Allozyme relationships among ten species of Rhodniini, showing paraphyly of Rhodnius including Psammolestes

Genetic relationships among 10 species of bugs belonging to the tribe Rhodniini (Hemiptera: Reduviidae), including some important vectors of Chagas disease, were inferred from allozyme analysis of 12 enzyme loci (out of 21 enzyme systems examined), using agarose gel electrophoresis. These species formed two clusters: one comprising Rhodnius brethesi, R. ecuadoriensis, R. pallescens and R. pictipes; the other with Psammolestes tertius, Rhodnius domesticus and the Rhodnius prolixus group comprising R. nasutus, R. neglectus, R. prolixus and R. robustus. The resulting tree was [((R. ecuadoriensis, R. pallescens) R. brethesi) R. pictipes], [R. domesticus (P. tertius [(R. nasutus, R. neglectus) (R. prolixus, R. robustus)])]. Rhodnius nasutus and R. neglectus differed by only one locus, whereas no diagnostic loci were detected between R. prolixus and R. robustus (22 loci were analysed for these four species), despite considerable DNA sequence divergence between species in each of these pairs. Allozymes of the R. prolixus group showed greater similarity with Psammolestes tertius than with other Rhodnius spp., indicating that Rhodnius is paraphyletic and might include Psammolestes.     

Feeding and defecation of Rhodnius (hemiptera: Reduviidae) fed human blood

Feeding and defecation behavior of Rhodnius prolixus Stal, 1859, R. robustus Larrousse, 1927, R. neivai Lent, 1953 and R. pictipes Stal, 1872, artificially fed on human blood, were studied under laboratory conditions. In all species, first instar nymphs did not defecate in the first 30 minutes after feeding. R. pictipes did not accept artificial feeding but fed directly on humans. Nymph and adult R. prolixus had a higher defecation index (DI) than other species; third instar nymphs had the highest DI = 1.62. In all instars, most individuals accepted the food in 3 Pounds minutes and finished feeding in less than 15 minutes.     

Ribosomal DNA ITS-1 Intergenic Spacer Polymorphism in Triatomines (Triatominae, Heteroptera)

The length polymorphism of ribosomal DNA ITS-1 intergenic spacer was analyzed in eight species of triatomines belonging to Triatoma, Rhodnius, and Panstrongylus genera. The analyzed species were Rhodnius domesticus, R. neivai, R. robustus, Triatoma brasiliensis, T. infestans, T. vitticeps, Panstrongylus megistus, and P. herreri. These insects are vectors of Chagas' disease, one of the most prominent public health problems among South American countries. This work allowed the differentiation between species of the Triatomini and Rhodniini tribes through the analysis of ITS-1 length polymorphism by PCR and RFLP techniques. The species of the Triatoma and Panstrongylus genera presented an amplified ITS-1 fragment between 600 and 1000 bp, whereas Rhodnius presented a less variable ITS-1 length fragment, around 300 bp, which could reflect the monophyletic origin of the Rhodniini tribe. Species belonging to this genus were further differentiated by RFLP with HaeIII and AluI endonucleases. Our results corroborate the hypothesis of polyphyletic origin in this group of insects and contribute to knowledge about evolutionary relationships in triatomines.     

Isozyme variability and differentiation between Rhodnius prolixus, R.robustus and R.pictipes, vectors of Chagas disease in Venezuela

Enzyme polymorphism in triatomine bugs of the genus Rhodnius (Hemiptera: Reduviidae), vectors of Chagas disease, is analysed using both starch and polyacrylamide gel electrophoresis. Out of forty-five enzymes assayed, the electromorphs of seventeen of them: AO, CA, DIA, ES, ES-A, FH, GPD, G6PD, GPI, MDH, ME, 6PGD, PGM, ACON, ACPH, LAP and SOD, involving twenty-two putative structural loci, were scorable. These gene-enzyme systems were therefore selected for routine characterization of R.prolixus Stal adults from different strains. The first thirteen enzymes, involving sixteen structural loci, were also analysed in first instar nymphs of the three species, R.prolixus, R.robustus Larrousse and R.pictipes Stal. Allelic frequencies were calculated for three R.prolixus strains: three to five loci appeared to be polymorphic. The proportion of polymorphic loci (22%) and the average heterozygosity (0.06) indicated low genetic variability, with significant differences between the strains at individual loci. Rhodnius prolixus and R.robustus were found to have identical isozymic patterns. R.pictipes was genetically well differentiated, with twelve diagnostic loci.     

Molecular phylogeography of the Amazonian Chagas disease vectors Rhodnius prolixus and R. robustus

The phylogeographical structure of the closely related species Rhodnius prolixus and R. robustus is presented based on a 663-base pair (bp) fragment of the mitochondrial cytochrome b gene. Twenty haplotypes were recovered from 84 samples examined, representing 26 populations from seven Latin American countries. The resulting phylogenetic tree is composed of five major reciprocally monophyletic clades, one representing R. prolixus and four representing R. robustus. While R. prolixus is a very homogeneous assemblage, R. robustus has deeper clades and is paraphyletic, with the clade comprising R. robustus from Venezuela (Orinoco region) more closely related to the R. prolixus clade than to the other R. robustus populations from the Amazon region. The R. robustus paraphyly was supported further by the analysis of a nuclear gene (D2 region of the 28S RNA) for a subset of specimens. The data support the view that R. robustus represents a species complex. Levels of sequence divergence between clades within each region are compatible with a Pleistocene origin. Nucleotide diversity (pi) for all R. prolixus populations was extremely low (0.0008), suggesting that this species went through a recent bottleneck, and was subsequently dispersed by man.     

Rhodnius robustus in Bolivia identified by its wings.

Wings of a Rhodnius specimen from Alto Beni (Bolivia) was examined for identification and compared with R. stali, R. robustus, (certified Bolivian species), R. pictipes and R. prolixus (suspected Bolivian species). A projection of the unidentified wings as supplementary data into a discriminant analysis of shape revealed clear cut differences with R. stali and R. pictipes, less differences with R. prolixus, and none with R. robustus. Combining global size and shape of the wings, the unknown specimen was identified as R. robustus. Thus, this study confirmed the presence of R. robustus in Bolivia. It also highlighted the possibility of morphometrics to taxonomically interpret one individual, or even one piece of an individual, when related species data are available for comparison.     

Molecular cloning,over expression and characterization of thermoalkalophilic esterases isolated from <Emphasis Type="Italic">Geobacillus</Emphasis> sp.

Due to potential use for variety of biotechnological applications, genes encoding thermoalkalophilic esterase from three different Geobacillus strains isolated from thermal environmental samples in Balçova (Agamemnon) geothermal site were cloned and respective proteins were expressed in Escherichia coli (E.coli) and characterized in detail. Three esterases (Est1, Est2, Est3) were cloned directly by PCR amplification using consensus degenerate primers from genomic DNA of the strains Est1, Est2 and Est3 which were from mud, reinjection water and uncontrolled thermal leak, respectively. The genes contained an open reading frame (ORF) consisting of 741 bp for Est1 and Est2, which encoded 246 amino acids and ORF of Est3 was 729 bp encoded 242 amino acids. The esterase genes were expressed in E. coli and purified using His-Select HF nickel affinity gel. The molecular mass of the recombinant enzyme for each esterase was approximately 27.5 kDa. The three esterases showed high specific activity toward short chain p-NP esters. Recombinant Est1, Est2, Est3 have exhibited similar activity and the highest esterase activity of 1,100 U/mg with p-nitrophenyl acetate (pNPC₂) as substrate was observed with Est1. All three esterase were most active around 65°C and pH 9.5–10.0. The effect of organic solvents, several metal ions, inhibitors and detergents on enzyme activity for purified Est1, Est2, Est3 were determined separately and compared.     

Structural and biochemical characterization of a metagenome‐derived esterase with a long N‐terminal extension

The genes encoding six novel esterolytic/lipolytic enzymes, termed LC‐Est1～6, were isolated from a fosmid library of a leaf‐branch compost metagenome by functional screening using tributyrin agar plates. These enzymes greatly vary in size and amino acid sequence. The highest identity between the amino acid sequence of each enzyme and that available from the database varies from 44 to 73%. Of these metagenome‐derived enzymes, LC‐Est1 is characterized by the presence of a long N‐terminal extension (LNTE, residues 26–283) between a putative signal peptide (residues 1–25) and a C‐terminal esterase domain (residues 284–510). A putative esterase from Candidatus Solibacter usitatus (CSu‐Est) is the only protein, which shows the significant amino acid sequence identity (46%) to the entire region of LC‐Est1. To examine whether LC‐Est1 exhibits activity and its LNTE is important for activity and stability of the esterase domain, LC‐Est1 (residues 26–510), LC‐Est1C (residues 284–510), and LC‐Est1C* (residues 304–510) were overproduced in E. coli, purified, and characterized. LC‐Est1C* was only used for structural analysis. The crystal structure of LC‐Est1C* highly resembles that of the catalytic domain of Thermotoga maritima esterase, suggesting that LNTE is not required for folding of the esterase domain. The enzymatic activity of LC‐Est1C was lower than that of LC‐Est1 by 60%, although its substrate specificity was similar to that of LC‐Est1. LC‐Est1C was less stable than LC‐Est1 by 3.3°C. These results suggest that LNTE of LC‐Est1 rather exists as an independent domain but is required for maximal activity and stability of the esterase domain.     

Studies on Trypanosoma rangeli Tejera, 1920. VII--Its effect on the survival of infected triatomine bugs

The pathological effects of Trypanosoma rangeli on Rhodnius prolixus and R. robustus, and the relation of mortality to infection, were studied under laboratory conditions. Frequent observations revealed that when the first instar nymphs of R. prolixus and R. robustus were infected with T. rangeli, survival of the bugs during the stages of development to the adult stage decreased. This decrease was statistically significant when compared with uninfected control-bugs, indicating that T. rangeli is pathogenic for both species of triatomine. In R. prolixus the most affected nymphal stages were the first, second and fifth instars, where a higher mortality was also observed. In R. robustus a progressive increase of the mortality from the first to fifth instars, was observed. The pathogenicity of T. rangeli as measured by overall mortality was the same in R. prolixus and R. robustus. The possible pathogenic mechanism of T. rangeli in triatomine-bugs and its epidemiological implications, are discussed.     

Resistance to starvation of Rhodnius neivai Lent, 1953 (Hemiptera: Reduviidae: Triatominae) under experimental conditions

The period of resistance to starvation and the loss of weight until death of Rhodnius neivai in all stages of development were studied. Work was based on experiments conducted under controlled laboratory conditions. One hundred specimens of each nymphal instar were observed: 50 were fed on chicken and 50 on rabbit. Adult females and males were kept together and fed on each host. All bugs were weighed weekly until death. Laid eggs were collected weekly and observed during five weeks to obtain hatchability. Resistance to starvation was similar with both hosts and increased with the evolutionary stage, excepting the 5th nymphal instar and adults. With both hosts, loss of weight was abrupt in the first week and steady in the following weeks. In adults, on the first weeks after eating, there was little or no mortality, after which mortality increased rapidly with the starving time. Reproductive output was higher in the bugs fed on rabbit. R. neivai is among the least resistant triatomine species.     

Cytogenetics as a tool for triatomine species distinction (Hemiptera-Reduviidae).

Several cytogenetic traits were tested as species diagnostic characters on five triatomine species: Rhodnius pictipes, R. nasutus, R. robustus, Triatoma matogrossensis and T. pseudomaculata. Four of them are described for the first time. The detailed analysis of the meiotic process and the application of C-banding allowed us to identify seven cytogenetic characters which result useful to characterize and differentiate triatomine species.     

The genetics and expression of an esterase locus inAnopheles gambiae

The main polymorphic system of esterase isoenzymes in adults of the G3 laboratory strain ofAnopheles gambiae consists of two to five major bands of activity per individual. The bands are designated 5S, 5F, 13, 14, and 15. In genetic crosses, the genes which coded for the bands assorted as three codominant alleles, Est A, Est B, and Est C, at a single autosomal locus. Homozygotes for the Est C allele were significantly underrepresented among backcross progeny. The developmental pattern of esterase expression was examined. Esterase gene expression in embryos was first detectable between 2 and 12 hr after oviposition. The initiation or termination of expression of some of the bands corresponded to boundaries between developmental stages. Most of the esterase fractions were not specifically localized within the tissues tested, with the exception of a series of bands which were restricted largely to adult male testes.     

Linkage of genes, controlling morphological signs with isozyme markers of rye chromosomes

Data on linkage of 12 rye genes controlling morphological traits (el, Vs, ln, w, np, ct2, Hs, Ddw, cb, mn, vil, mp) with one or several isozyme markers of individual rye chromosomes (2R-7R) are presented. Linkage of the following gene pairs was established: chromosome 2R: Est3/5-el, el-beta-Glu, Sod2-el, Sod2-Vs; chromosome 3R: ln-Got4; chromosome 4R: w-Got1, np-Got1; chromosome 5R: Est4-ct2, Est6/9-ct2, ct2-Est2, ct2-Aco2, Est2-Hs, Aco2-Hs, Est2-Ddw, Aco2-Ddw; chromosome 6R: Lap2-cb, cb-Aco1, Est10-mn; chromosome 7R: Acph2/3-vi1, Got2-vi1, mp-Acph2/3. The reasons for mapping a very small number of genes in rye in spite of high intraspecific variability of this species are discussed. An approach is suggested to improve this situation by simultaneous identification and mapping of all diverse spontaneous mutations maintained in heterozygous state in various rye cultivars.     

Biology of nymphs of Rhodnius robustus Larrousse, 1927 (Hemiptera, Reduviidae), fed on pigeon or on Swiss mouse blood in laboratory conditions.

The duration of the life cycle of Rhodnius robustus Larrousse, 1927 as well as the mortality rate of each nymphal instar were studied comparing groups fed on pigeon or on mouse blood weekly or fortnightly. This species showed a better development and lower mortality rate when fed on swiss mouse. The intervals between feedings apparently did not have influence on the shortening of the cycle. We suggest that laboratory colonies and experiments with R. robustus are better maintained when these triatomines are fed on swiss mouse.     

Differential susceptibility of triatomines of the genus Rhodnius to Trypanosoma rangeli strains from different geographical origins

The susceptibility of four Rhodnius species to different Trypanosoma rangeli strains was evaluated using both intracoelomic inoculation and oral infection. Rhodnius prolixus, Rhodnius domesticus, Rhodnius neglectus and Rhodnius nasutus were infected with Trypanosoma rangeli Macias (Venezuela), Choachi (Colombia) and SC-58 (Brazil) strains, revealing distinct haemolymph and salivary glands infection rates. The obtained infection rates were revealed to be dependent on the method of infection and the triatomine species. Our results suggest the existence of a high adaptation between the strain and the local vector.     

ESTERASE VARIATION AND GENETIC STRUCTURE IN THREE GEOGRAPHIC POPULATIONS OF SITODIPLOSIS MOSELLANA (GEHIN) (DIPTERA: CECIDOMYIIDAE) IN WESTERN CHINA*

Abstract This paper investigates the esterase variation and genetic structure in three geographic populations of Sitodiplosis mosellana (Géhin) in western China by PAGE. The localities surveyed are Gaolan (36.3°N, 103.9°E) and Wuwei (37.9°N, 102.6°E) in spring wheat region and Chang'an (34. 1°N, 108.9° E) in winter wheat region. The results suggest that the esterase is coded by two loci: Est‐1 and Est‐2. Est‐1 is coded by a plastogene producing only one band that is the fastest on the gel among all bands. The Est‐2 is duplicated loci with 8 alleles, namely, a, b, c, d, e, f, g, h, which produce altogether 8 bands in all the populations and 1–4 bands in individual samples. There are 19 zymogram types observed in the three geographic populations. Seventeen zymogram types emerge in Chang'an population, but 5 and 4 zymogram types are found respectively in Gaolan and Wuwei populations. II₂ zymogram type is the commonest in all the populations. The alleles that had the highest frequencies in all the populations are d, e, g. All 8 alleles at the Est2 were observed in Chang'an population, but only total 3 alleles‐d, e, g at the Est‐2 appeared in Gaolan and Wuwei populations. The analysis of genetic identity and cluster (UPGMA) on the alloenzyme indicates that the relationship between the two populations of spring wheat region seems to be closer, as compared with the relationship between spring wheat population and winter wheat population. It is evident that there exists some infraspecific variation caused mainly by genetic drift in S. mosellana and the gene flow among the populations possibly took place to some extent.     

Feeding behaviour of morphologically similar Rhodnius species: influence of mechanical characteristics and salivary function

Despite their morphological similarities, very similar Rhodnius species (R. prolixus, R. robustus, R. nasutus and R. neglectus) displayed a distinct feeding behaviour when fed on artificial feeder, pigeon or mouse. On pigeon hosts, these species showed distinct groups in terms of cumulative probing time - quicker species (R. prolixus and R. neglectus) followed by R. nasutus and finally a much slower species (R. robustus). On mouse hosts, R. nasutus showed quicker probing time compared to the other three species. Moreover, R. prolixus displayed quicker probing time compared to R. robustus and R. neglectus. Except for R. nasutus, the mean total ingestion rate tended to have different values between feeding sources (artificial feeder>pigeon>mouse). The volume ingested by each cibarial pump contraction and maximum frequency obtained using the artificial feeder are expected to be related to intrinsic mechanical characteristics of the insect feeding apparatus. However, probing time and the modulation of cibarial pump frequency on live hosts may be related to salivary function. R. prolixus showed high mechanical and salivary efficiency, achieving high values of total ingestion rate when fed on artificial feeder or either of the hosts. Comparative analysis suggests that species which possess higher total ingestion rates tend to achieve higher nutritional status, allowing them to reach higher densities.     

Cloning and identification of a new group esterase (Est5S) from noncultured rumen bacterium

The gene encoding an esterase enzyme was cloned from a metagenomic library of cow rumen bacteria. The esterase gene (est5S) was 1,026 bp in length, encoding a protein of 366 amino acid residues with a calculated molecular mass of 40,168 Da. The molecular mass of the enzyme was estimated to be 40,000 Da. The Est5S protein contains the Gly-X-Ser-X-Gly motif found in most bacterial and eukaryotic serine hydrolases. However, the Asp or Glu necessary for the catalytic triad [Ser-Asp-(Glu)-His] was not present, indicating Est5S represents a novel member of the GHSQG family of esterolytic enzymes. BlastP in the NCBI database analysis of Est5S revealed homology to hypothetical proteins and it had no homology to previous known lipases and esterases. Est5S was optimally active at pH 7.0 and 40 degrees C. Among the p-nitrophenyl acylesters tested, high enzymatic activities were observed on the short-chain p-nitrophenyl acylesters, such as p-nitrophenyl acetate, etc. The conserved serine residue (Ser190) was shown to be important for Est5S activity. The primers that amplified the est5S gene did not show any relative band with 49 species of culturable rumen bacteria. This implies that a new group esterase gene, est5S, may have come from a noncultured cow rumen bacterium.     

Differentiation and genetic analysis of Rhodnius prolixus and Rhodnius colombiensis by rDNA and RAPD amplification.

Domiciliated Rhodnius prolixus and sylvatic R. colombiensis were analyzed in order to confirm their genetic divergence and verify the risk that the latter represents in the domiciliation process, and to provide tools for identifying the sources of possible reinfestation by triatomines in human dwellings allowing control programs to be undertaken. Comparison of random amplified polymorphic DNA amplification patterns and cluster analysis suggests reproductive discontinuity between the two species. The calculated statistical F value of 0.24 and effective migration rate of 0.6 individuals per generation are insufficient to maintain genetic homogeneity between them and confirm the absence of present genetic flow. R. colombiensis presents higher intrapopulation variability. Polymerase chain reaction of ribosomal DNA supports these findings. The low genetic flow between the two species implies that R. colombiensis do not represent an epidemiological risk for the domiciliary transmission of Trypanosoma cruzi in the Tolima Department. The lower variability of the domiciliated R. prolixus could result in greater susceptibility to the use of pesticides in control programs.     

Aspects of classification of Hemiptera hemocytes from six triatomine species.

The objective of this work was to characterize, and compare different morphological types of hemocytes of Rhodnius prolixus, Rhodnius robustus, Rhodnius neglectus, Triatoma infestans, Panstrongylus megistus, and Dipetalogaster maximus. This information provides the basis for studying the cellular immune systems of these insects. Seven morphological hemocyte types were identified by phase-contrast microscopy: prohemocytes, plasmatocytes, granular cells, cystocytes, oenocytoids, adipohemocytes and giant cells. All seven types of hemocytes are not present in every species. For example, adipohemocytes and oenocytoids were not observed in P. megistus and P. infestans, and giant cells were rarely found in any of the species studied. The hemocytes of Rhodnius and Dipetalogaster are more similar to each other than those from Triatoma and Panstronglus which in turn closely resemble each other. Emphasis is placed on methodological problems arising in this work which are discussed in detail.