Laboratory and field evaluation of an oviposition trap for Culex quinquefasciatus(Diptera: Culicidae)

An ovitrap (BR-OVT) based on physical and chemical stimuli for attracting gravid Culex quinquefasciatus (Diptera: Culicidae) females was developed and evaluated under laboratory and field conditions. Attractants were assayed using alternative chamber bioassays prior to being used in the BR-OVT oviposition trap. A significant preference of gravid females for sites containing conspecific egg rafts was observed, as a response to the natural oviposition pheromone, as well as for sites treated with the synthetic pheromone erythro-6-acetoxy-5-hexadecanolide. Five- to 20-day old grass infusion was strongly attractive to gravid females for laying eggs. On the other hand, entomopathogenic Bacillus sphaericus (Bs) did not influence the choice of an oviposition site when used in combination with grass infusion and can therefore be used as a larvicide in ovitraps. Results from field trials showed that the BR-OVT with grass infusion and with or without Bs works as a preferred oviposition site for Cx. quinquefasciatus. The BR-OVT was more effective for egg collection when placed indoors and comparison with the number of egg rafts laid in cesspits over 40 days indicates that this very simple ovitrap may be a useful tool for monitoring populations of the most important of the vectors of bancroftian filariasis.

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.

Culex quinquefasciatus vitellogenesis: morphological and biochemical aspects

The vitellogenic process in Culex quinquefasciatus, which is triggered by a blood meal, involves the synthesis, distribution and storage of the nutrients necessary for embryo development. The fat body of an adult female Cx. quinquefasciatus revealed two cell types: large trophocytes and small, eosinophilic, "oenocyte-like" cells, which show no morphological changes throughout the gonotrophic cycle. Trophocytes, which only begin to synthesise vitellogenin (Vg) 12 h post-blood meal (PBM), undergo a series of morphological changes following engorgement. These changes include the expansion of the rough endoplasmic reticulum (RER) and Golgi complex, which are later destroyed by autophagosomes. At 84 h PBM, trophocytes return to their pre-engorgement morphology. The ovarian follicles of non-blood-fed Cx. quinquefasciatus contain a cluster of eight undifferentiated cells surrounded by follicular epithelium. After engorgement, the oocyte membrane facing the perioocytic space increases its absorptive surface by microvilli development; large amounts of Vg and lipids are stored between 24 and 48 h PBM. Along with yolk storage in the oocyte, follicular cells exhibit the development of RER cisternae and electron-dense granules begin to fill the perioocytic space, possibly giving rise to endochorion. Later in the gonotrophic cycle, electron-dense vesicles, which are possible exochorion precursors, fuse at the apical membrane of follicular cells. This fusion is followed by follicular cell degeneration.

Genetic-morphometric variation in Culex quinquefasciatus from Brazil and La Plata, Argentina

Variation among natural populations of Culex (Culex) quinquefasciatus Say is associated with different vectorial capacities. The species Cx. quinquefasciatus is present in the equatorial, tropical and subtropical zones in the Brazilian territory, with intermediate forms between Cx. quinquefasciatus and Culex pipiens occurring in regions of latitudes around 33°-35°S. Herein, we studied geographically distinct populations of Cx. quinquefasciatus by genetic characterization and analysis of intra-specific wing morphometrics. After morphological analysis, molecular characterization of Cx. quinquefasciatus and intermediate forms was performed by polymerase chain reaction of the polymorphic nuclear region of the second intron of the acetylcholinesterase locus. Additionally, the morphology of adult female wings collected from six locations was analyzed. Wing centroid sizes were significantly different between some geographical pairs. Mean values of R2/R2+3 differed significantly after pairwise comparisons. The overall wing shape represented by morphometric characters could be divided into two main groupings. Our data suggest that Brazilian samples are morphologically and genetically distinct from the Argentinean samples and also indicated a morphological distinction between northern and southern populations of Brazilian Cx. quinquefasciatus. We suggest that wing morphology may be used for preliminary assessment of population structure of Cx. quinquefasciatusin Brazil.

Monitoring temporal fluctuations of Culex quinquefasciatus using oviposition traps containing attractant and larvicide in an urban environment in Recife, Brazil

The use of attractants and larvicides in oviposition traps is of practical interest for the surveillance and control of urban mosquitoes. In addition to increasing the safety of the traps, this combination is essential for an attract-and-kill control strategy based on trapping mosquito eggs. The combination of Bacillus thuringiensis var. israelensis (Bti) and grass infusion (GI) vs. GI alone were tested for their ability to attract in paired BR-OVT traps in the backyards of 10 houses in Recife, Brazil, for a period of 45 days. Results show that females prefer to oviposit in traps containing Bti (363 compared with 251 egg rafts over 45 days). Results from a one-year trial on the efficacy of BR-OVT traps loaded with GI and Bti as a sampling tool to monitor temporal fluctuations in the population densities of Culex quinquefasciatus in an urban environment are also reported. From December 2006-January 2007, one trap per home was installed and maintained for 348 consecutive days in 134-151 houses located in three urban blocks. Throughout the one-year field trial a total of 43,151 Culex egg rafts were collected in the traps. The data show that BR-OVT loaded with GI and Bti is sensitive enough to demonstrate continuous reproductive activity of Cux. quinquefasciatus in the study area throughout the year and to monitor temporal fluctuations in population density.

Establishment and characterisation of a new cell line derived from Culex quinquefasciatus (Diptera: Culicidae)

Insect cell cultures are an important biotechnological tool for basic and applied studies. The objective of this work was to establish and characterise a new cell line from Culex quinquefasciatus embryonic tissues. Embryonated eggs were taken as a source of tissue to make explants that were seeded in L-15, Grace's, Grace's/L-15, MM/VP12, Schneider's and DMEM culture media with a pH range from 6.7-6.9 and incubated at 28ºC. The morphological, cytogenetic, biochemical and molecular characteristics of the cell cultures were examined by observing the cell shapes, obtaining the karyotypes, using a cellulose-acetate electrophoretic system and performing random amplified polymorphic DNA-polymerase chain reaction analysis, respectively. The Grace's/L-15 medium provided the optimal nutritional conditions for cell adhesion and proliferation. Approximately 40-60 days following the explant procedure, a confluent monolayer was formed. Cellular morphology in the primary cultures and the subcultures was heterogeneous, but in the monolayer the epithelioid morphology type predominated. A karyotype with a diploid number of six chromosomes (2n = 6) was observed. Isoenzymatic and molecular patterns of the mosquito cell cultures matched those obtained from the immature and adult forms of the same species. Eighteen subcultures were generated. These cell cultures potentially constitute a useful tool for use in biomedical applications.

The impact of CO2 on collection of Aedes aegypti (Linnaeus) and Culex quinquefasciatus Say by BG-Sentinel(r) traps in Manaus, Brazil

Carbon dioxide (CO2) is an important component for activating and attracting host-seeking mosquitoes. The BG-Sentinel(r) trap is a well-established monitoring tool for capturing Culicidae, but CO2 role for the trap effectiveness has not been evaluated in highly urbanised areas. The objective was to evaluate the effectiveness of BG-Sentinel traps baited with and without CO2 for capturing urban mosquitoes. Fifteen areas were selected within the city of Manaus, Brazil, where four BG-Sentinels were operated for 24 h, two of them with CO2 and two without CO2. Captured Aedes aegypti females were dissected for the determination of their parity status. A significantly higher proportion of traps (from 32-79%) were positive for female Ae. aegypti when using the BG-Sentinel with CO2 (χ2 = 11.0271, p ≤ 0.001). Catches of female Culex spp were six times higher in CO2 traps (Mann-Whitney U test = 190.5; p = 0.001). Parity rates were similar for both traps. This study showed that CO2 has primarily an enhancing effect on the efficacy of BG-Sentinel for capturing Culex spp in Manaus. For Ae. aegypti, the positivity rate of the trap was increased, when CO2 was added.

DsRed2 transient expression in Culex quinquefasciatus mosquitoes

Culex quinquefasciatus mosquitoes have been successfully genetically modified only once, despite the efforts of several laboratories to transform and establish a stable strain. We have developed a transient gene expression method, in Culex, that delivers plasmid DNA directly to the mosquito haemolymph and additional tissues. We were able to express DsRed2 fluorescent protein in adult Cx. quinquefasciatus mosquitoes by injecting plasmids directly into their thorax. The expression of DsRed2 in adult Cx. quinquefasciatus mosquitoes is an important stepping stone to genetic transformation and the potential use of new control strategies and genetic interactions.

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.

Aspectos ecológicos da população de Culex quinquefasciatus Say (Diptera, Culicidae) em abrigos situados no Parque Ecológico do Tietê, São Paulo, SP

O estudo objetivou conhecer, ao longo das estações do ano, aspectos ecológicos da população adulta de Culex quinquefasciatus em abrigos. As coletas dos adultos foram realizadas mensalmente, cobrindo-se o período de um ano, na vegetação da margem de um canal de circunvalação. Os insetos foram capturados com o auxílio de um aspirador à bateria. O material foi acondicionado e transportado ao laboratório e, posteriormente, a espécie de interesse foi identificada; machos e fêmeas foram quantificados e o desenvolvimento ovariano foi identificado segundo o critério de Sella. Dados metereológicos foram coletados visando correlacioná-los com a variação sazonal da população de mosquitos. Foram coletados 8.298 mosquitos da espécie Culex quinquefasciatus. Desse total, 6.313 (76%) eram machos e 1.985 (24%) fêmeas. As fêmeas foram encontradas com o abdome vazio (90,1%), com sangue (7,4%) ou com óvulos maduros (2,5%). As correlações de número de mosquitos coletados versus temperatura mensal média e precipitação mensal total não indicaram influência forte e positiva desses fatores na freqüência de mosquitos. A maior freqüência de machos pode ser explicada pela dispersão das fêmeas e pela permanência dos machos nos abrigos, representados pela vegetação na margem do canal favorecendo o acúmulo de mosquitos.

Aspectos da distribuição de Culex (Culex) quinquefasciatus Say (Diptera, Culicidae) na região do rio Pinheiros, na cidade de São Paulo, Estado de São Paulo, Brasil

A distribuição de Culex (Culex) quinquefasciatus Say (1823) ao longo das margens do rio Pinheiros e os principais fatores que levam à proliferação da espécie foram estudados efetuando-se coletas semanais de mosquitos adultos, no período de um ano, em três pontos eqüidistantes às margens do rio. Para as coletas de mosquitos utilizou-se aspirador à bateria, por um período de cinco minutos. Os mosquitos foram identificados, diferenciados segundo o sexo e contados. Para verificação do estado fisiológico, as fêmeas foram separadas em vazias, com sangue e com ovos. Foram coletados 35.684 mosquitos, todos identificados como Cx. quinquefasciatus, sendo 39,4% fêmeas e 60,6% machos. As freqüências tomaram proporções diferentes entre os pontos de coletas e, em uma série temporal. O ambiente impactado do rio Pinheiros representa um excelente criadouro de Cx. quinquefasciatus, confirmado pela ocorrência de picos acentuados na freqüência de mosquitos, com desenvolvimento de forma explosiva e sobreposições entre as gerações, após as chuvas e em épocas de verão.

Observações sobre aplicações de controle em ecótopo com alta infestação de Culex quinquefasciatus Say (Diptera, Culicidae)

Com o objetivo de conhecer aspectos da infestação do mosquito Culex quinquefasciatus no rio Pinheiros, São Paulo, SP, Brasil, e avaliar a eficácia das aplicações de controle efetuadas no local, foram realizadas atividades de monitoramento da população de mosquitos em dez pontos de amostragens, localizados ao longo das duas margens do rio Pinheiros. Os mosquitos foram coletados por meio de técnica de aspiração à bateria, durante o período de um ano e encaminhados ao laboratório para identificação, contagem e tratamento dos dados de freqüência com as datas de aplicações de controle químico e biológico da população de mosquitos. As aplicações de controle apresentam efeitos parciais na freqüência populacional da espécie, o que abre discussão sobre as peculiaridades do ecótopo, como possíveis barreiras na eficiência dos produtos aplicados no meio aquático e na vegetação marginal, ao longo da calha do rio.

Vector competence of Culex quinquefasciatus Say, 1823 exposed to different densities of microfilariae of Dirofilaria immitis (Leidy, 1856)

The metropolitan region of Recife, Brazil is endemic for Dirofilaria immitis and has an environment favorable to the development of Culex quinquefasciatus. The goal of this study was to evaluate the vector competence of the Cx. quinquefasciatus RECIFE population for D. immitis transmission. A total of 2,104 females of Cx. quinquefasciatus RECIFE population were exposed to different densities of D. immitis microfilariae blood meals, ranging from 1,820 to 2,900 mf/ml of blood, in a natural membrane apparatus. The results showed a variation between 92.3% and 98.8% of females fed. The exposure of the Cx. quinquefasciatus RECIFE population to different densities of microfilariae did not influence the mortality of the mosquitoes. Infective larvae from D. immitis were observed in the Malpighian tubules beginning on the 12th day, whereas larvae were observed in the head and proboscis beginning on the 13th day following infection. The vector efficiency index (VEI) presented by the mosquitoes ranged from 7.8 to 56.5. The data demonstrates that the Cx. quinquefasciatus RECIFE population has great potential for the transmission of D. immitis, as it allowed the development of the filarid until the infectious stage at the different densities of microfilariae to which it was exposed.

Wing diagnostic characters for Culex quinquefasciatus and Culex nigripalpus (Diptera, Culicidae)

Wing diagnostic characters for Culex quinquefasciatus and Culex nigripalpus (Diptera, Culicidae). Culex quinquefasciatus and Culex nigripalpus are mosquitoes of public health interest, which can occur sympatrically in urban and semi-urban localities. Morphological identification of these species may be difficult when specimens are not perfectly preserved. In order to suggest an alternative taxonomical diagnosis, wings of these species were comparatively characterized using geometric morphometrics. Both species could be distinguished by wing shape with accuracy rates ranging from 85-100%. Present results indicate that one can identify these species relying only on wing characters when traditional taxonomical characters are not visible.

Control y efectos morfológicos de Culex quinquefasciatus Say (Diptera: culicidae) en contenedores artificiales en el campo agrícola experimental del ITESM en Apodaca, Nuevo León, México

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) UANL