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Social insects exhibit a high degree of intraspecific behavioral variation. Moreover, they often harbor specialized microbial communities in their gut. Recent studies suggest that these two characteristics of social insects are interlinked: insect behavioral phenotypes affect their gut microbiota composition, partly through exposure to different environments and diet, and in return, the gut microbiota has been shown to influence insect behavior. Here, we discuss the bidirectional relationship existing between intraspecific variation in gut microbiota composition and behavioral phenotypes in social insects., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2021 The Authors. Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences.)

<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.

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., (Copyright © 2020 American Society for Microbiology.)

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.)

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.

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.

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., (© The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

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., (© 2015 The Authors. Insect Science published by Wiley Publishing Asia Pty Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences.)

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.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (Copyright © 2015 The British Mycological Society. All rights reserved.)

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.

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.

Effects of the fungal endophytes Beauveria bassiana (isolates ICIPE 279, G1LU3, S4SU1) and Hypocrea lixii (isolate F3ST1) on the life-history of Phaedrotoma scabriventris and Diglyphus isaea, parasitoids of the pea leafminer Liriomyza huidobrensis, were studied in the laboratory. Parasitoids were allowed to parasitize 2(nd) and 3(rd) instar L. huidobrensis larvae reared on endophytically-inoculated faba bean, Vicia faba. In the control, parasitoids were reared on non-inoculated host plants. Parasitism, pupation, adult emergence and survival were recorded. No significant difference was observed between the control and the endophyte-inoculated plants in terms of parasitism rates of P. scabriventris (p = 0.68) and D. isaea (p = 0.45) and adult' survival times (p = 0.06). The survival period of the F1 progeny of P. scabriventris was reduced (p<0.0001) in B. bassiana S4SU1 to 28 days as compared to more than 40 days for B. bassiana G1LU3, ICIPE 279 and H. lixii F3ST1. However, no significant difference (p = 0.54) was observed in the survival times of the F1 progeny of D. isaea. This study has demonstrated that together, endophytes and parasitoids have beneficial effects in L. huidobrensis population suppression.

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.

Winged males of leaf-cutting ants are considered an ephemeral reproductive caste only produced before the mating flight season. Although much is known about the yeast diversity found in fungus gardens of attine ants, no study has focused on the yeasts associated with males of leaf-cutting ants. Here, we surveyed the yeasts on the integuments of males of Atta sexdens rubropilosa and assessed their potential role in the attine ant-microbe symbiosis. Using culture-dependent techniques, we found yeasts to be abundant on the integuments of males (54.5 %, n = 200 alates). A total of 242 yeast strains were obtained representing six orders, ten genera and 25 species. Strains of Aureobasidium, Cryptococcus, Hannaella and Rhodotorula were prevalent on the integuments and likely originated from the fungus garden of the parental nest or from the soil. The majority of strains (87.1 %) produced at least one of the evaluated enzymes: pectinase, polygalacturonase, cellulase, xylanase, ligninases and lipase. Aureobasidium pullulans accounted for the highest number of strains that produced all enzymes. In addition, yeasts showed the ability to assimilate the resulting oligosaccharides, supporting observations of other studies that yeasts may be involved in the plant biomass metabolism in the fungus gardens. Because winged males harbor several yeasts with putative functional roles, these fungi may take part and be beneficial in the microbial consortia of the new incipient nest.

Chandler and Turelli postulate that intrinsic hybrid dysfunction underscores hybrid lethality in Nasonia. Although it is a suitable conception for examining hybrid incompatibilities, their account of the evidence is factually inaccurate and leaves out the evolutionary process for why lethality became conditional on nuclear-microbe interactions. Hybrid incompatibilities in the context of phylosymbiosis are resolved by hologenomic principles and exemplify this emerging postmodern synthesis., (Copyright © 2014, American Association for the Advancement of Science.)

Brucker and Bordenstein (Reports, 9 August 2013, p. 667) claim that adaptive codivergence of gut bacteria with hosts contributes to hybrid lethality. Yet, they provide no evidence for coadaptation of bacteria and Nasonia hosts. Their data on hybrid viability suggest that bacteria contribute to inviability only because intrinsic hybrid dysfunction increases susceptibility to free-living bacteria. Hologenomic speciation remains testable speculation without experimental support., (Copyright © 2014, American Association for the Advancement of Science.)

Background: 'Candidatus Streptomyces philanthi' is a monophyletic clade of formerly uncultured bacterial symbionts in solitary digger wasps of the genera Philanthus, Philanthinus and Trachypus (Hymenoptera, Crabronidae). These bacteria grow in female-specific antennal reservoirs and - after transmission to the cocoon - produce antibiotics protecting the host larvae from fungal infection. However, the symbionts' refractoriness to cultivation has thus far hampered detailed in vitro studies on their physiology and on the evolutionary changes in metabolic versatility in response to the host environment., Results: Here we isolated in axenic culture 22 'Streptomyces philanthi' biovars from different host species. Sequencing of gyrB revealed no heterogeneity among isolates within host individuals, suggesting low levels of (micro)diversity or even clonality of the symbionts in individual beewolf antennae. Surprisingly, however, isolates from different host species differed strongly in their physiology. All biovars from the Eurasian/African Philanthus and the South American Trachypus host species had high nutritional demands and were susceptible to most antibiotics tested, suggesting a tight association with the hosts. By contrast, biovars isolated from the genus Philanthinus and the monophyletic North American Philanthus clade were metabolically versatile and showed broad antibiotic resistance. Concordantly, recent horizontal symbiont transfer events - reflected in different symbiont strains infecting the same host species - have been described only among North American Philanthus species, altogether indicative of facultative symbionts potentially capable of a free-living lifestyle. Phylogenetic analyses reveal a strong correlation between symbiont metabolic versatility and host phylogeny, suggesting that the host environment differentially affects the symbionts' evolutionary fate. Although opportunistic bacteria were occasionally isolated from the antennae of different host species, only filamentous Actinobacteria (genera Streptomyces, Amycolatopsis and Nocardia) could replace 'S. philanthi' in the antennal gland reservoirs., Conclusion: Our results indicate that closely related bacteria from a monophyletic clade of symbionts can experience very different evolutionary trajectories in response to the symbiotic lifestyle, which is reflected in different degrees of metabolic versatility and host-dependency. We propose that the host-provided environment could be an important factor in shaping the degenerative metabolic evolution in the symbionts and deciding whether they evolve into obligate symbionts or remain facultative and capable of a host-independent lifestyle.

The diamondback moth (DBM) Plutella xylostella (L.) has traditionally been managed using synthetic insecticides. However, the increasing resistance of DBM to insecticides offers an impetus to practice integrated pest management (IPM) strategies by exploiting its natural enemies such as pathogens, parasitoids, and predators. Nevertheless, the interactions between pathogens and parasitoids and/or predators might affect the effectiveness of the parasitoids in regulating the host population. Thus, the parasitism rate of Nosema-infected DBM by Cotesia vestalis (Haliday) (Hym., Braconidae) can be negatively influenced by such interactions. In this study, we investigated the effects of Nosema infection in DBM on the parasitism performance of C. vestalis. The results of no-choice test showed that C. vestalis had a higher parasitism rate on non-infected host larvae than on Nosema-treated host larvae. The C. vestalis individuals that emerged from Nosema-infected DBM (F1) and their progeny (F2) had smaller pupae, a decreased rate of emergence, lowered fecundity, and a prolonged development period compared to those of the control group. DBM infection by Nosema sp. also negatively affected the morphometrics of C. vestalis. The eggs of female C. vestalis that developed in Nosema-infected DBM were larger than those of females that developed in non-infected DBM. These detrimental effects on the F1 and F2 generations of C. vestalis might severely impact the effectiveness of combining pathogens and parasitoids as parts of an IPM strategy for DBM control.

Background: Detecting intracellular bacterial symbionts can be challenging when they persist at very low densities. Wolbachia, a widespread bacterial endosymbiont of invertebrates, is particularly challenging. Although it persists at high titers in many species, in others its densities are far below the detection limit of classic end-point Polymerase Chain Reaction (PCR). These low-titer infections can be reliably detected by combining PCR with DNA hybridization, but less elaborate strategies based on end-point PCR alone have proven less sensitive or less general., Results: We introduce a multicopy PCR target that allows fast and reliable detection of A-supergroup Wolbachia--even at low infection titers--with standard end-point PCR. The target is a multicopy motif (designated ARM: A-supergroup repeat motif) discovered in the genome of wMel (the Wolbachia in Drosophila melanogaster). ARM is found in at least seven other Wolbachia A-supergroup strains infecting various Drosophila, the wasp Muscidifurax and the tsetse fly Glossina. We demonstrate that end-point PCR targeting ARM can reliably detect both high- and low-titer Wolbachia infections in Drosophila, Glossina and interspecific hybrids., Conclusions: Simple end-point PCR of ARM facilitates detection of low-titer Wolbachia A-supergroup infections. Detecting these infections previously required more elaborate procedures. Our ARM target seems to be a general feature of Wolbachia A-supergroup genomes, unlike other multicopy markers such as insertion sequences (IS).

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., (© The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America.)

A novel actinomycete, designated strain NEAU-ycm1(T), was isolated from an edible Chinese black ant (Polyrhachis vicina Roger) and characterized with a polyphasic approach. The organism was found to have morphological and chemotaxonomic characteristics typical of streptomycetes. Phylogenetic analysis based on the almost complete 16S rRNA gene sequence show that the novel isolate belongs to the genus Streptomyces and forms a separate subclade. The closest phylogenetic relatives were identified as the type strains of Streptomyces intermedius NBRC 13049(T) (97.74 %), Streptomyces aureoverticillatus NRRL B-3326(T) (97.69 %), Streptomyces rutgersensis NBRC 12819(T) (97.68 %), Streptomyces gougerotii NBRC 3198(T) (97.68 %) and Streptomyces diastaticus subsp. diastaticus NBRC 3714(T) (97.68 %). Similarities to other type strains of the genus Streptomyces were lower than 97.55 %. A comparison between strain NEAU-ycm1(T) and the closest related Streptomyces type strains revealed that it is different from them in morphological, physiological and biochemical characteristics. Therefore, it is proposed that NEAU-ycm1(T) (=CGMCC 4.7094(T) = DSM 42102(T)) represents a novel species of the genus of Streptomyces, for which the name Streptomyces polyrhachii sp. nov. is proposed.

Wolbachia are maternally inherited endosymbiotic bacteria of arthropods and nematodes. In arthropods, they manipulate the reproduction of their hosts to facilitate their own spread in host populations, causing cytoplasmic incompatibility, parthenogenesis induction, feminization of genetic males and male-killing. In this study, we investigated Wolbachia infection and studied wsp (Wolbachia surface protein) sequences in three wasp species associated with the unisexual galls of A. mukaigawae with the aim of determining the transmission mode and the reason for multiple infections of Wolbachia. Frequency of Wolbachia infected populations for A. mukaigawae, Synergus japonicus (inquiline), and Torymus sp. (parasitoid) was 75%, 100%, and 100%, respectively. Multiple Wolbachia infections were detected in A. mukaigawae and S. japonicus, with 5 and 8 Wolbachia strains, respectively. The two host species shared 5 Wolbachia strains and were infected by identical strains in several locations, indicating horizontal transmission of Wolbachia. The transmission potentially takes place through gall tissues, which the larvae of both wasps feed on. Furthermore, three recombination events of Wolbachia were observed: the strains W8, W2 and W6 apparently have derived from W3 and W5a, W6 and W7, W4 and W9, respectively. W8 and W2 and their respective parental strains were detected in S. japonicus. W6 was detected with only one parent (W4) in S. japonicus; W9 was detected in Torymus sp., suggesting horizontal transmission between hosts and parasitoids. In conclusion, our research supports earlier studies that horizontal transmission of Wolbachia, a symbiont of the Rickettsiales order, may be plant-mediated or take place between hosts and parasitoids. Our research provides novel molecular evidence for multiple recombination events of Wolbachia in gall wasp communities. We suggest that genomic recombination and potential plant-mediated horizontal transmission may be attributable to the high levels of multiple Wolbachia infections observed in A. mukaigawae and S. japonicus.

Although the gut microbiome influences numerous aspects of organismal fitness, its role in animal evolution and the origin of new species is largely unknown. Here we present evidence that beneficial bacterial communities in the guts of closely related species of the genus Nasonia form species-specific phylosymbiotic assemblages that cause lethality in interspecific hybrids. Bacterial constituents and abundance are irregular in hybrids relative to parental controls, and antibiotic curing of the gut bacteria significantly rescues hybrid survival. Moreover, feeding bacteria to germ-free hybrids reinstates lethality and recapitulates the expression of innate immune genes observed in conventionally reared hybrids. We conclude that in this animal complex, the gut microbiome and host genome represent a coadapted "hologenome" that breaks down during hybridization, promoting hybrid lethality and assisting speciation.

Mutualism between microbes and insects is common and alignment of the reproductive interests of microbial symbionts with this lifestyle typically involves clonal reproduction and vertical transmission by insect partners. Here the Amylostereum fungus-Sirex woodwasp mutualism was used to consider whether their prolonged association and predominance of asexuality have affected the mating system of the fungal partner. Nucleotide information for the pheromone receptor gene rab1, as well as the translation elongation factor 1α gene and ribosomal RNA internal transcribed spacer region were utilized. The identification of rab1 alleles in Amylostereum chailletii and Amylostereum areolatum populations revealed that this gene is more polymorphic than the other two regions, although the diversity of all three regions was lower than what has been observed in free-living Agaricomycetes. Our data suggest that suppressed recombination might be implicated in the diversification of rab1, while no evidence of balancing selection was detected. We also detected positive selection at only two codons, suggesting that purifying selection is important for the evolution of rab1. The symbiotic relationship with their insect partners has therefore influenced the diversity of this gene and influenced the manner in which selection drives and maintains this diversity in A. areolatum and A. chailletii., (Copyright © 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The animal gut is a habitat for diverse communities of microorganisms (microbiota). Honeybees and bumblebees have recently been shown to harbour a distinct and species poor microbiota, which may confer protection against parasites. Here, we investigate diversity, host specificity and transmission mode of two of the most common, yet poorly known, gut bacteria of honeybees and bumblebees: Snodgrassella alvi (Betaproteobacteria) and Gilliamella apicola (Gammaproteobacteria). We analysed 16S rRNA gene sequences of these bacteria from diverse bee host species across most of the honeybee and bumblebee phylogenetic diversity from North America, Europe and Asia. These focal bacteria were present in 92% of bumblebee species and all honeybee species but were found to be absent in the two related corbiculate bee tribes, the stingless bees (Meliponini) and orchid bees (Euglossini). Both Snodgrassella alvi and Gilliamella apicola phylogenies show significant topological congruence with the phylogeny of their bee hosts, albeit with a considerable degree of putative host switches. Furthermore, we found that phylogenetic distances between Gilliamella apicola samples correlated with the geographical distance between sampling locations. This tentatively suggests that the environmental transmission rate, as set by geographical distance, affects the distribution of G. apicola infections. We show experimentally that both bacterial taxa can be vertically transmitted from the mother colony to daughter queens, and social contact with nest mates after emergence from the pupa greatly facilitates this transmission. Therefore, sociality may play an important role in vertical transmission and opens up the potential for co-evolution or at least a close association of gut bacteria with their hosts., (© 2013 Blackwell Publishing Ltd.)

Lactobacilli (Lactobacillales: Lactobacillaceae) are well known for their roles in food fermentation, as probiotics, and in human health, but they can also be dominant members of the microbiota of some species of Hymenoptera (ants, bees, and wasps). Honey bees and bumble bees associate with host-specific lactobacilli, and some evidence suggests that these lactobacilli are important for bee health. Social transmission helps maintain associations between these bees and their respective microbiota. To determine whether lactobacilli associated with social hymenopteran hosts are generally host specific, we gathered publicly available Lactobacillus 16S rRNA gene sequences, along with Lactobacillus sequences from 454 pyrosequencing surveys of six other hymenopteran species (three sweat bees and three ants). We determined the comparative secondary structural models of 16S rRNA, which allowed us to accurately align the entire 16S rRNA gene, including fast-evolving regions. BLAST searches and maximum-likelihood phylogenetic reconstructions confirmed that honey and bumble bees have host-specific Lactobacillus associates. Regardless of colony size or within-colony oral sharing of food (trophallaxis), sweat bees and ants associate with lactobacilli that are closely related to those found in vertebrate hosts or in diverse environments. Why honey and bumble bees associate with host-specific lactobacilli while other social Hymenoptera do not remains an open question. Lactobacilli are known to inhibit the growth of other microbes and can be beneficial whether they are coevolved with their host or are recruited by the host from environmental sources through mechanisms of partner choice.

Venoms from Hymenoptera display a wide range of functions and biological roles. These notably include manipulation of the host, capture of prey and defense against competitors and predators thanks to endocrine and immune systems disruptors, neurotoxic, cytolytic and pain-inducing venom components. Recent works indicate that many hymenopteran species, whatever their life style, have also evolved a venom with properties which enable it to regulate microbial infections, both in stinging and stung animals. In contrast to biting insects and their salivary glands, stinging Hymenoptera seem to constitute an under-exploited ecological niche for agents of vector-borne disease. Few parasitic or mutualistic microorganisms have been reported to be hosted by venom-producing organs or to be transmitted to stung animals. This may result from the presence of potent antimicrobial molecules in venoms, histological features of venom apparatuses and selective effects of venoms on immune defenses of targeted organisms. The present paper reviews for the first time the venom antimicrobial potential of solitary and social Hymenoptera in molecular, ecological, and evolutionary perspectives., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Bumblebees are of profound ecological importance because of the pollination services they provide in natural and agricultural ecosystems. Any decline of these pollinators is therefore of great concern for ecosystem functioning. Increased parasite pressures have been discussed as a major factor for the loss of pollinators. One of the main parasites of bumblebees is Nosema bombi, an intracellular microsporidian parasite with considerable impact on the vitality of the host. Here we study the effect of host colony density and host genetic variability on N. bombi infections in natural populations of the bumblebee Bombus terrestris. We sampled males and workers from six B. terrestris populations located in an agricultural landscape in Middle Sweden to determine the prevalence and degree of N. bombi infections. All individuals were genotyped with five microsatellite markers to infer the colony densities in the sampled populations and the genetic variability of the host population. We confirmed that genetic variability and sex significantly correlate with the degree of infection with N. bombi. Males and workers with lower genetic variability had significantly higher infection levels than average. Also colony density had a significant impact on the degree of infection, with high density populations having higher infected individuals., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Wolbachia are endosymbiotic bacteria known to manipulate the reproduction of their hosts. Some populations of the parasitoid wasp Asobara japonica are infected with Wolbachia and reproduce parthenogenetically, while other populations are not infected and reproduce sexually. Wolbachia-infected A. japonica females regularly produce small numbers of male offspring. Because all females in the field are infected and infected females are not capable of sexual reproduction, male production seems to be maladaptive. We investigated why these females nevertheless produce males. We tested three hypotheses: high rearing temperatures could result in higher offspring sex ratios (more males), low Wolbachia titer of the mother could lead to higher offspring sex ratios and/or the Wolbachia infection is of relatively recent origin and not enough time has passed to allow complete coadaptation between Wolbachia and host. In all, 33% of the Wolbachia-infected females produced males and 56% of these males were also infected with Wolbachia. Neither offspring sex ratio nor male infection frequency was significantly affected by rearing temperature or Wolbachia concentration of the mother. The mitochondrial DNA sequence of one of the uninfected populations was identical to that of two of the infected populations. Therefore, the initial Wolbachia infection of A. japonica must have occurred recently. Mitochondrial sequence variation among the infected populations suggests that the spread of Wolbachia through the host populations involved horizontal transmission. We conclude that the occasional male production by Wolbachia-infected females is most likely a maladaptive side effect of incomplete coevolution between symbiont and host in this relatively young infection.

Integrative taxonomy is a recently developed approach that uses multiple lines of evidence such as molecular, morphological, ecological and geographical data to test species limits, and it stands as one of the most promising approaches to species delimitation in taxonomically difficult groups. The Pnigalio soemius complex (Hymenoptera: Eulophidae) represents an interesting taxonomical and ecological study case, as it is characterized by a lack of informative morphological characters, deep mitochondrial divergence, and is susceptible to infection by parthenogenesis-inducing Rickettsia. We tested the effectiveness of an integrative taxonomy approach in delimiting species within the P. soemius complex. We analysed two molecular markers (COI and ITS2) using different methods, performed multivariate analysis on morphometric data and exploited ecological data such as host-plant system associations, geographical separation, and the prevalence, type and effects of endosymbiont infection. The challenge of resolving different levels of resolution in the data was met by setting up a formal procedure of data integration within and between conflicting independent lines of evidence. An iterative corroboration process of multiple sources of data eventually indicated the existence of several cryptic species that can be treated as stable taxonomic hypotheses. Furthermore, the integrative approach confirmed a trend towards host specificity within the presumed polyphagous P. soemius and suggested that Rickettsia could have played a major role in the reproductive isolation and genetic diversification of at least two species., (© 2012 Blackwell Publishing Ltd.)

Insects engage in symbiotic associations with a large diversity of beneficial microorganisms. While the majority of well-studied symbioses have a nutritional basis, several cases are known in which bacteria protect their host from pathogen infestation. Solitary wasps of the genera Philanthus and Trachypus (beewolves; Hymenoptera, Crabronidae) cultivate the actinomycete "Candidatus Streptomyces philanthi" in specialized antennal gland reservoirs. The symbionts are transferred to the larval cocoon, where they provide protection against pathogenic fungi by producing at least nine different antibiotics. Here we investigated the closest relatives of Philanthus and Trachypus, the rare genus Philanthinus, for the presence of antennal gland reservoirs and symbiotic streptomycetes. Molecular analyses identified "Ca. Streptomyces philanthi" in reservoirs of Philanthinus quattuordecimpunctatus. Phylogenies based on the 16S rRNA gene suggest that P. quattuordecimpunctatus may have acquired "Ca. Streptomyces philanthi" by horizontal transfer from other beewolf species. In histological sections and three-dimensional reconstructions, the antennal gland reservoirs were found to occupy six antennal segments (as opposed to only five in Philanthus and Trachypus) and to be structurally less complex than those of the evolutionarily more derived genera of beewolves. The presence of "Ca. Streptomyces philanthi" in antennal glands of Philanthinus indicates that the symbiosis between beewolves and Streptomyces bacteria is much older than previously thought. It probably evolved along the branch leading to the monophyletic tribe Philanthini, as it seems to be confined to the genera Philanthus, Trachypus, and Philanthinus, which together comprise 172 described species of solitary wasps.

The most conspicuous ants in all of tropical America are those that belong to the tribe Attini which cultivate fungus. The objective of the present study is to verify the alterations that occur in the cuticle of the worker larvae from Myrmicocrypta, Mycetarotes and Trachymyrmex with the purpose of trying to establish the degree of this association. Attine ants from the Atta genus were used as a control group. The analysis of histological sections showed results about the distribution of the cells and tissues on the epidermis of A. sexdens rubropilosa ant larvae and Mycetarotes parallelus, Trachymyrmex fuscus and Myrmicocrypta sp. A cuticle covering the ants was observed in the larvae of all the species, and this is formed by a simple cubic epithelium, whose cells possibly change its shape to prismatic, depending on their secretory activity. Just under this epithelium large adipose cells with reserve granules in their cytoplasm were found. The presence of a space filled by granulose and acellular material was also observed, indicating that the larvae were in a molt period. The presence of fungal hyphae is observed both on the external side of the basal attine larvae as well as emitting projections to the interior of the cuticle reaching the epithelium and the adipocyte cells. Data obtained in the present study demonstrated that the fungus deposited on the surface of immature ants from attine basal species maintain a close relationship with them, once the fungus hyphae have the ability to disorganize the cuticle lamellas, penetrating the interior of the insect cells through the emission of prolongations transporting the cuticle and epithelium barriers and making substances exchanges between larvae and fungus., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The identification of micro-organisms carried by ticks is an important issue for human and animal health. In addition to their role as pathogen vectors, ticks are also the hosts for symbiotic bacteria whose impact on tick biology is poorly known. Among these, the bacterium Wolbachia pipientis has already been reported associated with Ixodes ricinus and other tick species. However, the origins of Wolbachia in ticks and their consequences on tick biology (known to be very diverse in invertebrates, ranging from nutritional symbionts in nematodes to reproductive manipulators in insects) are unknown. Here we report that the endoparasitoid wasp Ixodiphagus hookeri (Hymenoptera, Chalcidoidea, Encyrtidae)--strictly associated with ticks for their development--infested at almost 100% prevalence by a W. pipientis strain belonging to a Wolbachia supergroup that has already been reported as associated with other hymenopteran parasitoids. In a natural population of I. ricinus that suffers high parasitism rates due to I. hookeri, we used specific PCR primers for both hymenopteran and W. pipientis gene fragments to show that all unfed tick nymphs parasitized by I. hookeri also harbored Wolbachia, while unparasitized ticks were Wolbachia-free. We demonstrated experimentally that unfed nymphs obtained from larvae exposed to I. hookeri while gorging on their vertebrate host also harbor Wolbachia. We hypothesize that previous studies that have reported W. pipientis in ticks are due to the cryptic presence of the endoparasitoid wasp I. hookeri. This association has remained hidden until now because parasitoids within ticks cannot be detected until engorgement of the nymphs brings the wasp eggs out of diapause. Finally, we discuss the consequences of this finding for our understanding of the tick microbiome, and their possible role in horizontal gene transfer among pathogenic and symbiotic bacteria.

Background: Ixodiphagus hookeri is a parasitic wasp of ixodid ticks around the world. It has been studied as a potential bio-control agent for several tick species. We suspected that the presence of Wolbachia infected I. hookeri eggs in ticks is responsible for incidental detection of Wolbachia DNA in tick samples., Methods: The 28S rRNA and 16S rRNA genes of a specimen of I. hookeri was amplified and sequenced. PCR on part of the 28S rRNA gene was used to detect parasitic wasp DNA in 349 questing Ixodes ricinus ticks from various sampling sites. Furthermore, the wsp gene of Wolbachia was sequenced from the I. hookeri specimen and a subset of ticks was tested using this marker., Results: Several sequences from tick specimens were identical to the Wolbachia sequence of the I. hookeri specimen. Ixodiphagus hookeri was detected in 9.5% of all tested ticks, varying between 4% and 26% depending on geographic location. Ten out of eleven sampling sites throughout the Netherlands were positive for I. hookeri. Eighty-seven percent of I. hookeri-positive but only 1.6% of I. hookeri-negative ticks were Wolbachia positive. Detection of I. hookeri DNA was strongly associated with the detection of Wolbachia in ticks., Conclusion: This is the first reported case of I. hookeri in the Netherlands. Furthermore I. hookeri harbours Wolbachia species and is broadly distributed in the Netherlands. While detection of Wolbachia DNA in ticks might often be due to parasitism with this wasp, other sources of Wolbachia DNA in ticks might exist as well., (© 2011 Tijsse-Klasen et al; licensee BioMed Central Ltd.)

The Eurasian Sirex noctilio-Amylostereum areolatum complex was discovered and has become established close to the North American Great Lakes in the 2000s. This invasive forest insect pest represents a very high risk to native and exotic pines in North America. We investigated the geographical origin of clonal lineages of the fungal symbiont A. areolatum in the recently pest-colonized eastern Canadian region by analyzing mitochondrial and nuclear sequence variations and comparing the genetic diversity of a worldwide collection of fungal symbionts among six countries where the Sirex complex is native and four countries from which the insect-fungal complex has been introduced. In total, 102 isolates were analyzed. While 12 multilocus genotypes (MLGs) are observed in the areas where S. noctilio is native, only two MLGs are retrieved from areas where S. noctilio is not native, indicating the wide spread of clonal lineages in the introduced fungal symbiont of S. noctilio. MLG2 comprises 26% of the Canadian isolates and is also observed in Chile and South Africa, where the insect-fungal complex has also been introduced. MLG3 comprises 74% of the Canadian isolates and is also observed in the USA, but nowhere else in our worldwide collection. Thus, at least one of the Canadian clonal lineages shares a common origin with A. areolatum isolates from the Southern Hemisphere. The source of the second clonal lineage is still unknown, but phylogenetic analyses show that MLG3 is isolated. More extended sampling is necessary to determine the origin of this fungal clonal lineage and investigate its probable symbiotic association with native North American Sirex., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

Transferring endosymbiotic bacteria between different host species can perturb the coordinated regulation of the host and bacterial genomes. Here we use the most common maternally transmitted bacteria, Wolbachia pipientis, to test the consequences of host genetic background on infection densities and the processes underlying those changes in the parasitoid wasp genus Nasonia. Introgressing the genome of Nasonia giraulti into the infected cytoplasm of N. vitripennis causes a two-order-of-magnitude increase in bacterial loads in adults and a proliferation of the infection to somatic tissues. The host effect on W. pipientis distribution and densities is associated with a twofold decrease in densities of the temperate phage WO-B. Returning the bacteria from the new host species back to the resident host species restores the bacteria and phage to their native densities. To our knowledge, this is the first study to report a host-microbe genetic interaction that affects the densities of both W. pipientis and bacteriophage WO-B. The consequences of the increased bacterial density include a reduction in fecundity, an increase in levels of cytoplasmic incompatibility (CI), and unexpectedly, male-to-female transfer of the bacteria to uninfected females and an increased acceptance of densely infected females to interspecific mates. While paternal inheritance of the W. pipientis was not observed, the high incidence of male-to-female transfer in the introgressed background raises the possibility that paternal transmission could be more likely in hybrids where paternal leakage of other cytoplasmic elements is also known to occur. Taken together, these results establish a major change in W. pipientis densities and tissue tropism between closely related species and support a model in which phage WO, Wolbachia, and arthropods form a tripartite symbiotic association in which all three are integral to understanding the biology of this widespread endosymbiosis.

Parthenogenesis-inducing (PI) Wolbachia belong to a class of intracellular symbionts that distort the offspring sex ratio of their hosts toward a female bias. In many PI Wolbachia-infected species sex ratio distortion has reached its ultimate expression-fixation of infection and all-female populations. This is only possible with thelytokous PI symbionts as they provide an alternative form of reproduction and remove the requirement for males and sexual reproduction. Many populations fixed for PI Wolbachia infection have lost the ability to reproduce sexually, even when cured of the infection. We examine one such population in the species Trichogramma pretiosum. Through a series of backcrossing experiments with an uninfected Trichogramma pretiosum population we were able to show that the genetic basis for the loss of female sexual function could be explained by a dominant nuclear effect. Male sexual function had not been completely lost, though some deterioration of male sexual function was also evident when males from the infected population (created through antibiotic curing of infected females) were mated to uninfected females. We discuss the dynamics of sex ratio selection in PI Wolbachia-infected populations and the evolution of non-fertilizing mutations.

By the method of ISSR, the genetic heterogeneity of 111 Beauveria bassiana isolates in a pure Masson' s pine plantation in Magu Mountains of Anhui Province was analyzed. A total of 58 loci were obtained with 7 ISSR primers, 54 of which (93.10%) were polymorphic. For the isolates sampled at different time, their Nei's gene diversity (He) was 0.2552, Shannon's diversity index (I) was 0.3825, coefficient of gene differentiation (Gst) was 0.2269, and gene flow Nm was 0.8518. The genetic distance between the isolates sampled in May and July was the shortest (0.0408). For the isolates grouped based on host orders, their He was 0.2623, I was 0.3884, Gst was 0.1964, and Nm was 1.0223. The genetic distance between the isolates from Coleopteran and Hymenoteran was the shortest (0.0163). All the results suggested that the genetic heterogeneity of B. bassiana population in the test plantation was rich, and the genetic variation between B. bassiana sub-populations was relatively low but that within the sub-populations was high, and none of the isolates were identical or highly similar with each other.

Wolbachia are symbiotic intracellular bacteria, which are classified as reproductive parasites. Although generally facultative, Wolbachia is necessary for Asobara tabida (Hymenoptera), because aposymbiotic females do not produce any offspring. Interestingly, the ovarian phenotype of aposymbiotic females is variable: some females do not produce any eggs, whereas others do produce some eggs, but these are aborted. Here, we show that the ovarian phenotype of aposymbiotic females is highly polymorphic within populations, although dependence remains complete in both cases. We also identified some lines in which aposymbiotic females were able to produce a very few viable offspring, further extending the range of variation observed. These results suggest that various factors actively maintain polymorphism. We demonstrated that Wolbachia is necessary to trigger oogenetic processes, but that the ovarian phenotype was determined by the host only. Phenotypic variation was also correlated with the differential expression of genes controlling iron homeostasis and oxidative stress, which are potentially involved in the evolution of dependence. This suggests that variation in the ovarian phenotype could reflect selection for different levels of compensatory mechanisms in response to Wolbachia infection, and that polymorphism is maintained through selection on different antagonist traits influenced by oxidative stress., (© 2010 The Author(s). Journal compilation © 2010 The Society for the Study of Evolution.)

The molecular and ultrastructural profiles of the symbionts found in the midgut and ileum of Cephalotes atratus, Cephalotes clypeatus, and Cephalotes pusillus were determined using the V3 region of the bacterial 16S rDNA gene and transmission electron microscopy (T.E.M.). Two samples of C. atratus, three of C. clypeatus, and six of C. pusillus were analyzed. The coefficients of similarity ranged from 80% to 94% for the samples of symbionts from C. clypeatus and C. atratus, despite being collected in geographically distant sites. The variability within symbionts found in the samples of C. pusillus varied from 29% to 55%, in samples geographically close as well as distant. PCR-DGGE was effective for the purpose of this study and can be considered a versatile tool to analyze gut microbiota. Details of the ultrastructural aspect of these bacteria are presented., (Copyright 2010 Elsevier Ltd. All rights reserved.)