Absence of complementary sex determination in two Leptopilina species (Figitidae, Hymenoptera) and a reconsideration of its incompatibility with endosymbiont-induced thelytoky.

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.

Aphytis lepidosaphes (Hymenoptera: Aphelinidae) as an Effective Parasitoid for Controlling the Lepidosaphes tapleyi (Williams).

<b>Background and Objective:</b> The guava long scale insect <i>Lepidosaphes tapleyi</i> (Williams) (Hemiptera: Diaspididae) is considered one of the main destructive pests of guava around the world. Biological control represents a sustainable alternative for saving control of <i>L. tapleyi</i>. The main objective of the present work was to study the seasonal activity and evaluate the impacts of climatic factors on populations of the parasitoid, <i>Aphytis</i> <i>lepidosaphes</i>, during two successive years (2017/2018 and 2018/2019) in Esna district, Luxor Governorate, Egypt. <b>Materials and Methods:</b> Estimation of the relationship between the population density of <i>L. tapleyi</i> and <i>A. lepidosaphes</i> activity, by using different models of correlation and regression analyses. The estimate of the effects of climatic factors (daily mean max. temp., min. temp., mean of % relative humidity and mean of dew point) on seasonal activity of the parasitoid, <i>A. lepidosaphes</i>, during two successive years (2017/2018 and 2018/2019). <b>Results:</b> The results showed that the relationship between the population density of <i>L. tapleyi</i> and <i>A. lepidosaphes </i>activity was positive during both years. Furthermore, simple regression analysis indicated that the abundance of <i>A. lepidosaphes </i>was more highly correlated with the <i>L. tapleyi</i> population density in each whole year during the two successive years. The percentages of explained variance EV (%) indicated that all tested variables, i.e. daily mean maximum temperature, minimum temperature, relative humidity and dew point were responsible for 76.26 and 65.40% of the changes in parasitoid, respectively. Furthermore, the dew point was the most effective variable for the change in the parasitoid populations by 33.61 and 18.62%. <b>Conclusion:</b> The results showed that <i>A. lepidosaphes </i>had three peaks of seasonal abundance over the entire year. As well, the activity of <i>A. lepidosaphes</i> was more highly correlated with the <i>L. tapleyi </i>population size over the two successive years.

Proteomic Characterization of Lignocellulolytic Enzymes Secreted by the Insect-Associated Fungus Daldinia decipiens oita, Isolated from a Forest in Northern Japan.

Wood-devastating insects utilize their symbiotic microbes with lignocellulose-degrading abilities to extract energy from recalcitrant woods. It is well known that free-living lignocellulose-degrading fungi secrete various carbohydrate-active enzymes (CAZymes) to degrade plant cell wall components, mainly cellulose, hemicellulose, and lignin. However, CAZymes from insect-symbiotic fungi have not been well documented except for a few examples. In this study, an insect-associated fungus, Daldinia decipiens oita, was isolated as a potential symbiotic fungus of female Xiphydria albopicta captured from Hokkaido forest. This fungus was grown in seven different media containing a single carbon source, glucose, cellulose, xylan, mannan, pectin, poplar, or larch, and the secreted proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 128 CAZymes, including domains of 92 glycoside hydrolases, 15 carbohydrate esterases, 5 polysaccharide lyases, 17 auxiliary activities, and 11 carbohydrate-binding modules, were identified, and these are involved in degradation of cellulose and hemicellulose but not lignin. Together with the results of polysaccharide-degrading activity measurements, we concluded that D. decipiens oita tightly regulates the expression of these CAZymes in response to the tested plant cell wall materials. Overall, this study described the detailed proteomic approach of a woodwasp-associated fungus and revealed that the new isolate, D. decipiens oita, secretes diverse CAZymes to efficiently degrade lignocellulose in the symbiotic environment.IMPORTANCE Recent studies show the potential impacts of insect symbiont microbes on biofuel application with regard to their degradation capability of a recalcitrant plant cell wall. In this study, we describe a novel fungal isolate, D. decipiens oita, as a single symbiotic fungus from the Xiphydria woodwasp found in the northern forests of Japan. Our detailed secretome analyses of D. decipiens oita, together with activity measurements, reveal that this insect-associated fungus exhibits high and broad activities for plant cell wall material degradation, suggesting potential applications within the biomass conversion industry for plant mass degradation.

Effects of temperature and superparasitism on quality and characteristics of thelytokous Wolbachia-infected Trichogramma dendrolimi Matsumura (Hymenoptera: Trichogrammatidae) during mass rearing.

Thelytokous Wolbachia-infected Trichogramma spp. are widely used egg parasitoids against lepidopteran pests in biological control programs. Wolbachia may manipulate host wasps for superparasitism and is sensitive to temperature. To explore effects of temperature and superparasitism, we compared fitness parameters and Wolbachia-mediated phenotype of thelytokous Wolbachia-infected Trichogramma dendrolimi between those emerging from superparasitised or single-parasitised hosts at 17, 21, 25, or 29 °C. Infected mothers of T. dendrolimi showed reduced superparasitism and parasitism increased with temperature. Wolbachia titre decreased with temperature when females emerged from singly-parasitised hosts, but there was no correlation in superparasitised hosts. Females showed higher Wolbachia titres at 21, 25, or 29 °C when developing from superparasitised hosts. The daily male ratio of offspring increased with temperature, and the day-age threshold for 5%, 50%, or 95% daily male ratio decreased with temperature in both parasitism forms. Females that emerged from superparasitised hosts had a shorter life span and reduced fecundity. These results indicate that Wolbachia may affect host behaviour by increasing superparasitism to enhance its spread, but this has negative effects on thelytokous Wolbachia-infected T. dendrolimi.

A systems biology approach for studying Wolbachia metabolism reveals points of interaction with its host in the context of arboviral infection.

Wolbachia are alpha-proteobacteria known to infect arthropods, which are of interest for disease control since they have been associated with improved resistance to viral infection. Although several genomes for different strains have been sequenced, there is little knowledge regarding the relationship between this bacterium and their hosts, particularly on their dependency for survival. Motivated by the potential applications on disease control, we developed genome-scale models of four Wolbachia strains known to infect arthropods: wAlbB (Aedes albopictus), wVitA (Nasonia vitripennis), wMel and wMelPop (Drosophila melanogaster). The obtained metabolic reconstructions exhibit a metabolism relying mainly on amino acids for energy production and biomass synthesis. A gap analysis was performed to detect metabolic candidates which could explain the endosymbiotic nature of this bacterium, finding that amino acids, requirements for ubiquinone precursors and provisioning of metabolites such as riboflavin could play a crucial role in this relationship. This work provides a systems biology perspective for studying the relationship of Wolbachia with its host and the development of new approaches for control of the spread of arboviral diseases. This approach, where metabolic gaps are key objects of study instead of just additions to complete a model, could be applied to other endosymbiotic bacteria of interest.

Microbiota of Honeybees, Apis mellifera Adansonii (Hymenoptera: Apidae) from Selected Ecozones, South West Nigeria.

BACKGROUND AND OBJECTIVES: Honeybees explore their environment in search of nectar and pollens including water whose sources are contaminated by variety of microbes. This has both negative and positive economic implications on health status of honeybees, the consumers of the products including the quality of the products. Hence, the study was designed to investigate the effect of geographical locations on the occurrence and diversity of microbiota on the external integument and the digestive gut of adult worker honeybees, Apis mellifera adansonii. MATERIALS AND METHODS: The honeybee samples were sourced from Ondo, Ogun, Oyo and Osun of South West Nigeria. The samples were subjected to microbiological analyses using standard techniques. Two-ways ANOVA was used for statistical test of the data. RESULTS: The microbiota of the studied samples includes Corynebacterium kutsceri, Corynebacterium xerosis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus fermentum, Micrococcus luteus, Micrococcus varians and Staphylococcus aureus which were gram-positive bacteria, while gram-negative bacteria include Aeromonas veronii, Citrobacter diversus, Citrobacter freundii, Klebsiella oxytoca, Klebsiella pneumonia and Pseudomonas aeruginosa. Fungi isolates include Aspergillus parasiticus, Aspergilus flavus, Aspergilus fumigatus, Aspergilus niger and Rhizopus stolonifer. Most microbiota were not cosmopolitan across selected ecozones of south west, Nigeria except for Aspergillus fumigatus. The number of identified species of microbiota either in the digestive guts or on the external integument of worker honeybees from selected ecozones varies from one ecozone to another. There was significant species diversity of microbiota on external integument than in digestive guts of adult workers of A. mellifera adansonii from selected ecozones in South West, Nigeria. CONCLUSION: The study showed that both the external integument and the digestive guts of A. mellifera adansonii exhibited variety of microbial flora providing research exploration opportunity for probable microbes of economic and geographical importance.

Infection of the chalcid parasitoid Pteromalus venustus Walker (Hymenoptera: Pteromalidae) with the male-killing symbiont Arsenophonus nasoniae (Gamma-Proteobacteria: Enterobacteriaceae).

The male-killing symbiont Arsenophonus nasoniae is a gamma-proteobacterium that infects parasitic wasps; the male progeny of infected females exhibit increased embryonic death. In this study, we examined methods to horizontally infect Pteromalus venustus (a parasitoid infesting populations of the alfalfa leafcutting bee Megachile rotundata) with A. nasoniae. We then tested the success of these methods via semi-quantitative PCR and quantitative digital PCR, using a molecular marker specific to A. nasoniae. Controlled parasitoid mating experiments were then undertaken to determine whether infections of A. nasoniae in P. venustus induce the male-killing phenotype as has been reported for other host species; evidence of this male-killing phenotype was observed in the current study. Over the course of the eight parasitoid generations following introduction of A. nasoniae infection in P. venustus, the male component of the parasitoid sex ratio was substantially reduced in the infected population (1.05 ♂: 1.00 ♀) compared to the control population (2.46 ♂: 1.00 ♀). Establishment of stable A. nasoniae infections in P. venustus populations could lower the proportion of male progeny, thus negatively impacting the mating success of females, and reducing overall populations of the parasitoid in alfalfa leafcutting bee populations.

Within the fortress: A specialized parasite is not discriminated against in a social insect society.

Social insect colonies function cohesively due, in part, to altruistic behaviors performed towards related individuals. These colonies can be affected by parasites in two distinct ways, either at the level of the individual or the entire colony. As such, colonies of social insects can experience conflict with infected individuals reducing the cohesiveness that typifies them. Parasites of social insects therefore offer us a framework to study conflicts within social insect colonies in addition to the traditionally viewed conflicts afforded by groups of low genetic relatedness due to multiple mating for example. In our study, we use the behavior manipulating fungal pathogen, Ophiocordyceps kimflemingiae (= unilateralis) and its host, Camponotus castaneus, to ask if colony members are able to detect infected individuals. Such detection would be optimal for the colony since infected workers die near foraging trails where the fungus develops its external structures and releases spores that infect other colony members. To determine if C. castaneus workers can detect these future threats, we used continuous-time point observations coupled with longer continuous observations to discern any discrimination towards infected individuals. After observing 1,240 hours of video footage we found that infected individuals are not removed from the colony and continuously received food during the course of fungal infection. We also calculated the distances between workers and the nest entrance in a total of 35,691 data points to find infected workers spent more time near the entrance of the nest. Taken together, these results suggest healthy individuals do not detect the parasite inside their nestmates. The colony's inability to detect infected individuals allows O. kimflemingiae to develop within the colony, while receiving food and protection from natural enemies, which could damage or kill its ant host before the parasite has completed its development.

Potential Side Effects of the Entomopathogenic Fungus Metarhizium anisopliae on the Egg Parasitoid Trichogramma pretiosum (Hymenoptera: Trichogrammatidae) Under Controlled Conditions.

The purpose was to evaluate the side effects of strains Metarhizium anisopliae (Metsch.) Sorokin sensu lato Unioeste 43 and M. anisopliae sensu stricto ESALQ 1641 on Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) under controlled conditions. A free-choice test for parasitism was performed, with the confinement of T. pretiosum females mated with cards (1 × 5 cm) containing age-standardized Anagasta kuehniella Zeller eggs, either sprayed with a fungal strain (109 conidia/ml) or 0.01% v/v Tween 80 (control). For the no-choice tests, T. pretiosum females mated were confined with cards sprayed with fungal strains before or after parasitism, and cards with fungal applications at different times. The number of parasitized eggs, percentage of emergence, longevity, egg-to-adult period, sex ratio, total and confirmed mortality by the fungus, and longevity of females that parasitized previously sprayed eggs, were assessed. Histological analysis of immature phases was also performed. The fungus was repellent to T. pretiosum in the free-choice test, while in the no-choice test, fungal applications before and after parasitism did not affect the number of eggs parasitized by T. pretiosum or the sex ratio of emerging adults. However, both strains affected adult emergence rates, the egg-to-adult period, and longevity. Overall, both M. anisopliae strains had minor effects on these biological parameters of T. pretiosum under controlled conditions. Hyphae were not detected in histological observations of immature stages of the parasitoid.

Wolbachia infections of the butterfly Eurema mandarina interfere with embryonic development of the sawfly Athalia rosae.

Although maternally transmitted microorganisms such as Wolbachia are well known to have a variety of effects on the reproduction of diverse insect species, little is known about the underlying mechanisms of actions. Artificial transfer of Wolbachia between taxonomically distant host species may provide insights into Wolbachia-induced manipulations of hosts. Here we performed a cross-order transfer of feminizing Wolbachia derived from a butterfly, Eurema mandarina. The Wolbachia were propagated in the Eurema hecabe cell line, called NTU-YB, and then used to inject prepupal/pupal females of a Wolbachia-free hymenopteran sawfly, Athalia rosae. The 14 females that emerged as adults looked morphologically and behaviorally healthy, and ovarian development appeared normal on dissection. However, in contrast to the control, none of the 333 eggs harbored by the seven Wolbachia-injected females developed successfully. Similarly, none of the 140 eggs laid on host plant by the four Wolbachia-injected females, which were mated with males, showed any signs of development. Wolbachia infection was detected from whole-body samples of the inoculated individuals, but not from the eggs they produced. Disruption of embryonic development despite the absence of Wolbachia in the egg cytoplasm may represent a new phenotype involving maternal effects that result in female sterility.

Searching for particular traits of sawfly (Hymenoptera: Tenthredinidae) larvae that emit hemolymph as a defence against predators.

Easy bleeding is a defence strategy that allows the larvae of some Tenthredinidae sawfly species to emit deterrent hemolymph when attacked by a predator. However, a drawback of this defence is that hemolymph is frequently in contact with the exterior, thus potentially subjected to multiple microbial infections at any body's integumental spot. Here we aimed to identify physiological traits that are linked to easy bleeding. First, larvae of several sawfly species were subjected to daily experimental losses of hemolymph equivalent to 10% of their body weight, and changes in body weight and survival were recorded. Easy bleeders' survival rates were better compared to non-easy bleeders. Second, testing hemolymph melanisation revealed that nearly all sawfly hemolymph samples did not melanise over a 24h period. Third, inhibition zone tests against live Escherichia coli were conducted using hemolymph collected 24-48h after a sterile wounding and an infection with Micrococcus luteus, as well as from control, untouched individuals. Sterile wounding induced similar antibacterial activities compared to those detected in the control group. However, the activity was significantly enhanced upon infection in some species, similarly to other insects. Thus, easy bleeders have a tendency to compensate for hemolymph loss resulting from predator-prey interactions, whereas a non-melanising hemolymph is probably a characteristic of sawflies, and the antimicrobial activity can be high but is comparable in easy bleeders versus other insects.

Does Gnomoniopsis castanea contribute to the natural biological control of chestnut gall wasp?

Gnomoniopsis castanea has been reported as the causal agent of necrosis of chestnut wasp (Dryocosmus kuriphilus) galls. The fungus is frequently observed on galls in chestnut stands infested by the insect in Italy. In the present study the impact of gall necrosis and the dynamic of its development have been studied in mature and young Castanea sativa stands in Central Italy during spring and early summer, before the D. kuriphilus adult flies. Results suggest that gall necrosis develops from resident endophytic inoculum of G. castanea. During the 2 y of monitoring, no differences were found in incidence and severity of the disease. Gall necrosis increased exponentially during the season, reaching 75,4% of galls totally necrotized in the investigated site in mid July. Gall necrosis was shown to have a severe impact on D. kuriphilus vitality, mostly impacting the adults inside the galls. Gall necrosis by G. castanea appears to efficiently control gall wasp in chestnut stands, although the high virulence of the fungus to chestnut fruits precludes its use as biocontrol agent in biological control strategies.

Side effects of Bacillus thuringiensis var. kurstaki on the hymenopterous parasitic wasp Trichogramma chilonis.

Most of the detrimental effects of using conventional insecticides to control crop pests are now well identified and are nowadays major arguments for replacing such compounds by the use of biological control agents. In this respect, the bacterium Bacillus thuringiensis var. kurstaki and Trichogramma (Hymenoptera: Trichogrammatidae) parasitic wasp species are both effective against lepidopterous pests and can actually be used concomitantly. In this work, we studied the potential side effects of B. thuringiensis var. kurstaki on Trichogramma chilonis females. We first evidenced an acute toxicity of B. thuringiensis on T. chilonis. Then, after ingestion of B. thuringiensis at sublethal doses, we focused on life history traits of T. chilonis such as longevity, reproductive success and the time spent on host eggs patches. The reproductive success of T. chilonis was not modified by B. thuringiensis while a significant effect was observed on longevity and the time spent on host eggs patches. The physiological and ecological meanings of the results obtained are discussed.

Assemblage of filamentous fungi associated with aculeate hymenopteran brood in reed galls.

Monotypic stands of common reed and the reed-gall-associated insect assemblages are distributed worldwide. However, fungi associated with these assemblages have not been characterized in detail. Here we examined 5200 individuals (12 species) of immature aculeate hymenopterans or their parasitoids collected at 34 sampling sites in Central Europe. We noticed fungal outgrowth on exoskeletons of 83 (1.60%) larvae and pupae. The most common host was eudominant Pemphredon fabricii. However, the less abundant aculeate hymenopteran reed gall inquilines were infected at higher prevalence, these included Trypoxylon deceptorium, Trypoxylon minus, Hoplitis leucomelana and Hylaeus moricei (all considered new host records). We identified three fungal species, Penicillium buchwaldii (72% of cases), Aspergillus pseudoglaucus (22%) and Penicillium quebecense (6%). When multibrooded nests were affected, only a part of individuals was infected in 62% of cases. The sampling site-specific infection rate reached up to 13%, thus fungal infections should be considered an important variable driving the abundance of gall inquilines. Infections of generalist host species were more frequent than those of reed gall specialists, suggesting that suboptimal conditions decreased the immunocompetence of non-specialized species, which only occasionally nest in reed galls and feed in reed beds.

Soil application of Beauveria bassiana GHA against apple sawfly, Hoplocampa testudinea (Hymenoptera: Tenthredinidae): Field mortality and fungal persistence.

Low impact alternatives to synthetic insecticides for the control of apple sawfly (Hoplocampa testudinea Klug) are scarce encumbering pest management in organic apple orchards. We investigated the soil persistence and field efficacy of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (BotaniGard) against apple sawfly under common organic orchard practices. We also assessed the efficacy of B. bassiana GHA and Metarhizium brunneum Petch (indigenous strain) against sawfly in the laboratory. Larvae treated with either fungus in the laboratory died faster than control larvae and displayed 49.4%-68.4% mycosis. In the field, B. bassiana density remained high in the week after application, during larval descent to the soil. Fungal density decreased to 25% at 49 d after application and to 0.4% after 55 weeks. Molecular markers revealed that the majority of fungal isolates recovered comprised the applied B. bassiana strain GHA. Larvae pupating in soil cages in the orchard for 49 d displayed 17% mycosis. The high efficacy under laboratory conditions was not seen in the field. B. bassiana application resulted in densities above the upper natural background level during the growing season, but reversion to background levels occurred within a year. It remains to be investigated whether this has a detrimental effect on nontarget organisms. Additional work is needed to bridge the knowledge gap between laboratory and field efficacy in orchards.

Multilocus genotyping of Amylostereum spp. associated with Sirex noctilio and other woodwasps from Europe reveal clonal lineage introduced to the US.

Sirex noctilio is a woodwasp of Eurasian origin that was inadvertently introduced to the southern hemisphere in the 1900s and to North America over a decade ago. Its larvae bore in Pinus spp. and can cause significant mortality in pine plantations. S noctilio is associated with a symbiotic white rot fungus, Amylostereum areolatum, which females inject into trees when they oviposit and which is required for survival of developing larvae. We compared the genetic diversity of A. areolatum isolated from S. noctilio and other woodwasps collected from Europe and from northeastern North America to determine the origin of introduction(s) into the United States. Multilocus genotyping of nuclear ribosomal regions and protein coding genes revealed two widespread multilocus genotypes (MLGs) among the European samples, one of which is present in the US. The other two MLGs associated with S. noctilio in the US represented unique haplotypes. These latter two haplotypes were likely from unrepresented source populations, and together with the introduced widespread haplotype reveal multiple A. areolatum MLGs introduced by S. noctilio and indicate possible multiple S. noctilio introductions to North America from Europe. Our results also showed a lack of fidelity between woodwasp hosts and Amylostereum species.

16S rRNA Amplicon Sequencing Demonstrates that Indoor-Reared Bumblebees (Bombus terrestris) Harbor a Core Subset of Bacteria Normally Associated with the Wild Host.

A MiSeq multiplexed 16S rRNA amplicon sequencing of the gut microbiota of wild and indoor-reared Bombus terrestris (bumblebees) confirmed the presence of a core set of bacteria, which consisted of Neisseriaceae (Snodgrassella), Orbaceae (Gilliamella), Lactobacillaceae (Lactobacillus), and Bifidobacteriaceae (Bifidobacterium). In wild B. terrestris we detected several non-core bacteria having a more variable prevalence. Although Enterobacteriaceae are unreported by non next-generation sequencing studies, it can become a dominant gut resident. Furthermore the presence of some non-core lactobacilli were associated with the relative abundance of bifidobacteria. This association was not observed in indoor-reared bumblebees lacking the non-core bacteria, but having a more standardized microbiota compared to their wild counterparts. The impact of the bottleneck microbiota of indoor-reared bumblebees when they are used in the field for pollination purpose is discussed.

Highly effective bacterial agents against Cimbex quadrimaculatus (Hymenoptera: Cimbicidae): isolation of bacteria and their insecticidal activities.

Cimbex quadrimaculatus (Hymenoptera: Cimbicidae) is one of the serious pests of almonds in Turkey and worldwide. Since there is no effective control application against this pest, it has been a serious problem up to now. Therefore, we aimed to find an effective bacterium that can be utilized as a biocontrol agent against C. quadrimaculatus in pest management. We isolated seven bacteria from dead and live C. quadrimaculatus larvae, and evaluated the larvicidal potency of all isolates on the respective pest. Based on the morphological, physiological, biochemical and molecular properties (partial sequence of 16S rRNA gene), the isolates were identified to be Bacillus safensis (CQ1), Bacillus subtilis (CQ2), Bacillus tequilensis (CQ3), Enterobacter sp. (CQ4), Kurthia gibsonii (CQ5), Staphylococcus sp. (CQ6) and Staphylococcus sciuri (CQ7). The results of the larvicidal activities of these isolates indicated that the mortality value obtained from all treatments changed from 58 to 100 %, and reached 100 % with B. safensis (CQ1) and B. subtilis (CQ2) on the 3rd instar larvae within 10 days of application of 1.89 × 10(9) cfu/mL bacterial concentration at 25 °C under laboratory conditions. Findings from this study indicate that these isolates appear to be a promising biocontrol agent for C. quadrimaculatus.

Presence of Wolbachia in three hymenopteran species: Diprion pini (Hymenoptera: Diprionidae), Neodiprion sertifer (Hymenoptera: Diprionidae), and Dahlbominus fuscipennis (Hymenoptera: Eulophidae).

Sawflies are important pests of various plant species. Diprion pini (L.) and Neodiprion sertifer (Geoffroy) (Hymenoptera: Diprionidae) are two of the most important sawfly pests in Italy, and both species are parasitized by the hymenopteran parasitoid Dahlbominus fuscipennis (Zetterstedt). Bacterial endosymbionts are currently studied for their high potential in strategies of biocontrol in a number of insect species. In this study, we investigated the presence of symbiotic bacteria (Wolbachia and Cardinium) in the three species of hymenoptera mentioned earlier, both in wild and laboratory populations. Although all samples were negative for the presence of Cardinium, 100% prevalence for Wolbachia was detected, as all examined individuals resulted to be PCR positive. Furthermore, 16S rDNA and ftsZ gene sequencing indicated that all individuals from the three hymenopteran species are infected by a single Wolbachia strain. Additionally, we report the presence of gynandromorphic individuals in D. pini, both in wild and laboratory-reared populations. Heat treatments on D. pini colonies removed the Wolbachia symbionts, but they also prevented the development of adults.

Bacterial associates of seed-parasitic wasps (Hymenoptera: Megastigmus).

BACKGROUND: The success of herbivorous insects has been shaped largely by their association with microbes. Seed parasitism is an insect feeding strategy involving intimate contact and manipulation of a plant host. Little is known about the microbial associates of seed-parasitic insects. We characterized the bacterial symbionts of Megastigmus (Hymenoptera: Torymidae), a lineage of seed-parasitic chalcid wasps, with the goal of identifying microbes that might play an important role in aiding development within seeds, including supplementing insect nutrition or manipulating host trees. We screened multiple populations of seven species for common facultative inherited symbionts. We also performed culture independent surveys of larvae, pupae, and adults of M. spermotrophus using 454 pyrosequencing. This major pest of Douglas-fir is the best-studied Megastigmus, and was previously shown to manipulate its tree host into redirecting resources towards unfertilized ovules. Douglas-fir ovules and the parasitoid Eurytoma sp. were also surveyed using pyrosequencing to help elucidate possible transmission mechanisms of the microbial associates of M. spermotrophus. RESULTS: Three wasp species harboured Rickettsia; two of these also harboured Wolbachia. Males and females were infected at similar frequencies, suggesting that these bacteria do not distort sex ratios. The M. spermotrophus microbiome is dominated by five bacterial OTUs, including lineages commonly found in other insect microbiomes and in environmental samples. The bacterial community associated with M. spermotrophus remained constant throughout wasp development and was dominated by a single OTU - a strain of Ralstonia, in the Betaproteobacteria, comprising over 55% of all bacterial OTUs from Megastigmus samples. This strain was also present in unparasitized ovules. CONCLUSIONS: This is the first report of Ralstonia being an abundant and potentially important member of an insect microbiome, although other closely-related Betaproteobacteria, such as Burkholderia, are important insect symbionts. We speculate that Ralstonia might play a role in nutrient recycling, perhaps by redirecting nitrogen. The developing wasp larva feeds on megagametophyte tissue, which contains the seed storage reserves and is especially rich in nitrogen. Future studies using Ralstonia-specific markers will determine its distribution in other Megastigmus species, its mode of transmission, and its role in wasp nutrition.