The combined effects of ultrasound and plasma-activated water on silkworm pupae:Physicochemical properties, microbiological diversity and ultrastructure.

A novel technique was proposed for processing silkworm pupae by combining plasma- activated water (PAW) with ultrasound (US). The microbial diversity and quality characteristics of the silkworm pupae were also evaluated. The results of the microbial diversity analysis indicated that PAW combined with US treatment significantly reduced the relative abundance of Streptococcaceae, Leuconostocaceae, and Acetobacteraceae from 32%, 18% and 16% to 27%, 11% and 11%, respectively. Microstructural analysis demonstrated that the collapse of the internal structure of chitin in silkworm pupae facilitated the release of nutrients and flavour compounds including fatty acids, water-soluble proteins (WSP), amino acids, phenolics, and volatile compounds. Furthermore, the increase in antioxidant capacity and the decrease in catalase activity and malondialdehyde content confirmed the mechanism of quality change. These findings provide new insights into the possible mechanism of PAW combined with US to improve the quality of edible insects.

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.

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.

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.

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.

Diversity and dynamics of bacteria at the Chrysomya megacephala pupal stage revealed by third-generation sequencing.

Characterization of the microbial community is essential for understanding the symbiotic relationships between microbes and host insects. Chrysomya megacephala is a vital resource, a forensic insect, a pollinator, and a vector for enteric bacteria, protozoa, helminths, and viruses. However, research on its microbial community is incomprehensive, particularly at the pupal stage, which comprises approximately half of the entire larval development stage and is important entomological evidence in forensic medicine. For the first time, this study investigated the bacterial communities of C. megacephala pupae at different ages using third-generation sequencing technology. The results showed that C. megacephala has a diverse and dynamic bacterial community. Cluster analysis at ≥ 97% similarity produced 154 operational taxonomic units (OTUs) that belonged to 10 different phyla and were distributed into 15 classes, 28 orders, 50 families, 88 genera, and 130 species. Overall, the number of bacterial OTUs increased with the development of pupae, and the relative abundance of Wolbachia in the Day5 group was significantly lower than that in the other groups. Within the pupal stage, Proteobacteria, Firmicutes, and Bacteroidetes were the dominant phyla of bacteria. At the genus level, Wolbachia and Ignatzschineria coexisted, a rarely known feature. In addition, we found Erysipelothrix rhusiopathiae, the etiological agent of swine erysipelas, which is rarely identified in insects. This study enriches the understanding of the microbial community of C. megacephala and provides a reference for better utilization and control of C. megacephala.

Honey Bee Larval and Adult Microbiome Life Stages Are Effectively Decoupled with Vertical Transmission Overcoming Early Life Perturbations.

Microbiomes provide a range of benefits to their hosts which can lead to the coevolution of a joint ecological niche. However, holometabolous insects, some of the most successful organisms on Earth, occupy different niches throughout development, with larvae and adults being physiologically and morphologically highly distinct. Furthermore, transition between the stages usually involves the loss of the gut microbiome since the gut is remodeled during pupation. Most eusocial organisms appear to have evolved a workaround to this problem by sharing their communal microbiome across generations. However, whether this vertical microbiome transmission can overcome perturbations of the larval microbiome remains untested. Honey bees have a relatively simple, conserved, coevolved adult microbiome which is socially transmitted and affects many aspects of their biology. In contrast, larval microbiomes are more variable, with less clear roles. Here, we manipulated the gut microbiome of in vitro-reared larvae, and after pupation of the larvae, we inoculated the emerged bees with adult microbiome to test whether adult and larval microbiome stages may be coupled (e.g., through immune priming). Larval treatments differed in bacterial composition and abundance, depending on diet, which also drove larval gene expression. Nonetheless, adults converged on the typical core taxa and showed limited gene expression variation. This work demonstrates that honey bee adult and larval stages are effectively microbiologically decoupled, and the core adult microbiome is remarkably stable to early developmental perturbations. Combined with the transmission of the microbiome in early adulthood, this allows the formation of long-term host-microbiome associations. IMPORTANCE This work investigated host-microbiome interactions during a crucial developmental stage-the transition from larvae to adults, which is a challenge to both, the insect host and its microbiome. Using the honey bee as a tractable model system, we showed that microbiome transfer after emergence overrides any variation in the larvae, indicating that larval and adult microbiome stages are effectively decoupled. Together with the reliable vertical transfer in the eusocial system, this decoupling ensures that the adults are colonized with a consistent and derived microbiome after eclosion. Taken all together, our data provide additional support that the evolution of sociality, at least in the honey bee system tested here, is linked with host-microbiome relationships.

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.

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.

Pathogenicity of local and exotic entomopathogenic fungi isolates against different life stages of red palm weevil (Rhynchophorus ferrugineus).

Entomopathogenic fungi are regarded as effective biocontrol agents in pest management. Different fungi isolates exhibit varying degree of pathogenicity against red palm weevil [Rhynchophorus ferrugineus (Olivier)]. The pathogenicity of four native isolate from Saudi Arabia (three Beauveria bassiana named as BbSA-1, BbSA-2, BbSA-3 and one Metarhizium anisopliae regarded as MaSA-1) and three exotic isolates from Indonesia (B. bassiana coded as BbIDN-1 and M. anisopliae named as MaIDN-1 and MaIDN-2) was evaluated against red palm weevil under laboratory conditions. The isolates were applied to eggs (1 day old), larvae (3 and 35 days old), pupae (5 days old) and adults (10 days old). The average mortality rate of eggs and hatched larvae was 100% in all of the isolates except BbSA-2 and BbIDN-1, where mortality was 93.3 and 90%, respectively. The lowest mortality rate (73.3%) was recorded for BbSA-3 against 3-days-old larvae; however, all other isolates caused >80% larval mortality. Meanwhile, 93.3% mortality of 35-day-old larvae was noted for MaSA-1 isolate. The highest pupa mortality (80%) was observed for MaSA-1, while remaining isolates caused >60% mortality. The isolates BbSA-1 and MaSA-1 caused 61 and 74.3% mortality in adults, respectively. The tested fungi isolates exhibited high virulence against all life stages of red palm weevil. Local isolates had higher pathogenicity than exotic isolates. The findings of the current study suggest that entomopathogenic fungi could be used as biological control agents for the management of red palm weevil. However, field studies are needed to reach the sound conclusions and practical applications.

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.

Diversity of bacteria in different life stages and their impact on the development and reproduction of Zeugodacus tau (Diptera: Tephritidae).

Fruit flies usually harbor diverse communities of bacteria in their digestive systems, which are known to play a significant role in their fitness. However, little information is available on Zeugodacus tau, a polyphagous pest worldwide. This study reports the first extensive analysis of bacterial communities in different life stages and their effect on the development and reproduction of laboratory-reared Z. tau. Cultured bacteria were identified using the conventional method, and all bacteria were identified by high-throughput technologies (16S ribosomal RNA gene sequencing of V3-V4 region). A total of six bacterial phyla were identified in larvae, pupae, and male and female adult flies, which were distributed into 14 classes, 32 orders, 58 families and 96 genera. Proteobacteria was the most represented phylum in all the stages except larvae. Enterobacter, Klebsiella, Providencia, and Pseudomonas were identified by conventional and next-generation sequencing analysis in both male and female adult flies, and Enterobacter was found to be the main genus. After being fed with antibiotics from the first instar larvae, bacterial diversity changed markedly in the adult stage. Untreated flies laid eggs and needed 20 days before oviposition while the treated flies showed ovary development inhibited and were not able to lay eggs, probably due to the alteration of the microbiota. These findings provide the cornerstone for unexplored research on bacterial function in Z. tau, which will help to develop an environmentally friendly management technique for this kind of harmful insect.

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.

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.

Variation in the microbiota across different developmental stages of Aedes albopictus is affected by ampicillin exposure.

The microbiota plays an important role in the growth of mosquitoes and the transmission of mosquito-borne pathogens. The effects of changes in aquatic habitats in which mosquitoes live, as one of the major factors closely associated with the microbial communities of mosquitoes, on the microbiota of different developmental stages remain to be elucidated. Here, we compared the microbiota of larvae and pupae of Aedes albopictus exposed to different ampicillin concentrations and investigated the bacterial composition of adult females. The results demonstrate that the microbial community differed substantially between developmental stages and that samples of the same stages shared similarities, whereas differences were observed between adult females. Based on all observations, we hypothesize that the use of ampicillin caused dysbiosis rather than excluding bacteria from mosquitoes and that the disturbing effect of ampicillin was obvious in adults. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that most of the bacteria identified in this study were significantly associated with metabolism. Taken together, our results indicate that ampicillin can change the abundance of bacteria, while microbial communities of Ae. albopictus showed obvious stage-specific characteristics. Further investigations are needed to characterize specific bacterial components that are affected by ampicillin exposure and to quantify their functions, thereby providing a better understanding of the influence of antibiotics on microbial communities at different life stages.

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.

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.

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.

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.