Laboratory testing of a lethal ovitrap for Aedes aegypti

Laboratory tests were conducted to determine the feasibility of making the mosquito ovitrap lethal to Aedes aegypti (L.) when they attempt to oviposit in the trap. Heavy-weight velour paper strips (2.54 x 11 cm) were used as an alternative to the wooden paddle normally provided as a substrate for mosquito oviposition. The paper strips were pretreated with insecticide solutions and allowed to dry before being used in oviposition cups of 473 ml capacity, filled with water initially to within 2.5 cm of the brim. Insecticides chosen for their quick knock-down efficacy were bendiocarb 76% WP (1.06 mg a.i./strip) and four pyrethroids: permethrin 25% WP (0.16 mg a.i./strip), deltamethin 4.75% SC (0.87 mg a.i./strip), cypermethrin 40% WP (2.81 mg a.i./strip), and cyfluthrin 20% WP (0.57 mg a.i./ strip). For experimental evaluation, two oviposition cups (one with an insecticide-treated strip and one with an untreated strip) were placed in cages (cubic 30 cm) with gravid female Ae. aegypti mosquitoes (aged 6-8 days) from a susceptible laboratory strain. Mortality-rates of female mosquitoes were 45% for bendiocarb, 47% for permethrin, 98% for deltamethrin, 100% for cypermethrin, and 100% for cyfluthrin. Young instar larvae added to the treated cups died within 2h. After water evaporation from the cups for 38 days, fresh mosquito females had access to previously submerged portions of the velour paper paddle, and mortality rates of 59% or more occurred. Cups that had water (360 ml) dripped into them, to simulate rain, produced female mosquito mortality rates of > 50% and all larvae died within 3 h of being added. These tests demonstrate that the ovitrap can be made lethal to both adults and larvae by insecticidal treatment of the ovistrip. Field efficacy trials are underway in Brazil to access the impact of this simple, low-cost, environmentally benign approach on populations of the dengue vector Ae. aegypti.     

RNAi knock-downs support roles for the mucin-like (AeIMUC1) gene and short-chain dehydrogenase/reductase (SDR) gene in Aedes aegypti susceptibility to Plasmodium gallinaceum

The mosquito midgut represents the first barrier encountered by the Plasmodium parasite (Haemosporida: Plasmodiidae) when it is ingested in blood from an infected vertebrate. Previous studies identified the Aedes aegypti (L.) (Diptera: Culicidae) mucin-like (AeIMUC1) and short-chain dehydrogenase/reductase (SDR) genes as midgut-expressed candidate genes influencing susceptibility to infection by Plasmodium gallinaceum (Brumpt). We used RNA inference (RNAi) by double-stranded RNA (dsRNA) injections to examine ookinete survival to the oocyst stage following individual gene knock-downs. Double-stranded RNA gene knock-downs were performed 3 days prior to P. gallinaceum infection and oocyst development was evaluated at 7 days post-infection. Mean numbers of parasites developing to the oocyst stage were significantly reduced by 52.3% in dsAeIMUC1-injected females and by 36.5% in dsSDR-injected females compared with females injected with a dsβ-gal control. The prevalence of infection was significantly reduced in dsAeIMUC1- and dsSDR-injected females compared with females injected with dsβ-gal; these reductions resulted in a two- and three-fold increase in the number of uninfected individuals, respectively. Overall, these results suggest that both AeIMUC1 and SDR play a role in Ae. aegypti vector competence to P. gallinaceum.     

Competition and resistance to starvation in larvae of container-inhabiting Aedes mosquitoes

Abstract. 1. Hypotheses about declining populations of container-inhabiting Aedes mosquitoes following the invasion by additional species were tested.
2. The larval competition hypothesis was studied experimentally in pure and mixed cultures of Aedes aegypti (L.), A.albopictus (Skuse) and A.triseriatus (Say). The experiments used decomposing leaf litter in the laboratory, as opposed to most previous research which used non-natural food.
3. Resistance to starvation is introduced as a new measure of larval performance and competitiveness. The hypothesis is that more successful larvae store larger energy reserves and resist the lack of food longer.
4. Contrary to previous research showing better performance of A.aegypti in mixed cultures, A.albopictus developed faster and had greater survival when natural food was used.
5. Resistance to starvation was greater in the better performing species (i.e. A.aegypti with non-natural food and A.albopictus with leaf litter). Oxygen consumption by starved larvae was similar in the three container species, and in the ground-water mosquito, A.taeniorhynchus (Wied.), whose resistance to starvation was comparatively very low.     

Estimation of the number of full sibling families at an oviposition site using RAPD-PCR markers: applications to the mosquito Aedes aegypti

There are many species in which groups of individuals encountered in the field are known to consist of mixtures of full-sibling families. We describe a statistical technique, based on the use of random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) markers, that allows for the estimation of the number of families contained in these groups. We test the technique on full-sibling families of the mosquito Aedes aegypti, a species that distributes its eggs among several locations. Mixtures of 10 families with 15 individuals per family were analyzed using 40 RAPD-PCR loci amplified by 5 primers. Our analysis accurately estimated the number of families. The technique was accurate when the number of families was small or when family sizes were small and variable.     

Genetic relationships in the peregrine falcon (Falco peregrinus) analysed by microsatellite DNA markers

Microsatellite DNA markers were developed from a peregrine falcon (Falco peregrinus) and genetic relationships among peregrine falcons in southern Norway were analysed using the markers. The genomic DNA library was screened for the presence of dinucleotide microsatellite repeats. Twelve loci revealed polymorphism through the initial analysis of 24 unrelated peregrine falcons, and Mendelian inheritance was confirmed in two peregrine falcon families bred in captivity. The estimated mean probability of identical genotypes in two unrelated individuals was 3 x 10-8, and the combined exclusion probability for parentage testing was 0.99 and 0.94 for one or both parents unknown, respectively. The markers were used to investigate the parentage of peregrine broods from the same nest site from different breeding seasons, and subsequently the nest-site fidelity of the breeding peregrines. High nest-site fidelity was found by studying pairwise comparisons of relatedness (rxy) estimates among chicks at six nest sites from three different breeding seasons. Cross-species amplifications showed that most loci also appeared to amplify polymorphic products in the gyrfalcon (F. rusticolus), merlin (F. columbarius), hobby (F. subbuteo) and kestrel (F. tinnunculus), demonstrating that the loci will provide powerful genetic markers in these falcons too.     

Restriction Fragment Length Polymorphism Mapping of Quantitative Trait Loci for Malaria Parasite Susceptibility in the Mosquito Aedes Aegypti

Susceptibility of the mosquito Aedes aegypti to the malarial parasite Plasmodium gallinaceum was investigated as a quantitative trait using restriction fragment length polymorphisms (RFLP). Two F(2) populations of mosquitoes were independently prepared from pairwise matings between a highly susceptible and a refractory strain of A. aegypti. RFLP were tested for association with oocyst development on the mosquito midgut. Two putative quantitative trait loci (QTL) were identified that significantly affect susceptibility. One QTL, pgs[2,LF98], is located on chromosome 2 and accounted for 65 and 49% of the observed phenotypic variance in the two populations, respectively. A second QTL, pgs[3,MalI], is located on chromosome 3 and accounted for 14 and 10% of the observed phenotypic variance in the two populations, respectively. Both QTL exhibit a partial dominance effect on susceptibility, wherein the dominance effect is derived from the refractory parent. No indication of epistasis between these QTL was detected. Evidence suggests that either a tightly linked cluster of independent genes or a single locus affecting susceptibility to various mosquito-borne parasites and pathogens has evolved near the LF98 locus; in addition to P. gallinaceum susceptibility, this general genome region has previously been implicated in susceptibility to the filarial nematode Brugia malayi and the yellow fever virus.     

山东省假性肥大型肌营养不良的RFLP分析 Ⅰ．可疑携带者的基因型确定

本文选用10个DMD基因内部和桥探针,对山东省9个地区的21个DMD／BMD家系的173名成员进行RFLP分析,其中可疑携带者55人,多数家系应用1－3个探针,有基因重组家庭选用4－5个探针,便可完成多态分析,RFLP分析可以确定85．45％的可疑携带者（≥95％可信限）,即13人确定为携带者,30人排除携带者,另有4人风险大幅提高,通过这些探针的群体多态检测和不同家庭结构和类型的应用分析,我们提出了在中国人群中DMD／SMD基因诊断的基本程序。     

Effects of Toxorhynchites moctezuma larval predation on Aedes aegypti populations: experimental evaluation

Abstract. Predatory larvae of the mosquito Toxorhynchites moctezuma were used experimentally to control a standing crop of larvae of the dengue vector mosquito Ae.aegypti. Each week, fifty Ae.aegypti first instar larvae were introduced to each of five water-filled drums (220 litres) of the type commonly used for domestic water storage in Caribbean dwellings. At the beginning of the fourth week, a certain number (0, 1, 2, 5 or 10) of first instar Tx.moctezuma larvae were introduced to each drum and the daily yield of Ae.aegypti adults from each drum was monitored thereafter. The experiment was repeated three times. With only one or two Tx.moctezuma larvae, predation on Ae.aegypti larvae stopped the output of Ae.aegypti adults for 1 week. Five or ten Tx. moctezuma prevented any Ae.aegypti emergence for up to 16 weeks. Cannibalism among Tx. moctezuma larvae was seldom observed and appeared not to be a hindrance in using this species against Ae.aegypti. Thus Tx.moctezuma is regarded as a good candidate for the biological control of Ae.aegypti by augmentative releases.     

遗传不育技术在蚊媒疾病防控中的应用

疟疾、登革热等重大传染性蚊媒疾病严重危害人类健康,且目前缺乏有效的药物和疫苗,防治埃及伊蚊、冈比亚按蚊等媒介昆虫是控制和消除这些疾病的有效手段。化学杀虫剂的大规模使用在一定程度上控制了疾病的传播,但其抗药性和环境污染等问题也随之而来。分子生物学的飞速发展为昆虫不育技术(SIT)的更新及害虫防治提供了新的策略,由此发展起来的以释放携带显性致死基因昆虫(RIDL)为代表的一系列遗传不育技术为蚊虫种群防控提供了更加有效的选择。本文概述了遗传技术在蚊虫防控中的应用进展,包括蚊虫遗传防治的历史和策略,阐述了RIDL技术体系的原理,同时介绍了相关遗传控制品系和已经开展的田间释放研究,展示了遗传修饰不育技术在蚊媒疾病防治中的巨大潜力。     

State‐wide survey of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Florida

Aedes aegypti and Aedes albopictus are invasive mosquito species with geographic ranges that have oscillated within Florida since their presence was first documented. Local transmission of dengue, chikungunya, and Zika viruses serves as evidence of the public health importance of these two species. It is important to have detailed knowledge of their distribution to aid in mosquito control efforts and understand the risk of arbovirus transmission to humans. Through a partnership involving the University of Florida Institute of Food and Agricultural Sciences Cooperative Extension Service and the Florida Medical Entomology Laboratory; the Florida Department of Health; and mosquito control agencies throughout Florida, a container mosquito surveillance program involving all life stages was launched in the summer of 2016 to detect the presence of Ae. aegypti and Ae. albopictus. Results from this survey were mapped to provide a picture of the current known distribution of Ae. aegypti and Ae. albopictus in Florida. Aedes aegypti and/or Ae. albopictus were detected in the 56 counties that were part of the survey. Only Aedes albopictus was detected in 26 counties, primarily in the panhandle region of Florida. The results of this work underscore the importance of maintaining container mosquito surveillance in a state where chikungunya, dengue, and Zika viruses are present and where there is continued risk for exotic arbovirus introductions.     

Identification of a polymorphic mucin-like gene expressed in the midgut of the mosquito, Aedes aegypti, using an integrated bulked segregant and differential display analysis.

The identification of putative differentially expressed genes within genome regions containing QTL determining susceptibility of the mosquito, Aedes aegypti, to the malarial parasite, Plasmodium gallinaceum, was investigated using an integrated, targeted approach based on bulked segregant and differential display analysis. A mosquito F2 population was obtained from pairwise matings between the parasite-susceptible RED strain and the resistant MOYO-R substrain. DNA from female carcasses was used to genotype individuals at RFLP markers of known chromosomal position around the major QTL (pgs 1). Midguts, dissected 48 hr after an infected blood meal, were used to prepare two RNA bulks, each representing one of the parental genotypes at the QTL interval. The RNA bulks were compared by differential display PCR. A mucin-like protein gene (AeIMUC1) was isolated and characterized. The gene maps within the pgs 1 QTL interval and is expressed in the adult female midgut. AeIMUC1 RNA abundance decreased with time after blood meal ingestion. No differential expression was observed between the two mosquito strains but three different alleles with inter- and intrastrain allelic polymorphisms including indels and SNPs were characterized. The AeIMUC1 gene chromosome location and allelic polymorphisms raise the possibility that the protein might be involved in parasite-mosquito interactions.     

Assessment of Aedes albopictus (Skuse) (Diptera: Culicidae) clutch size in wild and laboratory populations

Aedes albopictus (Skuse) is an invasive mosquito species found across the southern U.S. with range expansion into many northern states. Intra‐ and interspecific larval competition have been evaluated for Ae. albopictus with respect to subsequent adult size, immature and adult survivability, and its capacity to vector pathogens as an adult. However, limited data are available on egg production as related to larval rearing conditions. Because Ae. albopictus is a container‐inhabiting mosquito that oviposits in resource‐limited habitats, it is found under variable density‐dependent conditions. Therefore, we examined the impact of specific rearing conditions on Ae. albopictus clutch size and adult body size; comparing the egg production values and wing lengths from known developmental densities to those from field‐collected populations. Field populations varied significantly among collection sites in mean clutch size (23 to 46). These clutch sizes were comparable to the mean clutch sizes of females reared at the larval densities of nine (20 eggs) and three (53 eggs) larvae per 3 ml of water in the laboratory. Field populations experienced density‐dependent effects impacting adult mosquito size. Mosquitoes from the four sample sites had mean wing lengths of 1.99, 2.47, 2.51, and 2.54 mm, which were less than the mean wing length of mosquitoes reared at larval densities of three larvae per 3 ml of water (2.57 mm).     

Horizontal transfer of the insect growth regulator pyriproxyfen to larval microcosms by gravid Aedes albopictus and Ochlerotatus triseriatus mosquitoes in the laboratory

The insect growth regulator (IGR) pyriproxyfen is highly active against mosquitoes (Diptera: Culicidae). Through continuous emersion of large larvae (instars 3-4) the concentration causing 50% inhibition of adult emergence (EI50) was determined as 0.200 p.p.b. for Aedes albopictus (Skuse) and 3.5 to 7 times less for Ochlerotatus triseriatus (Say): IE50 0.0288 p.p.b. As a possible method of application to larval microcosms of these species that oviposit in water containers and phytotelmata, the horizontal transfer of pyriproxyfen to larval microcosms by adult mosquitoes was evaluated under laboratory conditions. Gravid females were forced to walk on surfaces treated with pyriproxyfen (tarsal contact exposure) and then allowed to oviposit in larval microcosms. Using replicate bioassay cages, each with an oviposition container, and a factorial experimental design, we assessed Ae. albopictus for the effects of (i) pyriproxyfen concentration (0.2, 0.3 and 0.4 mg/cm2) contacted by gravid females, and (ii) the number of treated gravid females added to bioassay cages (one, three or five females/cage), on the mortality of larvae in oviposition containers. Only 0.2 mg/cm2 treatment rate was tested on Oc. triseriatus. A significant (P < 0.05) curvilinear response in inhibition of emergence (IE) was achieved on both species. Densities of one or three treated Oc. triseriatus females/cage yielded IE rates of only 21-27%, whereas five treated females/cage resulted in 70% inhibition. With Ae. albopictus, densities of three or five treated females/cage yielded 48-67% and 59-73% IE, respectively, whereas one treated female/cage gave only 4-30% inhibition. Use of IGR-treated oviposition containers to achieve horizontal transfer of pyriproxyfen to mosquito oviposition sites could be a field management technique based on mosquito biology and behaviour. In binary choice tests with Ae. albopictus, horizontal transfer of pyriproxyfen from a container with a treated ovistrip (0.3 or 0.4 mg/cm2) to an untreated microcosm resulted in 14-38% inhibition. In larval bioassays, pyriproxyfen activity declined markedly within 10 days. Forcibly exposing gravid female mosquitoes to pyriproxyfen-treated paper surface did not affect their fecundity. However, from the 1st to 2nd gonotrophic cycles the egg hatch rate declined by 30% (P < 0.05). Some variation of results could be due to interactions between females at the oviposition site, possibly causing disproportionate transfer of pyriproxyfen to larval microcosms. Comparative studies of the oviposition behaviour of each mosquito are warranted and would potentially provide information needed to improve the technique.     

Superior reproductive success on human blood without sugar is not limited to highly anthropophilic mosquito species

Anthropophilic mosquitoes such as Aedes aegypti L. (Diptera: Culicidae) have been shown to have superior reproductive success on human blood when sugar is not available. Life-table experiments were conducted with Aedes albopictus Skuse and Ae. aegypti to compare the effects of sugar availability on age-specific survivorship, lifetime and daily fecundity, and blood-feeding frequency when offered human blood daily. There were no significant interactions between the effects of sugar availability and mosquito species for these four variables, indicating similar effects of sugar availability for both species. Lifetime fecundity was not significantly affected by sugar availability, but sugar-deprived females had significantly reduced age-specific survivorship than did sugar-fed females. In absence of sugar, females took bloodmeals twice as often, resulting in a higher daily fecundity. The results indicate that superior reproductive success on human blood without sugar does not seem to be limited to highly anthropophilic mosquito species, such as Ae. aegypti. We conclude that evolution of a highly anthropophilic feeding strategy is not an inevitable result of the ability to thrive on human blood alone.     

Investigations of dengue-2 susceptibility and body size among Aedes aegypti populations

The mosquito Aedes aegypti (L.) (Diptera: Culicidae) is the primary global vector for dengue virus (DENV), yet considerable genetic variation exists among populations in terms of its competence to vector DENV. Variability in adult body size has also been observed among various mosquito populations and several studies have reported a relationship between body size and arbovirus dissemination, although most of these relied on artificially derived variation in body size. Here we examine the relationship between body size and disseminated DENV infection among 10 Ae. aegypti populations reared under optimum laboratory conditions. Body size variability was inferred from wing length measurements and DENV competence was evaluated as the proportion of individuals with disseminated infections following exposure to the dengue-2 JAM1409 strain. There were significant differences in mean wing lengths among populations (anova, F(9,22)= 7.10, P < 0.0001), ranging from 2.16 mm (Bangkok population) to 2.79 mm (MOYO-S [susceptible] population). We also observed significant differences among some populations in mean DENV infection rates (Waller-Duncan K-ratio t-test), ranging from 19.54% (MOYO-R [refractory] population) to 56.60% (MOYO-S population). However, we did not observe evidence for significant interactions between body size and DENV dissemination. We suggest that either the two traits are genetically independent or that our ability to detect interactions between them was limited by their respective inheritances as quantitative traits.     

Estimation of Hardy-Weinberg and pairwise disequilibrium in the apolipoprotein AI-CIII-AIV gene cluster.

Departures from Hardy-Weinberg (HW) equilibria and pairwise disequilibria were estimated in a sample of unrelated healthy individuals typed for six RFLPs in the apo AI-CIII-AIV gene region. The sample was composed of males and females, selected for health, from two populations, those of exclusively French-Canadian (FC) and those of some non-French-Canadian (NFC) ancestry. An approach suggested by Weir and Cockerham, which includes estimates of nonrandom association (disequilibria) between three and four alleles at two loci as well as the traditional associations between two alleles, at two loci was used. The pattern of departures from HW equilibria suggested that the genetic structures of the FC and NFC are different. Departure from HW equilibrium at an RFLP locus could not be predicted from information about other loci in the same gene region. Nonrandom associations were also evident from the pairwise analyses. Two pairs of loci had significant diallelic disequilibria, while two other pairs had significant triallelic disequilibria. All of the RFLP pairs had at least one measure of disequilibrium at its maximum value determined by allele frequencies. Inferences about pairwise disequilibria depended on the statistical approach used. Sizes of the pairwise disequilibria were not correlated with the physical distance between loci. The impact of these disequilibria on RFLP-phenotype association studies is discussed.     

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.     

Calibration and evaluation of field cage for oviposition study with Aedes (Stegomyia) aegypti female (L.) (Diptera: Culicidae)

Differences among results gathered from insect behavior studies conducted in laboratory and field situations are due to ambient variables that differ greatly between both environments. In laboratory studies the environmental conditions can be controlled whereas in field temperature, humidity and air velocity vary uncontrollably. The objective of this study was to calibrate and evaluate an experimental area (field cage) (14 x 7 x 3.5 m) subdivided into eight test cages (2.5 x 2.5 x 2 m) for use in behavioral oviposition tests of Aedes aegypti (L.) mosquitoes for developing a new methodology to assess attractants and oviposition traps. Test cage calibration involved: (1) minimal experiment duration tests; (2) optimal female release number per traps test and (3) trap placement tests. All tests used gravid A. aegypti females; 3-4 days post blood meal and the sticky trap MosquiTRAP to catch adults. Ninety percent of the females released were recaptured 2h after the beginning of the experiment, and this allowed up to 32 test repetitions/day to be conducted in the field cage. The minimum number of females necessary to conduct statistical analyses was 20 females/trap/test per cage. No significant difference was found in the behavioral response of gravid females to four different trap positions within test cages. Field trapping results with attractant were similar to those in the field cage. Therefore, the field cage could replace field trapping for evaluating at least mosquito traps and oviposition attractants for A. aegypti.     

Suppression of Aedes aegypti by predatory Toxorhynchites moctezuma in an island habitat

Larval populations of the mosquito Aedes aegypti were suppressed by predatory Toxorhynchites moctezuma mosquito larvae released systematically in a village on Union Island (Saint Vincent and the Grenadines) during March-December 1988. Eggs and larvae of Tx.moctezuma were transported from Trinidad and introduced into all semi-permanent and permanent water-holding containers in the experimental village at Clifton. The semi-isolated village of Ashton served as control. Base-line Ae.aegypti indices (house, ovitrap, Breteau, cistern/tank, drum/barrel, small containers) were obtained for the two villages over a 4-month period prior to the introduction of the predatory Tx.moctezuma mosquito larvae. After sustained releases of predators for 5 months, all indices of Ae.aegypti were lower in the treated village than in the untreated village during the last 3 months of the year.     

Assessing the impact of density dependence in field populations of Aedes aegypti

Although many laboratory studies of intra-specific competition have been conducted with Ae. aegypti, there have been few studies in natural environments and none that examined density dependence in natural containers at normal field densities. Additionally, current mathematical models that predict Ae. aegypti population dynamics lack empirically-based functions for density-dependence. We performed field experiments in Tapachula, Mexico, where dengue is a significant public health concern. Twenty-one containers with natural food and water that already contained larvae were collected from local houses. Each container was divided in half and the naturally occurring larvae were apportioned in a manner that resulted in one side of the container (high density) having four times the density of the second side (low density). Larvae were counted and pupae were removed daily. Once adults emerged, wing span was measured to estimate body size. Density had a significant impact on larval survival, adult body size, and the time taken to transition from 4(th) instar to pupation. Increased density decreased larval survival by 20% and decreased wing length by an average of 0.19 mm. These results provide a starting point for a better understanding of density dependence in field populations of Ae. aegypti.