Spatial and temporal variation in biting rates and parasite transmission potentials of onchocerciasis vectors in Ecuador.

The influence of spatial and temporal factors on onchocerciasis transmission by Simulium exiguum s.l. and S. quadrivittatum in Ecuador was investigated to help develop sampling protocols for entomological surveillance of ivermectin programmes. Flies were collected in alternate months (November 1995-November 1996) at four sites each in the hyperendemic communities of San Miguel and El Tigre. A fixed-effects analysis of variance was used to explore the influence on vector abundance of locality, site, month and hour. Infectivity rates detected by dissection and PCR assays were compared. Simulium exiguum s.l. predominated at El Tigre (75%) whereas S. quadrivittatum prevailed at San Miguel (62%). Vector abundance was highest on river banks and outside houses. Biting and infection rates peaked from March to July. Hourly activity patterns were bimodal in S. exiguum but unimodal in S. quadrivittatum. Annual transmission potentials (ATP) for both species combined were 385 and 733 third stage larvae/person in San Miguel and El Tigre respectively, with S. exiguum accounting for 80% of the combined ATP at both localities. We recommend protocols that may maximize detection of parasite transmission. Infection rates thus obtained must be linked with vector density estimates to assess meaningfully host exposure as treatment progresses.

Speciation and population structure in the morphospecies Lutzomyia longipalpis (Lutz & Neiva) as derived from the mitochondrial ND4 gene.

Recent studies have suggested that the phlebotomine sand fly Lutzomyia longipalpis (Diptera: Psychodidae), the principal vector of visceral leishmaniasis in the Neotropics, may consist of several allopatric sibling species. Phylogenetic and population genetic analyses of nucleotide variation in a 618-bp fragment of the mitochondrial ND4 gene were carried out on specimens of Lu. longipalpis from several locations in Central and South America. The analyses were concordant with previous findings, indicating that certain allopatric populations of Lu. longipalpis have become sufficiently differentiated as to represent sibling species. Phylogenetic analyses revealed deep genetic divisions between four clades represented by specimens from northern South America, Brazil, Central America, and an isolated Colombian population. Strong differentiation also was observed between certain populations within the first two clades. Partitioning of genetic diversity within and between Central American populations did not show the presence of more than one species in the region. However, distance, even within the 70-km range of the Honduran collection sites, was found to have a remarkably strong effect on gene flow. The highly subdivided population structure may be due to the patchiness of their distribution. F(ST) values comparing a Guatemalan population with several Honduran populations revealed a level of differentiation associated with a negligible rate of gene flow.

Phylogeny of nearctic members of the Anopheles maculipennis species group derived from the D2 variable region of 28S ribosomal RNA.

Phylogenetic affinities among taxa associated with the Nearctic component of the Anopheles maculipennis species group (subgenus Anopheles) were inferred from sequence divergence in the D2 variable region of 28S ribosomal RNA. The base composition of this region had a marked GC bias which ranged from 59.9% in Anopheles walkeri to 65.1% in Anopheles punctipennis E. Although over two-thirds of the base positions in the D2 region were double-stranded (stem), substitution frequencies at single-stranded (loop) positions (0.068 over all taxa) were 2.7 times greater than at stem positions (0.025). Most mutations were point mutations and were most frequent at loop positions. In the shortest trees generated by both parsimony and distance methods, the four American species traditionally identified with the maculipennis complex (Anopheles aztecus, Anopheles earlei, Anopheles freeborni, and Anopheles occidentalis) were monophyletic with A. punctipennis E and W as sister taxa. The latter two correspond to genetically distinct forms from the eastern United States and California, respectively. The sibling species of the Anopheles quadrimaculatus complex formed a distinct clade, and A. quadrimaculatus D, with six autapomorphies, was the most divergent of these taxa. Sequence divergence between A. walkeri and the other taxa included in the study was of such magnitude as to suggest only a distant affinity to these species.

Polymerase chain reaction species diagnostic assay for Anopheles quadrimaculatus cryptic species (Diptera: Culicidae) based on ribosomal DNA ITS2 sequences.

Species-specific differences in the nucleotide sequences of the 2nd internal transcribed spacer (ITS2) of nuclear ribosomal DNA (rDNA) were used to develop a diagnostic assay based on the polymerase chain reaction (PCR) that can distinguish 4 of the 5 cryptic sibling species in the common malaria mosquito, Anopheles quadrimaculatus Say, complex. The assay requires only a small amount of tissue from an individual mosquito and a mixture of 5 PCR primers. The plus strand universal primer is derived from a sequence in the 5.8S coding region that is identical in all members of the complex. The 4 minus strand primers were selected from species-unique sequences within the ITS2 region. PCR amplification produces a different sized fragment for each of the 4 species which can be visualized readily under ultraviolet light after electrophoresis through an ethidium bromide-containing agarose gel. The assay has been developed and tested only with An. quadrimaculatus complex specimens from Florida populations.

Variation in ribosomal DNA intergenic spacers among populations of Anopheles albimanus in South and Central America.

Variation in the length and copy number of intergenic spacers (IGS) of nuclear ribosomal DNA were examined to test for genetic differentiation among Anopheles albimanus populations. Extensive collections were made in Guatemala but populations were also sampled over a large range of its distribution in Central and South America. Discriminant analysis of IGS patterns in individual mosquitoes indicated that populations generally had unique sets of IGS length variants. The IGS patterns from populations on the Pacific side of Central America were distinct from those on the Atlantic side or from South America. Cluster analysis indicated a similar trend. The IGS diversity in Central America was 50% greater than in South America. These results suggest that barriers to gene flow exist among Atlantic and Pacific coast populations of An. albimanus. No gene flow barriers were detected among populations from Colombia, Ecuador, Peru, and Venezuela.

Sequence and secondary structure comparisons of ITS rDNA in mosquitoes (Diptera: Culicidae).

Sequences of the internal transcribed spacers (ITS1 and ITS2) of the mosquito Aedes aegypti, and the ITS2 of six related species, A. simpsoni, A. albopictus, A. vexans, A. triseriatus, Haemagogus mesodentatus, and Psorophora ferox are reported. Intraspecific variation in A. aegypti ITS1 is 1.07% among four clones from three individuals, and in the ITS2 is 1.17% among 15 clones from four individuals. In A. simpsoni, intraspecific ITS2 variation is 0.46% among 10 clones from a single individual. Alignment of the ITS2 sequence of the seven species reveals several homologous domains. Secondary structure predictions for the ITS2 region indicate that these domains base pair to form a core region central to several stem features. The sequence outside the ITS2 homologous domains tends to be GC-rich and characteristically slippage generated; these areas preserve or add to the stem length of the predicted secondary structures. These ITS2 intraspacer variable regions resemble previously described expansion segments of the 28S gene region. Evolutionary analysis of the ITS2 of these species, using both sequence and secondary structure information, leads to the prediction of divergence in the mosquito tribe Aedini that is not clearly reflected in current taxonomic designations.

Microepidemiology of onchocerciasis in Guatemala: dispersal and survival of Simulium ochraceum.

Wild Simulium ochraceum females, both blood engorged and non-blood engorged, were collected from human volunteers infected with Onchocerca volvulus, marked with fluorescent dyes, and released from the same locality as they were collected during February and March 1989. A small hyperendemic village located within 0.5 km of streams supporting large populations of S. ochraceum larvae served as the site for both collection and release of adult females. Fifteen sites for the recapture of flies were located within this same village, within two other villages located 1.0 and 3.7 km from it, and at other places spaced approximately 0.25-3.5 km within a coffee agroecosystem. Flies from both groups were recaptured at distances ranging to 3.5 km from the point of release. Non-blood-engorged flies, however, exhibited a greater tendency to disperse away from the release site. Of the total number of blood-engorged flies recaptured, 51.9% were collected at the release point, 25.7% at 1.0 km, and 1.6% at 3.3-3.5 km. The corresponding percentages for non-blood-engorged flies were 26.9%, 40.4%, and 4.4%, respectively. No flies from either group were recaptured at the most distant site, a large village that was 3.7 km away. Marked flies from both groups were recaptured 12-14 days after release, which was sufficient time for the development of infective O. volvulus larvae. A survival rate (4.7%) of marked, blood-engorged flies over the second and third gonotrophic cycles was estimated from the slope of the regression line of the log number of flies recaptured.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of Leishmania braziliensis from Lutzomyia ovallesi (Diptera:Psychodidae) in Guatemala.

Leishmania braziliensis is endemic in Guatemala and Belize in Central America. To help identify the vector(s) of this parasite in Guatemala, phlebotomine sand flies that were aspirated from the clothing of collectors at Tikal National Park in the Department of the Peten were examined for flagellates. Lutzomyia ovallesi was found infected with flagellates that were identified as L. braziliensis by isoenzyme electrophoresis. The isoenzyme profile of this isolate matched those from humans from the same area.

Species-diagnostic differences in a ribosomal DNA internal transcribed spacer from the sibling species Anopheles freeborni and Anopheles hermsi (Diptera:Culicidae).

Approximately 460 base pairs (bp) of DNA sequence that included the second internal transcribed spacer (ITS2) and some flanking 5.8S and 28S ribosomal RNA coding regions were compared between the two closely related and morphologically indistinguishable mosquito species Anopheles freeborni and A. hermsi and a third related species, A. occidentalis. Sequences were determined from 14 clones of polymerase chain reaction (PCR)-amplified DNA obtained from four colonies of A. freeborni, two colonies of A. hermsi, and one individual A. occidentalis. Four clones showed independent single bp differences from the consensus for the relevant species. Eleven sites differed between the consensus sequences of A. hermsi and A. freeborni; 28 sites differed between A. hermsi and A. occidentalis. With the exception of a single bp mismatch in the 5.8S and two single bp mismatches near the undetermined junction of the ITS2 and 28S regions, all differences were confined to the ITS2 region. A PCR-based species-diagnostic assay for the cryptic species A. hermsi and A. freeborni was developed; it uses four synthetic oligonucleotides, two derived from areas of interspecies sequence difference in the ITS2, and two derived from highly conserved regions in the flanking coding sequences. Small amounts of mosquito DNA amplified in the presence of these four primers produce fragments of diagnostic size for each species: 900 bp for A. freeborni, 350 bp for A. hermsi, and approximately 1.2-1.4 kb for various other Anopheles species tested. We believe that this general approach to the development of species-diagnostic assays can be extended easily to other complexes of closely related, morphologically indistinguishable species.

Diagnostic characterization of Anopheles freeborni and An. hermsi by hybrid crosses, frequencies of polytene X chromosomes and rDNA restriction enzyme fragments.

A polytene chromosome analysis was prepared from Anopheles freeborni collected from 25 locations in north and central California, and parts of Washington and Oregon. The X chromosome banding pattern, thought previously to be specific to An. hermsi, was common in mosquitoes collected from foothill regions in California, and in all samples from Washington and Oregon. At some of these locations, many mosquitoes had heterokaryotypes for the inversion that distinguishes the X chromosome of An. freeborni from that of An. hermsi. Use of rDNA restriction site analysis, and the results from crossing of different strains bearing either type of X chromosome, showed that An. hermsi does not have a unique or diagnostic X chromosome. Anopheles hermsi was collected in San Mateo County, CA, which is now the northernmost known limit of this species. Crossing studies, or the examination of rDNA restriction enzyme profiles, are presently the only means of identifying An. hermsi.

Susceptibility of Anopheles hermsi to Plasmodium vivax.

Two outbreaks of Plasmodium vivax malaria have occurred recently in southern California, and Anopheles hermsi, a newly described species closely related to A freeborni, has been implicated as the vector. To assess the competence of A. hermsi as a vector, its susceptibility to P. vivax was compared with that of the efficient vector A. freeborni by allowing 150 females of each species to feed to repletion on infected squirrel monkeys. Oocyst density was greater in A. hermsi than in A. freeborni, and the frequency and density of sporozoites were similar. A. hermsi was susceptible to P. vivax and readily supported development to the sporozoite stage.

Comparison of rDNA and mtDNA in the sibling species Anopheles freeborni and A. hermsi.

Comparison of the ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) of Anopheles freeborni and A. hermsi, 2 morphologically indistinguishable mosquito species in the North American A. maculipennis complex, revealed restriction enzyme site variation in both DNA families. Diagnostically useful interspecific differences in the rDNA were observed in the external transcribed spacer (ETS) and internal transcribed spacer (ITS) regions. The regions encoding rRNA, however, were indistinguishable with respect to the enzymes used. Intraspecific site and genome length variations were present in the mtDNA of 5 colonies of A. freeborni and 3 colonies of A. hermsi, but no species-specific differences were observed.

Development of a penile prosthesis. The Hydroflex.

The development and commercialization of a penile prosthesis is a long and involved process that is influenced by a large number of factors including the patient, surgeon, FDA, commercial viability, and competitive environment. The process is illustrated in this article by historically tracing some of the factors involved in the development of the American Medical Systems self-contained penile prosthesis, the Hydroflex. This device was introduced into unrestricted commercial distribution in May 1985.

Transmission of Onchocerca volvulus by secondary vectors in Guatemala.

A comprehensive study of the transmission of Onchocerca volvulus at 4 locations in Guatemala with different prevalence rates of onchocerciasis included observations on potential secondary vectors, the most prevalent of which were Simulium metallicum, S. callidum, and S. gonzalezi. Filariae encountered in S. metallicum were primarily of a Dipetalonema-like species, but third-stage larvae indistinguishable from O. volvulus were found in 4 flies of this species. Our findings suggest that O. volvulus may occasionally be transmitted by S. metallicum, but such transmission is likely limited to areas having both a high parasite prevalence maintained by S. ochraceum and a relatively high host-seeking density of S. metallicum. Two third-stage larvae that could not be differentiated from O. volvulus were found once in S. gonzalezi; however, transmission by this species appears to be inconsequential.

Vector density, parasite prevalence, and transmission of Onchocerca volvulus in Guatemala.

Transmission of Onchocerca volvulus at 4 locations with different prevalences of human onchocerciasis in the Atitlán region of Guatemala is described in relation to vector density and infection rates. The percentages of residents with skin biopsies positive for microfilariae of O. volvulus at these locations were 13.8%, 33.3%, 65.4%, and 89.6%. The following variables associated with transmission were calculated from our observations (the values are presented in an order that corresponds with the above prevalence rates): frequency of third-stage larvae (calculated on an annual basis) in parous Simulium ochraceum, 0, 0.004, 0.005, and 0.004; estimated daily biting density of S. ochraceum, 23, 24, 254, and 1,509 flies per day; and estimated annual infective biting density (based on S. ochraceum), 0, 18, 185, and 1,101 potentially infective bites per year. The frequencies of third-stage larvae are very small compared with those observed in Africa, and suggest that transmission of O. volvulus in Guatemala depends on high vector density. Locations with low, and perhaps tolerable, levels of onchocerciasis (less than 15% of female residents with skin biopsies positive for microfilariae) have mean daily biting densities for S. ochraceum of less than or equal to 24 flies, and infected residents normally have mean microfilarial densities of less than or equal to 3 microfilariae per mg of skin. Stratification of prevalence rates by age group proved useful for assessing current transmission within a village.

Biting activity of black flies in Guatemala: parity rates and differences between localities and habitats.

Host-seeking activity of anthropophilic black flies at 4 localities in Guatemala with different prevalence rates of onchocerciasis was assessed using human attractants and semimonthly catches over one year. Density of host-seeking Simulium ochraceum was greatest at the locality with the highest incidence of onchocerciasis and very reduced at the 2 localities with low levels of human infection. The overall percentage of parous host-seeking S. ochraceum at the 4 localities ranged from 41%-49%. Host-seeking activity appeared to be concentrated near streams containing immature stages, and few females dispersed as far as 3 km away. S. metallicum was the second most frequently captured species; however, at the locality with the highest prevalence of onchocerciasis, its host-seeking density was much less than that of S. ochraceum. For S. metallicum, the overall percentage of parous females ranged from 28%-34% at the 4 localities. S. metallicum were consistently taken in much greater numbers in coffee cultivation areas than in housing areas. Host-seeking S. callidum and S. gonzalezi also were captured.

Seasonality of adult black flies and Onchocerca volvulus transmission in Guatemala.

Transmission of Onchocerca volvulus, principally by Simulium ochraceum, was studied over a 14-month period in the Atitlán region of Guatemala. Semimonthly catches of black flies were made on human volunteers at 4 localities with different prevalences of human onchocerciasis. Host-seeking activity of S. ochraceum reached its greatest magnitude in the early dry season (October-January) but then declined rapidly and was lowest during the late dry season (February-May). The frequency of O. volvulus larvae in parous host-seeking S. ochraceum also varied seasonally, and this variation was most pronounced for third stage larvae. At a hyperendemic locality, the highest frequency of this larval stage occurred during the February-March period (0.0142 in 1979 and 0.0095 in 1980). From June-January, the frequency of third stage larvae in S. ochraceum was less than 0.003. The frequency of early first stage larvae exhibited the least seasonal variation, ranging from 0.0354 in August-September to 0.0628 in April-May. The transmission rate of O. volvulus by S. ochraceum also varied seasonally. At the same hyperendemic locality, infective biting density of S. ochraceum attained its greatest magnitude in February-March. The survival rate of female S. ochraceum from one gonotrophic cycle to the next was estimated from the ratio of flies with early first stage larvae to those with infective stage larvae. These rates varied seasonally and ranged from 0.2132 to 0.3974, with the highest rates occurring in the late dry season.