Insect Immunity

En este trabajo se estudia la hibridación en condiciones de laboratorio entre Phoracantha semipunctata Fabricius y Phoracantha recurva Newman (Coleoptera: Cerambycidae) a fin de evaluar la viabilidad y vigor de dichos híbridos, así como el porcentaje de parasitismo de A. longoi Siscaro (Hymenoptera: Encyrtidae) sobre la

Article history: Received: 03 September, 2020 Accepted: 07 January, 2021 Online: 05 February, 2021 Capturing similarity in gene sequences of a target organism to detect significant regions of comparison will most likely occur because genes share a related descendant. Local sequence alignment for the targeted organisms can help preserve associations among sequences of related organisms. Such homologous genes possess identical sequences with common ancestral genes. The genes may be similar to common traits, and varying purposes, but they descend from a common ancestor. Basic local alignment search tool (BLAST) from the National Center for Biotechnology Information. (NCBI) has been used by different researchers to resolve the various forms of alignment problems. However, much literature to bare the efficacy of standard proteinprotein BLAST (BLASTp) on the MATLAB platform has not been seen. In this study, a positionspecific iteration BLASTp of 20 anopheles insecticide target protein seq...

Xenorhabdus nematophila, a bacterium pathogenic for insects associated with the nematode Steinernema carpocapsae, releases high quantities of proteases, which may participate in the virulence against insects. Zymogram assays and cross-reactions of antibodies suggested that two distinct proteases were present. The major one, protease II, was purified and shown to have a molecular mass of 60 kDa and an estimated isoelectric point of 8.5. Protease II digested the chromogenic substrate N-tosyl-Gly-Pro-Arg-paranitroanilide (pNA) with V max and K m values of 0.0551 M/min and 234 M, respectively, and the substrate DL-Val-Leu-Arg-pNA with V max and K m values of 0.3830 M/min and 429 M, respectively. Protease II activity was inhibited 93% by Pefabloc SC and 45% by chymostatin. The optimum pH for protease II was 7, and the optimum temperature was 23°C. Proteolytic activity was reduced by 90% at 60°C for 10 min. Sequence analysis was performed on four internal peptides that resulted from the digestion of protease II. Fragments 29 and 45 are 75 and 68% identical to alkaline metalloproteinase produced by Pseudomonas aeruginosa. Fragment 29 is 79% identical to a metalloprotease of Erwinia amylovora and 75% identical to the protease C precursor of Erwinia chrysanthemi. Protease II showed no toxicity to hemocytes but destroyed antibacterial activity on the hemolymph of inoculated insects' larvae and reduced 97% of the cecropin A bacteriolytic activity.

During in vitro incubations, the nematobacterial complex Steinernema carpocapsae-Xenorhabdus nematophilus produces different factors having toxic activities in vitro towards haemocytes, the insect cells responsible for cellular immune defense reactions. Among others, two effects were evident on haemocyte monolayers; one of them was a cytotoxic activity while the other was an unsticking effect. The factors responsible for cytotoxic activity and unsticking effect, were separated from each other by a single chromatography on anion exchange column. These two effects on haemocytes were lost after heat treatment at 57°C for 1 h and 45°C for 30 min, respectively. Both factors were recovered after dialysis in a 10 4 Da cut off membrane. The cytotoxic activity was susceptible to proteases. Cytotoxic and unsticking factors did not show any lipase or lecithinase activity but the unsticking factor had protease activity. Lipopolysaccharides, purified from the bacteria harvested after incubation of the complex, did not have cytotoxic or unsticking effect on the insect cells in vitro.

Background: Endoparasitoid wasps inject venom proteins at oviposition to alter host immunity. Results: The venom of Leptopilina boulardi, but not of a related species, contains an active extracellular SOD. Recombinant SODs inhibit the Drosophila host phenoloxidase activity. Conclusion: An extracellular SOD is secreted and active in an insect fluid. Significance: SODs may be used as immune suppressive virulence factors by parasitoid wasps.

P252, a 252-kDa Bombyx mori protein located on the larval midgut membrane, has been shown to bind strongly with Bacillus thuringiensis Cry1A toxins (Hossain et al. Appl Environ Microbiol 70:4604-4612, 2004). P252 was also shown to bind chlorophyllide (Chlide) to form red fluorescence-emitting complex Bm252RFP with significant antimicrobial activity (Pandian et al. Appl Environ Microbiol 74:1324-1331, 2008). In this article, we show that Cry1A toxin bound with Bm252RFP and Bm252RFP-Cry1A macrocomplex, with both antimicrobial and insecticidal activities, was formed. The insecticidal activity of Bm252RFP-Cry1Ab was reduced from an LD 50 of 1.62 to 5.05 lg, but Bm252RFP-Cry1Aa and Bm252RFP-Cry1Ac did not show such reduction. On the other hand, the antimicrobial activity of Bm252RFP-Cry1Ab was shown to retain almost the same activity as Bm252RFP, while the other two complexes lost around 30% activity. The intensity of photo absorbance and fluorescence emission of Bm252RFP-Cry1Ab were significantly reduced compared to those of the other two complexes. Circular dichroism showed that the contents of Cry1Ab a-helix was significantly decreased in Bm252RFP-Cry1Ab but not in the other two toxins. These data suggested that the reduction of contents of a-helix in Cry1Ab affected the insecticidal activity of the macrocomplex but did not alter the antimicrobial moiety in the macrocomplex of Bm252RFP-Cry1Ab.

Ceratotoxins (Ctxs) are a family of antibacterial sex-specific peptides expressed in the female reproductive accessory glands of the Mediterranean fruit fly Ceratitis capitata. As a first step in the study of molecular evolution of Ctx genes in Ceratitis, partial genomic sequences encoding four distinct Ctx precursors have been determined. In addition, anti-Escherichia coli activity very similar to that of the accessory gland secretion from C. capitata was found in the accessory gland secretion from Ceratitis (Pterandrus) rosa. SDS-PAGE analysis of the female reproductive accessory glands from C. rosa showed a band with a molecular mass (3 kDa) compatible with that of Ctx peptides, also slightly reacting with an anti-Ctx serum. Four nucleotide sequences encoding Ctx-like precursors in C. rosa were determined. Sequence and phylogenetic analyses show that Ctxs from C. rosa fall into different groups as C. capitata Ctxs. Our results suggest that the evolution of the ceratotoxin gene family might be viewed as a combination of duplication events that occurred prior to and following the split between C. capitata and C. rosa. Genomic hybridization demonstrated the presence of multiple Ctx-like sequences in C. rosa, but low-stringency Southern blot analyses failed to recover members of this gene family in other tephritid flies.

Microbial consortia accompanied to all eukaryotes can be inherited from ancestors, environment, and/or from various food source. Gut microbiota study is an emerging discipline of biological sciences that expands our understanding of the ecological and functional dynamics of gut environments. Microorganisms associated with honey bees play an important role in food digestion, colony performance, immunity, pollination, antagonistic effect against different pathogens, amelioration of food and many more. Although, many repots about honey bee gut microbiota are well documented, microbiome with other key components of honey bees such as larvae, adults, their food (pollen, beebread, and honey), honey combs, and floral nectar are poorly understood. Mutual interactions and extent of the roles of microbial communities associated with honey bees are still unclear and demand for more research on the nutritional physiology and health benefits of this ecologically and economically important group. Here in this study, we highlighted all the honey bee microbiome that harbored from different life stages and other relevant components. The anatomical parts of honey bee (larvae, adults), food source (pollen, beebread, and honey), honey combs, and floral nectar were highly flourished by numerous microorganisms like bacteria (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Actinomycetes, Bacilli, Bacteroidetes, Cocci, Clostridia, Coliforms, Firmicutes, Flavobacteriia, Mollicutes) and fungi (Dothideomycetes, Eurotiomycetes, Mucormycotina, Saccharomycetes, Zygomycetes, Yeasts, Molds). Some distinctive microbial communities of a taxonomically constrained species have coevolved with social bees. This contribution is to enhance the understanding of honey bee gut microbiota, to accelerate bee microbiota and microbiome research in general and to aid design of future experiments in this growing field.

We describe eicosanoid biosynthesis by microsomai-enriched preparations of hemocytes from larvae of the tobacco hornworm Manduca sexta. Four major prostaglandins, PGA2, PGE2, PGD2 and PGF~, and a lipoxygenase product that co-chromatographed with 15-hydroxyeicosatetraenoic acid (HETE) were synthesized under most conditions. The HETE's fraction was the predominant product. Eicosanoid biosynthesis was sensitive to experimental conditions, including incubation time, temperature, and protein concentration. Optimal biosynthesis was observed with 1.5 mg of microsomal-enriched protein, incubated at 30°C for 2 min. The hemocyte preparation is sensitive to low dosages of naproxin and esculetin. As in mammals, most lipoxygenase activity (87%) was localized in the eytosolie fraction of hemocytes. Unlike mammals, in which PGH synthase is associated with intraceHular membranes, the hemocytie activity was detected in microsomal (59%), eytosolie (35%) and mitochondrial fractions (5%).

Prostaglandin levels were determined by fluorometric HPLC analysis of hemolymph collected from larvae of the true armyworm, Pseudaletia unipuncta, that had been injected with bacteria. Prostaglandins were extracted and derivatized with the fluorogenic compound 9-anthryldiazomethane and detected by fluorescence-HPLC. One of the prostaglandins produced was identified as prostaglandin F 2a based on HPLC retention time. The chemical identity of prostaglandin F 2a was confirmed by isolation and derivatization followed by gas chromatography mass spectrometry analysis. Larvae injected with heat-killed bacteria, Serratia marcescens, produced about 4 times as much prostaglandin F 2a as larvae injected with saline. In a separate experiment, larvae injected with bacteria and the prostaglandin precursor arachidonic acid produced still higher levels of prostaglandin F 2a . The production of prostaglandin was inhibited with phenidone, a dual cyclooxygenase and lipoxygenase inhibitor. These data indicate that bacterial injections stimulate increased eicosanoid biosynthesis in true armyworms, particularly biosynthesis of prostaglandin F 2a . Our findings add considerable support to the hypothesis that eicosanoids mediate insect cellular immune reactions to bacterial infections. Arch. Insect Biochem.

Studies on ecological immunology are crucial in determining how the immune responses of insects against both abiotic and biotic factors are affected. In this study, the effects of phenolic compounds in plants on the immune responses and antioxidant enzyme activities of Hyphantria cunea larvae infected by Bacillus thuringiensis subsp. kurstaki were investigated. For this purpose, phenolic compounds present in mulberry, apple, walnut and plum plants (preferred by H. cunea and having economic importance) were determined. H. cunea culture was obtained from larvae collected in Bafra, Samsun in 2019. It was found that the haemocyte counts of both control and infected larvae fed in the walnut plant containing the highest gallotannin amount were the highest. It was found that catechin and rutin flavonoids caused minimum phenoloxidase activity. It was determined that the highest superoxide dismutase activities were in the plum-fed groups and the lowest catalase activities were in the larvae fed in the walnut groups both in the control and infected groups. As a result, it was determined that immune responses and antioxidant enzyme activities can be changed due to both phenolic compounds present in plants and infection.

This study aimed to investigate the changes in total consumption amounts, survival rates, and phenoloxidase (PO) activities in Malacosoma neustria (L., 1758) (Lepidoptera: Lasiocampidae) larvae infected with Bacillus thuringiensis subsp. kurstaki Berliner. Malacosoma neustria eggs were collected from Bafra District of Samsun, Turkey, in 2018. Malacosoma neustria larvae that hatched from the eggs fed on the host plants, Coryllus maxima Mill., Elaeagnus rhamnoides (L.) A. Nelson, Quercus cerris L. 1753, and Crataegus monogyna Jacq. Except for the control groups, the experiment continued by spraying Bacillus thuringiensis on the plants. The amounts of consumption and survival rates of both the control and infected larvae fed on E. rhamnoides leaves with the highest amounts of protein, phenolic, and gallotannin, were higher than in other groups. In all groups, both the consumption amounts and the survival rates decreased with a bacterial infection. When compared to the control groups, the infected groups had the higher phenoloxidase activities (except C. monogyna). As a result of this study, it was thought that the changes in total consumption amount, survival rates, and PO activities in larvae were related to both bacterial infection and the amounts of protein, phenolic, and gallotannin in the plants.

The aim of this study was to investigate the effect of tannic acid, some heavy metals (nickel, zinc and copper) and their combinations, and Bacillus thuringiensis subsp. kurstaki infection on the catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) enzymes and hemocyte count of Hyphantria cunea larvae. For this purpose, 1 diet was prepared by adding tannic acid to 5% concentration of dry weight in the control diet. Nickel, zinc, and copper solutions were prepared and 3 diets were made and 3 diets were made with tannic acid and heavy metal combinations. All analyzes were determined by ANOVA-Dunnet test. The hemocyte count was the highest in the larvae fed with the control diet. Hemocyte counts of all groups increased with infection compared to control groups. SOD, CAT and GPx activities of the larvae fed with heavy metals and tannic acid diet increased compared to the larvae fed with the control diet. Synergistic and antogonistic effects were observed in some groups where heavy metals and tannic acid were combined.

Pollen samples collected in the spring of 2002 in 8 south-western Slovakia localities and 40 live individuals of bumblebee were analyzed for the presence of bacteria and microscopic fungi. Microorganisms occurring on pollen and bumblebees were identified using cultivation and microscopic methods supplemented with biochemical tests. In the pollen were found fermenting and non-fermenting Gram-negative rods, Gram-positive sporulating cocci and non-haemolytic Gram-positive cocci. Analyses of microscopic fungi on bumblebee bodies showed the presence of only four species -Acremonium murorum, Aspergillus penicilloides, Fusarium oxysporum, Harpografium fasciculatum representing Fungi imperfecti. The highest amount of microscopic fungi occurred on drone, lower numbers on queen and worker bees. In the pollen samples 21 fungal species forming 13 genera of microscopic fungi were detected. The highest number of mould species was classified in the genera of Mucor, Rhizopus, Aspergillus, Alternaria and Paecilomyces, the species of other genera occurred in lower frequency. As the majority of the identified micromycetes represent the mitosporic fungal group of saprophytic microorganisms inhabiting soil or the organic residues of plants lacking pathogenic effects, it could be concluded, the tested pollen samples may be declared for safe resource of food and/or feed.

Objective. To characterize the native strains associated with the intestinal tract of the species Panaque cochliodon by microbiology. Materials and methods. Three adult specimens were used. These were captured in the Magdalena River, transported, and sacrificed under animal welfare regulations. Dissection of the intestinal tract was performed, obtaining samples for microbiological isolation, using culture media, purifying the microorganisms, and performing metabolic biochemical tests and API 20E (Biomeriux) tests for their identification. Results. Information on the microbial population structure was obtained, reporting the phylum Proteobacteria with the species Pantoea sp., Erwinia sp., Providencia stuarti, Providencia alcalifaciens, Serratia ficaria, Citrobacter koseri, and the phylum Firmicutes with the species Bacillus sphaericus, Bacillus subtilis, Bacillus thuringiensis, Bacillus mycoides, Bacillus coagulans, and Bacillus circulans. Conclusions. The predominant culturable microorganisms in P. cohliodon belong to the phylum Proteobacteria and the phylum Firmicutes.

The introduction of novel biochemical, genetic, molecular and cell biology tools to the study of insect immunity has generated an information explosion in recent years. Due to the biodiversity of insects, complementary model systems have been developed. The conceptual framework built based on these systems is used to discuss our current understanding of mosquito immune responses and their implications for malaria transmission. The areas of insect and vertebrate innate immunity are merging as new information confirms the remarkable extent of the evolutionary conservation, at a molecular level, in the signaling pathways mediating these responses in such distant species. Our current understanding of the molecular language that allows the vertebrate innate immune system to identify parasites, such as malaria, and direct the acquired immune system to mount a protective immune response is very limited. Insect vectors of parasitic diseases, such as mosquitoes, could represent excellent models to understand the molecular responses of epithelial cells to parasite invasion. This information could broaden our understanding of vertebrate responses to parasitic infection and could have extensive implications for anti-malarial vaccine development.

Aphid is a vector for various plant pathogenic viruses and gives severe damage various economically important crops and vegetables. However, they are hardly controlled by biopesticides as they are living with phloem sap supplied by a sucking the li quid. We in vestigated the aphidicidal activity of Bacillus thuringiensis toxin Cyt2Aa. The green peach aphid. Myzus persicae (Sulzer), were collected from field and reared with leaves of radish in a plastic column. In the bioassay, 100 III of Cyt2Aa was given with sucrose solution at 200 Ilg/ ml as final concentration through Parafilm and significant kill effects was observed with 38% mortality at 72 h after the start of rearing. On the other hand, Cry1Aa. Cry1Ab and Cry1Ac as well as PBS showed no activity. Admire, chemical pesticide (Bayer Cropsience. Monheim, Germany) which was used as positive control kill all aphids tested. This is the first report showing aphidicidal activity of Cyt2Aa. Kill mechanism are also briefly discussed.

In this study, we report the analysis of the immune-related transcriptome from an apterygote insect, the firebrat Thermobia domestica (Zygentoma, Lepismatidae), which currently emerges as a suitable model insect for evolutionary and developmental studies. The suppression subtractive hybridization method was used for targeted screening of genes that are up-regulated in response to injected bacterial lipopolysaccharide (LPS). A subtracted cDNA library enriched in immune-inducible genes was constructed and analysis of 288 cDNAs resulted in identification of 26 novel genes in T. domestica. Among these immune-related transcripts we found homologues of genes from other insects which are involved in the regulation of signaling cascades and six novel putative antimicrobial peptides. The identified genes implicate the presence of sophisticated regulatory mechanisms in insect immune signaling and give insight into evolutionarily conserved features of insect innate immunity.

The red flour beetle, Tribolium castaneum, is an established genetically tractable model insect for evolutionary and developmental studies. Therefore, it may also represent a valuable model for comparative analysis of insect immunity. Here, we used the suppression subtractive hybridization method to identify Tribolium genes that are transcriptionally induced in response to injection of crude lipopolysaccharide (LPS). Determined genes encode proteins that share sequence similarities with counterparts from other insects known to mediate sensing of infection (e.g. Toll and PGRP) or to represent potential antimicrobial effectors (e.g. ferritin, c-type lysozyme, serine proteinase inhibitors, and defensins). Especially significant is the identification of thaumatin-like peptides, representing ancient antifungal peptides originally reported from plants, that are absent from the genomes of many other insects such as Drosophila, Anopheles, and Apis. We produced recombinant thaumatin-1 in bacteria and we found that it represents an antimicrobial peptide against filamentous fungi in Tribolium. Additionally, septic injury induces expression of genes involved in stress adaptation (e.g. heat-shock proteins) or insecticide resistance (e.g. cytochrome P450s) in Tribolium, suggesting that there may be crosstalk between the immune and stress responses.

The first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes. Here, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data. Lessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination.

A greenhouse experiment was performed to analyze a potential effect of genetically modified potatoes expressing antibacterial compounds (attacin/cecropin, T4 lysozyme) and their nearly isogenic, nontransformed parental wild types on rhizosphere bacterial communities. To compare plant transformation-related variations with commonly accepted impacts caused by altered environmental conditions, potatoes were cultivated under different environmental conditions, for example using contrasting soil types. Further, plants were challenged with the blackleg pathogen Erwinia carotovora ssp. atroseptica. Rhizosphere soil samples were obtained at the stem elongation and early flowering stages. The activities of various extracellular rhizosphere enzymes involved in the C-, P-and N-nutrient cycles were determined as the rates of fluorescence of enzymatically hydrolyzed substrates containing the highly fluorescent compounds 4-methylumbelliferone or 7-amino-4-methyl coumarin. The structural diversity of the bacterial communities was assessed by 16S rRNA-based terminal restriction fragment length polymorphism analysis, and 16S rRNA gene clone libraries were established for the flowering conventional and T4 lysozyme-expressing Desirée lines grown on the chernozem soil, each line treated with and without E. carotovora ssp. atroseptica. Both genetic transformation events induced a differentiation in the activity rates and structures of associated bacterial communities. In general, T4 lysozyme had a stronger effect than attacin/cecropin. In comparison with the other factors, the impact of the genetic modification was only transient and minor, or comparable to the dominant variations caused by soil type, plant genotype, vegetation stage and pathogen exposure.

The present study seeks to investigate the comparative estimation and electrophoretic profile of haemolymph proteins among the different semi domesticated morphs (green, blue, orange) and the wild (bivoltine) morph of Antheraea assamensis Helfer in order to investigate their differential gene expression. The proteomic approach was utilized to investigate the proteome of the haemolymph of early, mid and late stages of 5th instar (larval stage) and to improve the understanding of their important bioprocess and gene expression situation. The results suggest that the quantitative approach is useful to arrive at a conclusion of genetic variation among the morphs. SDS-PAGE electrophoresis of protein associated with haemolymph revealed separation of 16 distinct protein bands among which some of them represent the storage and immunity related proteins.

Insect pests such as termites cause damages to crops and man-made structures estimated at over $30 billion per year, imposing a global challenge for the human economy. Here, we report a strategy for compromising insect immunity that might lead to the development of nontoxic, sustainable pest control methods. Gram-negative bacteria binding proteins (GNBPs) are critical for sensing pathogenic infection and triggering effector responses. We report that termite GNBP-2 (tGNBP-2) shows β( 1 , 3 )-glucanase effector activity previously unknown in animal immunity and is a pleiotropic pattern recognition receptor and an antimicrobial effector protein. Termites incorporate this protein into the nest building material, where it functions as a nest-embedded sensor that cleaves and releases pathogenic components, priming termites for improved antimicrobial defense. By means of rational design, we present an inexpensive, nontoxic small molecule glycomimetic that blocks tGNBP-2, thus exposing term...

Proteomic Application to Study Crustacean Innate Immune Response against Pathogenes Tahereh Alinejad1*, Shabnam Modarressi2, Subha Bhassu1, and Rofi na Yasmin Othman1 1Department of Genetics and molecular biology, Institute of Biological Science, Faculty of Science, & Center for Research in Biotechnology for Agriculture (CEBAR), Research management and innovation complex University of Malaysia, Malaysia 2Department of food Science, Food microbiology, University of Copenhagen *Address for Correspondence: Tahereh Alinejad, Department of Genetics and molecular biology, Institute of Biological Science, Faculty of Science & Center for Research in Biotechnology for Agriculture (CEBAR), Research management and innovation complex University of Malaya, 50603, Kuala Lumpur, Malaysia; E-mail: Submitted: 18 December 2020; Approved: 23 December 2020; Published: 04 January 2021

The pollen stores of bumble bees host diverse microbiota that influence overall colony fitness. Yet, the taxonomic identity of these symbiotic microbes is relatively unknown. In this descriptive study, we characterized the microbial community of pollen provisions within captive-bred bumble bee hives obtained from two commercial suppliers located in North America. Findings from 16S rRNA and ITS gene-based analyses revealed that pollen provisions from the captive-bred hives shared several microbial taxa that have been previously detected among wild populations. While diverse microbes across phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Ascomycota were detected in all commercial hives, significant differences were detected at finer-scale taxonomic resolution based on the supplier source. The causative agent of chalkbrood disease in honey bees, Ascosphaera apis, was detected in all hives obtained from one supplier source, although none of the hives showed symptoms...

As pollen and nectar foragers, bees have long been considered strictly herbivorous. Their pollen provisions, however, are host to abundant microbial communities, which feed on the pollen before and/or while it is consumed by bee larvae. In the process, microbes convert pollen into a complex of plant and microbial components. Since microbes are analogous to metazoan consumers within trophic hierarchies, the pollen-eating microbes are, functionally, herbivores. When bee larvae consume a microbe-rich pollen complex, they ingest proteins from plant and microbial sources and thus should register as omnivores on the trophic "ladder." We tested this hypothesis by examining the isotopic compositions of amino acids extracted from native bees collected in North America over multiple years. We measured bee trophic position across the six major bee families. Our findings indicate that bee trophic identity was consistently and significantly higher than that of strict herbivores, providing the first evidence that omnivory is ubiquitous among bee fauna. Such omnivory suggests that pollen-borne microbes represent an important protein source for larval bees, which introduces new questions as to the link between floral fungicide residues and bee development.

Very little is known about the functional basis of plasmatocyte motile bohaviour. In this study we documented the effects of cytochalasin B (CB) on the cytoskeletal organisation and behaviour of plasmatocytes during spreading on a planar surface in vitro, and during in vitro encapsulation. CB produced a reversible and dose-dependent effect on the rate of plasmatocyte spreading. Moreover, incubation of fully spread plasmatocytes in CB caused their microflaments to coalesce and the cells to arborise and eventually revert to the less adhesive spindle-shaped morphology characteristic of free plasmatocytes in circulation. The use of cytochalasins B and D on haemocytes encapsulating cotton fbre loops in vitro resulted in the development of abnormally flocculent capsules. The data demonstrate the importance of microfilament-dependent PL motility, both during the secondary "compaction phase" of encapsulation and in the laboratory manifestation of this process, spreading on a planar substratum. 354 MICROFILAMENT-DEPENDENT PL MOTILITY Vol. ii, No. 2 MATERIALS AND METHODS Haemocytes. Larvae of three species of Lepidoptera were used: the small tortoiseshell butterfly Aglais urticae (O. Nymphalidae), the Mediterranean flour moth Ephestia kuehniella (O. Pyralidae) and the tobacco budworm Heliothis virescens (O. Noctuidae). Haemolymph was extracted from CO 2-anaesthetised final instar larvae by proleg amputation (or integumental puncture for E. kuehniella). The PLs from these insects, while differing in size, were similar in morphology and motile behaviour. A. urticae (collected from the field and reared on nettles) have large PLs and these were used for the cytoskeletal studies. The other species were laboratory bred. E. kuehniella, reared on a equal mixture of flour and rolled oats (3), were used for the PL spreading assays. Budworms were reared in the laboratory on an artificial medium (4) and used for the in vitro encapsulation experiments (5); their PLs participate in encapsulation both in vitro (5) and in vivo (6). The PLs ofE. kuehniella have also been shown to encapsulate foreign implants (7).

Red flour beetle, Tribolium castaneum Herbst. (Coleoptera: Tenebrionidae) tolerate the heat treatment in the food processing facilities and storage godowns. The anhydrobiotic character to tolerate the heat treatment of the insect is due to some special metabolites. Characterization of the insect homogenate showed that protein was one of the major constituent imparting the heat tolerance. Using SDS-PAGE (10%) analysis we could decipher the protein with molecular weight of the 70KDa (heat shock protein) act as molecular chaperone in protecting the normal protein in cells of the heat tolerant stages of the beetle. The identity of the heat shock protein (HSP) has been confirmed by the N-terminal sequencing. Further theoretical analysis of the protein sequence shows that the protein is stable and composed of four conserved domains.

Although alkaloids are frequent in the poison glands of ants of the genus Aphaenogaster, this is not the case for A. iberica. Hypothesizing that in the genus Aphaenogaster, alkaloids are produced by symbiotic bacteria, except for A. iberica, we treated an experimental lot of both A. iberica and a 'classical' Aphaenogaster species, A. senilis, with an antibiotic. Compared to workers from a control lot, this treatment reduced considerably alkaloid production in A. senilis, whereas A. iberica did not react to the treatment. Furthermore, the treatment induced an increase in cuticular hydrocarbon quantities in A. senilis, but not in A. iberica. An analysis of the ant microbiota will be the next step to confirm our hypothesis.

This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Animal biology and pathology/Biologie et pathologie animales Blochmannia endosymbionts and their host, the ant Camponotus fellah: Cuticular hydrocarbons and melanization Relations entre endosymbiotes Blochmannia et leur hôte, la fourmi Camponotus fellah avec hydrocarbures cuticulaires et me´lanisation

Although alkaloids are frequent in the poison glands of ants of the genus Aphaenogaster, this is not the case for A. iberica. Hypothesizing that in the genus Aphaenogaster, alkaloids are produced by symbiotic bacteria, except for A. iberica, we treated an experimental lot of both A. iberica and a 'classical' Aphaenogaster species, A. senilis, with an antibiotic. Compared to workers from a control lot, this treatment reduced considerably alkaloid production in A. senilis, whereas A. iberica did not react to the treatment. Furthermore, the treatment induced an increase in cuticular hydrocarbon quantities in A. senilis, but not in A. iberica. An analysis of the ant microbiota will be the next step to confirm our hypothesis.

This study investigated both bacterial and fungal communities in corbicular pollen and hive-stored bee bread of two commercial honey bees, Apis mellifera and Apis cerana, in China. Although both honey bees favor different main floral sources, the dynamics of each microbial community is similar. During pH reduction in hive-stored bee bread, results from conventional culturable methods and next-generation sequencing showed a declining bacterial population but a stable fungal population. Different honey bee species and floral sources might not affect the core microbial community structure but could change the number of bacteria. Corbicular pollen was colonized by the Enterobacteriaceae bacterium (Escherichia-Shiga, Panteoa, Pseudomonas) group; however, the number of bacteria significantly decreased in hive-stored bee bread in less than 72 h. In contrast, Acinetobacter was highly abundant and could utilize protein sources. In terms of the fungal community, the genus Cladosporium remained abundant in both corbicular pollen and hive-stored bee bread. This filamentous fungus might encourage honey bees to reserve pollen by releasing organic acids. Furthermore, several filamentous fungi had the potential to inhibit both commensal/contaminant bacteria and the growth of pathogens. Filamentous fungi, in particular, the genus Cladosporium, could support pollen preservation of both honey bee species.

This study investigated different bacterial communities in three intestinal parts (foregut, midgut and hindgut) of Xylocopatenuiscapa to understand the roles of gut bacteria. Our phylogenetic analysis revealed that X. tenuiscapa is closely related to Xylocopa latipes. The 16S rRNA gene in the genomic DNA samples from the gut was examined by illumina (Solexa) and a total of 998 operational taxonomic unit (OTUs) clusters were found. Taxonomic classification identified 16 bacterial phyla and unclassified bacteria. The dominant bacteria taxa in the three parts of X. tenuiscapa gut were Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. In the foregut, Lactobacillales and Enterobacteriaceae were predominantly found. The population in the midgut was similar to that in the foregut, with the addition of Gilliamella, which was also abundant. The most dominant bacteria identified in the hindgut were similar to those in the midgut and Lactobacillales, Enterobacteriaceae, Gilliamella...

In insect immunity, antibacterial proteins are an important part of the immune system. These proteins are mostly produced by epithelial cells and released through hemolymph. Antibacterial proteins in insects belong to the five major families of cecropins, defensins, attacin-like proteins, proline-rich peptides, and lysozymes. Considering the importance of these proteins in fighting infections, the aim of this study was to evaluate and identify these proteins in silkworms (Bombyx mori). In the present work, profiling of the proteins present in the hemolymph of control silkworms versus, those infected with bacteria was performed by SDS-PAGE, 2D gel electrophoresis, and image analysis. We also used MALDI-TOF and MS/MS to investigate novel and uncharacterized immune protein. For this aim, the silkworm hemolymph after inoculation and infection with Staphylococcus aureus bacteria was analyzed using SDS-PAGE, 2-dimensional gel electrophoresis, MALDI-TOF and MS/MS to identify the immune proteins. The Swiss-Prot and NCBI databases were used for protein identification. A novel protein with a molecular weight of 31.9 KDa was discovered on the fourth day of exposure to bacteria. The expressed protein showed effective activity against S. aureus, which was infected silkworms. In the MALDI-TOF/MS result and protein identification analysis, 90 numbers of mass values were searched, the mass values matched 16, the total sequence coverage was 46%, and their score was 57. According to the analyses, the expressed protein belongs to the hemolysin secretion protein (HlyD). The results led to the identification of a new protein with antimicrobial properties in silkworm, although more information is needed.

Males and females of a Bt-tolerant mulberry silkworm (Bombyx mori L.) population were crossed with females and males of a Bt-susceptible population, to produce Bt-tolerant silkworm hybrids, and to determine the expression of the Bt-tolerance pattern in the F1 hybrids. It was observed that when a Bt-tolerant (42% larval mortality) female (BtT) silkworm was crossed with a Bt-susceptible (85% larval mortality) male (BtS), the resultant F1 offspring showed lower levels of Bt-tolerance (87% larval mortality). On the other hand, when a Bt-tolerant male (BtT) was crossed with a Bt-susceptible female (BtS), the F1 hybrid showed higher levels of Bt-tolerance (35% larval mortality) characteristic. The probit statistics showed that both hybrids expressed Bt-tolerance or susceptible levels similar to their male parents. These different patterns of Bt-tolerance in F1 hybrids might be due to the transferring of a Bttolerant gene, from the parents to offspring, through the homozygotic male (ZZ) silkworm.

Considering the economic and environmental role played by bees and their present threats it is necessary to develop food supplements favoring their health. The aim of this work was to isolate and characterize an immunomodulating probiotic capable to improve the health of honeybee colonies. For this purpose, bacterial strains were isolated from Apis mellifera bees (N = 180) obtained at three apiaries. A total of 44 strains were isolated and 9 of them were identified as Lactobacillus having the capacity to grow under saccharose osmotic stress, at pH 4.0 and possessing a wide susceptibility to antibiotics. Results allowed to select two strains but finally only one of them, strain A14.2 showed a very significant immunomodulating activity. This strain increased the expression of mRNA codifying the antimicrobial peptides 24 h postadministration. We evaluated its growth kinetics under aerobic and microaerobic conditions and its survival in the presence of high concentrations of saccharose. Results demonstrated that Lactobacillus casei A14.2 strain was highly tolerant to oxygen and that it was able to adapt to saccharose enriched environments (50% and 100% w/v). Finally, L. casei A14.2 strain was administered monthly during summer and early fall to 4 honeybee colonies (2 controls and 2 treatments). The results showed a gradual sustained decrease of infestation (p < 0.05) by the pathogenic Nosema spp. but no reduction in the infestation by the mite Varroa destructor. These results suggest that the administration of this potential probiotic, may increase the resistance of honeybee colonies to infectious diseases caused by Nosema spp.

Recent studies have greatly increased understanding of how the immune system of insects responds to infection, whereas much less is known about how pathogens subvert immune defenses. Key regulators of the insect immune system are Rel proteins that form Nuclear Factor-kB (NF-kB) transcription factors, and inhibitor kB (IkB) proteins that complex with and regulate NF-kBs. Major mortality agents of insects are parasitoid wasps that carry immunosuppressive polydnaviruses (PDVs). Most PDVs encode ank genes that share features with IkBs, while our own prior studies suggested that two ank family members from Microplitis demolitor bracovirus (MdBV) (Ank-H4 and Ank-N5) behave as IkB mimics. However, the binding affinities of these viral mimics for Rel proteins relative to endogenous IkBs remained unclear. Surface plasmon resonance (SPR) and co-immunoprecipitation assays showed that the IkB Cactus from Drosophila bound Dif and Dorsal homodimers more strongly than Relish homodimers. Ank-H4 and-N5 bound Dif, Dorsal and Relish homodimers with higher affinity than the IkB domain of Relish (Rel-49), and also bound Relish homodimers more strongly than Cactus. Ank-H4 and-N5 inhibited processing of compound Relish and reduced the expression of several antimicrobial peptide genes regulated by the Imd signaling pathway in Drosophila mbn2 cells. Studies conducted in the natural host Pseudoplusia includens suggested that parasitism by M. demolitor also activates NF-kB signaling and that MdBV inhibits this response. Overall, our data provide the first quantitative measures of insect and viral IkB binding affinities, while also showing that viral mimics disable Relish processing.

Recent studies have greatly increased understanding of how the immune system of insects responds to infection, whereas much less is known about how pathogens subvert immune defenses. Key regulators of the insect immune system are Rel proteins that form Nuclear Factor-kB (NF-kB) transcription factors, and inhibitor kB (IkB) proteins that complex with and regulate NF-kBs. Major mortality agents of insects are parasitoid wasps that carry immunosuppressive polydnaviruses (PDVs). Most PDVs encode ank genes that share features with IkBs, while our own prior studies suggested that two ank family members from Microplitis demolitor bracovirus (MdBV) (Ank-H4 and Ank-N5) behave as IkB mimics. However, the binding affinities of these viral mimics for Rel proteins relative to endogenous IkBs remained unclear. Surface plasmon resonance (SPR) and co-immunoprecipitation assays showed that the IkB Cactus from Drosophila bound Dif and Dorsal homodimers more strongly than Relish homodimers. Ank-H4 and-N5 bound Dif, Dorsal and Relish homodimers with higher affinity than the IkB domain of Relish (Rel-49), and also bound Relish homodimers more strongly than Cactus. Ank-H4 and-N5 inhibited processing of compound Relish and reduced the expression of several antimicrobial peptide genes regulated by the Imd signaling pathway in Drosophila mbn2 cells. Studies conducted in the natural host Pseudoplusia includens suggested that parasitism by M. demolitor also activates NF-kB signaling and that MdBV inhibits this response. Overall, our data provide the first quantitative measures of insect and viral IkB binding affinities, while also showing that viral mimics disable Relish processing.

Maize (zea mays) is the most important cereal crop after rice in Bangladesh. During storage, maize grains are severely destroyed by insect pests. Most of the farmers of Bangladesh do not know how to preserve corn seed properly due to insufficient knowledge of storage pest of maize. An experiment was conducted to assess the major pests associated with stored maize, the damage and losses they cause in Bangladesh during July to December 2017. Sixty (60) farmer's farms from fifteen major maize growing districts of Bangladesh were assessed. Sixteen Arthropods, Fifteen Coleopterans, Five Lepidopterans and one Acarina were documented from the collected samples. Maize weevil (Sitophilus zeamais) followed by maize grain moth (Sitotroga cereallela), rice weevil (Sitophilus oryzae), flour beetle (Tribolium confusum) and Areca nut weevil (A. fasciculatus) were the major pests of stored maize in the study area. Among them, Maize weevil (S. zeamais) is the dominant, destructive and most important pest of maize in Bangladesh. This pest was found in abundance in all areas assessed damaging maize grain. Quantitative average grain damage of 75.85% and losses of 51 to 85% are common in the store within five to six months after storage. An integrated weevil management method should be implemented to prevent the high quantitative and qualitative grain losses incurred to contribute towards family food security in Bangladesh.

Ceratotoxins are antibacterial peptides produced in the female reproductive accessory glands of the medfly Ceratitis capitata. Their expression is not affected by bacterial infection, but is enhanced after mating and is modulated by juvenile hormone. Three different peptides, named ceratotoxins A, B and C, have been previously purified from the female accessory gland secretion and their amino acid and cDNA sequences have been determined. We report here the complete nucleotide sequences of four genes encoding closely related ceratotoxin peptides. One of them encodes a novel peptide, which we named ceratotoxin D. Restriction and nucleotide sequence analysis indicate that these ceratotoxin genes are organized in a large cluster spanning more than 26 kilobases of DNA. All ceratotoxin genes are coordinately expressed. Ceratotoxin transcripts appear in 2-3 day old adult females, and they reach a maximum in 6-7 day old females. The presence of highly conserved motifs in the upstream regions of all the sequenced ceratotoxin genes suggests the presence of common regulatory elements for all ceratotoxins.

Prior infection of Manduca sexta caterpillars with the non-pathogenic bacterium Escherichia coli elicits effective immunity against subsequent infection by the usually lethal and highly virulent insect pathogen Photorhabdus luminescens TT01. Induction of this protective effect is associated with up-regulation of both microbial pattern recognition protein genes (hemolin, immulectin-2 and peptidoglycan recognition protein) and anti-bacterial effector genes (attacin, cecropin, lebocin, lysozyme and moricin). We used RNA interference to knock down over-transcription of members of both these sets of genes one at a time. Interfering with expression of individual recognition proteins had a drastic adverse effect on the E. coli elicited immunity. RNAi knock-down of immulectin-2 caused the greatest reduction in immunity, followed by hemolin and peptidoglycan recognition protein (PGRP) in that order, to the extent that knock-down of any one of these three proteins left the insects more susceptible to P. luminescens infection than insects that had not experienced prior infection with E. coli. Interfering with the expression of individual antibacterial effector proteins and peptides had a much less marked effect on immunity. Knock-down of attacin, cecropin or moricin caused treated insects to be more susceptible to P. luminescens infection than controls that had been pre-infected with E. coli but which had not received the specific RNAi reagents, but they were still less susceptible than insects that had not been pre-infected with E. coli. RNAi knock-down with expression of lebocin or lysozyme had no effect on E. coli-induced immunity to P. luminescens, indicating that these effectors are not involved in the response. By bleeding pre-infected caterpillars and growing the pathogen directly within cell-free insect haemolymph, we showed that at least part of the protection elicited by previous exposure to E. coli is due to the presence of factors within the blood plasma that inhibit the growth of P. luminescens. The production of these factors is inhibited by RNAi treatment with ds-RNA reagents that knock down hemolin, immulectin-2, and PGRP. These results demonstrate that the insect immune system can be effectively primed by prior infection with nonpathogenic bacteria against subsequent infection by a highly virulent pathogen. Given the continuous normal exposure of insects to environmental and symbiotic bacteria, we suggest that prior infection is likely to play a significant and underestimated role in determining the level of insect immunity found in nature.

Prior infection of Manduca sexta caterpillars with the non-pathogenic bacterium Escherichia coli elicits effective immunity against subsequent infection by the usually lethal and highly virulent insect pathogen Photorhabdus luminescens TT01. Induction of this protective effect is associated with up-regulation of both microbial pattern recognition protein genes (hemolin, immulectin-2 and peptidoglycan recognition protein) and anti-bacterial effector genes (attacin, cecropin, lebocin, lysozyme and moricin). We used RNA interference to knock down over-transcription of members of both these sets of genes one at a time. Interfering with expression of individual recognition proteins had a drastic adverse effect on the E. coli elicited immunity. RNAi knock-down of immulectin-2 caused the greatest reduction in immunity, followed by hemolin and peptidoglycan recognition protein (PGRP) in that order, to the extent that knock-down of any one of these three proteins left the insects more susceptible to P. luminescens infection than insects that had not experienced prior infection with E. coli. Interfering with the expression of individual antibacterial effector proteins and peptides had a much less marked effect on immunity. Knock-down of attacin, cecropin or moricin caused treated insects to be more susceptible to P. luminescens infection than controls that had been pre-infected with E. coli but which had not received the specific RNAi reagents, but they were still less susceptible than insects that had not been pre-infected with E. coli. RNAi knock-down with expression of lebocin or lysozyme had no effect on E. coli-induced immunity to P. luminescens, indicating that these effectors are not involved in the response. By bleeding pre-infected caterpillars and growing the pathogen directly within cell-free insect haemolymph, we showed that at least part of the protection elicited by previous exposure to E. coli is due to the presence of factors within the blood plasma that inhibit the growth of P. luminescens. The production of these factors is inhibited by RNAi treatment with ds-RNA reagents that knock down hemolin, immulectin-2, and PGRP. These results demonstrate that the insect immune system can be effectively primed by prior infection with nonpathogenic bacteria against subsequent infection by a highly virulent pathogen. Given the continuous normal exposure of insects to environmental and symbiotic bacteria, we suggest that prior infection is likely to play a significant and underestimated role in determining the level of insect immunity found in nature.

The larval stages of the tobacco budworm, Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae), are parasitized by the endophagous parasitoid wasp, Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae). During the injections of eggs, this parasitoid wasp also injects into the host body the secretion of the venom gland and the calyx fluid, which contains a polydnavirus (T. nigriceps BracoVirus: TnBV) and the Ovarian calyx fluid Proteins (OPs). The effects of the OPs on the host immune system have recently been described. In particular, it has been demonstrated that the OPs cause hemocytes to undergo a number of changes, such as cellular oxidative stress, actin cytoskeleton modifications, vacuolization, and the inhibition of hemocyte encapsulation capacity, which results in both a loss of hemocyte functionality and cell death. In this study, by using a combined transcriptomic and proteomic analysis, the main components of T. nigriceps ovarian calyx fluid proteins were identifie...

Toxoneuron nigriceps is an endophagous parasitoid of larval stages of the noctuid moth Heliothis virescens. As all parasitoids, this wasp avoid host immune reaction by a combination of several passive and active mechanisms. Secretions injected by ovipositing females, which contain venom, calyx fluid and polydnaviruses, are the most probably factors actively disrupting Heliothis virescens immune system. This paper describes the main alterations of the host immune response observed shortly after oviposition by T. nigriceps. A transient block of prophenoloxidase activity is registered along with changes in hemocyte number, adhesion and structure, which suggest the occurrence of apoptosis. In contrast, the host plasmatocytes appear structurally unaltered, but unable to produce a capsule in vitro.

Heliothis virescens (F.) larvae parasitized by the endophagous braconid Cardiochiles nigriceps Viereck fail to attain the pupal stage. This developmental alteration is caused by both an inactivation of prothoracic glands of last-instar larvae and an altered ecdysone metabolism. Decrease in ecdysteroidogenesis in vitro was already evident in glands explanted from larvae that have attained the early cell formation stage (day 4 of fifth instar), 6 h after parasitoid oviposition. Ecdysteroidogenesis nearly ceased by 24 h after parasitoid oviposition. The degree of this biosynthetic depression increased as the time between parasitization and gland dissection increased. A time-course study allowed us to determine if both the degree of phosphorylation of regulatory target proteins, the rate of general protein synthesis and ecdysteroidogenesis decreased in concert over time. The results provide further evidence in support of the hypothesis that these cellular activities in prothoracic gland cells are functionally correlated in steroidogenic responses. Treatment with calyx fluid and venom of C. nigriceps duplicates the parasitism-induced inactivation of host prothoracic glands. A 6-h conditioning in vitro of pupally committed host prothoracic glands with these parasitoid female reproductive secretions resulted in a significant depression of their ecdysteroid production. However, glands lost their sensitivity to calyx fluid and venom treatment when explanted from hosts that had already attained the cell formation stage. This was further supported by the fact that nearly all the host larvae parasitized on day 4 of fifth instar (cell formation stage) pupated, while parasitization on day 3 resulted in only 11% pupation. The coupled trioxsalen/UV irradiation treatment of C. nigriceps calyx fluid and venom eliminated their negative effect on biosynthetic activity in vitro by host prothoracic glands. This result indirectly demonstrates that C. nigriceps polydnavirus is the major regulating factor involved in the host prothoracic gland inactivation. Arch.

Entomopathogenic nematodes are used as biological control agents against a broad range of insect pests. We ascribed the pathogenicity of these organisms to the excretory/secretory products (ESP) released by the infective nematode. Our group characterized different virulence factors produced by Steinernema carpocapsae that underlie its success as an insect pathogen. A novel ShK-like peptide (ScK1) from this nematode that presents high sequence similarity with the ShK peptide from a sea anemone was successfully produced recombinantly in Escherichia coli. The secondary structure of ScK1 appeared redox-sensitive, exhibiting a far-UV circular dichroism spectrum consistent with an alpha-helical secondary structure. Thermal denaturation of the ScK1 allowed estimating the melting temperature to 59.2 ± 0.1 °C. The results from toxicity assays using Drosophila melanogaster as a model show that injection of this peptide can kill insects in a dose-dependent manner with an LD50 of 16.9 µM per ad...