Niveles de expresión de citocromos P450 (CYP6AA7, CYP4C52v1, CYP6BY3, CYP9J34, CYP9M10, CYP9J40 y CYP9AL1) determinados por RT-qPCR en larvas de Culex quinquefasciatus expuestas a diversos insecticidas.

En el presente trabajo, se describen los niveles de expresión de siete transcritos de Culex quinquefasciatus que codifican para citocromos P450, estos medidos por qPCR en larvas expuestas a dos concentraciones del insecticida permetrina (0.51 y 0.71 mg) comparadas con un grupo control sin exponer, las cuales fueron colectadas en distintas localidades del noreste de México. Debido a que los genes analizados no se encontraban reportados, fue necesario diseñar oligonucleótidos utilizando secuencias trazas producto de la secuenciación del genoma del mosquito antes mencionado. Con los oligonucleótidos diseñados y utilizando DNA complementario, sintetizado a partir de RNA total de las larvas fueron amplificados por PCR los mensajeros, se procedió a clonar cada uno de estos genes en nueve poblaciones analizadas, posteriormente fueron secuenciados y éstas fueron comparadas para deducir los porcentajes de similitud. Se diseñaron sondas taqman en segmentos conservados, y utilizando el DNA complementario previamente sintetizado fueron deducidos los niveles de expresión de cada uno de los genes analizados bajo las condiciones citadas anteriormente para las poblaciones. Con el trabajo realizado, reportamos las secuencias nucleótidicos y aminoacídicas de cada uno de los genes analizados de cada una de las poblaciones estudiadas y sus niveles de expresión. Concluimos que los genes analizados son una herramienta potente para ahondar en el estatus de resistencia que han desarrollado las poblaciones silvestres de Culex quinquefasciatus en localidades del noreste de México.

Larvicidal, pupicidal and insecticidal activities of Cosmos bipinnatus , Foeniculum vulgare and Tagetes minuta against Culex quinquefasciatus mosquitoes

Purpose: To evaluate the larvicidal, pupicidal and insecticidal activities of Cosmos bipinnatus , Foeniculum vulgare and Tagetes minuta against Culex quinquefasciatus mosquitoes. Methods: The leaves of the plants were extracted with distilled water, ethanol (95 %), and hexane and the extracts screened for their phytochemical profile. While larvicidal and pupicidal activities were assayed at concentrations ranging from 0.1 - 10 mg/mL, insecticidal property was tested at varying amounts (0.25 - 2 g) of the plant sample. The respective larval mortality was thereafter evaluated using Probit analysis. Results: Saponins, terpenoids, flavonoids and steroids were detected in the plant extracts. The ethanol extracts of F. vulgare, T. minuta and C. bipinnatus exhibited larvicidal activity half-maximal lethal concentration (LC50) of 0.10, 1.17 and 1.18 mg/mL, followed by hexane extracts with LC50 value of 1.03, 1.01 and 1.27 mg/mL, respectively, against the larvae of C. quinquefasciatus mosquito. Hexane extracts displayed pupicidal activity with LC50 of 1.07, 1.12 and 1.16 mg/mL against F. vulgare, T. minuta and C. bipinnatus, respectively, while the ethanol extracts of T. minuta, C. bipinnatus and F. vulgare displayed pupicidal activity at LC50 of 1.11, 1.14 and 1.31 mg/mL respectively, against pupa of C. quinquefasciatus mosquito. The aqueous extracts had no (p > 0.05) lethal effects on both larvae and pupa of C. quinquefasciatus at all evaluated concentrations. F. vulgare had the highest (p < 0.05) half-maximal knock-down effect (KD50 = 7.52 min-1), followed by T. minuta (KD50 = 8.64 min-1) on adult C. quinquefasciatus mosquitoes after 6 h of exposure. F. vulgare and T. minuta killed all evaluated mosquito adults within 12 h with LD99 = 0.25 g/air, while the leaves of C. bipinnatus had no (p > 0.05) knock-down or lethal effects on the adult mosquito. Conclusion: C. bipinnatus, F. vulgare and T. minuta possess larvicidal and pupicidal properties against C. quinquefasciatus, whereas only F. vulgare and T. minuta displayed insecticidal properties. Consequent upon these findings, all the plants can be considered naturally potent larvicidal and pupicidal agents against C. quinquefasciatus.

Culicidae (Diptera, Culicomorpha) from the western Brazilian Amazon: Juami-Japurá Ecological Station

With 312 trap-hours of sampling effort, 1554 specimens of Culicidae (Diptera) were collected, using CDC and Malaise traps, in nine different locations along the Juami River, within the Juami-Japurá Ecological Station, Amazonas State, Brazil. A list of mosquito species with 54 taxa is presented, which includes three new distributional records for the state of Amazonas. The species found belong to the genera Anopheles, Aedeomyia, Aedes, Psorophora, Culex, Coquillettidia, Sabethes, Wyeomyia and Uranotaenia.

Biotin/avidin sandwich enzyme-linked immunosorbent assay for Culicidae mosquito blood meal identification

The knowledge of mosquitoes Culicidae host feeding patterns is basic to understand the roles of different species and to indicate their importance in the epidemiology of arthropod-borne diseases. A laboratory assay was developed aiming at standardizing the biotin-avidin sandwich enzyme-linked immunosorbent assay, which was unprecedented for mosquito blood meal identification. The enzyme-linked immunosorbent assay (ELISA) activity was evaluated by the detection of titers on each sample of the 28 blood-fed Culex quinquefasciatus. In light of the high sensitivity that the technique permits, by means of small quantities of specific antibodies commercially provided and phosphatase substrate which reinforces additional dilutions, human and rat blood meals were readily identified in all laboratory-raised Culex quinquefasciatus tested. The assay was effective to detect human blood meal dilutions up to 1:4,096, which enables the technique to be applied in field studies. Additionally, the present results indicate a significant difference between the detection patterns recorded from human blood meal which corroborate the results of host feeding patterns.

Culicídeos em bromélias: diversidade de fauna segundo influência antrópica, litoral de São Paulo

OBJETIVO: Comparar a diversidade da fauna de culicídeos em bromélias de solo segundo ambientes urbano, periurbano e mata primitiva. MÉTODOS: O estudo foi realizado no município de Ilhabela, litoral norte do estado de São Paulo, em tanques de bromélias de ambientes urbano, periurbano e mata. Realizaram-se coletas de imaturos quinzenalmente, de março de 1998 a julho de 1999. A presença e freqüência de espécies nos diferentes ambientes foram comparadas com base em estimativas da diversidade para medir a riqueza, dominância e análise de variância (ANOVA). RESULTADOS: Coletaram-se 31.134 formas imaturas de mosquitos nas bromélias, distribuídas em sete gêneros e 37 espécies. O ambiente urbano registrou maior abundância, 14.575 indivíduos, seguido do periurbano com 10.987, e a mata, com o menor número de exemplares, 5.572. Foram coletadas 30 espécies no habitat urbano, 32 no periurbano e 33 na mata. As espécies dominantes foram Culex (Microculex) pleuristriatus nos ambientes urbano e periurbano, e Culex ocellatus na mata. De acordo com teste ANOVA a freqüência de mosquitos em bromélias não foi diferente entre os ambientes pesquisados (F=0,5564; p=0,5769). A diversidade de espécies na mata foi maior, e semelhante entre periurbano e urbano. CONCLUSÕES: A composição específica de culicídeos em bromélias de solo mostrou-se diversificada, sendo maior naquelas de ambiente de mata. As espécies dominantes foram Cx. (Mcx.) pleuristriatus e Cx. ocellatus.

Presença de mosquitos (Diptera: Culicidae) em piscinões na zona leste de São Paulo

INTRODUÇÃO: Alterações no ambiente vêm contribuindo com mudanças climáticas, como o aumento do volume de chuvas, que acarreta as inundações. Medidas estão sendo tomadas no enfrentamento das inundações, como a implantação dos reservatórios de contenção de cheias (piscinões). Neste trabalho, foi avaliada a fauna de culicídeos, de importância epidemiológica, nos piscinões Caguaçu e Inhumas. MÉTODOS: Foram realizadas coletas mensais nos piscinões Caguaçu e Inhumas, situados na região leste de São Paulo, de março de 2006 a fevereiro de 2007, empregando-se os métodos de concha entomológica e aspirador. Para análise dos dados, foram realizadas análises estatísticas descritivas e a regressão linear simples. RESULTADOS: Foram coletados 8.917 culicídeos, destacando-se Culex (Culex) quinquefasciatus, que representou 98,9% dos espécimes no Inhumas e 95,2% no Caguaçu. No Caguaçu, a maior frequência de imaturos foi observada no vertedouro (61%) e no Inhumas na canaleta (42,6%). A precipitação prediz 87% da abundância numérica de larvas de terceiro e de quarto estágio no Caguaçu e 60% do número de pupas coletadas. No Inhumas, a precipitação explicou 36% da abundância numérica de larvas e 18% do número de pupas. CONCLUSÕES: Culex quinquefasciatus, vetor de agentes da filariose, arboviroses e fator de incômodo à população, foi a espécie mais frequente nos dois ambientes. Medidas de controle da espécie nos piscinões estudados se fazem necessárias tendo em vista seu potencial epidemiológico.

Presença de culicídeos em município de porte médio do Estado de São Paulo e risco de ocorrência de febre do Nilo Ocidental e outras arboviroses

INTRODUÇÃO: O objetivo deste estudo foi mensurar a diversidade de espécies de culicídeos, descrever sua abundância e variação sazonal em áreas urbanas e matas de São José do Rio Preto, SP, e discutir o risco de ocorrência de arboviroses. MÉTODOS: Coletas de larvas e de mosquitos adultos foram realizadas mensalmente, em 2006 e 2007, em área urbana e em quatro fragmentos de mata. No perímetro urbano, coletaram-se larvas nos sítios mais prováveis de oviposição para mosquitos do gênero Culex e nas matas foi realizada a coleta de mosquitos adultos, sendo que em duas utilizaram-se armadilhas CDC à noite e, em duas, aspirador de Nasci de dia. RESULTADOS: Na área urbana identificaram-se 34 espécies de culicídeos em um total de 8.683 exemplares; destes, 80,7% corresponderam ao Culex quinquefasciatus, 9,6% ao Culex grupo Coronator, 3,2% ao Aedes albopictus (3,2%) e 1,1% ao Ochlerotatus fluviatilis. A abundância de larvas de Cx. quinquefasciatus correlacionou-se negativamente com a chuva. Nas quatro matas, foram coletados 2.268 mosquitos distribuídos entre 10 gêneros, 46 espécies ou grupos. As mais abundantes foram Aedeomyia squamipennis, Culex. coronator, Culex (Mel.) seção Melanoconion, Culex declarator, Ochlerotatus scapularis, Anopheles triannulatus, Culex bidens/interfor e Culex habilitator/pseudojhantinosoma. CONCLUSÕES: A abundância de Cx. quinquefasciatus na área urbana e a presença de outros culicídeos nas áreas urbanas e de matas apontam para a possibilidade de transmissão do vírus do Nilo Ocidental e de outras arboviroses em São José do Rio Preto e outras cidades do Brasil, sendo fundamental o estabelecimento de medidas visando à vigilância destas arboviroses.

Culicidae (Diptera) em área de barragem em Santa Catarina e no Rio Grande do Sul

A composição de Culicidae do lago de Barra Grande, situado entre os municípios de Esmeralda (Rio Grande do Sul) e Anita Garibaldi (Santa Catarina), foi realizada com coletas mensais. Vinte e quatro espécies foram identificadas dentre um total de 1.185 espécimes (74,7% adultos e 25,3% imaturos), sendo Aedes fluviatilis Lutz a espécie mais frequente. Algumas espécies constituem novos registros e são interesse em saúde pública. O fato sugere que mudanças ambientais na área podem alterar a relação entre humanos e mosquitos vetores

Biotin/Avidin sandwich enzyme-linked immunosorbent assay for Culicidae mosquito blood meal identification

The knowledge of mosquitoes Culicidae host feeding patterns is basic to understand the roles of different species and to indicate their importance in the epidemiology of arthropod-borne diseases. A laboratory assay was developed aiming at standardizing the biotin-avidin sandwich enzyme-linked immunosorbent assay, which was unprecedented for mosquito blood meal identification. The enzyme-linked immunosorbent assay (ELISA) activity was evaluated by the detection of titers on each sample of the 28 blood-fed Culex quinquefasciatus. In light of the high sensitivity that the technique permits, by means of small quantities of specific antibodies commercially provided and phosphatase substrate which reinforces additional dilutions, human and rat blood meals were readily identified in all laboratory-raised Culex quinquefasciatus tested. The assay was effective to detect human blood meal dilutions up to 1:4,096, which enables the technique to be applied in field studies. Additionally, the present results indicate a significant difference between the detection patterns recorded from human blood meal which corroborate the results of host feeding patterns

The Genus Wolbachia

Numerous invertebrate species form long lasting symbioses with bacteria (Buchner, 1949; Buchner, 1965). One of the most common of these bacterial symbionts is Wolbachia pipientis, which has been estimated to infect anywhere from 15–75% of all insect species (Werren et al., 1995a; West et al., 1998; Jeyaprakash and Hoy, 2000; Werren and Windsor, 2000) as well as many species of arachnids, terrestrial crustaceans and filarial nematodes (O’Neill et al., 1997a; Bandi et al., 1998). In most arthropod associations, Wolbachia act as reproductive parasites manipulating the reproduction of their hosts to enhance their own vertical transmission. There appears to be little direct fitness cost to the infected host besides the costs arising from the reproductive manipulations. However instances have been reported where Wolbachia can be either deleterious (Min and Benzer, 1997; Bouchon et al., 1998) or beneficial (Girin and Boultreau, 1995; Stolk and Stouthamer, 1995; Wade and Chang, 1995; Vavre et al., 1999b; Dedeine et al., 2001) to their hosts. Wolbachia were first described as intracellular Rickettsia-like organisms (RLOs), infecting the gonad cells of the mosquito, Culex pipiens (Hertig and Wolbach, 1924), and were later named 'Wolbachia pipientis' (Hertig, 1936). It was not until the work of Yen and Barr (Yen and Barr, 1971; Yen and Barr, 1973) that Wolbachia were implicated in causing crossing incompatibilities between different mosquito populations (Laven, 1951; Ghelelovitch, 1952). When polymerase chain reaction (PCR) diagnostics for Wolbachia became available, it became clear that this agent was both extremely widespread and also responsible for a range of different reproductive phenotypes in the different hosts it infected (O’Neill et al., 1992; Rousset et al., 1992; Stouthamer et al., 1993). The most common of these are cytoplasmic incompatibility, inducing parthenogenesis, overriding host sex-determination, and male-killing (O’Neill et al., 1997a). As of the time of this writing, more than 450 different Wolbachia strains with unique gene sequences, different phenotypes, and infecting different hosts have been deposited in GenBank and the Wolbachia host database (http://www.wolbachia.sols. uq.edu.au).

Wolbachia infections are distributed throughout insect somatic and germ line tissues

Wolbachia are intracellular microorganisms that form maternally-inherited infections within numerous arthropod species. These bacteria have drawn much attention, due in part to the reproductive alterations that they induce in their hosts including cytoplasmic incompatibility (CI), feminization and parthenogenesis. Although Wolbachia's presence within insect reproductive tissues has been well described, relatively few studies have examined the extent to which Wolbachia infects other tissues. We have examined Wolbachia tissue tropism in a number of representative insect hosts by western blot, dot blot hybridization and diagnostic PCR. Results from these studies indicate that Wolbachia are much more widely distributed in host tissues than previously appreciated. Furthermore, the distribution of Wolbachia in somatic tissues varied between different Wolbachia/host associations. Some associations showed Wolbachia disseminated throughout most tissues while others appeared to be much more restricted, being predominantly limited to the reproductive tissues. We discuss the relevance of these infection patterns to the evolution of Wolbachia/host symbioses and to potential applied uses of Wolbachia.

Acetylcholinesterase cDNA of the cattle tick, Boophilus microplus: characterisation and role in organophosphate resistance

Acetylcholinesterase is the target of organophosphate and carbamate pesticides. Organophosphate resistance is widespread in the cattle tick, Boophilus microplus, in Australia. We have isolated a cDNA of acetylcholinesterase from B. microplus and show that it would encode a protein 62 kDa in size. The predicted amino acid sequence contains all the residues characteristic of an acetylcholinesterase. Alternative splicing of the transcript was detected at both the 5' and 3' ends. Alternative splicing at the 5' end would result in two proteins differing by six amino acids. This is the first report of alternative splicing of the N-terminal coding region in a cholinesterase. No point mutations were detected in the acetylcholinesterase gene from organophosphate resistant strains of B. microplus. Alternative explanations for resistance to organophosphates in B. microplus are discussed. (C) 1998 Elsevier Science Ltd. All rights reserved.

Experience-induced preference for oviposition repellents derived from a non-host plant by a specialist herbivore

Foraging adults of phytophagous insects are attracted by host-plant volatiles and supposedly repelled by volatiles from non-host plants. In behavioural control of pest insects, chemicals derived from non-host plants applied to crops are expected to repel searching adults and thereby reduce egg laying. How experience by searching adults of non-host volatiles affects their subsequent searching and oviposition behaviour has been rarely tested. In laboratory experiments, we examined the effect of experience of a non-host-plant extract on the oviposition behaviour of the diamondback moth (DBM), Plutella xylostella, a specialist herbivore of cruciferous plants. Naive ovipositing DBM females were repelled by an extract of dried leaves of Chrysanthemum morifolium, a non-host plant of DBM, but experienced females were not repelled. Instead they were attracted by host plants treated with the non-host-plant extract and laid a higher proportion of eggs on treated than on untreated host plants. Such behavioural changes induced by experience could lead to host-plant range expansion in phytophagous insects and play an important role in determining outcome for pest management of some behavioural manipulation methods.

Identification of Plasmodium relictum causing mortality in penguins (Spheniscus magellanicus) from Sao Paulo Zoo, Brazil

This study reports avian malaria caused by Plasmodium relictum in Magellanic Penguins (Spheniscus magellanicus) from Sao Paulo Zoo. The disease was highly infective among the birds and was clinically characterized by its acute course and high mortality. The penguins of Sao Paulo Zoo were housed for at least 2 years without malaria; however, they had always been maintained in an enclosure protected from mosquito exposure during the night period. When they presented pododermatitis, they were freed at night for a short period. sao Paulo Zoo is located in one of the last forest remnants of the city, an area of original Atlantic forest. In the winter, the space destined for Zoo birds is shared with migratory species. Hence the possibility exists that the disease was transmitted to the penguins by mosquitoes that had previously bitten infected wild birds. Avian malaria parasites are transmitted mainly by mosquitoes of the genera Aedes and Culex, common vectors in the Atlantic forest. In this study, one Culex (Cux.) sp. was found, infected with P. relictum. There are diverse problems in housing distinct species of animals in captivity, principally when occupying the same enclosure, since it facilitates the transmission of diseases with indirect cycles, as is the case of Plasmodium spp., because certain species that cause discrete infections in some bird species can become a serious danger for others, especially penguins, which do not possess natural resistance. Thus, serious implications exist for periodically testing and administrating malaria therapy in captive penguins potentially exposed to mosquitoes during the night period, as well as other captive birds from Sao Paulo Zoo. (C) 2010 Elsevier B.V. All rights reserved.