Immmunological clearance of Batrachochytrium dendrobatidis infection at a pathogen-optimal temperature in the hylid frog Hypsiboas crepitans.

Amphibian declines worldwide have been linked to the fungal disease chytridiomycosis. Its causative agent (Batrachochytrium dendrobatidis, hereafter Bd), however, also infects many nondeclining species. Experimental infections have shown species-specific and temperature-dependent frog responses to Bd infection. Although Bd infection may be eliminated by housing amphibians at temperatures above those tolerated by the fungus, the question of whether frogs can eliminate infection under more favorable conditions remains unanswered. Repeated diagnostics using real-time polymerase chain reaction (rt-PCR) assays of postmetamorphic individuals at 28, 38, 45, 53, and 62 days after exposure demonstrated that Hypsiboas crepitans is able to clear infection within a few weeks at 23°C. Thus, we demonstrate a temperature-independent and likely immunological mechanism for the clearance of Bd in a resistant amphibian species. Future studies are needed to determine the generality of this mechanism among amphibians and to describe the immune factors affecting different outcomes of Bd exposure including resistance to infection, tolerance of infection, and clearance of infection.

Ability of cellulose acetate and polyacrylamide enzyme electrophoresis to separate 13 species of phlebotomine sand flies (Diptera: Psychodidae) from Venezuela.

The detection of cryptic species by biochemical methods indicates that within the phlebotomine fauna morphological data are not always adequate for species diagnosis. Cellulose acetate and polyacrylamide enzyme electrophoresis methods were compared for their effectiveness in identifying 13 species of Venezuelan phlebotomine sand flies and resolving alleles. Eight diagnostic loci unambiguously separated these 13 species of sand flies. Although acrylamide was as effective as cellulose acetate in species separation, differences were detected in the resolution of some alleles. Cellulose acetate identified more alleles at Ak and Fum, and resolved better at Pgm, whereas acrylamide identified more alleles at Gpi, Mdh, and Me. Therefore, erroneous species diagnoses may occur, if diagnostic loci detected by one technique are used by a second technique without adequate reference standards.

Phlebotomine sandflies in Venezuela. V. Review of the genus Brumptomyia (Diptera: Psychodidae), with description of the female of Brumptomyia devenanzii, re-description of the male and isozymatic profile.

Recently collected data on the Venezuelan species of the genus Brumptomyia are used to produce an updated review of these sandflies. At present, four species are recognized in Venezuela: B. devenanzii, B. beaupertuyi, B. avellari and B. pintoi. A key for the males is given and the geographical distribution of each of these species is outlined. The previously unknown female of B. devenanzii is described, the male is re-described, and the genetic variability (based on 11 enzymatic loci) of this species and of B. beaupertuyi (a sympatric species in Rancho Grande, the type locality of B. devenanzii) are reported. Fixed allelic differences in one diagnostic locus (adenylate kinase; Ak), between sympatric and allopatric populations, allowed for the unequivocal separation of both sexes of B. beaupertuyi from those of B. devenanzii. Significant inter-specific differences were also detected in the allele frequencies of malate dehydrogenase (Mdh-2) and decarboxylating malate dehydrogenase (Me). For B. devenanzii, mean heterozygosity and mean number of alleles per locus ranged from 2.0%-3.1% and 1.1-1.5, respectively. The corresponding values for B. beaupertuyi were 3.8% and 1.2.

Gene flow between natural and domestic populations of Lutzomyia longipalpis (Diptera: Psychodidae) in a restricted focus of American visceral leishmaniasis in Venezuela.

The epidemiology of the visceral leishmaniasis in the Americas is associated with both a natural and a domestic cycle. The existence of reproductively isolated populations of Lutzomyia longipalpis (Lutz & Neiva), and the scarcity of records of this species from natural habitats in areas where it has been associated with domestic habitats indicated that natural populations could be genetically distinct from domestic ones. Therefore, we compared the genetic structure and estimated the gene flow between L. longipalpis from domestic and peridomestic habitat and from an adjacent undisturbed natural environment along a 1.2-km transect. The analyses were performed on electrophoretic data from eight isozyme loci. The absence of fixed differences in the diagnostic loci Ak and Hk indicated that all specimens belonged to one of the two cryptic species identified in Venezuela. The average number of alleles per locus ranged from 2.0 to 2.9 and the average heterozygosity ranged from 7.8 to 13.4%. No differences were detected in the genetic structure of this species from domestic or peridomestic habitats and those trapped as far as 1.2 km from human dwellings. Nm, estimated from Wright's Fst, indicated that at least 208 individuals per generation migrated between the peridomestic habitat and a 1.2-km distant point to maintain the observed similarities in allelic frequencies. This high rate of gene flow indicated that this species has high migration rates between domestic and natural environments, and has the potential to transport for Leishmania from natural to domestic environments.

A possible role of bats as a blood source for the Leishmania vector Lutzomyia longipalpis (Diptera: Psychodidae).

Some evidence suggests that bats may provide an alternative blood source for Lutzomyia longipalpis, the main vector of American visceral leishmaniasis. Feeding trials were conducted to determine whether L. longipalpis feeds on captive bats. The high feeding success indicated that L. longipalpis is capable of feeding on at least four species of bats. Implications for the epidemiology of leishmaniases are discussed.

Occurrence of sibling species of Lutzomyia longipalpis (Diptera: Psychodidae) in Venezuela: first evidence from reproductively isolated sympatric populations.

The delimitation of cryptic species within the main vector of the American visceral leishmaniasis, Lutzomyia longipalpis, remains a topic of controversy. An analysis of genetic variability based on 8 enzymatic loci revealed fixed differences in 2 diagnostic loci, adenylate kinase (Ak) and hexokinase (Hk), between sympatric and allopatric populations at 4 localities in Venezuela. The absence of heterozygotes for these 2 loci within 1 locality indicates, for the first time, the presence of 2 sympatric reproductively isolated populations or cryptic species within L. longipalpis. Significant differences were also detected between these cryptic species in the allele frequencies of glucose-6-phosphate isomerase (Gpi) and malate dehydrogenase, decarboxylating (Me). One species showed mean heterozygosities that ranged between 6.6% and 6.7%, with 1.6-1.9 alleles detected per locus, while the other had mean heterozygosities that ranged from 4.3% to 6.3%, with 1.3-1.6 alleles per locus. Comparisons of isozyme profiles with published data suggests that 1 species is similar to the L. longipalpis described in Colombian and Brazilian populations, whereas the other has not been previously reported.

Population genetic structure of a three-host tick, Amblyomma dissimile, in eastern Venezuela.

Patterns of genetic variation for the tick Amblyomma dissimile were analyzed from a total of 200 ticks collected on 12 toads (Bufo marinus), 14 snakes (Boa constrictor), and 8 lizards (Iguana iguana) at 11 localities. The analyses were performed on electrophoretic data from 8 isozyme loci. Mean heterozygosity per locus was 6% (+/-3.1) per population. Differences in allelic frequencies among ticks from different individual hosts were the major source of genetic variability in this study. Host species was a smaller source of genetic variation. Genetic distances between localities varied according to which host species was present in each locality, and these appeared to be related to the extent of habitat overlap between host species. The smallest genetic distances between samples from different host species were recorded for I. iguana and B. constrictor. In contrast, the genetic distances between tick samples from B. marinus and either of the reptile species were significantly larger than between tick samples from this amphibian species. Ecological variables or the geographic distance did not explain the local patterns of differentiation observed in A. dissimile. Major genetic differences between island and mainland sites (0.03702) suggested an association between genetic distances and geographic isolation. The consistency between patterns of genetic variation and those of host home range overlap suggests that host dispersion is the main force structuring the genetic variation within this tick species.

Genetic markers for the identification of two tick species, Amblyomma dissimile and Amblyomma rotundatum.

Genetic markers are described for 2 species of reptile and amphibian ticks, Amblyomma dissimile and Amblyomma rotundatum, by allozyme electrophoresis. Fixed allelic differences in 4 out of the 8 examined loci allowed the unequivocal separation of both of these species. A strong correlation was found between these genetic markers and the relative size of the spurs in coxae IV but not with the punctuation pattern of the scutum. Moreover, no overlap was found in the distribution of relative spur sizes between samples of both species. The percent polymorphic loci and the mean percent heterozygosity per locus for A. rotundatum was considerably lower than that for A. dissimile. Differences in the amount of genetic variability may be related to their modes of reproduction.