Loop‐mediated isothermal amplification (LAMP) for the identification of invasive Aedes mosquito species

Invasive Aedes mosquito species (Diptera: Culicidae) are of public health concern in Europe because they are either recognized or potential vectors of pathogens. Loop‐mediated isothermal amplification (LAMP) is a rapid and simple method for amplifying DNA with high specificity and efficiency, with the technique having potential for application in the field, including in high‐throughput format. Specific LAMP assays based on rDNA internal transcribed spacers 1 or 2 sequences, considering intraspecies variability at these loci, were developed for Aedes aegypti, Aedes albopictus, Aedes japonicus, Aedes koreicus and the indigenous Aedes geniculatus. No such assays could be developed for Aedes atropalpus and Aedes triseriatus because both loci were too short to serve as target. The assays rely on the clearly visible colour change from violet to sky blue after successful amplification. Sensitivity of egg detection was confirmed with ratios of up to one mosquito egg in 99 other eggs. Simple sample preparation of adults or eggs by mechanical homogenization in water required an additional heat treatment or centrifugation step to avoid non‐specific colour changes. Thus, further technical improvements are needed to render these assays truly field‐applicable, which would greatly facilitate surveillance of these invasive mosquito species and allow for prompt implementation of control measures.     

Satyrization without evidence of successful insemination from interspecific mating between invasive mosquitoes

Previous research has documented low frequencies of interspecific mating in nature between the invasive vectors Aedes aegypti and Aedes albopictus. It is also known that heterospecific male accessory gland substances transferred during mating sterilize A. aegypti but not A. albopictus females, leading to satyrization, a form of reproductive interference. This paper demonstrates that satyrization of A. aegypti by A. albopictus may occur without evidence of successful insemination. Our results show that A. aegypti females, previously exposed to A. albopictus males, are rendered refractory to subsequent conspecific mating even though their spermathecae contain no heterospecific sperm. Additional experiments demonstrating transfer of labelled semen from A. albopictus males to A. aegypti females and low production of viable eggs of females housed with conspecific males, following exposure to A. albopictus males, confirm higher incidences of satyrization than expected, based on heterospecific insemination rates. We conclude that frequencies of satyrization based on detection of interspecific sperm in spermathecae may underestimate the impact of this form of reproductive interference.     

Pyrethroid resistance in Aedes aegypti and Aedes albopictus from Port‐au‐Prince,Haiti

In Port‐au‐Prince, Haiti, the status of insecticide resistance has not recently been evaluated for Aedes aegypti (L) and Aedes albopictus (Skuse) populations. No prophylactics exist for dengue, so prevention is only through vector control methods. An earthquake occurred in Haiti on January 12, 2010, with a magnitude of 7.0 Mw that devastated the area. Dengue became a major concern for the humanitarian relief workers that entered the country. Bottle bioassays were conducted in the field on adult mosquitoes reared from larvae collected from the grounds of the U.S. Embassy and from an adjacent neighborhood in eastern Port‐au‐Prince, Haiti. At the CDC, Fort Collins, CO, bioassays, molecular, and biochemical assays were performed on mosquitoes reared from field‐collected eggs. A small percentage of the population was able to survive the diagnostic dose in bioassays run in Haiti. Mosquitoes tested at the CDC demonstrated no phenotypic resistance. A variety of factors could be responsible for the discrepancies between the field and lab data, but temperature and larval nutrition are probably most important. Knowledge of localized resistance and underlying mechanisms helps in making rational decisions in selection of appropriate and effective insecticides in the event of a dengue outbreak.     

Effect of mating period and time‐of‐day for bloodmeal on rearing of Asian tiger mosquito (Aedes albopictus) in laboratory conditions

Colonization and maintenance of mosquitoes in the laboratory is required to study physiology, ecology, and behavior of mosquitoes and interactions between mosquito and pathogens. Artificial blood feeding systems have been widely used to maintain the laboratory colony of Aedes albopictus. In this study, we investigated the effects of mating period (1, 3, 6, and 10 days) and time‐of‐day for bloodmeal (08:00, 13:00, and 18:00) in the use of an artificial feeding system on blood‐feeding rate, female fecundity, egg hatching rate, and developmental time of the Asian tiger mosquito, A. albopictus. Younger females mated for three or fewer days reproduced more eggs compared to those of oldest females mated for ten days. Similar to the result for eggs laid, the mean egg‐hatching rate was significantly higher from the offspring of younger females than from those of older females. However, mating period and time‐of‐day for bloodmeal had no effect on the blood feeding rate and developmental time. Taken together, we suggest that three‐day mating with bloodmeal at 18:00 is optimal for maintaining colonies of A. albopictus in laboratory conditions.     

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.     

The challenge of invasive mosquito vectors in the U.K. during 2016–2018: a summary of the surveillance and control of Aedes albopictus

Mosquito‐borne diseases resulting from the expansion of two key vectors, Aedes aegypti and Aedes albopictus (Diptera: Culicidae), continue to challenge whole regions and continents around the globe. In recent years there have been human cases of disease associated with Chikungunya, dengue and Zika viruses. In Europe, the expansion of Ae. albopictus has resulted in local transmission of Chikungunya and dengue viruses. This paper considers the risk that Ae. aegypti and Ae. albopictus represent for the U.K. and details the results of mosquito surveillance activities. Surveillance was conducted at 34 points of entry, 12 sites serving vehicular traffic and two sites of used tyre importers. The most common native mosquito recorded was Culex pipiens s.l. (Diptera: Culicidae). The invasive mosquito Ae. albopictus was detected on three occasions in southern England (September 2016, July 2017 and July 2018) and subsequent control strategies were conducted. These latest surveillance results demonstrate ongoing incursions of Ae. albopictus into the U.K. via ground vehicular traffic, which can be expected to continue and increase as populations in nearby countries expand, particularly in France, which is the main source of ex‐continental traffic.     

The effect of photoperiod on life history and blood‐feeding activity in Aedes albopictus and Aedes aegypti (Diptera: Culicidae)

Several studies have examined how climatic variables such as temperature and precipitation may affect life history traits in mosquitoes that are important to disease transmission. Despite its importance as a seasonal cue in nature, studies investigating the influence of photoperiod on such traits are relatively few. This study aims to investigate how photoperiod alters life history traits, survival, and blood‐feeding activity in Aedes albopictus (Skuse) and Aedes aegypti (Linnaeus). We performed three experiments that tested the effects of day length on female survival, development time, adult size, fecundity, adult life span, and propensity to blood feed in Ae. albopictus and Ae. aegypti. Each experiment had three photoperiod treatments: 1) short‐day (10L:14D), 2) control (12L:12D), and 3) long‐day (14L:10D). Aedes albopictus adult females were consistently larger in size when reared in short‐day conditions. Aedes aegypti adult females from short‐day treatments lived longer and were more likely to take a blood meal compared to other treatments. We discuss how species‐specific responses may reflect alternative strategies evolved to increase survival during unfavorable conditions. We review the potential impacts of these responses on seasonal transmission patterns, such as potentially increasing vectorial capacity of Ae. aegypti during periods of shorter day lengths.     

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.     

登革热媒介昆虫抗药性的研究进展

近年来,登革热在全球的流行迅速增长,埃及伊蚊Aedes aegypti(L.)和白纹伊蚊Aedes albopictus(Skuse)是登革热传播的主要媒介。目前,化学防治仍然是防治蚊虫的主要手段,每年化学杀虫剂的大量使用直接或间接地导致其产生了抗药性。关于埃及伊蚊和白纹伊蚊的抗药性研究,主要集中于DDT、有机磷和拟除虫菊酯类杀虫剂导致的抗药性,并已深入到抗药性分子机理的研究。文章就埃及伊蚊和白纹伊蚊对杀虫剂诱导的抗药性现状及抗性机理的研究进展进行综述。     

Frequency of pyrethroid resistance in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Thailand

Thirty‐two Aedes aegypti populations collected throughout Thailand and five populations of Aedes albopictus from southern Thailand were subjected to standard WHO contact bioassays to assess susceptibility to three commonly used synthetic pyrethroids: permethrin, deltamethrin, and lambda‐cyhalothrin. A wide degree of physiological response to permethrin was detected in Ae. aegypti, ranging from 56.5% survival (Lampang, northern Thailand) to only 4% (Kalasin in northeastern and Phuket in southern Thailand). All 32 populations of Ae. aegypti were found to have evidence of incipient resistance (62.5%) or levels of survival deemed resistant (37.5%) to permethrin. Four populations of Ae. albopictus were found with incipient resistance (97 – 80% mortality) and one with resistance (< 80%) to permethrin. The majority of Ae. aegypti populations (68.7%) was susceptible (> 98% mortality) to deltamethrin, with incipient resistance (observed 97–82% mortality) in other localities. In contrast, all populations of Ae. aegypti were completely susceptible (100% mortality) to the recommended operational dosage of lambda‐cyhalothrin. All five populations of Ae. albopictus were found completely susceptible to both deltamethrin and lambda‐cyhalothrin. Evidence of defined incipient or resistance to synthetic pyrethroids mandates appropriate response and countermeasures to mitigate further development and spread of resistance. In light of these findings, we conclude that routine and comprehensive susceptibility monitoring of dengue mosquito vectors to synthetic pyrethroids should be a required component of resistance management policies and disease control activities.     

Experience‐ and age‐mediated oviposition behaviour in the yellow fever mosquito Stegomyia aegypti (=Aedes aegypti)

In repeated behaviours such as those of feeding and reproduction, past experiences can inform future behaviour. By altering their behaviour in response to environmental stimuli, insects in highly variable landscapes can tailor their behaviour to their particular environment. In particular, female mosquitoes may benefit from plasticity in their choice of egg‐laying site as these sites are often temporally variable and clustered. The opportunity to adapt egg‐laying behaviour to past experience also exists for mosquito populations as females typically lay eggs multiple times throughout their lives. Whether experience and age affect egg‐laying (or oviposition) behaviour in the mosquito Stegomyia aegypti (=Aedes aegypti) (Diptera: Culicidae) was assessed using a wind tunnel. Initially, gravid mosquitoes were provided with a cup containing either repellent or well water. After ovipositing in these cups, the mosquitoes were blood‐fed and introduced into a wind tunnel. In this wind tunnel, an oviposition cup containing repellent was placed in the immediate vicinity of the gravid mosquitoes. A cup containing well water was placed at the opposite end of the tunnel so that if the females flew across the chamber, they encountered the well water cup, in which they readily laid eggs. Mosquitoes previously exposed to repellent cups became significantly more likely to later lay eggs in repellent cups, suggesting that previous experience with suboptimal oviposition sites informs mosquitoes of the characteristics of nearby oviposition sites. These results provide further evidence that mosquitoes modify behaviour in response to environmental information and are demonstrated in a vector species in which behavioural plasticity may be ecologically and epidemiologically meaningful.     

Assessing natural infection with Zika virus in the southern house mosquito,Culex quinquefasciatus,during 2016 in Puerto Rico

The epidemic of Zika in the Western hemisphere has led to intense investigations of all species important in the transmission of Zika virus (ZikV), including putative mosquito vectors. Although evidence points to Stegomyia (= Aedes) (Diptera: Culicidae) mosquitoes as the primary vectors in nature among humans, there remains the possibility that other common mosquito species may be implicated in the rapid spread of the virus. Herein, field‐caught Culex quinquefasciatus (Diptera: Culicidae) collected during June 2016 in different neighbourhoods in San Juan, Puerto Rico were examined for the presence of natural infection with ZikV. Stegomyia aegypti (= Aedes aegypti) from the same locations were also analysed. None of the Cx. quinquefasciatus tested showed natural infection for ZikV, whereas S. aegypti tested positive at seven sites. The present results suggest that Cx. quinquefasciatus was not involved in the transmission of ZikV in San Juan, Puerto Rico in 2016.     

Pathogenicity of entomopathogenic fungus,Metarhizium anisopliae MET-GRA4 isolate on dengue vectors,Aedes albopictus and Aedes aegypti mosquito larvae (Diptera: Culicidae)

Considering the rapid transmission of the dengue virus, substantial efforts need to be conducted to ward-off the epidemics of dengue viruses. The control effort is depending on chemical insecticides and had aroused undesirable conflicts of insecticide resistance. Here, we study the entomopathogenic fungus, Metarhizium anisopliae as a promising new biological control agent for vector control. The pathogenicity effects of Metarhizium anisopliae against field and laboratory strains of Aedes albopictus and Aedes aegypti larvae were tested using the larvicidal bioassay technique. The results demonstrate that the treatments using M. anisopliae isolate MET-GRA4 were highly effective and able to kill 100% of both Ae. albopictus and Ae. aegypti mosquito larvae at a conidia concentration of 1 × 10?/ml within 7 days of the treatment period. The fungus displayed high larvicidal activity against laboratory and field strain of Ae. aegypti larvae with LC₅₀ values (9.6 × 10³/ml, 1.3 × 10³/ml) and LC₉₅ values (1.2 × 10?/ml, 5.5 × 10⁵/ml) respectively. For Ae. albopictus, LC₅₀ values for laboratory and field strains were (1.7 × 10⁴/ml, 2.7 × 10⁴/ml) and the LC₉₅ values were (2.1 × 10?/ml, 7.0 × 10⁵/ml) respectively. Interestingly, the susceptibility of field strain towards M. anisopliae was higher as compared to the laboratory strain Aedes larvae. In which, the causative agents of all the dead larvae were verified by the virulence of M. anisopliae and caused morphological deformities on larval body. The findings from this study identify this isolate could be an effective potential biocontrol agent for vector mosquitoes in Malaysia.     

Impact of inter‐ and intra‐specific competition among larvae on larval,adult, and life‐table traits of Aedes aegypti and Aedes albopictus females

1. Few studies have taken a comprehensive approach of measuring the impact of inter‐ and intra‐specific larval competition on adult mosquito traits. In this study, the impact of competition among Aedes aegypti (L.) and A. albopictus (Skuse) was quantified over the entire life of a cohort. 2. Competitive treatments affected hatch‐to‐adult survivorship and the development time to adulthood of females for both species but affected the median wing length of females only for A. albopictus. Competitive treatments had no significant effect on the median adult female longevity nor were there any effects on other individual traits related to blood feeding and reproductive success. 3. Analysis of life table traits revealed no effect of competitive treatment on the net reproductive rate (R₀) but there were significant effects on the cohort generation time (T_c) and the cohort rate of increase (r) for both species. 4. Inter‐ and intra‐specific competition among Aedes larvae may produce individual and population‐level effects that are manifest in adults; however, benign conditions may enable resulting adults to compensate for some impacts of competition, particularly those affecting blood‐feeding success, fecundity, and the net reproductive rate, R₀. The effect of competition, therefore, affects primarily larva‐to‐adult survivorship and the larval development time, which in turn impacts the cohort generation time, T_c, and ultimately the cohort rate of increase, r. 5. The lack of effects of the larval rearing environment on adult longevity suggests that effects on vectorial capacity owing to longevity may be limited if adults have easy access to sugar and bloodmeals.     

Electronic mosquito repellers induce increased biting rates in Aedes aegypti mosquitoes (Diptera: Culicidae)

Studies have demonstrated that electronic mosquito repellers are useless and that some of them could even increase the attraction of mosquitoes. While testing some electronic repellers, we noted that they also promoted an increase in biting rates. The present work has evaluated three commercial devices and a computer program working on five different sound frequencies. In a test chamber, the number of Aedes aegypti L. bite attempts was computed during four cycles of 3 min each by alternately turning the devices off and on. The mosquito biting rates for five sound frequencies (ranging from 9.6 kHz to 18.2 kHz) initially demonstrated a significant increase (ranging from around 20% to 50%), which decreased from 8.3% to 25.1% when the repellers were turned off. The biting rate significantly increased at 11.8 kHz (33.7%) when the device was turned on again. The danger of using electronic repellers and the role of sound frequencies stimulating mosquito biting are discussed.     

Swarming mechanisms in the yellow fever mosquito: aggregation pheromones are involved in the mating behavior of Aedes aegypti

Mosquitoes of various species mate in swarms comprised of tens of thousands of flying males. In this study, we examined Aedes aegypti swarming behavior and identified associated chemical cues. Novel evidence is provided that Ae. aegypti females aggregate by means of olfactory cues, such as aggregation pheromones. Isolation of Ae. aegypti aggregation pheromones was achieved by aeration of confined mosquitoes and collection of associated volatiles by glass filters. The collected volatiles were identified through gas chromatography mass spectrometry (GCMS). Three aggregation pheromones were collected and identified as 2,6,6‐trimethylcyclohex‐2‐ene‐1,4‐dione (ketoisophorone) (CAS# 1125–21–9, t_R = 18.75), 2,2,6‐trimethylcyclohexane‐1,4‐dione (the saturated analog of ketoisophorone) (CAS# 20547–99–3, t_R = 20.05), and 1‐(4‐ethylphenyl) ethanone (CAS# 937–30–4, t_R = 24.22). Our biological studies revealed that the identified compounds stimulated mosquito behavior under laboratory conditions. The mechanism of mosquito swarm formation is discussed in light of our behavioral study findings. A preliminary field trial demonstrated the potential application of the isolated aggregation pheromones in controlling Ae. aegypti.     

The community ecology of Aedes egg hatching: implications for a mosquito invasion

Abstract. 1. A recently introduced treehole mosquito from Asia, Aedes albopictus , is spreading throughout eastern North America, especially in tyre-refuse piles. Previous studies have identified inhibitory effects of larvae on egg hatch as a potential population regulatory mechanism within Aedes. Larva-egg interactions may also occur between species. This experiment assesses the ability of larvae of A. albopictus and two possible competitors in North America, A.triseriatus and A.aegypti , to suppress hatching of conspecific and congeneric eggs.
2. We exposed eggs of each species to varying combinations of larval species and density for 24h and assessed subsequent hatch rates. Aedes albopictus eggs exhibited the lowest level of inhibition when exposed to high larval densities; moreover, at the lowest larval density they imposed the most intense interspecific hatch inhibition.
3. Discretionary hatching in response to larval density may influence community composition by promoting the spread of A.albopictus , perhaps even leading to its dominance within North American Aedes communities.     

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.     

Effects of temperature and diet on development and interspecies competition in Aedes aegypti and Aedes albopictus

We asked whether climate change might affect the geographic distributions of Aedes aegypti (L.) and Aedes albopictus (Skuse) (Diptera: Culicidae). We tested the effects of temperature, diet and the presence of congeneric species on the performance of immature stages of these two aedine species in the laboratory. Mosquitoes in three different species-density combinations were reared at four constant temperatures (20 °C, 25 °C, 30 °C, 35 °C) on low- or high-level diets. Of the four temperatures tested, mortality increased only at 35 °C in both species. Mortality was higher on the high-level diet than on the low-level diet at 35 °C, but not at other temperatures. The presence of congeneric species had a significant positive effect on mortality in Ae. albopictus, but not in Ae. aegypti. Both species developed more quickly at higher temperatures within the range of 20-30 °C; development was not enhanced at 35 °C. Population growth of Ae. albopictus was more stable, regardless of diet and temperature; that of Ae. aegypti varied more according to these two factors. These species-specific attributes may help to explain the latitudinal distribution of the mosquitoes and degree of species dominance where they are sympatric.