Tire-breeding mosquitoes of public health importance along an urbanisation gradient in Buenos Aires, Argentina

Used vehicle tires are a source of mosquito vectors and a means of their introduction and expansion. With the aim of assessing the effects of urbanisation on the main mosquito vectors in temperate Argentina, the infestation levels of Aedes aegypti (L.) and Culex pipiens L. were studied in used tires from highly urbanised cities to low-urbanised small towns in Buenos Aires. Immatures of both species accounted for 96% of the 9,722 individuals collected; the total individuals collected represented seven species. The percentage of water-filled tires containing mosquitoes [container index (CI)] was 33% and the percentage of infested sites [site index (SI)] was 65.2%. These indexes decreased significantly from low to high urbanisation levels for both mosquito species. The relative abundance (RA) of Ae. aegypti immatures was slightly higher toward large cities, but showed no difference for Cx. pipiens. The CI of shaded tires was significantly higher than the CI of exposed tires for both mosquito species. There was no difference in RA values between shaded and sunlit tires. The CI and the SI were highest during the summer across the urbanisation levels, except for Cx. pipiens, which continued to increase during the autumn in small towns. Results related to urbanisation gradient, sunlit exposure and seasonality are discussed.     

Impact of population age structure on Wolbachia transgene driver efficacy: ecologically complex factors and release of genetically modified mosquitoes

Wolbachia symbionts hold theoretical promise as a way to drive transgenes into insect vector populations for disease prevention. For simplicity, current models of Wolbachia dynamics and spread ignore ecologically complex factors such as the age structure of vector populations and overlapping vector generations. We developed a model including these factors to assess their impact on the process of Wolbachia spread into populations of three mosquito species (Anopheles gambiae, Aedes aegypti and Culex pipiens). Depending on the mosquito species, Wolbachia parameters, released mosquito life stage and initial age structure of the target population, the number of Wolbachia-infected mosquitoes that we predict would need to be released ranged from less than the threshold calculated by the simple model to a 10-30-fold increase. Transgenic releases into age-structured populations, which is an expectation for wild mosquitoes, will be difficult and depending on the circumstances may not be economically or logistically feasible due to the large number of infected mosquitoes that must be released. Our results support the perspective that understanding ecological factors is critical for designing transgenic vector-borne disease control strategies.     

The effect of parasitism by the mermithid Romanomermis culicivorax on the dry weight and hemolymph soluble protein content of three species of mosquitoes

The dry weight, hemolymph soluble protein composition, and content of three species of mosquitoes, Culex pipiens, Aedes taeniorhynchus, and Anopheles quadrimaculatus were examined to determine the effects of parasitism by the mermithid nematode Romanomermis culicivorax. The dry weights of infected fourth-stage larvae of all three species were significantly lower than controls. The differences in weight found between infected early and late C. pipiens and A. quadrimaculatus larvae were attributed to the weight of the parasite itself. This difference was not noticeable in A. taeniorhynchus larvae. Hemolymph proteins were severely depleted in all three mosquito species during parasitism by R. culicivorax. Analysis of protein composition by PAGE showed that these depletions were accompanied by a reduction in the number of proteins. Differences between protein composition concentrations were evident between early and late fourthstage control larvae of C. pipiens and A. quadrimaculatus. The concentration of some low-molecular-weight proteins (below 68,000) remained constant between infected and control samples of all three mosquito species.     

High-pressure liquid chromatographic analysis of hemolymph plasma catecholamines in immune-reactive Aedes aegypti

Tyrosine and catecholamines have been implicated as substrates for the encapsulation reactions involved in the immune response of mosquitoes to microfilariae (mff). Identification and quantitation of tyrosine and catecholamines present in Aedes aegypti hemolymph plasma were accomplished by ion-pair high-pressure liquid chromatography with electrochemical detection at either +650 or +850 mV vs Ag/AgCl. Tyrosine, dopamine, and N-beta-alanyldopamine were detected in the hemolymph plasma of naive A. aegypti. Although no differences in these compounds were observed in hemolymph plasma from A. aegypti inoculated with Dirofilaria immitis mff, the chromatogram showed a single major peak (PI) (65 microM, expressed as dopamine equivalents) that was not present in naive hemolymph plasma. Saline-inoculated controls contained only 5% of the PI in immune reactive hemolymph plasma. A high concentration of PI (127 +/- 39 microM) was also detected after treatment of hemolymph plasma with mild alkaline conditions (pH 9.0), indicating that it is normally present as an electrochemically inert form in naive mosquitoes. High concentrations of PI were also detected in the naive hemolymph plasma from three other mosquito species, but no PI was found in A. trivittatus under any conditions. PI did not cochromatograph with any of the catecholamines commonly thought to be involved in immune responses of dipterans against metazoan parasites, suggesting that it may be a unique substrate for these reactions. The biological relevance of PI was evidenced by its appearance in the hemolymph plasma of two strains of D. immitis-inoculated A. aegypti.     

Further characterization of refractoriness in Aedes aegypti (L.) to infection by Dirofilaria immitis (Leidy)

Factors which control the expression of the refractory or susceptible condition to infection with Dirofilaria immitis in the mosquito. Aedes aegypti, were investigated using three protocols. (1) Microfilariae and prelarvae were injected into the hemocoel of susceptible A. aegypti. Some microfilariae and prelarvae developed to the L1 larval stage but they failed to complete development to the infective stage. (2) Enema of microfilariae and prelarvae from infected susceptible and refractory donor females were given into the midgut of uninfected susceptible and refractory recipient females. The results indicate that the conditions which inhibit the initiation of development are present in the Malpighian tubules and not in the midgut of the refractory mosquitoes. (3) Transplants of infected Malpighian tubules from susceptible and refractory donor females were made into the abdominal hemocoel of uninfected susceptible and refractory recipient females. The results showed that the refractory condition depends on the genetic makeup of the donor, not the recipient, mosquito. The above results taken as a whole indicate that the factors which control refractoriness are not present in the midgut but are present in the Malpighian tubule cells of refractory A. aegypti.     

Parasite-induced suppression of the immune response in Aedes aegypti by Brugia pahangi

The melanization response against intrathoracically inoculated Brugia pahangi and Dirofilaria immitis microfilariae (mff) isolated from vertebrate host blood was evaluated in both uninfected Aedes aegypti black-eyed Liverpool strain and in mosquitoes harboring a developing B. pahangi infection. The immune response against inoculated mff of either species was significantly reduced by 28-47% in infected as compared with uninfected mosquitoes. Attempts to passively transfer this suppression factor(s) by inoculating naive mosquitoes with 0.1-0.2 microliter of hemolymph from B. pahangi-infected mosquitoes produced equivocal results. The role this parasite-induced immune suppression might play in aiding parasite survival in compatible vectors is discussed.     

Plasmodium gallinaceum: sporozoite activity in immune mosquito hemolymph

Sporozoites of Plasmodium gallinaceum are relatively inactive in Hanks' balanced salt solution or in the hemolymph of the susceptible Aedes aegypti mosquito. They become agitated and very active in the presence of the hemolymph of the innately immune mosquito, Culex pipiens. Sporozoites from the latter are more infective in chicks than the former. This behavior is likely the result of stimulation or irritation by the adverse environment.     

Responses of Anopheles gambiae, Anopheles stephensi, Aedes aegypti, and Culex pipiens mosquitoes (Diptera: Culicidae) to cool and humid refugium conditions

Like all terrestrial arthropods, mosquitoes must cope with the threat of desiccation. To gain insight into their survival strategies, we recorded the behavioral responses of Anopheles gambiae, Anopheles stephensi, Aedes aegypti, and Culex pipiens offered zones of different microclimatic conditions in laboratory cages. The cooled refugium was at 25.6 degrees C, 86% RH and the control was at 28.5 degrees C, 75% RH, i.e., a difference in saturation deficit of 3.9 mm Hg between the two zones. We show that newly-emerged adults, with no access to water or sugar, prefer the cooler and more humid refugium with a saturation deficit half that in the control and where the mosquitoes could reduce their metabolic rate. This response is delayed in Ae. aegypti, perhaps because the energy reserves accumulated as larvae are higher in this species. This study shows that mosquitoes under stress can use their thermohygroreceptor cells to guide them to locations that facilitate survival.     

Hemolymph of Anopheles stephensi from noninfected and Plasmodium berghei-infected mosquitoes. 1. Collection procedure and physical characteristics.

Hemolymph was collected from adult female Anopheles stephensi by centrifugation of incised mosquitoes. Approximately 0.1 muliter was collected from each recently emerged mosquito, although smaller amounts were recovered with increasing age of the mosquito. Determinations were made of the pH, osmotic pressure, and specific gravity of this hemolymph at various times during the life of the adult mosquito. The values obtained were within the ranges found for other insects. Hemolymph collected from mosquitoes fed on hamsters infected with Plasmodium berghei had different values than hemolymph from mosquitoes fed on noninfected hamsters. This probably was due to differences between the quality of these 2 types of blood meals, rather than to the direct effects of the malaria parasite on the infected mosquito itself.     

Hematophagous insects as vectors for frog trypanosomes

Experimental infections of three hematophagous arthropods (Rhodnius prolixus, Aedes aegypti, and Culex pipiens) with a trypanosome of the Trypanosoma rotatorium complex found in the frogs Hyla crepitans and Leptodactylus insularum revealed that A. aegypti is a good host for the flagellate; the course of development in the intestinal tract of the mosquito is described from 15 minutes to 168 hours. C. pipiens showed only low intestinal infections and R. prolixus did not permit development of the parasite. It is postulated that, in addition to the transmission of T. rotatorium by leeches, batrachophilic mosquitoes may transmit the parasite to frogs of more terrestrial habits by being ingested by these anurans.     

Relationship of hemolymph phenol oxidase and mosquito age in Aedes aegypti.

Monophenol oxidase (MPO) and diphenol oxidase (DPO) activity in hemocytes and cell-free plasma perfused from 7-, 14-, 21-, and 28-day-old Aedes aegypti mosquitoes were compared. A progressive decrease of enzyme activity was detected as mosquito age increased, and this decrease was significant in both hemocytes and cell-free plasma when mosquitoes were 28 days old as compared with that found in 7-day-old mosquitoes. There was no significant difference in total hemolymph protein as mosquito age increased. Although this decreased MPO and DPO activity might be partially responsible for the reduced immune response against filarial worms previously reported for older mosquitoes, other factors undoubtedly play a significant role.     

A two-year survey on mosquitoes of Lebanon

A total of 6,500 mosquitoes were identified during a two-year survey (1999-2001) in Lebanon, and these belonged to twelve species: Culex pipiens, Cx. laticinctus, Cx. mimeticus, Cx. hortensis, Cx. judaicus, Aedes aegypti, Ae. cretinus, Ochlerotatus caspius, Oc. geniculatus, Oc. pulchritarsis, Culiseta longiareolata and Anopheles claviger. Culex pipiens was the most predominant species in Lebanon, collected indoors and outdoors. It was continuously abundant and active throughout the year. Culex judaicus was a small and rare mosquito and it is reported to occur for the first time in Lebanon. On the coastal areas, Ochlerotatus caspius was very common, and proved to be a complex of species as two forms were detected. One of the vectors of malaria, Anopheles claviger, was collected from May to September, from eight sites in Lebanon. Its breeding sites were restricted to fresh, cool, and clean water in pools and wells. Most of these breeding sites were in the populated Metn County where a few indigenous cases of malaria were reported from 1997-2000. This shows that the reported malaria cases were not imported, but caused by the bites of locally infected Anopheles females.     

Parasitoid-induced behavioral alterations of Aedes aegypti mosquito larvae infected with mermithid nematodes (Nematoda: Mermithidae).

A wide range of parasites are known to cause behavioral changes in their hosts and parasitized insects are especially amenable to the study of such changes. The majority of studies addressing parasite-induced behavioral alterations have focused on parasites with complex life cycles and the adaptive nature of such changes. Behavioral changes caused by parasitoids, single-host parasites that kill their host upon emergence, have been studied less and the adaptive nature of these changes is likely to be different than those in complex life cycles. I investigated behavioral alterations in Aedes aegypti mosquito larvae infected with parasitoid nematodes (family Mermithidae). I conducted several experiments in which I tested the following hypotheses: 1) Mermithid nematodes induce behavioral changes in mosquito larvae and the changes are density dependent. 2) Different species of mermithid nematodes induce similar changes in mosquito larvae behavior. 3) Behavioral alterations vary with mermithid developmental stage. 4) Mosquito larvae infected with mermithid nematodes behave similarly to uninfected food-deprived mosquito larvae. I found that 4th instar Ae. aegypti infected with Romanomermis culicivorax or Strelkovimermis spiculatus exhibited resting behaviors significantly more often than uninfected controls but that intensity of infection did not affect activity levels. In earlier instars, infected mosquito larvae were more active than uninfected control larvae in some behaviors associated with feeding. There was no significant difference between infected and uninfected food-deprived mosquitoes in nine of the ten behaviors observed. The decrease in activity of late instar Ae. aegypti larvae infected with mermithids may be a parasitoid adaptation that reduces the risk of predation and thus increases host and parasitoid survival. The increase in feeding activity in earlier instars as well as the similarity between uninfected food-deprived and infected Ae. aegypti behavior may indicate that these behaviors are adaptive for the parasitoid, increasing nutritional acquisition for successful parasitoid development.     

Characterization of phospholipase B of Culex pipiens fatigans

Phospholipase B has been found in the mosquito Culex pipiens fatigans, and some of its properties have been studied. The enzyme had a high optimum temperature (45 degrees C) and broad alkaline pH optimum (8-9). It was inactive toward diacylphospholipids, and hydrolyzed lysolecithin at a higher rate than lysophosphatidyl ethanolamine. The enzyme was heat labile, but lysolecithin protected it against heat inactivation. Phosphatidyl ethanolamine, phosphatidyl choline, deoxycholate, Fe(+++), and Hg(++) inhibited the enzyme markedly. The enzyme was present mainly in larvae; little enzyme activity was detected in pupae or adults. The total and specific activities were highest in IV instar (6 day) and I instar (1st day) larvae, respectively. It was localized in the microsomal fraction and was distributed mainly in the abdomen and thorax of the insect. The enzyme was present at much higher levels of activity in larvae of the mosquitoes Anopheles stephensi and Aedes aegypti.     

Plasmodium gallinaceum: antibodies to circumsporozoite protein prevent sporozoites from invading the salivary glands of Aedes aegypti.

A circumsporozoite protein-specific monoclonal antibody (N2H6D5) was injected into malaria-infected mosquitoes to determine its effect on the sporogonic cycle. After injection of antibody into mosquitoes (100 ng each), positive immunofluorescence (measured on air-dried sporozoites) reactions in hemolymph extracts were observed at a dilution of 1:1000. At 72 hr postinjection the levels dropped to 1:10. Sporozoites coinjected with antibody did not invade the salivary glands. In naturally infected mosquitoes, sporozoites were released over a period of 3 to 4 days. Therefore, mosquitoes were injected twice. The first injection was a day before the beginning of sporozoite release and the second, 2 days later. Sporozoite invasion of the salivary glands was assessed 3 days after the second injection, by microscopic examination of dissected glands. At this stage, all oocysts had completed maturation and released the sporozoites. Salivary gland infections were totally prevented in mosquitoes given two injections of 100 ng N2H6D5. Hence, sustained presence of anti-circumsporozoite antibodies in the hemolymph can render female Aedes aegypti refractory to Plasmodium gallinaceum.     

Hemolymph of Anopheles stephensi from noninfected and Plasmodium berghei-infected mosquitoes. 3. Carbohydrates.

Determinations were made of carbohydrates in hemolymph collected from adult female mosquitoes (Anopheles stephensi). First the hemolymph was fractionated by extraction and precipitation procedures, after which qualitative and quantitative determinations of carbohydrates were made by thin layer chromatography. The most abundant sugars found in the hemolymph were glucose and trehalose, though maltose, glucuronic acid, and inositol could be found after the mosquitoes took blood meals. After the mosquitoes ingested a noninfected blood meal, their hemolymph sugar levels rose almost 4-fold. There was less of an increase following a blood meal infected with the rodent malaria parasite, Plasmodium berghei. Depletion of sugars in the hemolymph of infected mosquitoes may result from direct utilization of sugar by the malaria parasite developing within the mosquito.     

Midgut barrier imparts selective resistance to filarial worm infection in Culex pipiens pipiens

Mosquitoes in the Culex pipiens complex thrive in temperate and tropical regions worldwide, and serve as efficient vectors of Bancroftian lymphatic filariasis (LF) caused by Wuchereria bancrofti in Asia, Africa, the West Indies, South America, and Micronesia. However, members of this mosquito complex do not act as natural vectors for Brugian LF caused by Brugia malayi, or for the cat parasite B. pahangi, despite their presence in South Asia where these parasites are endemic. Previous work with the Iowa strain of Culex pipiens pipiens demonstrates that it is equally susceptible to W. bancrofti as is the natural Cx. p. pipiens vector in the Nile Delta, however it is refractory to infection with Brugia spp. Here we report that the infectivity barrier for Brugia spp. in Cx. p. pipiens is the mosquito midgut, which inflicts internal and lethal damage to ingested microfilariae. Following per os Brugia exposures, the prevalence of infection is significantly lower in Cx. p. pipiens compared to susceptible mosquito controls, and differs between parasite species with <50% and <5% of Cx. p. pipiens becoming infected with B. pahangi and B. malayi, respectively. When Brugia spp. mf were inoculated intrathoracically to bypass the midgut, larvae developed equally well as in controls, indicating that, beyond the midgut, Cx. p. pipiens is physiologically compatible with Brugia spp. Mf isolated from Cx. p. pipiens midguts exhibited compromised motility, and unlike mf derived from blood or isolated from the midguts of Ae. aegypti, failed to develop when inoculated intrathoracically into susceptible mosquitoes. Together these data strongly support the role of the midgut as the primary infection barrier for Brugia spp. in Cx. p. pipiens. Examination of parasites recovered from the Cx. p. pipiens midgut by vital staining, and those exsheathed with papain, suggest that the damage inflicted by the midgut is subcuticular and disrupts internal tissues. Microscopic studies of these worms reveal compromised motility and sharp bends in the body; and ultrastructurally the presence of many fluid or carbohydrate-filled vacuoles in the hypodermis, body wall, and nuclear column. Incubation of Brugia mf with Cx. p. pipiens midgut extracts produces similar internal damage phenotypes; indicating that the Cx. p. pipiens midgut factor(s) that damage mf in vivo are soluble and stable in physiological buffer, and inflict damage on mf in vitro.     

Four species of mosquito as possible vectors for Dirofilaria immitis piedmont rice-fields

Wild mosquitoes were allowed to feed, during two nights, on a heartworm-infected dog with a very high microfilaraemia (72,000 and 78,000 microfilariae/ml just prior to be allocated in the live trap). A heartworm-free dog was used as control bait in the second night. Engorged mosquitoes (Aedes caspius, Anopheles maculipennis s.l., Culex modestus, and Cx pipiens) were kept under laboratory conditions, artificially fed until day 10 post-infection (PI) and then dissected for the presence of Dirofilaria immitis larvae. Mortality of Ae. caspius and Cx modestus was significantly higher than controls on day 3 PI (89.4 and 80.3%, respectively), but survival rates were similar in the following period. Third-stage larvae were observed from day 12 to 17 PI in the four mosquito species studied. However, vector efficiency was significantly higher in Ae. caspius which produced 102 (73.9%) of the 138 infective larvae found. Although less efficient vectors, the other 3 species may contribute to D. immitis transmission in the study area due to their abundance (Cx modestus) or higher resistance to the negative effects of infection (An. maculipennis s.l., Cx pipiens). As far as Cx pipiens is concerned, this mosquito, which luckily fed the least frequently on the dog, confirmed to act as powerful vector in iperendemic areas. The risks for veterinary and medical health, related to the zoo-antropophylic host-feeding pattern of the studied species, are discussed.     

Polymorphism of mitochondrial DNA and infection with symbiotic cytoplasmic bacterium Wolbachia pipientis in mosquitoes of the Culex pipiens complex from Russia

A total of 208 mosquitoes of the Culex pipiens complex from 15 basement and terrestrial populations collected in different regions of the European part of Russia and Siberia were examined by genetic methods. Among these, two major mitotypes, M and P, were identified. These mitotypes differed by six substitutions in the 246-bp mitochondrial DNA cytochrome oxidase I gene fragment examined. Urban basement mosquito ecotype C. pipiens (form molestus) were characterized by the presence of mitotype M and infection with the endosymbiotic bacteria of the genus Wolbachia. Mosquitoes of the C. pipiens complex inhabiting opened biotopes harbored mitotype P, or its variety, mitotype P1, and were not infected with Wolbachia. Thus, in natural conditions marked linkage disequilibrium between cytoplasmic elements, mitochondrial DNA and Wolbachia, can be observed. Similarity of mitotypes in form molestus mosquito from different geographical localities favors the hypothesis on the common ancestry of urban mosquitoes.     

广州地区荷木夜间树干液流补水的影响因子及其对蒸腾的贡献

明确树木夜间水分补充现象有助于提高总蒸腾量和冠层气孔导度估算的精确度,进一步认识冠层蒸腾与树干液流之间存在的时滞关系.本研究采用热消散探针法测定了广州地区的荷木树干液流密度,同步监测了主要的环境因子,从不同时间尺度分析了树干夜间液流的水分补充现象.结果表明：与白天相比,荷木夜间液流密度较小,旱季变化幅度比湿季大;夜间水分补充的时间段主要在前半夜(18:00-22:00);年内各季节夜间水分补充量之间没有显著差异,与环境因子之间的偏相关关系不显著,但与胸径、树高、冠幅、树干生物量、冠层生物量的回归曲线拟合很好,表明树形特征和生物量能更好地解释夜间补水的变化;各季节夜间水分补充量对总蒸腾量的贡献有显著差异,旱季明显高于湿季.