Molecular Phylogeny and Historical Biogeography of the Butterfly Tribe Aeromachini Tutt (Lepidoptera: Hesperiidae) from China.

: The butterfly tribe Aeromachini Tutt, 1906 is a large group of skippers. In this study, a total of 10 genera and 45 species of putative members of this tribe, which represent most of the generic diversity and nearly all the species diversity of the group in China, were sequenced for two mitochondrial genes and three nuclear genes (2093 bp). The combined dataset was analyzed with maximum likelihood inference using IQtree. We found strong support for monophyly of Aeromachini from China and support for the most recent accepted species in the tribe. Two paraphyletic genera within Aeromachini are presented and discussed. The divergence time estimates with BEAST and ancestral-area reconstructions with RASP provide a detailed description about the historical biogeography of the Aeromachini from China. The tribe very likely originated from the Hengduan Mountains in the late Ecocene and expanded to the Himalaya Mountains and Central China Regions. A dispersal-vicariance analysis suggests that dispersal events have played essential roles in the distribution of extant species, and geological and climatic changes have been important factors driving current distribution patterns.

Influence of Wolbachia infection on mitochondrial DNA variation in the genus Polytremis (Lepidoptera: Hesperiidae).

The maternally inherited obligate bacteria Wolbachia is known for infecting the reproductive tissues of a wide range of arthropods and can contribute to phylogenetically discordant patterns between mtDNA and nDNA. In this study, we tested for an association between mito-nuclear discordance in Polytremis and Wolbachia infection. Six of the 17 species of Polytremis were found to be infected with Wolbachia. Overall, 34% (70/204) of Polytremis specimens were Wolbachia positive and three strains of Wolbachia identified using a wsp marker were further characterized as six strains based on MLST markers. Wolbachia acquisition in Polytremis appears to occur mainly through horizontal transmission rather than codivergence based on comparison of the divergence times of Wolbachia and Polytremis species. At the intraspecific level, one of the Wolbachia infections (wNas1) is associated with reduced mtDNA polymorphism in the infected Polytremis population. At the interspecific level, there is one case of mito-nuclear discordance likely caused by introgression of P. fukia mtDNA into P. nascens driven by another Wolbachia strain (wNas3). Based on an absence of infected males, we suspect that one Wolbachia strain (wNas2) affects sex ratio, but the phenotypic effects of the other strains are unclear. These data reveal a dynamic interaction between Polytremis and Wolbachia endosymbionts affecting patterns of mtDNA variation.

Costs of Three Wolbachia Infections on the Survival of Aedes aegypti Larvae under Starvation Conditions.

The mosquito Aedes aegypti, the principal vector of dengue virus, has recently been infected experimentally with Wolbachia: intracellular bacteria that possess potential as dengue biological control agents. Wolbachia depend on their hosts for nutrients they are unable to synthesize themselves. Consequently, competition between Wolbachia and their host for resources could reduce host fitness under the competitive conditions commonly experienced by larvae of Ae. aegypti in the field, hampering the invasion of Wolbachia into natural mosquito populations. We assess the survival and development of Ae. aegypti larvae under starvation conditions when infected with each of three experimentally-generated Wolbachia strains: wMel, wMelPop and wAlbB, and compare their fitness to wild-type uninfected larvae. We find that all three Wolbachia infections reduce the survival of larvae relative to those that are uninfected, and the severity of the effect is concordant with previously characterized fitness costs to other life stages. We also investigate the ability of larvae to recover from extended food deprivation and find no effect of Wolbachia on this trait. Aedes aegypti larvae of all infection types were able to resume their development after one month of no food, pupate rapidly, emerge at a large size, and exhibit complete cytoplasmic incompatibility and maternal transmission. A lowered ability of Wolbachia-infected larvae to survive under starvation conditions will increase the threshold infection frequency required for Wolbachia to establish in highly competitive natural Ae. aegypti populations and will also reduce the speed of invasion. This study also provides insights into survival strategies of larvae when developing in stressful environments.

Genetic structure of the whitefly Bemisia tabaci populations in Colombia following a recent invasion.

The whitefly Bemisia tabaci (Gennadius) is one of the most important pests causing economic losses in a variety of cropping systems around the world. This species was recently found in a coastal region of Colombia and has now spread inland. To investigate this invasive process, the genetic structure of B. tabaci was examined in 8 sampling locations from 2 infested regions (coastal, inland) using 9 microsatellite markers and the mitochondrial COI gene. The mitochondrial analysis indicated that only the invasive species of the B. tabaci complex Middle East-Asia Minor 1 (MEAM 1 known previously as biotype B) was present. The microsatellite data pointed to genetic differences among the regions and no isolation by distance within regions. The coastal region in the Caribbean appears to have been the initial point of invasion, while the inland region in the Southwest showed genetic variation among populations most likely reflecting founder events and ongoing changes associated with climatic and topographical heterogeneity. These findings have implications for tracking and managing B. tabaci.

Using Wolbachia-based release for suppression of Aedes mosquitoes: insights from genetic data and population simulations.

A novel strategy for suppressing disease transmission by Aedes aegypti, the main vector of dengue, uses releases of mosquitoes infected with the bacterium Wolbachia pipientis. Wolbachia are currently released to interfere with viral transmission, but there is also potential to use strains in mosquito suppression and elimination programs via the deleterious effects of the bacterium on the host. Mosquito suppression depends on target areas being relatively isolated to prevent reinvasion and on local climatic conditions. Here we explored the opportunity for suppression of A. aegypti in central Queensland, Australia, by using microsatellite data and simulations based on CIMSiM models of local weather conditions and breeding container data. Our results indicate that Wolbachia-induced extinctions in central Queensland are possible, although they may eventually be compromised by ongoing mosquito migration between towns until these sources are also suppressed. The results highlight a novel use of deleterious Wolbachia infections to achieve ecological as well as disease-related endpoints.

Larval competition extends developmental time and decreases adult size of wMelPop Wolbachia-infected Aedes aegypti.

The intracellular endosymbiont Wolbachia has been artificially transinfected into the dengue vector Aedes aegypti, where it is being investigated as a potential dengue biological control agent. Invasion of Wolbachia in natural populations depends upon the fitness of Wolbachia-infected Ae. aegypti relative to uninfected competitors. Although Wolbachia infections impose fitness costs on the adult host, effects at the immature stages are less clear, particularly in competitive situations. We look for effects of two Wolbachia infections, wMel and wMelPop, on intra-strain and inter-strain larval competition in Ae. aegypti. Development of Wolbachia-infected larvae is delayed in mixed cohorts with uninfected larvae under crowded-rearing conditions. Slow developing wMelPop-infected larvae have reduced adult size compared with uninfected larvae, and larvae with the wMel infection are somewhat larger and have greater viability relative to uninfected larvae when in mixed cohorts. Implications for successful invasion by these Wolbachia infections under field conditions are considered.

Assessing quality of life-shortening Wolbachia-infected Aedes aegypti mosquitoes in the field based on capture rates and morphometric assessments.

BACKGROUND: Recent releases have been carried out with Aedes aegypti mosquitoes infected with the wMelPop mosquito cell-line adapted (wMelPop-CLA) strain of Wolbachia. This infection introduced from Drosophila provides strong blockage of dengue and other arboviruses but also has large fitness costs in laboratory tests. The releases were used to evaluate the fitness of released infected mosquitoes, and (following termination of releases) to test for any effects of wMelPop-CLA on wing size and shape when mosquitoes were reared under field conditions. METHODS: We monitored gravid females via double sticky traps to assess the reproductive success of wMelPop-CLA-infected females and also sampled the overall mosquito population post-release using Biogent Sentinel traps. Morphometric analyses were used to evaluate infection effects on wing shape as well as size. RESULTS: Oviposition success as assessed through double sticky traps was unrelated to size of released mosquitoes. However, released mosquitoes with lower wing loading were more successful. Furthermore, wMelPop-CLA-infected mosquitoes had 38.3% of the oviposition success of uninfected mosquitoes based on the predicted infection frequency after release. Environmental conditions affected wing shape and particularly size across time in uninfected mosquitoes, but not in naturally-reared wMelPop-CLA-infected mosquitoes. Although the overall size and shape do not differ between naturally-reared wMelPop-CLA-infected and uninfected mosquitoes, the infected mosquitoes tended to have smaller wings than uninfected mosquitoes during the cooler November in comparison to December. CONCLUSION: These results confirm the lower fitness of wMelPop-CLA infection under field conditions, helping to explain challenges associated with a successful invasion by this strain. In the long run, invasion may depend on releasing strains carrying insecticide resistance or egg desiccation resistance, combined with an active pre-release population suppression program.

Body size and wing shape measurements as quality indicators of Aedes aegypti mosquitoes destined for field release.

There is increasing interest in rearing modified mosquitoes for mass release to control vector-borne diseases, particularly Wolbachia-infected Aedes aegypti for suppression of dengue. Successful introductions require release of high quality mosquitoes into natural populations. Potential indicators of quality are body size and shape. We tested to determine if size, wing/thorax ratio, and wing shape are associated with field fitness of Wolbachia-infected Ae. aegypti. Compared with field-collected mosquitoes, released mosquitoes were larger in size, with lower size variance and different wing shape but similar in wing-thorax ratio and its associated variance. These differences were largely attributed to nutrition and to a minor extent to wMel Wolbachia infection. Survival potential of released female mosquitoes was similar to those from the field. Females at oviposition sites tended to be larger than those randomly collected from BG-Sentinel traps. Rearing conditions should thus aim for large size without affecting wing/thorax ratios.

Evidence of cryptic genetic lineages within Aedes notoscriptus (Skuse).

Aedes notoscriptus (Skuse), a mosquito from the southwest Pacific region including Australia, has been implicated as a vector of arboviruses, but its status as a species is unclear. To investigate the taxonomic situation, we assessed genetic variation and phylogenetic relationships among Ae. notoscriptus from the east coast of Australia, Western Australia and New Zealand. Phylogenetic analyses of DNA sequence data from mitochondrial markers indicate that Ae. notoscriptus is a complex of divergent genetic lineages, some of which appear geographically restricted, while others are widespread in eastern Australia. Samples from New Zealand and Western Australia were related to populations from one southern Australian lineage. Nuclear markers show no evidence of genetic isolation by geographic distance in the overall sample of mosquitoes, but strong isolation by distance is obvious within two of the lineages, supporting their status as isolated gene pools. The morphological character of wing centroid size variation is also associated with genetic lineage. These findings point to the possibility that Ae. notoscriptus is a complex of species, highlighting the need to understand physiological and ecological differences that may influence future control strategies.

Population genetic structure of Aedes (Stegomyia) aegypti (L.) at a micro-spatial scale in Thailand: implications for a dengue suppression strategy.

BACKGROUND: The genetic population structure of Aedes (Stegomyia) aegypti (L.), the main vector of dengue virus, is being investigated in areas where a novel dengue suppression program is to be implemented. The aim of the program is to release and establish mosquito populations with impaired virus transmission capabilities. To model effects of the release and devise protocols for its implementation, information about the genetic structure of populations at a range of spatial scales is required. METHODOLOGY/PRINCIPAL FINDINGS: This study investigates a potential release site in the Hua Sam Rong Subdistrict of Plaeng Yao District, Chachoengsao Province, in eastern Thailand which comprises a complex of five villages within a 10 km radius. Aedes aegypti resting indoors was sampled at four different times of year from houses within the five villages. Genetic markers were used to screen the mosquitoes: two Exon Primed Intron Crossing (EPIC) markers and five microsatellite markers. The raw allele size was determined using several statistical software packages to analyze the population structure of the mosquito. Estimates of effective population size for each village were low, but there was no evidence of genetic isolation by geographic distance. CONCLUSIONS: The presence of temporary genetic structure is possibly caused by genetic drift due to large contributions of adults from a few breeding containers. This suggests that the introduction of mosquitoes into an area needs to proceed through multiple releases and targeting of sites where mosquitoes are emerging in large numbers.

High-throughput PCR assays to monitor Wolbachia infection in the dengue mosquito (Aedes aegypti) and Drosophila simulans.

We have developed and validated two new fluorescence-based PCR assays to detect the Wolbachia wMel strain in Aedes aegypti and the wRi and wAu strains in Drosophila simulans. The new assays are accurate, informative, and cost-efficient for large-scale Wolbachia screening.