Structure and expression of Rhodnius prolixus GH18 chitinases and chitinase-like proteins: Characterization of the physiological role of RpCht7, a gene from subgroup VIII, in vector fitness and reproduction.

Chitinases are enzymes responsible for the hydrolysis of glycosidic linkages within chitin chains. In insects, chitinases are typically members of the multigenic glycoside hydrolase family 18 (GH18). They participate in the relocation of chitin during development and molt, and in digestion in detritivores and predatory insects, and they control the peritrophic membrane thickness. Chitin metabolism is a promising target for developing vector control strategies, and knowledge of the roles of chitinases may reveal new targets and illuminate unique aspects of their physiology and interaction with microorganisms. Rhodnius prolixus is an important vector of Chagas disease, which is caused by the parasite Trypanosoma cruzi. In this study, we performed annotation and structural characterization of nine chitinase and chitinase-like protein genes in the R. prolixus genome. The roles of their corresponding transcripts were studied in more depth; their physiological roles were studied through RNAi silencing. Phylogenetic analysis of coding sequences showed that these genes belong to different subfamilies of GH18 chitinases already described in other insects. The expression patterns of these genes in different tissues and developmental stages were initially characterized using RT-PCR. RNAi screening showed silencing of the gene family members with very different efficiencies. Based on the knockdown results and the general lack of information about subgroup VIII of GH18, the RpCht7 gene was chosen for phenotype analysis. RpCht7 knockdown doubled the mortality in starving fifth-instar nymphs compared to dsGFP-injected controls. However, it did not alter blood intake, diuresis, digestion, molting rate, molting defects, sexual ratio, percentage of hatching, or average hatching time. Nevertheless, female oviposition was reduced by 53% in RpCht7-silenced insects, and differences in oviposition occurred within 14-20 days after a saturating blood meal. These results suggest that RpCht7 may be involved in the reproductive physiology and vector fitness of R. prolixus.

Ecdysone modulates both ultrastructural arrangement of hindgut and attachment of Trypanosoma cruzi DM 28c to the rectum cuticle of Rhodnius prolixus fifth-instar nymph.

Studies on the effects of azadirachtin treatment, ecdysone supplementation and ecdysone therapy on both the ultrastructural organization of the rectum in 5th-instar nymph of Rhodnius prolixus and the ex vivo attachment behavior of Trypanosoma cruzi under these experimental conditions were carried out. Control insects had a typical and significant organization of the rectum cuticle consisted of four main layers (procuticle, inner epicuticle, outer epicuticle, and wax layer) during the entire period of the experiment. Both azadirachtin treatment and ecdysone supplementation avoid the development of both outer epicuticle and wax layer. Oral therapy with ecdysone partially reversed the altered organization and induce the development of the four main rectal cuticle layers. In the same way, the ex vivo attachment of T. cruzi to rectal cuticle was blocked by azadirachtin treatment but ecdysone therapy also partially recovered the parasite adhesion rates to almost those detected in control insects. These results point out that ecdysone may be a factor responsible - directly or indirectly - by the modulation of rectum ultrastructural arrangement providing a superficial wax layer to the attachment followed by metacyclogenesis of T. cruzi in the rectum of its invertebrate hosts.

Determination of Chitin Content in Insects: An Alternate Method Based on Calcofluor Staining.

Chitin is an aminopolysaccharide present in yeast cells and arthropod cuticle and is one of the most abundant biopolymers. The conventional methods for the quantitation of chitin content in biological samples are based on its hydrolysis (acid or enzymatic), and the assessment of the byproduct, glucosamine. However, previously described methodologies are time-consuming, laborious, low throughput, and not applicable to insect samples in many cases. Here we describe a new approach to chitin content quantitation based on calcofluor fluorescent brightener staining of samples, followed by microplate fluorescence readings. Calcofluor is a specific chitin stain commonly used for topological localization of the polymer. The protocol was tested in three important disease vector species, namely Lutzomyia longipalpis, Aedes aegypti, and Rhodnius prolixus, and then compared to a classic colorimetric chitin assessment method. Results show that chitin content in the tested insects can vary largely in a range of 8-4600 micrograms of chitin per insect, depending on species, sex, and instar. Comparisons between measurements from the previous protocol and calcofluor method showed statistically significant differences in some samples. However, the difference might be due to interference in the classic method from non-chitin sources of glucosamine and reducing agents. Furthermore, chitinase hydrolysis reduces the total chitin mass estimated between 36 and 74%, consolidating the fluorescent measurements as actual stained chitin in the same extent that was observed with the standard protocol. Therefore, the calcofluor staining method revealed to be a fast and reliable technique for chitin quantitation in homogenized insect samples.

Characterization of the Temporal Pattern of Blood Protein Digestion in Rhodnius prolixus: First Description of Early and Late Gut Cathepsins.

Rhodnius prolixus is one important vector for the parasite Trypanosoma cruzi in Latin America, where Chagas disease is a significant health issue. Although R. prolixus is a model for investigations of vector-parasite interaction and transmission, not much has been done recently to further comprehend its protein digestion. In this work, gut proteolysis was characterized using new fluorogenic substrates, including optimum pH, inhibition profiles, and tissue and temporal expression patterns. Each protease possessed a particular tissue prevalence and activity cycle after feeding. Cathepsin L had a higher activity in the posterior midgut lumen, being characterized by a plateau of high activities during several days in the intermediate phase of digestion. Cathepsin D showed high activity levels in the tissue homogenates and in the luminal content of the posterior midgut, with a single peak 5 days after blood feeding. Aminopeptidases are highly associated with the midgut wall, where the highest activity is located. Assays with proteinaceous substrates as casein, hemoglobin, and serum albumin revealed different activity profiles, with some evidence of biphasic temporal proteolytic patterns. Cathepsin D genes are preferentially expressed in the anterior midgut, while cathepsin L genes are mainly located in the posterior portion of the midgut, with specific sets of genes being differently expressed in the initial, intermediate, or late phases of blood digestion. Significance Statement This is the first description in a non-dipteran hematophagous species of a sequential protease secretion system based on midgut cathepsins instead of the most common insect digestive serine proteases (trypsins and chymotrypsins). The midgut of R. prolixus (Hemiptera) shows a different temporal expression of proteases in the initial, intermediate, and late stages of blood digestion. In this respect, a different timing in protease secretion may be an example of adaptative convergence in blood-sucking vectors from different orders. Expanding the knowledge about gut physiology in triatomine vectors may contribute to the development of new control strategies, aiming the blocking of parasite transmission.

Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels.

Glycoside Hydrolases (GHs) are enzymes able to recognize and cleave glycosidic bonds. Insect GHs play decisive roles in digestion, in plant-herbivore, and host-pathogen interactions. GH activity is normally measured by the detection of a release from the substrate of products as sugars units, colored, or fluorescent groups. In most cases, the conditions for product release and detection differ, resulting in discontinuous assays. The current protocols result in using large amounts of reaction mixtures for the obtainment of time points in each experimental replica. These procedures restrain the analysis of biological materials with limited amounts of protein and, in the case of studies regarding small insects, implies in the pooling of samples from several individuals. In this respect, most studies do not assess the variability of GH activities across the population of individuals from the same species. The aim of this work is to approach this technical problem and have a deeper understanding of the variation of GH activities in insect populations, using as models the disease vectors Rhodnius prolixus (Hemiptera: Triatominae) and Lutzomyia longipalpis (Diptera: Phlebotominae). Here we standardized continuous assays using 4-methylumbelliferyl derived substrates for the detection of α-Glucosidase, ß-Glucosidase, α-Mannosidase, N-acetyl-hexosaminidase, ß-Galactosidase, and α-Fucosidase in the midgut of R. prolixus and L. longipalpis with results similar to the traditional discontinuous protocol. The continuous assays allowed us to measure GH activities using minimal sample amounts with a higher number of measurements, resulting in data that are more reliable and less time and reagent consumption. The continuous assay also allows the high-throughput screening of GH activities in small insect samples, which would be not applicable to the previous discontinuous protocol. We applied continuous GH measurements to 90 individual samples of R. prolixus anterior midgut homogenates using a high-throughput protocol. α-Glucosidase and α-Mannosidase activities showed the normal distribution in the population. ß-Glucosidase, ß-Galactosidase, N-acetyl-hexosaminidase, and α-Fucosidase activities showed non-normal distributions. These results indicate that GHs fluorescent-based high-throughput assays apply to insect samples and that the frequency distribution of digestive activities should be considered in data analysis, especially if a small number of samples is used.

Lethal and sublethal effects of essential oil of Lippia sidoides (Verbenaceae) and monoterpenes on Chagas´ disease vector Rhodnius prolixus

The aim of this study was to identify the composition of the essential oil from leaves of Lippia sidoides (EOLS), a typical shrub commonly found in the dry northeast of Brazil, popularly known as “alecrim-pimenta”. Additionally, we investigated the nymphicidal, ovicidal, phagoinhibitory and excretion effects of EOLS, its major constituent thymol and its isomer carvacrol, on fourth instar nymphs and eggs of Rhodnius prolixus, the Chagas’ disease vector. The nymphicidal and ovicidal activity of thymol, carvacrol, and EOLS was assessed by tests using impregnated Petri dishes. The lethal concentration values (LC50) for EOLS, carvacrol, and thymol were 54.48, 32.98, and 9.38 mg/cm2, respectively. The ovicidal test showed that both carvacrol and thymol (50 mg/cm2) inhibited hatching (50% and 23.3%, respectively), while treatments with 10 mg/cm2 or 50 mg/cm2 EOLS did not affect the hatching rate at all (80% and 90%, respectively). We observed an anti-feeding effect in insects fed with blood containing natural products at the higher concentrations (100 µg/mL). Finally, excretion rate was affected by EOLS and carvacrol, but not by thymol. These findings offer novel insights into basic physiological processes that make the tested natural compounds interesting candidates for new types of insecticides.

Lethal and sublethal effects of essential oil of Lippia sidoides (Verbenaceae) and monoterpenes on Chagas' disease vector Rhodnius prolixus.

The aim of this study was to identify the composition of the essential oil from leaves of Lippia sidoides (EOLS), a typical shrub commonly found in the dry northeast of Brazil, popularly known as "alecrim-pimenta". Additionally, we investigated the nymphicidal, ovicidal, phagoinhibitory and excretion effects of EOLS, its major constituent thymol and its isomer carvacrol, on fourth instar nymphs and eggs of Rhodnius prolixus, the Chagas' disease vector. The nymphicidal and ovicidal activity of thymol, carvacrol, and EOLS was assessed by tests using impregnated Petri dishes. The lethal concentration values (LC50) for EOLS, carvacrol, and thymol were 54.48, 32.98, and 9.38 mg/cm2, respectively. The ovicidal test showed that both carvacrol and thymol (50 mg/cm2) inhibited hatching (50% and 23.3%, respectively), while treatments with 10 mg/cm2 or 50 mg/cm2 EOLS did not affect the hatching rate at all (80% and 90%, respectively). We observed an anti-feeding effect in insects fed with blood containing natural products at the higher concentrations (100 µg/mL). Finally, excretion rate was affected by EOLS and carvacrol, but not by thymol. These findings offer novel insights into basic physiological processes that make the tested natural compounds interesting candidates for new types of insecticides.

Genome Wide Mapping of Peptidases in Rhodnius prolixus: Identification of Protease Gene Duplications, Horizontally Transferred Proteases and Analysis of Peptidase A1 Structures, with Considerations on Their Role in the Evolution of Hematophagy in Triatominae.

Triatominae is a subfamily of the order Hemiptera whose species are able to feed in the vertebrate blood (i.e., hematophagy). This feeding behavior presents a great physiological challenge to insects, especially in Hemipteran species with a digestion performed by lysosomal-like cathepsins instead of the more common trypsin-like enzymes. With the aim of having a deeper understanding of protease involvement in the evolutionary adaptation for hematophagy in Hemipterans, we screened peptidases in the Rhodnius prolixus genome and characterized them using common blast (NCBI) and conserved domain analyses (HMMER/blast manager software, FAT, plus PFAM database). We compared the results with available sequences from other hemipteran species and with 18 arthropod genomes present in the MEROPS database. Rhodnius prolixus contains at least 433 protease coding genes, belonging to 71 protease families. Seven peptidase families in R. prolixus presented higher gene numbers when compared to other arthropod genomes. Further analysis indicated that a gene expansion of the protease family A1 (Eukaryotic aspartyl protease, PF00026) might have played a major role in the adaptation to hematophagy since most of these peptidase genes seem to be recently acquired, are expressed in the gut and present putative secretory pathway signal peptides. Besides that, most R. prolixus A1 peptidases showed high frequencies of basic residues at the protein surface, a typical structural signature of Cathepsin D-like proteins. Other peptidase families expanded in R. prolixus (i.e., C2 and M17) also presented significant differences between hematophagous (higher number of peptidases) and non-hematophagous species. This study also provides evidence for gene acquisition from microorganisms in some peptidase families in R. prolixus: (1) family M74 (murein endopeptidase), (2) family S29 (Hepatitis C virus NS3 protease), and (3) family S24 (repressor LexA). This study revealed new targets for studying the adaptation of these insects for digestion of blood meals and their competence as vectors of Chagas disease.

Everybody loves sugar: first report of plant feeding in triatomines.

BACKGROUND: Triatomines, which are the vectors of Trypanosoma cruzi, have been considered to be exclusive blood feeders for more than 100 years, since the discovery of Chagas disease. METHODS: We offered artificial sugar meals to the laboratory model-insect Rhodnius prolixus, which is considered a strict haematophagous insect. We registered feeding by adding colorant to sugar meals. To assess putative phytophagy, fruits of the tomato Solanum lycopersicum were offered to R. prolixus and the presence of tomato DNA was assessed in the insects using PCR. We also assessed longevity, blood feeding and urine production of fruit-exposed triatomines and control insects. RESULTS: All instars of R. prolixus ingested sugar from artificial sugar meals in laboratory conditions. First instar R. prolixus ingested plant tissue from S. lycopersicum fruits, and this increased the amount of blood ingested and urine excreted. Decreased mortality was also observed after blood feeding. Exposure to S. lycopersicum increased longevity and reduced weight loss caused by desiccation. CONCLUSIONS: We describe here the first report of sugar feeding and phytophagy in a species that was considered to be a strict blood-feeder for over a century. We suggest that local plants might be not merely shelters for insects and vertebrate hosts as previously described, but may have a nutritional role for the maintenance of the triatomine vectors. The description of sugar and plant meals in triatomines opens new perspectives for the study and control of Chagas Disease.

Genome of Rhodnius prolixus, an insect vector of Chagas disease, reveals unique adaptations to hematophagy and parasite infection.

Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (∼ 702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods.

Isolation of aerobic cultivable cellulolytic bacteria from different regions of the gastrointestinal tract of giant land snail Achatina fulica.

The enzymatic hydrolysis of cellulose by cellulases is one of the major limiting steps in the conversion of lignocellulosic biomass to yield bioethanol. To overcome this hindrance, significant efforts are underway to identify novel cellulases. The snail Achatina fulica is a gastropod with high cellulolytic activity, mainly due to the abundance of glycoside hydrolases produced by both the animal and its resident microbiota. In this study, we partially assessed the cellulolytic aerobic bacterial diversity inside the gastrointestinal tract of A. fulica by culture-dependent methods and evaluated the hydrolytic repertoire of the isolates. Forty bacterial isolates were recovered from distinct segments of the snail gut and identified to the genus level by 16S rRNA gene sequence analysis. Additional phenotypic characterization was performed using biochemical tests provided by the Vitek2 identification system. The overall enzymatic repertoire of the isolated strains was investigated by enzymatic plate assays, containing the following substrates: powdered sugarcane bagasse, carboxymethylcellulose (CMC), p-nitrophenyl-ß-D-glucopyranoside (pNPG), p-nitrophenyl-ß-D-cellobioside (pNPC), 4-methylumbelliferyl-ß-D-glucopyranoside (MUG), 4-methylumbelliferyl-ß-D-cellobioside (MUC), and 4-methylumbelliferyl-ß-D-xylopyranoside (MUX). Our results indicate that the snail A. fulica is an attractive source of cultivable bacteria that showed to be valuable resources for the production of different types of biomass-degrading enzymes.

Rhodnius prolixus interaction with Trypanosoma rangeli: modulation of the immune system and microbiota population.

BACKGROUND: Trypanosoma rangeli is a protozoan that infects a variety of mammalian hosts, including humans. Its main insect vector is Rhodnius prolixus and is found in several Latin American countries. The R. prolixus vector competence depends on the T. rangeli strain and the molecular interactions, as well as the insect's immune responses in the gut and haemocoel. This work focuses on the modulation of the humoral immune responses of the midgut of R. prolixus infected with T. rangeli Macias strain, considering the influence of the parasite on the intestinal microbiota. METHODS: The population density of T. rangeli Macias strain was analysed in different R. prolixus midgut compartments in long and short-term experiments. Cultivable and non-cultivable midgut bacteria were investigated by colony forming unit (CFU) assays and by 454 pyrosequencing of the 16S rRNA gene, respectively. The modulation of R. prolixus immune responses was studied by analysis of the antimicrobial activity in vitro against different bacteria using turbidimetric tests, the abundance of mRNAs encoding antimicrobial peptides (AMPs) defensin (DefA, DefB, DefC), prolixicin (Prol) and lysozymes (LysA, LysB) by RT-PCR and analysis of the phenoloxidase (PO) activity. RESULTS: Our results showed that T. rangeli successfully colonized R. prolixus midgut altering the microbiota population and the immune responses as follows: 1 - reduced cultivable midgut bacteria; 2 - decreased the number of sequences of the Enterococcaceae but increased those of the Burkholderiaceae family; the families Nocardiaceae, Enterobacteriaceae and Mycobacteriaceae encountered in control and infected insects remained the same; 3 - enhanced midgut antibacterial activities against Serratia marcescens and Staphylococcus aureus; 4 - down-regulated LysB and Prol mRNA levels; altered DefB, DefC and LysA depending on the infection (short and long-term); 5 - decreased PO activity. CONCLUSION: Our findings suggest that T. rangeli Macias strain modulates R. prolixus immune system and modifies the natural microbiota composition.

Characterization of the microbiota in the guts of Triatoma brasiliensis and Triatoma pseudomaculata infected by Trypanosoma cruzi in natural conditions using culture independent methods.

BACKGROUND: Chagas disease is caused by Trypanosoma cruzi, which is transmitted by triatomine vectors. The northeastern region of Brazil is endemic for Chagas disease and has the largest diversity of triatomine species. T. cruzi development in its triatomine vector depends on diverse factors, including the composition of bacterial gut microbiota. METHODS: We characterized the triatomines captured in the municipality of Russas (Ceará) by sequencing the cytochrome c oxidase subunit I (COI) gene. The composition of the bacterial community in the gut of peridomestic Triatoma brasiliensis and Triatoma pseudomaculata was investigated using culture independent methods based on the amplification of the 16S rRNA gene by polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), DNA fragment cloning, Sanger sequencing and 454 pyrosequencing. Additionally, we identified TcI and TcII types of T. cruzi by sequencing amplicons from the gut metagenomic DNA with primers for the mini-exon gene. RESULTS: Triatomines collected in the peridomestic ecotopes were diagnosed as T. pseudomaculata and T. brasiliensis by comparing their COI sequence with GenBank. The rate of infection by T. cruzi in adult triatomines reached 80% for T. pseudomaculata and 90% for T. brasiliensis. According to the DNA sequences from the DGGE bands, the triatomine gut microbiota was primarily composed of Proteobacteria and Actinobacteria. However, Firmicutes and Bacteroidetes were also detected, although in much lower proportions. Serratia was the main genus, as it was encountered in all samples analyzed by DGGE and 454 pyrosequencing. Members of Corynebacterinae, a suborder of the Actinomycetales, formed the next most important group. The cloning and sequencing of full-length 16S rRNA genes confirmed the presence of Serratia marcescens, Dietzia sp., Gordonia terrae, Corynebacterium stationis and Corynebacterium glutamicum. CONCLUSIONS: The study of the bacterial microbiota in the triatomine gut has gained increased attention because of the possible role it may play in the epidemiology of Chagas disease by competing with T. cruzi. Culture independent methods have shown that the bacterial composition of the microbiota in the guts of peridomestic triatomines is made up by only few bacterial species.

Detection and preliminary physico-chemical properties of antimicrobial components in the native excretions/secretions of three species of Chrysomya (Diptera, Calliphoridae) in Brazil.

Antibiotic-resistant bacteria in hospitals and communities increasingly threaten public health in Brazil and the rest of the World. There is an urgent need for additional antimicrobial drugs. Calliphorid blowfly larvae are a rich source of antimicrobial factors but the potential of Neotropical species has been neglected. This preliminary study evaluates the antimicrobial activity of the native excretions/secretions of larvae of three species of Brazilian calliphorids, Chrysomya megacephala, Chrysomya albiceps and Chrysomya putoria. Native excretions/secretions were collected from third instar larvae, sterile filtered and tested for antibacterial activity against Staphylococcus aureus 9518, Escherichia coli K12 4401 and Serratia marcescens 365. Turbidometric assays were made in micro-plates, using an ELISA reader, with readings taken up to 22 h. Bacterial suspensions at the start and end of each experiment were also serially diluted, spread on nutrient agar plates and then colony forming units counted. The physico-chemical characteristics of the native excretions/secretions were also tested by freezing/thawing, boiling, and protease digestion. The native excretions/secretions of larvae from these three Chrysomya species significantly inhibited bacterial growth. Therefore, Brazilian calliphorid flies could potentially provide new classes of antibiotics.

Humoral responses in Rhodnius prolixus: bacterial feeding induces differential patterns of antibacterial activity and enhances mRNA levels of antimicrobial peptides in the midgut.

BACKGROUND: The triatomine, Rhodnius prolixus, is a major vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. It has a strictly blood-sucking habit in all life stages, ingesting large amounts of blood from vertebrate hosts from which it can acquire pathogenic microorganisms. In this context, the production of antimicrobial peptides (AMPs) in the midgut of the insect is vital to control possible infection, and to maintain the microbiota already present in the digestive tract. METHODS: In the present work, we studied the antimicrobial activity of the Rhodnius prolixus midgut in vitro against the Gram-negative and Gram-positive bacteria Escherichia coli and Staphylococcus aureus, respectively. We also analysed the abundance of mRNAs encoding for defensins, prolixicin and lysozymes in the midgut of insects orally infected by these bacteria at 1 and 7 days after feeding. RESULTS: Our results showed that the anterior midgut contents contain a higher inducible antibacterial activity than those of the posterior midgut. We observed that the main AMP encoding mRNAs in the anterior midgut, 7 days after a blood meal, were for lysozyme A, B, defensin C and prolixicin while in the posterior midgut lysozyme B and prolixicin transcripts predominated. CONCLUSION: Our findings suggest that R. prolixus modulates AMP gene expression upon ingestion of bacteria with patterns that are distinct and dependent upon the species of bacteria responsible for infection.

An insight into the transcriptome of the digestive tract of the bloodsucking bug, Rhodnius prolixus.

The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.

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.

Trypanosoma cruzi TcSMUG L-surface mucins promote development and infectivity in the triatomine vector Rhodnius prolixus.

BACKGROUND: TcSMUG L products were recently identified as novel mucin-type glycoconjugates restricted to the surface of insect-dwelling epimastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. The remarkable conservation of their predicted mature N-terminal region, which is exposed to the extracellular milieu, suggests that TcSMUG L products may be involved in structural and/or functional aspects of the interaction with the insect vector. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we investigated the putative roles of TcSMUG L mucins in both in vivo development and ex vivo attachment of epimastigotes to the luminal surface of the digestive tract of Rhodnius prolixus. Our results indicate that the exogenous addition of TcSMUG L N-terminal peptide, but not control T. cruzi mucin peptides, to the infected bloodmeal inhibited the development of parasites in R. prolixus in a dose-dependent manner. Pre-incubation of insect midguts with the TcSMUG L peptide impaired the ex vivo attachment of epimastigotes to the luminal surface epithelium, likely by competing out TcSMUG L binding sites on the luminal surface of the posterior midgut, as revealed by fluorescence microscopy. CONCLUSION AND SIGNIFICANCE: Together, these observations indicate that TcSMUG L mucins are a determinant of both adhesion of T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine, and the infection of the insect vector, R. prolixus.

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.

Miniaturization of hydrolase assays in thermocyclers.

We adapted the protocols of reducing sugar measurements with dinitrosalicylic acid and bicinchoninic acid for thermocyclers and their use in enzymatic assays for hydrolases such as amylase and ß-1,3-glucanase. The use of thermocyclers for these enzymatic assays resulted in a 10 times reduction in the amount of reagent and volume of the sample needed when compared with conventional microplate protocols. We standardized absorbance readings from the polymerase chain reaction plates, which allowed us to make direct readings of the techniques above, and a ß-glycosidase assay was also established under the same conditions. Standardization of the enzymatic reaction in thermocyclers resulted in less time-consuming temperature calibrations and without loss of volume through leakage or evaporation from the microplate. Kinetic parameters were successfully obtained, and the use of the thermocycler allowed the measurement of enzymatic activities in biological samples from the field with a limited amount of protein.