Bacterial community composition shifts in the gut of Periplaneta americana fed on different lignocellulosic materials.

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.

Microbial community diversity in the gut of the South American termite Cornitermes cumulans (Isoptera: Termitidae).

Termites inhabit tropical and subtropical areas where they contribute to structure and composition of soils by efficiently degrading biomass with aid of resident gut microbiota. In this study, culture-independent molecular analysis was performed based on bacterial and archaeal 16S rRNA clone libraries to describe the gut microbial communities within Cornitermes cumulans, a South American litter-feeding termite. Our data reveal extensive bacterial diversity, mainly composed of organisms from the phyla Spirochaetes, Bacteroidetes, Firmicutes, Actinobacteria, and Fibrobacteres. In contrast, a low diversity of archaeal 16S rRNA sequences was found, comprising mainly members of the Crenarchaeota phylum. The diversity of archaeal methanogens was further analyzed by sequencing clones from a library for the mcrA gene, which encodes the enzyme methyl coenzyme reductase, responsible for catalyzing the last step in methane production, methane being an important greenhouse gas. The mcrA sequences were diverse and divided phylogenetically into three clades related to uncultured environmental archaea and methanogens found in different termite species. C. cumulans is a litter-feeding, mound-building termite considered a keystone species in natural ecosystems and also a pest in agriculture. Here, we describe the archaeal and bacterial communities within this termite, revealing for the first time its intriguing microbiota.

Transcriptome and gene expression profile of ovarian follicle tissue of the triatomine bug Rhodnius prolixus.

Insect oocytes grow in close association with the ovarian follicular epithelium (OFE), which escorts the oocyte during oogenesis and is responsible for synthesis and secretion of the eggshell. We describe a transcriptome of OFE of the triatomine bug Rhodnius prolixus, a vector of Chagas disease, to increase our knowledge of the role of FE in egg development. Random clones were sequenced from a cDNA library of different stages of follicle development. The transcriptome showed high commitment to transcription, protein synthesis, and secretion. The most abundant cDNA was a secreted (S) small, proline-rich protein with maximal expression in the vitellogenic follicle, suggesting a role in oocyte maturation. We also found Rp45, a chorion protein already described, and a putative chitin-associated cuticle protein that was an eggshell component candidate. Six transcripts coding for proteins related to the unfolded-protein response (UPR) by were chosen and their expression analyzed. Surprisingly, transcripts related to UPR showed higher expression during early stages of development and downregulation during late stages, when transcripts coding for S proteins participating in chorion formation were highly expressed. Several transcripts with potential roles in oogenesis and embryo development are also discussed. We propose that intense protein synthesis at the FE results in reticulum stress (RS) and that lowering expression of a set of genes related to cell survival should lead to degeneration of follicular cells at oocyte maturation. This paradoxical suppression of UPR suggests that ovarian follicles may represent an interesting model for studying control of RS and cell survival in professional S cell types.

Microscopic and molecular characterization of ovarian follicle atresia in Rhodnius prolixus Stahl under immune challenge.

In this work we characterized the degenerative process of ovarian follicles of the bug Rhodnius prolixus challenged with the non-entomopathogenic fungus Aspergillus niger. An injection of A. niger conidia directly into the hemocoel of adult R. prolixus females at the onset of vitellogenesis caused no effect on host lifespan but elicited a net reduction in egg batch size. Direct inspection of ovaries from the mycosed insects revealed that fungal challenge led to atresia of the vitellogenic follicles. Light microscopy and DAPI staining showed follicle shrinkage, ooplasm alteration and disorganization of the monolayer of follicle cells in the atretic follicles. Transmission electron microscopy of thin sections of follicle epithelium also showed nuclei with condensed chromatin, electron dense mitochondria and large autophagic vacuoles. Occurrence of apoptosis of follicle cells in these follicles was visualized by TUNEL labeling. Resorption of the yolk involved an increase in protease activities (aspartyl and cysteinyl proteases) which were associated with precocious acidification of yolk granules and degradation of yolk protein content. The role of follicle atresia in nonspecific host-pathogen associations and the origin of protease activity that led to yolk resorption are discussed.

Prostaglandin signaling and ovarian follicle development in the silkmoth, Bombyx mori.

Previous work on in vitro culturing of silkmoth (Bombyx mori) ovarian follicles has shown that starting from middle vitellogenesis, follicles develop according to an endogenous developmental program that does not require the presence of extra-ovarian factors. In this paper, we are reporting on our investigation for a possible involvement of autocrine/paracrine signaling by prostaglandins in the control of silkmoth ovarian follicle development. Using an initial rapid test that evaluates the formation of a protective eggshell around the oocyte, we are showing that aspirin and indomethacin, potent inhibitors of prostaglandin biosynthesis, block the transition of cultured vitellogenic follicles into choriogenesis. More detailed studies involving analyses of temporal expression patterns of genes known to be expressed in follicular epithelium cells at specific stages of ovarian development revealed that inhibition of prostaglandin biosynthesis arrests stages of follicle development from middle vitellogenesis to late choriogenesis. The arrest could be reversed by the addition of exogenous prostaglandins or cAMP into the culture media leading to the conclusion that the production of prostaglandins triggers cAMP-mediated intracellular signaling that allows the developmental progression of the follicles. Finally, because neither prostaglandins nor cAMP is capable of rescuing a developmental block effected at mid-vitellogenesis by the ecdysone agonist tebufenozide, we are proposing that prostaglandins have a role in the maintenance of normal physiological homeostasis in the ovarian follicles rather than a more specific role in developmental decision-making at distinct stages of follicle development.

Oogenesis and egg development in triatomines: a biochemical approach

Em triatomíneos, assim como em outros insetos, o acúmulo de vitelo é um processo no qual um tecido extraovariano, o corpo gorduroso, produz proteínas que são empacotadas no interior de um ovo. A principal proteína, sintetizada pelo corpo gorduroso, que é acumulada no interior de um ovócito, é a vitelogenina. Este processo é também conhecido por vitelogênese. Existem crescentes evidências em triatomíneos, que além do corpo gorduroso, o ovário também produz proteínas de vitelo. A forma como estas proteínas de vitelo entram nos ovócitos será aqui comentada. O vitelo é um material complexo composto por proteínas, lipídeos, carboidratos e outros compostos minoritários que são empacotados de uma maneira organizada no interior dos ovócitos. A fertilização dispara a embriogênese, um processo que culmina com o desenvolvimento do embrião. Durante a embriogênese o vitelo será utilizado para a construção de um novo indivíduo, a ninfa de primeiro estádio. O desafio para a próxima década é entender onde e como estas proteínas de vitelo são utilizadas junto com os seus componentes não protéicos, em compasso com o programa genético do embrião, que comanda a diferenciação celular (fase inicial da embriogênese) e diferenciação do embrião (fase final da embriogênese) no interior do ovo.

Oogenesis and egg development in triatomines: a biochemical approach.

In triatomines, as well as in other insects, accumulation of yolk is a process in which an extra-ovarian tissue, the fat body, produces yolk proteins that are packed in the egg. The main protein, synthesized by the fat body, which is accumulated inside the oocyte, is vitellogenin. This process is also known as vitellogenesis. There are growing evidences in triatomines that besides fat body the ovary also produces yolk proteins. The way these yolk proteins enter the oocyte will be discussed. Yolk is a complex material composed of proteins, lipids, carbohydrates and other minor components which are packed inside the oocyte in an organized manner. Fertilization triggers embryogenesis, a process where an embryo will develop. During embryogenesis the yolk will be used for the construction of a new individual, the first instar nymph. The challenge for the next decade is to understand how and where these egg proteins are used up together with their non-protein components, in pace with the genetic program of the embryo, which enables cell differentiation (early phase of embryogenesis) and embryo differentiation (late phase) inside the egg.

The role of eicosanoids on Rhodnius heme-binding protein (RHBP) endocytosis by Rhodnius prolixus ovaries.

The participation of eicosanoids and second messengers on the regulation of RHBP endocytosis by the ovaries was investigated, using [(125)I]RHBP in experiments in vivo and in vitro. Addition of PGE(2) (one of the products of the cyclooxygenase pathway) decreased in vitro the uptake of RHBP by 35%. The rate of RHBP endocytosis increased in the presence of indomethacin, a potent cyclooxigenase inhibitor, up to 50% in vitro and up to 55% in vivo, thus giving support to the role of cyclooxygenase derivatives on endocytosis regulation. The amount of PGE(2) secreted to the culture medium by the cells of Rhodnius prolixus ovaries was 1.1 ng/ovary following RHBP uptake assay. The amount of PGE(2) decreases approximately 25% in the presence of 5 microM indomethacin. Using a scanning electron microscope we have observed that neither the surface area nor the patencies of follicle cells were affected by treatment with indomethacin, thus suggesting that, its effect is elicited in the oocyte. Finally, we have identified two ovarian peptides that were dephosphorylated after the indomethacin treatment (18 and 25 kDa). Taken together these data show that local mediators such as eicosanoids act upon the oocytes controlling RHBP endocytosis, perhaps using the protein phosphorylation signal transduction pathway.