Investigation of the aedes (Stegomyia) flavopictus complex (Diptera: Culicidae) in Japan by sequence analysis of the internal transcribed spacers of ribosomal DNA

Aedes (Stegomyia) flavopictus Yamada is widely distributed in Japan and Korea. The species comprises three subspecies based on current morphological taxonomy: Aedes flavopictus in the Palearctic region of Japan, Ae. f downsi Bohart & Ingram from Amami and Okinawa Islands, and Ae. f miyarai Tanaka, Mizusawa & Ingram from Ishigaki and Iriomote Islands of the Ryukyu Archipelago. These subspecies designations are based on observations of a combination of several morphological characters, none of which, by itself is diagnostic for discriminating among the three subspecies. To further study the relationships in this group, we examined the nucleic acid sequence divergence in the internal transcribed spacer regions (ITSI and ITS2) of the ribosomal DNA gene array of Ae. flavopictus individuals collected at five sites from three geographic regions in Japan. Analysis of sequence data by distance and maximum parsimony methods produced phylogenetic trees that showed separation of the specimens into three major clades, corresponding to both subspecies and geographic region. These results were consistent with and support the current classification and geographic distribution of the three subspecies.     

Differential survival of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) larvae exposed to low temperatures in Taiwan

Aedes aegypti (L.) and Aedes albopictus (Skuse) differ in their distribution in Taiwan. The former species is distributed in the south of Taiwan, whereas the latter is found throughout the island. One possible explanation proposes that low temperatures in the winter prevent the expansion of Ae. aegypti. Hence, the impact of low temperatures on immatures of both species was studied in the laboratory and in the field. Our study showed that, under most conditions, Ae. aegypti were more sensitive to low temperatures than Ae. albopictus both in the laboratory and in the field. The survival rates at 10 degrees C for the first and fourth instars of Ae. albopictus were significantly better than those of Ae. aegypti. At 2.5 and 5.0 degrees C, the first instars of Ae. albopictus survived better than the same stadium of Ae. aegypti, but the fourth instars of Ae. aegypti survived better. Short exposures to low temperatures did not affect the acclimatization of Ae. aegypti immatures but longer exposures did increase the physiological adaptation to low temperatures. For Ae. albopictus, exposure to low temperatures increases the acclimatization of this species. In field experiments, Ae. aegypti larvae had a significantly higher mortality than Ae. albopictus during exposures to cold fronts in the 2004 winter. We conclude that low temperatures in northern and central Taiwan have a negative impact on the distribution of Ae. aegypti, but this factor alone is not sufficient to prevent this species from occupying the rest of Taiwan.     

Interspecific competition among Aedes aegypti, Ae. albopictus, and Ae. triseriatus (Diptera: Culicidae): larval development in mixed cultures

Interactions among the larvae of Aedes aegypti (L.), Ae. albopictus (Skuse), and Ae. triseriatus (Say) were studied in trispecific and bispecific mixed populations under laboratory and field conditions. Competitive stress (as evidenced by the average time to first, 50, and 75% pupation and the total pupation periods for mixed populations of each species in comparison with their single species controls) was more pronounced in mixed cultures reared in glass jars in the laboratory than in tires under field conditions. In the laboratory, the larval development of Ae. aegypti reared together with Ae. albopictus or Ae. triseriatus, or both, larvae was accelerated significantly. Conversely, the time to pupation for Ae. albopictus and Ae. triseriatus was delayed when reared with Ae. aegypti. However, the average wing length of female Ae. albopictus and Ae. triseriatus was greater in the mixed cultures than in single species cultures. These data indicated that the effect of intraspecific competition was greater than interspecific competition. Adequate food and higher temperature appeared to promote rapid development and higher survival of the immature stages of the three Aedes species in tires placed in the field. In general, Ae. triseriatus larvae required a longer period for larval development and had greater larval mortality than either Ae. aegypti or Ae. albopictus. In mixed populations of Ae, albopictus and Ae. aegypti in the jars and food-rich tires, the periods needed to attain first, 50, and 75% cumulative pupation were not significantly different than in single species controls. We conclude that no clear-cut displacement occurred in mixed experimental populations of Ae. aegypti and Ae. albopictus.     

Reproductive and metabolic differences between Aedes aegypti and Ae. albopictus (Diptera: Culicidae).

Aedes albopictus (Skuse) females were more likely to develop eggs after they ingested small blood meals than were Aedes aegypti (L.) when both species were maintained under the same suboptimal adult nutritional regimen. The longevity of adult female Ae. albopictus under conditions of starvation was also significantly greater than that of Ae. aegypti. Analyses of total body proteins, lipids, and glycogen indicated that the increased reproductive efficiency of Ae. albopictus may be a result of its greater reserves accumulated during a longer larval period.     

Ribosomal DNA structure in Aedes mosquitoes (Diptera: Culicidae) and their cell lines

The molecular organization of the ribosomal DNA repeat unit in laboratory colonies of Aedes albopictus (Skuse) and Aedes aegypti (L.) and in continuous cell lines derived from these species was examined using restriction enzyme mapping and Southern blotting techniques. In the rRNA coding regions, restriction enzyme sites were conserved in Ae. aegypti and Ae. albopictus. In particular, sites that had been mapped in rDNA cloned from the Mauritius strain of Ae. albopictus mosquitoes were confirmed in cloned DNA from cultured Ae. alboptictus cells. The intergenic spacer region of the ribosomal DNA repeat unit in Ae. albopictus was longer than that in Ae. aegypti; contained multiple recognition sites for AluI, SstI, PvuI, and XhoI; and was more heterogeneous in DNA from cultured cells than in DNA from inbred laboratory mosquitoes. The rDNA copy number was 3- to 4-fold lower in cell lines from both Ae. albopictus and Ae. aegypti than in the corresponding adult mosquitoes.     

Susceptibility of selected strains of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) to chikungunya virus.

The relative susceptibility of selected strains of Aedes aegypti (L.) and Aedes albopictus (Skuse) fed on a viremic monkey to infection with chikungunya virus was determined. Infection rates were consistently higher in 10 strains of Ae. albopictus tested than in 7 strains of Ae. aegypti tested, regardless of the geographic location from which the strains originated or the dose of virus ingested. Similarly, virus dissemination rates were higher in the Ae. albopictus strains compared with the Ae. aegypti strains. For nearly all (11 of 12) strains tested of both species, groups of mosquitoes with one or more females with a disseminated infection transmitted virus by bite to weanling mice. Based on these studies, Ae. albopictus appears to be a more competent laboratory vector of chikungunya virus than does Ae. aegypti.     

Detritus type alters the outcome of interspecific competition between Aedes aegypti and Aedes albopictus (Diptera: Culicidae)

Many studies of interspecific competition between Aedes albopictus (Skuse) and Aedes aegypti (L.) (Diptera: Culicidae) larvae show that Ae. albopictus are superior resource competitors to Ae. aegypti. Single-species studies indicate that growth and survival of Ae. albopictus and Ae. aegypti larvae are affected by the type of detritus present in containers, which presumably affects the amount and quality of microorganisms that the mosquito larvae consume. We tested whether different detritus types alter the intensity of larval competition by raising 10 different density/species combinations of Ae. albopictus and Ae. aegypti larvae under standard laboratory conditions, with one of four detritus types (oak, pine, grass, or insect) provided as a nutrient base. Intraspecific competitive effects on survival were present with all detritus types. Ae. albopictus survivorship was unaffected by interspecific competition in all treatments. Negative interspecific effects on Ae. aegypti survivorship were present with three of four detritus types, but absent with grass. Estimated finite rate of increase (lambda') was lower with pine detritus than with any other detritus type for both species. Furthermore, Ae. aegypti lambda' was negatively affected by high interspecific density in all detritus types except grass. Thus, our experiment confirms competitive asymmetry in favor of Ae. albopictus with oak, pine, or insect detritus, but also demonstrates that certain detritus types may eliminate interspecific competition among the larvae of these species, which may allow for stable coexistence. Such variation in competitive outcome with detritus type may help to account for observed patterns of coexistence/exclusion of Ae. albopictus and Ae. aegypti in the field.     

Comparative potential of Aedes triseriatus, Aedes albopictus, and Aedes aegypti (Diptera: Culicidae) to transovarially transmit La Crosse virus

Aedes triseriatus (Say) (Diptera: Culicidae), the major vector of La Crosse (LAC) virus, efficiently transmits LAC virus both horizontally and transovarially. We compared the vector competence and transovarial transmission ability of Ae. triseriatus, Aedes albopictus Skuse, and Aedes aegypti (L.) for LAC virus. Ae. triseriatus and Ae. albopictus were significantly more susceptible to oral infection with LAC virus than Ae. aegypti. The three species also differed in oral and disseminated infection rates (DIRs). Transovarial transmission (TOT) rates and filial infection rates (FIRs) were greater for Ae. triseriatus than either Ae. albopictus or Ae. aegypti. These measures were integrated into a single numerical score, the transmission amplification potential (TAP) for each species. Differences in TAP scores were due mainly to the differences in DIRs and FIRs among these mosquitoes. Although the TAP score for Ae. albopictus was lower than that of Ae. triseriatus, it was 10-fold greater than that for Ae. aegypti.     

Intraguild predation among larval treehole mosquitoes, Aedes albopictus, Ae. aegypti, and Ae. triseriatus (Diptera: Culicidae), in laboratory microcosms.

We compared the tendency for 4th-instar larvae to prey on newly hatched larvae, and the vulnerability of those 1st instars to such predation for Aedes triseriatus (Say), Ae. aegypti (L.), and Ae. albopictus (Skuse), all container-breeding mosquitoes. The latter 2 species were introduced to North America and are now sympatric with Ae. triseriatus, a native species in eastern North America. The experiment also enabled the assessment of species-specific influences of food supplements and spatial heterogeneity on predatory behavior. Ae. triseriatus was substantially more predatory and less susceptible to attack than the other 2 species. These differences were amplified in food-deprived and spatially simple conditions, indicating that Ae. triseriatus predatory behavior may have important retarding effects on the colonization of occupied treehole habitats by Ae. albopictus. Ae. aegypti and Ae. albopictus were similar in imposing little (Ae. aegypti) or almost no (Ae. albopictus) predation on 1st instars and in being susceptible to predation by Ae. triseriatus. The general lack of species-specific differences between Ae. aegypti and Ae. albopictus indicates that interspecific predation is not a likely explanation for the rapid displacement of Ae. aegypti by Ae. albopictus in domestic containers in the southeastern United States.     

Movement of dengue vectors between the human modified environment and an urban forest in Rio de Janeiro

The movement of Aedes aegypti (L.) and Aedes albopictus (Skuse) females between sylvatic and urban environments was investigated by marking, releasing, and recapturing adults and by identifying rubidium (Rb)-marked eggs of females that were released after taking a bloodmeal containing RbCI. When released in the forest, Ae. albopictus females flew as far as 1000 m and reached houses within 1 wk. When Ae. albopictus were released close to houses, most females were recaptured near the release point, and Rb-marked eggs were found 1000 m away in the forest only once, 35 d after the release. These differing patterns of movement may suggest a preference of Ae. albopictus for the human-modified environment. Ae. aegypti, however, showed low tendency to disperse into the forest. The capacity of Ae. albopictus females to disperse from a sylvatic into a human-modified environment suggests that this species may play a role in the dissemination of forest-restricted pathogens, such as yellow fever virus.     

Differences in energy metabolism and adult desiccation resistance among three Aedes (Stegomyia) species (Diptera: Culicidae) from South Sulawesi, Indonesia

To determine the mechanisms of adult desiccation resistance of Aedes (Stegomyia) species, we estimated the carbohydrate and lipid contents of newly emerged adult mosquitoes from South Sulawesi, Indonesia. Inter- and intraspecific differences in glycogen and free fatty acid accumulation were found in Aedes aegypti (L.), Ae. albopictus (Skuse), and Ae. paullusi (Stone & Farmer). High glycogen content was determined for both sexes in all Ae. aegypti strains and in the Ujung Pandang strain of Ae. albopictus. There were no differences among species in either trehalose or sorbitol content. A trend similar to glycogen was seen for lipid content, especially the free fatty acids of neutral lipids. These energy reserves of adult mosquitoes correlated with adult desiccation survival time at 90% RH calculated by Mogi et al. (1996). In both sexes, survival time was longer in strains with high glycogen and free fatty acid content, and low trehalose content. Ae aegypti, Ae. albopictus, and Ae. paullusi are species with high, intermediate, and low levels of glycogen and free fatty acids, respectively. However, longevity of these species under desiccation conditions is associated strongly with glycogen-trehalose conversion, and free fatty acid accumulation.     

Overwintering survival of Aedes albopictus (Diptera: Culicidae) eggs in Indiana

Eggs of 12 strains of Aedes albopictus (Skuse), two strains of A. aegypti L., and one strain of A. triseriatus (Say) were tested for ability to overwinter at three outdoor locations in Indiana. Some survival of A. albopictus eggs was observed during the winters of 1986-1987 and 1987-1988. A. triseriatus had greater overwintering ability than A. albopictus; A. aegypti did not survive at all locations. Prolonged cold-conditioning and photoperiodically induced diapause increased the overwintering ability of A. albopictus eggs. Strains of A. albopictus from northern Asia and North America showed higher overwintering survival rates than A. albopictus strains from tropical Asia, Hawaii, and Brazil; strains from Indiana had greater ability to overwinter than those from Texas, Louisiana, and Florida. Results are discussed in relation to the overwintering limits of this species in the United States.     

A polymerase chain reaction-based diagnostic to identify larvae and eggs of container mosquito species from the Australian region

Dengue outbreaks occur regularly in parts of northern Queensland, Australia, and there is concern that these outbreaks may spread with the introduction and range expansion of the two main vectors Aedes aegypti (L.) and Aedes albopictus (Skuse). Problems encountered in separating larvae of endemic and exotic container mosquito species resulted in the development of a polymerase chain reaction diagnostic procedure that uses a restriction enzyme to cut the internal transcribed spacer region 1 of the ribosomal DNA to separate Ae. aegypti and Ae. albopictus from a number of common local container mosquito species which can be used at any stage of the life cycle, including eggs up to 8 wk of age. Identification was possible using desiccated or alcohol-preserved specimens with a response time of < 24 h after receipt of the specimens.     

Differences in male mating response and female flight sounds in Aedes aegypti and Ae. albopictus (Diptera: Culicidae).

Aedes aegypti (L.) and Ae. albopictus (Skuse) showed similar rates and timing of insemination in the laboratory. Laboratory attempts at interspecific mating were unsuccessful. Because Ae. aegypti males are known to locate females by flight sounds, male response to sound was compared in the two species. Ae. aegypti males responded to female flight sounds with stereotypical orientation and mating behavior, whereas Ae. albopictus males seldom responded. Recorded flight sounds of females were sampled via computer digitization and compared. Ae. aegypti females produced louder sounds with more harmonics than Ae. albopictus. Males were tested for their ability to discriminate between the sounds of the two species. Ae. albopictus males did not respond to recordings of either Ae. albopictus or Ae. aegypti females. Ae. aegypti males responded preferentially to the recorded sounds of Ae. aegypti females. Thus, males of the two species use different mechanisms in locating mates. Ae. aegypti males rely more on sound than do Ae. albopictus males.     

Laboratory study of competition between United States strains of Aedes albopictus and Aedes aegypti (Diptera: Culicidae)

Mosquito abatement workers in Houston, Tex., and New Orleans, La., have observed that the recent introduction of Aedes albopictus (Skuse) has been accompanied by a decline and virtual disappearance of Ae. aegypti (L.). This suggests competitive displacement but contradicts the direction of displacement observed in native habitats of Ae. albopictus. The paper reports a series of laboratory studies on competition between U.S. strains of the two species. Larval survivorship, size, and development time were monitored in increasing densities of pure and mixed cultures of the two species under limited and optimal diets. Oviposition preferences were examined by offering gravid females cups containing first or fourth instars of either species. Ethological isolation was studied by simultaneously offering virgin females of one species to males of both species and monitoring courtship behavior as well as rates of oviposition, percentage of hatch, and embryonation. The results of the larval competition studies suggest that interspecific competition increased development time only slightly and had no effect on survival of Ae. albopictus. Competition increased survival in Ae. aegypti. Size of both species was influenced by interspecific competition, but the magnitude and direction of the effect was inconsistent between strains and diets such that neither species emerged consistently larger. The results of the oviposition study indicated a higher fecundity in Ae. aegypti but otherwise demonstrated that both species oviposit without regard to the presence, age, or species of larvae in the oviposition container. The ethological isolation tests demonstrated that Ae. aegypti males were more sexually aggressive than males or Ae. albopictus. However, Ae. albopitus females placed with increasing densities of Ae. aegypti males demonstrated no decrease in rates of oviposition, and eggs laid showed equal rates of hatch and embryonation. In general, the results do not suggest that the U.S Ae. albopictus population is inherently more competitive in the laboratory than Ae. aegypti. Other reasons for the observed decline in Ae. aegypti in the United States are discussed.     

Molecular and morphological characterization of Aedes albopictus in northwestern Greece and differentiation from Aedes cretinus and Aedes aegypti

The presence of Aedes albopictus (Skuse) was recently confirmed for the first time in northwestern Greece. This location is within the distribution range of a morphologically similar species, Aedes cretinus Edwards, and is a potentially favorable region for the reintroduction of Aedes aegypti (L.). It was thus compelling to use methods in addition to morphology-based keys to correctly identify specimens badly damaged, rubbed, or otherwise altered in their external characteristics. It was decided to use molecular techniques as a novel and reliable method for differentiating the three Stegomyia species. The nuclear internal transcribed spacer 2 (ITS2) fragments from morphologically identified Ae. albopictus and Ae. cretinus specimens were amplified, and their sequences were compared with those in GenBank for Ae. albopictus, Ae. cretinus, and Ae. aegypti. Also, mitochondrial cytochrome oxidase I (COI) fragments were amplified for Ae. albopictus and Ae. cretinus (so far not available in GenBank) and compared with Ae. aegypti fragments. ITS2 and COI sequences generated in our study were deposited in GenBank and could be useful in future studies of mosquitoes by other research workers.     

Geographic distribution and breeding site preference of Aedes albopictus and Aedes aegypti (Diptera: culicidae) in Cameroon, Central Africa

Presence in Cameroon of the recently introduced Aedes (Stegomyia) albopictus (Skuse) in association with the indigenous Aedes aegypti (L.) raises public heath concerns because it might alter the risk of arbovirus transmission. The breeding site and distribution of the two Stegomyia species are updated and reported following entomological surveys carried out in 22 localities throughout Cameroon, with a total of 1,353 containers with water visited. Ae. aegypti was found in every location sampled, showing higher infestation rates in northern Cameroon. Breeding populations of Ae. albopictus were observed in all 19 southern localities, up to the Adamaoua mountains, but the species was not recorded further north. In the area where both species are present, they were often sampled in the same larval developmental sites, suggesting convergent habitat segregation. The most frequently encountered artificial and natural breeding sites were used tires, discarded tins and plastic containers, abandoned car parts, brick holes, dead leaves on the ground, tree holes, and rock pools. Further monitoring of the demographic as well as geographic expansion of Ae. albopictus in this Afrotropical environment and its relationships with indigenous Ae. aegypti should provide insight into the biology of this highly invasive species and help to implement arboviruses surveillance programs in the area.     

Vector competence of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) for dengue virus in the Florida Keys

In 2009-2011, Monroe County in southern Florida experienced locally acquired and traveler-imported focal dengue outbreaks. Aedes aegypti (L.) is the primary vector of dengue virus (DENV) worldwide, is prevalent in Monroe County, and is the suspected vector in Florida. Ae. albopictus (Skuse) is also known to be an important vector of DENV and this species is ubiquitous in Florida; however, it is not yet established in Monroe County. Florida Ae. aegypti (Key West and Stock Island geographic colonies) and Ae. albopictus (Vero Beach geographic colony) were fed blood containing 3.7 Log10 plaque-forming unit equivalents of DENV serotype 1 isolated from a patient involved in the Key West, FL, outbreak in 2010. Mosquitoes were maintained at extrinsic incubation temperatures of 28 or 30 degrees C for an incubation period of 14 d. Vector competence was assessed using rates of infection (percent with virus-positive bodies), dissemination (percent infected with virus-positive legs), and transmission (percent infected with virus-positive saliva). No significant differences were observed in rates of infection or dissemination between Ae. aegypti or Ae. albopictus at either extrinsic incubation temperature. Transmission was observed only at 28 degrees C in both Ae. aegypti (Key West) and Ae. albopictus. The assessment of local mosquito populations for their DENV vector competence is essential and will aid mosquito control operators interested in pinpointing specific vector populations for control. The extent to which vector competence is affected by seasonal changes in temperature is discussed and provides baseline risk assessment data to mosquito control agencies.