GloPID-R report on chikungunya, o'nyong-nyong and Mayaro virus, part 5: Entomological aspects.

The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) chikungunya (CHIKV), o'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group has been established to investigate natural history, epidemiology and clinical aspects of infection by these viruses. Here, we present a report dedicated to entomological aspects of CHIKV, ONNV and MAYV. Recent global expansion of chikungunya virus has been possible because CHIKV established a transmission cycle in urban settings using anthropophilic vectors such as Aedes albopictus and Aedes aegypti. MAYV and ONNV have a more limited geographic distribution, being confined to Africa (ONNV) and central-southern America (MAYV). ONNV is probably maintained through an enzootic cycle that has not been characterized yet, with Anopheles species as main vectors and humans as amplification hosts during epidemics. MAYV is transmitted by Haemagogus species in an enzootic cycle using non-human primates as the main amplification and maintenance hosts, and humans becoming sporadically infected when venturing in or nearby forest habitats. Here, we focused on the transmission cycle and natural vectors that sustain circulation of these viruses in their respective locations. The knowledge of the natural ecology of transmission and the capacity of different vectors to transmit these viruses is crucial to understand CHIKV emergence, and to assess the risk that MAYV and ONNV will expand on wide scale using anthropophilic mosquito species not normally considered primary vectors. Finally, the experts identified knowledge gaps and provided adapted recommendations, in order to address future entomological investigations in the right direction.

Mosquitoes in Bromeliads at Ground Level of the Brazilian Atlantic Forest: the Relationship Between Mosquito Fauna, Water Volume, and Plant Type.

Water accumulating in the axils of bromeliads provides habitat for numerous invertebrates, frequently among them, immature mosquitoes. To evaluate mosquito richness in bromeliads and the relationship between mosquito presence and biotic and abiotic variables, we performed a study in the Parque Nacional do Itatiaia, Rio de Janeiro, Brazil. Mosquitoes of genus Culex were the most abundant and varied in species richness, among which nine belonged to subgenus Microculex, Culex (Microculex) neglectus Lutz and Culex ocellatus Theobald being the most frequent species. Sabethines of genera Wyeomyia and Runchomyia were found in low numbers. Wyeomyia (Spilonympha) airosai Lane and Cerqueira and Wyeomyia (Spilonympha) finlayi Lane and Cerqueira tend to proliferate in bromeliads of the genus Bilbergia which hold less than 50 ml of water and grow either alone or with Runchomyia frontosa (Theobald). The larger the volume of water, the greater the chance of finding Culex, Anopheles as well as Wyeomyia (Phoniomyia) species, which seems to be the more generalist as it is present in different bromeliad types with a large range of plant water holding capacities.

Lower survival rate, longevity and fecundity of Aedes aegypti (Diptera: Culicidae) females orally challenged with dengue virus serotype 2.

As the pathogenic effects of a parasite on its hosts can strongly influence its epidemiology, we compared the life-histories of dengue virus serotype 2 (DENV-2)-infected and uninfected Aedes aegypti females. Unexposed mosquitoes lived longer than exposed ones, but those infected lived longer than exposed but negative (as assayed by Real-Time quantitative Reverse Transcription PCR [qRT-PCR]) mosquitoes. Infected mosquitoes from a long-established laboratory colony presented more viral RNA copies at death than those from the F1-generation of a field population from Rio de Janeiro. The mortality of infected colony-mosquitoes was independent of the number of viral RNA copies at death, whereas in the field population, longevity decreased with the number of viral RNA copies, suggesting that F1 of field mosquitoes are less tolerant to infection than the laboratory-colony. Infected females had a lower fecundity than controls. F1 of field mosquitoes were more likely to lay eggs than the colony; egg-laying success was strongly affected by mosquito age for both mosquito populations: from 49.28 in the first clutch to 20.7 in the fifth. Overall, DENV-2 reduced Aedes aegypti survival and fecundity, clearly affecting vectorial capacity and consequently transmission intensity.

Influence of the spatial distribution of human hosts and large size containers on the dispersal of the mosquito Aedes aegypti within the first gonotrophic cycle.

It is generally accepted that Aedes aegypti (L.) (Diptera: Culicidae) has a short dispersal capacity, and that displacement can be influenced by the availability of oviposition sites in the surroundings of emergence or release sites. In the present article, we observed the influence of spatial heterogeneity of large containers and human hosts on the cumulative flight direction of Ae. aegypti females during the first gonotrophic cycle, testing the hypothesis that they aggregate in resource-rich areas, i.e. where there are higher concentrations of large containers and/or humans per habitation. We analysed data from pupal surveys and mark-release-recapture experiments (non-blood-fed females were released) carried out in two dengue endemic neighbourhoods of Rio de Janeiro, Brazil: Tubiacanga (a suburb, with a human density of 337 inhabitants/ha) and Favela do Amorim (a slum, with a human density of 901 inhabitants/ha). In both areas, host-seeking females of three different release cohorts showed an overall non-uniform and extensive dispersal from their release point within 1-2 days post-release. At 4-5 days post-release, when many of the released females would be expected to be gravid, in Tubiacanga most mosquitoes were collected in areas with a relatively higher density of containers/premise, independently of the density of residents/house, whereas in Favela do Amorim, almost half of the captured mosquitoes were collected in relatively resource-poorer areas. Although Ae. aegypti dispersal patterns varied between sites, overall the distances travelled from the release point and the cumulative flight directions were correlated with the density of containers and hosts, more markedly in Tubiacanga than in Favela do Amorim.

Measuring mosquito diversity patterns in an Amazonian terra firme rain forest.

We reanalyzed a dataset consisting of approximately 10,700 crepuscular and night-biting female mosquitoes (Culicidae) collected over 12 mo in the canopy and understorey of primary Amazonian rain forest. We investigate whether vertical habitat stratification and rainfall modified major ecological parameters of this mosquito ensemble, combining descriptive and hypothesis-testing statistics with species richness and diversity metrics in the analyses. A total of 31 species was recorded. Contrary to expectations, the host-seeking mosquito fauna was less diverse in the forest canopy than in the understorey. In particular, species diversity and evenness were higher in understorey samples, whereas species richness estimates were similar in both habitats. Only two out of 12 species tested for vertical stratification were clearly acrodendrophilic, and five preferred understorey habitats. The mosquito fauna was more diverse in the rainy than in the dry season. We propose the hypothesis that female mosquito density and host defensive behavior may promote host seeking in nonpreferred habitats by acrodendrophilic mosquito species. These results may be particularly relevant for understanding the dynamics of Plasmodium malariae/brasilianum and arboviral infections in Amazonian forested landscapes.

Temporal distribution of Aedes aegypti in different districts of Rio de Janeiro, Brazil, measured by two types of traps.

Dengue dynamics in Rio de Janeiro, Brazil, as in many dengue-endemic regions of the world, is seasonal, with peaks during the wet-hot months. This temporal pattern is generally attributed to the dynamics of its mosquito vector Aedes aegypti (L.). The objectives of this study were to characterize the temporal pattern of Ae. aegypti population dynamics in three neighborhoods of Rio de Janeiro and its association with local meteorological variables; and to compare positivity and density indices obtained with ovitraps and MosquiTraps. The three neighborhoods are distinct in vegetation coverage, sanitation, water supply, and urbanization. Mosquito sampling was carried out weekly, from September 2006 to March 2008, a period during which large dengue epidemics occurred in the city. Our results show peaks of oviposition in early summer 2007 and late summer 2008, detected by both traps. The ovitrap provided a more sensitive index than MosquiTrap. The MosquiTrap detection threshold showed high variation among areas, corresponding to a mean egg density of approximately 25-52 eggs per ovitrap. Both temperature and rainfall were significantly related to Ae. aegypti indices at a short (1 wk) time lag. Our results suggest that mean weekly temperature above 22-24 degrees C is strongly associated with high Ae. aegypti abundance and consequently with an increased risk of dengue transmission. Understanding the effects of meteorological variables on Ae. aegypti population dynamics will help to target control measures at the times when vector populations are greatest, contributing to the development of climate-based control and surveillance measures for dengue fever in a hyperendemic area.

Body size-associated survival and dispersal rates of Aedes aegypti in Rio de Janeiro.

The daily survival rate of Aedes aegypti (L) (Diptera: Culicidae) is one of the most important parameters in mathematical models of dengue transmission. In this report, we evaluate the effect of adult body size on the survival and dispersal rates of male and female Ae. aegypti, the primary dengue vector in Brazil. Independent of larval diet (i.e. size), the daily survival rate of females recaptured in the field was greater than that of males (smaller: t = 5.617; d.f. = 15; P < 0.05; larger: t = 4.241; d.f. = 16; P < 0.05). Larger males lived longer than smaller males (t = 2.2893; d.f. = 7; P < 0.05), but no size effect was observed for females (t =- 0.086; d.f. = 19; P= 0.932). The daily survival rate of smaller females was similar to that of larger females (0.712 and 0.730, respectively, as calculated by an exponential model, and 0.743 and 0.779, respectively, calculated by a non-linear model), and they dispersed further than larger females (mean distances travelled were 78.8 m and 40.9 m, respectively; t =- 10.22; d.f. = 323; P < 0.05). Adult body size did not influence male dispersal distances (t = 0.904; d.f. = 206; P= 0.367). Given our evidence that smaller females appear to have similar lifespans and evidence from other studies that they bite more frequently during a single gonotrophic cycle than larger females, our results suggest that smaller females have a higher vectorial capacity.

Molecular confirmation of the specific status of Anopheles halophylus (Diptera: Culicidae) and evidence of a new cryptic species within An. triannulatus in central Brazil.

Anopheles halophylus Silva-do-Nascimento & Lourenço-de-Oliveira was recently described using morphological and biological variants in specimens previously identified as Anopheles triannulatus (Neiva & Pinto). Because these two species occur in sympatry in central Brazil, we used allozymes to determine the extent of gene flow to confirm that they are different species. Of 11 allozyme loci analyzed, one (Mpi) was found to be diagnostic for An. halophylus and An. triannulatus, confirming their specific status. This locus revealed a second sibling species within An. triannulatus sensu lato. An. halophylus and the new undescribed species were confirmed using random amplified polymorphic DNA markers that showed moderate genetic divergence among these three sympatric and closely related taxa (D = 0.145-0.428). Moreover, this marker indicates that An. halophylus and the new species are more closely related to each other than either is to An. triannulatus.

Amazonian malaria vector anopheline relationships interpreted from ITS2 rDNA sequences.

Species identification of anopheline mosquitoes (Diptera: Culicidae) can be problematic because many of them belong to complexes of morphologically similar species, often with contrasted ecology, behaviour and vectorial importance. The application of DNA-based diagnostics has proved to be useful for distinguishing between such species. We determined ribosomal DNA sequences of the second internal transcribed spacer (ITS2) from samples of 16 species of Anopheles captured in the Amazon Basin, Brazil. Length of the ITS2 varied from 323 to 410 base pairs, with GC content ranging from 50.7% to 66.5% and sequence identity from 25% to 99% between species. Maximum-likelihood paup analysis separated two distinct groups of species conforming with the recognized subgenera Anopheles (represented by eiseni, mattogrossensis, mediopunctatus and peryassui) and Nyssorhynchus (represented by 12 spp.). For the latter group, the neighbour-joining tree generated from rDNA sequence ITS2 relationships is compatible with the morphological taxonomic key established for these Amazonian species: albitarsis, aquasalis, benarrochi, braziliensis, darlingi, deaneorum, dunhami, evansae, nuneztovari, oswaldoi, rangeli and triannulatus. These ITS2 sequence data proved to be a useful tool for species identification and, potentially, to solve taxonomic problems.

Aedes aegypti in Brazil: genetically differentiated populations with high susceptibility to dengue and yellow fever viruses.

Aedes aegypti was eliminated from Brazil in 1955, but re-infested the country in the 1970s. Dengue outbreaks have occurred since 1981 and became endemic in several cities in Brazil after 1986. Urban yellow fever has not occurred since 1942, and only jungle yellow fever cases have been reported. A population genetic analysis using isoenzyme variation combined with an evaluation of susceptibility to both yellow fever and dengue 2 viruses was conducted among 23 A. aegypti samples from 13 Brazilian states. We demonstrated that experimental infection rates of A. aegypti for both dengue and yellow fever viruses (YFV) are high and heterogeneous, and samples collected in the endemic and transition areas of sylvatic yellow fever were highly susceptible to yellow fever virus. Boa Vista, a border city between Brazil and Venezuela, and Rio de Janeiro in the Southeast region are considered as the most important entry points for dengue dissemination. Considering the high densities of A. aegypti, and its high susceptibility to dengue and yellow fever viruses, the risk of dengue epidemics and yellow fever urbanization in Brazil is more real than ever.

[Epidemiological research in Brazil]. / A pesquisa em epidemiologia no Brasil.

The current epidemiological research in Brazil is described. Secondary data sources were consulted, such as the year 2000 database of the Brazilian Directory of Research Groups and the National Board of Scientific and Technological Development (CNPq). The criterion to identify a group as a research one relies on the existence of at least one research line in the field of epidemiology, as defined by the group leader. After identifying the defined universe of epidemiological research, which included 176 groups and 320 different research lines, the following issues were presented and discussed: the relationships between research financing and health research, focusing on CAPES (Coordination Center for the Advance of University Professionals) graduation programs, public health research and epidemiological research, geographic and institutional distribution and outreach of the current epidemiological research, the researchers and students directly participating in epidemiological research, research topics and patterns of disseminating research findings; the journals where papers in its fullness were published; the financial support of the epidemiological research focusing on the 23 officially recognized graduate programs in public health field.

[Frequency of Aedes aegypti and Aedes albopictus larvae and pupae in traps, Brazil]. / Freqüência de larvas e pupas de Aedes aegypti e Aedes albopictus em armadilhas, Brasil.

OBJECTIVE: To evaluate the monthly frequency of larvae and pupae of Aedes albopictus, Aedes aegypti and other mosquitoe species in tires, and the influence of environmental factors on that. METHODS: The immature stages of mosquitoes were collected monthly from four tires in the municipality of Nova Iguaçu, Brazil, from November 1997 to October 1998. The following variables were measured: rainfall, temperature, water volume, water pH. The tires were arranged in a pyramid, one at the base (tire 1) and 3 others (2,3 e 4) laying over it. RESULTS: Were collected 10,310 larvae and 612 pupae. Aedes albopictus was the most common species in both the larval and pupal stages. Aedes aegypti and Aedes albopictus were collected throughout the year but were more frequent during in the rainy season. The number of Aedes aegypti was significantly correlated with the temperature, rainfall and water volume of the tires. The correlation between water pH and number of larvae was not significant. Aedes albopictus larvae were more frequent in tires left in the shade. CONCLUSIONS: Aedes albopictus was more abundant in tires than Aedes aegypti. Discarded tires seem to be an important source of both Aedes species throughout the year. The favored environmental conditions of the tires, such as water volume and exposure to sunlight differ for Aedes albopictus and Aedes aegypti.

Vectorial competence of Aedes aegypti (Linnaeus 1762) Rio de Janeiro strain, to Dirofilaria immitis (Leidy 1856).

Dirofilaria immitis (Leidy 1856), a nematode parasite, is the etiologic agent of canine heartworm disease and mosquitoes are essential intermediate hosts. Mosquito susceptibility to the worms differ with species, strains and also among individuals of the same strain. To evaluate the degree of susceptibility of Rio de Janeiro laboratory raised strain of Aedes aegypti, we fed mosquitoes on canine blood with different densities of microfilariae (mf). There was no significant difference in the rate of development among the three different densities of mf. Infective larvae were found in the head and proboscis of all mosquitoes provided bloodmeals with different densities of mf after the 11th day post-infection. The infection rate of mosquitoes after ingestion of blood containing 3,000 mf/ml, 5,000 mf/ml and 7,000 mf/ml were 55.3%, 66.7% and 100%, respectively. The vector efficiency indices ranged from 1.6 to 9.3. The finding of L3 stage larvae, high infection rates and vector efficiency indices suggest that Ae. aegypti, Rio de Janeiro laboratory strain, is a potential vector of D. immitis, although of low efficiency.

The subgenus Dendromyia theobald: a review with redescriptions of four species (Diptera: Culicidae).

A morphological study of larval, pupal and adult life stages (including genitalia characters) was performed in order to evaluate the classification of the subgenus Dendromyia, genus Wyeomyia Theobald. Six species are included: Wy. ypsipola Dyar, Wy. jocosa (Dyar & Knab), Wy. testei Senevet & Abonnenc, Wy. complosa (Dyar), Wy. luteoventralis Theobald and Wy. trifurcata Clastrier, the first four of which are redescribed. The descriptions include illustrations of the male and female genitalia, fourth-instar larva and pupa. The result firmly supports Dendromyia as a monophyletic group, with well defined characters. Keys for the identification of all life stages of species included in Dendromyia are provided.

Trypanosomes of non-human primates from the National Centre of Primates, Ananindeua, State of Pará, brazil.

Trypanosome infections were sought in 46 non-human primates captured principally in Amazonian Brazil. Twenty-two (47.8%) were infected with four Trypanosoma species: T. cruzi, T. minasense, T. devei and T. rangeli. These preliminary results confirmed the high prevalence and diversity of natural infections with trypanosomes in primates from Brazilian Amazon and were the first formal record of simian infections with trypanosomes in the State of Acre. The presence of T. cruzi-like and T. rangeli-like parasites are recorded in four new hosts.

Sequence analysis of the second internal transcribed spacer of ribosomal DNA in Anopheles oswaldoi (Diptera: Culicidae).

Sequence divergence in the second internal transcribed spacer (ITS2) of ribosomal DNA was examined for female specimens of Anopheles oswaldoi Peryassu from 7 localities in South America. The lengths of ITS2 for all mosquitoes ranged from 348 to 356 nucleotides. After alignment of these sequences, similarity ranged from 87 to 100%. Divergence was within the range of inter-specific differences for members of anopheline species complexes. Therefore, specimens were placed into 4 groups that may correspond to at least 4 cryptic species. One is probably related to An. oswaldoi sensu stricto and another to Anopheles konderi Galvão & Damasceno. The other 2 groups may correspond to species for which morphological identification remains to be clarified. These data provide evidence that An. oswaldoi comprise a complex of cryptic species and that DNA identification may help to resolve the taxonomic questions related to this group.

Polymorphism in trypomastigotes of Trypanosoma (Megatrypanum) minasense in the blood of experimentally infected squirrel monkey and marmosets.

Experimental infections by Trypanosoma (Megatrypanum) minasense were performed in primates - Saimiri sciureus and Callithrix penicillata - with the objective of searching for morphological variations of the blood trypomastigotes with respect to hosts and time of infection. We carried out morphological and morphometric analysis of blood trypomastigotes. Illustrations are given. Both the squirrel monkey and marmoset became infected after the injection of blood trypomastigotes of T. minasense, although the parasitaemia were briefer in the squirrel monkey. The parasites detected in the later host were narrower and shorter than those found in the inoculated marmoset. In the marmoset, the blood stream parasites derived from culture metacyclic trypomastigotes were considerably smaller than those derived from the inoculation of infected blood. Stronger evidence of polymorphism was found when, at the same time of infection, the blood trypomastigotes found in squirrel monkey had smaller length, body width and the distance from posterior end of the body to the kinetoplast almost four times smaller than the parasite found in the marmoset. Therefore, conflicting results on morphology and morphometry of T. minasense obtained by previous investigators could be due to polymorphism.

[Canine heartworm on São Luís Island, Northeastern Brazil: a potential zoonosis]. / Dirofilariose canina na Ilha de São Luís, Nordeste do Brasil: uma zoonose potencial.

A survey on the prevalence of canine heartworm was conducted in 1, 495 dogs from Maranhão Island, State of Maranhão, Northeastern Brazil, from 1991 to 1994, by testing for microfilariae in blood. Of the total, 1,358 (12.8% of which were infected) were dogs with no known history; they included 1,265 homeless animals (10.3% with microfilariae) and 93 kept by owners at the time the survey was conducted (37.8% of which were infected). Prevalence is high among dogs captured and/or living along the seashore. Examples of such high coastal prevalence rates were found in Olho d'Agua and Calhau (46% and 43%, respectively). The search for microfilariae in blood samples of 137 exclusively domiciliary dogs with a known history showed that 43% of these animals were infected, confirming transmission of heartworm on the island. This was the first survey formally published on canine dirofilariasis in Northeastern Brazil. Since D. immitis is infective to man and prevalence of the parasite is high, particularly along the coast of Maranhão Island, human cases of dirofilariasis may be expected. Local physicians should consider this parasite among the possible causes of solitary lesions in humans living in this area.

Wyeomyia (Prosopolepis) confusa (Lutz): subgeneric validation, species description, and recognition of Wyeomyia flui (Bonne-Wepster and Bonne) as the senior synonym of Wyeomyia kerri del Ponte and Cerqueira.

Prosopolepis Lutz is validated as a monotypic subgenus of Wyeomyia Theobald and the type species, Weomyia confusa (Lutz), is redescribed. The description includes illustrations of the male and female genitalia, the 4th-stage larva, and the pupa. Prosopolepis flui Bonne-Wepster and Bonne is resurrected from synonymy with Wy. confusa and recognized as the senior synonym of Wyeomyia kerri del Ponte and Cerqueira. Wyeomyia flui does not belong in the subgenus Prosopolepis and remains in the genus Wyeomyia without subgeneric placement. Trichoprosopon pusillum Lutz and Nuñez-Tovar is not synonymous with Wy. confusa and is provisionally regarded as a nomen dubium within Wyeomyia. The identity of Wy. confusa is fixed by neotype selection.