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BackgroundGlossinaspecies (tsetse flies), the sole vectors of African trypanosomes, maintained along their long evolutionary history a unique reproductive strategy, adenotrophic viviparity. Viviparity reduces their reproductive rate and, as such, imposes strong selective pressures on males for reproductive success. These species live in sub-Saharan Africa, where the distributions of the main sub-generaFusca,Morsitans, andPalpalisare restricted to forest, savannah, and riverine habitats, respectively. Here we aim at identifying the evolutionary patterns of the male reproductive genes of six species belonging to these three main sub-genera. We then interpreted the different patterns we found across the species in the light of viviparity and the specific habitat restrictions, which are known to shape reproductive behavior.ResultsWe used a comparative genomic approach to build consensus evolutionary trees that portray the selective pressure acting on the male reproductive genes in these lineages. Such trees reflect the long and divergent demographic history that led to an allopatric distribution of theFusca,Morsitans, andPalpalisspecies groups. A dataset of over 1700 male reproductive genes remained conserved over the long evolutionary time scale (estimated at 26.7 million years) across the genomes of the six species. We suggest that this conservation may result from strong functional selective pressure on the male imposed by viviparity. It is noteworthy that more than half of these conserved genes are novel sequences that are unique to theGlossinagenus and are candidates for selection in the different lineages.ConclusionsTsetse flies represent a model to interpret the evolution and differentiation of male reproductive biology under different, but complementary, perspectives. In the light of viviparity, we must take into account that these genes are constrained by a post-fertilization arena for genomic conflicts created by viviparity and absent in ovipositing species. This constraint implies a continuous antagonistic co-evolution between the parental genomes, thus accelerating inter-population post-zygotic isolation and, ultimately, favoring speciation. Ecological restrictions that affect reproductive behavior may further shape such antagonistic co-evolution.

The Mediterranean fruit fly (medfly), Ceratitis capitata, is a worldwide pest of agriculture able to use olfactory cues to locate habitat, food sources, mates and oviposition sites. The sensitivity of medfly olfaction has been exploited to develop olfactory-based attractants that are currently important tools for detection, control and eradication of its populations. Among these is Cera Trap® (BIOIBERICA, S.A.U.), a cost-effective bait. Here we used coupled gas chromatography/electroantennographic detection (GC-EAD) and GC/mass spectrometry (GC-MS) approaches to characterize the medfly antennally-active compounds released by this lure. We identified GC peaks corresponding to chemicals belonging to six different classes including heterocyclic aromatic compounds, aliphatic alcohols, aldehydes, esters, sesquiterpene hydrocarbons, and aromatic alcohols. We tested ten potential candidate volatiles belonging to these classes and predicted to be emitted by the lure and found that they were eliciting electroantennographic responses in medfly adults. These results will help in unravelling the physiological mechanisms of odor perception in both sexes, especially in relation to Cera Trap® attractant activity, which in the field has been shown to be female-specific. These findings and their developments will ultimately expand the toolbox for medfly control in the field.

BackgroundThe Oriental fruit fly,Bactrocera dorsalis, is a highly polyphagous invasive species with a high reproductive potential. In many tropical and subtropical parts of the world it ranks as one of the major pests of fruits and vegetables. Due to its economic importance, genetic, cytogenetic, genomic and biotechnological approaches have been applied to understand its biology and to implement the Sterile Insect Technique, currently a part of area-wide control programmes against this fly. Its chromosome complement includes five pairs of autosomes and the sex chromosomes. The X and Y sex chromosomes are heteromorphic and the highly heterochromatic and degenerate Y harbours the male factorBdMoY. The characterization of the Y chromosome in this fly apart from elucidating its role as primary sex determination system, it is also of crucial importance to understand its role in male biology. The repetitive nature of the Y chromosome makes it challenging to sequence and characterise.ResultsUsing Representational Difference Analysis, fluorescent in situ hybridisation on mitotic chromosomes and in silico genome resources, we show that theB. dorsalisY chromosome harbours transcribed sequences ofgyf,(typo-gyf) a homologue of theDrosophila melanogaster Gigyfgene, and of a non-LTR retrotransposon R1. Similar sequences are also transcribed on the X chromosome. Paralogues of theGigyfgene are also present on the Y and X chromosomes of the related speciesB. tryoni. Another identified Y-specific repetitive sequence linked toBdMoYappears to be specific toB. dorsalis.ConclusionsOur random scan of the Y chromosome provides a broad picture of its general composition and represents a starting point for further applicative and evolutionary studies. The identified repetitive sequences can provide a useful Y-marking system for molecular karyotyping of single embryos. Having a robust diagnostic marker associated withBdMoYwill facilitate studies on howBdMoYregulates the male sex determination cascade during the embryonic sex-determination window. The Y chromosome, despite its high degeneracy and heterochromatic nature, harbours transcribed sequences oftypo-gyfthat may maintain their important function in post-transcriptional mRNA regulation. That transcribed paralogous copies ofGigyfare present also on the X and that this genomic distribution is maintained also inB. tryoniraises questions on the evolution of sex chromosomes inBactroceraand other tephritids.

Simple Summary Tsetse flies, the sole transmitters of African Sleeping Sickness parasites, have a unique reproductive biology. They only develop one offspring at a time, they carry that offspring in their uterus for its entire immature development and provide nourishment for that offspring via milk-like secretions. This specialized reproductive biology has required dramatic modifications to the morphology of the reproductive organs in these and related flies. Here, we use phase contrast micro-Computed Tomography (Micro-CT) to visualize these adaptations in three dimensions for the first time. These adaptations include cuticular modifications allowing increased abdominal volume, expanded abdominal and uterine musculature, reduced egg development capacity, structural features of the male seminal secretions and detailed visualization of the gland responsible for synthesis and secretion of “milk” to feed intrauterine larvae. The ability to examine these tissues within the context of the rest of the organ systems in the fly provides new functional insights into how these changes have facilitated the evolution of the mating and reproductive biology of these flies. Abstract 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.

Background The Asian tiger mosquito, Aedes albopictus, is a highly dangerous invasive vector of numerous medically important arboviruses including dengue, chikungunya and Zika. In four decades it has spread from tropical Southeast Asia to many parts of the world in both tropical and temperate climes. The rapid invasion process of this mosquito is supported by its high ecological and genetic plasticity across different life history traits. Our aim was to investigate whether wild populations, both native and adventive, also display transcriptional genetic variability for functions that may impact their biology, behaviour and ability to transmit arboviruses, such as sensory perception. Results Antennal transcriptome data were derived from mosquitoes from a native population from Ban Rai, Thailand and from three adventive Mediterranean populations: Athens, Greece and Arco and Trento from Italy. Clear inter-population differential transcriptional activity was observed in different gene categories related to sound perception, olfaction and viral infection. The greatest differences were detected between the native Thai and the Mediterranean populations. The two Italian populations were the most similar. Nearly one million quality filtered SNP loci were identified. Conclusion The ability to express this great inter-population transcriptional variability highlights, at the functional level, the remarkable genetic flexibility of this mosquito species. We can hypothesize that the differential expression of genes, including those involved in sensory perception, in different populations may enable Ae. albopictus to exploit different environments and hosts, thus contributing to its status as a global vector of arboviruses of public health importance. The large number of SNP loci present in these transcripts represents a useful addition to the arsenal of high-resolution molecular markers and a resource that can be used to detect selective pressure and adaptive changes that may have occurred during the colonization process.

The mosquito Aedes albopictus is one of the most dangerous invasive species. Its worldwide spread has created health concerns as it is a major vector of arboviruses of public health significance such as chikungunya (CHIKV). Dynamics of different genetic backgrounds and admixture events may have impacted competence for CHIKV in adventive populations. Using microsatellites, we infer the genetic structure of populations across the expansion areas that we then associate with their competence for different CHIKV genotypes. Here we show that the demographic history of Ae. albopictus populations is a consequence of rapid complex patterns of historical lineage diversification and divergence that influenced their competence for CHIKV. The history of adventive populations is associated with CHIKV genotypes in a genotype-by-genotype interaction that impacts their vector competence. Thus, knowledge of the demographic history and vector competence of invasive mosquitoes is pivotal for assessing the risk of arbovirus outbreaks in newly colonized areas., Anubis Vega-Rúa, Michele Marconcini et al. study how the dynamics of genetics and admixture events of Aedes albopictus affect its competence for the CHIKV. Using microsatellites, they found that the demographic history influences the competence for the virus and is an important feature to assess the risk of outbreaks in new areas.

Tsetse flies (genus Glossina), the sole vectors of African trypanosomiasis, are distinct from other disease vectors, and most other insects, due to dramatic morphological and physiological adaptations required to support their unique life histories. These evolutionary adaptations are driven by demands associated with their strict dietary and reproductive requirements. Tsetse reproduce by obligate viviparity which entails obligate intrauterine larval development and provisioning of nutrients for the developing larvae. Viviparous reproduction reduces reproductive capacity/rate which also drives increased inter- and intra-sexual competition. Here, we use phase contrast microcomputed tomography (pcMicroCT) to perform a three-dimensional (3D) analysis of viviparity associated morphological adaptations of tsetse female reproductive tract. These include 1) abdominal modifications facilitating the extreme abdominal distention required during blood feeding and pregnancy; 2) abdominal and uterine musculature required for parturition of developed larvae; 3) reduction of ovarian structure and capacity; 4) structural features of the spermatophore form in the female uterus to enhance semen/sperm delivery and inhibition of insemination by competing males; 5) uterine morphological features facilitating expansion and contraction before, during and after pregnancy; 6) milk gland structural optimizations facilitating nutrient incorporation and transfer into the uterus. The use of pcMicroCT provides unprecedented opportunities for examination and discovery of internal morphological features not possible with traditional microscopy techniques and new opportunities for comparative morphological analyses over time and between species.

Background The 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. Methods A 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. Results We 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. Conclusions Here 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., Author summary Aedes albopictus is a highly invasive mosquito and a vector for a number of arboviruses. The arrival and establishment of Ae. albopictus in temperature regions of the world, such as Europe, has been accompanied by re-emergence of arboviral diseases. There are no effective therapeutic treatments for arboviruses meaning control of vector populations is the primary strategy to prevent arboviral disease transmission. Pyrethroids are frequently used for control of vectors based on their low mammalian toxicity and rapid knockdown effect on mosquitoes. The identification of mutations predictive of resistance phenotype in the para sodium channel gene, the target site of pyrethroids, has provided for molecular markers to test for resistance by genotyping wild-collected mosquitoes. Here we analysed all currently known predictive mutations for pyrethroid resistance in 512 geographic mosquitoes sampled in a span of seven years. Thus, we are able to add a temporal dimension to the analysis of geographic distribution of resistance alleles. Overall our data confirm a patchy distribution of mutations predictive of the resistance phenotype, but also reveal an alarming increase of resistance mutations in China, Greece and Italy. Resistance mutations appear to have arisen locally more than being spread through mosquito migration/invasions.

Tsetse flies (Glossina spp.) act as the sole vectors of the African trypanosome species that cause Human African Trypanosomiasis (HAT or African Sleeping Sickness) and Nagana in animals. These flies have undergone a variety of specializations during their evolution including an exclusive diet consisting solely of vertebrate blood for both sexes as well as an obligate viviparous reproductive biology. Alongside these adaptations, Glossina species have developed intricate relationships with specific microbes ranging from mutualistic to parasitic. These relationships provide fundamental support required to sustain the specializations associated with tsetse's biology. This chapter provides an overview on the knowledge to date regarding the biology behind these relationships and focuses primarily on four bacterial species that are consistently associated with Glossina species. Here their interactions with the host are reviewed at the morphological, biochemical and genetic levels. This includes: the obligate symbiont Wigglesworthia, which is found in all tsetse species and is essential for nutritional supplementation to the blood-specific diet, immune system maturation and facilitation of viviparous reproduction; the commensal symbiont Sodalis, which is a frequently associated symbiont optimized for survival within the fly via nutritional adaptation, vertical transmission through mating and may alter vectorial capacity of Glossina for trypanosomes; the parasitic symbiont Wolbachia, which can manipulate Glossina via cytoplasmic incompatibility and shows unique interactions at the genetic level via horizontal transmission of its genetic material into the genome in two Glossina species; finally, knowledge on recently observed relations between Spiroplasma and Glossina is explored and potential interactions are discussed based on knowledge of interactions between this bacterial Genera and other insect species. These flies have a simple microbiome relative to that of other insects. However, these relationships are deep, well-studied and provide a window into the complexity and function of host/symbiont interactions in an important disease vector.

International audience; Arboviral diseases such as chikungunya, dengue, and Zika viruses have been threatening the European countries since the introduction in 1979 of the major vector Aedes albopictus. In 2017, more than three hundred of CHIKV autochthonous cases were reported in Italy, highlighting the urgent need for a risk assessment of arboviral diseases in European countries. In this study, the vector competence for three major arboviruses were analyzed in eight Ae. albopictus populations from europe. Here we show that Southern european Ae. albopictus were susceptible to CHIKV, DENV-1 and ZIKV with the highest vector competence for CHIKV. Based on vector competence data and vector distribution, a prediction risk map for CHIKV was generated stressing the fear of CHIKV and to a lesser extent, of other arboviruses for Europe, calling us for new public health strategies. Recent epidemics involving several arboviral diseases such as chikungunya (CHIKV), dengue (DENV) and Zika (ZIKV) have received global attention as major public health issues 1,2. The Aedes spp. mosquitoes, main vectors of these arboviruses, have extended their distribution owing to human activities (trade and travels) and climate change; they are no longer restricted to tropical regions and have initiated the invasion of European regions 3-6. Although Europe was not considered prone to arboviral diseases, the presence of newly introduced competent mosquitoes coupled to a growing number of imported cases 7 led to local transmissions of chikungunya and den-gue fever in Croatia 8,9 , France 10-14 , and Italy 15. The 2017 CHIKV outbreak in Italy was attributed to few incident cases in Anzio in June, subsequently spreading to Guardavalle Marina later and to Rome in October. A total of 337 infections were reported, 61 of which occurred in the capital city of Rome 16. This outbreak exemplifies that the presence of Ae. albopictus in Italy favors the occurrence of CHIKV epidemics 16. Mosquitoes are able to transmit arboviruses by acquiring a viremic blood meal from an infected host. Along with blood, the ingested virus enters in the mosquito midgut and infects the epithelial cells that may in turn disseminate the virus to the mosquito internal tissues or organs. After the extrinsic incubation period (EIP) 17-19 , the virus may reach the salivary glands where the viral cycle in the vector ends up with the virus transmitted to the host by the mosquito saliva 20. The virus efficiency to cross each anatomical barrier (midgut and salivary glands) depends on several genetic or biological factors regulating mosquito antiviral immunity 21. Geographic populations of a same mosquito species may not share the same immunological background, leading to varying susceptibilities to transmit arboviruses 22. A vector competence analysis of European Ae. albopictus populations is critical for assessing the risk of arbovi-ral diseases outbreaks as the establishment of competent mosquitoes can become the breeding ground for various arboviruses. To this aim, we collected eight European Ae. albopictus populations from Croatia, Greece, Italy, Montenegro, and Switzerland, and experimentally infected them with three arboviruses, CHIKV (Alphavirus, Togaviridae), and DENV and ZIKV (Flavivirus, Flaviviridae). Based on data obtained, we elaborated a vector competence data-driven prediction for CHIKV transmission using computational modeling to assist in evaluating the current risk of arboviral diseases transmission in Southern Europe.

Background Tsetse flies (Glossina sp.) are the vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse flies are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood-specific diet by both sexes, and obligate bacterial symbiosis. This work describes the comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans, G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes), and Fusca (G. brevipalpis) which represent different habitats, host preferences, and vectorial capacity. Results Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex-linked scaffolds show increased rates of female-specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse-specific genes are enriched in protease, odorant-binding, and helicase activities. Lactation-associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other insects. Vision-associated Rhodopsin genes show conservation of motion detection/tracking functions and variance in the Rhodopsin detecting colors in the blue wavelength ranges. Conclusions Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.

Background: Tsetse flies (Glossina sp.) are the sole vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood specific diet by both sexes and obligate bacterial symbiosis. This work describes comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans (G.m. morsitans), G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes) and Fusca (G. brevipalpis) which represent different habitats, host preferences and vectorial capacity. Results: Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex linked scaffolds show increased rates of female specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse specific genes are enriched in protease, odorant binding and helicase activities. Lactation associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other characterized insects. Vision associated Rhodopsin genes show conservation of motion detection/tracking functions and significant variance in the Rhodopsin detecting colors in the blue wavelength ranges. Conclusions: Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.

In the Mediterranean fruit fly, Ceratitis capitata (Wiedemann)(Diptera: Tephritidae), mating has a strong impact on female biology, leading to a decrease in sexual receptivity and increased oviposition and fecundity. Previous studies suggest that sperm transfer may play a role in inducing these behavioural changes. Here we report the identification of a medfly innexin gene, Cc-inx5, whose expression is limited to the germ-line of both sexes. Through RNA interference of this gene, we generated males without testes and, consequently, sperm, but apparently retaining all the other reproductive organs intact. These sperm-less males were able to mate and, like their wild-type counterparts, to induce in their partners increased oviposition rates and refractoriness to remating. Interestingly, matings to sperm-less males results in oviposition rates higher than those induced by copulation with control males. In addition, the observed female post-mating behavioural changes were congruent with changes in transcript abundance of genes known to be regulated by mating in this species. Our results suggest that sperm transfer is not necessary to reduce female sexual receptivity and to increase oviposition and fecundity. These data pave the way to a better understanding of the role/s of seminal components in modulating female post-mating responses. In the long term, this knowledge will be the basis for the development of novel approaches for the manipulation of female fertility, and, consequently, innovative tools to be applied to medfly control strategies in the field.

Background Tsetse flies (Diptera, Glossinidae) display unique reproductive biology traits. Females reproduce through adenotrophic viviparity, nourishing the growing larva into their modified uterus until parturition. Males transfer their sperm and seminal fluid, produced by both testes and male accessory glands, in a spermatophore capsule transiently formed within the female reproductive tract upon mating. Both sexes are obligate blood feeders and have evolved tight relationships with endosymbionts, already shown to provide essential nutrients lacking in their diet. However, the partnership between tsetse and its symbionts has so far been investigated, at the molecular, genomic and metabolomics level, only in females, whereas the roles of microbiota in male reproduction are still unexplored. Results Here we begin unravelling the impact of microbiota on Glossina m. morsitans (G. morsitans) male reproductive biology by generating transcriptomes from the reproductive tissues of males deprived of their endosymbionts (aposymbiotic) via maternal antibiotic treatment and dietary supplementation. We then compared the transcriptional profiles of genes expressed in the male reproductive tract of normal and these aposymbiotic flies. We showed that microbiota removal impacts several male reproductive genes by depressing the activity of genes in the male accessory glands (MAGs), including sequences encoding seminal fluid proteins, and increasing expression of genes in the testes. In the MAGs, in particular, the expression of genes related to mating, immunity and seminal fluid components’ synthesis is reduced. In the testes, the absence of symbionts activates genes involved in the metabolic apparatus at the basis of male reproduction, including sperm production, motility and function. Conclusions Our findings mirrored the complementary roles male accessory glands and testes play in supporting male reproduction and open new avenues for disentangling the interplay between male insects and endosymbionts. From an applied perspective, unravelling the metabolic and functional relationships between tsetse symbionts and male reproductive physiology will provide fundamental information useful to understanding the biology underlying improved male reproductive success in tsetse. This information is of particular importance in the context of tsetse population control via Sterile Insect Technique (SIT) and its impact on trypanosomiasis transmission. Electronic supplementary material The online version of this article (10.1186/s12866-018-1289-2) contains supplementary material, which is available to authorized users.

The fruit fly species, Ceratitis rosa sensu stricto and Ceratitis quilicii, are sibling species restricted to the lowland and highland regions, respectively. Until recently, these sibling species were considered as allopatric populations of C. rosa with distinct bionomics. We used deep Next Generation Sequencing (NGS) technology on intact guts of individuals from the two sibling species to compare their transcriptional profiles and simultaneously understand gut microbiome and host molecular processes and identify distinguishing genetic differences between the two species. Since the genomes of both species had not been published previously, the transcriptomes were assembled de novo into transcripts. Microbe-specific transcript orthologs were separated from the assembly by filtering searches of the transcripts against microbe databases using OrthoMCL. We then used differential expression analysis of host-specific transcripts (i.e. those remaining after the microbe-specific transcripts had been removed) and microbe-specific transcripts from the two-sibling species to identify defining species-specific transcripts that were present in only one fruit fly species or the other, but not in both. In C. quilicii females, bacterial transcripts of Pectobacterium spp., Enterobacterium buttiauxella, Enterobacter cloacae and Klebsiella variicola were upregulated compared to the C. rosa s.s. females. Comparison of expression levels of the host transcripts revealed a heavier investment by C. quilicii (compared with C. rosa s.s.) in: immunity; energy production; cell proliferation; insecticide resistance; reproduction and proliferation; and redox reactions that are usually associated with responses to stress and degradation of fruit metabolites.

We used a population genetic approach to detect the presence of genetic diversity among six populations of A. fraterculus across Brazil. To this aim, we used Simple Sequence Repeat (SSR) markers, which may capture the presence of differentiative processes across the genome in distinct populations. Spatial analyses of molecular variance were used to identify groups of populations that are both genetically and geographically homogeneous while also being maximally differentiated from each other. The spatial analysis of genetic diversity indicates that the levels of diversity among the six populations vary significantly on an eco-geographical basis. Particularly, altitude seems to represent a differentiating adaptation, as the main genetic differentiation is detected between the two populations present at higher altitudes and the other four populations at sea level. The data, together with the outcomes from different cluster analyses, identify a genetic diversity pattern that overlaps with the distribution of the known morphotypes in the Brazilian area. Fil: Manni, Mosè. Università degli Studi di Pavia; Italia Fil: Lima, Kátia Manuela. Universidade Estadual de Santa Cruz; Brasil Fil: Rosalba Guglielmino, Carmela. Università degli Studi di Pavia; Italia Fil: Lanzavecchia, Silvia Beatriz. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Juri, Marianela Lucia. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Vera, Maria Teresa. Universidad Nacional de Tucumán. Facultad de Agronomía y Zootecnia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Cladera, Jorge. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Scolari, Francesca. Università degli Studi di Pavia; Italia Fil: Gomulski, Ludvik. Università degli Studi di Pavia; Italia Fil: Bonizzoni, Mariangela. Università degli Studi di Pavia; Italia Fil: Gasperi, Giuliano. Università degli Studi di Pavia; Italia Fil: Gomes Silva, Janisete. Universidade Estadual de Santa Cruz; Brasil Fil: Malacrida, Anna Rodolfa. Università degli Studi di Pavia; Italia

The Mediterranean fruit fly (medfly) Ceratitis capitata is a polyphagous pest of fruits and crops with a worldwide distribution. Its ability to use different larval hosts may have multiple effects, including impacts on adult reproductive biology. The male sex pheromone, which plays a key role in attracting both other males to lekking arenas and females for mating, is a mixture of chemical compounds including esters, acids, alkanes and terpenes known to differ between laboratory strains and wild-type populations. The relationship between larval diet and adult pheromone composition remains unexplored. Here, we investigated the effect of larval diet, including laboratory media and fresh fruits, on the composition of the male pheromone mixture. Using Headspace Solid Phase Microextraction we collected the pheromone emitted by males reared as larvae on different substrates and found both qualitative and quantitative differences. A number of alkanes appeared to be typical of the pheromone of males reared on wheat bran-based larval medium, and these may be cuticular hydrocarbons involved in chemical communication. We also detected differences in pheromone composition related to adult male age, suggesting that variations in hormonal levels and/or adult diet could also play a role in determining the chemical profile emitted. Our findings highlight the plasticity of dietary responses of C. capitata, which may be important in determining the interactions of this pest with the environment and with conspecifics. These results also have applied relevance to increase the mating competitiveness of mass-reared C. capitata used in Sterile Insect Technique programs.

Ecological specialization of insects involves the functional and morphological reshaping of olfactory systems. Little is known about the degree to which insect sensitivity to odorant compounds is conserved between genera, tribes, or families. Here we compared the olfactory systems of six tephritid fruit fly species spanning two tribes and the distantly related Drosophila melanogaster at molecular, functional, and morphological levels. Olfaction in these flies is mediated by a set of olfactory receptors (ORs) expressed in different functional classes of neurons located in distinct antennal regions. We performed a phylogenetic analysis that revealed both family-specific OR genes and putative orthologous OR genes between tephritids and Drosophila. With respect to function, we then used a current source density (CSD) analysis to map activity across antennae. Functional maps mirrored the intrinsic structure of antennae observed with scanning electron microscopy. Together, the results revealed partial conservation of the olfactory systems between tephritids and Drosophila. We also demonstrate that the mapping of olfactory responses is necessary to decipher antennal sensory selectivity to olfactory compounds. CSD analysis can be easily applied to map antennae of other species and therefore enables the rapid deriving of olfactory maps and the reconstructing of the target organisms’ history of evolution.

Bactrocera papayae Drew & Hancock, Bactrocera philippinensis Drew & Hancock, Bactrocera carambolae Drew & Hancock, and Bactrocera invadens Drew, Tsuruta & White are four horticultural pest tephritid fruit fly species that are highly similar, morphologically and genetically, to the destructive pest, the Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). This similarity has rendered the discovery of reliable diagnostic characters problematic, which, in view of the economic importance of these taxa and the international trade implications, has resulted in ongoing difficulties for many areas of plant protection and food security. Consequently, a major international collaborative and integrated multidisciplinary research effort was initiated in 2009 to build upon existing literature with the specific aim of resolving biological species limits among B. papayae, B. philippinensis, B. carambolae, B. invadens and B. dorsalis to overcome constraints to pest management and international trade. Bactrocera philippinensis has recently been synonymized with B. papayae as a result of this initiative and this review corroborates that finding; however, the other names remain in use. While consistent characters have been found to reliably distinguish B. carambolae from B. dorsalis, B. invadens and B. papayae, no such characters have been found to differentiate the latter three putative species. We conclude that B. carambolae is a valid species and that the remaining taxa, B. dorsalis, B. invadens and B. papayae, represent the same species. Thus, we consider B. dorsalis (Hendel) as the senior synonym of B. papayae Drew and Hancock syn.n. and B. invadens Drew, Tsuruta & White syn.n. A redescription of B. dorsalis is provided. Given the agricultural importance of B. dorsalis, this taxonomic decision will have significant global plant biosecurity implications, affecting pest management, quarantine, international trade, postharvest treatment and basic research. Throughout the paper, we emphasize the value of independent and multidisciplinary tools in delimiting species, particularly in complicated cases involving morphologically cryptic taxa.

The Mediterranean fruit fly (or medfly), Ceratitis capitata (Wiedemann; Diptera: Tephritidae), is a serious pest of agriculture worldwide, displaying a very wide larval host range with more than 250 different species of fruit and vegetables. Olfaction plays a key role in the invasive potential of this species. Unfortunately, the pheromone communication system of the medfly is complex and still not well established. In this study, we report the isolation of chemicals emitted by sexually mature individuals during the “calling” period and the electrophysiological responses that these compounds elicit on the antennae of male and female flies. Fifteen compounds with electrophysiological activity were isolated and identified in male emissions by gas chromatography coupled to electroantennography (GC–EAG). Within the group of 15 identified compounds, 11 elicited a response in antennae of both sexes, whilst 4 elicited a response only in female antennae. The binding affinity of these compounds, plus 4 additional compounds known to be behaviourally active from other studies, was measured using C. capitata OBP, CcapOBP83a-2. This OBP has a high homology to Drosophila melanogaster OBPs OS-E and OS-F, which are associated with trichoid sensilla and co-expressed with the well-studied Drosophila pheromone binding protein LUSH. The results provide evidence of involvement of CcapOBP83a-2 in the medfly's odorant perception and its wider specificity for (E,E)-α-farnesene, one of the five major compounds in medfly male pheromone emission. This represents the first step in the clarification of the C. capitata and pheromone reception pathway, and a starting point for further studies aimed towards the creation of new powerful attractants or repellents applicable in the actual control strategies., Graphical abstract, Highlights • The pheromone blend produced by medfly males during the calling period is investigated using air entrainment and GC–EAG. • Out of 15 chemicals, 11 elicited antennal responses in both sexes and 4 elicited the response only in female antennae. • The first medfly odorant binding protein CcapOBP83a-2 has been expressed and purified (87). • CcapOBP83a-2 showed binding affinity for (E,E)-α-farnesene, one of the components of medfly pheromone. • The ability of CcapOBP83a-2 to bind other electrophysiologically active compounds shows its broad binding specificity.

Bactrocera correcta is one of the most destructive pests of horticultural crops in tropical and subtropical regions. Despite the economic risk, the population genetics of this pest have remained relatively unexplored. This study explores population genetic structure and contemporary gene flow in B. correcta in Chinese Yunnan Province and attempts to place observed patterns within the broader geographical context of the species’ total range. Based on combined data from mtDNA cox1 sequences and 12 microsatellite loci obtained from 793 individuals located in 7 countries, overall genetic structuring was low. The expansion history of this species, including likely human-mediated dispersal, may have played a role in shaping the observed weak structure. The study suggested a close relationship between Yunnan Province and adjacent countries, with evidence for Western and/or Southern Yunnan as the invasive origin of B. correcta within Yunnan Province. The information gleaned from this analysis of gene flow and population structure has broad implications for quarantine, trade and management of this pest, especially in China where it is expanding northward. Future studies should concentrate effort on sampling South Asian populations, which would enable better inferences of the ancestral location of B. correcta and its invasion history into and throughout Asia.

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 A. albopictus to expand its geographical range.

Male Seminal Fluid Proteins (SFPs) transferred during copulation modulate female reproductive physiology and behavior, impacting sperm storage/use, ovulation, oviposition and remating receptivity. These capabilities make them ideal targets for developing novel methods of insect disease vector control. Little is known about the nature of SFPs in the viviparous tsetse flies (Diptera: Glossinidae), vectors of Human and Animal African trypanosomiasis. In tsetse, male ejaculate is assembled into a capsule-like spermatophore structure visible post-copulation in the female uterus. We applied high-throughput approaches to uncover the composition of the spermatophore in Glossina morsitans morsitans. We found that both male accessory glands and testes contribute to its formation. The male accessory glands produce a small number of abundant novel proteins with yet unknown functions, in addition to enzyme inhibitors and peptidase regulators. The testes contribute sperm in addition to a diverse array of less abundant proteins associated with binding, oxidoreductase/transferase activities, cytoskeletal and lipid/carbohydrate transporter functions. Proteins encoded by female-biased genes are also found in the spermatophore. About half of the proteins display sequence conservation relative to other Diptera and low similarity to SFPs from other studied species, possibly reflecting both their fast evolutionary pace and the divergent nature of tsetse’s viviparous biology.

31 p.-11 fig.-2 tab.+ Erratum (2 p.) Papanikolaou, Alexie et al., Background: The Mediterranean fruit fly (medfly), Ceratitis capitata, is a major destructive insect pest due to its broad host range, which includes hundreds of fruits and vegetables. It exhibits a unique ability to invade and adapt to ecological niches throughout tropical and subtropical regions of the world, though medfly infestations have been prevented and controlled by the sterile insect technique (SIT) as part of integrated pest management programs (IPMs). The genetic analysis and manipulation of medfly has been subject to intensive study in an effort to improve SIT efficacy and other aspects of IPM control., Results: The 479 Mb medfly genome is sequenced from adult flies from lines inbred for 20 generations. A highquality assembly is achieved having a contig N50 of 45.7 kb and scaffold N50 of 4.06 Mb. In-depth curation of more than 1800 messenger RNAs shows specific gene expansions that can be related to invasiveness and host adaptation, including gene families for chemoreception, toxin and insecticide metabolism, cuticle proteins, opsins, and aquaporins. We identify genes relevant to IPM control, including those required to improve SIT., Conclusions: The medfly genome sequence provides critical insights into the biology of one of the most serious and widespread agricultural pests. This knowledge should significantly advance the means of controlling the size and invasive potential of medfly populations. Its close relationship to Drosophila, and other insect species important to agriculture and human health, will further comparative functional and structural studies of insect genomes that should broaden our understanding of gene family evolution, Support of this project was provided by the U.S. Department of Agriculture(USDA), Agricultural Research Service (ARS), Animal and Plant Health Inspection Service (APHIS), and National Institute of Food and Agriculture(NIFA)-Biotechnology Risk Assessment Grants Program (grant #2011-39211-30769 to AMH) for funding the initial phase of this project, and to the National Institutes of Health (NIH)-National Human Genome Research Institute (NHGRI) for funding the medfly genome sequencing, assembly and Maker 2.0 automated annotation as part of the i5K 30 genome pilot project (grant #U54 HG003273 to RAG). The NIH Intramural Research Program, National Library of Medicine funded the NCBI Gnomon annotation and the USDA-National Agricultural Library (NAL) provided support for the WebApollo curation website, with support for manual curation training (to MM-T) provided by NIGMS (grant #5R01GM080203),NHGRI (grant #5R01HG004483), and the U.S. Department of Energy(contract #DE-AC02-05CH11231). Support was provided for: toxin metabolism and insecticide resistance gene studies from MINECO,Spain (AGL2013-42632-R to FO and PH-C); microRNAs, horizontal gene transfer and bacterial contaminant studies from the European Social Fund and National Strategic Reference Framework-THALES (MIS375869 to KB, GT, AGH, and KM) and the U.S. National Science Foundation(DEB 1257053 to JHW); cuticle protein gene studies from USDA-NIFA(grant #2016-67012-24652 to AJR); sex-determination studies from L.R. Campania (grant 5/02, 2008 to GS); male reproduction and sexual differentiation studies from the FAO/IAEA (Technical Contract No: 16966 to GGa) and Cariplo IMPROVE (to FS); and programmed cell death gene studies and genomic data analysis (to MFS) from the Emmy Noether program, DFG(SCHE 1833/1-1) and the LOEWE Center for Insect Biotechnology & Bioresources grant of the Hessen State Ministry of Higher Education, Research and the Arts(HMWK), Germany and from the USDA-NIFA-Biotechnology Risk Assessment Grants Program (grant #2015-33522-24094 to AMH).

Microsatellites, also referred to as short tandem repeats (STRs) or simple sequence repeats (SSRs) are short sequences of tandem repeats of 1–6 bp in length in clusters of less than 150 bp flanked by sections of non-repetitive unique sequences that are scattered throughout the nuclear genome. These markers are co-dominant and hypervariable, revealing many alleles per locus; they are inherited in Mendelian fashion making them useful for detecting genetic variability within species. Once isolated and characterized, microsatellites can also be used in closely related taxa. Microsatellites can be amplified, even from highly degraded DNA, and are very simple to score. More importantly, these markers are highly polymorphic due to the plethora of variations in the repeat motifs. Several studies have endorsed microsatellite markers as an effective genetic tool to determine the historical distinctiveness of populations, and hence, the designation of species. Being highly polymorphic and selectively neutral, microsatellite markers offer a powerful genetic tool for investigating population structure, colonization processes, temporal and spatial population dynamics and evolutionary trends of insect pests. Furthermore, these markers have been successfully applied to different invasive fruit fly species to infer the evolutionary aspects underlying their invasive processes. Microsatellite markers have offered an analytical tool for the study of fruit fly invasion genetics as exemplified for the Mediterranean fruit fly, Ceratitis capitata. Herein, a detailed utility of microsatellite markers in inferring invasion histories of key fruit flies of economic importance is given, with a special focus on invasion into Africa of Bactrocera dorsalis.

The oriental fruit fly, Bactrocera dorsalis (Hendel), is a key pest that causes reduction of the crop yield within the international fruit market. Fruit flies have been suppressed by two Area-Wide Integrated Pest Management programs in Thailand using Sterile Insect Technique (AW-IPM-SIT) since the late 1980s and the early 2000s. The projects’ planning and evaluation usually rely on information from pest status, distribution, and fruit infestation. However, the collected data sometimes does not provide enough detail to answer management queries and public concerns, such as the long term sterilization efficacy of the released fruit fly, skepticism about insect migration or gene flow across the buffer zone, and the re-colonisation possibility of the fruit fly population within the core area. Established microsatellite DNA markers were used to generate population genetic data for the analysis of the fruit fly sampling from several control areas, and non-target areas, as well as the mass-rearing facility. The results suggested limited gene flow (m < 0.100) across the buffer zones between the flies in the control areas and flies captured outside. In addition, no genetic admixture was revealed from the mass-reared colony flies from the flies within the control area, which supports the effectiveness of SIT. The control pests were suppressed to low density and showed weak bottleneck footprints although they still acquired a high degree of genetic variation. Potential pest resurgence from fragmented micro-habitats in mixed fruit orchards rather than pest incursion across the buffer zone has been proposed. Therefore, a suitable pest control effort, such as the SIT program, should concentrate on the hidden refuges within the target area.

Insect transgenesis is continuously being improved to increase the efficacy of population suppression and replacement strategies directed to the control of insect species of economic and sanitary interest. An essential prerequisite for the success of both pest control applications is that the fitness of the transformant individuals is not impaired, so that, once released in the field, they can efficiently compete with or even out-compete their wild-type counterparts for matings in order to reduce the population size, or to spread desirable genes into the target population. Recent research has shown that the production of fit and competitive transformants can now be achieved and that transgenes may not necessarily confer a fitness cost. In this article we review the most recent published results of the fitness assessment of different transgenic insect lines and underline the necessity to fulfill key requirements of ecological safety. Fitness evaluation studies performed in field cages and medium/large-scale rearing will validate the present encouraging laboratory results, giving an indication of the performance of the transgenic insect genotype after release in pest control programmes.

Tephritid fruit fly species cause major economical losses in crops worldwide. Genetic transformation of insect pests, which are targets of the Sterile Insect Technique (SIT), a key component of area-wide pest management, has been achieved for several years. For the medfly Ceratitis capitata as well as several Bactrocera and Anastrepha species, germline transformation can now be used to bioengineer strains that should increase the efficacy and cost-effectiveness of the SIT. Novel transformation vectors, robust genetic markers and diverse promoters to drive stage- and tissue-specific gene expression provide powerful tools to test the contribution that these technologies can make to current SIT programmes.

The Sterile Insect Technique (SIT) involving area-wide release of mass-reared and sterilized pest insects has proven successful to reduce, control and eradicate economically important pest species, such as the Mediterranean fruit fly (medfly). For the efficient application, effective monitoring to assess the number and mating success of the released medflies is essential. Here, we report sperm-specific marking systems based on the spermatogenesis-specific Ceratitis capitata beta2-tubulin (Ccbeta2t) promoter. Fluorescent sperm can be isolated from testes or spermathecae. The marking does not cause general disadvantages in preliminary laboratory competitiveness assays. Therefore, transgenic sperm marking could serve as a major improvement for monitoring medfly SIT programs. The use of such harmless transgenic markers will serve as an ideal initial condition to transfer insect transgenesis technology from the laboratory to field applications. Moreover, effective and easily recognizable sperm marking will make novel studies possible on medfly reproductive biology which will help to further improve SIT programs.

The phytophagous insects of the Tephritidae family offer different case histories of successful invasions. An example is Bactrocera dorsalis sensu stricto, the oriental fruit fly which has been recognized as a key pest of Asia and the Pacific. It is known to have the potential to establish adventive populations in various tropical and subtropical areas. Despite the economic risk associated with a putative stable presence of this fly, the genetic aspects of its invasion process have remained relatively unexplored. Using microsatellite markers we have investigated the population structure and genetic variability in 14 geographical populations across the four areas of the actual species range: Far East Asia, South Asia, Southeast Asia and the Pacific Area. Results of clustering and admixture, associated with phylogenetic and migration analyses, were used to evaluate the changes in population genetic structure that this species underwent during its invasion process and establishment in the different areas. The colonization process of this fly is associated with a relatively stable population demographic structure, especially in an unfragmented habitat, rich in intensive cultivation such as in Southeast Asia. In this area, the results suggest a lively demographic history, characterized by evolutionary recent demographic expansions and no recent bottlenecks. Cases of genetic isolation attributable to geographical factors, fragmented habitats and/or fruit trade restrictions were observed in Bangladesh, Myanmar and Hawaii. Regarding the pattern of invasion, the overall genetic profile of the considered populations suggests a western orientated migration route from China to the West.

Scaphoideus titanus Ball is a Nearctic leafhopper that was introduced for the first time in Europe probably at the beginning of the 20th century. In Europe, this species is a specialist on cultivated grapevines and is of great economic importance as the vector of Flavescence dorée (FD), a Grapevine Yellows disease caused by Candidatus Phytoplasma vitis. The Random Amplified Polymorphic DNA (RAPD) technique was employed to obtain genetic information about the diffusion and the structure of S. titanus populations. Two American and 14 European populations were analysed. A total of 188 reproducible bands, obtained from three arbitrary primers, were considered to assess the amount and the pattern of genetic variation within and among leafhopper populations. American populations showed high levels of intra-population polymorphism and dissimilarity and appeared to be the most isolated of all the tested samples. The results confirm the historical role of American samples as the sources for the more recently founded European populations. RAPD analyses revealed a weak genetic structure of European samples that could probably be explained invoking the human role in their diffusion. The non-natural spreading of S. titanus across Europe is in fact attributable to the exchange of grapevine canes and grafts carrying eggs that the insect laid under the bark to overwinter.

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 FST 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.

The draft genome sequence of Italian specimens of the Asian tiger mosquito Aedes (Stegomyia) albopictus (Diptera: Culicidae) was determined using a standard NGS (next generation sequencing) approach. The size of the assembled genome is comparable to that of Aedes aegypti; the two mosquitoes are also similar as far as the high content of repetitive DNA is concerned, most of which is made up of transposable elements. Although, based on BUSCO (Benchmarking Universal Single-Copy Orthologues) analysis, the genome assembly reported here contains more than 99% of protein-coding genes, several of those are expected to be represented in the assembly in a fragmented state. We also present here the annotation of several families of genes (tRNA genes, miRNA genes, the sialome, genes involved in chromatin condensation, sex determination genes, odorant binding proteins and odorant receptors). These analyses confirm that the assembly can be used for the study of the biology of this invasive vector of disease. © W. S. Maney & Son Ltd 2015.

The phytophagous insects of the Tephritidae family commonly referred to as "true fruit flies" offer different case histories of successful invasions. Mankind has played an important role in altering the distributions of some of the more polyphagous and oligophagous species. However, the question arises why only a few species have become major invaders. The understanding of traits underlying adaptation in different environments is a major topic in invasion biology. Being generalists or specialists, along the K-r gradient of the growth curve, make a difference in term of food resources exploitation and interspecies competition and displacement. The species of the genus Ceratitis are good examples of r-strategists. The genetic and biological data of the most notorious Ceratitis species, the Mediterranean fruit fly Ceratitis capitata (medfly), are reviewed to investigate the traits and behaviours that make the medfly an important invader. It can be learnt from medfly, that invasions in a modern global trade network tend to be due to multiple introductions. This fact allows a maintenance or enhancement of genetic variability in the adventive populations, which in turn increases their potential invasiveness. Our current knowledge of the medfly genome opens the way for future studies on functional genomics.

In many insect species, females can mate more than once and store sperm from more than one male. An assessment and understanding of polyandry in the field can be important for pest species with a high colonization potential, such as the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), which is also highly polyphagous and among the most destructive agricultural insects. The use of polymorphic microsatellite markers, combined with different statistical approaches, provides evidence that polyandry occurs in two C. capitata natural populations, one population from the Greek island of Chios and one population from Rehovot, in Israel. The observed different level of polyandry is discussed in relation to the genetic diversity, seasonality, and demography of the two populations. When polyandry is present, paternity analysis also indicates that one male, presumably the last, tends to sire most of the progeny. Polyandry and paternity skew may have important implications for the evolution of the species, in terms of maintenance of the genetic variability. Moreover, these aspects of the mating behavior, i.e., remating frequency and paternity skew, may locally affect the sterile insect technique, the most commonly applied control strategy against C. capitata.

We report here the molecular characterization of the female-specific FST (female-specific transcript) genes from the Mediterranean fruit fly (medfly) Ceratitis capitata. A genomic clone was isolated, containing a sequence coding for FST. Nucleotide analysis of the clone showed that the gene contains a putative unique intron located in the region encoding the signal peptide. Southern blotting and in situ hybridization analysis on polytene chromosomes suggested the presence of additional genes similar to FST in the genome of the medfly. A novel cDNA clone was isolated from an accessory gland cDNA library, encoding a product that shares 98% identity with the hypothetical translational product of the previously isolated FST cDNA. The novel cDNA was therefore named FST2. The analysis of mitotic and polytene chromosomes by in situ hybridization showed that FST genes map on the left arm of the 4th chromosome of C. capitata.Key words: FST, female-specific genes, C. capitata, medfly, FISH.

As a result of their rapid expansion and large larval host range, true fruit flies are among the world's most important agricultural pest species. Among them, Ceratitis capitata has become a model organism for studies on colonization and invasion processes. The genetic aspects of the medfly invasion process have already been analysed throughout its range, with the exception of Australia. Bioinvasion into Australia is an old event: medfly were first captured in Australia in 1895, near Perth. After briefly appearing in Tasmania and the eastern states of mainland Australia, medfly had disappeared from these areas by the 1940s. Currently, they are confined to the western coastal region. South Australia seems to be protected from medfly infestations both by the presence of an inhospitable barrier separating it from the west and by the limited number of transport routes. However, numerous medfly outbreaks have occurred since 1946, mainly near Adelaide. Allele frequency data at 10 simple sequence repeat loci were used to study the genetic structure of Australian medflies, to infer the historical pattern of invasion and the origin of the recent outbreaks. The combination of phylogeographical analysis and Bayesian tests showed that colonization of Australia was a secondary colonization event from the Mediterranean basin and that Australian medflies were unlikely to be the source for the initial Hawaiian invasion. Within Australia, the Perth area acted as the core range and was the source for medfly bioinvasion in both Western and South Australia. Incipient differentiation, as a result of habitat fragmentation, was detected in some localized areas at the periphery of the core range.

The medfly genome has been shown to contain a rich assortment of transposable elements from the mariner, Tc1, hAT and gypsy/Ty3 families. These elements display different levels of diversity, abundance and distribution in the genome. The presence of actively transposing elements in the medfly genome is revealed by hybrid dysgenesis phenomena, insertion site polymorphisms and other genetic instabilities. The medfly has been a target of transformation studies involving the exogenous elements Minos, Hermes and piggyBac from three families. The presence of active endogenous homologous elements can have important implications for the stability of such transgenic lines. The potential applications of endogenous elements for medfly population analysis and control are discussed.

The possibility to cross-species amplify microsatellites in fruit flies of the genusCeratitiswas tested with the polymerase chain reaction (PCR) by analysing 23Ceratitis capitata(Wiedemann) microsatellite markers on the genomic DNA of three other economically important, congeneric species:C. rosa(Karsch),C. fasciventris(Bezzi) andC. cosyra(Walker). Twenty-two primer pairs produced amplification products in at least one of the three species tested. The majority of the products were similar, if not identical in size to those expected inC. capitata. The structures of the repeat motifs and their flanking sequences were examined for a total of 79 alleles from the three species. Sequence analysis revealed the same repeat type as the homologousC. capitatamicrosatellites in the majority of the loci, suggesting their utility for population analysis across the species range. A total of seven loci were differentially present/absent inC. capitata,C. rosa,C. fasciventrisandC. cosyra, suggesting that it may be possible to differentiate these four species using a simple sequence repeat-based PCR assay. It is proposed that medfly-based microsatellite markers could be utilized in the identification and tracing of the geographical origins of colonist pest populations of the four tested species and in the assessment of their risk and invasive potentials; thereby assisting regulatory authorities in implementing quarantine restrictions and other pest control measures.