid parasitoids currently considered as generalist species (Aphelinus abdominalis, Aphidius ervi, Diaeretiella rapae). As expected, the three parasitoids species showed low selectivity, i.e., females stung all aphid species encountered (at least in some extent). However, depending on the parasitoid species, only 42%–58% of aphid species enabled producing parasitoid offspring. We did not find a correlation between the extent of preference and the performance of three generalist aphid parasitoids. For A. ervi, host phylogeny is also important as females showed higher attack and developmental rates on hosts closely related to the most suitable one. In addition, traits such as (a) the presence of protective secondary endosymbionts, for example, Hamiltonella defensa detected in Aphis fabae and Metopolophium dirhodum and (b) the sequestration of plant toxins as defense mechanism against parasitism, for example, in Aphis nerii and Brevicoryne brassicae, were likely at play to some extent in narrowing parasitoid host range. The lack of PPH relationship involved a low selectivity leading to a high adaptability, as well as selection pressure; the combination of which enabled the production of offspring in a new host species or a new environment. Testing for PPH relationships in parasitoids may provide useful cues to classify parasitoids in terms of specialization degree. [ABSTRACT FROM AUTHOR]

Drosophila suzukii (the spotted-wing Drosophila) appears to be unsuitable for the development of most Drosophila larval endoparasitoids, be they sympatric or not. Here, we questioned the physiological bases of this widespread failure by characterizing the interactions between D. suzukii and various parasitoid species (Asobara japonica, Leptopilina boulardi, Leptopilina heterotoma and Leptopilina victoriae) and comparing them with those observed with D. melanogaster, a rather appropriate host. All parasitoids were able to oviposit in L1 and L2 larval stages of both hosts but their propensity to parasitize was higher on D. melanogaster. A. japonica and, to a much lesser extent, L. heterotoma, were the two species able to successfully develop in D. suzukii, the failure of the parasitism resulting either in the parasitoid encapsulation (notably with L. heterotoma) or the host and parasitoid deaths (especially with L. boulardi and L. victoriae). Compared to D. melanogaster, encapsulation in D. suzukii was strongly delayed and led, if successful, to the production of much larger capsules in surviving flies and, in the event of failure, to the death of both partners because of an uncontrolled melanization. The results thus revealed a different timing of the immune response to parasitoids in D. suzukii compared to D. melanogaster with a lose-lose outcome for parasitoids (generally unsuccessful development) and hosts (high mortality and possible reduction of the fitness of survivors). Finally, these results might suggest that some European endoparasitoids of Drosophila interact with this pest in the field in an unmeasurable way, since they kill their host without reproductive success. [ABSTRACT FROM AUTHOR]

The spotted wing Drosophila, Drosophila suzukii (Ds), became a major economic pest for fruit production since its establishment in Europe and America. Among potential control methods, only classical biological control appears to be a mean of sustainably regulating Ds in both cultivated and natural habitats. In the frame of risk assessment, pre-release surveys were carried out in a restricted but highly heterogeneous area in the south-east of France using traps and deliberate field exposures of Ds and D. melanogaster larvae/pupae. Although Ds abundance varied according to sampling methods, it was found to be pervasive and to produce offspring and adults in most conditions (spatial and seasonal). Its main limits are some specific abiotic conditions (i.e., desiccation) as well as interspecific competition. Indeed, Ds mostly co-occurred with D. busckii and D. hydei, probably due to common phenology and/or ecological requirements. These two species thus deserve more attention for risk assessment. The main indigenous parasitoids collected belonged to two pupal species, Trichopria cf drosophilae and Pachycrepoideus vindemmiae, but their presence was observed late in the autumn and mainly in cultivated areas. Results are discussed in a comparison of the methodological approaches for monitoring Drosophilids and the benefits-risks assessment of classical biological control. [ABSTRACT FROM AUTHOR]

Aspartylglucosaminidase (AGA) is a low-abundance intracellular enzyme that plays a key role in the last stage of glycoproteins degradation, and whose deficiency leads to human aspartylglucosaminuria, a lysosomal storage disease. Surprisingly, high amounts of AGA-like proteins are secreted in the venom of two phylogenetically distant hymenopteran parasitoid wasp species, Asobara tabida (Braconidae) and Leptopilina heterotoma (Cynipidae). These venom AGAs have a similar domain organization as mammalian AGAs. They share with them key residues for autocatalysis and activity, and the mature α- and β-subunits also form an (αβ)2 structure in solution. Interestingly, only one of these AGAs subunits (α for AtAGA and β for LhAGA) is glycosylated instead of the two subunits for lysosomal human AGA (hAGA), and these glycosylations are partially resistant to PGNase F treatment. The two venom AGAs are secreted as fully activated enzymes, they have a similar aspartylglucosaminidase activity and are both also efficient asparaginases. Once AGAs are injected into the larvae of the Drosophila melanogaster host, the asparaginase activity may play a role in modulating their physiology. Altogether, our data provide new elements for a better understanding of the secretion and the role of venom AGAs as virulence factors in the parasitoid wasps’ success. [ABSTRACT FROM AUTHOR]

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases affecting a wide range of mammalian species. They are caused by prions, a proteinaceous pathogen essentially composed of PrPSc, an abnormal isoform of the host encoded cellular prion protein PrPC. Constrained steric interactions between PrPSc and PrPC are thought to provide prions with species specificity, and to control cross-species transmission into other host populations, including humans. Transgenetic expression of foreign PrP genes has been successfully and widely used to overcome the recognized resistance of mouse to foreign TSE sources. Rabbit is one of the species that exhibit a pronounced resistance to TSEs. Most attempts to infect experimentally rabbit have failed, except after inoculation with cell-free generated rabbit prions. To gain insights on the molecular determinants of the relative resistance of rabbits to prions, we generated transgenic rabbits expressing the susceptible V136R154Q171 allele of the ovine PRNP gene on a rabbit wild type PRNP New Zealand background and assessed their experimental susceptibility to scrapie prions. All transgenic animals developed a typical TSE 6–8 months after intracerebral inoculation, whereas wild type rabbits remained healthy more than 700 days after inoculation. Despite the endogenous presence of rabbit PrPC, only ovine PrPSc was detectable in the brains of diseased animals. Collectively these data indicate that the low susceptibility of rabbits to prion infection is not enciphered within their non-PrP genetic background. [ABSTRACT FROM AUTHOR]

Background Endoparasitoid wasps are important natural enemies of the widely distributed aphid pests and are mainly used as biological control agents. However, despite the increased interest on aphid interaction networks, only sparse information is available on the factors used by parasitoids to modulate the aphid physiology. Our aim was here to identify the major protein components of the venom injected at oviposition by Aphidius ervi to ensure successful development in its aphid host, Acyrthosiphon pisum. Results A combined large-scale transcriptomic and proteomic approach allowed us to identify 16 putative venom proteins among which three γ-glutamyl transpeptidases (γ-GTs) were by far the most abundant. Two of the γ-GTs most likely correspond to alleles of the same gene, with one of these alleles previously described as involved in host castration. The third γ-GT was only distantly related to the others and may not be functional owing to the presence of mutations in the active site. Among the other abundant proteins in the venom, several were unique to A. ervi such as the molecular chaperone endoplasmin possibly involved in protecting proteins during their secretion and transport in the host. Abundant transcripts encoding three secreted cystein-rich toxin-like peptides whose function remains to be explored were also identified. Conclusions Our data further support the role of γ-GTs as key players in A. ervi success on aphid hosts. However, they also evidence that this wasp venom is a complex fluid that contains diverse, more or less specific, protein components. Their characterization will undoubtedly help deciphering parasitoid-aphid and parasitoid-aphid-symbiont interactions. Finally, this study also shed light on the quick evolution of venom components through processes such as duplication and convergent recruitment of virulence factors between unrelated organisms. [ABSTRACT FROM AUTHOR]

Highlights: [•] RNAi was developed in an endoparasitoid wasp species, parasitoid of Drosophila. [•] The expression of a major venom protein was strongly and specifically silenced. [•] The silencing of the venom protein lasted the entire parasitoid lifetime. [ABSTRACT FROM AUTHOR]

Abstract: The arms race between immune suppressive parasites that produce virulence factors and hosts that evolve resistance to these factors is suggested to be a key driver for the diversification of both partners. However, little is known regarding the diversity of virulence factors in closely related parasites or the mechanisms underlying the variation of virulence. One of the best-described model to address this issue is the interaction between Leptopilina parasitic wasps and their Drosophila hosts, in which variation of virulence is well documented. Thanks to a combined transcriptomic and proteomic approach, we have identified the main secreted proteins in the venom of Leptopilina heterotoma (Gotheron strain, 66 proteins) and of two well-characterized strains of Leptopilina boulardi, ISm and ISy (65 and 49 proteins, respectively). Results revealed significant quantitative differences in venom components between the L. boulardi strains, in agreement with their different virulence properties. Strikingly, the two related Leptopilina species did not share any abundant venom protein. The main identified proteins in L. boulardi were RhoGAPs and serpins while an aspartylglucosaminidase (AGA) was found abundant in L. heterotoma. The extensive quantitative variation observed between these species may be related with their use of different virulence strategies and/or to differences in their host range (specialist versus generalist). Altogether, our data suggests that parasitoid venom can quickly evolve, mainly through rapid changes in regulation of gene expression. It also evidences venom evolutionary processes largely described in other venomous animals i.e. the convergent recruitment of venom proteins between phylogenetically unrelated organisms, and the role of duplications in the emergence of multigenic families of virulence factors. [Copyright &y& Elsevier]

Abstract: The venom gland is a conserved organ in Hymenoptera that shows adaptations associated with life-style diversification. Few studies have investigated venom components and function in the highly diverse parasitic wasps and all suggest that the venom regulates host physiology. We explored the venom of the endoparasitoid Hyposoter didymator (Campopleginae), a species with an associated polydnavirus produced in the ovarian tissue. We investigated the effects of the H. didymator venom on two physiological traits of the host Spodoptera frugiperda (Noctuidae): encapsulation response and growth rate. We found that H. didymator venom had no significant effect on host cellular immunity or development, suggesting that it does not contribute to parasitism success. The host physiology seemed to be modified essentially by the ovarian fluid containing the symbiotic polydnaviruses. Proteomic analyses indicated that the H. didymator venom gland produces a large variety of proteins, consistent with the classical hymenopteran venom protein signature, including: reprolysin-like, dipeptidyl peptidase IV, hyaluronidase, arginine kinase or allergen proteins. The venom extracts also contained novel proteins, encoded by venom genes conserved in Campopleginae ichneumonids, and proteins with similarities to active molecules identified in other parasitoid species, such as calreticulin, reprolysin, superoxide dismutase and serpin. However, some of these proteins appear to be produced only in small amounts or to not be secreted. Possibly, in Campopleginae carrying polydnaviruses, the host-modifying activities of venom became redundant following the acquisition of polydnaviruses by the lineage. [Copyright &y& Elsevier]

Abstract: Endoparasitoid wasps develop at the expense of other insects, leading to their death. Eggs deposited inside the host body induce an immune response, which results in the formation of a melanized cellular capsule around the egg. To evade or counteract this response, endoparasitoids have evolved different strategies, the most often reported being injection into the host of immunosuppressive factors, notably venom proteins, along with the egg. The analysis of venom components has been performed independently in species of different taxa, but the present picture is far from complete. Intriguingly, the question of the level of venom variability inside species has been neglected, although it may partly determine the potential for parasitoid adaptation. Here, we present a short review of our present knowledge of venom components in endoparasitoids, as well as of the only well-known example of intraspecific variability in a venom immune suppressive protein being responsible for variation in parasitoid virulence. We then present data evidencing inter-individual variation of venom protein profiles, using a gel electrophoresis approach, both in laboratory strains and field populations of a figitid and a braconid species. Whether occurrence of such variability may permit a selection of parasitoid venom components driven by the host remains to be tested, notably in the context of the production and use of biological control auxiliaries. [Copyright &y& Elsevier]

The acquisition of the ability of the male gamete to fertilize an ovum is the result of numerous and sequential steps of differentiation of spermatozoa that occur as they transit from the testis to the end of the epididymal tubule. The post gonadal sperm modifications are mostly related to motility, egg binding, and penetration processes. As the activity of the epididymis and its luminal fluid composition are believed to be directly related to 'sperm maturation', a review on epididymal proteins is presented. Comparative studies have shown that the epididymal activities are species specific. Nevertheless, for all mammalian species studied, similarities exist in the sequential proteomic changes of the luminal composition of the epididymal tubule and proteins on the sperm surface. The potential roles of these modifications are discussed. [ABSTRACT FROM AUTHOR]

Recent studies suggest that the pea aphid (Acyrthosiphon pisum) has low immune defenses. However, its immune components are largely undescribed, and notably, extensive characterization of circulating cells has been missing. Here, we report characterization of five cell categories in hemolymph of adults of the LL01 pea aphid clone, devoid of secondary symbionts (SS): prohemocytes, plasmatocytes, granulocytes, spherulocytes and wax cells. Circulating lipid-filed wax cells are rare; they otherwise localize at the basis of the cornicles. Spherulocytes, that are likely sub-cuticular sessile cells, are involved in the coagulation process. Prohemocytes have features of precursor cells. Plasmatocytes and granulocytes, the only adherent cells, can form a layer in vivo around inserted foreign objects and phagocytize latex beads or Escherichia coli bacteria injected into aphid hemolymph. Using digital image analysis, we estimated that the hemolymph from one LL01 aphid contains about 600 adherent cells, 35% being granulocytes. Among aphid YR2 lines differing only in their SS content, similar results to LL01 were observed for YR2-Amp (without SS) and YR2-Ss (with Serratia symbiotica), while YR2-Hd (with Hamiltonella defensa) and YR2(Ri) (with Regiella insecticola) had strikingly lower adherent hemocyte numbers and granulocyte proportions. The effect of the presence of SS on A. pisum cellular immunity is thus symbiont-dependent. Interestingly, Buchnera aphidicola (the aphid primary symbiont) and all SS, whether naturally present, released during hemolymph collection, or artificially injected, were internalized by adherent hemocytes. Inside hemocytes, SS were observed in phagocytic vesicles, most often in phagolysosomes. Our results thus raise the question whether aphid symbionts in hemolymph are taken up and destroyed by hemocytes, or actively promote their own internalization, for instance as a way of being transmitted to the next generation. Altogether, we demonstrate here a strong interaction between aphid symbionts and immune cells, depending upon the symbiont, highlighting the link between immunity and symbiosis. [ABSTRACT FROM AUTHOR]

Endoparasitoid wasps inject venom proteins with their eggs to protect them from the host immune response and ensure successful parasitism. Here we report identification of Cu,Zn superoxide dismutase (SOD) transcripts for both intracellular SOD1 and extracellular SOD3 in the venom apparatus of two Leptopilina species, parasitoids of Drosophila. Leptopilina SODs show sequence and structure similarity to human SODs, but phylogenetic analyses indicate that the extracellular SODs are more related to cytoplasmic vertebrate SODs than to extracellular SODs, a feature shared by predicted insect extracellular SODs. We demonstrate that L. boulardi SOD3 is indeed secreted and active as monomeric glycosylated forms in venom. Our results also evidence quantitative variation in SOD3 venom contents between closely related parasitoid species, as sod3 is 100-fold less expressed in Leptopilina heterotoma venom apparatus and no protein and SOD activity are detected in its venom. Leptopilina recombinant SOD3s as well as a mammalian SOD in vitro inhibit the Drosophila phenoloxidase activity in a dose-dependent manner, demonstrating that SODs may interfere with the Drosophila melanization process and, therefore, with production of cytotoxic compounds. Although the recombinant L. boulardi SOD3 quantity needed to observe this effect precludes a systemic effect of the wasp venom SOD3, it is still consistent with a local action at oviposition. This work provides the first demonstration that insect extracellular SODs are indeed secreted and active in an insect fluid and can be used as virulence factors to counteract the host immune response, a strategy largely used by bacterial and fungal pathogens but also protozoan parasites during infection. [ABSTRACT FROM AUTHOR]

Abstract: The epididymis plays a major role in the acquisition of sperm fertility. In order to shed light on specific features of epididymal function in mammalian species, we characterized the luminal proteins (luminal proteome) and secreted proteins (secretome) in the bovine epididymis. We identified 172 different luminal proteins in 9 distinct epididymal regions. The concentration and secretory activity of luminal proteins were quantified throughout the epididymis. Among the most abundant secreted proteins, we found lipocalin 5, (LCN5), NADP+dependent prostaglandin dehydrogenase (PTGDS), Niemann-Pick disease type C2 protein (NPC2), glutathione peroxidase type 5 (GPX 5), clusterin (CLU), hexosaminidase B (HEXB) and galactosidase (GLB1), each of which is released in distinct epididymal regions. Gelsolin, (GSN) previously not described in mammalian epididymal fluid, appeared to be a major protein secreted exclusively in the distal region of the bovine epididymis, where fully mature spermatozoa are stored. Although the major epididymal proteins are conserved between mammalian species, this study highlights the specificity and mechanisms of protein processing of epididymal secretion in the bull. In addition, this study provides a major insight into the sequential changes occurring in the sperm environment while gaining fertilizing capacity and could provide new information for the future identification of potential fertility markers. [ABSTRACT FROM AUTHOR]

Abstract: In all mammalian species, the final differentiation of the male germ cell occurs in the epididymal duct where the spermatozoa develop the ability to be motile and fertilize an ovum. Understanding of these biological processes is the key to understanding and controlling male fertility. Comparative studies between several mammals could be an informative approach to finding common sperm modifications which are not species-specific. The new global biological approaches such as transcriptomes and proteomes provide considerable information which can be used for such comparative approaches. This report summarizes our proteomic studies of the epididymis of several mammals, including humans. [Copyright &y& Elsevier]

Background: Mammalians gamete production takes place in the testis but when they exit this organ, although spermatozoa have acquired a specialized and distinct morphology, they are immotile and infertile. It is only after their travel in the epididymis that sperm gain their motility and fertility. Epididymis is a crescent shaped organ adjacent to the testis that can be divided in three gross morphological regions, head (caput), body (corpus) and tail (cauda). It contains a long and unique convoluted tubule connected to the testis via the efferent ducts and finished by joining the vas deferens in its caudal part. Results: In this study, the testis, the efferent ducts (vas efferens, VE), nine distinct successive epididymal segments and the deferent duct (vas deferens, VD) of four adult boars of known fertility were isolated and their mRNA extracted. The gene expression of each of these samples was analyzed using a pig generic 9 K nylon microarray (AGENAE program; GEO accession number: GPL3729) spotted with 8931 clones derived from normalized cDNA banks from different pig tissues including testis and epididymis. Differentially expressed transcripts were obtained with moderated t-tests and F-tests and two data clustering algorithms based either on partitioning around medoid (top down PAM) or hierarchical clustering (bottom up HCL) were combined for class discovery and gene expression analysis. Tissue clustering defined seven transcriptomic units: testis, vas efferens and five epididymal transcriptomic units. Meanwhile transcripts formed only four clusters related to the tissues. We have then used a specific statistical method to sort out genes specifically over-expressed (markers) in testis, VE or in each of the five transcriptomic units of the epididymis (including VD). The specific regional expression of some of these genes was further validated by PCR and Q-PCR. We also searched for specific pathways and functions using available gene ontology information. Conclusion: This study described for the first time the complete transcriptomes of the testis, the epididymis, the vas efferens and the vas deferens on the same species. It described new genes or genes not yet reported over-expressed in these boar tissues, as well as new control mechanisms. It emphasizes and fulfilled the gap between studies done in rodents and human, and provides tools that will be useful for further studies on the biochemical processes responsible for the formation and maintain of the epididymal regionalization and the development of a fertile spermatozoa. [ABSTRACT FROM AUTHOR]

[Display omitted] • Female Leptopilina parasitoid wasps inject venom at oviposition. • As pro-ovigenic species, they should have enough venom to protect their eggs. • L. boulardi but not L. heterotoma has enough venom to protect more than 100 eggs. • L. heterotoma must actively synthesize venom after emergence. • The venom may lose some of its effectiveness after repetitive egg laying. The Drosophila endoparasitoid wasps Leptopilina boulardi and L. heterotoma (Hymenoptera: Cynipidae) are pro-ovigenic 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. [ABSTRACT FROM AUTHOR]

In healthy Drosophila melanogaster larvae, plasmatocytes and crystal cells account for 95% and 5% of the hemocytes, respectively. A third type of hemocytes, lamellocytes, are rare, but their number increases after oviposition by parasitoid wasps. The lamellocytes form successive layers around the parasitoid egg, leading to its encapsulation and melanization, and finally the death of this intruder. However, the total number of lamellocytes per larva remains quite low even after parasitoid infestation, making direct biochemical studies difficult. Here, we used the Hop Tum-l mutant strain that constitutively produces large numbers of lamellocytes to set up a purification method and analyzed their major proteins by 2D gel electrophoresis and their plasma membrane surface proteins by 1D SDS-PAGE after affinity purification. Mass spectrometry identified 430 proteins from 2D spots and 344 affinity-purified proteins from 1D bands, for a total of 639 unique proteins. Known lamellocyte markers such as PPO3 and the myospheroid integrin were among the components identified with specific chaperone proteins. Affinity purification detected other integrins, as well as a wide range of integrin-associated proteins involved in the formation and function of cell-cell junctions. Overall, the newly identified proteins indicate that these cells are highly adapted to the encapsulation process (recognition, motility, adhesion, signaling), but may also have several other physiological functions (such as secretion and internalization of vesicles) under different signaling pathways. These results provide the basis for further in vivo and in vitro studies of lamellocytes, including the development of new markers to identify coexisting populations and their respective origins and functions in Drosophila immunity. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: We describe a simple and direct zymographic method for the detection of proteases using quenched fluorescent substrates. The proteases were separated using one- and two-dimensional electrophoresis, and the gel subsequently was incubated with the quenched fluorescent substrate. After a short incubation, the released fluorescence allowed the localization of the proteases directly using UV light. The protease spots could then be cut directly from the gel and processed for identification by mass spectrometry. This method could easily be used to develop or test whether a substrate is specific or not and also to detect the proteases that are able to cleave this substrate in a complex biological fluid. This also allowed direct identification of proteases without complex purification. [Copyright &y& Elsevier]

Bioinsecticides based on Bacillus thuringiensis (Bt) spores and toxins are increasingly popular alternative solutions to control insect pests, with potential impact of their accumulation in the environment on non-target organisms. Here, we tested the effects of chronic exposure to commercial Bt formulations (Bt var. kurstaki and israelensis) on eight non-target Drosophila species present in Bt-treated areas, including D. melanogaster (four strains). Doses up to those recommended for field application (~ 106 Colony Forming Unit (CFU)/g fly medium) did not impact fly development, while no fly emerged at ≥ 1000-fold this dose. Doses between 10- to 100-fold the recommended one increased developmental time and decreased adult emergence rates in a dose-dependent manner, with species-and strain-specific effect amplitudes. Focusing on D. melanogaster, development alterations were due to instar-dependent larval mortality, and the longevity and offspring number of adult flies exposed to bioinsecticide throughout their development were moderately influenced. Our data also suggest a synergy between the formulation compounds (spores, cleaved toxins, additives) might induce the bioinsecticide effects on larval development. Although recommended doses had no impact on non-target Drosophila species, misuse or local environmental accumulation of Bt bioinsecticides could have side-effects on fly populations with potential implications for their associated communities. [ABSTRACT FROM AUTHOR]

Simple Summary: The use of chemical pesticides is controversial mainly because of their detrimental effects on both the environment and human health. Public opinion along with government policies encourage reductions in their exploitation. An emerging solution is the use of biopesticides, which sales are currently increasing. Among biopesticides, the bacterium called Bacillus thuringiensis is the most used bioinsecticide both in organic and conventional farming to fight larval pests. One strain of Bacillus thuringiensis produces toxins that specifically kill lepidopteran (butterfly) larvae in two or three days. However, although not lethal for other insects such as bees or fruit flies, Bacillus thuringiensis may affect their development. Only scarce data are currently available on the unintended effects of Bacillus thuringiensis strains on insects other than those targeted. In this study, we characterized the adverse effects of Bacillus thuringiensis strains that target lepidopteran larvae, on the development of the fruit fly Drosophilamelanogaster, a dipteran non-target insect. We showed that amounts of Bacillus thuringiensis in the nutritive medium close to the amounts found on vegetables after treatment induced developmental and growth defects in Drosophila larvae. We further showed that these effects are due to the disturbance of the larval intestinal physiology, reducing protein digestion. Bioinsecticides made from the bacterium Bacillus thuringiensis (Bt) are the bestselling bioinsecticide worldwide. Among Bt bioinsecticides, those based on the strain Bt subsp. kurstaki (Btk) are widely used in farming to specifically control pest lepidopteran larvae. Although there is much evidence of the lack of acute lethality of Btk products for non-target animals, only scarce data are available on their potential non-lethal developmental adverse effects. Using a concentration that could be reached in the field upon sprayings, we show that Btk products impair growth and developmental time of the non-target dipteran Drosophila melanogaster. We demonstrate that these effects are mediated by the synergy between Btk bacteria and Btk insecticidal toxins. We further show that Btk bioinsecticides trigger intestinal cell death and alter protein digestion without modifying the food intake and feeding behavior of the larvae. Interestingly, these harmful effects can be mitigated by a protein-rich diet or by adding the probiotic bacterium Lactobacillus plantarum into the food. Finally, we unravel two new cellular mechanisms allowing the larval midgut to maintain its integrity upon Btk aggression: First the flattening of surviving enterocytes and second, the generation of new immature cells arising from the adult midgut precursor cells. Together, these mechanisms participate to quickly fill in the holes left by the dying enterocytes. [ABSTRACT FROM AUTHOR]

The pupal ectoparasitoid Pachycrepoideus vindemiae injects venom into its fly hosts prior to oviposition. We have shown that this venom causes immune suppression in Drosophila melanogaster pupa but the mechanism involved remained unclear. Here, we show using transgenic D. melanogaster with fluorescent hemocytes that the in vivo number of plasmatocytes and lamellocytes decreases after envenomation while it has a limited effect on crystal cells. After in vitro incubation with venom, the cytoskeleton of plasmatocytes underwent rearrangement with actin aggregation around the internal vacuoles, which increased with incubation time and venom concentration. The venom also decreased the lamellocytes adhesion capacity and induced nucleus fragmentation. Electron microscopy observation revealed that the shape of the nucleus and mitochondria became irregular after in vivo incubation with venom and confirmed the increased vacuolization with the formation of autophagosomes-like structures. Almost all venom-treated hemocytes became positive for TUNEL assays, indicating massive induced apoptosis. In support, the caspase inhibitor Z-VAD-FMK attenuated the venom-induced morphological changes suggesting an involvement of caspases. Our data indicate that P. vindemiae venom inhibits D. melanogaster host immunity by inducing strong apoptosis in hemocytes. These assays will help identify the individual venom component(s) responsible and the precise mechanism(s)/pathway(s) involved. [ABSTRACT FROM AUTHOR]

Parasitoid wasps rely primarily on venom to suppress the immune response and regulate the physiology of their host. Intraspecific variability of venom protein composition has been documented in some species, but its evolutionary potential is poorly understood. We performed an experimental evolution initiated with the crosses of two lines of Leptopilina boulardi of different venom composition to generate variability and create new combinations of venom factors. The offspring were maintained for 10 generations on two strains of Drosophila melanogaster differing in resistance/susceptibility to the parental parasitoid lines. The venom composition of individuals was characterized by a semi-automatic analysis of 1D SDS-PAGE electrophoresis protein profiles whose accuracy was checked by Western blot analysis of well-characterized venom proteins. Results made evident a rapid and differential evolution of the venom composition on both hosts and showed that the proteins beneficial on one host can be costly on the other. Overall, we demonstrated the capacity of rapid evolution of the venom composition in parasitoid wasps, important regulators of arthropod populations, suggesting a potential for adaptation to new hosts. Our approach also proved relevant in identifying, among the diversity of venom proteins, those possibly involved in parasitism success and whose role deserves to be deepened. [ABSTRACT FROM AUTHOR]