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BackgroundThe arboviral vector Aedes albopictus became established on all continents except Antarctica in the past 50 years. A consequence of its rapid global invasion is the transmission of diseases previously confined to the tropics and subtropics occurring in temperate regions of the world, including the re-emergence of chikungunya and dengue in Europe. Application of pyrethroids is among the most widely-used interventions for vector control, especially in the presence of an arboviral outbreak. Studies are emerging that reveal phenotypic resistance and monitor mutations at the target site, the para sodium channel gene, primarily on a local scale.MethodsA total of 512 Ae. albopictus mosquitoes from twelve geographic sites, including those from the native home range and invaded areas, were sampled between 2011 and 2018, and were analyzed at five codons of the para sodium channel gene with mutations predictive of resistance phenotype. Additionally, to test for the origin of unique kdr mutations in Mexico, we analyzed the genetic connectivity of southern Mexico mosquitoes with mosquitoes from home range, the Reunion Island, America and Europe.ResultsWe detected mutations at all tested positions of the para sodium channel gene, with heterozygotes predominating and rare instance of double mutants. We observed an increase in the distribution and frequency of F1534C/L/S mutations in the ancestral China population and populations in the Mediterranean Greece, the appearance of the V1016G/I mutations as early as 2011 in Italy and mutations at position 410 and 989 in Mexico. The analyses of the distribution pattern of kdr alleles and haplotype network analyses showed evidence for multiple origins of all kdr mutations.ConclusionsHere we provide the most-up-to-date survey on the geographic and temporal distribution of pyrethroid-predictive mutations in Ae. albopictus by combining kdr genotyping on current and historical samples with published data. While we confirm low levels of pyrethroid resistance in most analyzed samples, we find increasing frequencies of F1534C/S and V1016G in China and Greece or Italy, respectively. The observed patterns of kdr allele distribution support the hypothesis that on site emergence of resistance has contributed more than spread of resistance through mosquito migration/invasions to the current widespread of kdr alleles, emphasizing the importance of local surveillance programs and resistance management.

Tsetse flies (genus Glossina), the sole vectors of African trypanosomiasis, are distinct from most other insects, due to dramatic morphological and physiological adaptations required to support their unique biology. These adaptations are driven by demands associated with obligate hematophagy and viviparous reproduction. Obligate viviparity entails intrauterine larval development and the provision of maternal nutrients for the developing larvae. The reduced reproductive capacity/rate associated with this biology results in increased inter- and intra-sexual competition. Here, we use phase contrast microcomputed tomography (pcMicroCT) to analyze morphological adaptations associated with viviparous biology. These include (1) modifications facilitating abdominal distention required during blood feeding and pregnancy, (2) abdominal and uterine musculature adaptations for gestation and parturition of developed larvae, (3) reduced ovarian structure and capacity, (4) structural features of the male-derived spermatophore optimizing semen/sperm delivery and inhibition of insemination by competing males and (5) structural features of the milk gland facilitating nutrient incorporation and transfer into the uterus. Three-dimensional analysis of these features provides unprecedented opportunities for examination and discovery of internal morphological features not possible with traditional microscopy techniques and provides new opportunities for comparative morphological analyses over time and between species.

Abstract Background The dramatic worldwide expansion of Aedes albopictus (the Asian tiger mosquito) and its vector competence for numerous arboviruses represent a growing threat to public health security. Molecular markers are crucially needed for tracking the rapid spread of this mosquito and to obtain a deeper knowledge of population structure. This is a fundamental requirement for the development of strict monitoring protocols and for the improvement of sustainable control measures. Methods Wild population samples from putative source areas and from newly colonised regions were analysed for variability at the ribosomal DNA internal transcribed spacer 2 (ITS2). Moreover, a new set of 23 microsatellite markers (SSR) was developed. Sixteen of these SSRs were tested in an ancestral (Thailand) and two adventive Italian populations. Results Seventy-six ITS2 sequences representing 52 unique haplotypes were identified, and AMOVA indicated that most of their variation occurred within individuals (74.36%), while only about 8% was detected among populations. Spatial analyses of molecular variance revealed that haplotype genetic similarity was not related to the geographic proximity of populations and the haplotype phylogeny clearly indicated that highly related sequences were distributed across populations from different geographical regions. The SSR markers displayed a high level of polymorphism both in the ancestral and in adventive populations, and F ST estimates suggested the absence of great differentiation. The ancestral nature of the Thai population was corroborated by its higher level of variability. Conclusions The two types of genetic markers here implemented revealed the distribution of genetic diversity within and between populations and provide clues on the dispersion dynamics of this species. It appears that the diffusion of this mosquito does not conform to a progressive expansion from the native Asian source area, but to a relatively recent and chaotic propagule distribution mediated by human activities. Under this scenario, multiple introductions and admixture events probably play an important role in maintaining the genetic diversity and in avoiding bottleneck effects. The polymorphic SSR markers here implemented will provide an important tool for reconstructing the routes of invasion followed by this mosquito.

BACKGROUND:Invasive species represent a global concern for their rapid spread and the possibility of infectious disease transmission. This is the case of the global invader Aedes albopictus, the Asian tiger mosquito. This species is a vector of medically important arboviruses, notably chikungunya (CHIKV), dengue (DENV) and Zika (ZIKV). The reconstruction of the complex colonization pattern of this mosquito has great potential for mitigating its spread and, consequently, disease risks. METHODOLOGY/PRINCIPAL FINDINGS:Classical population genetics analyses and Approximate Bayesian Computation (ABC) approaches were combined to disentangle the demographic history of Aedes albopictus populations from representative countries in the Southeast Asian native range and in the recent and more recently colonized areas. In Southeast Asia, the low differentiation and the high co-ancestry values identified among China, Thailand and Japan indicate that, in the native range, these populations maintain high genetic connectivity, revealing their ancestral common origin. China appears to be the oldest population. Outside Southeast Asia, the invasion process in La Réunion, America and the Mediterranean Basin is primarily supported by a chaotic propagule distribution, which cooperates in maintaining a relatively high genetic diversity within the adventive populations. CONCLUSIONS/SIGNIFICANCE:From our data, it appears that independent and also trans-continental introductions of Ae. albopictus may have facilitated the rapid establishment of adventive populations through admixture of unrelated genomes. As a consequence, a great amount of intra-population variability has been detected, and it is likely that this variability may extend to the genetic mechanisms controlling vector competence. Thus, in the context of the invasion process of this mosquito, it is possible that both population ancestry and admixture contribute to create the conditions for the efficient transmission of arboviruses and for outbreak establishment.

In the last 40 years, the Asian tiger mosquito Aedes albopictus, indigenous to East Asia, has colonized every continent except Antarctica. Its spread is a major public health concern, given that this species is a competent vector for numerous arboviruses, including those causing dengue, chikungunya, West Nile and the recently emerged Zika fever. To acquire more information on the ancestral source(s) of adventive populations and the overall diffusion process from its native range, we analyzed the mitogenome variation of 27 individuals from representative populations of Asia, the Americas and Europe. Phylogenetic analyses revealed five haplogroups in Asia, but population surveys appear to indicate that only three of these (A1a1, A1a2 and A1b) were involved in the recent worldwide spread. We also found out that a derived lineage (A1a1a1) within A1a1, which is now common in Italy, most likely arose in North America from an ancestral Japanese source. These different genetic sources now coexist in many of the recently colonized areas, thus probably creating novel genomic combinations which might be one of the causes of the apparently growing ability of Ae. albopictus to expand its geographical range.

Tsetse flies (Glossina spp.) are the cyclical vectors of Trypanosoma spp., which are unicellular parasites responsible for multiple diseases, including nagana in livestock and sleeping sickness in humans in Africa. Glossina species, including Glossina morsitans morsitans (Gmm), for which the Whole Genome Sequence (WGS) is now available, have established symbiotic associations with three endosymbionts: Wigglesworthia glossinidia, Sodalis glossinidius and Wolbachia pipientis (Wolbachia). The presence of Wolbachia in both natural and laboratory populations of Glossina species, including the presence of horizontal gene transfer (HGT) events in a laboratory colony of Gmm, has already been shown. We herein report on the draft genome sequence of the cytoplasmic Wolbachia endosymbiont (cytWol) associated with Gmm. By in silico and molecular and cytogenetic analysis, we discovered and validated the presence of multiple insertions of Wolbachia (chrWol) in the host Gmm genome. We identified at least two large insertions of chrWol, 527,507 and 484,123 bp in size, from Gmm WGS data. Southern hybridizations confirmed the presence of Wolbachia insertions in Gmm genome, and FISH revealed multiple insertions located on the two sex chromosomes (X and Y), as well as on the supernumerary B-chromosomes. We compare the chrWol insertions to the cytWol draft genome in an attempt to clarify the evolutionary history of the HGT events. We discuss our findings in light of the evolution of Wolbachia infections in the tsetse fly and their potential impacts on the control of tsetse populations and trypanosomiasis.

The Mediterranean fruit fly, Ceratitis capitata (medfly), is an extremely invasive agricultural pest due to its extremely wide host range and its ability to adapt to a broad range of climatic conditions and habitats. Chemosensory behaviour plays an important role in many crucial stages in the life of this insect, such as the detection of pheromone cues during mate pursuit and odorants during host plant localisation. Thus, the analysis of the chemosensory gene repertoire is an important step for the interpretation of the biology of this species and consequently its invasive potential. Moreover, these genes may represent ideal targets for the development of novel, effective control methods and pest population monitoring systems. Expressed sequence tag libraries from C. capitata adult heads, embryos, male accessory glands and testes were screened for sequences encoding putative odorant binding proteins (OBPs). A total of seventeen putative OBP transcripts were identified, corresponding to 13 Classic, three Minus-C and one Plus-C subfamily OBPs. The tissue distributions of the OBP transcripts were assessed by RT-PCR and a subset of five genes with predicted proteins sharing high sequence similarities and close phylogenetic affinities to Drosophila melanogaster pheromone binding protein related proteins (PBPRPs) were characterised in greater detail. Real Time quantitative PCR was used to assess the effects of maturation, mating and time of day on the transcript abundances of the putative PBPRP genes in the principal olfactory organs, the antennae, in males and females. The results of the present study have facilitated the annotation of OBP genes in the recently released medfly genome sequence and represent a significant contribution to the characterisation of the medfly chemosensory repertoire. The identification of these medfly OBPs/PBPRPs permitted evolutionary and functional comparisons with homologous sequences from other tephritids of the genera Bactrocera and Rhagoletis.

Sexual maturation and mating in insects are generally accompanied by major physiological and behavioural changes. Many of these changes are related to the need to locate a mate and subsequently, in the case of females, to switch from mate searching to oviposition behaviour. The prodigious reproductive capacity of the Mediterranean fruit fly, Ceratitis capitata, is one of the factors that has led to its success as an invasive pest species. To identify the molecular changes related to maturation and mating status in male and female medfly, a microarray-based gene expression approach was used to compare the head transcriptomes of sexually immature, mature virgin, and mated individuals. Attention was focused on the changes in abundance of transcripts related to reproduction, behaviour, sensory perception of chemical stimulus, and immune system processes. Broad transcriptional changes were recorded during female maturation, while post-mating transcriptional changes in females were, by contrast, modest. In male medfly, transcriptional changes were consistent both during maturation and as a consequence of mating. Of particular note was the lack of the mating-induced immune responses that have been recorded for Drosophila melanogaster, that may be due to the different reproductive strategies of these species. This study, in addition to increasing our understanding of the molecular machinery behind maturation and mating in the medfly, has identified important gene targets that might be useful in the future management of this pest.

Glossina fuscipes fuscipes is the main vector of human and animal trypanosomiasis in Africa, particularly in Uganda. Attempts to control/eradicate this species using biological methods require knowledge of its reproductive biology. An important aspect is the number of times a female mates in the wild as this influences the effective population size and may constitute a critical factor in determining the success of control methods. To date, polyandry in G.f. fuscipes has not been investigated in the laboratory or in the wild. Interest in assessing the presence of remating in Ugandan populations is driven by the fact that eradication of this species is at the planning stage in this country.Two well established populations, Kabukanga in the West and Buvuma Island in Lake Victoria, were sampled to assess the presence and frequency of female remating. Six informative microsatellite loci were used to estimate the number of matings per female by genotyping sperm preserved in the female spermathecae. The direct count of the minimum number of males that transferred sperm to the spermathecae was compared to Maximum Likelihood and Bayesian probability estimates. The three estimates provided evidence that remating is common in the populations but the frequency is substantially different: 57% in Kabukanga and 33% in Buvuma.The presence of remating, with females maintaining sperm from different mates, may constitute a critical factor in cases of re-infestation of cleared areas and/or of residual populations. Remating may enhance the reproductive potential of re-invading propagules in terms of their effective population size. We suggest that population age structure may influence remating frequency. Considering the seasonal demographic changes that this fly undergoes during the dry and wet seasons, control programmes based on SIT should release large numbers of sterile males, even in residual surviving target populations, in the dry season.

BACKGROUND: Diptera have an extraordinary variety of sex determination mechanisms, and Drosophila melanogaster is the paradigm for this group. However, the Drosophila sex determination pathway is only partially conserved and the family Tephritidae affords an interesting example. The tephritid Y chromosome is postulated to be necessary to determine male development. Characterization of Y sequences, apart from elucidating the nature of the male determining factor, is also important to understand the evolutionary history of sex chromosomes within the Tephritidae. We studied the Y sequences from the olive fly, Bactrocera oleae. Its Y chromosome is minute and highly heterochromatic, and displays high heteromorphism with the X chromosome. METHODOLOGY/PRINCIPAL FINDINGS: A combined Representational Difference Analysis (RDA) and fluorescence in-situ hybridization (FISH) approach was used to investigate the Y chromosome to derive information on its sequence content. The Y chromosome is strewn with repetitive DNA sequences, the majority of which are also interdispersed in the pericentromeric regions of the autosomes. The Y chromosome appears to have accumulated small and large repetitive interchromosomal duplications. The large interchromosomal duplications harbour an importin-4-like gene fragment. Apart from these importin-4-like sequences, the other Y repetitive sequences are not shared with the X chromosome, suggesting molecular differentiation of these two chromosomes. Moreover, as the identified Y sequences were not detected on the Y chromosomes of closely related tephritids, we can infer divergence in the repetitive nature of their sequence contents. CONCLUSIONS/SIGNIFICANCE: The identification of Y-linked sequences may tell us much about the repetitive nature, the origin and the evolution of Y chromosomes. We hypothesize how these repetitive sequences accumulated and were maintained on the Y chromosome during its evolutionary history. Our data reinforce the idea that the sex chromosomes of the Tephritidae may have distinct evolutionary origins with respect to those of the Drosophilidae and other Dipteran families.