Unfed Female Mosquitoes Research Articles

Molecular xenomonitoring of Dirofilaria immitis and Dirofilaria repens in mosquitoes from north-eastern Italy by real-time PCR coupled with melting curve analysis

BackgroundDirofilaria immitis and Dirofilaria repens are transmitted by bloodsucking culicid mosquitoes belonging to Culex, Aedes, Ochlerotatus, Anopheles and Mansonia genera.The detection of filarioids in mosquitoes for assessing distribution of vectors and/or of pathogens in a given area (also known as “xenomonitoring”), when based on individual dissection of wild-caught female mosquitoes is time consuming and hardly applicable in large epidemiological surveys.Our study aimed to evaluate the recently developed duplex real-time PCR for screening large number of culicids and to assess their positivity for D. immitis and D. repens in an area where both species are endemic.MethodsA duplex real-time PCR was used to detect and differentiate D. immitis and D. repens in mosquitoes collected in six provinces of the Veneto region using 43 carbon dioxide-baited traps under the frame of an entomological surveillance program to monitor the vectors of West Nile disease. From early May till October 2010, unfed female mosquitoes (n = 40,892) were captured in 20 selected sites.ResultsMosquitoes identified as Culex pipiens, Ochlerotatus caspius, Aedes vexans and Culex modestus were grouped into 995 pools according to species, day and site of collection (from minimum of 1 to maximum of 57). Out of 955 pools, 23 (2.41 %) scored positive for Dirofilaria spp. of which, 21 (2.2 %) for D. immitis and two (0.21 %) for D. repens. An overall Estimated Rate of Infection (ERI) of 0.06 % was recorded, being higher in Och. caspius and Ae. vexans (i.e., 0.18 % and 0.14 %, respectively). At least one mosquito pool was positive for Dirofilaria spp. in each province with the highest ERI recorded in Vicenza and Padova provinces (i.e., 0.42% and 0.16 %, respectively). Mosquitoes collected in all provinces were positive for D. immitis whereas, only two (i.e., Padova and Rovigo) provinces scored positive for D. repens. All mosquito species, except for Cx. modestus, were positive for D. immitis, whereas D. repens was only found in Cx. pipiens.ConclusionsThe results suggest that both Dirofilaria species are endemic and may occur in sympatry in the examined area. The molecular approach herein used represents a powerful tool for surveillance programs of D. immitis and D. repens in the culicid vectors towards a better understanding of the epidemiology of the infections they cause and their seasonal transmission patterns.

Adulticidal activity of Ageratum houstonianum Mill. (Asteraceae) leaf extracts against three vector mosquito species (Diptera: Culicidae)

ObjectiveTo determine the intrinsic toxicity of hexane, ethyl acetate and methanol crude extracts of Ageratum houstonianum leaves against adult Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus mosquitoes. MethodsBioassay was performed in 2-day-old laboratory reared unfed adult female mosquitoes by topical application at concentrations of 0.01, 0.05, 0.10, 0.25 and 0.50 μg/mg female adult mosquito. ResultsAedes aegypti was found to be more susceptible to ethyl acetate and hexane extracts with LD50 value of 0.10 μg/mg, and both Anopheles stephensi and Culex quinquefasciatus were susceptible to methanol extract with LD50 values of 0.12 μg/mg female adult mosquito. ConclusionsThe results show promising adulticidal activity on topical application and further studies followed by in-depth laboratory and field bioassays are needed to screen, isolate and purify bioactive phytochemical constituents or compounds.

Knockdown resistance in Anopheles vagus, An. sinensis, An. paraliae and An. peditaeniatus populations of the Mekong region

BackgroundIn the Mekong region (Vietnam, Cambodia and Laos), a large investigation was conducted to assess the susceptibility of Anopheles species against DDT and pyrethroids. In this study, the resistance status of the potential malaria vectors An. vagus, An. sinensis, An. paraliae and An. peditaeniatus was assessed.MethodsBioassays were performed on field collected unfed female mosquitoes using the standard WHO susceptibility tests. In addition, the DIIS6 region of the para-type sodium channel gene was amplified and sequenced and four allele-specific PCR assays were developed to assess the kdr frequencies.ResultsIn Southern Vietnam all species were DDT and pyrethroid resistant, which might suggest the presence of a kdr resistance mechanism. Sequence-analysis of the DIIS6 region of the para-type sodium channel gene revealed the presence of a L1014S kdr mutation in An. vagus, An. sinensis and An. paraliae. In An. peditaeniatus, a low frequency L1014S kdr mutation was found in combination with a high frequency L1014F kdr mutation. For pyrethroids and DDT, no genotypic differentiation was found between survivors and non-survivors for any of these species. In the two widespread species, An. vagus and An. sinensis, kdr was found only in southern Vietnam and in Cambodia near the Vietnamese border.ConclusionsDifferent levels of resistance were measured in Laos, Cambodia and Vietnam. The kdr mutation in different Anopheles species seems to occur in the same geographical area. These species breed in open agricultural lands where malaria endemicity is low or absent and vector control programs less intensive. It is therefore likely that the selection pressure occurred on the larval stages by insecticides used for agricultural purposes.

Microsporidiosis (Microsporidia: Culicosporidae) Alters Blood-Feeding Responses and DEET Repellency in <I>Aedes aegypti</I> (Diptera: Culicidae)

Infection of Aedes aegypti (L.) (Diptera: Culicidae) with Edhazardia aedis (Microsporidia: Culicosporidae) reduced mean human host attraction and landing/probing rates in female mosquitoes by 53 and 62%, respectively, compared with rates in microsporidia-free females. Infection with E. aedis reduced the average weight of unfed female mosquitoes by 4%, caused them to imbibe 23% less blood, and to lay 30% fewer eggs than healthy females. In contrast, E. aedis-infected mosquitoes required 20% more time (>1 h) than healthy females to bite skin treated with 15% DEET. Statistically significant morbidity in E. aedis-infected females was indicated by reductions in host attraction and landing/probing responses, the mass of unfed and blood-engorged females, and fecundity, and by increased DEET repellency.

Effects of permethrin at different temperatures on pyrethroid-resistant and susceptible strains of Anopheles.

The influence of temperature (16, 22, 28, 37 degrees C) on effects of permethrin was investigated for susceptible and pyrethroid-resistant strains of the mosquitoes Anopheles gambiae and An. stephensi (Diptera: Culicidae). Young unfed female adult mosquitoes were exposed to 0.25% permethrin test papers or to polyester netting treated with permethrin 500mg a.i./m2. The time to 50% knockdown (KT50) declined as temperature increased, i.e. there was a positive temperature coefficient of this effect of the pyrethroid. Resistance ratios (comparing KT50 values) between resistant and susceptible An. stephensi ranged between 2.5 and 4.4 at the different temperatures. Comparative tests of pyrethroid tolerance of different strains would be valid over the 22-28 degrees C range but, when using a discriminating dose to detect resistance, more precise temperature control is desirable. Mortality 24h after exposure to 0.25% permethrin of both susceptible and resistant strains of An. stephensi showed a negative correlation with temperature between 16 and 22 degrees C and a positive correlation at higher temperatures. In An. gambiae, however, the correlation was positive over the whole range. Irritancy of permethrin-treated netting to Anopheles females (measured as time lapse until first flight take-off, and the number of take-offs during 7.5 min exposure) was positively correlated with temperature in all four strains and was much greater for the susceptible than the resistant strains.

Odour‐mediated host preference of Culex quinquefasciatus in Tanzania

AbstractField experiments to determine the host preference of Culex quinquefasciatus Say (Diptera: Culicidae) between man, calf and goat, were conducted in Muheza, north‐east Tanzania. The responses of the mosquito to the three hosts were also compared to their responses to carbon dioxide. A total of 2565 unfed female mosquitoes were collected, of which Cx quinquefasciatus accounted for 96.6% of the catch. A human‐baited tent caught a larger number of host‐seeking Cx quinquefasciatus than a calf‐baited or goat‐baited tent (P<0.05). The man:calf ratio was 7.8:1 while the man:goat ratio was 10:1. The difference in response to either a calf‐ or goat‐baited tent was not statistically significant (P>0.05). The number of mosquitoes attracted to a human‐baited tent was significantly larger than that attracted to carbon dioxide released at 300 ml min−1. Only 25.3% of the human host attractancy was attributed to carbon dioxide. The number of Cx quinquefasciatus responding to a tent baited with a calf or goat was not significantly different from the number responding to a tent baited with carbon dioxide. It can be concluded that with equal availability of the three vertebrate hosts, Cx quinquefasciatus would respond more to volatile cues from a human host than from either a calf or goat, thereby supporting earlier data about its high degree of anthropophily. The major olfactory cue from a goat or a calf to which this mosquito responds is probably carbon dioxide.

Constitutive and Blood Meal-Induced Trypsin Genes in Anopheles gambiae

Trypsin genes in Anopheles gambiae are arranged as a tightly clustered gene family consisting of seven related coding sequences, devoid of introns. The two blood meal-inducible members of this family, Antryp1 and Antryp2, were shown to play a crucial role in the breakdown of the blood meal constituents. The role of Antryp3,4,5,6, and Antryp7 in the process of blood meal digestion remains to be elucidated. We have examined the localization and the expression patterns of these trypsins as well as the functional interactions in blood meal digestion between trypsins and other gut-specific proteases. Northern blot and RT-PCR analysis indicated that the genes Antryp3,4,5,6, and Antryp7 are all constitutively expressed in unfed female mosquitoes. Soon after blood feeding the mRNA of these trypsin genes became undetectable and appeared again at the end of the gonotrophic cycle. The blood meal-inducible trypsin Antrypl was also constitutively expressed at low level in the gut of adult female mosquitoes. This trypsin was the only member of this gene family to be expressed in the gut of male and female pupae. By using antisera that specifically recognized recombinant Antryp4 we were able to show that the corresponding protein in Anopheles is synthesized and stored in the gut epithelium of unfed females as zymogen. Secretion and activation of this trypsin was shown to occur in the midgut lumen immediately after fluid ingestion and independently of the protein content of the meal, Recombinant trypsins expressed in Escherichia coli, with the exception of Antryp5 and Antryp6, were able to activate in vitro recombinant A. gambiae chymotrypsinogen, thus suggesting that blood meal ingestion is able to trigger a cascade of events leading to the activation of several proteases.

Isolation and characterization of mosquito ferritin and cloning of a cDNA that encodes one subunit.

Ferritin, an iron storage protein, was isolated from larvae and pupae of Aedes aegypti grown in an iron-rich medium. Mosquito ferritin is a high molecular weight protein composed of several different, relatively small, subunits. Subunits of molecular mass 24, 26, and 28 kDa are equally abundant, while that of 30 kDa is present only in small amounts. The N-terminal sequence of the 24 and 26 kDa subunits are identical for the first 30 amino acids, while that of the 28 kDa subunit differs. Studies using antiserum raised against a subunit mixture showed that the ferritin subunit were present in larvae, pupae, and adult females, and were increased in animals exposed to excess iron. The antiserum also was used to screen a cDNA library from unfed adult female mosquitoes. Nine clones were obtained that differed only in a 27 bp insertion in the 3' end. Rapid amplification of cDNA ends (RACE) was used to obtain the complete protein coding sequence. A putative iron-responsive element (IRE) is present in the 5'-untranslated region. The deduced amino acid sequence shows a typical leader sequence, consistent with the fact that most insect ferritins are secreted, rather than cytoplasmic proteins. The sequence encodes a mature polypeptide of 20,566 molecular weight, smaller than the estimated size of any of the subunits. However, the sequence exactly matches the N-terminal sequences of the 24 and 26 kDa subunits as determined by Edman degradation. Of the known ferritin sequences, that of the mosquito is most similar to that of somatic cells of a snail.

Esters of alpha-hydroxy-beta-phenyl aliphatic acids that attract female yellow-fever mosquitoes

An attiactant for the yellow-fever mosquito, Aedes aegypti (L.), is needed for its detection. In our screening of chemicals, several chlorohydrins and α-hydroxy esters showed significant attraction for unfed nongravid female mosquitoes.

A comparison of the range of attraction of animal baits and of carbon dioxide for some West African mosquitoes.

Experiments were carried out on the flight pattern of unfed female mosquitoes in an area of the Gambia adjacent to mangrove swamps. The mosquitoes were trapped in flight by the use of ramp-traps as they approached natural or simulated baits. The traps were arranged in series in four avenues of cleared bush, radiating out at right angles to each other. The baits were installed either at the centre of the cross formed by the avenue or at the outer end of one avenue. Trapping was carried out at six ranges simultaneously, extending from 5–80 yd (4·5–73 m) distance from the bait. The baits were either two small calves or carbon dioxide emitted from a cylinder at a rate equivalent to the expiration rate of the animals. A series of catches with no bait was also made.