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.

Early determination of the reproductive number for vector-borne diseases: the case of dengue in Brazil.

OBJECTIVE: To evaluate a new method of deriving the reproductive number for vector-borne diseases from the early epidemic curves for vector-borne diseases with incubations in the vectors and in the hosts. METHOD: We applied the model to several dengue epidemics in different climatic regions of Brazil: Brasilia, Belém, Fortaleza, Boa Vista. RESULTS: The new method leads to higher estimates of the reproductive number than previous models. CONCLUSION: At present, Aedes aegypti densities, the meeting of more compatible strains of viruses and mosquitoes, may lead to re-emergence of urban yellow fever epidemics.

Studies on populations of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) in Brazil.

Studies were performed on five Brazilian populations of Lutzomyia longipalpis: Salvaterra (PA), São José do Ribamar (MA), Canindé (CE), Natal (RN) and Gruta da Lapinha, Lagoa Santa (MG). No morphological differences were observed that could distinguish between these populations. Homogeneity tests showed that the allopatric populations display a certain heterogeneity and that the sympatric populations, with different patterns of spots, are homogeneous. The Student-Newman-Keuls test, represented by Euler-Venn diagrams, showed a disjunction between the populations from the north/northeast and the one from Gruta da Lapinha. Genetic distances between the four populations (excluding the Canindé population) were within the range of intrapopulational differences. The Gruta da Lapinha population displayed a heterozygotic deficiency that could be a consequence of high levels of inbreeding due to cryptic habits of living in a small cave. These results do not favor the hypothesis of a L. longipalpis species complex in Brazil, and the species should be considered high polymorphic.

Aspects related to productivity for four generations of a Lutzomyia longipalpis laboratory colony.

A closed colony of Lutzomyia longipalpis was established with specimens collected in the Raposa - Serra do Sol indian reservoir, one of the main foci of visceral leishmaniasis in the State of Roraima, Brazil. Biological observations were made on four generations of a L. longipalpis colony with emphasis on productivity. Aspects studied were the number of laid and retained eggs, and the number of adults (male and female) per generation. During the four generations the percentage of engorged females that laid eggs varied from 64.2% (third generation-F3) to 90.3% (second generation-F2). The mean number of eggs laid per female varied from 23.6 (F3) to 39. 9 (first generation-F1). The maximum number of eggs laid per female varied from 84 (F3) to 124 (F1). The mean number of retained eggs per female was 12.7 (parental generation-P and F1) to 22.1 (F2). The number of females exceeded the number of males in all generations. However, significant difference for male/female ratio was found only for F3. Fecundity rates were between 42.1 (F3) and 58.3 (F2). From a total of 439 blood-fed females, 355 females laid 12,257 eggs that yield 5,354 adults (2,525 males and 2,829 females) in four generations. F2 presented maximum productivity and fecundity rates.

Molecular population genetics of the primary neotropical malaria vector Anopheles darlingi using mtDNA.

Samples of the neotropical malaria vector Anopheles darlingi from Bolivia, Brazil, and Venezuela were analyzed to test for differences in mitochondrial haplotype frequencies. With the use of molecular variance components and F-statistics, significant genetic variability of An. darlingi was found apportioned primarily among populations within regions or within populations, with regions defined either as biomes (n = 5) or ecoregions (n = 2). The Mantel analysis resulted in a significant correlation [Prob (r) = 0.009] between genetic and geographic distances, evidence that these populations are genetically isolated by distance. Such isolation could reflect differences in phenotypes for factors affecting vector capacity.

Anopheles albitarsis eggs: ultrastructural analysis of chorion layers after permeabilization.

Construction of transgenic Anopheles mosquitoes refractory to Plasmodium requires knowledge of mosquito developmental biology. In order to study Anopheles embryology the removal or, alternatively, the permeabilization of the melanized and sclerotized egg chorion were attempted. The protocol classically used for chorion removal of Drosophila eggs was applied, with partial efficacy, to Anopheles albitarsis, a neotropical malaria vector. Each step was monitored by scanning electron microscopy and the results suggest differences in chorion composition between the two taxa. As an alternative to chorion removal, mosquito eggs were permeabilized with benserazide, an inhibitor of Dopa Decarboxylase, one of the enzymes needed for mosquito eggshell sclerotization. Embryo morphology and viability were not affected by this treatment. Permeabilization of the egg chorion allowed the ultrastructural observation of an internal homogeneous endochorion and an external compound exochorion, the latter consisting of a basal lamellar layer and protruding tubercles.

Mosquito embryos and eggs: polarity and terminology of chorionic layers.

The development of genetically modified vectors refractory to parasites is seen as a promising strategy in the future control of endemic diseases such as malaria. Nevertheless, knowledge of mosquito embryogenesis, a pre-requisite to the establishment of transgenic individuals, has been presently neglected. We have here studied the eggs from two neotropical malaria vectors. Eggs from Anopheles (Nyssorhynchus) albitarsis and Anopheles (Nyssorhynchus) aquasalis were analyzed by laser scanning microscopy and scanning electron microscopy and compared to those of Drosophila melanogaster. We verified basic conflicting data such as mosquito egg polarity and ultrastructure of eggshell layers. A 180 degrees rotation movement of the mosquito embryo along its longitudinal axis, a phenomenon not conserved among all Diptera, was confirmed. This early event is not taken into account by several present groups, leading to a non-consensual assignment of eggshell dorsal and ventral poles. Since embryo and egg polarities, defined during oogenesis, are the same, we propose to consider the flattened egg side as the dorsal one. The structure of Anopheles eggshell was also examined. Embryos are covered by a smooth endochorion or inner chorion layer. Outside this coat lies the compound exochorion or outer chorion layer, assembled by a thin basal lamellar layer and external tubercles. The terminology related to eggshell layers is discussed.

Cuticular hydrocarbons, isoenzymes and behavior of three populations of Anopheles darlingi from Brazil.

Three populations of Anopheles darlingi were studied for cuticular hydrocarbons, isoenzymes and patterns of peak biting activity. Differences were found in specimens from Costa Marques, a malaria endemic area; Dourado, a site with a very exophilic population and Juturnaíba, located near the type locality. Twelve hour collections from sunset to sunrise showed that An. darlingi from Costa Marques had a bimodal biting activity profile with a major peak at sunset and a minor peak at sunrise. At Dourado, the pattern was trimodal, with peaks at both morning and evening periods of twilight and near midnight. The Juturnaíba population showed a slight increase in activity near 2000 and 0100 h. Nei's genetic distances, determined by isoenzyme electrophoresis between pairs of populations, were low (D < or = 0.049). Using discriminant analysis for the cuticular hydrocarbons, 92.4% of the specimens from Costa Marques, 91.2% of the specimens from Dourado and 61.3% from Juturnaíba were correctly identified. Cuticular hydrocarbon and isoenzyme results matched very well: the smaller the Nei's distance, the more misidentifications occurred in the jackknife estimator used in the cuticular hydrocarbon analysis. This is the first report of cuticular hydrocarbon analysis in combination with isoenzymes to investigate neotropical anopheline species.