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

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.

Thermal acclimation of locomotor performance in tadpoles of the frog Limnodynastes peronii

Previous analyses of thermal acclimation of locomotor performance in amphibians have only examined the adult life history stage and indicate that the locomotor system is unable to undergo acclimatory changes to temperature. In this study, we examined the ability of tadpoles of the striped marsh frog (Limnodynastes peronii) to acclimate their locomotor system by exposing them to either 10 degrees C or 24 degrees C for 6 weeks and testing their burst swimming performance at 10, 24, and 34 degrees C. At the test temperature of 10 degrees C, maximum velocity (U-max) of the 10 degrees C-acclimated tadpoles was 47% greater and maximum acceleration (A(max)) 53% greater than the 24 degrees C-acclimated animals. At 24 degrees C, U-max was 16% greater in the 10 degrees C-acclimation group, while there was no significant difference in A(max) or the time taken to reach U-max (T-U-max). At 34 degrees C, there was no difference between the acclimation groups in either U-max or A(max), however T-U-max was 36% faster in the 24 degrees C-acclimation group. This is the first study to report an amphibian (larva or adult) possessing the capacity to compensate for cool temperatures by thermal acclimation of locomotor performance. To determine whether acclimation period affected the magnitude of the acclimatory response, we also acclimated tadpoles of L. peronii to 10 degrees C for 8 months and compared their swimming performance with tadpoles acclimated to 10 degrees C for 6 weeks. At the test temperatures of 24 degrees C and 34 degrees C, U-max and A(max) were significantly slower in the tadpoles acclimated to 10 degrees C for 8 months. At 10 degrees C, T-U-max was 40% faster in the 8-month group, while there were no differences in either U-max or A(max). Although locomotor performance was enhanced at 10 degrees C by a longer acclimation period, this was at the expense of performance at higher temperatures.

Analysis of the sequence and expression of a second putative acetylcholinesterase cDNA from organophosphate-susceptible and organophosphate-resistant cattle ticks

The cattle tick, Boophilus microplus, is a major pest of cattle in Australia, Central and South America, and parts of Africa and Asia. Control of ticks with organophosphates (OPs) and carbamates, which target acetylcholinesterases (AChE), led to evolution of resistance to these pesticides. Alleles at the locus studied here, AChE2, from OP-susceptible female ticks from Australia and Mexico differed at 46 of 1689 nucleotide positions (20 putative amino acid differences) whereas alleles from three strains of OP-resistant ticks from Australia differed with the allele from the Australian susceptible ticks at six to 13 nucleotide positions (three to six putative amino acid differences). However, the role, if any, of these polymorphisms in the OP-resistance phenotype is unknown. Certainly none of the polymorphisms correspond to sites in ACK that are involved in catalysis or binding of acetylcholine in other organisms. Both of the AChE loci of B. microplus, AChE1 and AChE2, are apparently expressed in synganglia; AChE1 is also expressed in salivary glands and ovaries, in OP-susceptible and OP-resistant ticks. This seems to contradict studies of enzyme kinetics, which indicated that only one form of AChE was present in the synganglia, the site of the action of OPs, in this species of tick. (C) 2002 Elsevier Science Ltd. All rights reserved.

Vector competence of Australian mosquitoes (Diptera : Culicidae) for Japanese encephalitis virus

Australian mosquitoes were evaluated for their ability to become infected with and transmit a Torres Strait strain of Japanese encephalitis virus. Mosquitoes, which were obtained from either laboratory colonies and collected using Centers for Disease Control and Prevention light traps baited with CO2 and octenol or reared from larvae, were infected by feeding on a blood/sucrose solution containing 10(4.5+/-0.1) porcine stable-equine kidney (PS-EK) tissue culture infectious dose(50)/ mosquito of the TS3306 virus strain. After 14 d, infection and transmission rates of 100% and 81%, respectively, were obtained for a southeast Queensland strain of Culex annulirostris Skuse, and 93% and 61%, respectively, for a far north Queensland strain. After 13 or more days, infection and transmission rates of > 90% and greater than or equal to 50%, respectively, were obtained for southeast Queensland strains of Culex sitiens Wiedemann and Culex quinquefasciatus Say, and a far north Queensland strain of Culex gelidus Theobald. Although infection rates were > 55%, only 17% of Ochlerotatus vigilax (Skuse) and no Cx. quinquefasciatus, collected from far north Queensland, transmitted virus. North Queensland strains of Aedes aegypti L., Ochlerotatus kochi (Donitz), and Verrallina funerea (Theobald) were relatively refractory to infection. Vertical transmission was not detected among 673 F, progeny of Oc. vigilax. Results of the current vector competence study, coupled with high field isolation rates, host feeding patterns and widespread distribution, confirm the status of Cx. annulirostris as the major vector of Japanese encephalitis virus in northern Australia. The relative roles of other species in potential Japanese encephalitis virus transmission cycles in northern Australia are discussed.

Flaviviruses in mosquitoes from Southern Portugal, 2009-2010

Os flavivírus são vírus pertencentes à família Flaviviridae, género Flavivirus. Estes formam um grande grupo caraterizado pela sua ampla distribuição e diversidade genética. Os flavivírus são, na sua maioria, transmitidos por artrópodes vectores incluíndo agentes patogénicos para humanos e animais que podem potencialmente provocar grandes epidemias e causar elevadas taxas de mortalidade e morbidade. Nos últimos anos, tem-se registado uma grande expansão a nível da distribuição geográfica dos flavivirus e diversidade dos seus hospedeiros. O vírus do Nilo Ocidental tem sido continuamente detectado em toda a Europa recentemente, e também isolado de mosquitos colhidos no Sul de Portugal, onde já foram registados casos humanos e animais. O principal objectivo deste trabalho é o rastreio de flavivírus em mosquitos colhidos em duas regiões do Sul de Portugal, onde os mesmos foram anteriormente detectados. As colheitas de mosquitos foram realizadas em 24 locais em zonas húmidas nos districtos de Faro e Setúbal, através de armadilhas luminosas tipo CDC com CO2 e aspiradores mecânicos manuais para colheita de mosquitos em repouso em abrigos de animais. Os mosquitos colhidos foram agrupados por lotes contendo aproximadamente 50 espécimens cada, e rastreados para a presença de flavivírus por heminested RT-PCR, direccionado à amplificação de um pequeno fragmento do gene NS5 usando oligonucleótidos degenerados específicos para flavivírus. Entre Abril e Outubro de 2009 e 2010 foram colhidos no total 36273 mosquitos pertencentes às seguintes espécies: Anopheles algeriensis, An.atroparvus, Aedes berlandi, Ae.caspius, Ae.detritus, Coquillettidia richiardii, Culex laticinctus, Cx.pipiens, Cx.theileri, Cx.univittatus, Culiseta annulata, Cs.longiareolata, Cs.subochrea, e Uranotaenia unguiculata. As espécies mais abundantes foram Ae.caspius, Cx.theileri e Cx.pipiens, respectivamente. Contudo, as densidades de mosquitos foram variáveis de acordo com o método de colheita e área de amostragem. As densidades de mosquitos colhidos em 2010 foram quatro vezes superior às registadas no ano anterior. No total foram analisados 745 lotes dos quais 31% testaram positivos para a presença de sequências de flavivirus. As espécies que apresentaram taxas de positividade mais elevadas foram: An.algeriensis com uma Taxa Mínima de Infecção (TMI) de 56/1000 no Algarve em 2009, Cs.annulata TMI =22/1000 no Algarve em 2010, Cx.theileri e Cx.pipiens em Setúbal em 2010, TMI =20/1000. An. atroparvus, Ae. caspius, Ae. detritus e Cx. univittatus também produziram lotes positives. No geral, a positividade foi maior no Algarve. Análise das sequências virais obtidas revelou homologia das nossas sequências virais com sequências de referência de flavivírus específicos de mosquitos depositadas em bases de dados de acesso livre. A análise filogenética reflectiu a variabilidade genética dos flavivírus e revelou a relação genética das nossas sequências com as de outros flavivírus, especialmente os específicos de insectos. Tendo em consideração os anteriores isolamentos do vírus do Nilo Ocidental, o aumento acentuado nas densidades de mosquitos, o aumento de temperaturas que se tem vindo a registar, os casos recentes de transmissão de flavivírus por toda a Europa e o padrão desconhecido e imprevisível dos surtos destes vírus, os programas contínuos de vigilância epidemiológica têm-se revelado uma ferramenta indispensável para a Saúde Pública.

Molluscicidal and Mosquitocidal Activities of the Essential oils of Thymus capitatus Hoff. et Link. and Marrubium vulgare L.

Steam distillation of essential oils of aerial parts of Thymus capitatus and Marrubium vulgare L. collected at North cost of Egypt yielded 0.5% and 0.2%, respectively. Results of Gas chromatography-mass spectrometry analyses of the two samples identified 96.27% and 90.19% of the total oil composition for T. capitatus and M. vulgare, respectively. The two oil samples appeared dominated by the oxygenated constituents (88.22% for T. capitatus and 57.50% for M. vulgare), composed of phenols, mainly carvacrol (32.98%) and thymol (32.82%) in essential oil of T. capitatus, and thymol (34.55%) in essential oil of M. vulgare. It was evaluated the molluscicidal activity of T. capitatus and M. vulgare essential oils on adult and eggs of Biomphalaria alexandrina as well as their mosquitocidal activity on Culex pipiens. The LC50 and LC90 of T. capitatus essential oil against adult snails was 200 and 400 ppm/3hrs, respectively, while for M. vulgare it was 50 and 100 ppm/3hrs, respectively. Moreover, M. vulgare showed LC100 ovicidal activity at 200 ppm/24 hrs while T. capitatus oil showed no ovicidal activity. It was verified mosquitocidal activity, with LC50 and LC90 of 100 and 200 ppm/12hrs respectively for larvae, and 200 and 400 ppm/12hrs respectively for pupae of C. pipiens.

Low genetic diversity in Wolbachia-Infected Culex quinquefasciatus (Diptera: Culicidae) from Brazil and Argentina

Culex quinquefasciatus is a vector of human pathogens, including filarial nematodes and several viruses. Although its epidemiological relevance is known to vary across geographical regions, an understanding of its population genetic structure is still incipient. In light of this, we evaluated the genetic diversity of Cx. quinquefasciatus and Cx. pipiens x Cx. quinquefasciatus hybrids collected from nine localities in Brazil and one site in Argentina. We used mitochondrial genes cox1 and nd4, along with the coxA and wsp genes of the maternally-inherited Wolbachia endosymbiont. The nd4 fragment was invariant between samples, whilst cox1 exhibited four haplotypes that separated two types of Cx. quinquefasciatus, one clustered in southern Brazil. Low sequence diversity was generally observed, being discussed. Both Brazilian and Argentinian mosquitoes were infected with a single Wolbachia strain. As reported in previous studies with these populations, cox1 and nd4 diversity is not congruent with the population structure revealed by nuclear markers or alar morphology. Future Cx. quinquefasciatus research should, if possible, evaluate mtDNA diversity in light of other markers.

Estudo dos culicídeos (Diptera: Culicidae) nos cemitérios das ilhas da Madeira e Porto Santo.

O estudo bioecológico dos culicídeos é de grande importância, pois deste modo, é possível detectar a presença de várias espécies de mosquitos vectores de agentes patogénicos para o Homem e outros animais. Além disso, os culicídeos podem ser considerados factor de incomodidade para as populações humanas, devido às reacções alérgicas provocadas pelas suas picadas e pela dor causada. De Janeiro a Agosto de 2009 foram examinadas 415 armadilhas de oviposição e foram prospectados 700 recipientes artificiais nos 52 cemitérios das ilhas da Madeira e do Porto Santo. Das 415 armadilhas de oviposição examinadas, 148 (35,7%) continham formas imaturas, e, apesar dos cemitérios serem considerados as principais fontes de certas espécies de mosquitos, apenas foram detectados 45 (6,4%) criadouros larvares nos 700 recipientes artificiais com água investigados. Foram identificadas 4 espécies de mosquitos: Aedes (Finlaya) eatoni (Edwards, 1916), Culiseta (Allotheobaldia) longiareolata (Macquart, 1838), Culex (Culex) pipiens (Linnaeus, 1758) e Culex (Culex) theileri (Theobald, 1903). Verificou-se a predominância de Aedes eatoni em relação às outras espécies. A distribuição dos mosquitos variou com a altitude e com a temperatura do ar, encontrando-se em maior abundância nos 200-300 metros e entre os 22-26ºC. Os recipientes de metal (2=156,9; p=0,00<0,05) e aqueles com capacidade até 0,5 litros (2=9,925; p=0,0019<0,05) foram os preferidos pelas formas imaturas. No entanto, os imaturos não mostraram preferência em relação à exposição solar dos recipientes (2=0,799; p=0,671). ix Em relação à qualidade da água dos criadouros, verificou-se que a temperatura da água (t=- 1,39; p=0,16) e o teor em oxigénio dissolvido (z=-1,21; p=0,23) não tiveram qualquer influência na presença dos imaturos, mas tiveram-no o pH (z=-2,12; p=0,03<0,05) e a salinidade (z=-3,089; p=0,002<0,05) que influenciaram a sua presença tendo os respectivos valores variado entre 6-8 e 0,1-1,7 g/L, respectivamente. Verificou-se a existência de uma associação larvar entre Culiseta longiareolata e Culex pipiens (2=32,05; p=0,00<0,05) e entre Culex pipiens e Culex theileri (2=118,71; p=0,00<0,05). O mosquito Aedes aegypti não foi encontrado durante o período de estudo que ocorreu de Janeiro a Agosto de 2009. No entanto, foi detectado nos cemitérios de Câmara de Lobos e do Caniço no decorrer de duas campanhas, nas quais a autora participou, nos meses de Outubro e Novembro do mesmo ano.

Caso de um mosquito intensamente parasitado por larvas de ácaros aquáticos

One female of Culex fatigans Wied., 1828, was collected in a light trap at CENA-ESA«LQ»-USP, Piracicaba, at about 500 meters off the Piracicaba River banks. This mosquito exhibited 15 partially engorged pionid larvae of water mites (Prostigmata, Hydrachnellae) of the genus Arrenurus, attached to the articular membranes of the abdominal segments; no mites were found on the thorax or coxae. The larva is drawn, dorsad and ventrad. Since species cannot be identified from larvae, no specific name is given.

Caracterización del consumo de larvas de culícidos (Diptera) en dos especies de peces indígenas de la zona central de Argentina

Los mosquitos representan una amenaza para la salud del hombre y de los animales debido a que actúan como vectores de distintas enfermedades. Especies de peces nativos son potenciales candidatos a tenerse en cuenta para control biológico de poblaciones de culícidos. Experiencias de consumo de media hora y de 24 horas por Cnesterodon decemmaculatus (Jenyns, 1842) y Jenynsia multidentata (Jenyns, 1842) se llevaron a cabo con larvas de Culex pipiens (Linnaeus, 1758), midiéndose longitud estándar, ancho de boca y peso en individuos de las dos especies. En ambas pruebas, J. multidentata consumió más C. pipiens que C. decemmaculatus, consumiendo las hembras de esta última especie más que los machos (e igual a ambos sexos de J. multidentata en la prueba de 24 horas de duración). Estos resultados no variaron cuando se compararon tantos consumos absolutos o relativos para las pruebas de media hora, sin embargo cuando se compararon los consumos relativos al peso no se encontraron diferencias entre las especies para las pruebas de 24 horas. Análisis de regresión entre las tasas de consumo versus las variables morfométricas y el peso mostraron poco valor explicativo en las pruebas de media hora de duración, mientras que en las pruebas de 24 horas de duración los análisis de regresión tuvieron un mayor valor explicativo, especialmente con el ancho de la boca. Por último, pruebas de media hora de duración fueron llevadas a cabo exponiendo a hembras de C. decemmaculatus con larvas de C. pipiens y Aedes aegypti (Linnaeus, 1742) observándose una fuerte preferencia por las últimas. Este trabajo permitió evidenciar que las especies de peces en estudio presentan grandes diferencias en las tasas de consumo de C. pipiens en periodos cortos. Estas diferencias se atenuaron cuando las tasas de consumo se prolongaron y hasta llegar a desaparecer cuando el peso se tuvo en cuenta.

O virus do mixoma no coelho do mato (Sylvilagus minenses), sua transmissão pelos Aedes scapularis e aegypti

The brazilian wild rabbit (Sylvilagus minensis) is sensible to the virus of the mixomatosis but the desease takes on it a mild character, lasts for long time and generally do not kill the animal. The tumors are generally smaller and less numerous than those of the domestic rabbit, but sometimes there were noted large and flat lesions (fig. 3). The natural infection of the wild rabbit may be quite common not only because many rabbits caught in the country were found to be immune as also because it was found among the animals caught in the country near Rio, one that was infected with mixomatosis. The experimental infection of the Sylvilagus may be easily obtained by cutan, subcutan or conjuntival way and also when a health wild rabbit is placed in the same cage with a sick domestic animal. It is also possible to obtain the infection of the wild and domestic rabbits by the bite of infected blood sucking insects as fleas and mosquitoes. The infected mosquito can transmit the disease 2 or 3 times til 17 days after an infective meal on a sick rabbit. The transmission is a mecanical one and only the proboscis of the insect contains the virus as it was shown by the inoculation of emulsions of the proboscis, thorax and abdomen of the mosquito. Though mecanical this kind of transmission acts as an important epidemiological mean of dissemination of the deseasse and splains the suddendly outbreaks of mixomatosis in rabbits breedings where no new rabbits were introduced since very long time. The transmition of mixomatosis by fleas (Slenopsylla) was at first demonstrated by us, then S. Torres pointed out the capacity of Culex fatigans to transmit the desease and now we have proved that Aedes scapularis and Aedes aegypti were also able to transmit it (Foto 1 and 2). The virus of the mixomatosis (Chlamidozoon mixoma) is seen on the smeavs of the tumors of the wild reabbit with the same morphology, as in the material of the domestic animal.