Investigation of Fungal Community Structure in the Gut of the Stag Beetle Dorcus hopei (Coleoptera; Lucanidae): Comparisons Among Developmental Stages.

Stag beetles, recognized as common saproxylic insects, are valued for their vibrant coloration and distinctive morphology. These beetles play a crucial ecological role in decomposition and nutrient cycling, serving as a vital functional component in ecosystem functioning. Although previous studies have confirmed that stag beetles are predominantly fungivores, the fluctuations in their intestinal fungal communities at different developmental stages remain poorly understood. In the current study, high-throughput sequencing was employed to investigate the dynamic changes within intestinal fungal communities at various developmental stages in the stag beetle Dorcus hopei. Results showed that microbial diversity was higher during the larval stage than during the pupal and adult stages. Furthermore, significant differences were identified in the composition of the intestinal fungal communities across the larval, pupal, and adult stages, suggesting that developmental transitions may be crucial factors contributing to variations in fungal community composition and diversity. Dominant genera included Candida, Scheffersomyces, Phaeoacremonium, and Trichosporon. Functional predictions indicated a greater diversity and relative abundance of endosymbiotic fungi in the larval gut, suggesting a potential dependency of larvae on beneficial gut fungi for nutrient acquisition. Additionally, the application of abundance-based ß-null deviation and niche width analyses revealed that the adult gut exerted a stronger selection pressure on its fungal community, favoring certain taxa. This selection process culminates in a more robust co-occurrence network of fungal communities within the adult gut, thereby enhancing their adaptability to environmental fluctuations. This study advances our understanding of the intestinal fungal community structure in stag beetles, providing a crucial theoretical foundation for the development of saproxylic beetle resources, biomass energy utilization, plastic degradation strategies, and beetle conservation efforts.

Gut fungal diversity across different life stages of the onion fly Delia antiqua.

A significant number of microorganisms inhabit the intestinal tract or the body surface of insects. While the majority of research on insect microbiome interaction has mainly focused on bacteria, of late multiple studies have been acknowledging the importance of fungi and have started reporting the fungal communities as well. In this study, high-throughput sequencing was used to compare the diversity of intestinal fungi in Delia antiqua (Diptera: Anthomyiidae) at different growth stages, and effect of differential fungi between adjacent life stages on the growth and development of D. antiqua was investigated. The results showed that there were significant differences in the α and ß diversity of gut fungal communities between two adjacent growth stages. Among the dominant fungi, genera Penicillium and Meyerozyma and family Cordycipitaceae had higher abundances. Cordycipitaceae was mainly enriched in the pupal and adult (male and female) stages, Penicillium was mainly enriched in the pupal, 2nd instar and 3rd instar larval stages, and Meyerozyma was enriched in the pupal stage. Only three fungal species were found to differ between two adjacent growth stages. These three fungal species including Fusarium oxysporum, Meyerozyma guilliermondii and Penicillium roqueforti generally inhibited the growth and development of D. antiqua, with only P. roqueforti promoting the growth and development of female insects. This study will provide theoretical support for the search for new pathogenic microorganisms for other fly pests control and the development of new biological control strategies for fly pests.

Bacteria isolated from Aedes aegypti with potential vector control applications.

Highly anthropophilic and adapted to urban environments, Aedes aegypti mosquitoes are the main vectors of arboviruses that cause human diseases such as dengue, zika, and chikungunya fever, especially in countries with tropical and subtropical climates. Microorganisms with mosquitocidal and larvicidal activities have been suggested as environmentally safe alternatives to chemical or mechanical mosquito control methods. Here, we analyzed cultivable bacteria isolated from all stages of the mosquito life cycle for their larvicidal activity against Ae. aegypti. A total of 424 bacterial strains isolated from eggs, larvae, pupae, or adult Ae. aegypti were analyzed for the pathogenic potential of their crude cultures against larvae of this same mosquito species. Nine strains displayed larvicidal activity comparable to the strain AM65-52, reisolated from commercial BTi-based product VectoBac® WG. 16S rRNA gene sequencing revealed that the set of larvicidal strains contains two representatives of the genus Bacillus, five Enterobacter, and two Stenotrophomonas. This study demonstrates that some bacteria isolated from Ae. aegypti are pathogenic for the mosquito from which they were isolated. The data are promising for developing novel bioinsecticides for the control of these medically important mosquitoes.

Study on the virulence of Metarhizium anisopliae against Spodoptera frugiperda (J. E. Smith, 1797).

This study examined the impact of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) conidia on the eggs, larvae, pupae, and adults of Spodoptera frugiperda. The results showed that eggs, larvae, pupae, and adults exhibited mortality rates that were dependent on the dose. An increased amount of conidia (1.5 × 109 conidia/mL) was found to be toxic to larvae, pupae, and adults after 9 days of treatment, resulting in a 100% mortality rate in eggs, 98% in larvae, 76% in pupae, and 85% in adults. A study using earthworms as bioindicators found that after 3 days of exposure, M. anisopliae conidia did not cause any harmful effects on the earthworms. In contrast, the chemical treatment (positive control) resulted in 100% mortality at a concentration of 40 ppm. Histopathological studies showed that earthworm gut tissues treated with fungal conidia did not show significant differences compared with those of the negative control. The gut tissues of earthworms treated with monocrotophos exhibited significant damage, and notable differences were observed in the chemical treatment. The treatments with 70 and 100 µg/mL solutions of Eudrilus eugeniae epidermal mucus showed no fungal growth. An analysis of the enzymes at a biochemical level revealed a decrease in the levels of acetylcholinesterase, α-carboxylesterase, and ß-carboxylesterase in S. frugiperda larvae after exposure to fungal conidia. This study found that M. anisopliae is effective against S. frugiperda, highlighting the potential of this entomopathogenic fungus in controlling this agricultural insect pest.

Novel report on soil infection with Metarhizium rileyi against soil-dwelling life stages of insect pests.

Entomopathogenic fungi are the most effective control remedy against a wide range of medical and agricultural important pests. The present study aimed to isolate, identify, and assess the virulence of Metarhizium rileyi against Spodoptera litura and Spodoptera frugiperda pupae under soil conditions. The biotechnological methods were used to identify the isolate as M. rileyi. The fungal conidial pathogenicity (2.0 × 107, 2.0 × 108, 2.0 × 109, 2.0 × 1010, and 2.0 × 1011 conidia/mL-1) was tested against prepupae of S. litura and S. frugiperda at 3, 6, 9, and 12 days after treatments. Additionally, the artificial soil-conidial assay was performed on a nontarget species earthworm Eudrilus eugeniae, using M. rileyi conidia. The present results showed that the M. rileyi caused significant mortality rates in S. litura pupae (61-90%), and S. litura pupae were more susceptible than S. frugiperda pupae (46%-73%) at 12 day posttreatment. The LC50 and LC90 of M. rileyi against S. litura, were 3.4 × 1014-9.9 × 1017 conidia/mL-1 and 6.6 × 105-4.6 × 1014 conidia/mL-1 in S. frugiperda, respectively. The conidia of M. rileyi did not exhibit any sublethal effect on the adult stage of E. eugeniae, and Artemia salina following a 12-day treatment period. Moreover, in the histopathological evaluation no discernible harm was observed in the gut tissues of E. eugeniae, including the lumen and epithelial cells, as well as the muscles, setae, nucleus, mitochondria, and coelom. The present findings provide clear evidence that M. rileyi fungal conidia can be used as the foundation for the development of effective bio-insecticides to combat the pupae of S. litura and S. frugiperda agricultural pests.

Pathogenicity of Metarhizium rileyi (Hypocreales: Clavicipitaceae) against Tenebrio molitor (Coleoptera: Tenebrionidae).

Tenebrio molitor L., also known as the mealworm, is a polyphagous insect pest that infests various stored grains worldwide. Both the adult and larval stages can cause significant damage to stored grains. The present study focused on isolating entomopathogenic fungi from an infected larval cadaver under environmental conditions. Fungal pathogenicity was tested on T. molitor larvae and pupae for 12 days. Entomopathogenic fungi were identified using biotechnological methods based on their morphology and the sequence of their nuclear ribosomal internal transcribed spacer (ITS). The results of the insecticidal activity indicate that the virulence of fungi varies between the larval and pupal stages. In comparison to the larval stage, the pupal stage is highly susceptible to Metarhizium rileyi, exhibiting 100% mortality rates after 12 days (lethal concentration 50 [LC50] = 7.8 × 106 and lethal concentration 90 (LC90) = 2.1 × 1013 conidia/mL), whereas larvae showed 92% mortality rates at 12 days posttreatment (LC50 = 1.0 × 106 and LC90 = 3.0 × 109 conidia/mL). The enzymatic analyses revealed a significant increase in the levels of the insect enzymes superoxide dismutase (4.76-10.5 mg-1) and glutathione S-transferase (0.46-6.53 mg-1) 3 days after exposure to M. rileyi conidia (1.5 × 105 conidia/mL) compared to the control group. The findings clearly show that M. rileyi is an environmentally friendly and effective microbial agent for controlling the larvae and pupae of T. molitor.

Pathogenic fungi infection attributes of malarial vectors Anopheles maculipennis and Anopheles superpictus in central Iran.

BACKGROUND: Due to the effect of synthetic and commercial insecticides on non-target organisms and the resistance of mosquitoes, non-chemical and environmentally friendly methods have become prevalent in recent years. The present study was to isolate entomopathogenic fungi with toxic effects on mosquitoes in natural larval habitats. METHODS: Larvae of mosquitoes were collected from Central, Qamsar, Niasar, and Barzok Districts in Kashan County, Central Iran by standard dipping method, from April to late December 2019. Dead larvae, live larvae showing signs of infection, and larvae and pupae with a white coating of fungal mycelium on the outer surface of their bodies were isolated from the rest of the larvae and sterilized with 10% sodium hypochlorite for 2 min, then washed twice with distilled water and transferred to potato-dextrose-agar (PDA) and water-agar (WA) media and incubated at 25 ± 2 °C for 3-4 days. Larvae and fungi were identified morphologically based on identification keys. RESULTS: A total of 9789 larvae were collected from urban and rural areas in Kashan County. Thirteen species were identified which were recognized to belong to three genera, including Anopheles (7.89%), Culiseta (17.42%) and Culex (74.69%). A total of 105 larvae, including Anopheles superpictus sensu lato (s.l), Anopheles maculipennis s.l., Culex deserticola, Culex perexiguus, and Culiseta longiareolata were found to be infected by Nattrassia mangiferae, Aspergillus niger, Aspergillus fumigatus, Trichoderma spp., and Penicillium spp. Of these, Penicillium spp. was the most abundant fungus isolated and identified from the larval habitats, while An. superpictus s.l. was the most infected mosquito species. CONCLUSIONS: Based on the observations and results obtained of the study, isolated fungi had the potential efficacy for pathogenicity on mosquito larvae. It is suggested that their effects on mosquito larvae should be investigated in the laboratory. The most important point, however, is the proper way of exploiting these biocontrol agents to maximize their effect on reducing the population of vector mosquito larvae without any negative effect on non-target organisms.

A switch of microbial flora coupled with ontogenetic niche shift in Leptinotarsa decemlineata.

In Leptinotarsa decemlineata, a final-instar wandering larva typically undergoes an ontogenetic niche shift (ONS), from potato plant during the foraging stage to its pupation site below ground. Using high-throughput sequencing of the bacterial 16S ribosomal RNA gene, we determined the hypothesis that the L. decemlineata pupae harbor stage-specific bacteria to meet the physiological requirements for underground habitat. We identified 34 bacterial phyla, comprising 73 classes, 208 orders, 375 families, and 766 genera in the collected specimens. Microbes across phyla Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were enriched in the pupae, while those in the phylum Proteobacteria, Tenericutes, Firmicutes, and Bacteroidetes dominated in the larvae and adults. A total of 18 genera, including Blastococcus, Corynebacterium_1, Gordonia, Microbacterium, Nocardia, Nocardioides, Rhodococcus, Solirubrobacter, Tsukamurella, Enterococcus, Acinetobacter, Escherichia_Shigella, Lysobacter, Pseudomonas, and Stenotrophomonas, were specifically distributed in pupae. Moreover, soil sterilizing removed a major portion of bacteria in pupae. Specifically, both Enterococcus and Pseudomonas were eliminated in the soil sterilizing and antibiotic-fed beetle groups. Furthermore, the pupation rate and fresh pupal weight were similar, whereas the emergence rate and adult weight were decreased in the antibiotic-fed beetles, compared with controls. The results demonstrate that a switch of bacterial communities occurs in the pupae; the pupal-specific bacteria genera are mainly originated from soil; this bacterial biodiversity improves pupa performance in soil. Our results provide new insight into the evolutionary fitness of L. decemlineata to different environmental niches.

Antifungal roles of adult-specific cuticular protein genes of the red flour beetle, Tribolium castaneum.

The insect cuticle is a composite structure that can further be divided into a few sub-structural layers. Its large moiety comprises a lattice of chitin fibrils and structural proteins, both of which are stabilized by covalent bonding among them. The cuticle covers the whole surface of insect body, and thus has long been suggested for the involvement in defense against entomopathogens, especially entomopathogenic fungi that infect percutaneously. We have been addressing this issue in the past few years and have so far demonstrated experimentally that chitin synthase 1, laccase2 as well as benzoquinone synthesis-related genes of Tribolium castaneum have indispensable roles in the antifungal host defense. In the present study we focused on another major component of the insect cuticular integument, structural cuticular proteins. We chose three genes coding for adult-specific cuticular proteins, namely CPR4, CPR18 and CPR27, and examined their roles in forming immunologically sound adult cuticular integuments. Analyses of developmental expression revealed that the three genes showed high level expression in the pupal stage. These results are consistent with their proposed roles in constructing cuticle of adult beetles. The RNA interference-mediated gene knockdown was employed to silence these genes, and the administration of double strand RNAs in pupae resulted in the adults with malformed elytra. The single knockdown of the three genes attenuated somewhat the defense of the resulting adult beetles against Beauveria bassiana and Metarhizium anisopliae, but statistical analyses indicated no significant differences from controls. In contrast, the double or triple knockdown mutant beetles displayed a drastic disruption of the host defense against the two entomopathogenic fungal species irrespective of the combination of targeted cuticular protein genes, demonstrating the important roles of the three cuticular protein genes in conferring robust antifungal properties on the adult cuticle. Scanning electron microscopic observation revealed that the germination of conidia attached on the adult body surface was still suppressed after the gene knockdown as in the case of wild-type beetles, suggesting that the weakened antifungal phenotypes resulted from the combined knockdown of the adult-specific cuticular protein genes could not be accounted for by the disfunction of secretion/retention of fungistatic benzoquinone derivatives.

Novel associations in antibiosis stemming from an insect pupal cell.

The pupal soil cell of the pecan weevil, Curculio caryae (Coleoptera: Curculionidae), was reported previously to exhibit antibiosis to an entomopathogenic fungus, Beauveria bassiana. The objectives of this study were to examine 1) if the antimicrobial effect occurs in other insects that form pupal cells, 2) whether the effect extends to plant pathogenic fungi, and 3) identify the source of antibiosis in pupal soil cells of C. caryae. Antibiosis of pupal cells against B. bassiana was confirmed in-vitro in three additional curculionids, Diaprepes abbreviatus, Conotrachelus nenuphar, and Pissodes nemorensis, all of which had fewer fungal colonies relative to controls. Pupal soil cells were found to suppress phytopathogenic fungi in-vitro, including suppression of Alternaria solani by D. abbreviatus pupal cell, and that of Monilinia fructicola by C. caryae. The detection of antibiosis of soil cells formed by surface-sterilized insects using sterile soil implies the antimicrobial effect stemmed from inside the insect. Further, a novel biotic mechanism was identified: a bacterium related to Serratia nematodiphila was isolated from C. caryae pupal soil cells and was found to be associated with antibiosis. The bacterial cultures with or without autoclave had similar effects but were not as potent as pupal soil cells for suppressing B. bassiana. Also, autoclaved soil cells and autoclaved bacterial culture suppressed M. fructicola but were not as inhibitory as non-autoclaved soil cells. This indicates that antibiosis may be due to bacterial metabolites, although other factors may also be involved. Our findings suggest potential to develop the antibiotic compounds as novel bio-fungicides to control plant diseases.

European Foulbrood in stingless bees (Apidae: Meliponini) in Brazil: Old disease, renewed threat.

Stingless bees (Apidae: Meliponini) are a group of bees with vestigial stings showing a high level of social organization. They are important pollinators in tropical and subtropical regions, and, in the last decades, stingless beekeeping has increased rapidly in Brazil. Bee-collected pollen and honey of Apis mellifera can be an important source of disease when used as supplements to feed stingless bee colonies, a common and increasing practice adopted by stingless beekeepers. Here, we aimed to investigate the presence of pathogens commonly found in honey bees in diseased colonies of Melipona species in Espírito Santo and São Paulo States, Southeast Brazil. We detected, for the first time, the bacterium Melissococcus plutonius and symptoms of European foulbrood in Melipona spp., associated with brood death and colony losses in some cases. In addition, we tested for the presence of the bacterium Paenibacillus larvae and the fungus Aschosphaera apis, as well as the six more common honey bee viruses in Brazil (BQCV, ABPV, DWV, KBV, IAPV, CBPV) and the microsporidia Nosema apis and Nosema ceranae. However, only one sample of brood was infected with N. ceranae and all other pathogens, with the exception of Melissococcus plutonius, were absent in the analyzed brood. Lastly, we looked for toxic pollen in all food fed to diseased colonies, but none was present.

Benzoquinone synthesis-related genes of Tribolium castaneum confer the robust antifungal host defense to the adult beetles through the inhibition of conidial germination on the body surface.

Insects fight against invading microbial pathogens through various immune-related measures that comprise 'internal', 'external' as well as 'social' immunities. The defenses by external immunity associated with the cuticular integument are supposed to be of particular importance in repelling entomopathogenic fungi that infect host insects transcutaneously. Among such integument-related defenses, external secretions of benzoquinone derivatives typical of tenebrionid beetles have been suggested to play important roles in the antimicrobial defenses. In the present study, by utilizing the experimental infection system composed of the red flour beetle Tribolium castaneum and generalist ascomycete entomopathogens Beauveria bassiana and Metarhizium anisopliae, we performed the functional assays of the three T. castaneum genes whose involvement in benzoquinone synthesis in the adults has been reported, namely GT39, GT62 and GT63. Observations by scanning electron microcopy (SEM) revealed that the conidia of the two fungal species did not germinate on the wild-type adult body surface but did on the pupae. The expression analyses demonstrated that the levels of GT39 and GT62 mRNA increased from middle pupae and reached high in early adults while GT63 did not show a clear adult-biased expression pattern. The RNA interference-based knockdown of any of the three genes in pupae resulted in the adults compromised to the infection of the both fungal species. SEM observations revealed that the gene silencing allowed the conidial germination on the body surface of the knockdown beetles, thereby impairing the robust antifungal defense of adult beetles. Thus, we have provided direct experimental evidence for the functional importance in vivo of these benzoquinone synthesis-related genes that support the antifungal defense of tenebrionid beetles.

Activity of Metarhizium brunneum and Beauveria bassiana against early developmental stages of the false codling moth Thaumatotibia leucotreta.

This study evaluated the efficacy of two entomopathogenic Hypocrealean fungi, Metarhizium brunneum (laboratory isolate) and Beauveria bassiana (the commercial product Botanigard), for preventative control of the false codling moth, Thaumatotibia leucotreta. The mortality of eggs and first instar larvae was studied in three different assays. First, fungal virulence was examined under optimal laboratory conditions (25 °C, 85% RH) by placing T. leucotreta eggs on conidia-impregnated filter paper. One-day-old eggs and first instar larvae were susceptible to both fungi. In contrast, 5-day-old eggs (advanced embryo development) were susceptible to M. brunneum, but not to B. bassiana. The activity of both fungi against eggs was assessed under two humidity regimes: 85% RH-optimal for fungal germination, and 60% RH-the average humidity in the laboratory. Pieces of parchment paper serving as oviposition surfaces were treated with each of the fungi and introduced to gravid females at different time points after inoculation (0, 2, 7 and 14 days). Although the tested fungal species differed in their virulence to T. leucotreta eggs, both reduced hatching rate under both humidity regimes to 8.3-58.3%, compared to 71.7-83.3% in the control treatments. To evaluate reduction of T. leucotreta infestation of fruit, 'Ori' citrus fruit (easy peeler Citrus sinensis) were treated with each of the fungi. Eggs were placed on the fruit peels and the fruit were maintained under room conditions (25 °C, 60% RH). Between 41.7% and 54.1% of fruit in control groups were infested by the T. leucotreta larvae. Treatments with either of the fungi resulted in about 16% infestation of the fruit with larvae, a marked (3.3-fold) reduction.

Molecular and morphological characterisation of a novel microsporidian species, Tubulinosema suzukii, infecting Drosophila suzukii (Diptera: Drosophilidae).

A microsporidium showing morphological characteristics typical of a Tubulinosema species was discovered in Drosophila suzukii. All developmental stages were diplokaryotic and grew in direct contact with the host cell cytoplasm. Spores from fresh preparations were ovoid to slightly pyriform and measured 4.29 × 2.47 µm in wet mount preparations. The spore wall consisted of a 125 nm thick endospore covered by a double layered exospore of 39 nm and 18 nm. The polar filament measured 67 µm in length, was slightly anisofilar and was arranged in ten coils in one or rarely two rows. The two posterior coils were 95 nm in diameter while the anterior coils were 115 nm in diameter. Early developmental stages were surrounded by electron-dense, 35.3 nm diameter, surface ornaments scattered over the membrane. Tubular elements with diameters of approximately 75 nm were seen attaching to the periphery of meronts and sporonts. Tissues infected included fat body, midgut and muscle. A 1915 bp rDNA fragment, covering the small subunit (SSU), the internal transcribed spacer (ITS) and the 5' end of the large subunit ribosomal DNA, was amplified by PCR and sequenced. Phylogenetic analyses of the SSU rDNA fragment revealed closest relationship to Tubulinosema pampeana (Host: Bombus atratus, South America) and Tubulinosema loxostegi (Host: Loxostege sticticalis, ubiquitous), but using the complete dataset of SSU-ITS-LSU rDNA genes revealed T. hippodamiae (Host: Hippodamiae convergens) as the most closely related species. Based on the morphological and genetic features a new species, Tubulinosema suzukii sp. nov., is proposed for this microsporidium isolated from D. suzukii.

Side effects of Bacillus thuringiensis on the parasitoid Palmistichus elaeisis (Hymenoptera: Eulophidae).

The endoparasitoid wasp Palmistichus elaeisis Delvare & LaSalle (Hymenoptera: Eulophidae) is used to control defoliating lepidopteran pests. Chemical insecticides are not compatible with natural enemies, but bioinsecticides, such as Bacillus thuringiensis Berliner (Bt), have great potential for use in integrated pest management. However, interactions between Bt and P. elaeisis still need to be investigated. This study aimed to evaluate the effects of Bt on parental and first-generation P. elaeisis parasitizing Bt-susceptible and -resistant Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae). An additional aim was to determine the toxicity of Bt to susceptible third-instar S. frugiperda larvae. Larvae were exposed to lethal concentrations (LC50 and LC90) of Bt and then allowed to be parasitized by P. elaeisis. Parasitoid longevity, immature production, reproductive performance, and behavioral responses were evaluated. Bt repelled P. elaeisis and reduced immature production. Parental and first filial generation parasitoids of both sexes emerged from Bt-treated larvae showed lower survivorship than controls. Parasitoids had poorer reproductive performance in Bt-susceptible and -resistant pupae than in untreated pupae. Palmistichus elaeisis emerged from Bt-susceptible and -resistant S. frugiperda showed altered host-searching behavior and reproductive parameters, which indicates low compatibility between the bioinsecticide agent and the parasitoid wasp.

Emergence of Walnut Husk Maggot Adults in Central Illinois and Potential for Control with Metarhizium brunneum.

The walnut husk maggot, Rhagoletis sauvis (Loew) (Diptera: Tephritidae), causes damage to walnuts when maggots feed inside the husk. September applications of the entomopathogenic fungi Metarhizium brunneum F52 as microsclerotia laced granules to the soil in Illinois were evaluated for pest control based on adult emergence during the following summer. Over 3 yr in central Illinois, adult emergence began near 1 July, peaked before 23 July, and emergence extended as late as 23 August. One summer application of fungus (30 June) when pupae were present, did not reduce fly emergence. Of two September applications that targeted maggots as they move to the soil to pupate, one significantly reduced the number of flies emerging from treated plots when compared with untreated plots for one 7-d sample collected 29 July 2020. Emergence trap data show a defined peak adult emergence in July for central Illinois while September applications of granules containing Metarhizium brunneum (Petch) (Hypocreales: Clavicipitaceae) show shows potential to reduced subsequent fly emergence.

Construction of a Baculovirus Derivative to Produce Linearized Antheraea pernyi (Lepidoptera: Saturniidae) Multicapsid Nucleopolyhedrovirus Genomic DNA.

In the Antheraea pernyi multicapsid nucleopolyhedrovirus (AnpeNPV)-based expression vector system, the frequency of homologous recombination events between wild-type AnpeNPV DNA and the transfer vector is low, resulting in a small amount of recombinant virus. Previous reports have indicated that linearized baculovirus DNA can increase the proportion of recombinant virus relative to the total progeny. To improve the recombination efficiency, we constructed a linearized derivative of AnpeNPV, referred to as AnpeNPVPhEGFP-AvrII, in which egfp flanked by AvrII restriction sites was located at the polyhedrin locus and driven by the polyhedrin promoter. Linear AnpeNPV DNA was obtained by the treatment of AnpeNPVPhEGFP-AvrII genomic DNA with AvrII endonuclease. The infectivity and recombinogenic activity between the linearized and circular viral DNA were evaluated by quantitative real-time polymerase chain reactions. We demonstrated that the linearized AnpeNPV DNA produced only small numbers of infectious budded viruses, accounting for approximately 4.5% of the budded virus production of wild-type AnpeNPV DNA in A. pernyi pupae. However, the linearized AnpeNPV DNA substantially increased recombinant virus production after cotransfection with an appropriate transfer vector; relative abundance of the recombinant virus was approximately 5.5-fold higher than that of the wild-type AnpeNPV DNA in A. pernyi pupae. The linearization of AnpeNPV DNA will facilitate the purification of recombinant viruses using the AnpeNPV-based expression vector system and the construction of an AnpeNPV-based bacmid system.

Interaction Between Beauveria bassiana (Hypocreales: Cordycipitaceae) and Coptera haywardi (Hymenoptera: Diapriidae) for the Management of Anastrepha obliqua (Diptera: Tephritidae).

The interaction between the entomopathogenic fungus Beauveria bassiana (Balsamo) and the parasitoid Coptera haywardi (Oglobin), as potential biological control agents for Anastrepha obliqua (Macquart) fruit flies, was evaluated under laboratory and semi-protected field cage conditions. The effects of the parasitoids and fungus were individually and jointly assessed in Plexiglas cages. Application of B. bassiana dry conidia to soil produced 40% mortality in A. obliqua adults. However, mortality was lower (21.2%) on evaluation under field cage conditions. According to the multiple decrement life table analysis, the probability of death of A. obliqua was 88% when C. haywardi parasitoids and B. bassiana conidia were used in conjunction, 89% when only C. haywardi parasitoids were released and 23% when only B. bassiana conidia were applied. These results demonstrate that no synergistic, additive or antagonistic interaction took place with the simultaneous use of these natural enemies, since the presence of B. bassiana had no effect on the C. haywardi parasitism. These results indicate that the parasitoid is a better natural enemy for the control of A. obliqua, and show that, although the two biological control agents can be used simultaneously, their joint application will not produce increased control.

Microbial Communities in Different Developmental Stages of the Oriental Fruit Fly, Bactrocera dorsalis, Are Associated with Differentially Expressed Peptidoglycan Recognition Protein-Encoding Genes.

The insect microbiota can change dramatically to enable adaptation of the host in different developmental stages and environments; however, little is known about how the host maintains its microbiota to achieve such adaptations. In this study, 16S rRNA sequencing revealed that the microorganisms in larvae and adults of the Oriental fruit fly, Bactrocera dorsalis, are primarily Gram-negative bacteria but that the major components in pupae are Gram-positive bacteria. Using suppression subtractive hybridization (SSH) and transcriptome analysis, we screened two specifically expressed genes encoding peptidoglycan recognition proteins (PGRP-LB and PGRP-SB1) and analyzed their relationship to B. dorsalis microbial communities. Knockdown of the PGRP-LB gene in larvae and adults led to increased ratios of Gram-positive bacteria; knockdown of the PGRP-SB1 gene in pupae led to increased ratios of Gram-negative bacteria. Our results suggest that maintenance of the microbiota in different developmental stages of B. dorsalis may be associated with the PGRP-LB and PGRP-SB1 genes.IMPORTANCE Microorganisms are ubiquitous in insects and have widespread impacts on multiple aspects of insect biology. However, the microorganisms present in insects can change dramatically in different developmental stages, and it is critical to maintain the appropriate microorganisms in specific host developmental stages. Therefore, analysis of the factors associated with the microbiota in specific development stages of the host is needed. In this study, we applied suppression subtractive hybridization (SSH) combined with transcriptome analysis to investigate whether the microbiota in development stages of the Oriental fruit fly, Bactrocera dorsalis, is associated with expression of PGRP genes. We found that two different PGRP genes were specifically expressed during development and that these genes may be associated with changes in microbial communities in different developmental stages of B. dorsalis.

In-hive variation of the gut microbial composition of honey bee larvae and pupae from the same oviposition time.

BACKGROUND: Knowledge of microbiota composition, persistence, and transmission as well as the overall function of the bacterial community is important and may be linked to honey bee health. This study aimed to investigate the inter-individual variation in the gut microbiota in honey bee larvae and pupae. RESULTS: Individual larvae differed in the composition of major bacterial groups. In the majority of 5th instar bees, Firmicutes showed predominance (70%); however, after larval defecation and during pupation, the abundance decreased to 40%, in favour of Gammaproteobacteria. The 5th instar larvae hosted significantly more (P < 0.001) Firmicutes than black pupae. Power calculations revealed that 11 and 18 replicate-individuals, respectively, were required for the detection of significant differences (P < 0.05) in the Bacteroidetes and Firmicutes abundance between stages, while higher numbers of replicates were required for Actinobacteria (478 replicates) and Gammaproteobacteria (111 replicates). CONCLUSIONS: Although sample processing and extraction protocols may have had a significant influence, sampling is very important for studying the bee microbiome, and the importance of the number of individuals pooled in samples used for microbiome studies should not be underestimated.