Species Identification and Resistance Status of Anopheles gambiae s.l. (Diptera: Culicidae) Mosquitoes in Guinea.

Insecticide resistance is one of the primary threats to the recent gains in malaria control. This is especially true in Guinea, where long-lasting insecticidal nets are currently the primary vector control intervention. To better inform the national malaria control program on the current status of insecticide resistance in Guinea, resistance bioassays were conducted, using Anopheles gambiae s.l. Giles, in three sites. Molecular analyses were also done on An. gambiae s.l. to determine the species and find whether the target-site mutations kdr and Ace1R were present. Susceptibility tests revealed resistance to DDT and pyrethroids, although mosquitoes were susceptible to deltamethrin in two of the three sites tested. Mosquitoes were susceptible to bendiocarb, except in Kissidougou, Guinea. The kdr-west mutation was widespread and the frequency was 60% or more in all sites. However, the Ace1R mutation was present in low levels. Insecticide susceptibility should continue to be monitored in Guinea to ensure insecticide-based vector control methods remain effective.

CHARACTERISTICS OF PHLEBOTOMINE SANDFLIES IN SELECTED AREAS OF SRI LANKA.

Cutaneous leishmaniasis (CL) is an endemic disease in Sri Lanka. Studies on vector aspects, although important for better understanding of disease transmission dynamics, are still limited. The present study describes the species distribution and behavioral patterns of sandflies within selected disease-prevalent zones in the country. Adult sandflies were collected from several field sites over a two-year duration in Sri Lanka using cattle-baited net traps, CDC light traps and manual methods. Species identification was performed using standard keys. Leishmania donovani and source of blood meal in blood-fed female sandflies DNA were identified using PCR-based methods. Aggregation period of adult sandflies during overnight collections was also noted. The collected sandflies were identified as Phlebotomus argentipes glaucus (previously known as morphospecies A) and a non-vector species, Sergentomyia zeylanica. Presence of L. donovani DNA was found in 2/634 female sandflies. The parasite ITS1 region of SSU rDNA had 99% sequence similarity with L. donovani from Bangladesh and India. The peak aggregation period of sandflies within cattle-traps was between 8:00 PM to 11:00 PM, indicating that vector control strategies could be conducted during this time period. As Sergentomyia zeylanica is likely to be merely a biting nuisance and showed more of an anthropophilic behavior, whereas the probable vector of CL in Sri Lanka (P. argentipes glaucus) demonstrated zoophilic behavior, has implications for the planning of future vector control strategies.

Genetic variability, phylogenetic relationships and gene flow in Triatoma infestans dark morphs from the Argentinean Chaco.

The recent discovery of sylvatic populations of Triatoma infestans outside the Andean Valleys of Bolivia prompted an evolutionary question about the putative ancestral area of origin and dispersal of the species, and an epidemiological question regarding the possible role of these sylvatic populations in the recolonization process of insecticide-treated houses. The finding of a population of sylvatic melanic T. infestans (dark morphs) in the Argentinean dry Chaco at 7 km from a peridomestic bug population of typical coloration gave us the opportunity to test both questions simultaneously by employing phylogenetic and population genetic approaches. For this purpose we analyzed sylvatic and peridomestic bugs using sequence-based mitochondrial and nuclear markers (mtCOI and ITS-1) and microsatellites. Sylvatic bugs were confirmed to be T. infestans and not hybrids, and showed high levels of genetic variability and departures from neutral expectations for mtCOI variation. New ITS-1 and mtCOI haplotypes were recorded, as well as haplotypes shared with peridomestic and/or domestic bugs from previous records. The peridomestic population was invariant for ITS-1 and mtCOI, but showed variability for microsatellites and signatures of a population bottleneck, probably due to a limited number of founders. Phylogenetic analyses were consistent with the presence of ancestral haplotypes in sylvatic bugs. According to F-statistics and assignment methods there was a significant differentiation between sylvatic and peridomestic bugs and gene flow was low and asymmetric, with more bugs moving from the peridomicile to the sylvatic environment. These results support the hypothesis of the Chaco region as the area of origin of T. infestans, and a limited role of sylvatic melanic T. infestans in peridomestic infestation in the Argentinean Chaco.

Molecular population genetics and phylogeography of the Chagas disease vector Triatoma infestans in South America.

Knowledge of the genetic variability, population structure, and evolutionary history of Triatoma infestans may be useful for developing rational vector control strategies. A 661-bp fragment of the mitochondrial gene cytochrome oxidase I (COI) was sequenced and analyzed in bugs from Argentina, Uruguay, Peru, and Bolivia, including peridomestic, domestic, Andean, and Chaco sylvatic bugs. A total of 48 polymorphic sites among 37 haplotypes were described. Nucleotide variation fluctuated among samples, with the highest nucleotide diversity observed in seven Argentinean provinces. Within this group, some populations showed patterns of variability compatible with population expansions and/or fine-scale population structure, whereas others suggested population bottlenecks and/or population admixture processes. A maximum parsimony analysis of the haplotypes showed the presence of a Bolivian/Peruvian and an Argentinean/Uruguayan clade. Bolivian sequences were further divided in Chaco sylvatic and Andean domestic and sylvatic. Two different nested clades were found within the Argentinean/Uruguayan cluster. Analysis of molecular variance (AMOVA) and K(ST)* analysis supported a strong population structure in Argentina, where genetic differentiation was correlated with geographic distance. Departures from neutrality expectations and a nested cladistic analysis suggest a recent population expansion of T. infestans in Argentina, followed by restricted gene flow and patterns of isolation by distance. This expansion could have taken place as a two-wave process, as was shown by the phylogenetic analysis and signatures of population admixture in the southern most Argentinean populations.

Genetic structure of Triatoma infestans populations in rural communities of Santiago del Estero, northern Argentina.

To gain an understanding of the genetic structure and dispersal dynamics of Triatoma infestans populations, we analyzed the multilocus genotype of 10 microsatellite loci for 352 T. infestans collected in 21 houses of 11 rural communities in October 2002. Genetic structure was analyzed at the community and house compound levels. Analysis revealed that vector control actions affected the genetic structure of T. infestans populations. Bug populations from communities under sustained vector control (core area) were highly structured and genetic differentiation between neighboring house compounds was significant. In contrast, bug populations from communities with sporadic vector control actions were more homogeneous and lacked defined genetic clusters. Genetic differentiation between population pairs did not fit a model of isolation by distance at the microgeographical level. Evidence consistent with flight or walking bug dispersal was detected within and among communities, dispersal was more female-biased in the core area and results suggested that houses received immigrants from more than one source. Putative sources and mechanisms of re-infestation are described. These data may be use to design improved vector control strategies.

Identification and characterization of microsatellite markers in the Chagas disease vector Triatoma infestans (Heteroptera: Reduviidae).

Triatoma infestans, the main vector of Chagas disease in the southern cone countries, is the principal target of a regional elimination program. A better understanding of its dispersal, sources of reinfestation, and insecticide resistance is key to an effective control program. To address such problems, we identified and characterized 13 microsatellite loci of T. infestans. For each locus, primer sequences and PCR conditions are presented. Allele variability and frequency were analyzed in 59 T. infestans specimens from different rural communities in northwestern Argentina; nine loci were considered suitable for population genetic studies. Departure from Hardy-Weinberg equilibrium was detected in 10/13 loci with F(IS) values ranging from 0.04 to 0.91, indicating heterozygote deficit and a possible grade of sub-structure in the sample analyzed. Presence of null alleles in some loci cannot be discarded. The present work provides a promising tool to develop a population genetic study of natural populations of T. infestans in tandem with field studies and analyses of bug dispersal and the reinfestation process.

Sequence and organization of the mitochondrial genome of the Chagas disease vector, Triatoma dimidiata.

The 17 019 bp mitochondrial genome of Triatoma dimidiata is composed of thirteen protein coding sequences, twenty-two tRNAs, small and large ribosomal units, and a control region. The gene order and orientation are identical to that of Drosophila yakuba. The nucleotide composition is biased toward adenine and thymine (69.5% A + T). The 2.1 kb putative control region, known as the A + T rich region in most insects, has an A + T bias of 66%, but contains a 400 bp sequence that is 77.5% A + T and two other distinct regions: (1) one with a lower A + T bias (60.1%) and (2) a region of eight tandem repeat units. The identified 1.4 kb nuclear copy of mitochondrial sequences encompasses the string of Gs and the beginning of the cytochrome c oxidase 1 gene but lacks the 1.8 kb region spanning the eight tandem repeats and the 5' end of the NADH dehydrogenase subunit II gene.

Bacterial symbiosis and paratransgenic control of vector-borne Chagas disease.

The triatomine vectors of Chagas disease are obligate haematophagous insects, feeding on vertebrate blood throughout their entire developmental cycle. As a result of obtaining their nutrition from a single food source, their diet is devoid of certain vitamins and nutrients. Consequently, these insects harbour populations of bacterial symbionts within their intestinal tract, which provide the required nutrients that are lacking from their diet. We have isolated and characterised symbiont cultures from various triatomine species and developed a method for genetically transforming them. We can then reintroduce them into their original host species, thereby producing stable paratransgenic insects in which we are able to express heterologous gene products. Using this methodology, we have generated paratransgenic Rhodnius prolixus that are refractory for infection with Trypanosoma cruzi. Two examples of potentially refractory genes are currently being expressed in paratransgenic insects. These include the insect immune peptide cecropin A and active single chain antibody fragments. We have also developed an approach that would allow introduction of genetically modified bacterial symbionts into natural populations of Chagas disease vectors. This approach utilises the coprophagic behaviour of these insects, which is the way in which the symbionts are transmitted among bug populations in nature. The production and ultimate release of transgenic or paratransgenic insects for public health applications is potentially very promising but also worthy of much careful consideration with respect to environmental, political, and human safety concerns.

Phylogeny and molecular taxonomy of the Rhodniini derived from mitochondrial and nuclear DNA sequences.

Eleven species of Rhodnius and one of Psammolestes were compared by DNA sequence analysis of fragments of the mitochondrial large subunit ribosomal RNA (mtlsurRNA), the mitochondrial cytochrome b (mtCytb), and the D2 variable region of the 28S nuclear RNA (D2), totaling 1,429 base pairs. The inferred phylogeny, using Triatoma infestans as an outgroup, revealed two main clades within the Rhodniini--one, including the prolixus group of species (Rhodnius prolixus, Rhodnius robustus, Rhodnius neglectus, and Rhodnius nasutus) together with Rhodnius domesticus and Rhodnius neivai, and the other comprising two groups formed by Rhodnius pictipes plus Rhodnius brethesi, and Rhodnius ecuadoriensis plus Rhodnius pallescens. Psammolestes tertius appeared most closely related to the prolixus group. The analysis strongly supports the validity of R. robustus as a species distinct from others of the prolixus group, but suggests higher genetic structuring of R. robustus populations compared to the other species. Although R. robustus has been found naturally infected by Trypanosoma cruzi, the fact that it is apparently entirely sylvatic and unable to establish in homes suggests that it is of no great importance as a Chagas disease vector in humans.

A family of pupal-specific cuticular protein genes in the mosquito Anopheles gambiae.

We have cloned and sequenced members of a cuticular protein multi-gene family from the mosquito Anopheles gambiae. Three genes (agcp2a-c), each approximately 1 kb in length, were found in a 17.4 kb genomic phage clone. Analysis of ten cDNAs revealed that at least four related genes are present. The open reading frame of the genes and cDNAs showed 95% sequence identity. Divergence was observed in the sequence of the 3' ends and the number of copies of two repeated coding sequences. In situ hybridizations with a probe prepared from one of these circular protein genes physically mapped to two loci, 26B on chromosome 2L and 37A on 3R. Transcription of these An. gambiae cuticular protein genes appears to be limited to pharate pupae and the expressed protein(s) is found in early pupae. The deduced amino acid sequence of these proteins contains a hydrophilic region with significant similarity to other cuticular proteins including the pupal-specific cuticular protein, EDG84, of Drosophila melanogaster (Apple and Fristrom).

Laboratory life cycle of Ixodes woodi (Acari: Ixodidae).

A 24-mo study of the laboratory life cycle of the tick Ixodes woodi Bishopp was conducted. No other such study of any species in the subgenus Ixodiopsis Filippova has been reported. Three generations of I. woodi were examined. Larvae and nymphs fed for an average of 4 d; approximately 8-9 d were required for females to engorge. Females laid approximately 900 eggs that required an average of 37.33 d to hatch.

A recombinant protein from Schistosoma mansoni useful for the detection of S. mansoni and Schistosoma haematobium antibodies.

A recombinant Schistosoma mansoni protein has been identified as a useful antigen for the detection of S. mansoni and Schistosoma haematobium antibodies. The purified recombinant protein, Sm22.3, was assayed using an enzyme-linked immunosorbent assay format against a battery of 491 well defined sera, including S. mansoni, S. haematobium, and Schistosoma japonicum infection sera, normal human sera, sera from 9 other parasitic infections, and sera from 2 additional infections. The sensitivity for detecting S. mansoni and S. haematobium infections with this single recombinant protein is 80.1%. The specificity is 94.8%. However, 15 of the 16 cross-reactive sera are malaria infection sera, and we have data suggesting that these malaria sera are actually recognizing an epitope on the vector-derived 6Xhistidine tag of recombinant Sm22.3. If this is the case, then, the actual specificity of the assay is 99.6%.

The Tryptophan oxygenase gene of Anopheles gambiae.

The Anopheles gambiae gene encoding tryptophan oxygenase, a homolog of the Drosophila melanogaster vermilion gene, has been molecularly cloned and characterized. Unlike Drosophila, where it is X-linked, the A. gambiae gene maps to chromosome 2R, subdivision 12E, by in situ hybridization to the polytene chromosomes. Of the six introns present, four are positioned identically to those of the Drosophila homolog, one is similarly positioned, and one is novel. A 1 955 nt cDNA potentially encodes a 392 amino acid protein of an estimated 45 kDa. Amino acid comparisons between the deduced protein and previously known tryptophan oxygenases revealed 74% identity between Anopheles and Drosophila, and 53% identity between Anopheles and nematode or mammalian proteins. Northern analysis detected a developmentally regulated transcript about 2 kb in length. Since this gene is known to control adult eye color in other flies, its cloning from A. gambiae provides the basis for a dominant phenotypic marker for germline transformation, one whose expression, unlike that of white, is not cell autonomous.

Ecdysteroid titers during postembryonic development of Dermanyssus gallinae (Acari: Dermanyssidae).

Ecdysteroid titers in methanolic extracts of protonymphal and deutonymphal stages of the chicken mite, Dermanyssus gallinae (De Geer), were measured using radioimmunoassay. Highest titers of ecdysteroids were present 24 h after feeding. Further studies using high-performance liquid chromatography radioimmunoassay showed that ecdysteroid immunopositive material had the same retention time as 20-hydroxyecdysone and ecdysone. Ecdysteroid conjugates appear to be present in polar and apolar fractions.

Reduced longevity of Dermacentor variabilis and Ixodes scapularis (Acari: Ixodidae) molting from immatures fed on Rattus norvegicus.

Nymphal and adult Dermacentor variabilis (Say) molting from larvae and nymphs, respectively, that fed on a Holtzman strain of Rattus norvegicus demonstrated reduced longevity compared with those from immature stages that fed on albino laboratory mice, Mus musculus. The decrease in survivorship did not appear to be caused by host-acquired resistance; lower feeding weights, longer feeding times, and reduced molting were not observed. In addition, reduced survivorship was not limited to ticks fed on Holtzman rats. It was also demonstrated in adult D. variabilis molting from nymphs that fed on Wistar strain rats, but the effect was not as severe as with Holtzman rats. Moreover, reduced survivorship occurred in Ixodes scapularis Say that fed on rats, but, again, the effect as less severe than on D. variabilis. Nymphal feeding on mice appeared to counteract the negative effect of larval feeding on rats in both adult D. variabilis and I. scapularis.

Ecdysteroid titre and metabolism and cuticle deposition during embryogenesis of the ixodid tick Amblyomma hebraeum (Koch).

Three embryonic cuticles are formed before larval cuticle deposition during embryonic development of Amblyomma hebraeum. The quantity of radioimmunoassay-positive material varied between 50 and 200 pg ecdysone equivalents per mg, but no significant peaks were detected. Maternally incorporated [3H]-20-hydroxyecdysone and [3H]-ecdysone contained in freshly laid eggs appear to be conjugated to C-22 fatty acid esters and 3 alpha epimers of those esters, and, thus, appear doubly inactivated. In addition, ecdysone is converted to an unknown product called 2'. The role of these maternally derived ecdysteroids is unknown.

Correlation between ecdysteroids and cuticulogenesis in nymphs of the tick Ornithodoros parkeri (Acari: Argasidae).

Ecdysteroids and cuticulogenesis were studied in third-instar nymphs of the tick Ornithodoros parkeri Cooley. Ecdysteroid titer increases slightly during the postfeeding intermolt period, which correlates with the deposition of additional procuticle lamellae. The titer increases at the time of apolysis, peaks at the time of epicuticle deposition, and then drops to a low but detectable level from the time of procuticle deposition until after ecdysis. Ecdysone and 20-hydroxyecdysone are the two major ecdysteroids present during the postfeeding intermolt period and the period of epicuticle deposition. After ecdysis, the ecdysteroid immunoreactive material is composed principally of an unknown compound more polar than 20,26-dihydroxyecdysone and is found only in the midgut. This compound is possibly a metabolite of ecdysteroids left over from the previous instar.

Localization of insulin-like immunoreactivity in the synganglion of nymphal and adult Dermacentor variabilis (Acari: Ixodidae).

Immunocytochemical staining based on a peroxidase-antiperoxidase method showed neurosecretory cells (NSC) reactive to bovine insulin in five of 18 paraldehyde fuchsin-positive neurosecretory regions (NSR) in the synganglion of unfed adult Dermacentor variabilis. This is the first report of a neuropeptide in an ixodid tick. The insulin-specific immunoreactive cells included the posterior medial group of the protocerebral center, posterior group of dorsal opisthosomal center, anterior lateral group of the dorso-lateral cheliceral center, dorsal group of the frontal stomodeal center, and anterior group of the ventral palpal center. After feeding and mating, females no longer had immunoreactive cells in three of five NSR found in virgin, unfed females. However, two cells of the posterior group in dorsal opisthosomal center and anterior lateral group of the dorso-lateral cheliceral center remained immunoreactive throughout feeding. Fed, mated males continued to display immunoreactive cells in four of five NSR found in the virgin, unfed males. All developmental stages of nymphs examined had insulin-specific immunoreactive cells in two of the five NSR found in unfed adults, including two positively stained cells of the posterior group in dorsal opisthosomal center and anterior group of ventral palpal neurosecretory center.

Metabolism of [3H]-ecdysone in embryos and larvae of the tick Ornithodoros moubata.

The fate of [3H]-ecdysone ([3H]-E) was investigated in hanging drop cultures of embryos and larvae of the tick Ornithodoros moubata using HPLC. The hormone was metabolized more slowly during described periods of increasing endogenous ecdysteroid (ES) titers than during periods of low titers except for young embryos. Three different classes of metabolites were produced: 1) apolar products (AP) corresponding to C-22 fatty acid ester conjugates of E and, in some cases, of 20-hydroxyecdysone (20E), 2) unidentified polar products (PP) more polar than E, one peak of which had the same retention times as 20,26-dihydroxyecdysone, and finally, 3) 20E verified by comigration of cold standards on RP-18 and silica columns. Hydroxylation of E to 20E first became evident in cultures of 2 day old embryos and was present in all cultures of older animals. Highest production of free 20E occurred during increasing endogenous ES titers in embryos and during the ES peak in larvae. Conjugation of E to AP occurred in all stages investigated, but was more pronounced during periods of low endogenous ES titers, and may correspond to a detoxification mechanism. In contrast, PP were produced during high 20E production in embryos and during periods of high and decreasing endogenous titers in larvae.

Conspecificity of the ticks Ixodes scapularis and I. dammini (Acari: Ixodidae).

Reciprocal crosses between Ixodes dammini Spielman, Clifford, Piesman & Corwin from Massachusetts and Ixodes scapularis Say from Georgia produced offspring through the F3 generation when the experiment was discontinued. Reciprocal I. dammini x Ixodes pacificus Cooley & Kohls (California) and I. scapularis x I. pacificus crosses produced F1 progeny; however, all progeny were sterile. Assortative mating experiments between I. dammini and I. scapularis indicated that males and females of both species mated with the opposite sex of heterospecific or conspecific ticks when there was a choice. Conventional discriminant analysis of morphometric measurements of ticks from Georgia, North Carolina, Maryland, Massachusetts, and two populations of F1 hybrids indicated that there were recognizable differences. However, size-free (sheared) discriminant analysis indicated that these differences were largely size-dependent, with much overlap of the four eastern and two hybrid populations but no overlap with I. pacificus from California. Analysis of chromosomes (morphology and C band) indicated no differences between the Georgia and Massachusetts populations but showed a difference between them and the California population of I. pacificus. Analysis of isozymes showed that the genetic identity value for the Georgia and Massachusetts populations was within the normal range for conspecific populations, whereas the California population indicated congeneric but not conspecific relatedness to the Georgia and Massachusetts populations. Life cycle data collected under similar laboratory conditions showed no differences in length of feeding and molting periods among Georgia, Massachusetts, and California populations. These data and results of the work of other authors on tick host preferences and vector competence indicate that I. dammini is not a valid species separate from I. scapularis. Because the name Ixodes scapularis Say, 1821, has priority over the name Ixodes dammini Spielman, Clifford, Piesman & Corwin, 1979, I. dammini is relegated to a junior subjective synonym of I. scapularis (based on Article 23 of the International Code of Zoological Nomenclature).