Susceptibility of Anopheles campestris-like and Anopheles barbirostris species complexes to Plasmodium falciparum and Plasmodium vivax in Thailand

Nine colonies of five sibling species members of Anopheles barbirostris complexes were experimentally infected with Plasmodium falciparum and Plasmodium vivax. They were then dissected eight and 14 days after feeding for oocyst and sporozoite rates, respectively, and compared with Anopheles cracens. The results revealed that Anopheles campestris-like Forms E (Chiang Mai) and F (Udon Thani) as well as An. barbirostris species A3 and A4 were non-potential vectors for P. falciparum because 0 percent oocyst rates were obtained, in comparison to the 86.67-100 percent oocyst rates recovered from An. cracens. Likewise, An. campestris-like Forms E (Sa Kaeo) and F (Ayuttaya), as well as An. barbirostris species A4, were non-potential vectors for P. vivax because 0 percent sporozoite rates were obtained, in comparison to the 85.71-92.31 percent sporozoite rates recovered from An. cracens. An. barbirostris species A1, A2 and A3 were low potential vectors for P. vivax because 9.09 percent, 6.67 percent and 11.76 percent sporozoite rates were obtained, respectively, in comparison to the 85.71-92.31 percent sporozoite rates recovered from An. cracens. An. campestris-like Forms B and E (Chiang Mai) were high-potential vectors for P. vivax because 66.67 percent and 64.29 percent sporozoite rates were obtained, respectively, in comparison to 90 percent sporozoite rates recovered from An. cracens.

Salivary gland proteins of the human malaria vector, Anopheles dirus B (Diptera: Culicidae)

Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3 - 10 days was approximately 1.08 ± 0.04 æg/female and 0.1 ± 0.05 æg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy.

Proteínas das glândulas salivares do Anopheles dirus B (Diptera: Culicidae), vetor da malária humana foram determinadas e analisadas. A quantidade de proteínas das glândulas salivares em mosquitos com três a 10 dias de idade foi de aproximadamente 1,08 ± 0,04 æg/ fêmea e de 0,1 ± 0,05 æg/macho. As glândulas salivares de ambos os sexos mostraram organização morfológica semelhante à de outros mosquitos anofelinos. Em fêmeas, apirase acumula-se nas regiões distais, enquanto alfa-glucosidase foi encontrada na região proximal dos lóbulos laterais. Esta distribuição diferencial das enzimas analisadas reflete a especialização de diferentes regiões para alimentação de açucares e sangue. Análise SDS-PAGE revelou que pelo menos sete proteínas foram encontradas nas glândulas salivares de fêmeas, das quais cada região morfológica continha diferentes proteínas principais. Perfis eletroforéticos de proteínas semelhantes foram detectados comparando-se mosquitos não alimentados e alimentados por sangue, sugerindo que não existe proteína específica induzida pelo mesmo. Análise por gel poliacrilamida bi-dimensional mostrou a mais abundante proteína de glândulas salivares com aproximadamente 35 kilodaltons de massa molecular e ponto isoelétrico de aproximadamente 4,0. Estes resultados dão informações básicas que levariam a estudos adicionais sobre o papel das proteínas salivares do An. dirus B na transmissão da doença e hematofagia.

Susceptibility of two karyotypic forms of Anopheles aconitus (Diptera: Culicidae) to Plasmodium falciparum and P. vivax

Quatro colônias desenvolvidas em laboratório, de duas formas cariotípicas de Anopheles aconitus i.e. forma B (cepa Chiang Mai e Phet Buri) e C (Cepa Chiang Mai e Mae Hong Son), foram infectadas experimentalmente com Plasmodium falciparum e P. vivax usando técnica de alimentação com membrana artificial e dissecados oito e 12 dias após alimentação da média de oocistos e esporozoitos, respectivamente. Os resultados revelaram que An. aconitus formas B e C foram suscetíveis ao P. falciparum e P. vivax isto é, forma B (cepa Chiang Mai e Phet Buri/P. falciparum e P. vivax) e forma C (cepa Chiang Mai e Mae Hong Son/P. vivax). Análises estatísticas comparativas das taxas de oocistos, número médio de oocistos por intestino médio infectado e taxas de esporozoitos entre todas as cepas de An. aconitus formas B e C ao grupo interno de vetores controles, An. minimus A e C, não exibiram nenhuma diferença significante, confirmando o alto potencial vetor das duas espécies de Plamodium. Os cristais semelhantes a esporozoitos encontrados no lobo médio das glândulas salivares que poderiam ser um fator enganoso na identificação de esporozoitos verdadeiros nas glândulas salivares foram encontrados em ambos An. aconitus formas B e C.

Laboratory colonization of Aedes lineatopennis.

Aedes lineatopennis, a species member of the subgenus Neomelaniconion, could be colonized for more than 10 successive generations from 30 egg batches [totally 2,075 (34-98) eggs] of wild-caught females. The oviposited eggs needed to be incubated in a moisture chamber for at least 7 days to complete embryonation and, following immersion in 0.25-2% hay-fermented water, 61-66% of them hatched after hatching stimulation. Larvae were easily reared in 0.25-1% hay-fermented water, with suspended powder of equal weight of wheat germ, dry yeast, and oatmeal provided as food. Larval development was complete after 4-6 days. The pupal stage lasted 3-4 days when nearly all pupae reached the adult stage (87-91%). The adults had to mate artificially, and 5-day-old males proved to be the best age for induced copulation. Three to five-day-old females, which were kept in a paper cup, were fed easily on blood from an anesthetized golden hamster that was placed on the top-screen. The average number of eggs per gravid female was 63.56 +/- 22.93 (22-110). Unfed females and males, which were kept in a paper cup and fed on 5% multivitamin syrup solution, lived up to 43.17 +/- 12.63 (9-69) and 15.90 +/- 7.24 (2-39) days, respectively, in insectarium conditions of 27 +/- 2 degrees C and 70-80% relative humidity.

Sequence analyses of three nuclear ribosomal loci and a mitochondrial locus in cytologically different forms of Thai Anopheles aconitus mosquitoes.

Ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) of Anopheles aconitus mosquitoes were examined to investigate intra- and inter-species variation amongst the members of the Minimus group of Anopheles subgenus Cellia. Three rDNA loci (ITS1, ITS2 and D3 regions) and a mtDNA locus (cytochrome oxidase II) were analyzed in An. aconitus Form B and Form C collected in Chiang Mai Province, Thailand. The results show that the consensus sequences of the four loci of the two forms are consistent with those of mosquitoes in the genus Anopheles. No intraindividual variation was detected, but intrapopulation variation was present with polymorphic sequences in some forms for each gene examined. The variation rates were approximately 0.15 to 0.8%. These data indicate that An. aconitus Form B and Form C in Chiang Mai, Thailand are conspecific. In this study, the complete ITS1 sequence of An. aconitus is reported for the first time. The region showed a high variation rate (approximately 55%), compared to the closely related species An. minimus C. It is suggested that this rDNA locus may provide sequence information to differentiate the members of the Minimus group of Anopheles subgenus Cellia.

Comparative morphometry and morphology of Anopheles aconitus Form B and C eggs under scanning electron microscope

Estudos comparativos morfométricos e morfológicos de ovos à microscopia eletrônica de varredura (SEM) foram efetuados nas três linhagens de duas formas cariotípicas de Anopheles aconitus, isto é, Forma B (linhagens Chiang Mai e Phet Buri) e Forma C (linhagens Chiang Mai e Mae Hong Son). Exame morfométrico revelou a variação intraespecífica com respeito à largura de superfície [36,77 ± 2,30 µm (Forma C: linhagem Chiang Mai) = 38,49 ± 2,78 µm (Forma B: linhagem Chiang Mai) = 39,06 ± 2,37 µm (Forma B: linhagem Phet Buri) > 32,40 ± 3,52 µm (Forma C: linhagem Mae Hong Son)] e número de tubérculos posteriores sobre a superfície livre [2,40 ± 0,52 (Forma B: linhagem Phet Buri) = 2,70 ± 0,82 (Forma B: linhagem Chiang Mai) < 3,10 ± 0,32 (Forma C: linhagem Chiang Mai) = 3,20 ± 0,42 (Forma C: linhagem Mae Hong Son)] embora a topografia de superfície dos ovos entre as três linhagens de duas formas cariotípicas tenham sido morfologicamente semelhantes.

Comparative studies on the biology and filarial susceptibility of selected blood-feeding and autogenous Aedes togoi sub-colonies

Blood-feeding and autogenous sub-colonies were selected from a laboratory, stock colony of Aedes togoi, which was originally collected from Koh Nom Sao, Chanthaburi province, Southeast Thailand. Comparative biology and filarial susceptibility between the two sub-colonies (blood-feeding: F11, F13; autogeny: F38, F40) were investigated to evaluate their viability and vectorial capacity. The results of comparison on biology revealed intraspecific differences, i.e., the average egg deposition/gravid female (F11/F38; F13/F40), embryonation rate (F13/F40), hatchability rate (F11/F38; F13/F40), egg width (F11/F38), wing length of females (F13/F40), and wing length and width of males (F11/F38) in the blood-feeding sub-colony were significantly greater than that in the autogenous sub-colony; and egg length (F11/F38) and width (F13/F40), and mean longevity of adult females (F11/F38) and males (F13/F40) in the blood-feeding sub-colony were significantly less than that in the autogenous sub-colony. The results of comparison on filarial susceptibility demonstrated that both sub-colonies yielded similar susceptibilities to Brugia malayi [blood-feeding/autogeny = 56.7 percent (F11)/53.3 percent(F38), 60 percent(F13)/83.3 percent(F40)] and Dirofilaria immitis [blood-feeding/autogeny = 85.7 percent(F11)/75 percent(F38), 45 percent(F13)/29.4 percent(F40)], suggesting autogenous Ae. togoi sub-colony was an efficient laboratory vector in study of filariasis