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Invertebrates can provide a valuable alternative to traditional vertebrate animal models for studying bacterial and fungal infections. This study aimed to establish the larvae of the coleoptera Tenebrio molitor (mealworm) as an in vivo model for evaluating virulence and horizontal gene transfer between Staphylococcus spp. After identifying the best conditions for rearing T. molitor, larvae were infected with different Staphylococcus species, resulting in dose-dependent killing curves. All species tested killed the insects at higher doses, with S. nepalensis and S. aureus being the most and least virulent, respectively. However, only S. nepalensis was able to kill more than 50% of larvae 72 h post-infection at a low amount of 105 CFU. Staphylococcus infection also stimulated an increase in the concentration of hemocytes present in the hemolymph, which was proportional to the virulence. To investigate T. molitor's suitability as an in vivo model for plasmid transfer studies, we used S. aureus strains as donor and recipient of a plasmid containing the gentamicin resistance gene aac(6')-aph(2″). By inoculating larvae with non-lethal doses of each, we observed conjugation, and obtained transconjugant colonies with a frequency of 1.6 × 10-5 per donor cell. This study demonstrates the potential of T. molitor larvae as a reliable and cost-effective model for analyzing the virulence of Staphylococcus and, for the first time, an optimal environment for the plasmid transfer between S. aureus carrying antimicrobial resistance genes.

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Biogenesis and consumption of the yolk are well-conserved aspects of the reproductive biology in oviparous species. Most egg-laying animals accumulate yolk proteins within the oocytes thus creating the source of nutrients and energy that will feed embryo development. Yolk accumulation drives the generation of a highly specialized oocyte cytoplasm with maternal mRNAs, ribosomes, mitochondria, and, mainly, a set of organelles collectively referred to as yolk granules (Ygs). Following fertilization, the Ygs are involved in regulated mechanisms of yolk degradation to fuel the anabolic metabolism of the growing embryo. Thus, yolk accumulation and degradation are essential processes that allow successful development in many species. Nevertheless, the molecular machinery and mechanisms dedicated to the programmed yolk mobilization throughout development are still enigmatic and remain mostly unexplored. Moreover, while the Ygs functional biology as a nutritional source for the embryo has been acknowledged, several reports have suggested that Ygs cargoes and functions go far beyond yolk storage. Evidence of the role of Ygs in gene expression, microbiota harboring, and paracrine signaling has been proposed. In this study, we summarize the current knowledge of the Ygs functional biology pointing to open questions and where further investigation is needed.

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Chagas disease is an important disease affecting millions of patients in the New World and is caused by a protozoan transmitted by haematophagous kissing bugs. It can be treated with drugs during the early acute phase; however, effective therapy against the chronic form of Chagas disease has yet to be discovered and developed. We herein tested the activity of solenopsin alkaloids extracted from two species of fire ants against the protozoan parasite Trypanosoma cruzi, the aetiologic agent of Chagas disease. Although IC50 determinations showed that solenopsins are more toxic to the parasite than benznidazole, the drug of choice for Chagas disease treatment, the ant alkaloids presented a lower selectivity index. As a result of exposure to the alkaloids, the parasites became swollen and rounded in shape, with hypertrophied contractile vacuoles and intense cytoplasmic vacuolization, possibly resulting in osmotic stress; no accumulation of multiple kinetoplasts and/or nuclei was detected. Overexpressing phosphatidylinositol 3-kinase-an enzyme essential for osmoregulation that is a known target of solenopsins in mammalian cells-did not prevent swelling and vacuolization, nor did it counteract the toxic effects of alkaloids on the parasites. Additional experimental results suggested that solenopsins induced a type of autophagic and programmed cell death in T. cruzi. Solenopsins also reduced the intracellular proliferation of T. cruzi amastigotes in infected macrophages in a concentration-dependent manner and demonstrated activity against Trypanosoma brucei rhodesiense bloodstream forms, which is another important aetiological kinetoplastid parasite. The results suggest the potential of solenopsins as novel natural drugs against neglected parasitic diseases caused by kinetoplastids.

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Biofilm formation on exposed surfaces is a serious issue for the food industry and medical health facilities. There are many proposed strategies to delay, reduce, or even eliminate biofilm formation on surfaces. The present study focuses on the applicability of fire ant venom alkaloids (aka 'solenopsins', from Solenopsis invicta) tested on polystyrene and stainless steel surfaces relative to the adhesion and biofilm-formation by the bacterium Pseudomonas fluorescens. Conditioning with solenopsins demonstrates significant reduction of bacterial adhesion. Inhibition rates were 62.7% on polystyrene and 59.0% on stainless steel surfaces. In addition, solenopsins drastically reduced cell populations already growing on conditioned surfaces. Contrary to assumptions by previous authors, solenopsins tested negative for amphipathic properties, thus understanding the mechanisms behind the observed effects still relies on further investigation.

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In Brazil, the use of transgenic plants expressing the insect-toxic Bacillus thuringiensis endotoxin has been successfully used as pest control management since 2013 in transgenic soybean lineages against pest caterpillars such as Helicoverpa armigera. These toxins, endogenously expressed by the plants or sprayed over the crops, are ingested by the insect and bind to receptors in the midgut of these animals, resulting in disruption of digestion and lower insect survival rates. Here, we identified and characterized a membrane-associated alkaline phosphatase (ALP) in the midgut of Anticarsia gemmatalis, the main soybean defoliator pest in Brazil, and data suggested that it binds to Cry1Ac toxin in vitro. Our data showed a peak of ALP activity in homogenate samples of the midgut dissected from the 4th and 5th instars larvae. The brush border membrane vesicles obtained from the midgut of these larvae were used to purify a 60 kDa ALP, as detected by in-gel activity and in vitro biochemical characterization using pharmacological inhibitors and mass spectrometry. When Cry1Ac toxin was supplied to the diet, it was efficient in decreasing larval weight gain and survival. Indeed, in vitro incubation of Cry1Ac toxin with the purified ALP resulted in a 43% decrease in ALP specific activity and enzyme-linked immunosorbent assay showed that ALP interacts with Cry1Ac toxin in vitro, thus suggesting that ALP could function as a Cry toxin ligand. This is a first report characterizing an ALP in A. gemmatalis.

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The termite gut is an efficient decomposer of polyphenol-rich diets, such as lignocellulosic biomasses, and it has been proposed that non-enzymatic oxidative mechanisms could be involved with the digestive process in these animals. However, oxidant levels are completely unknown in termites, as well as protective mechanisms against oxidative damage to the termite gut and its microbiota. As the first step in investigating the role oxidants plays in termite gut physiology, this work presents oxidant levels, antioxidant enzymatic defenses, cell renewal and microbiota abundance along the litter-feeding termite Cornitermes cumulans gut compartments (foregut, midgut, mixed segment and hindgut p1, p3, p4, and p5 segments) and salivary glands. The results show variable levels of oxidants along the C. cumulans gut, the production of antioxidant enzymes, gut cell renewal as potential defenses against oxidative injuries and the profile of microbiota distribution (being predominantly inverse to oxidant levels). In this fashion, the oxidative challenges imposed by polyphenol-rich diet seem to be circumvented by the C. cumulans gut, ensuring efficiency of the digestive process together with preservation of tissue homoeostasis and microbiota growth. These results present new insights into the physicochemical properties of the gut in a litter-feeding termite, expanding our view in relation to termites' digestive physiology.

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Fire ants are widely studied, invasive and venomous arthropod pests. There is significant biomedical interest in immunotherapy against fire ant stings. However, mainly due to practical reasons, the physiological effects of envenomation has remained poorly characterized. The present study takes advantage of a recently-described venom protein extract to delineate the immunological pathways underlying the allergic reaction to fire ant venom toxins. Mice were injected with controlled doses of venom protein extract. Following sensitization and a second exposure, a marked footpad swelling was observed. Based on eosinophil recruitment and production of Th2 cytokines, we hereby establish that fire ant proteins per se can lead to an allergic response, which casts a new light into the mechanism of action of these toxins.

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The genome of the subsocial cockroach Nauphoeta cinerea was partially sequenced in one-twelfth of an Illumina HiSeq lane. The mitochondrial genome was assembled using MIRA software, yielding a circular molecule of 15,923 bp in length and deposited in GenBank under the accession number KY212743. As expected, the mitogenome contained 13 protein-coding genes, 22 transfer tRNAs and 2 ribosomal RNAs. The molecule was assembled using 35,163 sequencing reads of 120 bp each, resulting in ∼286.9× coverage of uniformly distributed reads along the genome. All the 6 complete mitochondrial genomes available for the roaches from the superfamily Blaberoidea were downloaded and compared with the mitogenome of N. cinerea. We also downloaded complete mitochondrial genomes from the superfamily Blattoidea, including 6 mitochondrial genomes of Termitoidae, 2 mitogenomes of Cryptocercoidae and 3 from Blattoidae. A supermatrix dataset presenting the concatenated alignment of all mitochondrial genes was used as input for a maximum likelihood phylogeny. The phylogenomic tree obtained was consistent for most clades, with a relevant exception in the position of the Corydioidea species E. sinensis. Mitochondrial gene information suggests that superfamily Corydioidea should be classified as a clade inside Blattoidea. Nuclear markers and other Corydioidea mitogenomes should be studied to confirm the evolutionary relationships of Blattodea superfamilies.

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ABSTRACT: Cockroaches are insects that can accommodate diets of different composition, including lignocellulosic materials. Digestion of these compounds is achieved by the insect's own enzymes and also by enzymes produced by gut symbionts. The presence of different and modular bacterial phyla on the cockroach gut tract suggests that this insect could be an interesting model to study the organization of gut bacterial communities associated with the digestion of different lignocellulosic diets. Thus, changes in the diversity of gut associated bacterial communities of insects exposed to such diets could give useful insights on how to improve hemicellulose and cellulose breakdown systems. In this work, through sequence analysis of 16S rRNA clone libraries, we compared the phylogenetic diversity and composition of gut associated bacteria in the cockroach Periplaneta americana collected in the wild-types or kept on two different diets: sugarcane bagasse and crystalline cellulose. These high fiber diets favor the predominance of some bacterial phyla, such as Firmicutes, when compared to wild-types cockroaches. Our data show a high bacterial diversity in P. americana gut, with communities composed mostly by the phyla Bacteroidetes, Firmicutes, Proteobacteria and Synergistetes. Our data show that the composition and diversity of gut bacterial communities could be modulated by diet composition. The increased presence of Firmicutes in sugarcane bagasse and crystalline cellulose-fed animals suggests that these bacteria are strongly involved in lignocellulose digestion in cockroach guts. BACKGROUND: Cockroaches are omnivorous animals that can incorporate in their diets food of different composition, including lignocellulosic materials. Digestion of these compounds is achieved by the insect's own enzymes and also by enzymes produced by gut symbiont. However, the influence of diet with different fiber contents on gut bacterial communities and how this affects the digestion of cockroaches is still unclear. The presence of some bacterial phyla on gut tract suggests that cockroaches could be an interesting model to study the organization of gut bacterial communities during digestion of different lignocellulosic diets. Knowledge about the changes in diversity of gut associated bacterial communities of insects exposed to such diets could give interesting insights on how to improve hemicellulose and cellulose breakdown systems. METHODOLOGY/PRINCIPAL FINDINGS: We compared the phylogenetic diversity and composition of gut associated bacteria in the cockroach P. americana caught on the wild or kept on two different diets: sugarcane bagasse and crystalline cellulose. For this purpose we constructed bacterial 16S rRNA gene libraries which showed that a diet rich in cellulose and sugarcane bagasse favors the predominance of some bacterial phyla, more remarkably Firmicutes, when compared to wild cockroaches. Rarefaction analysis, LIBSHUFF and UniFrac PCA comparisons showed that gene libraries of wild insects were the most diverse, followed by sugarcane bagasse fed and then cellulose fed animals. It is also noteworthy that cellulose and sugarcane bagasse gene libraries resemble each other. CONCLUSION/SIGNIFICANCE: Our data show a high bacterial diversity in P. americana gut, with communities composed mostly by the phyla Bacteroidetes, Firmicutes, Proteobacteria and Synergistetes. The composition and diversity of gut bacterial communities could be modulated by font of diet composition. The increased presence of Firmicutes in sugarcane bagasse and crystalline cellulose-fed animals suggests that these bacteria are strongly involved in lignocellulose digestion in cockroach guts.

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Lipophorin (Lp) is the main haemolymphatic lipoprotein in insects and transports lipids between different organs. In adult females, lipophorin delivers lipids to growing oocytes. In this study, the interaction of this lipoprotein with the ovaries of Rhodnius prolixus was characterised using an oocyte membrane preparation and purified radiolabelled Lp (125I-Lp). Lp-specific binding to the oocyte membrane reached equilibrium after 40-60 min and when 125I-Lp was incubated with increasing amounts of membrane protein, corresponding increases in Lp binding were observed. The specific binding of Lp to the membrane preparation was a saturable process, with a Kdof 7.1 ± 0.9 x 10-8M and a maximal binding capacity of 430 ± 40 ng 125I-Lp/µg of membrane protein. The binding was calcium independent and pH sensitive, reaching its maximum at pH 5.2-5.7. Suramin inhibited the binding interaction between Lp and the oocyte membranes, which was completely abolished at 0.5 mM suramin. The oocyte membrane preparation from R. prolixus also showed binding to Lp from Manduca sexta. When Lp was fluorescently labelled and injected into vitellogenic females, the level of Lp-oocyte binding was much higher in females that were fed whole blood than in those fed blood plasma.

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