SCANNING ELECTRON-MICROSCOPY OF THE RHODNIUS-NEGLECTUS (HEMIPTERA) LABIAL SALIVARY-GLANDS AFTER STARVATION

Labial salivary glands are found in the majority of insects. They are relatively large, extend back into the thorax, and in Rhodnius, they are cherry red in color due to a pigment derived from traces of hemoglobin absorbed form the gut. In most insects they are acinous shaped, with long excretion channels that present differentiated regions which from salivary reservoirs. The glands may be relatively simple or complexly branched and convoluted. In Rhodnius they are described as being unilobed with no traces of division. The main duct leaves the gland at its anterior extremity. The acini have different kinds of cells but all of them are seen as sources of secretion. Our material has a different shape due to the fact that the animals spent 20 days under starvation conditions. New data are also obtained through treatment with collagenase and HCl. The importance of the study of these glands lies in the fact that it will further understanding of the transmission of Chagas' disease.

Esterase patterns in species of the triatome bug Rhodnius

The aim of the present study was to analyse esterase patterns in three triatomine species of Rhodnius genus. Four loci, Est 1, Est 2, Est 3 and Est 4, were found. The corresponding enzymes were characterized as carboxylesterases (E.C. 3.1.1.1) or cholinesterases (E.C. 3.1.1.8) based on inhibitory experiments, using eserine sulphate, malathion, mercury chloride, p-chloromercuribenzoate (pCMB) and iodoacetamide. Low genetic variability was observed: Est 1, Est 2 and Est 3 were monomorphic in Rhodnius domesticus, Rhodnius robustus and Rhodnius neivai, whereas locus Est 4 was polymorphic in the first two species. The UPGMA analysis based on esterase genotypic frequencies indicated greater similarity between R. domesticus and R. robustus when compared with R. neivai. The present study expands our knowledge about genetic variability among triatomines and accords with the hypothesis that R. domesticus is a species derived from R. robustus.

Nucleolar changes in malpighian tubules cells of Rhodnius prolixus during starvation

This study focused on nuclear and nucleolar area changes in Malpighian tubule cells of Rhodnius prolixus, an hematophagous insect, vector of Chagas' disease. Male and female adult insects were dissected after a 28-day starvation period, as well as insects which had been fed again after a 30-day starvation period. Malpighian tubules were fixed and silver stained. In both, males and females, nucleolar fusions and regions of nucleolar corpuscles were observed to be differentially impregnated by silver during feeding stress. In the males and females that were fed again, nucleolar corpuscles were partially fusioned, indicating a slight recovery upon the 30-day starvation period. The changes observed in the nucleolar phenotype and in both nuclear and nucleolar areas indicated that, as a result of stress, a more intense, compensatory activity occurred to supply the metabolic rate. The mechanism of this phenomenon may be associated to the decondensation and activation of chromatin that carries rDNA in order to increase rRNA transcription, and,consequently, protein synthesis.

Description of the nucleolar activity and karyotype in germinative cell lines of Rhodnius domesticus (Triatominae, Heteroptera)

The purpose of this work was to study the karyotype, spermatogenesis and nucleolar activity at meiosis, in the species Rhodnius domesticus (Heteroptera, Triatominae). The testicular tubules were cytologically prepared by the conventional method of cell crushing and subsequent application of cytogenetic staining techniques with lacto-acetic orcein and silver-ion impregnation. The species under study presented karyotype 2n= 20A+XY, the modal number of the subfamily Triatominae. The chromosomes presented no primary constriction and were therefore characterized as holocentric. It was observed that the sex chromosomes sometimes were located at the periphery, close to the ring formed by autosomes, at first meiotic division. At metaphases II, sex chromosomes were positioned in the center of the autosomal ring, thus evidencing a postreductional behavior. These same chromosomes showed late migration at anaphases and were clearly impregnated with silver-ions, suggesting they bore Nucleolar Organizer Regions. Dispersed nucleolar corpuscles in cytoplasm until telophase II and small dots in spermatids strongly impregnated with silver, could be seen. Thus, it may be inferred that, in triatomines, the nucleolus does not completely disappear but remains in the form of small corpuscles that have a role in cell differentiation.

Estudo biológico e morfométrico dos estádios ninfais de Rhodnius neglectus Lent, 1954 (Hemiptera, Reduviidae)

The following parameters of the biological cycle of the Rhodnius neglectus were determined under a temperature of 28°C and relative humidity of the air varying between 52-94%: a) average period of incubation: 13.67 days; b) rate of the eggs eclosion: 86.18%; c) average period of growth of the 1st, 2nd, 3rd, 4th, 5th instar nymphs: 18.91, 15.19, 21.38, 21.34, 30.43 days, respectively; d) percentage of death in the instar nymphs: 13.82, 18.26, 9.95, 8.78, 15.06% respectively; e) percentage of changes per instar nymphs: 86.18, 81.74, 90.05, 91.22, 84.94% respectively. The morphometric study with the stereoscopic microscope and Leica image analyses system, using the Qwin software, of the five instar nymphs showed that in the five instar nymphs a gradual growth of the head, thorax and abdomen occurs. In all the instars the abdomen is the largest segment. In the four first instars the head is larger than the thorax. In the fifth instar the head and the thorax present equivalent sizes.

Estudo biológico e biométrico dos estádios ninfais de Rhodnius prolixus Stäl, 1859 (Hemiptera, Reduviidae) sob condições laboratoriais

Eggs and nymphs originated from couples of Rhodnius prolixus obtained from nymphs of the 5th instar were used for biological cycle and biometric studies. The following biological cycle parameters were determined under a temperature of 28°C and relative humidity, varying between 52 - 94% : medium period of incubation: 13.01 days; rate of eggs eclosion: 77.6%; medium period of development of the 1st, 2nd, 3rd, 4 th, 5th instar nymphs : 19.33, 19.09, 20.38, 24.37, 38.14 days, respectively; percentual of deaths in the nymph instar: 26.70, 14.00, 18.26, 17.02, 35.47% respectively; percentual of changes per instar nymphs; 73.30, 86.03, 81.73, 82.97, 64.52%, respectively. Biometric measurements performed, showed that in all the instars the abdomen is the largest segment. In the four first instars, the head is larger than the thorax. In the fifth instar, the head and thorax present are about the same size.

Estudo de sete espécies de Rhodnius (Hemiptera, Reduviidae, Triatominae) por meio de dois genes nucleares e um mitocondrial

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A novel association between Rhodnius neglectus and the Livistona australis palm tree in an urban center foreshadowing the risk of Chagas disease transmission by vectorial invasions in Monte Alto City, Sao Paulo, Brazil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Study of the external female genitalia of 14 Rhodnius species (Hemiptera, Reduviidae, Triatominae) using scanning electron microscopy

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização do padrão eletroforético de glândulas salivares de Triatoma lecticularia e Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae) em diferentes tempos pós repasto sanguíneo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytochemical study of Rhodnius neglectus and Rhodnius prolixus salivary gland cells (Hemiptera, Triatominae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Entoepidemiology of Chagas disease in the Western region of the State of Sao Paulo from 2004 to 2008, and cytogenetic analysis in Rhodnius neglectus (Hemiptera, Triatominae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The prophenoloxidase system and in vitro interaction of Trypanosoma rangeli with Rhodnius prolixus and Triatoma infestans haemolymph

The presence of the prophenoloxidase (proPO) system in the haemolymph of Rhodnius prolixus and Triatoma infestans and the role played by Trypanosoma rangeli in the in vitro activation of proPO were tested. Both R. prolixus and T. infestans whole blood preparations showed a very active ProPO system. The proPO cascade of the two insect species were differentially activated by microbial-derived extracts: laminarin was a better activator of T. infestans haemolymph than of R. prolixus blood, and lipopolysaccharides from Shigella flexneri or Pseudomonas aeroginosa caused significant proPO activation of T. infestans haemolymph but not of R. prolixus preparations. For the two insect species, neither T. rangeli from culture nor parasite lysates were able to trigger proPO activation. The presence of the parasite in R. prolixus haemolymph/laminarin assays, however, significantly reduced the level of proPO activation to that of spontaneous activating controls. The immobilization of T. rangeli in vitro in haemolymph preparations occurred in both insect species and was dependent on the proPO activation intensity. Our results suggest that the susceptibility of R. prolixus to T. rangeli haemocoel infection may be explained, at least in part, by the suppression of the insect immune defence system i.e., inhibition of proPO in the presence of this protozoan parasite.

Comparing de novo and reference-based transcriptome assembly strategies by applying them to the blood-sucking bug Rhodnius prolixus

High Throughput Sequencing capabilities have made the process of assembling a transcriptome easier, whether or not there is a reference genome. But the quality of a transcriptome assembly must be good enough to capture the most comprehensive catalog of transcripts and their variations, and to carry out further experiments on transcriptomics. There is currently no consensus on which of the many sequencing technologies and assembly tools are the most effective. Many non-model organisms lack a reference genome to guide the transcriptome assembly. One question, therefore, is whether or not a reference-based genome assembly gives better results than de novo assembly. The blood-sucking insect Rhodnius prolixus-a vector for Chagas disease-has a reference genome. It is therefore a good model on which to compare reference-based and de novo transcriptome assemblies. In this study, we compared de novo and reference-based genome assembly strategies using three datasets (454, Illumina, 454 combined with Illumina) and various assembly software. We developed criteria to compare the resulting assemblies: the size distribution and number of transcripts, the proportion of potentially chimeric transcripts, how complete the assembly was (completeness evaluated both through CEGMA software and R. prolixus proteome fraction retrieved). Moreover, we looked for the presence of two chemosensory gene families (Odorant-Binding Proteins and Chemosensory Proteins) to validate the assembly quality. The reference-based assemblies after genome annotation were clearly better than those generated using de novo strategies alone. Reference-based strategies revealed new transcripts, including new isoforms unpredicted by automatic genome annotation. However, a combination of both de novo and reference-based strategies gave the best result, and allowed us to assemble fragmented transcripts.

Transposition burst of mariner-like elements in the sequenced genome of Rhodnius prolixus

Transposable elements (TEs) are widespread in insect´s genomes. However, there are wide differences in the proportion of the total DNA content occupied by these repetitive sequences in different species. We have analyzed the TEs present in R. prolixus (vector of the Chagas disease) and showed that 3.0% of this genome is occupied by Class II TEs, belonging mainly to the Tc1-mariner superfamily (1.65%) and MITEs (1.84%). Interestingly, most of this genomic content is due to the expansion of two subfamilies belonging to: irritans himar, a well characterized subfamily of mariners, and prolixus1, one of the two novel subfamilies here described. The high amount of sequences in these subfamilies suggests that bursts of transposition occurred during the life cycle of this family. In an attempt to characterize these elements, we performed an in silico analysis of the sequences corresponding to the DDD/E domain of the transposase gene. We performed an evolutionary analysis including network and Bayesian coalescent-based methods in order to infer the dynamics of the amplification, as well as to estimate the time of the bursts identified in these subfamilies. Given our data, we hypothesized that the TE expansions occurred around the time of speciation of R. prolixus around 1.4 mya. This suggestion lays on the Transposon Model of TE evolution, in which the members of a TE population that are replicative active are present at multiple loci in the genome, but their replicative potential varies, and of the Life Cycle Model that states that when present-day TEs have been involved in amplification bursts, they share an ancestral copy that dates back to this initial amplification.