Novel molecular evidence of population structure in Anopheles (Kerteszia) bellator from Brazilian Atlantic Forest.

Anopheles bellator is a primary malaria vector in the Atlantic Forest. Partial sequences of timeless and Clock genes were used to assess the genetic differentiation of five Brazilian populations, which showed strong population structure (e.g. high F ST values and fixed differences) in all pairwise comparisons between Bahia sample and the others from Paraná, São Paulo and Rio de Janeiro states. Also, the resulting phylogenetic trees clearly grouped the sequences from Bahia in a different cluster with high bootstrap values. Among southern and southeastern populations low levels of genetic differentiation were found suggesting a general stability of the genetic structure.

Novel molecular evidence of population structure in Anopheles (Kerteszia) bellator from Brazilian Atlantic Forest

Anopheles bellator is a primary malaria vector in the Atlantic Forest. Partial sequences of timeless and Clock genes were used to assess the genetic differentiation of five Brazilian populations, which showed strong population structure (e.g. high F ST values and fixed differences) in all pairwise comparisons between Bahia sample and the others from Paraná, São Paulo and Rio de Janeiro states. Also, the resulting phylogenetic trees clearly grouped the sequences from Bahia in a different cluster with high bootstrap values. Among southern and southeastern populations low levels of genetic differentiation were found suggesting a general stability of the genetic structure.

Cryptic diversity in an Atlantic Forest malaria vector from the mountains of South-East Brazil.

BACKGROUND: Anopheles (Kerteszia) cruzii is the primary vector of human and simian malarias in Brazilian regions covered by the Atlantic Rainforest. Previous studies found that An. cruzii presents high levels of behavioural, chromosomal and molecular polymorphisms, which led to the hypothesis that it may be a complex of cryptic species. Here, An. cruzii specimens were collected in five sites in South-East Brazil located at different altitudes on the inner and coastal slopes of two mountain ranges covered by Atlantic Rainforest, known as Serra do Mar and Serra da Mantiqueria. Partial sequences for two genes (Clock and cpr) were generated and compared with previously published sequences from Florianópolis (southern Brazil). Genetic diversity was analysed with estimates of population structure (F ST ) and haplotype phylogenetic trees in order to understand how many species of the complex may occur in this biome and how populations across the species distribution are related. RESULTS: The sequences from specimens collected at sites located on the lower coastal slopes of Serra do Mar (Guapimirim, Tinguá and Sana) clustered together in the phylogenetic analysis, while the major haplotypes from sites located on higher altitude and at the continental side of the same mountains (Bocaina) clustered with those from Serra da Mantiqueira (Itatiaia), an inner mountain range. These two An. cruzii lineages showed statistically significant genetic differentiation and fixed characters, and have high F ST values typical of between species comparisons. Finally, in Bocaina, where the two lineages occur in sympatry, we found deviations from Hardy-Weinberg equilibrium due to a deficit of heterozygotes, indicating partial reproductive isolation. These results strongly suggest that at least two distinct lineages of An. cruzii (provisorily named "Group 1" and "Group 2") occur in the mountains of South-East Brazil. CONCLUSIONS: At least two genetically distinct An. cruzii lineages occur in the Atlantic Forest covered mountains of South-East Brazil. The co-occurrence of distinct lineages of An. cruzii (possibly incipient species) in those mountains is an interesting biological phenomenon and may have important implications for malaria prevalence, Plasmodium transmission dynamics and control.

Analysis of the Effect of Cyclophosphamide and Methotrexate on Chrysomya megacephala (Diptera: Calliphoridae).

Forensic entomotoxicology investigates the effects of chemical substances in the development of scavenger insects and the reflection on estimating the minimum postmortem interval (PMI). To evaluate the impact of the chemotherapeutic drugs cyclophosphamide (CF) and methotrexate (MTX) on the postembryonic development of Chrysomya megacephala, controlled experiments were performed by rearing the larvae on spiked minced beef with different concentrations of these drugs. The results indicated that CF significantly decreased their developmental rate up to 28 h but had no significant effect on larval and adult sizes, survival rate, and sex ratio, whereas MTX decreased larval and adult sizes, survival rate, and there was a deviation in the expected sex ratio toward females in MTX-exposed larvae but had no significant impact on developmental rate. These negative interference factors should be considered in cases of suspected death of people that have undergone chemotherapy.

Ectoparasites of the critically endangered insular cavy, Cavia intermedia (Rodentia: Caviidae), southern Brazil.

Cavia intermedia is a rodent species critically endangered and is found only on a 10 hectare island off the southern Brazilian coast. To identify the ectoparasites of C. intermedia, 27 specimens (14 males and 13 females), representing approximately 65% of the estimated total population, were captured and examined. A total of 1336 chewing lice of two species were collected: Gliricola lindolphoi (Amblycera: Gyropidae) and Trimenopon hispidum (Amblycera: Trimenoponidae). In addition, chiggers Arisocerus hertigi (Acari: Trombiculidae) and Eutrombicula sp. (Acari: Trombiculidae) were collected from the ears of all captured animals. This low species richness compared to those for other Cavia species is expected for island mammals. Although the results presented here are not conclusive about the relationship between C. intermedia and ectoparasites, this low species richness found might be reflected in a low level of investment by the hosts in the basal immune defense, since investments in white blood cell production by mammals are influenced by the diversity of parasites in the environment. Additionally, considering that it might result in host vulnerability to other parasites that might be introduced through exotic or migratory host species, the monitoring of C. intermedia, including parasitological and immunological assessments, is recommended as a key component of conservation efforts.

Updated canine infection rates for Dirofilaria immitis in areas of Brazil previously identified as having a high incidence of heartworm-infected dogs.

BACKGROUND: Canine heartworm infections were frequently diagnosed in Brazil before the new millennium. After the year 2000, the frequency of diagnosis showed a sharp decline; however, a few years later, new evidence indicated that the parasite was still present and that canine infection rates seemed to be increasing. Therefore, an updated survey of canine heartworm prevalence was conducted in several locations in south, southeast, and northeast Brazil. METHODS: Dogs from 15 locations having previously reported a high prevalence of heartworm infection were included in the survey according to defined criteria, including the absence of treatment with a macrocyclic lactone for at least 1 year. Blood samples from 1531 dogs were evaluated by an in-clinic immunochromatography test kit (Witness® Heartworm, Zoetis, USA) for detection of Dirofilaria immitis antigen. At each location, epidemiologic data, including physical characteristics and clinical signs reported by owners or observed by veterinarians, were recorded on prepared forms for tabulation of results by location, clinical signs, and physical characteristics. RESULTS: The overall prevalence of canine heartworm infection was 23.1%, with evidence of heartworm-infected dogs detected in all 15 locations studied. There was a tendency for higher prevalence rates in environmentally protected areas, despite some locations having less-than-ideal environmental temperatures for survival of vector mosquitoes. Among physical characteristics, it was noted that dogs with predominantly white hair coats and residing in areas with a high (≥20%) prevalence of heartworm were less likely to have heartworm infection detected by a commercial heartworm antigen test kit than were dogs with other coat colors. In general, dogs older than 2 years were more frequently positive for D. immitis antigen than were younger dogs. Clinical signs of heartworm infections were rare or owners were unable to detect them, and could not be used for reliable prediction of the presence of heartworm. CONCLUSIONS: These results indicate that the prevalence of D. immitis has increased in these areas of Brazil over the past few years. Small animal practitioners in these areas should include routine screening tests for heartworm infections in every dog's annual evaluation protocol and make sure to have uninfected dogs on prevention.

Characterization of Trypanosoma cruzi isolated from humans, vectors, and animal reservoirs following an outbreak of acute human Chagas disease in Santa Catarina State, Brazil.

During March 2005, 24 cases of acute human Chagas disease were detected in Santa Catarina State, southern Brazil, all of them related to the ingestion of Trypanosoma cruzi-contaminated sugar cane juice. Following field studies allowed the isolation of 13 T. cruzi strains from humans, opossums (Didelphis aurita and Didelphis albiventris), and vectors (Triatoma tibiamaculata). The isolated strains were characterized by multilocus enzyme electrophoresis (MLEE) and analysis of the spliced-leader and 24Salpha rRNA genes. The assays revealed that all strains isolated from humans belong to the TcII group but revealed a TcII variant pattern for the phosphoglucomutase enzyme. Strains isolated from opossums also showed a TcI profile in all analysis, but strains isolated from triatomines revealed a mixed TcI/TcII profile by MLEE. No indication of the presence of Trypanosoma rangeli was observed in any assay. Considering that mixed strains (TcI/TcII) were isolated from triatomines in an area without active vectorial transmission to humans and that all strains isolated from humans belong to the TcII group, our results show that T. cruzi TcI and TcII groups are circulating among reservoirs and vectors in southern Brazil and indicate that selection toward TcII group in humans may occur after ingestion of a mixed (TcI/TcII) T. cruzi population.

Isoenzimatic analysis of four Anopheles (Kerteszia) cruzii (Diptera: Culicidae) populations of Brazil.

Anopheles cruzii is a small sylvatic mosquito and primary human Plasmodium vector in Southern Brazil. The distribution of this bromeliad-breeding mosquito follows the Atlantic forest coastal distribution, where bromeliads are abundant. Morphological, genetic, and molecular polymorphisms among different populations have been reported and it has recently been suggested that An. cruzii is a complex of cryptic species. The aim of this work is to analyze the gene flow between different populations of An. cruzii collected in four localities within the geographic distribution range of the species, and to examine if An. cruzii is a complex of cryptic species. The genetic distances show that populations of the states of Santa Catarina, Sao Paulo, and Rio de Janeiro are genetically closer (0.032 to 0.083) than populations of Bahia (0.364 to 0.853) based on profiles from 10 distinct isoenzyme loci. The Fst was lower (0.077) when the Bahia population was excluded than when it was included (0.300) in the analyses. The inferred number of migrants per generation was 2.99 individuals among populations from the states of Santa Catarina, Sao Paulo, and Rio de Janeiro and 0.58 migrants per generation among all populations. Results suggest that An. cruzii is a complex of species and that the specimens of state of Bahia can be considered as belonging to a species that is distinct from other three closely-related populations studied.

Isoenzimatic analysis of four Anopheles (Kerteszia) cruzii (Diptera: Culicidae) populations of Brazil

Anopheles cruzii is a small sylvatic mosquito and primary human Plasmodium vector in Southern Brazil. The distribution of this bromeliad-breeding mosquito follows the Atlantic forest coastal distribution, where bromeliads are abundant. Morphological, genetic, and molecular polymorphisms among different populations have been reported and it has recently been suggested that An. cruzii is a complex of cryptic species. The aim of this work is to analyze the gene flow between different populations of An. cruzii collected in four localities within the geographic distribution range of the species, and to examine if An. cruzii is a complex of cryptic species. The genetic distances show that populations of the states of Santa Catarina, São Paulo, and Rio de Janeiro are genetically closer (0.032 to 0.083) than populations of Bahia (0.364 to 0.853) based on profiles from 10 distinct isoenzyme loci. The Fst was lower (0.077) when the Bahia population was excluded than when it was included (0.300) in the analyses. The inferred number of migrants per generation was 2.99 individuals among populations from the states of Santa Catarina, São Paulo, and Rio de Janeiro and 0.58 migrants per generation among all populations. Results suggest that An. cruzii is a complex of species and that the specimes of state of Bahia can be considered as belonging to a species that is distinct from other three closely-related populations studied.

Isoenzymatic analysis of four Anopheles (Kerteszia) bellator Dyar & Knab (Diptera: Culicidae) populations

Anopheles bellator is a small silvatic bromelia-breeding mosquito and is a primary human malaria vector species in Southern Brazil. The bromelia-breeding habitat of the species should accompany the Atlantic forest coastal distribution, where bromeliads are abundant. Nonetheless, records on An. bellator collections show a gap in the species geographical distribution. An. bellator has been recorded in Southern Brazil and in the Brazilian states of Bahia and Paraíba. It appers again in the island of Trinidad, in Trinidad and Tobago. The aim of this work was to measure gene flow between different populations of An. bellator collected in the northern and southern extremes of the geographic distribution of this species. Mosquitoes were captured in forest borders in Santa Catarina, São Paulo, and Bahia states in Brazil and in the island of Trinidad in Republic of Trinidad and Tobago. Genetic distances varied between 0.076 and 0.680, based on enzymatic profiles from 11 distinct isoenzymes. Results indicate the existence of low-level gene flow between Brazilian populations of An. bellator, and a gene flow was even lower between the Brazilian and the Trinidad populations. This finding lead us to hypothesize that An. bellator did not spread along the coast, but reached northeastern areas through inland routes.

Isoenzymatic analysis of four Anopheles (Kerteszia) bellator Dyar & Knab (Diptera: Culicidae) populations.

Anopheles bellator is a small silvatic bromelia-breeding mosquito and is a primary human malaria vector species in Southern Brazil. The bromelia-breeding habitat of the species should accompany the Atlantic forest coastal distribution, where bromeliads are abundant. Nonetheless, records on An. bellator collections show a gap in the species geographical distribution. An. bellator has been recorded in Southern Brazil and in the Brazilian states of Bahia and Paraíba. It appears again in the island of Trinidad, in Trinidad and Tobago. The aim of this work was to measure gene flow between different populations of An. bellator collected in the northern and southern extremes of the geographic distribution of this species. Mosquitoes were captured in forest borders in Santa Catarina, São Paulo, and Bahia states in Brazil and in the island of Trinidad in Republic of Trinidad and Tobago. Genetic distances varied between 0.076 and 0.680, based on enzymatic profiles from 11 distinct isoenzymes. Results indicate the existence of low-level gene flow between Brazilian populations of An. bellator, and a gene flow was even lower between the Brazilian and the Trinidad populations. This finding lead us to hypothesize that An. bellator did not spread along the coast, but reached northeastern areas through inland routes.

Trapping Triatominae in silvatic habitats.

Large-scale trials of a trapping system designed to collect silvatic Triatominae are reported. Live-baited adhesive traps were tested in various ecosystems and different triatomine habitats (arboreal and terrestrial). The trials were always successful, with a rate of positive habitats generally over 20% and reaching 48.4% for palm trees of the Amazon basin. Eleven species of Triatominae belonging to the three genera of public health importance (Triatoma, Rhodnius and Panstrongylus) were captured. This trapping system provides an effective way to detect the presence of triatomines in terrestrial and arboreal silvatic habitats and represents a promising tool for ecological studies. Various lines of research are contemplated to improve the performance of this trapping system.

Trapping Triatominae in silvatic habitats

Large-scale trials of a trapping system designed to collect silvatic Triatominae are reported. Live-baited adhesive traps were tested in various ecosystems and different triatomine habitats (arboreal and terrestrial). The trials were always successful, with a rate of positive habitats generally over 20 percent and reaching 48.4 percent for palm trees of the Amazon basin. Eleven species of Triatominae belonging to the three genera of public health importance (Triatoma, Rhodnius and Panstrongylus) were captured. This trapping system provides an effective way to detect the presence of triatomines in terrestrial and arboreal silvatic habitats and represents a promising tool for ecological studies. Various lines of research are contemplated to improve the performance of this trapping system