Germline transformation of Aedes fluviatilis (Diptera:Culicidae) with the piggyBac transposable element

The technique to generate transgenic mosquitoes requires adaptation for each target species because of aspects related to species biology, sensitivity to manipulation and rearing conditions. Here we tested different parameters on the microinjection procedure in order to obtain a transgenic Neotropical mosquito species. By using a transposon-based strategy we were able to successfully transform Aedes fluviatilis (Lutz), which can be used as an avian malaria model. These results demonstrate the usefulness of the piggyBac transposable element as a transformation vector for Neotropical mosquito species and opens up new research frontiers for South American mosquito vectors.

Long-term storage and viability of Ochlerotatus albifasciatus (Diptera: Culicidae)

The viability of Ochlerotatus albifasciatus (Macquart) eggs stored at room temperature and at 5ºC was studied over 31 months. After 12, 18 and 31 months of storage, eggs were acclimatized at 22ºC for ten days, and then inundated twice every seven days. The effect of the storage period on the percentage of hatching was analyzed by one way ANOVA. Differences on the hatching response between the first and second flooding were analyzed by paired t-test. Differences on the hatching response between the two storage conditions were analyzed by Mann-Whitney rank test. Results showed that (1) Oc. albifasciatus eggs were able to survive and hatch over 31 months; (2) the percent hatching of eggs stored at 5ºC was higher than that of eggs stored at room temperature; and (3) low temperatures and long periods without water favor installment hatching.

Mosquito traps designed to capture Aedes aegypti (Diptera: Culicidae) females: preliminary comparison of Adultrap, MosquiTRAP and backpack aspirator efficiency in a dengue-endemic area of Brazil

In this report, the efficiency of Adultrap under field conditions is compared to a CDC backpack aspirator and to MosquiTRAP. An urban dengue-endemic area of Rio de Janeiro was selected to evaluate the efficiency of mosquito traps in capturing Aedes aegypti females. Adultrap and aspirator captured similar numbers of Ae. aegypti females, with the former showing high specificity to gravid individuals (93.6%). A subsequent mark-release-recapture experiment was conducted to evaluate Adultrap and MosquiTRAP efficiency concomitantly. With a 6.34% recapture rate, MosquiTRAP captured a higher mean number of female Ae. aegypti per trap than Adultrap (Ç2 = 14.26; df = 1; p < 0,05). However, some MosquiTRAPs (28.12%) contained immature Ae. aegypti after 18 days of exposure in the field and could be pointed as an oviposition site for female mosquitoes. Both trapping methods, designed to collect gravid Ae. aegypti females, seem to be efficient, reliable and may aid routine Ae. aegypti surveillance.

Ecology of Haemagogus sp. and Sabethes sp. (Diptera: Culicidae) in relation to the microclimates of the Caxiuanã National Forest, Pará, Brazil

This study was conducted in a meteorological tower located in the Caxiuanã Forest (municipality of Melgaço, Pará, Brazil) with the aim of assessing the vertical stratification of species of Haemagogus and Sabethes, potential vectors of the yellow fever virus. To investigate the role of microclimates in mosquito stratification, bimonthly collections were conducted at ground level (0 m), 8 m, 16 m and 30 m (canopy level), with the aid of entomological nets and suction tubes, from July 2005-April 2006. A total of 25,498 mosquitoes were collected; specimens of Sabethes sp. and Haemagogus janthinomyswere found mostly at heights of 16 m and 30 m while Hg. leucocelaenus was most frequently observed at ground level. The largest number of vector species was collected during the rainiest months, but this difference between seasons was not statistically significant. However, the number of Hg. janthinomys was positively correlated with variations in temperature and relative humidity.

Species composition and natural infectivity of anthropophilic Anopheles (Diptera: Culicidae) in the states of Córdoba and Antioquia, Northwestern Colombia

Malaria is a serious health problem in the states of Córdoba and Antioquia, Northwestern Colombia, where 64.4% of total Colombian cases were reported in 2007. Because little entomological information is available in this region, the aim of this work was to identify the Anopheles species composition and natural infectivity of mosquitoes distributed in seven localities with highest malaria transmission. A total of 1,768 Anopheles mosquitoes were collected using human landing catches from March 2007-July 2008. Ten species were identified; overall, Anopheles nuneztovari s.l. was the most widespread (62%) and showed the highest average human biting rates. There were six other species of the Nyssorhynchus subgenus: Anopheles albimanus (11.6%), Anopheles darlingi (9.8%), Anopheles braziliensis (6.6%), Anopheles triannulatus s.l. (3.5%), Anopheles albitarsis s.l. and Anopheles oswaldoi s.l. at < 1%; and three of the Anopheles subgenus: Anopheles punctimacula, Anopheles pseudopunctipennis s.l. and Anopheles neomaculipalpusat < 1% each. Two species from Córdoba, An. nuneztovari and An. darlingi, were found to be naturally infected by Plasmodium vivax VK247, as determined by ELISA and confirmed by nested PCR. All species were active indoors and outdoors. These results provide basic information for targeted vector control strategies in these localities.

Molecular comparison of topotypic specimens confirms Anopheles (Nyssorhynchus) dunhami Causey (Diptera: Culicidae) in the Colombian Amazon

The presence of Anopheles (Nyssorhynchus) dunhami Causey in Colombia (Department of Amazonas) is confirmed for the first time through direct comparison of mtDNA cytochrome c oxidase I (COI) barcodes and nuclear rDNA second internal transcribed spacer (ITS2) sequences with topotypic specimens of An. dunhami from Tefé, Brazil. An. dunhami was identified through retrospective correlation of DNA sequences following misidentification as Anopheles nuneztovari s.l. using available morphological keys for Colombian mosquitoes. That An. dunhami occurs in Colombia and also possibly throughout the Amazon Basin, is of importance to vector control programs, as this non-vector species is morphologically similar to known malaria vectors including An. nuneztovari, Anopheles oswaldoi and Anopheles trinkae. Species identification of An. dunhami and differentiation from these closely related species are highly robust using either DNA ITS2 sequences or COI DNA barcode. DNA methods are advocated for future differentiation of these often sympatric taxa in South America.

Confirmation of Anopheles (Anopheles) calderoni Wilkerson, 1991 (Diptera: Culicidae) in Colombia and Ecuador through molecular and morphological correlation with topotypic material

The morphologically similar taxa Anopheles calderoni, Anopheles punctimacula, Anopheles malefactor and Anopheles guarao are commonly misidentified. Isofamilies collected in Valle de Cauca, Colombia, showed morphological characters most similar to An. calderoni, a species which has never previously been reported in Colombia. Although discontinuity of the postsubcostal pale spots on the costa (C) and first radial (R1) wing veins is purportedly diagnostic for An. calderoni, the degree of overlap of the distal postsubcostal spot on C and R1 were variable in Colombian specimens (0.003-0.024). In addition, in 98.2% of larvae, seta 1-X was located off the saddle and seta 3-C had 4-7 branches in 86.7% of specimens examined. Correlation of DNA sequences of the second internal transcribed spacer and mtDNA cytochrome c oxidase subunit I gene (COI) barcodes (658 bp of the COI gene) generated from Colombian progeny material and wild-caught mosquitoes from Ecuador with those from the Peruvian type series of An. calderoni confirmed new country records. DNA barcodes generated for the closely related taxa, An. malefactor and An. punctimacula are also presented for the first time. Examination of museum specimens at the University of the Valle, Colombia, revealed the presence of An. calderoni in inland localities across Colombia and at elevations up to 1113 m.

Potential impact of a presumed increase in the biting activity of dengue-virus-infected Aedes aegypti (Diptera: Culicidae) females on virus transmission dynamics

Recently, we showed that infection with dengue virus increases the locomotor activity of Aedes aegypti females. We speculate that the observed increased locomotor activity could potentially increase the chances of finding a suitable host and, as a consequence, the relative biting rate of infected mosquitoes. We used a mathematical model to investigate the impact of the increased locomotor activity by assuming that this activity translated into an increased biting rate for infected mosquitoes. The results show that the increased biting rate resulted in dengue outbreaks with greater numbers of primary and secondary infections and with more severe biennial epidemics.

Field optimisation of MosquiTRAP sampling for monitoring Aedes aegypti Linnaeus (Diptera: Culicidae)

A sticky trap designed to capture gravid Aedes (Stegomyia) aegypti mosquitoes, MosquiTRAP, has been evaluated for monitoring this species in Brazil. However, the effects of trap densities on the capture rate of Ae. aegypti females and the sensitivity of vector detection are still unknown. After a preliminary study has identified areas of high and low female mosquito abundance, a set of experiments was conducted in four neighbourhoods of Belo Horizonte (state of Minas Gerais, Brazil) using densities of 1, 2, 4, 8, 16, 32 and 64 traps per block. Trap sensitivity (positive MosquiTRAP index) increased significantly when 1-8 MosquiTRAPs were installed per block in both high and low abundance areas. A strong fit was obtained for the total number of mosquitoes captured with increasing trap densities through a non-linear function (Box-Lucas) (r² = 0,994), which likely exhibits saturation towards an equilibrium level. The capacity of the Mean Female Aedes Index to distinguish between areas of high and low Ae. aegypti abundance was also investigated; the achieved differentiation was shown to be dependent on the MosquiTRAP density.

Culicidae (Diptera) selection of humans, chickens and rabbits in three different environments in the province of Chaco, Argentina

Studies were conducted to determine the selection of humans, chickens and rabbits by Culicidae in three different environments in the province of Chaco, Argentina. Mosquitoes were collected fortnightly using cylindrical metal traps containing animal bait (chickens and rabbits). The mosquitoes were collected between June 2001-May 2002. During the same period and with the same frequency, mosquitoes biting the human operators of the traps were collected during the first 15 min of exposure within different time intervals: from 09:00 am-11:00 am, 01:00 pm-03:00 pm, 05:00 pm-07:00 pm and 09:00 pm-10:00 pm. A total of 19,430 mosquitoes of 49 species belonging to 10 genera were collected. Culex species mainly selected chicken bait and Wyeomyia species selected rabbit bait. Ochlerotatus and Psorophora species were more abundant in rabbit-baited traps. Anopheles triannulatus, Coquillettidia nigricans, Ochlerotatus scapularis, Mansonia titillans and Psorophora albigenu showed a strong attraction for human bait. The Anopheles, Coquillettidia, Culex and Mansonia species were more active between 05:00 pm-09:00 pm, while Ochlerotatus, Psorophora, Haemagogus and Wyeomyia were most active from 09:00 am-07:00 pm. This study provides additional information about the biology and ecology of arbovirus vectors in Chaco.

Characterisation of Culex quinquefasciatus (Diptera: Culicidae) larval habitats at ground level and temporal fluctuations of larval abundance in Córdoba, Argentina

The aims of this study were to characterise the ground-level larval habitats of the mosquito Culex quinquefasciatus, to determine the relationships between habitat characteristics and larval abundance and to examine seasonal larval-stage variations in Córdoba city. Every two weeks for two years, 15 larval habitats (natural and artificial water bodies, including shallow wells, drains, retention ponds, canals and ditches) were visited and sampled for larval mosquitoes. Data regarding the water depth, temperature and pH, permanence, the presence of aquatic vegetation and the density of collected mosquito larvae were recorded. Data on the average air temperatures and accumulated precipitation during the 15 days prior to each sampling date were also obtained. Cx. quinquefasciatus larvae were collected throughout the study period and were generally most abundant in the summer season. Generalised linear mixed models indicated the average air temperature and presence of dicotyledonous aquatic vegetation as variables that served as important predictors of larval densities. Additionally, permanent breeding sites supported high larval densities. In Córdoba city and possibly in other highly populated cities at the same latitude with the same environmental conditions, control programs should focus on permanent larval habitats with aquatic vegetation during the early spring, when the Cx. quinquefasciatus population begins to increase.

Mosquitoes of eastern Amazonian Ecuador: biodiversity, bionomics and barcodes

Two snapshot surveys to establish the diversity and ecological preferences of mosquitoes (Diptera: Culicidae) in the terra firme primary rain forest surrounding the Tiputini Biodiversity Station in the UNESCO Yasuní Biosphere Reserve of eastern Amazonian Ecuador were carried out in November 1998 and May 1999. The mosquito fauna of this region is poorly known; the focus of this study was to obtain high quality link-reared specimens that could be used to unequivocally confirm species level diversity through integrated systematic study of all life stages and DNA sequences. A total of 2,284 specimens were preserved; 1,671 specimens were link-reared with associated immature exuviae, all but 108 of which are slide mounted. This study identified 68 unique taxa belonging to 17 genera and 27 subgenera. Of these, 12 are new to science and 37 comprise new country records. DNA barcodes [658-bp of the mtDNA cytochrome c oxidase ( COI ) I gene] are presented for 58 individuals representing 20 species and nine genera. DNA barcoding proved useful in uncovering and confirming new species and we advocate an integrated systematics approach to biodiversity studies in future. Associated bionomics of all species collected are discussed. An updated systematic checklist of the mosquitoes of Ecuador (n = 179) is presented for the first time in 60 years.

Morphology of the larvae, male genitalia and DNA sequences of Anopheles (Kerteszia) pholidotus (Diptera: Culicidae) from Colombia

Since 1984, Anopheles (Kerteszia) lepidotus has been considered a mosquito species that is involved in the transmission of malaria in Colombia, after having been incriminated as such with epidemiological evidence from a malaria outbreak in Cunday-Villarrica, Tolima. Subsequent morphological analyses of females captured in the same place and at the time of the outbreak showed that the species responsible for the transmission was not An. lepidotus, but rather Anopheles pholidotus. However, the associated morphological stages and DNA sequences of An. pholidotus from the foci of Cunday-Villarrica had not been analysed. Using samples that were caught recently from the outbreak region, the purpose of this study was to provide updated and additional information by analysing the morphology of female mosquitoes, the genitalia of male mosquitoes and fourth instar larvae of An. pholidotus, which was confirmed with DNA sequences of cytochrome oxidase I and rDNA internal transcribed spacer. A total of 1,596 adult females were collected in addition to 37 larval collections in bromeliads. Furthermore, 141 adult females, which were captured from the same area in the years 1981-1982, were analysed morphologically. Ninety-five DNA sequences were analysed for this study. Morphological and molecular analyses showed that the species present in this region corresponds to An. pholidotus. Given the absence of An. lepidotus, even in recent years, we consider that the species of mosquitoes that was previously incriminated as the malaria vector during the outbreak was indeed An. pholidotus, thus ending the controversy.

Estimation of Aedes aegypti (Diptera: Culicidae) population size and adult male survival in an urban area in Panama

Traditional mosquito control strategies rely heavily on the use of chemical insecticides. However, concerns about the efficiency of traditional control methods, environmental impact and emerging pesticide resistance have highlighted the necessity for developing innovative tools for mosquito control. Some novel strategies, including release of insects carrying a dominant lethal gene (RIDL®), rely on the sustained release of modified male mosquitoes and therefore benefit from a thorough understanding of the biology of the male of the species. In this report we present the results of a mark-release-recapture study aimed at: (i) establishing the survival in the field of laboratory-reared, wild-type male Aedes aegypti and (b) estimating the size of the local adult Ae. aegypti population. The study took place in Panama, a country where recent increases in the incidence and severity of dengue cases have prompted health authorities to evaluate alternative strategies for vector control. Results suggest a life expectancy of 2.3 days for released male mosquitoes (confidence interval: 1.78-2.86). Overall, the male mosquito population was estimated at 58 males/ha (range 12-81 males/ha), which can be extrapolated to an average of 0.64 pupae/person for the study area. The practical implications of these results are discussed.

Dynamics of the control of Aedes (Stegomyia) aegypti Linnaeus (Diptera, Culicidae) by Bacillus thuringiensis var israelensis, related with temperature, density and concentration of insecticide

The dynamics of the control of Aedes (Stegomyia) aegypti Linnaeus, (Diptera, Culicidae) by Bacillus thuringiensis var israelensis has been related with the temperature, density and concentration of the insecticide. A mathematical model for biological control of Aedes aegypti with Bacillus thuringiensis var israelensis (Bti) was constructed by using data from the literature regarding the biology of the vector. The life cycle was described by differential equations. Lethal concentrations (LC50 and LC95) of Bti were determined in the laboratory under different experimental conditions. Temperature, colony, larvae density and bioinsecticide concentration presented marked differences in the analysis of the whole set of variables; although when analyzed individually, only the temperature and concentration showed changes. The simulations indicated an inverse relationship between temperature and mosquito population, nonetheless, faster growth of populations is reached at higher temperatures. As conclusion, the model suggests the use of integrated control strategies for immature and adult mosquitoes in order to achieve a reduction of Aedes aegypti.