Optimizing CRE and PhiC31 mediated recombination in Aedes aegypti.

Introduction: Genetic manipulation of Aedes aegypti is key to developing a deeper understanding of this insects' biology, vector-virus interactions and makes future genetic control strategies possible. Despite some advances, this process remains laborious and requires highly skilled researchers and specialist equipment. Methods: Here we present two improved methods for genetic manipulation in this species. Use of transgenic lines which express Cre recombinase and a plasmid-based method for expressing PhiC31 when injected into early embryos. Results: Use of transgenic lines which express Cre recombinase allowed, by simple crossing schemes, germline or somatic recombination of transgenes, which could be utilized for numerous genetic manipulations. PhiC31 integrase based methods for site-specific integration of genetic elements was also improved, by developing a plasmid which expresses PhiC31 when injected into early embryos, eliminating the need to use costly and unstable mRNA as is the current standard. Discussion: Here we have expanded the toolbox for synthetic biology in Ae. aegypti. These methods can be easily transferred into other mosquito and even insect species by identifying appropriate promoter sequences. This advances the ability to manipulate these insects for fundamental studies, and for more applied approaches for pest control.

Silencing RNAs expressed from W-linked PxyMasc "retrocopies" target that gene during female sex determination in Plutella xylostella.

The Lepidoptera are an insect order of cultural, economic, and environmental importance, representing ∼10% of all described living species. Yet, for all but one of these species (silkmoth, Bombyx mori), the molecular genetics of how sexual fate is determined remains unknown. We investigated this in the diamondback moth (Plutella xylostella), a globally important, highly invasive, and economically damaging pest of cruciferous crops. Our previous work uncovered a regulator of male sex determination in P. xylostella-PxyMasc, a homolog of B. mori Masculinizer-which, although initially expressed in embryos of both sexes, is then reduced in female embryos, leading to female-specific splicing of doublesex. Here, through sequencing small RNA libraries generated from early embryos and sexed larval pools, we identified a variety of small silencing RNAs (predominantly Piwi-interacting RNAs [piRNAs]) complementary to PxyMasc, whose temporal expression correlated with the reduction in PxyMasc transcript observed previously in females. Analysis of these small RNAs showed that they are expressed from tandemly arranged, multicopy arrays found exclusively on the W (female-specific) chromosome, which we term "Pxyfem". Analysis of the Pxyfem sequences showed that they are partial complementary DNAs (cDNAs) of PxyMasc messenger RNA (mRNA) transcripts, likely integrated into transposable element graveyards by the noncanonical action of retrotransposons (retrocopies), and that their apparent similarity to B. mori feminizer more probably represents convergent evolution. Our study helps elucidate the sex determination cascade in this globally important pest and highlights the "shortcuts" that retrotransposition events can facilitate in the evolution of complex molecular cascades, including sex determination.

Intron-derived small RNAs for silencing viral RNAs in mosquito cells.

Aedes aegypti and Ae. albopictus are the main vectors of mosquito-borne viruses of medical and veterinary significance. Many of these viruses have RNA genomes. Exogenously provided, e.g. transgene encoded, small RNAs could be used to inhibit virus replication, breaking the transmission cycle. We tested, in Ae. aegypti and Ae. albopictus cell lines, reporter-based strategies for assessing the ability of two types of small RNAs to inhibit a chikungunya virus (CHIKV) derived target. Both types of small RNAs use a Drosophila melanogaster pre-miRNA-1 based hairpin for their expression, either with perfect base-pairing in the stem region (shRNA-like) or containing two mismatches (miRNA-like). The pre-miRNA-1 stem loop structure was encoded within an intron; this allows co-expression of one or more proteins, e.g. a fluorescent protein marker tracking the temporal and spatial expression of the small RNAs in vivo. Three reporter-based systems were used to assess the relative silencing efficiency of ten shRNA-like siRNAs and corresponding miRNA-like designs. Two systems used a luciferase reporter RNA with CHIKV RNA inserted either in the coding sequence or within the 3' UTR. A third reporter used a CHIKV derived split replication system. All three reporters demonstrated that while silencing could be achieved with both miRNA-like and shRNA-like designs, the latter were substantially more effective. Dcr-2 was required for the shRNA-like siRNAs as demonstrated by loss of inhibition of the reporters in Dcr-2 deficient cell lines. These positive results in cell culture are encouraging for the potential use of this pre-miRNA-1-based system in transgenic mosquitoes.

Insights into the Karyotype Evolution of Charinidae, the Early-Diverging Clade of Whip Spiders (Arachnida: Amblypygi).

Whip spiders (Amblypygi) represent an ancient order of tetrapulmonate arachnids with a low diversity. Their cytogenetic data are confined to only a few reports. Here, we analyzed the family Charinidae, a lineage almost at the base of the amblypygids, providing an insight into the ancestral traits and basic trajectories of amblypygid karyotype evolution. We performed Giemsa staining, selected banding techniques, and detected 18S ribosomal DNA and telomeric repeats by fluorescence in situ hybridization in four Charinus and five Sarax species. Both genera exhibit a wide range of diploid chromosome numbers (2n = 42-76 and 22-74 for Charinus and Sarax, respectively). The 2n reduction was accompanied by an increase of proportion of biarmed elements. We further revealed a single NOR site (probably an ancestral condition for charinids), the presence of a (TTAGG)n telomeric motif localized mostly at the chromosome ends, and an absence of heteromorphic sex chromosomes. Our data collectively suggest a high pace of karyotype repatterning in amblypygids, with probably a high ancestral 2n and its subsequent gradual reduction by fusions, and the action of pericentric inversions, similarly to what has been proposed for neoamblypygids. The possible contribution of fissions to charinid karyotype repatterning, however, cannot be fully ruled out.

Large-scale comparative analysis of cytogenetic markers across Lepidoptera.

Fluorescence in situ hybridization (FISH) allows identification of particular chromosomes and their rearrangements. Using FISH with signal enhancement via antibody amplification and enzymatically catalysed reporter deposition, we evaluated applicability of universal cytogenetic markers, namely 18S and 5S rDNA genes, U1 and U2 snRNA genes, and histone H3 genes, in the study of the karyotype evolution in moths and butterflies. Major rDNA underwent rather erratic evolution, which does not always reflect chromosomal changes. In contrast, the hybridization pattern of histone H3 genes was well conserved, reflecting the stable organisation of lepidopteran genomes. Unlike 5S rDNA and U1 and U2 snRNA genes which we failed to detect, except for 5S rDNA in a few representatives of early diverging lepidopteran lineages. To explain the negative FISH results, we used quantitative PCR and Southern hybridization to estimate the copy number and organization of the studied genes in selected species. The results suggested that their detection was hampered by long spacers between the genes and/or their scattered distribution. Our results question homology of 5S rDNA and U1 and U2 snRNA loci in comparative studies. We recommend the use of histone H3 in studies of karyotype evolution.

Cas13b-dependent and Cas13b-independent RNA knockdown of viral sequences in mosquito cells following guide RNA expression.

Aedes aegypti and Aedes albopictus mosquitoes are vectors of the RNA viruses chikungunya (CHIKV) and dengue that currently have no specific therapeutic treatments. The development of new methods to generate virus-refractory mosquitoes would be beneficial. Cas13b is an enzyme that uses RNA guides to target and cleave RNA molecules and has been reported to suppress RNA viruses in mammalian and plant cells. We investigated the potential use of the Prevotella sp. P5-125 Cas13b system to provide viral refractoriness in mosquito cells, using a virus-derived reporter and a CHIKV split replication system. Cas13b in combination with suitable guide RNAs could induce strong suppression of virus-derived reporter RNAs in insect cells. Surprisingly, the RNA guides alone (without Cas13b) also gave substantial suppression. Our study provides support for the potential use of Cas13b in mosquitoes, but also caution in interpreting CRISPR/Cas data as we show that guide RNAs can have Cas-independent effects.

The genetic architecture of a host shift: An adaptive walk protected an aphid and its endosymbiont from plant chemical defenses.

Host shifts can lead to ecological speciation and the emergence of new pests and pathogens. However, the mutational events that facilitate the exploitation of novel hosts are poorly understood. Here, we characterize an adaptive walk underpinning the host shift of the aphid Myzus persicae to tobacco, including evolution of mechanisms that overcame tobacco chemical defenses. A series of mutational events added as many as 1.5 million nucleotides to the genome of the tobacco-adapted subspecies, M. p. nicotianae, and yielded profound increases in expression of an enzyme that efficiently detoxifies nicotine, both in aphid gut tissue and in the bacteriocytes housing the obligate aphid symbiont Buchnera aphidicola. This dual evolutionary solution overcame the challenge of preserving fitness of a mutualistic symbiosis during adaptation to a toxic novel host. Our results reveal the intricate processes by which genetic novelty can arise and drive the evolution of key innovations required for ecological adaptation.

Sex Chromosome Turnover in Moths of the Diverse Superfamily Gelechioidea.

Sex chromosomes play a central role in genetics of speciation and their turnover was suggested to promote divergence. In vertebrates, sex chromosome-autosome fusions resulting in neo-sex chromosomes occur frequently in male heterogametic taxa (XX/XY), but are rare in groups with female heterogamety (WZ/ZZ). We examined sex chromosomes of seven pests of the diverse lepidopteran superfamily Gelechioidea and confirmed the presence of neo-sex chromosomes in their karyotypes. Two synteny blocks, which correspond to autosomes 7 (LG7) and 27 (LG27) in the ancestral lepidopteran karyotype exemplified by the linkage map of Biston betularia (Geometridae), were identified as sex-linked in the tomato leafminer, Tuta absoluta (Gelechiidae). Testing for sex-linkage performed in other species revealed that while LG7 fused to sex chromosomes in a common ancestor of all Gelechioidea, the second fusion between the resulting neo-sex chromosome and the other autosome is confined to the tribe Gnoreschemini (Gelechiinae). Our data accentuate an emerging pattern of high incidence of neo-sex chromosomes in Lepidoptera, the largest clade with WZ/ZZ sex chromosome system, which suggest that the paucity of neo-sex chromosomes is not an intrinsic feature of female heterogamety. Furthermore, LG7 contains one of the major clusters of UDP-glucosyltransferases, which are involved in the detoxification of plant secondary metabolites. Sex chromosome evolution in Gelechioidea thus supports an earlier hypothesis postulating that lepidopteran sex chromosome-autosome fusions can be driven by selection for association of Z-linked preference or host-independent isolation genes with larval performance and thus can contribute to ecological specialization and speciation of moths.

Recent advances in threshold-dependent gene drives for mosquitoes.

Mosquito-borne diseases, such as malaria, dengue and chikungunya, cause morbidity and mortality around the world. Recent advances in gene drives have produced control methods that could theoretically modify all populations of a disease vector, from a single release, making whole species less able to transmit pathogens. This ability has caused both excitement, at the prospect of global eradication of mosquito-borne diseases, and concern around safeguards. Drive mechanisms that require individuals to be released at high frequency before genes will spread can therefore be desirable as they are potentially localised and reversible. These include underdominance-based strategies and use of the reproductive parasite Wolbachia Here, we review recent advances in practical applications and mathematical analyses of these threshold-dependent gene drives with a focus on implementation in Aedes aegypti, highlighting their mechanisms and the role of fitness costs on introduction frequencies. Drawing on the parallels between these systems offers useful insights into practical, controlled application of localised drives, and allows us to assess the requirements needed for gene drive reversal.

Complementary sex determination in the parasitic wasp Diachasmimorpha longicaudata.

We studied the sex determination in Diachasmimorpha longicaudata, a parasitoid braconid wasp widely used as biological control agent of fruit pest tephritid flies. We tested the complementary sex determination hypothesis (CSD) known in at least 60 species of Hymenoptera. According to CSD, male or female development depends on the allelic composition of one sex locus (single-locus CSD) or multiple sex loci (multiple-locus CSD). Hemizygote individuals are normal haploid males, and heterozygotes for at least one sex locus are normal diploid females, but homozygotes for all the sex loci are diploid males. In order to force the occurrence of diploid males in D. longicaudata, we established highly inbred lines and examined their offspring using chromosome counting, flow cytometry, and sex ratio analysis. We found that when mother-son crosses were studied, this wasp produced about 20% of diploid males out of the total male progeny. Our results suggest that this parasitoid may represent the second genus with multiple-locus CSD in Hymenoptera. Knowledge about the sex determination system in D. longicaudata is relevant for the improvement of mass rearing protocols of this species. This information also provides the necessary background for further investigations on the underlying molecular mechanisms of sex determination in this species, and a better insight into the evolution of this pathway in Hymenoptera in particular and insects in general.

Mapping of single-copy genes by TSA-FISH in the codling moth, Cydia pomonella.

BACKGROUND: We work on the development of transgenic sexing strains in the codling moth, Cydia pomonella (Tortricidae), which would enable to produce male-only progeny for the population control of this pest using sterile insect technique (SIT). To facilitate this research, we have developed a number of cytogenetic and molecular tools, including a physical map of the codling moth Z chromosome using BAC-FISH (fluorescence in situ hybridization with bacterial artificial chromosome probes). However, chromosomal localization of unique, single-copy sequences such as a transgene cassette by conventional FISH remains challenging. In this study, we adapted a FISH protocol with tyramide signal amplification (TSA-FISH) for detection of single-copy genes in Lepidoptera. We tested the protocol with probes prepared from partial sequences of Z-linked genes in the codling moth. RESULTS: Using a modified TSA-FISH protocol we successfully mapped a partial sequence of the Acetylcholinesterase 1 (Ace-1) gene to the Z chromosome and confirmed thus its Z-linkage. A subsequent combination of BAC-FISH with BAC probes containing anticipated neighbouring Z-linked genes and TSA-FISH with the Ace-1 probe allowed the integration of Ace-1 in the physical map of the codling moth Z chromosome. We also developed a two-colour TSA-FISH protocol which enabled us simultaneous localization of two Z-linked genes, Ace-1 and Notch, to the expected regions of the Z chromosome. CONCLUSIONS: We showed that TSA-FISH represents a reliable technique for physical mapping of genes on chromosomes of moths and butterflies. Our results suggest that this technique can be combined with BAC-FISH and in the future used for physical localization of transgene cassettes on chromosomes of transgenic lines in the codling moth or other lepidopteran species. Furthermore, the developed protocol for two-colour TSA-FISH might become a powerful tool for synteny mapping in non-model organisms.