Seasonal Synchronization of Diapause Phases in Aedes albopictus (Diptera: Culicidae)

In temperate areas, population dynamics of the invasive Asian tiger mosquito Aedes albopictus are strongly affected by winter. The work we present here analyzes the adaptive synchronization of the diapause process in the wintry generation of A. albopictus, where the egg stage is exposed to adverse winter conditions. The seasonal pattern of egg laying activity of a French Mediterranean population of the Asian tiger mosquito was monitored weekly for 2 years with ovitraps. The field diapause incidence and the critical photoperiod (CPP, i.e. the maternal day length inducing diapause in 50% of the eggs), were determined by hatching experiments on the collected eggs. The period of diapause termination was estimated by a field survey of the first hatchings for both years. The CPP is equal to 13.5 hours of light and occurs in the field on the 25^th of August. Thus, it is on September 11^th, 17 days after the CPP, that 50% of the eggs are in a prediapause stage in the field. The egg diapause rate increases rapidly during September, whereas the mean number of eggs laid decreases sharply after mid-September. Surprisingly, after having reached a peak of 95% at the end of September, from mid-October the diapause incidence declined and stayed below 50%. Indeed, both years the diapause initiates before the rapid decrease of the environmental temperature. This leaves a sufficient period of time to the complete development of one generation of A. albopictus with effective induction of diapause in the laid eggs. The very first larvae hatched were sampled both years in the first half of March. With 20 to 26 weeks in the egg stage and about 7 weeks in the larval stages, the first annual generation spends a long time in immature stages. On a practical point of view, this long development time represents a wide window for eggs and larvae control in early spring.     

Temporal Patterns of Abundance of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) and Mitochondrial DNA Analysis of Ae. albopictus in the Central African Republic

The invasive Asian tiger mosquito Aedes albopictus (Diptera: Culicidae) was first reported in central Africa in 2000, in Cameroon, with the indigenous mosquito species Ae. aegypti (Diptera: Culicidae). Today, this invasive species is present in almost all countries of the region, including the Central African Republic (CAR), where it was first recorded in 2009. As invasive species of mosquitoes can affect the distribution of native species, resulting in new patterns of vectors and concomitant risk for disease, we undertook a comparative study early and late in the wet season in the capital and the main cities of CAR to document infestation and the ecological preferences of the two species. In addition, we determined the probable geographical origin of invasive populations of Ae. albopictus with two mitochondrial DNA genes, COI and ND5. Analysis revealed that Ae. aegypti was more abundant earlier in the wet season and Ae. albopictus in the late wet season. Used tyres were the most heavily colonized productive larval habitats for both species in both seasons. The invasive species Ae. albopictus predominated over the resident species at all sites in which the two species were sympatric. Mitochondrial DNA analysis revealed broad low genetic diversity, confirming recent introduction of Ae. albopictus in CAR. Phylogeographical analysis based on COI polymorphism indicated that the Ae. albopictus haplotype in the CAR population segregated into two lineages, suggesting multiple sources of Ae. albopictus. These data may have important implications for vector control strategies in central Africa.     

Temporal variation in the ribosomal DNA nontranscribed spacer of Aedes albopictus (Diptera: Culicidae)

Length variation in ribosomal DNA (rDNA) nontranscribed spacer within and among populations of the mosquito Aedes albopictus was studied over a 3-year period in eight different populations from Texas. Statistically significant variation was detected among populations at each sampling date. Significant, but nondirectional, changes were observed within populations over time. An average of 78% of the total variance in the frequencies of spacer variants was attributable to the within-population component, suggesting local differentiation. The results corroborate those from a previous study on spacer length diversity in populations of A. albopictus from around the world and are similar to those from a study on temporal variation in allozyme frequencies in some of the same populations and sampling dates. We propose that a high rate of production of length variants and localized breeding structure could explain the observed high levels of within- and among-population variation in A. albopictus.     

Biological cycle of Aedes albopictus (Diptera, Culicidae), in the laboratory]

Twelve colonies of Aedes albopictus were maintained at laboratory conditions, with the following results: Insectary conditions--Air humidity: 90-95%; Temperature: 25 degrees C; Blood source: human; Breeding places: Plastic glass or bamboo; Number eggs per female: 36.37; Incubation time: 1 to 6 days; Larval period: 4 to 9 days; Pupal period: 1 to 5 days; Adults life span: females--30.8 days and males--26.6 days.     

Aedes albopictus (Diptera: Culicidae) in Rome: experimental study of relevant control strategy parameters

Since 1997, Aedes albopictus has colonised and then rapidly invaded the city of Rome (Italy) and its peripheral areas. Presently, the control of this mosquito in Italy relies mainly on larvicidal treatment of street storm sewer catch basins with the organophosphate temephos. We have therefore obtained baseline data on the susceptibility to temephos of the Roman Ae. albopictus population by laboratory bioassays on F1 fourth-instar larvae following standard WHO protocols. Estimated lethal concentrations were 0.014 mg/l (LC50) and 0.022 mg/l (LC90) indicating a lack of resistance to this compound. The persistence of temephos in sewer catch basins was evaluated by follow-up of catch basins treated with a dose of 1.5 mg of active ingredient. Mosquito larvae were recovered in 10% and 50% of the treated basins at 9 and 18 days posttreatment, respectively. In order to understand the relative contribution of this larval habitat to adult populations, we conducted a survey in the Zoo of Rome to estimate the larval density of mosquitoes breeding in sewer catch basins. A complete census of a 16.5 ha area mapped 243 catch basins, but only 25 (10.3%) contained water; of the latter 8 (32.0%) hosted mosquito larvae. All positive catch basins contained larvae of Culex pipiens, which were associated with Culiseta longiareolata and/or Ae. albopictus in 6 and 3 cases, respectively. A longitudinal survey of one catch basin over 4 months showed that the mean larval density of Ae. albopictus was markedly lower than that of Cx pipiens and Cs. Iongiareolata, ranging between 0 and 1.3 larvae/dip as compared to 0-33.2 and 0-22.7 larvae/dip, respectively. However, adult densities of Ae. albopictus in this area estimated during the same period with 20 ovitraps showed consistently high values (proportion of positive ovitraps around 100%). These preliminary observations suggest that whenever alternative larval biotopes other than sewer catch basins are widely available, they might be more productive and/or preferred substrates to catch basins for Ae. albopictus breeding.     

Crouching Tiger,Hidden Trouble: Urban Sources of Aedes albopictus (Diptera: Culicidae) Refractory to Source-Reduction

Our ultimate objective is to design cost-effective control strategies for Aedes albopictus, the Asian tiger mosquito, an important urban nuisance and disease vector that expanded worldwide during the last 40 years.  We conducted mosquito larval surveys from May through October 2009 in the City of Trenton, New Jersey, USA, while performing intensive monthly source-reduction campaigns that involved removing, emptying, or treating all accessible containers with larvicides and pupicides. We examined patterns of occurrence of Ae. albopictus and Culex pipiens, another urban mosquito, among different container types by comparing observed and expected number of positive containers of each type. Expected use was based on the relative frequency of each container type in the environment. Aedes albopictus larvae and pupae were found significantly more often than expected in medium volumes of water in buckets and plant saucers but were rarely collected in small volumes of water found in trash items such as discarded cups and cans. They were also absent from large volumes of water such as in abandoned swimming pools and catch basins, although we consistently collected Cx. pipiens from those habitats. The frequency of Ae. albopictus in tires indicated rapid and extensive use of these ubiquitous urban containers. Standard larval-based indices did not correlate with adult catches in BG-Sentinel traps, but when based only on Ae. albopictus key containers (buckets, plant saucers, equipment with pockets of water, and tires) they did. Although we found that only 1.2% of the 20,039 water-holding containers examined contained immature Ae. albopictus (5.3% if only key containers were counted), adult populations were still above nuisance action thresholds six times during the 2009 mosquito season. We conclude that in urban New Jersey, effective source reduction for Ae. albopictus control will require scrupulous and repeated cleaning or treatment of everyday use containers and extensive homeowner collaboration.     

The Sterile Insect Technique for Controlling Populations of Aedes albopictus (Diptera: Culicidae) on Reunion Island: Mating Vigour of Sterilized Males

Reunion Island suffers from high densities of the chikungunya and dengue vector Aedes albopictus. The sterile insect technique (SIT) offers a promising strategy for mosquito-borne diseases prevention and control. For such a strategy to be effective, sterile males need to be competitive enough to fulfil their intended function by reducing wild mosquito populations in natura. We studied the effect of irradiation on sexual maturation and mating success of males, and compared the sexual competitiveness of sterile versus wild males in the presence of wild females in semi-field conditions. For all untreated or sterile males, sexual maturation was completed within 13 to 20 h post-emergence and some males were able to inseminate females when 15 h old. In the absence of competition, untreated and sterile males were able to inseminate the same number of virgin females during 48 h, in small laboratory cages: an average of 93% of females was inseminated no matter the treatment, the age of males, and the sex ratio. Daily mating success of single sterile males followed the same pattern as for untreated ones, although they inseminated significantly fewer females after the ninth day. The competitiveness index of sterile males in semi-field conditions was only 0.14 when they were released at 1-day old, but improved to 0.53 when the release occurred after a 5-day period in laboratory conditions. In SIT simulation experiments, a 5∶1 sterile to wild male ratio allowed a two-fold reduction of the wild population’s fertility. This suggests that sterile males could be sufficiently competitive to mate with wild females within the framework of an SIT component as part of an AW-IPM programme for suppressing a wild population of Ae. albopictus in Reunion Island. It will be of interest to minimise the pre-release period in controlled conditions to ensure a good competitiveness without increasing mass rearing costs.     

Laboratory evaluation of Toxorhynchites splendens (Diptera: Culicidae) for predation of Aedes albopictus mosquito larvae

Biology of the mosquito Toxorhynchites splendens (Wiedemann) was studied in the laboratory to provide baseline data for using the predatory larvae of this species against those of Aedes albopictus (Skuse) in a biological control programme. The mean incubation time of Tx.splendens eggs was 43.8 h and the time required for newly-hatched larvae to initiate predation was 2.5 h. Mean numbers of prey larvae consumed and killed by each Tx.splendens larva totalled 389 and 345 respectively. The larval period of Tx.splendens was not significantly different for rearing individually or in groups of nine, with equal prey density, and duration of larval development was proportional to prey density. In mass rearing, larval cannibalism was usually observed during days 1-3 post-eclosion. The incidence of cannibalism decreased sharply on the fourth day after hatching when some larvae became fourth-instar. Adult female Tx.splendens usually commenced oviposition on day 4 after emergence. The number of eggs laid daily increased on day 7 and the peak oviposition of 6.3 eggs/female/day occurred on day 11. When oviposition containers were provided only intermittently, gravid females of Tx.splendens scattered most of their eggs on the dry floor of the cage. Viability of eggs laid by females aged 4-14 days was high (60-90%) but decreased to less than 40% as the females aged.     

Experimental infection of Aedes albopictus (Diptera: Culicidae) Larvae with the Xiphidiocercariae of a hematolechid

Aedes albopictus larvae were exposed, either individually or in groups, to different concentrations of xiphidiocercariae of Haematoloechus sp. for parasitological studies. It was observed the acute lethal effect and some aspects of the host-parasite relationship, such as delay or progress in the host life cycle, the number and location of the metacercariae in the host, adult host malformations and the amount of metacercariae required to cause death. A delay in the cycle and a high mortality rate was in general observed. Inside the larvae, the metacercariae were found predominantly in the thorax, abdominal segments and in the head, along with a reduced number in the anal lobe and cervix. It was shown that in addition to the quantity of metacercariae present, their location in the larvae was also relevant in the determination of mortality and anomalies. Malformed adults developed from larvae containing from one to three metacercariae.     

Reproductive Strategies of Aedes albopictus (Diptera: Culicidae) and Implications for the Sterile Insect Technique

Male insects are expected to optimize their reproductive strategy according to the availability of sperm or other ejaculatory materials, and to the availability and reproductive status of females. Here, we investigated the reproductive strategy and sperm management of male and virgin female Aedes albopictus, a mosquito vector of chikungunya and dengue viruses. The dynamics of semen transfer to the female bursa inseminalis and spermathecae were observed. Double-mating experiments were conducted to study the effect of time lapsed or an oviposition event between two copulations on the likelihood of a female double-insemination and the use of sperm for egg fertilization; untreated fertile males and radio-sterilised males were used for this purpose. Multiple inseminations and therefore the possibility of sperm competition were limited to matings closely spaced in time. When two males consecutively mated the same female within a 40 min interval, in ca. 15% of the cases did both males sire progeny. When the intervals between the copulations were longer, all progeny over several gonotrophic cycles were offspring of the first male. The mating behavior of males was examined during a rapid sequence of copulations. Male Ae. albopictus were parceling sperm allocation over several matings; however they would also attempt to copulate with females irrespective of the available sperm supply or accessory gland secretion material. During each mating, they transferred large quantities of sperm that was not stored for egg fertilization, and they attempted to copulate with mated females with a low probability of transferring their genes to the next generation. The outcomes of this study provided in addition some essential insights with respect to the sterile insect technique (SIT) as a vector control method.     

Purification and expression of glutathione S-transferase from a Malaysian population of Aedes albopictus (Diptera: Culicidae)

Glutathione S-transferase (GST) from the 4^th instar larvae of the dengue vector Aedes albopictus was purified by glutathione-agarose affinity chromatography and characterised using SDS-PAGE. The expression of the purified enzyme in the life stages and insecticide treated populations of Ae. albopictus as well as its cross-reactivity with larval GST of two dipteran species Aedes aegypti and Batrocera papayae were observed using western blotting. The purified GST had a specific activity of 196.0 ± 11 μmol/min/mg with a purification fold and yield of 28 and 69%, respectively. The SDS-PAGE analysis of the purified GST depicted a single band size of 23 kDa. The GST was expressed in all the larval and adult stages of Ae. albopictus with the exception of the pupal stage. However, the expression level in the adult stage was visibly reduced as compared to the larval stages. Western blotting analysis showed no cross-reactivity with the GST of Ae. aegypti (4^th instar) and B. papayae (3^rd instar) larvae. The expression of this enzyme was not inducible by exposure to the insecticides dichlorodiphenyltrichloroethane (1.25 mg/L) and malathion (0.3125 mg/L).     

Conservation of a highly repeated DNA family of Aedes albopictus among mosquito genomes (Diptera: Culicidae)

Summary The genomic organization and chromosomal localization of a cloned 0.79-kb highly repeated DNA fragment, H-115, isolated from Aedes albopictus has been examined. The cloned fragment is a part of a larger unit of 1.86 kb that is tandemly repeated in the Ae. albopictus genome. The H-115 family of sequences are located at the intercalary position on chromosome 1 in Ae. albopictus. Similar patterns of in situ and Southern blot hybridization results are obtained in Ae. aegypti, Ae. seatoi, Ae. flavopictus, Ae. polynesiensis, Ae. Alcasidi, and Ae. katherinensis. The H-115 sequences are widely conserved in Culicidae and are found in Haemagogus equinus, Tripteroides bambusa, and Anopheles quadrimaculatus by hybridization under high stringency conditions. The H-115 sequences are also tandemly repeated in Hg. equinus with a monomer unit of 1.86 kb and in Tp. bambusa with a slightly diverged monomer unit of 1.90kb. In Anopheles quadrimaculatus, the H-115 sequences are dispersed throughout the genome. Partial sequence analysis shows that the H-115 insert is 62% AT and contains two perfect inverted repeats and numerous perfect direct repeats. The occurrence of inverted repeats with potential to form intrastrand palindromic structure suggests that the H-115 family of sequences may be involved in chromatin condensation.     

Differential identification of Ascogregarina species (Apicomplexa: Lecudinidae) in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) by polymerase chain reaction

We report 2 polymerase chain reaction (PCR)-based methods for distinguishing morphologically similar gregarine species based on amplification of variable regions of the internal transcribed spacer region of ribosomal DNA. The gregarines we investigated were Ascogregarina barretti (Vavra), A. culicis (Ross), and A. taiwanensis (Lien and Levine), parasites of the mosquitoes Ochlerotatus triseriatus (Say), Aedes aegypti (Linnaeus), and Ae. albopictus (Skuse), respectively. These 3 important vector mosquitoes often utilize the same container habitats, where larval development and infection by the parasite occurs, leaving ample opportunity for cross-species gregarine infection. Because previous studies have shown that the parasites A. culicis and A. taiwanensis variably affect fitness in both normal and abnormal mosquito hosts, distinguishing parasite infection and species is important. The task is complicated by the fact that these 2 parasite species are virtually identical in morphology, whereas A. barretti is morphologically distinct. Of the 2 PCR-based assays reported here, the first provides a rapid, sensitive, and straight-forward means of general ascogregarine detection based on a single PCR amplification. The second method provides a means of differentiation between A. culicis and A. taiwanensis based on a species-specific PCR assay. Together, these assays allow whole mosquitoes to be tested for the presence of Ascogregarina species as well as identification of both A. culicis and A. taiwanensis singly or in dual infections.     

Eclosion rate, development and survivorship of Aedes albopictus (Skuse)(Diptera: Culicidae) under different water temperatures

In tropical areas, where vector insects populations are particularly numerous, temperature usually range between 25 degrees C and 35 degrees C. Considering the importance of such temperature variation in determining mosquitoes population dynamics, in this work the developmental, eclosion and survival rates of the immature stages of Aedes albopictus (Skuse) were compared under constant 25, 30 and 35 degrees C (using acclimatized chambers) and environmental (25 degrees C to 29 degrees C) temperatures. The hatching rate was considered as total number of larvae recovered after 24h. The development period as well as larval and pupal survival rate were evaluated daily. Eclosion rate was significantly higher under environmental temperature than under the studied constant temperatures, suggesting that temperature variation may be an eclosion-stimulating factor. The mean eclosion time increased with the temperature, ranging from 2.8h (25 degrees C) to 5.2h (35 degrees C). The larval period was greatly variable inside each group, although it did not differ significantly amongst groups (11.0 +/- 4.19 days), with individuals showing longer larval stages in water at 35 degrees C (12.0 +/- 4.95 days) and environmental temperature (13.6 +/- 5.98 days). Oppositely, survival was strongly affected by the higher temperature, where only one individual lived through to adult phase. The results suggest that population of Ae. albopictus from Recife may be adapting to increasing of environmental temperatures and that the limiting temperature to larval development is around 35 degrees C.     

Differentiation of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) with egg surface morphology and morphometrics using scanning electron microscopy

Aedes aegypti and Aedes albopictus are potential arboviral vectors leading to high human fatality worldwide. Efforts in the present study were made to differentiate the eggs of A. aegypti and A. albopictus morphologically and morphometrically using scanning electron microscopy (SEM). Morphometrically, these species’ eggs were 48.48% significantly different of the 33 attributes including egg dimensions, micropylar apparatus, dimensions and density of outer chorionic cells (OCCs), tubercles and width of exochorionic network. In comparison to A. aegypti eggs, A. albopictus eggs were significantly smaller and more tapered at the posterior end; however, the micropylar disc of A. aegypti was wider and had incomplete circular sectors whereas it was a narrower polygon without sectors in A. albopictus. These species were also significantly different with regards to OCC which enclose both large central and small peripheral tubercles. Specifically, the exochorionic networks in A. aegypti were interwoven, reticulated and extensively wide whereas they were narrow, prominent and solid-wall-like in A. albopictus. This feature may strengthen A. albopictus eggs against desiccation, when they are laid in containers. The morphometrical and morphological analysis of the egg’s attributes of A. aegypti and A. albopictus may be helpful in understanding egg biology as well as in species confirmation.     

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.     

Reappearance of Aedes aegypti (Diptera: Culicidae) in Lima, Peru

We report here the reappearance of Aedes aegypti in the Rimac district, and summarize the history of this mosquito species in Peru since its first detection in 1852. On March 17 2000 were found Ae. aegypti and Culex quinquefasciatus in Mariscal Castilla town, Flor de Amancaes, San Juan de Amancaes, El Altillo and Santa Rosa in the Rimac district, Lima Province.