Karyotypic variation and geographic distribution of Anopheles campestris-like (Diptera: Culicidae) in Thailand

Seventy-one isolines of Anopheles campestris-like were established from wild-caught females collected from human-biting and animal-biting traps at 12 locations in Thailand. All isolines had an average branch summation of seta 2-VI pupal skins ranging from 20.3-30.0 branches, which is in the range of An. campestris (17-58 branches). They showed three different karyotypes based on the amount of extra heterochromatin in the sex chromosomes, namely Forms B (X2, Y2), E (X1, X2, X3, Y5) and a new karyotypic Form F (X2, X3, Y6). Form B has been found only in Chaing Mai and Kamphaeng Phet populations, while Forms E and F are widely distributed throughout the species range. Genetic crosses between the 12 isolines, which were arbitrarily selected as representatives of An. campestris-like Forms B, E and F, revealed genetic compatibility that provided viable progeny through F2 generations, suggesting a conspecific nature of these karyotypic forms. These results are supported by the very low intraspecies variation (genetic distance < 0.005) of ITS2, COI and COII from genomic DNA of the three karyotypic forms.

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.

Adulticidal activity against Stegomyia aegypti ( Diptera: Culicidae ) of three Piper spp

Três espécies de Piper, Piper longum, P. ribesoides e P. sarmentosum, foram selecionadas para investigação da potencialidade contra Stegomyia aegypti adultos, principal vetor de dengue e febre do dengue hemorrágico. Sucessivas extrações por maceração com etanol a 95% mostraram uma porcentagem de extratos etanólicos, derivados de P. longum, P. ribesoides e P. sarmentosum, de 8,89, 3,21 e 5,30% (w/w), respectivamente. Todos os extratos de Piper mostraram atividade adulticida expressiva quando testados contra fêmeas de mosquitos através de aplicação tópica. A suscetibilidade das fêmeas do St. aegypt ao extrato de Piper etanólico foi dose dependente e variou entre as espécies de plantas. O mais elevado efeito adulticida foi demonstrado a partir do P. sarmentosum, seguido pelo P. ribesoides e P. longum, valores LD50 de 0,14, 0,15 e 0,26 µg/fêmea, respectivamente. O potencial destas espécies de Piper, como possíveis mosquiticidas, estabeleceu atividade convincente para futuras pesquisas a fim de desenvolver substâncias naturais para o combate a mosquitos adultos.

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 assessment of the in vitro sensitivity of Brugia malayi infective larvae to albendazole, diethylcarbamazine and ivermectin alone and in combination.

The antifilaricidal drugs ivermectin (IVM), diethylcarbamazine (DEC), and albendazole (ALB), used alone or in combinations against infective third-stage larvae (L3) of nocturnally subperiodic (NSP) Brugia malayi (Narathiwat strain), were tested in vitro for sensitivity, for 7 days. IVM alone reduced larval motility at concentrations of 10(-7), 10(-6), and 10(-5) M on day 3. DEC alone also had this effect at concentrations of 10(-6). 10(-5), and 10(-4) M on day 2. ALB alone did not have this effect throughout the experiment, at various concentrations. However, it had greater effect when used in combination with either DEC or IVM. The result also indicated that DEC or IVM, when used in combination with ALB at concentrations of 10(-6) M/10(-6) M, and 10(-5) M/10(-5) M was effectively better than each drug used alone at those concentrations. When both drug combinations were compared, ALB/DEC seemed to be more effective than ALB/IVM at a concentration of 10(-6) M/10(-6) M on day 3. Although IVM and DEC can reduce larval motility when used alone or in combination with ALB, they cannot kill these larvae in an in vitro cultivation, even at a high concentration (10(-5) M).

Evaluation of crude antigen of Dirofilaria immitis third-stage larva for detection of antibody against Wuchereria bancrofti infection by indirect ELISA.

Dirofilaria immitis is an important heart worm in dogs. An immunodiagnostic test is frequently applied to use an alternative antigen from other parasites. A crude antigen from infective third stage larva (L3) of D. immitis was employed in detecting the antibody to Bancroftian filariasis in humans by indirect ELISA. It was shown that 25 cases of Bancroftian filariasis (76%) at a cut-off value of 0.230, were positive. Cross-reactivity was tested using available sera of other helminthic infections. These sera were 47% (23/49) positive. They comprised a major intestinal helminthic infection, 7 from 15 (46%) strongyloidiasis sera, none from 5 (0%) hookworm infection sera, 6 from 10 (60%) trichinosis sera, 2 from 10 (20%) cysticercosis sera and 8 from 9 (88%) gnathostomiasis sera. The mean OD of sera from Bancroftian filariasis patients was not significantly different from that of the other helminthic infections (p>0.05). In this study, crude antigen may be valuable for the serodiagnosis of Wuchereria bancrofti when subjects do not have tissue helminth infections. However, the crude antigen should be purified to obtain a better sensitivity and specificity of the test.

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

Intraspecific hybridization of two karyotypic forms of Anopheles vagus (Diptera: Culicidae) and the related egg surface topography.

Hybridization tests of the two karyotypic forms (Form A and B) of laboratory-raised, isolines of Anopheles vagus, were conducted by induced copulation. The results of reciprocal- and back-crosses indicated that they were genetically compatible, providing viable progeny. Comparative egg morphometry and morphology, aided by scanning electron microscopy (SEM), revealed that the eggs of the two karyotypic forms were morphometrically and morphologically similar.

Intraspecific hybridization of Anopheles minimus (Diptera: Culicidae) species A and C in Thailand.

Hybridization tests of laboratory-raised, isolines of Anopheles minimus, species A and C were conducted by induced copulation. The three isolines were established based on three morphological variants of wild-caught, fully engorged females and two distinct types of metaphase chromosomes. They were An. minimus species A: V form (X1,Y1), M form (X2,Y1); species C: P form (X3,Y2). The results of reciprocal and back crosses indicated that the two morphologically variant forms of species A were genetically compatible, providing viable progeny and completely synaptic salivary gland polytene chromosomes, whereas they were genetically incompatible with species C and/or the P form. Hybrid progeny was only obtained from both forms of species A females x species C males, but asynaptic salivary gland polytene chromosomes on 3L and partial development of ovarian follicles in females were seen. Back crosses of F1 hybrid males with parental species A females provided viable progeny, while back crosses of F1 hybrid females with parental species C males provided progeny of low viability and adult males with abnormal spermatozoa, suggesting the partial reproductive isolation of An. minimus species A and C.

A simple technique for the in vitro cultivation of nocturnally subperiodic Brugia malayi infective larvae.

A simple system for the in vitro cultivation of nocturnally subperiodic Brugia malayi was developed. The manner of cultivation consisted of a 1:1 (v/v) mixture of Iscove's Modified Dulbecco's medium and NCTC-135 medium supplemented with 20% fetal bovine serum by using candle jar incubation at 37 degrees C instead of CO2 incubator. Changing the media: every 2 days, 3 days and changing media on day 7, then every 2 days produced a larval survival rate of 50% (70/140) on day 10, 49% (82/166) on day 6, and 53% (105/200) on day 9. With this technique, up to 50% of the infective stage larvae (L3) survived for up to 10 days and had long life for at least 27 days in all experiments with low larval survival rate in the fourth week. In addition, the culture system promoted molting L3 to fourth stage larvae (L4) after 7 days, as shown by light microscope.