First isolation of microorganisms from the gut diverticulum of Aedes aegypti (Diptera: Culicidae): new perspectives for an insect-bacteria association

We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fed Aedes aegypti was dissected under aseptic conditions, homogenized and plated on BHI medium. Microbial isolates were identified by sequencing of 16S rDNA for bacteria and 28S rDNA for yeast. A direct DNA extraction from Ae. aegypti gut diverticulum was also performed. The bacterial isolates were: Bacillus sp., Bacillus subtilis and Serratia sp. The latter was the predominant bacteria found in our isolations. The yeast species identified was Pichia caribbica.

Systematics of Anopheles lanei Galvão and Amaral, a species of the subgenus Nyssorhynchus Blanchard (Diptera: Culicidae)

Anopheles (Nyssorhynchus) lanei Galvão and Amaral is here redescribed using morphological characteristics of adult, male and female, fourth instar larva and pupa. The larva, pupa, and male genitalia are illustrated. Diagnostic morphological characters of adults, male genitalia, fourth instar larva and pupa are provided to distinguish An. lanei from other species of the Argyritarsis section. Species distribution data are based on the published literature records and bionomics data are based on both literature records and field data.

Diverse population dynamics of three Anopheles species belonging to the Triannulatus Complex (Diptera: Culicidae)

The Triannulatus Complex of Anopheles (Nyssorhynchus) consists of at least three sibling species, namely Anopheles triannulatus s.s., Anopheles halophylus and a third undescribed member herein referred to as An. triannulatus "C". Sympatric anophelines belonging to species complexes, even though closely related, may exploit different environments such as larval habitats. In this paper we hypothesize that rainfall and seasonal flooding would distinctly influence the availability of larval habitats and consequently the seasonal population dynamics of sympatric members of the Triannulatus Complex. A reflection of this is distinct seasonal biting frequencies exhibited by three members of the Triannulatus Complex at a site in Central Brazil. Population dynamics seem to be influenced by the water level in the local rivers, although biting frequency of all three species was negatively influenced by rainfall. An. triannulatus s.s. was more abundant following the end of the rainy season, but notably 30 to 60 days after flooding. On the other hand, An. halophylus and An. triannulatus C peaked during the middle of the dry season, when water impoundments have no inflow, are somewhat reduced in size and the water becomes brackish. Differences in population dynamics were greater between An. triannulatus s.s. and An. halophylus and An. triannulatus C than between An. halophylus and An. triannulatus C. This might reflect differences in larval habitat exploitation and therefore spatial segregation among these members of the complex.

Morphological analysis of three populations of Anopheles (Nyssorhynchus) nuneztovari Gabaldón (Diptera: Culicidae) from Colombia

Based on the results of comparative analyses of 1,039 specimens of several progenies of Anopheles nuneztovarifrom three localities in Colombia, eight costal wing spot patterns were observed. Patterns I and III were the most frequent: 77.96% and 11.36%, respectively. Using the diagnostic characters ratio of the length of the basal dark area of hind tarsomere II/length of hind tarsomere II, ratio of the length of the humeral pale spot/length of the pre-humeral dark spot, and the ratio of the length of the subcostal pale spot/length of the distal sector dark spot (DS-III2/Ta-III2, HP/PHD, SCP/DSD) approximately 5% of the adult females were misidentified as a species of Nyssorhynchus, different from An. nuneztovari. Approximately 5% of the specimens showed DS-III2/Ta-III2 ratio less than 0.25 (range 0.21 - 0.24), and among them 3.34% shared a HP/PHD ratio less than 1.50. Consequently, 1.52% of An. nuneztovari individuals can be misidentified as Anopheles oswaldoi. In those specimens with the DS-III2/Ta-III2 ratios higher than 0.25, 34.45% displayed SCP/DSD values greater than 0.50 and of these, 3.65% displayed HP/PHD values greater than 1.8. This combination of characters could lead one to misidentify samples of An. nuneztovari as Anopheles rangeli. Similarly, 2.43% of the females could be identified erroneously as either Anopheles aquasalis or Anopheles benarrochi. Individuals with a HP/PHD ratio greater than 2.0, could be misidentified as Anopheles trinkae, Anopheles strodei or Anopheles evansae. A distinct combination of diagnostic characters for An. nuneztovari from Colombia is proposed.

Characterization of the 3-HKT gene in important malaria vectors in India, viz: Anopheles culicifacies and Anopheles stephensi (Diptera: Culicidae)

The 3-hydroxykynurenine transaminase (3-HKT) gene plays a vital role in the development of malaria parasites by participating in the synthesis of xanthurenic acid, which is involved in the exflagellation of microgametocytes in the midgut of malaria vector species. The 3-HKT enzyme is involved in the tryptophan metabolism of Anophelines. The gene had been studied in the important global malaria vector, Anopheles gambiae. In this report, we have conducted a preliminary investigation to characterize this gene in the two important vector species of malaria in India, Anopheles culicifacies and Anopheles stephensi. The analysis of the genetic structure of this gene in these species revealed high homology with the An. gambiae gene. However, four non-synonymous mutations in An. stephensi and seven in An. culicifacies sequences were noted in the exons 1 and 2 of the gene; the implication of these mutations on enzyme structure remains to be explored.

Anopheles darlingi bionomics and transmission of Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae in Amerindian villages of the Upper-Maroni Amazonian forest, French Guiana

French Guiana is one of the areas in South America most affected by malaria and where the disease has become a serious public health problem. In spite of this situation, little recent entomological data are available from the main localities where the disease occurs, even though they are crucial for development of an effective vector control strategy. A longitudinal entomological survey was carried out from March 2000-February 2002 in three Amerindian villages, namely Twenké, Taluène and Cayodé, located in the Amazonian forest of the Upper-Maroni area, to assess anopheline mosquitoes and malaria transmission dynamics. Anopheles darlingi (Diptera: Culicidae) was the most abundant mosquito species caught during the study. This efficient American malaria vector was active the entire year, but showed an evident peak of abundance during the main rainfall season, from April-June, with an average human biting rate of 255.5 bites per person per night. Parity rates were homogeneous all year, indicating no significant seasonal variability in female survival rates. Estimated vectorial capacity indices were higher during the rainy season, even though the risk of transmission was present throughout the year (VCI > 1). A total of 14 An. darlingi were found infected with Plasmodium falciparum, Plasmodium vivax or Plasmodium malariae. The annual circumsporozoite indices were 0.15, 0.14 and 0.05, and the entomological inoculation rates were 22.8, 27.4 and 14.4 infected bites per person per year in Twenké, Taluène and Cayodé, respectively. An. darlingiwas endo-exophagic and rather exophilic in these localities. The species was collected throughout the night but was more aggressive between 21:30-03:30 h and after 05:30 h. Parity rates were homogeneous during the entire night. Impregnated hammock and/or bed nets, coupled with the use of mosquito repellents, as well as the early treatment of malarial cases, appear to be the most suitable tools for fighting malaria in these Amerindian villages since the spraying of residual insecticides is inefficient because of vector biology and the housing structure.

Resurrection of Anopheles goeldii from synonymy with Anopheles nuneztovari (Diptera, Culicidae) and a new record for Anopheles dunhami in the Brazilian Amazon

Nucleotide sequences of the internal transcribed spacer 2 (ITS2) rDNA and partial sequences of the cytochrome coxidase subunit I (COI) mtDNA and white gene nDNA were obtained from specimens of Anopheles nuneztovari A collected in Macapá (state of Amapá), Óbidos, Prainha and Almeirim (state of Pará), Itacoatiara and Parintins (state of Amazonas), Brazil, and compared with previously published sequences of A. nuneztovari s.l. Results of the Bayesian phylogenetic analyses performed using either COI or combined ITS2, COI and white gene sequences suggest that An. nuneztovari B/C is distinct from specimens obtained in the Amazonas/Solimões River basin. Anopheles goeldii, currently in synonymy with An. nuneztovari, was described from individuals collected in Belterra (= Fordlândia) in the Tapajós River, state of Pará, Southern Amazonas River. Morphological comparisons of the characteristics of the male genitalia indicated that An. nuneztovari A and An. goeldii are similar but distinct from An. nuneztovariB/C by the apex of the aedeagus. In considering the results of the phylogenetic analyses and morphological comparisons, An. goeldii is resurrected from synonymy with An. nuneztovari. Additionally, Anopheles dunhamiis reported for the first time in Parintins. This species can be distinguished from An. goeldiiby characters of the male genitalia and molecular data.

Night and crepuscular mosquitoes and risk of vector-borne diseases in areas of piassaba extraction in the middle Negro River basin, state of Amazonas, Brazil

A study of crepuscular and night-biting mosquitoes was conducted at remote settlements along the Padauiri River, middle Negro River, state of Amazonas, Brazil. Collections were performed with human bait and a CDC-light trap on three consecutive days per month from June 2003-May 2004. In total, 1,203 h of collection were performed, of which 384 were outside and 819 were inside houses. At total of 11,612 specimens were captured, and Anophelinae (6.01%) were much less frequent than Culicinae (93.94%). Anopheles darlingi was the most frequent Anophelinae collected. Among the culicines, 2,666 Culex (Ae.) clastrieri Casal & Garcia, 2,394 Culex. (Mel.) vomerifer Komp, and 1,252 Culex (Mel.) eastor Dyar were the most frequent species collected. The diversity of insects found reveals the receptivity of the area towards a variety of diseases facilitated by the presence of vectors involved in the transmission of Plasmodium, arboviruses and other infectious agents.

Embryonic development of Aedes aegypti (Diptera: Culicidae): influence of different constant temperatures

Despite its vector importance little attention is given to Aedes aegypti embryonic development. In this study, temperature influence on time course of Ae. aegypti larvae hatching and egg viability were evaluated. The dormancy state at the end of embryogenesis could be interrupted with a proper stimulus. Temperatures tested ranged between 12-36°C; the maximum temperature limit is 35°C and the minimum one is below 12°C. Egg viability between 16-31°C was above 80%. The definition of physiological embryonic parameters at this temperature range corroborates Ae. aegypti presence on tropical and subtropical world regions.

Karyotypic variation and geographic distribution of Anopheles campestris-like (Diptera: Culicidae) in Thailand

Seventy-one isolines of Anopheles campestris-like were established from wild-caught females collected from human-biting and animal-biting traps at 12 locations in Thailand. All isolines had an average branch summation of seta 2-VI pupal skins ranging from 20.3-30.0 branches, which is in the range of An. campestris (17-58 branches). They showed three different karyotypes based on the amount of extra heterochromatin in the sex chromosomes, namely Forms B (X2, Y2), E (X1, X2, X3, Y5) and a new karyotypic Form F (X2, X3, Y6). Form B has been found only in Chaing Mai and Kamphaeng Phet populations, while Forms E and F are widely distributed throughout the species range. Genetic crosses between the 12 isolines, which were arbitrarily selected as representatives of An. campestris-like Forms B, E and F, revealed genetic compatibility that provided viable progeny through F2 generations, suggesting a conspecific nature of these karyotypic forms. These results are supported by the very low intraspecies variation (genetic distance < 0.005) of ITS2, COI and COII from genomic DNA of the three karyotypic forms.

Seasonal dynamics of Aedes aegypti (Diptera: Culicidae) in the northernmost state of Brazil: a likely port-of-entry for dengue virus 4

Roraima is the northernmost state of Brazil, bordering both Venezuela and Guyana. Appropriate climate and vector conditions for dengue transmission together with its proximity to countries where all four dengue serotypes circulate make this state, particularly the capital Boa Vista, strategically important for dengue surveillance in Brazil. Nonetheless, few studies have addressed the population dynamics of Aedes aegypti in Boa Vista. In this study, we report temporal and spatial variations in Ae. aegypti population density using ovitraps in two highly populated neighbourhoods; Centro and Tancredo Neves. In three out of six surveys, Ae. aegypti was present in more than 80% of the sites visited. High presence levels of this mosquito suggest ubiquitous human exposure to the vector, at least during part of the year. The highest infestation rates occurred during the peak of the rainy seasons, but a large presence was also observed during the early dry season (although with more variation among years). Spatial distribution of positive houses changed from a sparse and local pattern to a very dense pattern during the dry-wet season transition. These results suggest that the risk of dengue transmission and the potential for the new serotype invasions are high for most of the year.

Development of physiological resistance and its stage specificity in Culex quinquefasciatus after selection with deltamethrin in Assam, India

The study investigated the development and stage specificity of physiological resistance to insecticides in a colony of Culex quinquefasciatus Say (Diptera: Culicidae) mosquitoes, which are vectors of bancroftian filariasis in India, after selection with deltamethrin. Resistance was selected by exposing the larvae to the concentration of deltamethrin that caused 50% mortality in the tested population (i.e., LC50). Under continuous selection pressure, the LC50 increased steadily in subsequent generations. The estimated LC50 for the F0 generation was 0.409 μg/L; the LC50 first displayed a substantial increase in the F5 generation (5.616 μg/L) and reached 121.902 μg/L in the F10 generation. The objective of this study was to establish a deltamethrin-resistant colony to develop a research programme that will study the evolution of physiological resistance patterns and stage-specific resistance responses in Cx. quinquefasciatus larvae and adults under laboratory conditions. An approximately 298-fold increase in resistance was recorded after 10 generations, as evidenced by the resistance ratio (RR50). The progress and effect of the selection pressure in the adult stage was monitored with the World Health Organisation (WHO) diagnostic test. The mortality, as observed using the WHO diagnostic test, declined significantly from the F5 generation (85%) onwards and the highest rate of survival (65%) was observed in the F10 generation.

The Anopheles albitarsis complex with the recognition of Anopheles oryzalimnetes Wilkerson and Motoki, n. sp. and Anopheles janconnae Wilkerson and Sallum, n. sp. (Diptera: Culicidae)

The Anopheles (Nyssorhynchus) albitarsis complex includes six species: An. albitarsis, Anopheles oryzalimnetes Wilkerson and Motoki, n. sp., Anopheles marajoara, Anopheles deaneorum, Anopheles janconnae Wilkerson and Sallum, n. sp. and An. albitarsis F. Except for An. deaneorum, species of the complex are indistinguishable when only using morphology. The problematic distinction among species of the complex has made study of malaria transmission and ecology of An. albitarsis s.l. difficult. Consequently, involvement of species of the An. albitarsis complex in human Plasmodium transmission is not clear throughout its distribution range. With the aim of clarifying the taxonomy of the above species, with the exception of An. albitarsis F, we present comparative morphological and morphometric analyses, morphological redescriptions of three species and description of two new species using individuals from populations in Brazil, Paraguay, Argentina and Venezuela. The study included characters from adult females, males, fourth-instar larvae, pupae and male genitalia of An. albitarsis, An. marajoara, An. deaneorum and An. oryzalimnetes n. sp. For An. janconnae n. sp. only characters of the female, male and male genitalia were analyzed. Fourth-instar larvae, pupae and male genitalia characteristics of all five species are illustrated. Bionomics and distribution data are given based on published literature records.

Container productivity, daily survival rates and dispersal of Aedes aegypti mosquitoes in a high income dengue epidemic neighbourhood of Rio de Janeiro: presumed influence of differential urban structure on mosquito biology

Different urban structures might affect the life history parameters of Aedes aegypti and, consequently, dengue transmission. Container productivity, probability of daily survival (PDS) and dispersal rates were estimated for mosquito populations in a high income neighbourhood of Rio de Janeiro. Results were contrasted with those previously found in a suburban district, as well as those recorded in a slum. After inspecting 1,041 premises, domestic drains and discarded plastic pots were identified as the most productive containers, collectively holding up to 80% of the total pupae. In addition, three cohorts of dust-marked Ae. aegypti females were released and recaptured daily using BGS-Traps, sticky ovitraps and backpack aspirators in 50 randomly selected houses; recapture rate ranged from 5-12.2% within cohorts. PDS was determined by two models and ranged from 0.607-0.704 (exponential model) and 0.659-0.721 (non-linear model), respectively. Mean distance travelled varied from 57-122 m, with a maximum dispersal of 263 m. Overall, lower infestation indexes and adult female survival were observed in the high income neighbourhood, suggesting a lower dengue transmission risk in comparison to the suburban area and the slum. Since results show that urban structure can influence mosquito biology, specific control strategies might be used in order to achieve cost-effective Ae. aegypti control.

Bromeliad-inhabiting mosquitoes in an urban botanical garden of dengue endemic Rio de Janeiro - Are bromeliads productive habitats for the invasive vectors Aedes aegypti and Aedes albopictus?

Immatures of both Aedes aegypti and Aedes albopictus have been found in water-holding bromeliad axils in Brazil. Removal of these plants or their treatment with insecticides in public and private gardens have been undertaken during dengue outbreaks in Brazil despite uncertainty as to their importance as productive habitats for dengue vectors. From March 2005-February 2006, we sampled 120 randomly selected bromeliads belonging to 10 species in a public garden less than 200 m from houses in a dengue-endemic neighborhood in Rio de Janeiro. A total of 2,816 mosquito larvae and pupae was collected, with an average of 5.87 immatures per plant per collection. Culex (Microculex) pleuristriatus and Culex spp of the Ocellatus Group were the most abundant culicid species, found in all species of bromeliads; next in relative abundance were species of the genus Wyeomyia. Only two individuals of Ae. aegypti (0.07%) and five of Ae. albopictus(0.18%) were collected from bromeliads. By contrast, immatures of Ae. aegypti were found in manmade containers in nearly 5% of nearby houses. These results demonstrate that bromeliads are not important producers of Ae. aegypti and Ae. albopictus and, hence, should not be a focus for dengue control. However, the results of this study of only one year in a single area may not represent outcomes in other urban localities where bromeliads, Ae. aegypti and dengue coincide in more disturbed habitats.