Proof of concept for a novel insecticide bioassay based on sugar feeding by adult Aedes aegypti (Stegomyia aegypti)

Aedes aegypti L. (Stegomyia aegypti) (Diptera: Culicidae) is the principal vector of dengue and yellow fever viruses in tropical and subtropical regions of the world. Disease management is largely based on mosquito control achieved by insecticides applied to interior resting surfaces and through space sprays. Population monitoring to detect insecticide resistance is a significant component of integrated disease management programmes. We developed a bioassay method for assessing insecticide susceptibility based on the feeding activity of mosquitoes on plant sugars. Our prototype sugar‐insecticide feeding bioassay system was composed of inexpensive, disposable components, contained minimal volumes of insecticide, and was compact and highly transportable. Individual mosquitoes were assayed in a plastic cup that contained a sucrose‐permethrin solution. Trypan blue dye was added to create a visual marker in the mosquito's abdomen for ingested sucrose‐permethrin solution. Blue faecal spots provided further evidence of solution ingestion. With the sugar‐insecticide feeding bioassay, the permethrin susceptibility of Ae. aegypti females from two field‐collected strains was characterized by probit analysis of dosage‐response data. The field strains were also tested by forced contact of females with permethrin residues on filter paper. Dosage‐response patterns were similar, indicating that the sugar‐insecticide feeding bioassay had appropriately characterized the permethrin susceptibility of the two strains.     

Genetic structure among Thai populations of Aedes aegypti mosquitoes

Thirty‐one field populations of Aedes aegypti (L.) were compared using isozyme starch gel electrophoresis to characterize genetic variation between populations. Ae. aegypti were collected from seven provinces in Thailand. Thirty‐one isozyme encoding loci, including 19 polymorphic loci, were characterized. Only small levels of genetic differentiation were observed among the 31 district populations in the seven provinces. Isolation by distance among populations from the seven provinces showed no correlation between genetic variation and geographical distance.     

Pathogenicity of Spiroplasma taiwanense for larval Aedes aegypti mosquitoes

Helical replicative forms, but not the persistent non-replicative forms, of Spiroplasma taiwanense Abalain-Colloc et al. (isolated from the mosquito Anopheles sinensis Wiedemann in Taiwan) were shown to reduce significantly the survival of Aedes aegypti (L.) mosquito larvae reared in 10 ml of water with 0.3 ml of S.taiwanense suspensions added on days 0 and 3. The suspensions contained, respectively, helical forms at a concentration of 10(9) Colour Change Units (CCU)/ml and persistent forms at 10(6) CCU/ml. It is suggested that S.taiwanense, or toxins produced from it, are potentially useful for use in integrated mosquito control programmes.     

Insecticide susceptibility status in Mexican populations of Stegomyia aegypti (= Aedes aegypti): a nationwide assessment

In Mexico, mosquito vector‐borne diseases are of public health concern as a result of their impact on human morbidity and mortality. The use of insecticides against adult mosquitoes is one of the most common ways of controlling mosquito population densities. However, the use of these compounds has resulted in the development of insecticide resistance. The aim of this study was to estimate susceptibility to six pyrethroids, two carbamates and two organophosphates in Mexican populations of Stegomyia aegypti (Linnaeus, 1762) (= Aedes aegypti) (Diptera: Culicidae) mosquitoes. Bottle insecticide susceptibility tests, with 1 h exposure, were performed on adult mosquitoes from 75 localities across 28 states. At 30 min of exposure, the proportion of fallen mosquitoes was recorded. After 60 min of exposure, mosquitoes were recovered in non‐treated containers and mortality was determined at 24 h after the set‐up of the experiment. In general, the carbamate insecticides represented the most effective group in terms of the proportion of mosquitoes fallen at 30 min (72–100%) and 24‐h mortality (97–100%). High and widespread resistance to pyrethroids Types I and II and, to a lesser extent, to organophosphates was observed. Insecticide susceptibility among and within states was highly variable.     

Aedes aegypti (L.) mosquitoes in Trinidad,West Indies: longevity case studies

Few laboratory and field studies have reported long survival periods for Ae. aegypti females and even fewer have designed experiments to characterize this important life history trait. This study was conducted under laboratory conditions to determine the number of blood meals taken by individual females, the number of eggs laid per individual female, the length of the gonotrophic cycle, and the duration of female survival. The results showed individual females oviposited between 670 and 1,500 eggs throughout their lifetimes, females undergoing large numbers of gonotrophic cycles and surviving up to 224 days. These results are discussed in the context of vector competence, unique alternating high and low oviposition patterns observed after week 14, and resource partitioning/allocation by older Ae. aegypti females after blood feeding.     

Effect of body size and sugar meals on oviposition of the yellow fever mosquito,Aedes aegypti (Diptera: Culicidae)

The effects of dietary sugar and body size on the oviposition of Ae. aegypti were studied under laboratory conditions. In female mosquitoes provided with sugar, the start of maximum fecundity was significantly delayed and the oviposition period was longer than in females provided with water. The peak of oviposition was also delayed in sugar‐fed females. Large females oviposited more eggs per day than small females at maximum fecundity and during eight days of observations. Large females also visited significantly more water‐containing cups in their cages per day than small females at maximum fecundity. During the eight days of observations, large females and sugar‐fed females visited more water‐containing cups in their cages than water‐fed small females. Both large females and sugar‐fed females oviposited their eggs at sites higher above the water line than water‐fed small females. These results suggested that large and sugar‐fed female Ae. aegypti mosquitoes had more energy reserves and oviposited their eggs at higher sites, which would lead to a time lag in hatching.     

Insecticide resistance,associated mechanisms and fitness aspects in two Brazilian Stegomyia aegypti (= Aedes aegypti) populations

In Brazil, insecticide resistance in Stegomyia aegypti (= Aedes aegypti) (Diptera: Culicidae) populations to pyrethroids and to the organophosphate (OP) temephos is disseminated. Currently, insect growth regulators (IGRs) and the OP malathion are employed against larvae and adults, respectively. Bioassays with mosquitoes from two northeast municipalities, Crato and Aracaju, revealed, in both populations, susceptibility to IGRs and malathion (RR₉₅ ≤ 2.0), confirming the effectiveness of these compounds. By contrast, temephos and deltamethrin (pyrethroid) resistance levels were high (RR₉₅ > 10), which is consistent with the use of intense chemical control. In Crato, RR₉₅ values were > 50 for both compounds. Knock‐down‐resistant (kdr) mutants in the voltage‐gated sodium channel, the pyrethroid target site, were found in 43 and 32%, respectively, of Aracaju and Crato mosquitoes. Biochemical assays revealed higher metabolic resistance activity (esterases, mixed function oxidases and glutathione‐S‐transferases) at Aracaju. With respect to fitness aspects, mating effectiveness was equivalently impaired in both populations, but Aracaju mosquitoes showed more damaging effects in terms of longer larval development, decreased bloodmeal acceptance, reduced engorgement and lower numbers of eggs laid per female. Compared with mosquitoes in Crato, Aracaju mosquitoes exhibited lower OP and pyrethroid RR₉₅, increased activity of detoxifying enzymes and greater effect on fitness. The potential relationship between insecticide resistance mechanisms and mosquito viability is discussed.     

Effects of intraspecific larval competition on adult longevity in the mosquitoes Aedes aegypti and Aedes albopictus

Abstract Larval competition is common in container‐breeding mosquitoes. The impact of competition on larval growth has been thoroughly examined and findings that larval competition can lead to density‐dependent effects on adult body size have been documented. The effects of larval competition on adult longevity have been less well explored. The effects of intraspecific larval densities on the longevity of adults maintained under relatively harsh environmental conditions were tested in the laboratory by measuring the longevity of adult Aedes aegypti (L.) and Aedes albopictus (Skuse) (Diptera: Culicidae) that had been reared under a range of larval densities and subsequently maintained in high‐ or low‐humidity regimes (85% or 35% relative humidity [RH], respectively) as adults. We found significant negative effects of competition on adult longevity in Ae. aegypti, but not in Ae. albopictus. Multivariate analysis of variance suggested that the negative effect of the larval environment on the longevity of Ae. aegypti adults was most strongly associated with increased development time and decreased wing length as adults. Understanding how larval competition affects adult longevity under a range of environmental conditions is important in establishing the relationship between models of mosquito population regulation and epidemiological models of vector‐borne disease transmission.     

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.     

Renewal of sexual receptivity in mated female mosquitoes, Aedes aegypti

ABSTRACT. Females of Aedes aegypti (L.) (Diptera, Culicidae) were not receptive to mating 1 h after the initial insemination or at later times throughout the first gonotrophic cycle. A significant proportion of these females (75–90%) were inseminated again prior to the second gonotrophic cycle. Insemination was determined by the detection in females of sperm labelled with ³ H(G)-adenine or ³²PO₄ after matings with radiolabeled males. Females deprived of males after the first insemination displayed an abnormal reduction in fertility throughout successive gonotrophic cycles.     

Insecticide susceptibility status of Aedes aegypti (Diptera: Culicidae) from Townsville

Insecticide susceptibility of a 1995 Townsville Aedes aegypti (L.) population was investigated in the laboratory according to World Health Organization guidelines. Using baseline data from two Townsville populations (1955 and 1989), larval bioassays detected significant increases in susceptibility to synthetic pyrethroids and malathion, and significant reductions in susceptibility to most organophosphates and propoxur. Adult bioassays detected significant resistance to bendiocarb and DDT. Comparison of larval data with the international reference ROCK strain showed substantial resistance to have developed to malathion and fenthion. Further analysis revealed the presence of distinct substrains in the baseline 1989 population, which displayed varying levels of temephos susceptibility. We concluded that susceptibility investigations should assess mosquito populations collected from many sites within an area rather than taking a single population from one site, and that the 1989 Ae. aegypti colony would be unsuitable for use as a susceptible reference population.     

Lessons of Aedes aegypti control in Thailand

Abstract. The incidence of dengue haemorrhagic fever (DHF) in Thailand has increased cyclically since the first recognized outbreak in 1958. Without an effective vaccine against dengue, and considering the clinical difficulty of treating DHF cases, vector control is needed to prevent dengue transmission. Since the establishment of the WHO Aedes Research Unit in 1964, continued since 1973 as the WHO Collaborating Centre at the Department of Medical Research in Bangkok, much operational research has been carried out in Thailand on the bionomics and control of dengue vectors: Aedes aegypti and Ae.albopictus. This review shows that, as in most other countries, dengue vector control programmes in Thailand make little use of the procedures arising from research, nor have they reduced the upward trend of dengue or prevented DHF outbreaks. Implications of the reluctance to use results of operational research on vector control are considered and remedial suggestions made.     

Susceptibility of adult mosquitoes to insecticides in aqueous sucrose baits

Mosquitoes characteristically feed on plant‐derived carbohydrates and honeydew just after emergence and intermittently during their lives. Development of toxic baits focusing on this carbohydrate‐seeking behavior may potentially contribute to localized control. In the present study, ten insecticides were fed to female Culex quinquefasciatus, Anopheles quadrimaculatus, and Aedes taeniorhynchus in a 10% sucrose solution. Active ingredients representative of five classes of insecticides (pyrethroids, phenylpyroles, pyrroles, neonicotinoids, and macrocyclic lactones) were selected for comparison with commercial formulations used to facilitate incorporation of active ingredients into aqueous sucrose solutions. Sucrose as a phagostimulant significantly enhanced mortality to toxicants. In general, the most effective active ingredients were fipronil, deltamethrin and imidacloprid, followed by spinosad, thiamethoxam, bifenthrin, permethrin, and cyfluthrin. The least effective ingredients were chlorfenapyr and ivermectin. For some of the ingredients tested, Cx. quinquefasciatus was the least susceptible species. One‐day‐old male Cx. quinquefasciatus were more susceptible than females; however, no differences existed between one‐ and seven‐day‐old mosquitoes. There were no differences in susceptibility between unfed and gravid ten‐day‐old female Cx. quinquefasciatus to bifenthrin. In conclusion, several pesticides from different classes of compounds have potential for use in development of toxic baits for mosquitoes.     

Evaluation of the role of ATP-binding cassette transporters as a defence mechanism against temephos in populations of Aedes aegypti

The role of ATP-binding cassette (ABC) transporters in the efflux of the insecticide, temephos, was assessed in the larvae of Aedes aegypti. Bioassays were conducted using mosquito populations that were either susceptible or resistant to temephos by exposure to insecticide alone or in combination with sublethal doses of the ABC transporter inhibitor, verapamil (30, 35 and 40 μM). The best result in the series was obtained with the addition of verapamil (40 μM), which led to a 2x increase in the toxicity of temephos, suggesting that ABC transporters may be partially involved in conferring resistance to the populations evaluated.     

Virulence of entomopathogenic fungi isolated from wild mosquitoes against Aedes aegypti

The entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana are highly virulent control tools for insect pests and have been under evaluation for the control of globally important mosquito vectors such as Aedes aegypti. Here, we identified and isolated other virulent entomopathogenic fungi against Ae. aegypti. We collected 7 species of mosquitoes by human landing catch in 5 municipalities in Central and Northern Mexico and isolated 28 species of fungi. We harvested fungal conidia from six and assessed virulence against Ae. aegypti females. We observed variation in virulence of fungi in Ae. aegypti with the most virulent being Aspergillus tamarii, with a LT₅₀ of 6.4 (±0.65) days and the least virulent was Trichoderma euskadiense with a LT₅₀ of 16.3 (±1.5) days. Additional assays evaluated the impact of the fungi on Ae. aegypti fecundity and fertility and A. tamarii had the highest for both, resulting in 60% and 37% decrease, respectively. These results provide support for the potential utility of A. tamarii as an entomopathogenic control tool for the dengue vector, Ae. aegypti, pending further evaluations of environmental and nontarget safety.     

Biochemical screening of Lactobacilli in midgut of wild Aedes aegypti mosquitoes. A step toward paratransgenesis candidate

This study was conducted to isolate and identify lactobacilli from larval and adult midgut of wild Aedes aegypti (Ae. aegypti) to find candidate bacteria for paratransgenic control. Characterization of the bacterial symbionts was done using Gram staining, motility test, catalase test, and biochemical tests, among others, and the morphological features were confirmed using a standard scheme that simplifies the identification of lactic acid bacteria. A total of 174 strains were isolated and identified, 135 strains from larval midgut and 39 strains from adult midgut (mean ± SE, 4.00 ± 0.72; P = 0.00). The isolated species were confirmed to be Lactobacillus fermentum, L. casei, L. acidophilus, L. viridescens, L. brevis and L. gasseri. It can be concluded that Ae. aegypti has the potential of harboring the cultivable bacterial symbionts. In conclusion, the isolated species were nominated for paratransgenic control, particularly L. fermentum, being found in large numbers from both larval and adulxt midgut.