Reproductive physiology of Anopheles gambiae and Anopheles atroparvus.

When exposed to a human host, Anopheles gambiae started probing 4 h post-eclosion, but 95% successfully blood-fed by 16-20 h with maximal blood volumes of 5- 10 microl per female. When fed sugar, the 95% feeding was not observed until 36-40 h post-eclosion; sugar meals appeared to interfere with blood meals. Similarly in An. atroparvus, maximum volumes were 10 microl when starved but only 6 microl when fed sugar. This species did not bite before 2 d, and 95% biting was by 4 d. Given single blood meals to water-kept An. gambiae, a threshold body size for oogenesis was detected. With wing lengths below 2.8 mm, eggs never matured, but when sugar-fed, females of all sizes matured eggs including the synthesis of maternal deposits. Although sugar feeding interfered with blood feeding, more lipid was transferred to the yolk. In water-kept An. atroparvus only 5% of the females produced eggs. When sugar-fed for 4 d, all females matured eggs, so in this species sugar feeding appeared to be essential for oogenesis. An. gambiae always took multiple blood meals, tested at any time after the first ones, leading to 120 mature eggs/female. Yolk composition was 3.9 mcal protein and 3.8 mcal lipid/oocyte when kept on water, but 2.8 meal protein and 4.3 mcal lipid/oocyte with intermittent sugar meals, thus marking a surprising flexibility in synthesis of yolk protein and lipid that strongly depends on additional carbohydrates sources. Only 80% of water-fed An. atroparvus re-fed 2 d after a first blood meal with small females taking three blood meals but they still showed reduced fecundity. Only the large water-fed females matured eggs, with blood volumes higher than 9-12 microl. When fed sugar, the blood meal input was reduced, but oogenesis was possible, whereas water-fed females required three blood meals to reach the caloric level comparable to pre-feeding sugar-fed females. Water-fedAn. gambiae could survive on daily blood meals alone, but survival was further extended by intermittent sugar meals. When offered a blood donor daily, there was a behavioral difference. Females maintained alone showed a more or less regular 3 d feeding and oviposition activity, while females kept in groups fed daily followed a daily oviposition pattern, suggesting gonotrophic discordance.     

Characterization of larval habitats of Anopheles albimanus, Anopheles pseudopunctipennis, Anopheles punctimacula, and Anopheles oswaldoi s.l. populations in lowland and highland Ecuador

Recent collection data indicate that at least four potential malaria vectors occupy more widespread distributions within the Andean highlands than in the past. Since habitat elimination is an important aspect of malaria control, it is vital to characterize larval habitats for Anopheles species within both lowland and highland sites. To that end, 276 sites within Ecuador were surveyed between 2008 and 2010. Characteristics of Anopheles‐present sites for four species were compared to Anopheles‐absent sites within the same geographical range and also to Anopheles‐absent sites within a highland range representing potential future habitats. Thermochron iButtons^© were used to describe the daily temperature variation within a subset of potential habitats. Anopheles albimanus (W.) was positively associated with permanent habitats, sand substrates, floating algae (cyanobacterial mats), and warmer temperatures in both comparisons. Anopheles pseudopunctipennis (T.) was associated with floating algae (cyanobacterial mats), warmer temperatures, and higher water clarity in both comparisons. Anopheles punctimacula (D.&K.) was negatively associated with floating algae and positively associated with dissolved oxygen in both comparisons. Anopheles oswaldoi s.l. (P.) was not significantly associated with any parameters more often than expected given larval‐absent sites. The results indicate that minimum water temperatures might limit the upper altitudinal distribution of An. albimanus (18.7° C) and An. pseudopunctipennis (16.0° C).     

Flight performance of the malaria vectors Anopheles gambiae and Anopheles atroparvus.

The flight potential and metabolism of two malaria vectors, Anopheles gambiae s.str. and An. atroparvus, were analyzed on flightmills. The flight distance, the flight time, and individual flight activities of females were recorded during 22 h flight trials. The glycogen and lipid before flight, after flight, and of unflown controls were measured for starved, sugar-, or blood-fed females. Maximal flight distances of An. gambiae were 9 km when sugar-fed and 10 km when blood-fed, while in starved females it was below 3 km and the average speed was around 1 km/h. In Anopheles atroparvus, the maximal flight distances were 10-12 km when sugar-fed, 4.5 km when blood-fed, and below 3.5 km when starved, with an average speed of 1.3 km/h. Flight performances consisted of 1-4 h intervals of continuous flights, but mainly of bouts shorter than one h, randomly distributed during the long flight trials in both species. An. gambiae utilized an average of 47% of its pre-flight carbohydrate reserves for survival and 38% for flight at a rate of 0.07 cal/h/female. After a blood meal they utilized 11% for survival and 61% for flight at a rate of 0.04 cal/h. At the same time, 25% of the pre-flight lipid was mobilized for flight at a rate of 0.09 cal/h when sugar-fed and 22% when blood-fed at a rate of 0.06 cal/h; lipid was barely mobilized for survival. An. atroparvus differed: carbohydrate mobilization was 28% for survival and 41% for flight at a rate of 0.15 cal/h when sugar-fed; lipid mobilization for flight was only 13% at a rate of 0.06 cal/h. After a blood meal only 2% of the pre-flight lipid was used (0.02 cal/h). The contribution of carbohydrate reserves for flight metabolism at the high rate of 0.21 cal/h could not be fully elucidated because its decrease coincided with a pronounced resynthesis from the blood meal. An. atroparvus always depended on sugar meals for its flight activities and barely utilized lipid reserves. An. gambiae was independent of sugar sources for strong flights due to its early blood feeding and because of its equicaloric lipid mobilization during flights. Strong evidence for lipid oxidation during its flight is discussed.     

Multimodal pyrethroid resistance in malaria vectors, Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. in western Kenya

Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. are the most important species for malaria transmission. Pyrethroid resistance of these vector mosquitoes is one of the main obstacles against effective vector control. The objective of the present study was to monitor the pyrethroid susceptibility in the 3 major malaria vectors in a highly malaria endemic area in western Kenya and to elucidate the mechanisms of pyrethroid resistance in these species. Gembe East and West, Mbita Division, and 4 main western islands in the Suba district of the Nyanza province in western Kenya were used as the study area. Larval and adult collection and bioassay were conducted, as well as the detection of point mutation in the voltage-gated sodium channel (1014L) by using direct DNA sequencing. A high level of pyrethroid resistance caused by the high frequency of point mutations (L1014S) was detected in An. gambiae s.s. In contrast, P450-related pyrethroid resistance seemed to be widespread in both An. arabiensis and An. funestus s.s. Not a single L1014S mutation was detected in these 2 species. A lack of cross-resistance between DDT and permethrin was also found in An. arabiensis and An. funestus s.s., while An. gambiae s.s. was resistant to both insecticides. It is noteworthy that the above species in the same area are found to be resistant to pyrethroids by their unique resistance mechanisms. Furthermore, it is interesting that 2 different resistance mechanisms have developed in the 2 sibling species in the same area individually. The cross resistance between permethrin and DDT in An. gambiae s.s. may be attributed to the high frequency of kdr mutation, which might be selected by the frequent exposure to ITNs. Similarly, the metabolic pyrethroid resistance in An. arabiensis and An. funestus s.s. is thought to develop without strong selection by DDT.     

Evaluation of Insecticides Susceptibility and Malaria Vector Potential of Anopheles annularis s.l. and Anopheles vagus in Assam,India

During the recent past, development of DDT resistance and reduction to pyrethroid susceptibility among the malaria vectors has posed a serious challenge in many Southeast Asian countries including India. Current study presents the insecticide susceptibility and knock-down data of field collected Anopheles annularis sensu lato and An. vagus mosquito species from endemic areas of Assam in northeast India. Anopheles annularis s.l. and An. vagus adult females were collected from four randomly selected sentinel sites in Orang primary health centre (OPHC) and Balipara primary health centre (BPHC) areas, and used for testing susceptibility to DDT, malathion, deltamethrin and lambda-cyhalothrin. After insecticide susceptibility tests, mosquitoes were subjected to VectorTest^™ assay kits to detect the presence of malaria sporozoite in the mosquitoes. An. annularis s.l. was completely susceptible to deltamethrin, lambda-cyhalothrin and malathion in both the study areas. An. vagus was highly susceptible to deltamethrin in both the areas, but exhibited reduced susceptibility to lambda-cyhalothrin in BPHC. Both the species were resistant to DDT and showed very high KDT₅₀ and KDT₉₉ values for DDT. Probit model used to calculate the KDT₅₀ and KDT₉₉ values did not display normal distribution of percent knock-down with time for malathion in both the mosquito species in OPHC (p<0.05) and An. vagus in BPHC (χ² = 25.3; p = 0.0), and also for deltamethrin to An. vagus in BPHC area (χ² = 15.4; p = 0.004). Minimum infection rate (MIR) of Plasmodium sporozoite for An. vagus was 0.56 in OPHC and 0.13 in BPHC, while for An. annularis MIR was found to be 0.22 in OPHC. Resistance management strategies should be identified to delay the expansion of resistance. Testing of field caught Anopheles vectors from different endemic areas for the presence of malaria sporozoite may be useful to ensure their role in malaria transmission.     

The distribution of two major malaria vectors, Anopheles gambiae and Anopheles arabiensis, in Nigeria.

The distribution of Anopheles gambiae and An. arabiensis across the ecological zones of Nigeria (arid savanna in the north gradually turns into humid forest in the south) was investigated. Results of the present study were compared to the distributions determined from samples of indoor-resting females reported by an earlier study over 20 years ago. Larvae were sampled in the rainy seasons of 1997 and 1999 from 24 localities, 10 of which were sampled in both years. Specimens were identified by the polymerase chain reaction method. Results showed that species composition changed significantly among the 10 localities in both years (chi2=13.62, P = 0.0002), but this change was significant in only four of the 10 localities. The identity of the prevalent (more abundant) species changed between 1997 and 1999 in only three of 10 localities. An. arabiensis was prevalent in several localities in the southern Guinea savanna, an area where it was virtually absent over 20 years ago. The data suggest that An. arabiensis has extend its range, although differences in sampling technique (larval sampling versus adult collection) can not be ruled out as a possible explanation.     

Rapid Discrimination between Anopheles gambiae s.s. and Anopheles arabiensis by High-Resolution Melt (HRM) Analysis

There is a need for more cost-effective options to more accurately discriminate among members of the Anopheles gambiae complex, particularly An. gambiae and Anopheles arabiensis. These species are morphologically indistinguishable in the adult stage, have overlapping distributions, but are behaviorally and ecologically different, yet both are efficient vectors of malaria in equatorial Africa. The method described here, High-Resolution Melt (HRM) analysis, takes advantage of minute differences in DNA melting characteristics, depending on the number of incongruent single nucleotide polymorphisms in an intragenic spacer region of the X-chromosome-based ribosomal DNA. The two species in question differ by an average of 13 single-nucleotide polymorphisms giving widely divergent melting curves. A real-time PCR system, Bio-Rad CFX96, was used in combination with a dsDNA-specific dye, EvaGreen, to detect and measure the melting properties of the amplicon generated from leg-extracted DNA of selected mosquitoes. Results with seven individuals from pure colonies of known species, as well as 10 field-captured individuals unambiguously identified by DNA sequencing, demonstrated that the method provided a high level of accuracy. The method was used to identify 86 field mosquitoes through the assignment of each to the two common clusters with a high degree of certainty. Each cluster was defined by individuals from pure colonies. HRM analysis is simpler to use than most other methods and provides comparable or more accurate discrimination between the two sibling species but requires a specialized melt-analysis instrument and software.     

Identification of bloodmeals in Anopheles quadrimaculatus and Anopheles punctipennis from eastern equine encephalitis virus foci in northeastern U.S.A.

The host‐feeding patterns of Anopheles quadrimaculatus Say and Anopheles punctipennis (Say) were examined in order to evaluate their potential contributions to the transmission of eastern equine encephalitis virus (EEEv) and other arboviruses in the northeastern U.S.A. Engorged mosquitoes of the two species were collected from EEEv foci in central New York (NY) and throughout New Jersey (NJ), and their bloodmeals were identified using a polymerase chain reaction (PCR)‐based assay and sequencing portions of the mitochondrial cytochrome b gene. Analysis of 131 An. quadrimaculatus and 107 An. punctipennis from NY revealed that 97.7% and 97.2%, respectively, had acquired blood solely from mammalian hosts. Similarly, examination of 288 An. quadrimaculatus and 127 An. punctipennis from NJ showed 100% and 96.0%, respectively, contained mammalian‐derived bloodmeals. Mosquitoes containing mixed bloodmeals from both avian and mammalian hosts were detected in 1.6% of An. quadrimaculatus from NY, and 2.8% and 4.0% of An. punctipennis from NY and NJ, respectively. White‐tailed deer (Odocoileus virginianus) constituted the most common vertebrate host for these anopheline mosquitoes, accounting for 85.8–97.7% of all bloodmeals identified. The predominance of white‐tailed deer as a source of bloodmeals supports enzootic amplification of deer‐associated arboviruses in this region, including Jamestown Canyon, Cache Valley and Potosi viruses. One horse‐ and two human‐derived bloodmeals were also detected in An. quadrimaculatus collected in NJ. Limited avian‐derived bloodmeals were detected from mourning dove (Zenaida macroura), sharp‐shinned hawk (Accipiter striatus) and house finch (Carpodacus mexicanus), mostly in mixed bloodmeals. Occasional feeding on avian hosts suggests that these mosquitoes may participate as epizootic–epidemic bridge vectors of EEEv from viraemic birds to mammalian hosts of concern, including horses and humans. An isolate of EEEv was recovered from the head and thorax of an An. punctipennis mosquito collected in NY.     

Temperature influence on embryonic development of Anopheles albitarsis and Anopheles aquasalis

Temperature influence on the embryonic development of Anopheles aquasalis and An. albitarsis was investigated. At 26 degrees C, 75% and 60% of respectively An. aquasalis and An. albitarsis eggs hatched, with one peak of eclosion, between the 2nd and 3rd day after oviposition. At 20 +/- 2 degrees C, around 66-70% of An. aquasalis eggs hatched, with one eclosion peak, on the 5th day. On the other hand, An. albitarsis eclosion at 21+/- 2 degrees C decreased to 10-22%, with two eclosion peaks, on the 4th-5th day and on the 9th-12th day. These data indicate a stronger temperature influence over An.albitarsis than over An. aquasalis embryos.     

Hemolymph volume of noninfected and Plasmodium berghei-infected Anopheles stephensi.

The hemolymph volume of Anopheles stephensi adult female mosquitoes was determined by a radioisotope dilution technique. [carboxy-¹⁴C]Inulin was injected into the hemocoels of mosquitoes with a calibrated capillary needle. After sufficient time for thorough mixing, the labeled hemolymph was collected from groups of 50 mosquitoes by a centrifugation technique. Total hemolymph volume was calculated by a conventional formula for radioisotope dilution. The mean hemolymph volume of the newly emerged adult female mosquitoes was 336 nl/mosquito. The ratio of hemolymph volume to body weight was 0.25 μl/mg body wt. By 14 days after emergence, hemolymph volume had dropped to 190 nl/mosquito. Infection of mosquitoes with the rodent malaria parasite, Plasmodium berghei, had no significant effect on hemolymph volume of the mosquito.     

Evidence for recent population expansion in the evolutionary history of the malaria vectors Anopheles arabiensis and Anopheles gambiae.

Gene flow in malaria vectors is usually estimated based on differentiation indices (e.g., F(ST)) in order to predict the contemporary spread of genes such as those conferring resistance to insecticides. This approach is reliant on a number of assumptions, the most crucial, and the one most likely to be violated in these species, being mutation-migration-drift equilibrium. Tests of this assumption for the African malaria vectors Anopheles gambiae and Anopheles arabiensis are the focus of this study. We analyzed variation at 18 microsatellite loci and the ND5 region of the mitochondrial genome in two populations of each species. Equilibrium was rejected by six of eight tests for the A. gambiae population from western Kenya and by three tests in eastern Kenya. In western Kenya, all departures from equilibrium were consistent with a recent population expansion, but in eastern Kenya, there were traces of a recent expansion and a bottleneck. Equilibrium was also rejected by two of the eight tests for both A. arabiensis populations; the departure from equilibrium was consistent with an expansion. These multiple-locus tests detected a genomewide effect and therefore a demographic event rather than a locus-specific effect, as would be caused by selection. Disequilibrium due to a recent expansion in these species implies that rates of gene flow, as inferred from differentiation indices, are overestimates as they include a historical component. We argue that the same effect applies to the majority of pest species due to the correlation of their demography with that of humans.     

Comparative susceptibility of three important malaria vectors Anopheles stephensi, Anopheles fluviatilis, and Anopheles sundaicus to Plasmodium vivax

The 3 laboratory-colonized malaria vectors, i.e., Anopheles stephensi, An. sundaicus, and An. fluviatilis, were studied for their comparative susceptibility to Plasmodium vivax sporogony. There was no significant difference in oocyst and sporozoite recruitment by these 3 species, whereas the geometric mean (GM) of the oocyst number per midgut was significantly lower in An. fluviatilis as compared with that in the other 2 species. There was no difference in the GM of oocyst between An. stephensi and An. sundaicus. Adaptability to laboratory conditions and susceptibility to plasmodial infection suggest that An. fluviatilis and An. sundaicus can also be used as a vector model for vector-parasite interaction studies.     

Hut entry and exit by Anopheles gambiae and Anopheles funestus in an unsprayed village near Kaduna in Northern Nigeria.

Hut entry and exit by An. gambiae and An. funestus were studied in an unsprayed village near Kaduna in Northern Nigeria. A high turn-over of indoor resting mosquitoes of all blood digestion stages was noted throughout the night. The behaviour of both species was similar. The entry pattern was more uniform than the exit which showed two distinct peaks around sunset and after midnight. Most of the daytime resting mosquitoes left the huts during the first hour after sunset, and therefore the mosquitoes escaping during the subsequent hours of the night were mainly those that had entered the same night and spent only a limited period inside the huts. The significance of the movement of mosquitoes is discussed in relation to entomological evaluation of the impact of residual insecticides.