Evolutionary Plasticity in Insular Lizard, Adapting over Reproduction, Metabolism, and Color Variation.

The Italian wall lizard (Podarcis siculus) living on islets exhibits a melanic skin coloration and a suite of adaptive traits lacking in nearby mainland populations. On islets, the unpredictable environmental conditions and highly fluctuating population densities are believed to have produced reversed island syndrome (RIS). Several physiological, behavioral, and life-history changes based on the RIS could result from positive selection on increased activity of melanocortins. We hypothesize that phenotypes on islets are the product of a plastic variation depending on the regulation of specific genes. Focusing on control systems that determine the insular-adapted phenotype, we demonstrated that reproductive markers, involved in the hypothalamus-hypophysis-gonadal axis, and metabolism markers, flags for hypophysis-melanocortin receptors, are all up-regulated in island lizards under the RIS. This behavior, combined with the observed limited variation in the mitochondrial genome, agrees with the hypothesis that plasticity enables populations to persist in novel environmental conditions and that over time, natural selection will "fine-tune" the population to the environment by modifying the phenotype under selection. We believe that analysis of the transcriptome and the single gene expression, such that all the variations observed in the island populations, can be useful to shed light on evolutionary plasticity as a process affecting animals' populations in general.

Female Sex Determination Factors in Ceratitis capitata: Molecular and Structural Basis of TRA and TRA2 Recognition.

In the model system for genetics, Drosophila melanogaster, sexual differentiation and male courtship behavior are controlled by sex-specific splicing of doublesex (dsx) and fruitless (fru). In vitro and in vivo studies showed that female-specific Transformer (TRA) and the non-sex-specific Transformer 2 (TRA2) splicing factors interact, forming a complex promoting dsx and fru female-specific splicing. TRA/TRA2 complex binds to 13 nt long sequence repeats in their pre-mRNAs. In the Mediterranean fruitfly Ceratitis capitata (Medfly), a major agricultural pest, which shares with Drosophila a ~120 million years old ancestor, Cctra and Cctra2 genes seem to promote female-specific splicing of Ccdsx and Ccfru, which contain conserved TRA/TRA2 binding repeats. Unlike Drosophila tra, Cctra autoregulates its female-specific splicing through these putative regulatory repeats. Here, a yeast two-hybrid assay shows that CcTRA interacts with CcTRA2, despite its high amino acid divergence compared to Drosophila TRA. Interestingly, CcTRA2 interacts with itself, as also observed for Drosophila TRA2. We also generated a three-dimensional model of the complex formed by CcTRA and CcTRA2 using predictive approaches based on Artificial Intelligence. This structure also identified an evolutionary and highly conserved putative TRA2 recognition motif in the TRA sequence. The Y2H approach, combined with powerful predictive tools of three-dimensional protein structures, could use helpful also in this and other insect species to understand the potential links between different upstream proteins acting as primary sex-determining signals and the conserved TRA and TRA2 transducers.

The draft genome sequence of the Japanese rhinoceros beetle Trypoxylus dichotomus septentrionalis towards an understanding of horn formation.

The Japanese rhinoceros beetle Trypoxylus dichotomus is a giant beetle with distinctive exaggerated horns present on the head and prothoracic regions of the male. T. dichotomus has been used as a research model in various fields such as evolutionary developmental biology, ecology, ethology, biomimetics, and drug discovery. In this study, de novo assembly of 615 Mb, representing 80% of the genome estimated by flow cytometry, was obtained using the 10 × Chromium platform. The scaffold N50 length of the genome assembly was 8.02 Mb, with repetitive elements predicted to comprise 49.5% of the assembly. In total, 23,987 protein-coding genes were predicted in the genome. In addition, de novo assembly of the mitochondrial genome yielded a contig of 20,217 bp. We also analyzed the transcriptome by generating 16 RNA-seq libraries from a variety of tissues of both sexes and developmental stages, which allowed us to identify 13 co-expressed gene modules. We focused on the genes related to horn formation and obtained new insights into the evolution of the gene repertoire and sexual dimorphism as exemplified by the sex-specific splicing pattern of the doublesex gene. This genomic information will be an excellent resource for further functional and evolutionary analyses, including the evolutionary origin and genetic regulation of beetle horns and the molecular mechanisms underlying sexual dimorphism.

Characterisation of the antiviral RNA interference response to Toscana virus in sand fly cells.

Toscana virus (TOSV) (Bunyavirales, Phenuiviridae, Phlebovirus, Toscana phlebovirus) and other related human pathogenic arboviruses are transmitted by phlebotomine sand flies. TOSV has been reported in nations bordering the Mediterranean Sea among other regions. Infection can result in febrile illness as well as meningitis and encephalitis. Understanding vector-arbovirus interactions is crucial to improving our knowledge of how arboviruses spread, and in this context, immune responses that control viral replication play a significant role. Extensive research has been conducted on mosquito vector immunity against arboviruses, with RNA interference (RNAi) and specifically the exogenous siRNA (exo-siRNA) pathway playing a critical role. However, the antiviral immunity of phlebotomine sand flies is less well understood. Here we were able to show that the exo-siRNA pathway is active in a Phlebotomus papatasi-derived cell line. Following TOSV infection, distinctive 21 nucleotide virus-derived small interfering RNAs (vsiRNAs) were detected. We also identified the exo-siRNA effector Ago2 in this cell line, and silencing its expression rendered the exo-siRNA pathway largely inactive. Thus, our data show that this pathway is active as an antiviral response against a sand fly transmitted bunyavirus, TOSV.

Rat and fish peripheral blood leukocytes respond distinctively to Anisakis pegreffii (Nematoda, Anisakidae) crude extract.

Infective third-stage larvae (L3) of the marine nematode Anisakis pegreffii cause inflammation and clinical symptoms in humans, their accidental host, that subside and self-resolve in a couple of weeks after L3 die. To characterise the differences in an early immune response of a marine vs. terrestrial host, we stimulated peripheral blood leukocytes (PBLs) of fish (paratenic host) and rat (accidental, human-model host) with A. pegreffii crude extract and analysed PBL transcriptomes 1 and 12 h post-stimulation. Fish and rat PBLs differentially expressed 712 and 493 transcripts, respectively, between 1 and 12 h post-stimulation (false discovery rate, FDR <0.001, logFC >2). While there was a difference in the highest upregulated transcripts between two time-points, the same Gene Ontologies, biological processes (intracellular signal transduction, DNA-dependent transcription, and DNA-regulated regulation of transcription), and molecular functions (ATP and metal ion binding) were enriched in the two hosts, showing an incrementing dynamic between 1 and 12 h. This suggests that the two distinct hosts employ qualitatively different transcript cascades only to achieve the same effect, at least during an early innate immunity response. Activation of later immunity elements and/or a combination of other host's intrinsic conditions may contribute to the death of L3 in the terrestrial host.

Unraveling the role of male reproductive tract and haemolymph in cantharidin-exuding Lydus trimaculatus and Mylabris variabilis (Coleoptera: Meloidae): a comparative transcriptomics approach.

BACKGROUND: Meloidae (blister beetles) are known to synthetize cantharidin (CA), a toxic and defensive terpene mainly stored in male accessory glands (MAG) and emitted outward through reflex-bleeding. Recent progresses in understanding CA biosynthesis and production organ(s) in Meloidae have been made, but the way in which self-protection is achieved from the hazardous accumulation and release of CA in blister beetles has been experimentally neglected. To provide hints on this pending question, a comparative de novo assembly transcriptomic approach was performed by targeting two tissues where CA is largely accumulated and regularly circulates in Meloidae: the male reproductive tract (MRT) and the haemolymph. Differential gene expression profiles in these tissues were examined in two blister beetle species, Lydus trimaculatus (Fabricius, 1775) (tribe Lyttini) and Mylabris variabilis (Pallas, 1781) (tribe Mylabrini). Upregulated transcripts were compared between the two species to identify conserved genes possibly involved in CA detoxification and transport. RESULTS: Based on our results, we hypothesize that, to avoid auto-intoxication, ABC, MFS or other solute transporters might sequester purported glycosylated CA precursors into MAG, and lipocalins could bind CA and mitigate its reactivity when released into the haemolymph during the autohaemorrhaging response. We also found an over-representation in haemolymph of protein-domains related to coagulation and integument repairing mechanisms that likely reflects the need to limit fluid loss during reflex-bleeding. CONCLUSIONS: The de novo assembled transcriptomes of L. trimaculatus and M. variabilis here provided represent valuable genetic resources to further explore the mechanisms employed to cope with toxicity of CA in blister beetle tissues. These, if revealed, might help conceiving safe and effective drug-delivery approaches to enhance the use of CA in medicine.

Aedes albopictus bionomics data collection by citizen participation on Procida Island, a promising Mediterranean site for the assessment of innovative and community-based integrated pest management methods.

In the last decades, the colonization of Mediterranean Europe and of other temperate regions by Aedes albopictus created an unprecedented nuisance problem in highly infested areas and new public health threats due to the vector competence of the species. The Sterile Insect Technique (SIT) and the Incompatible Insect Technique (IIT) are insecticide-free mosquito-control methods, relying on mass release of irradiated/manipulated males, able to complement existing and only partially effective control tools. The validation of these approaches in the field requires appropriate experimental settings, possibly isolated to avoid mosquito immigration from other infested areas, and preliminary ecological and entomological data. We carried out a 4-year study in the island of Procida (Gulf of Naples, Italy) in strict collaboration with local administrators and citizens to estimate the temporal dynamics, spatial distribution, and population size of Ae. albopictus and the dispersal and survival of irradiated males. We applied ovitrap monitoring, geo-spatial analyses, mark-release-recapture technique, and a citizen-science approach. Results allow to predict the seasonal (from April to October, with peaks of 928-9,757 males/ha) and spatial distribution of the species, highlighting the capacity of Ae. albopictus population of Procida to colonize and maintain high frequencies in urban as well as in sylvatic inhabited environments. Irradiated males shown limited ability to disperse (mean daily distance travelled <60m) and daily survival estimates ranging between 0.80 and 0.95. Overall, the ecological characteristics of the island, the acquired knowledge on Ae. albopictus spatial and temporal distribution, the high human and Ae. albopictus densities and the positive attitude of the resident population in being active parts in innovative mosquito control projects provide the ground for evidence-based planning of the interventions and for the assessment of their effectiveness. In addition, the results highlight the value of creating synergies between research groups, local administrators, and citizens for affordable monitoring (and, in the future, control) of mosquito populations.

Correction: Fixation of genetic variation and optimization of gene expression: The speed of evolution in isolated lizard populations undergoing Reverse Island Syndrome.

[This corrects the article DOI: 10.1371/journal.pone.0224607.].

Targeting the autosomal Ceratitis capitata transformer gene using Cas9 or dCas9 to masculinize XX individuals without inducing mutations.

BACKGROUND: Females of the Mediterranean fruit fly Ceratitis capitata (Medfly) are major agricultural pests, as they lay eggs into the fruit crops of hundreds of plant species. In Medfly, female sex determination is based on the activation of Cctransformer (Cctra). A maternal contribution of Cctra is required to activate Cctra itself in the XX embryos and to start and epigenetically maintain a Cctra positive feedback loop, by female-specific alternative splicing, leading to female development. In XY embryos, the male determining Maleness-on-the-Y gene (MoY) blocks this activation and Cctra produces male-specific transcripts encoding truncated CcTRA isoforms and male differentiation occurs. RESULTS: With the aim of inducing frameshift mutations in the first coding exon to disrupt both female-specific and shorter male-specific CcTRA open reading frames (ORF), we injected Cas9 ribonucleoproteins (Cas9 and single guide RNA, sgRNA) in embryos. As this approach leads to mostly monoallelic mutations, masculinization was expected only in G1 XX individuals carrying biallelic mutations, following crosses of G0 injected individuals. Surprisingly, these injections into XX-only embryos led to G0 adults that included not only XX females but also 50% of reverted fertile XX males. The G0 XX males expressed male-specific Cctra transcripts, suggesting full masculinization. Interestingly, out of six G0 XX males, four displayed the Cctra wild type sequence. This finding suggests that masculinization by Cas9-sgRNA injections was independent from its mutagenic activity. In line with this observation, embryonic targeting of Cctra in XX embryos by a dead Cas9 (enzymatically inactive, dCas9) also favoured a male-specific splicing of Cctra, in both embryos and adults. CONCLUSIONS: Our data suggest that the establishment of Cctra female-specific autoregulation during the early embryogenesis has been repressed in XX embryos by the transient binding of the Cas9-sgRNA on the first exon of the Cctra gene. This hypothesis is supported by the observation that the shift of Cctra splicing from female to male mode is induced also by dCas9. Collectively, the present findings corroborate the idea that a transient embryonic inactivation of Cctra is sufficient for male sex determination.

ZanzaMapp: A Scalable Citizen Science Tool to Monitor Perception of Mosquito Abundance and Nuisance in Italy and Beyond.

Mosquitoes represent a considerable nuisance and are actual/potential vectors of human diseases in Europe. Costly and labour-intensive entomological monitoring is needed to correct planning of interventions aimed at reducing nuisance and the risk of pathogen transmission. The widespread availability of mobile phones and of massive Internet connections opens the way to the contribution of citizen in complementing entomological monitoring. ZanzaMapp is the first mobile "mosquito" application for smartphones specifically designed to assess citizens' perception of mosquito abundance and nuisance in Italy. Differently from other applications targeting mosquitoes, ZanzaMapp prioritizes the number of records over their scientific authentication by requesting users to answer four simple questions on perceived mosquito presence/abundance/nuisance and geo-localizing the records. The paper analyses 36,867 ZanzaMapp records sent by 13,669 devices from 2016 to 2018 and discusses the results with reference to either citizens' exploitation and appreciation of the app and to the consistency of the results obtained with the known biology of main mosquito species in Italy. In addition, we provide a first small-scale validation of ZanzaMapp data as predictors of Aedes albopictus biting females and examples of spatial analyses and maps which could be exploited by public institutions and administrations involved in mosquito and mosquito-borne pathogen monitoring and control.

Comparative Transcriptomics Reveals Clues for Differences in Pathogenicity between Hysterothylacium aduncum, Anisakissimplex sensu stricto and Anisakis pegreffii.

Ascaridoid nematodes are widespread in marine fishes. Despite their major socioeconomic importance, mechanisms associated to the fish-borne zoonotic disease anisakiasis are still obscure. RNA-Seq and de-novo assembly were herein applied to RNA extracted from larvae and dissected pharynx of Hysterothylacium aduncum (HA), a non-pathogenic nematode. Assembled transcripts in HA were annotated and compared to the transcriptomes of the zoonotic species Anisakis simplex sensu stricto (AS) and Anisakis pegreffii (AP). Approximately 60,000,000 single-end reads were generated for HA, AS and AP. Transcripts in HA encoded for 30,254 putative peptides while AS and AP encoded for 20,574 and 20,840 putative peptides, respectively. Differential gene expression analyses yielded 471, 612 and 526 transcripts up regulated in the pharynx of HA, AS and AP. The transcriptomes of larvae and pharynx of HA were enriched in transcripts encoding collagen, peptidases, ribosomal proteins and in heat-shock motifs. Transcripts encoding proteolytic enzymes, anesthetics, inhibitors of primary hemostasis and virulence factors, anticoagulants and immunomodulatory peptides were up-regulated in AS and AP pharynx. This study represents the first transcriptomic characterization of a marine parasitic nematode commonly recovered in fish and probably of negligible concern for public health.

Evolutionary Conservation of the Orchid MYB Transcription Factors DIV, RAD, and DRIF.

The MYB transcription factors DIVARICATA (DIV), DIV-and-RAD-Interacting-Factor (DRIF), and the small interfering peptide RADIALIS (RAD) can interact, forming a regulatory module that controls different plant developmental processes. In the snapdragon Antirrhinum majus, this module, together with the TCP transcription factor CYCLOIDEA (CYC), is responsible for the establishment of floral dorsoventral asymmetry. The spatial gene expression pattern of the OitDIV, OitDRIF, and OitRAD homologs of Orchis italica, an orchid with zygomorphic flowers, has suggested a possible conserved role of these genes in bilateral symmetry of the orchid flower. Here, we have identified four DRIF genes of orchids and have reconstructed their genomic organization and evolution. In addition, we found snapdragon transcriptional cis-regulatory elements of DIV and RAD loci generally conserved within the corresponding orchid orthologues. We have tested the biochemical interactions among OitDIV, OitDRIF1, and OitRAD of O. italica, showing that OitDRIF1 can interact both with OitDIV and OitRAD, whereas OitDIV and OitRAD do not directly interact, as in A. majus. The analysis of the quantitative expression profile of these MYB genes revealed that in zygomorphic orchid flowers, the DIV, DRIF1, and RAD transcripts are present at higher levels in the lip than in lateral inner tepals, whereas in peloric orchid flowers they show similar expression levels. These results indicate that MYB transcription factors could have a role in shaping zygomorphy of the orchid flower, potentially enriching the underlying orchid developmental code.

Fixation of genetic variation and optimization of gene expression: The speed of evolution in isolated lizard populations undergoing Reverse Island Syndrome.

The ecological theory of island biogeography suggests that mainland populations should be more genetically divergent from those on large and distant islands rather than from those on small and close islets. Some island populations do not evolve in a linear way, but the process of divergence occurs more rapidly because they undergo a series of phenotypic changes, jointly known as the Island Syndrome. A special case is Reversed Island Syndrome (RIS), in which populations show drastic phenotypic changes both in body shape, skin colouration, age of sexual maturity, aggressiveness, and food intake rates. The populations showing the RIS were observed on islets nearby mainland and recently raised, and for this they are useful models to study the occurrence of rapid evolutionary change. We investigated the timing and mode of evolution of lizard populations adapted through selection on small islets. For our analyses, we used an ad hoc model system of three populations: wild-type lizards from the mainland and insular lizards from a big island (Capri, Italy), both Podarcis siculus siculus not affected by the syndrome, and a lizard population from islet (Scopolo) undergoing the RIS (called P. s. coerulea because of their melanism). The split time of the big (Capri) and small (Scopolo) islands was determined using geological events, like sea-level rises. To infer molecular evolution, we compared five complete mitochondrial genomes for each population to reconstruct the phylogeography and estimate the divergence time between island and mainland lizards. We found a lower mitochondrial mutation rate in Scopolo lizards despite the phenotypic changes achieved in approximately 8,000 years. Furthermore, transcriptome analyses showed significant differential gene expression between islet and mainland lizard populations, suggesting the key role of plasticity in these unpredictable environments.

Maleness-on-the-Y (MoY) orchestrates male sex determination in major agricultural fruit fly pests.

In insects, rapidly evolving primary sex-determining signals are transduced by a conserved regulatory module controlling sexual differentiation. In the agricultural pest Ceratitis capitata (Mediterranean fruit fly, or Medfly), we identified a Y-linked gene, Maleness-on-the-Y (MoY), encoding a small protein that is necessary and sufficient for male development. Silencing or disruption of MoY in XY embryos causes feminization, whereas overexpression of MoY in XX embryos induces masculinization. Crosses between transformed XY females and XX males give rise to males and females, indicating that a Y chromosome can be transmitted by XY females. MoY is Y-linked and functionally conserved in other species of the Tephritidae family, highlighting its potential to serve as a tool for developing more effective control strategies against these major agricultural insect pests.

Identification of sex determination genes and their evolution in Phlebotominae sand flies (Diptera, Nematocera).

BACKGROUND: Phlebotomine sand flies (Diptera, Nematocera) are important vectors of several pathogens, including Leishmania parasites, causing serious diseases of humans and dogs. Despite their importance as disease vectors, most aspects of sand fly biology remain unknown including the molecular basis of their reproduction and sex determination, aspects also relevant for the development of novel vector control strategies. RESULTS: Using comparative genomics/transcriptomics data mining and transcriptional profiling, we identified the sex determining genes in phlebotomine sand flies and proposed the first model for the sex determination cascade of these insects. For all the genes identified, we produced manually curated gene models, developmental gene expression profile and performed evolutionary molecular analysis. We identified and characterized, for the first time in a Nematocera species, the transformer (tra) homolog which exhibits both conserved and novel features. The analysis of the tra locus in sand flies and its expression pattern suggest that this gene is able to autoregulate its own splicing, as observed in the fruit fly Ceratitis capitata and several other insect species. CONCLUSIONS: Our results permit to fill the gap about sex determination in sand flies, contribute to a better understanding of this developmental pathway in Nematocera and open the way for the identification of sex determining orthologs in other species of this important Diptera sub-order. Furthermore, the sex determination genes identified in our work also provide the opportunity of future biotechnological applications to control natural population of sand flies, reducing their impact on public health.

Positive selection in Europeans and East-Asians at the ABCA12 gene.

Natural selection acts on genetic variants by increasing the frequency of alleles responsible for a cellular function that is favorable in a certain environment. In a previous genome-wide scan for positive selection in contemporary humans, we identified a signal of positive selection in European and Asians at the genetic variant rs10180970. The variant is located in the second intron of the ABCA12 gene, which is implicated in the lipid barrier formation and down-regulated by UVB radiation. We studied the signal of selection in the genomic region surrounding rs10180970 in a larger dataset that includes DNA sequences from ancient samples. We also investigated the functional consequences of gene expression of the alleles of rs10180970 and another genetic variant in its proximity in healthy volunteers exposed to similar UV radiation. We confirmed the selection signal and refine its location that extends over 35 kb and includes the first intron, the first two exons and the transcription starting site of ABCA12. We found no obvious effect of rs10180970 alleles on ABCA12 gene expression. We reconstructed the trajectory of the T allele over the last 80,000 years to discover that it was specific to H. sapiens and present in non-Africans 45,000 years ago.

Tissue-specific transcriptomes of Anisakis simplex (sensu stricto) and Anisakis pegreffii reveal potential molecular mechanisms involved in pathogenicity.

BACKGROUND: Larval stages of the sibling species of parasitic nematodes Anisakis simplex (sensu stricto) (s.s.) (AS) and Anisakis pegreffii (AP) are responsible for a fish-borne zoonosis, known as anisakiasis, that humans aquire via the ingestion of raw or undercooked infected fish or fish-based products. These two species differ in geographical distribution, genetic background and peculiar traits involved in pathogenicity. However, thus far little is known of key molecules potentially involved in host-parasite interactions. Here, high-throughput RNA-Seq and bioinformatics analyses of sequence data were applied to the characterization of the whole sets of transcripts expressed by infective larvae of AS and AP, as well as of their pharyngeal tissues, in a bid to identify transcripts potentially involved in tissue invasion and host-pathogen interplay. RESULTS: Approximately 34,000,000 single-end reads were generated from cDNA libraries for each species. Transcripts identified in AS and AP encoded 19,403 and 10,424 putative peptides, respectively, and were classified based on homology searches, protein motifs, gene ontology and biological pathway mapping. Differential gene expression analysis yielded 226 and 339 transcripts upregulated in the pharyngeal regions of AS and AP, respectively, compared with their corresponding whole-larvae datasets. These included proteolytic enzymes, molecules encoding anesthetics, inhibitors of primary hemostasis and virulence factors, anticoagulants and immunomodulatory peptides. CONCLUSIONS: This work provides the scientific community with a list of key transcripts expressed by AS and AP pharyngeal tissues and corresponding annotation information which represents a ready-to-use resource for future functional studies of biological pathways specifically involved in host-parasite interplay.

First evidence of resistance to pyrethroid insecticides in Italian Aedes albopictus populations 26 years after invasion.

BACKGROUND: Aedes albopictus has spread during the last few decades all over the world. This has increased significantly the risk of exotic arbovirus transmission (e.g. chikungunya, dengue, and Zika) also in temperate areas, as demonstrated by the Chikungunya 2007 and 2017 outbreaks in northeastern and central Italy. Insecticides are an important tool for limiting the circulation of these mosquito-borne viruses. The aim of the present study was to address the gap in current knowledge of pyrethroid insecticide resistance of European Ae. albopictus populations, focusing on populations from Italy, Albania and Greece. RESULTS: Bioassays for resistance to permethrin (0.75%), α-cypermethrin (0.05%) or deltamethrin (0.05%) were performed according to World Health Organization (WHO) protocols and showed reduced susceptibility (<90% mortality) of some Italian populations to permethrin and α-cypermethrin, but not to deltamethrin. CONCLUSION: This study reports the first evidence of resistance to pyrethroids in adult Italian Ae. albopictus populations. Results refer to the season preceding the Chikungunya 2017 outbreak in central Italy and highlight the need to increase efforts to monitor the spread of insecticide resistance and the need to develop strategies to limit the spread of insecticide resistance, particularly in areas where extensive treatments have been carried out to contain disease outbreaks. © 2017 Society of Chemical Industry.

Deciphering the olfactory repertoire of the tiger mosquito Aedes albopictus.

BACKGROUND: The Asian tiger mosquito Aedes albopictus is a highly invasive species and competent vector of several arboviruses (e.g. dengue, chikungunya, Zika) and parasites (e.g. dirofilaria) of public health importance. Compared to other mosquito species, Ae. albopictus females exhibit a generalist host seeking as well as a very aggressive biting behaviour that are responsible for its high degree of nuisance. Several complex mosquito behaviours such as host seeking, feeding, mating or oviposition rely on olfactory stimuli that target a range of sensory neurons localized mainly on specialized head appendages such as antennae, maxillary palps and the mouthparts. RESULTS: With the aim to describe the Ae. albopictus olfactory repertoire we have used RNA-seq to reveal the transcriptome profiles of female antennae and maxillary palps. Male heads and whole female bodies were employed as reference for differential expression analysis. The relative transcript abundance within each tissue (TPM, transcripts per kilobase per million) and the pairwise differential abundance in the different tissues (fold change values and false discovery rates) were evaluated. Contigs upregulated in the antennae (620) and maxillary palps (268) were identified and relative GO and PFAM enrichment profiles analysed. Chemosensory genes were described: overall, 77 odorant binding proteins (OBP), 82 odorant receptors (OR), 60 ionotropic receptors (IR) and 30 gustatory receptors (GR) were identified by comparative genomics and transcriptomics. In addition, orthologs of genes expressed in the female/male maxillary palps and/or antennae and involved in thermosensation (e.g. pyrexia and arrestin1), mechanosensation (e.g. piezo and painless) and neuromodulation were classified. CONCLUSIONS: We provide here the first detailed transcriptome of the main Ae. albopictus sensory appendages, i.e. antennae and maxillary palps. A deeper knowledge of the olfactory repertoire of the tiger mosquito will help to better understand its biology and may pave the way to design new attractants/repellents.

Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein complexes.

The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economic impact and has become an emerging model for developing new genetic control strategies as an alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific single guide RNAs (sgRNA) into early embryos. When targeting the eye pigmentation gene white eye (we), a high rate of somatic mosaicism in surviving G0 adults was observed. Germline transmission rate of mutated we alleles by G0 animals was on average above 52%, with individual cases achieving nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (Ccprd) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end-joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations in C. capitata will significantly advance the design and development of new effective strategies for pest control management.