Carla Gentile

Circadian Expression of Clock Genes in Two Mosquito Disease Vectors: cry2 Is Different

Different mosquito species show a full range of activity patterns, including diurnal, crepuscular, and nocturnal behaviors. Although activity and blood-feeding rhythms are controlled by the circadian clock, it is not yet known whether such species-specific differences in behavior are controlled directly by core clock genes or instead reflect differences in how the information of the central clock is translated into output signals. The authors have analyzed the circadian expression of clock genes in two important mosquito vectors of tropical diseases, Aedes aegypti and Culex quinquefasciatus . Although these two species show very different locomotor activity patterns and are estimated to have diverged more than 22 million years ago, they show conserved circadian expression patterns for all major cycling clock genes except mammalian-like cryptochrome2 (cry2). The results indicate that different mechanisms for cry2 regulation may exist for the two species. The authors speculate that the correlation between the differences in behavior between Ae. aegypti and Cx. quinquefasciatus and their corresponding cry2 mRNA profiles suggests a potential role for this clock gene in controlling species-specific rhythmic behavior. However, further work is needed to establish that this is the case as the different cry2 expression patterns might reflect differences between the Aedes and Culex lineages that are not directly related to changes in behavior.

Prevalência de Anofelinos (Diptera: Culicidae) no Crepúsculo Vespertino em Áreas da Usina Hidrelétrica de Itaipu, no Município de Guaíra, Estado do Paraná, Brasil

Systematic collections of anophelines were conducted from November 1994 to August 1995 from 18:00 to 20:00 hr using Shannon traps and human-bait along the lake margin which forms the Itaipu Hydroelectric reservoir, State of Parana, Brazil. Species prevalence was studied at 15 min intervals. Anopheles albitarsis sensu latu and An. galvaoi, were the most frequently collected mosquitoes. All Anopheles species populations peaked between 18:45 and 19:30 hr. The observations illustrate the existence of a haematophagic activity cycle during the early evening hours: exogenous stimulus (the beginning of sunset) ® Shannon trap (light attraction) ® human bait (haematophagy) ® rest and digestion ® exogenous stimulus ® Shannon trap or surrounding vegetation. The greater abundance of An. albitarsis collected in human-bait and Shannon trap suggests it may be a potential malaria vector in the region

Isolation of a fragment homologous to the rp49 constitutive gene of Drosophila in the Neotropical malaria vector Anopheles aquasalis (Diptera: Culicidae)

The constitutive ribosomal gene rp49 is frequently used as an endogenous control in Drosophila gene expression experiments. Using the degenerate primer PCR technique we have cloned a fragment homologous to this gene in Anopheles aquasalis Curry, a Neotropical vector of malaria. In addition, based on this first sequence, a new primer was designed, which allowed the isolation of fragments of rp49 in two other species, Aedes aegypti (Linnaeus) and Culex quinquefasciatus Say, suggesting that it could be used to clone fragments of this gene in a number of other mosquito species. Primers were also designed to specifically amplify rp49 cDNA fragments in An. aquasalis and Ae. aegypti, showing that rp49 could be used as a good constitutive control in gene expression studies of these and other vectorially important mosquito species.

Ecology of mosquitoes (Diptera: Culicidae) in areas of Serra do Mar State Park, State of São Paulo, Brazil. II - Habitat distribution

The mosquito (Diptera: Culicidae) ecology was studied in areas of Serra do Mar State Park, State of São Paulo, Brazil. Systematized biweekly human bait collections were made three times a day, for periods of 2 or 3 h each, in sylvatic and rural areas for 24 consecutive months (January 1991 to December 1992). A total of 24, 943 adult mosquitoes belonging to 57 species were collected during 622 collective periods. Aedes scapularis, Coquillettidia chrysonotum, Cq. venezuelensis, Wyeomyia dyari, Wy. longirostris, Wy. theobaldi and Wy. palmata were more frequently collected at swampy and at flooded areas. Anopheles mediopunctatus, Culex nigripalpus, Ae. serratus, Ae. fulvus, Psorophora ferox, Ps. albipes and the Sabethini in general, were captured almost exclusively in forested areas. An. cruzii, An. oswaldoi and An. fluminensis were captured more frequently in a residence area. However, Cx. quinquefasciatus was the only one truly eusynanthropic. An. cruzii and Ae. scapularis were captured feeding on blood inside and around the residence, indicating that both species, malaria and arbovirus vectors respectively, may be involved in the transmission of these such diseases in rural areas.

Ecology of mosquitoes (Diptera: Culicidae) in areas of Serra do Mar State Park, State of São Paulo, Brazil. I - Monthly frequency and climatic factors

The ecology of mosquitoes were studied (Diptera: Culicidae) in areas of Serra do Mar State Park, State of São Paulo, Brazil. Systematized monthly human bait collections were made three times a day, for periods of 2 or 3 h each, in sylvatic and rural areas for 24 consecutive months (January 1991 to December 1992). A total of 24, 943 specimens of adult mosquitoes belonging to 57 species were collected during 622 collective periods. Coquillettidia chrysonotum was the most frequent collected mosquito (45.8%) followed by Aedes serratus (6.8%), Cq. venezuelensis (6.5%), Psorophora ferox (5.2) and Ps. albipes (3.1%). The monthly averages of temperature and relative humidity were inserted in the ten-year average limits of maximum and minimum of the previous ten-years. Rainfall accompanied the curve of the ten-year averages. Those climatic factors were influential in the incidence of some species; temperature: Anopheles cruzii, An. mediopunctatus, Ae. scapularis, Ae. fulvus, Cq. chrysonotum, Cq. venezuelensis, Runchomyia reversa, Wyeomyia dyari, Wy. confusa, Wy. shannoni, Wy. theobaldi and Limatus flavisetosus; relative humidity: Ae. serratus, Ae. scapularis, Cq. venezuelensis and Ru. reversa; rainfall: An. cruzii, Ae. scapularis, Ae. fulvus, Cq. venezuelensis Ru. reversa, Wy. theobaldi and Li. flavisetosus.

Ecologia de mosquitos em áreas do Parque Nacional da Serra da Bocaina: II -- Freqüência mensal e fatores climáticos

OBJETIVO: Estabelecer a influencia exercida pelos fatores climaticos na frequencia mensal da fauna de mosquitos em areas do Parque Nacional da Serra da Bocaina, Vale do Paraiba, SP. METODOS: Foram realizadas capturas mensais por meio de isca humana, em tres diferentes periodos do dia, durante 24 meses consecutivos (janeiro de 1991 a dezembro de 1992). RESULTADOS: Foram capturados 11.808 especimes adultos pertencentes a 28 especies. Anopheles cruzii, Runchomyia reversa e Ru. frontosa foram as especies mais presentes em todos os meses do ano; An. cruzii ocorreu preferencialmente de outubro a fevereiro; Ru. reversa e Ru. frontosa, em setembro; An. lutzi, Chagasia fajardoi, Coquillettidia chrysonotum, Aedes serratus, Trichoprosopon simile, Wyeomyia theobaldi, Ru. humboldti e Ru. theobaldi, pertencentes ao segundo grupo de especies mais abundantes, ocorreram preferencialmente nos meses mais quentes, umidos e chuvosos. A temperatura e as precipitacoes pluviometricas influenciaram positivamente na incidencia da maioria das especies. CONCLUSOES: A cobertura vegetal de Mata Atlântica bem preservada, as precipitacoes pluviometricas e a temperatura foram determinantes para a incidencia da fauna de mosquitos no parque. Os meses mais chuvosos, umidos e quentes contribuiram para o aumento da diversidade e da densidade. O periodo de setembro a marco foi o mais favoravel. Cerca de 70% das especies chegaram a desaparecer no periodo de abril a agosto, os meses mais frios e secos do ano.

Ecology of Anopheline (Diptera, Culicidae), malaria vectors around the Serra da Mesa Reservoir, State of Goiás, Brazil: 1 - Frequency and climatic factors

The ecology of anopheline species (Diptera, Culicidae) was studied in the vicinity of the Serra da Mesa Reservoir, State of Goiás, Brazil. Climatic factors and frequency of anopheline populations were analyzed. Bimonthly human-bait and Shannon trap captures were conducted for 36 consecutive months (January 1997 through December 1999). A total of 5,205 adult anophelines belonging to five species were collected. Anopheles darlingi was the most frequently collected anopheline (61.4%), followed by An. albitarsis s.l. (35.4%), An. triannulatus. (2.5%), An. oswaldoi (0.4%), and An. evansae (0.2%). The water level and vegetation along the banks of the reservoir were crucial to the frequency of the various anopheline species. Climatic factors had a secondary influence. The reservoirs water-level stability, increased frequency of An. darlingi, and the arrival of gold prospectors were responsible for the increase in malaria cases.

Ecologia de mosquitos (Diptera: Culicidae) em áreas do Parque Nacional da Serra da Bocaina, Brasil.I - Distribuição por habitat

OBJECTIVE To assess the mosquito fauna in Serra da Bocaina National Park (PNSB), by collecting information through a general survey, and investigating the population behavior in habitats within the park with different vegetation. METHODS Human bait collections were conducted once a month for both the forest and households, in diurnal and nocturnal periods, three time a day, throughout 24 months, from January 1991 to December 1992. RESULTS A total of 11, 808 adult mosquitoes belonging to 28 species were collected. Runchomyia reversa and Anopheles cruzii were the most abundant, reaching 52.5% and 17.9% of the total collected specimens, respectively. In the dense forest, Ru. reversa comprised 59.4% of the total, followed by Ru. frontosa with 10.5%, and An. cruzii with 9.9%. In the high altitude fields and in gallery forest, An. cruzii was the most abundant (48.1%) followed by Ru. reversa (28.1%). Inside households An. cruzii was also the most prominent species, representing 73.7% of the total for that location. Coquillettidia chrysonotum was the only species mainly seen in the household surroundings, where its distribution was: 14.9% (indoors), 19.4% (close to the house), and 65.7% (outdoors). An. cruzii and Ru. reversa were found throughout the whole year and captured every month. CONCLUSIONS Mosquitoes in PNSB present an assynanthropic behavior, except for Cq. chrysonotum which lives preferentially in the household environment. Though An. cruzii is an assynantropic species it may approaches live near households and even invades and infest them for the blood meals. The occurrence of Aedes serratus in the household vicinity emphasizes its epidemiological importance as a potential vector of arboviruses. Sabethini are all exclusively sylvatic species.OBJETIVO: Estabelecer a influencia exercida por tres diferentes biotopos em areas do Parque Nacional da Serra da Bocaina (PNSB) sobre a fauna local de mosquitos. METODOS: Foram realizadas capturas mensais em ambiente silvestre e domiciliar, em isca humana, durante tres diferentes periodos do dia, pelo periodo de 24 meses consecutivos, de janeiro de 1991 a dezembro de 1992. RESULTADOS: Foram capturados 11.808 especimes adultos, pertencentes a 28 especies. Ru. reversa e An. cruzii foram predominantes, respectivamente 52,5% e 17,9% do total de mosquitos. Ru. reversa representou 59,4% do total de especimes no ambiente de mata fechada, seguida por Ru. frontosa com 10,5% e An. cruzii com 9,9%. No ambiente formado por campos de altitude e matas de galeria, o An. cruzii predominou com 48,1%, seguido por Ru. reversa com 28,1%. No ambiente modificado pelo homem, o An. cruzii predominou com 73,7% dos especimes. Coquillettidia chrysonotum foi a unica que se apresentou preferencialmente nesse biotopo: 14,9% no intra, 19,4% no peri e 65,7% no extradomicilio. An. cruzii e Ru. reversa foram constantes em todos os ambientes ao longo do ano. CONCLUSOES: Com excecao de Cq. chrysonotum, com preferencia pelo ambiente modificado pelo homem, os mosquitos apresentam habitos assinantropicos no PNSB. An. cruzii, embora assinantropico, se aproxima e adentra o domicilio para realizar a hematofagia. A presenca do Ae. serratus no extra e peridomicilio reforca a importância epidemiologica como vetora potencial de arboviroses. Os Sabethini apresentaram-se exclusivamente silvestres.

Ecology of mosquitoes (Diptera: Culicidae) in areas of Serra do Mar State Park, State of São Paulo, Brazil. III. Daily biting rhythms and lunar cycle influence.

The ecology of mosquito species (Diptera: Culicidae) was studied in areas of the Serra do Mar State Park, State of São Paulo, Brazil. The influence of the lunar cycle and the daily biting rhythms of mosquito populations were analyzed. Systematized biweekly human bait collections were made in a silvatic environment for 24 consecutive months (January 1991 to December 1992). A total of 20,591 specimens of adult mosquitoes belonging to 55 species were collected from 545 catches. Sabethini species were captured exclusively during daylight periods, with the exception of Trichoprosopon digitatum, while members of Anophelinae predominated during nocturnal hours. Members of the subfamily Culicinae that were collected primary during nocturnal periods included Culex nigripalpus, Coquillettidia chrysonotum and Cq. venezuelensis while daytime catches included Psorophora ferox and Ps. albipes. Others members of culicines mosquitoes that were collected during both day and night included: Aedes serratus, Ae. scapularis and Ae. fulvus. Lunar cycles did not appear to influence the daily biting rhythms of most mosquito species in the area, but larger numbers of mosquitoes were collected during the new moon. Ae. scapularis were captured mainly during the full moon.

Analysis of cycle gene expression in Aedes aegypti brains by in situ hybridization.

Even though the blood-sucking mosquito Aedes aegypti is one of the most important disease vectors, relatively little is known about the molecular mechanisms underlying processes involved in the temporal pattern of its activity and host seeking behavior. In this study, we analyzed the expression of the cycle (cyc) gene, one of the core components of the circadian clock, in Ae. aegypti brains by in situ hybridization at two different time points in light-dark conditions and compared the results with those obtained using a quantitative PCR assay (qPCR). Within the brain, differential labeling was detected according to distinct areas empirically pre-defined. Six out of seven of these areas showed significantly higher staining at ZT3 (three hours after light-on) compared to ZT11 (one before light-off), which is consistent with the qPCR data. Predominant staining was observed in three of those areas which correspond to positions of the optical and antennal lobes, as well as the region where the neurons controlling activity rhythms are presumably localized.