Whitefly-Plant Interactions: An Integrated Molecular Perspective.

The rapid advances in available transcriptomic and genomic data and our understanding of the physiology and biochemistry of whitefly-plant interactions have allowed us to gain new and significant insights into the biology of whiteflies and their successful adaptation to host plants. In this review, we provide a comprehensive overview of the mechanisms that whiteflies have evolved to overcome the challenges of feeding on phloem sap. We also highlight the evolution and functions of gene families involved in host perception, evaluation, and manipulation; primary metabolism; and metabolite detoxification. We discuss the emerging themes in plant immunity to whiteflies, focusing on whitefly effectors and their sites of action in plant defense-signaling pathways. We conclude with a discussion of advances in the genetic manipulation of whiteflies and the potential that they hold for exploring the interactions between whiteflies and their host plants, as well as the development of novel strategies for the genetic control of whiteflies.

Geographical survey of the mycobiome and microbiome of Southern California glassy-winged sharpshooters.

The glassy-winged sharpshooter, Homalodisca vitripennis Germar, is an invasive xylem-feeding leafhopper with a devastating economic impact on California agriculture through transmission of the plant pathogen, Xylella fastidiosa. While studies have focused on X. fastidiosa or known symbionts of H. vitripennis, little work has been done at the scale of the microbiome (the bacterial community) or mycobiome (the fungal community). Here, we characterize the mycobiome and the microbiome of H. vitripennis across Southern California and explore correlations with captivity and host insecticide resistance status. Using high-throughput sequencing of the ribosomal internal transcribed spacer 1 region and the 16S rRNA gene to profile the mycobiome and microbiome, respectively, we found that while the H. vitripennis mycobiome significantly varied across Southern California, the microbiome did not. We also observed a significant difference in both the mycobiome and microbiome between captive and wild H. vitripennis. Finally, we found that the mycobiome, but not the microbiome, was correlated with insecticide resistance status in wild H. vitripennis. This study serves as a foundational look at the H. vitripennis mycobiome and microbiome across Southern California. Future work should explore the putative link between microbes and insecticide resistance status and investigate whether microbial communities should be considered in H. vitripennis management practices. IMPORTANCE The glassy-winged sharpshooter is an invasive leafhopper that feeds on the xylem of plants and transmits the devastating pathogen, Xylella fastidiosa, resulting in significant economic damage to California's agricultural system. While studies have focused on this pathogen or obligate symbionts of the glassy-winged sharpshooter, there is limited knowledge of the bacterial and fungal communities that make up its microbiome and mycobiome. To address this knowledge gap, we explored the composition of the mycobiome and the microbiome of the glassy-winged sharpshooter across Southern California and identified differences associated with geography, captivity, and host insecticide resistance status. Understanding sources of variation in the microbial communities associated with the glassy-winged sharpshooter is an important consideration for developing management strategies to control this invasive insect. This study is a first step toward understanding the role microbes may play in the glassy-winged sharpshooter's resistance to insecticides.

Transcriptome and population structure of glassy-winged sharpshooters (Homalodisca vitripennis) with varying insecticide resistance in southern California.

BACKGROUND: Homalodisca vitripennis Germar, the glassy-winged sharpshooter, is an invasive insect in California and a critical threat to agriculture through its transmission of the plant pathogen, Xylella fastidiosa. Quarantine, broad-spectrum insecticides, and biological control have been used for population management of H. vitripennis since its invasion and subsequent proliferation throughout California. Recently wide-spread neonicotinoid resistance has been detected in populations of H. vitripennis in the southern portions of California's Central Valley. In order to better understand potential mechanisms of H. vitripennis neonicotinoid resistance, we performed RNA sequencing on wild-caught insecticide-resistant and relatively susceptible sharpshooters to profile their transcriptome and population structure. RESULTS: We identified 81 differentially expressed genes with higher expression in resistant individuals. The significant largest differentially expressed candidate gene linked to resistance status was a cytochrome P450 gene with similarity to CYP6A9. Furthermore, we observed an over-enrichment of GO terms representing functions supportive of roles in resistance mechanisms (cytochrome P450s, M13 peptidases, and cuticle structural proteins). Finally, we saw no evidence of broad-scale population structure, perhaps due to H. vitripennis' relatively recent introduction to California or due to the relatively small geographic scale investigated here. CONCLUSIONS: In this work, we characterized the transcriptome of insecticide-resistant and susceptible H. vitripennis and identified candidate genes that may be involved in resistance mechanisms for this species. Future work should seek to build on the transcriptome profiling performed here to confirm the role of the identified genes, particularly the cytochrome P450, in resistance in H. vitripennis. We hope this work helps aid future population management strategies for this and other species with growing insecticide resistance.

Gene Editing and Genetic Control of Hemipteran Pests: Progress, Challenges and Perspectives.

The origin of the order Hemiptera can be traced to the late Permian Period more than 230 MYA, well before the origin of flowering plants 100 MY later in during the Cretaceous period. Hemipteran species consume their liquid diets using a sucking proboscis; for phytophagous hemipterans their mouthparts (stylets) are elegant structures that enable voracious feeding from plant xylem or phloem. This adaptation has resulted in some hemipteran species becoming globally significant pests of agriculture resulting in significant annual crop losses. Due to the reliance on chemical insecticides for the control of insect pests in agricultural settings, many hemipteran pests have evolved resistance to insecticides resulting in an urgent need to develop new, species-specific and environmentally friendly methods of pest control. The rapid advances in CRISPR/Cas9 technologies in model insects such as Drosophila melanogaster, Tribolium castaneum, Bombyx mori, and Aedes aegypti has spurred a new round of innovative genetic control strategies in the Diptera and Lepidoptera and an increased interest in assessing genetic control technologies for the Hemiptera. Genetic control approaches in the Hemiptera have, to date, been largely overlooked due to the problems of introducing genetic material into the germline of these insects. The high frequency of CRISPR-mediated mutagenesis in model insect species suggest that, if the delivery problem for Hemiptera could be solved, then gene editing in the Hemiptera might be quickly achieved. Significant advances in CRISPR/Cas9 editing have been realized in nine species of Hemiptera over the past 4 years. Here we review progress in the Hemiptera and discuss the challenges and opportunities for extending contemporary genetic control strategies into species in this agriculturally important insect orderr.

Efficient CRISPR/Cas9-mediated genome modification of the glassy-winged sharpshooter Homalodisca vitripennis (Germar).

CRISPR/Cas9 technology enables the extension of genetic techniques into insect pests previously refractory to genetic analysis. We report the establishment of genetic analysis in the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, which is a significant leafhopper pest of agriculture in California. We use a novel and simple approach of embryo microinjection in situ on the host plant and obtain high frequency mutagenesis, in excess of 55%, of the cinnabar and white eye pigmentation loci. Through pair matings, we obtained 100% transmission of w and cn alleles to the G3 generation and also established that both genes are located on autosomes. Our analysis of wing phenotype revealed an unexpected discovery of the participation of pteridine pigments in wing and wing-vein coloration, indicating a role for these pigments beyond eye color. We used amplicon sequencing to examine the extent of off-target mutagenesis in adults arising from injected eggs, which was found to be negligible or non-existent. Our data show that GWSS can be easily developed as a genetic model system for the Hemiptera, enabling the study of traits that contribute to the success of invasive pests and vectors of plant pathogens. This will facilitate novel genetic control strategies.

Improved draft reference genome for the Glassy-winged Sharpshooter (Homalodisca vitripennis), a vector for Pierce's disease.

Homalodisca vitripennis (Hemiptera: Cicadellidae), known as the glassy-winged sharpshooter, is a xylem feeding leafhopper and an important agricultural pest as a vector of Xylella fastidiosa, which causes Pierce's disease in grapes and a variety of other scorch diseases. The current H. vitripennis reference genome from the Baylor College of Medicine's i5k pilot project is a 1.4-Gb assembly with 110,000 scaffolds, which still has significant gaps making identification of genes difficult. To improve on this effort, we used a combination of Oxford Nanopore long-read sequencing technology combined with Illumina sequencing reads to generate a better assembly and first-pass annotation of the whole genome sequence of a wild-caught Californian (Tulare County) individual of H. vitripennis. The improved reference genome assembly for H. vitripennis is 1.93-Gb in length (21,254 scaffolds, N50 = 650 Mb, BUSCO completeness = 94.3%), with 33.06% of the genome masked as repetitive. In total, 108,762 gene models were predicted including 98,296 protein-coding genes and 10,466 tRNA genes. As an additional community resource, we identified 27 orthologous candidate genes of interest for future experimental work including phenotypic marker genes like white. Furthermore, as part of the assembly process, we generated four endosymbiont metagenome-assembled genomes, including a high-quality near complete 1.7-Mb Wolbachia sp. genome (1 scaffold, CheckM completeness = 99.4%). The improved genome assembly and annotation for H. vitripennis, curated set of candidate genes, and endosymbiont MAGs will be invaluable resources for future research of H. vitripennis.

Can CRISPR gene drive work in pest and beneficial haplodiploid species?

Gene drives based on CRISPR/Cas9 have the potential to reduce the enormous harm inflicted by crop pests and insect vectors of human disease, as well as to bolster valued species. In contrast with extensive empirical and theoretical studies in diploid organisms, little is known about CRISPR gene drive in haplodiploids, despite their immense global impacts as pollinators, pests, natural enemies of pests, and invasive species in native habitats. Here, we analyze mathematical models demonstrating that, in principle, CRISPR homing gene drive can work in haplodiploids, as well as at sex-linked loci in diploids. However, relative to diploids, conditions favoring the spread of alleles deleterious to haplodiploid pests by CRISPR gene drive are narrower, the spread is slower, and resistance to the drive evolves faster. By contrast, the spread of alleles that impose little fitness cost or boost fitness was not greatly hindered in haplodiploids relative to diploids. Therefore, altering traits to minimize damage caused by harmful haplodiploids, such as interfering with transmission of plant pathogens, may be more likely to succeed than control efforts based on introducing traits that reduce pest fitness. Enhancing fitness of beneficial haplodiploids with CRISPR gene drive is also promising.

The whole genome sequence of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), reveals insights into the biology and adaptive evolution of a highly invasive pest species.

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 high-quality 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.

hAT Transposable Elements.

hAT transposons are ancient in their origin and they are widespread across eukaryote kingdoms. They can be present in large numbers in many genomes. However, only a few active forms of these elements have so far been discovered indicating that, like all transposable elements, there is selective pressure to inactivate them. Nonetheless, there have been sufficient numbers of active hAT elements and their transposases characterized that permit an analysis of their structure and function. This review analyzes these and provides a comparison with the several domesticated hAT genes discovered in eukaryote genomes. Active hAT transposons have also been developed as genetic tools and understanding how these may be optimally utilized in new hosts will depend, in part, on understanding the basis of their function in genomes.

Structural basis of hAT transposon end recognition by Hermes, an octameric DNA transposase from Musca domestica.

Hermes is a member of the hAT transposon superfamily that has active representatives, including McClintock's archetypal Ac mobile genetic element, in many eukaryotic species. The crystal structure of the Hermes transposase-DNA complex reveals that Hermes forms an octameric ring organized as a tetramer of dimers. Although isolated dimers are active in vitro for all the chemical steps of transposition, only octamers are active in vivo. The octamer can provide not only multiple specific DNA-binding domains to recognize repeated subterminal sequences within the transposon ends, which are important for activity, but also multiple nonspecific DNA binding surfaces for target capture. The unusual assembly explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and suggests how the avidity provided by multiple sites of interaction could allow a transposase to locate its transposon ends amidst a sea of chromosomal DNA.

IPB7 transposase behavior in Drosophila melanogaster and Aedes aegypti.

Transposons are used in insect science as genetic tools that enable the transformation of insects and the identification and isolation of genes though their ability to insert in or near to them. Four transposons, piggyBac, Mos1, Hermes and Minos are commonly used in insects beyond Drosophila melanogaster with piggyBac, due to its wide host range and frequency of transposition, being the most commonly chosen. The utility of these transposons as genetic tools is directly proportional to their activity since higher transposition rates would be expected to lead to higher transformation frequencies and higher frequencies of insertion throughout the genome. As a consequence there is an ongoing need for hyperactive transposases for use in insect genetics, however these have proven difficult to obtain. IPB7 is a hyperactive mutant of the piggyBac transposase that was identified by a genetic screen performed in yeast, a mammalian codon optimized version of which was then found to be highly active in rodent embryonic stem cells with no apparent deleterious effects. Here we report the activity of IPB7 in D. melanogaster and the mosquito, Aedes aegypti. Somatic transposition assays revealed an increase in IPB7's transposition rate from wild-type piggyBac transposase in D. melanogaster but not Ae. aegypti. However the use of IPB7 in D. melanogaster genetic transformations produced a high rate of sterility and a low transformation rate compared to wild-type transposase. This high rate of sterility was accompanied by significant gonadal atrophy that was also observed in the absence of the piggyBac vector transposon. We conclude that IPB7 has increased activity in the D. melanogaster germ-line but that a component of the sterility associated with its activity is independent of the presence of the piggyBac transposon.

A resurrected mammalian hAT transposable element and a closely related insect element are highly active in human cell culture.

Chromosome structure and function are influenced by transposable elements, which are mobile DNA segments that can move from place to place. hAT elements are a superfamily of DNA cut and paste elements that move by excision and integration. We have characterized two hAT elements, TcBuster and Space Invaders (SPIN), that are members of a recently described subfamily of hAT elements called Buster elements. We show that TcBuster, from the red flour beetle Tribolium castaneum, is highly active in human cells. SPIN elements are currently inactive elements that were recently highly active in multiple vertebrate genomes, and the high level of sequence similarity across widely diverged species and patchy phylogenetic distribution suggest that they may have moved between genomes by horizontal transfer. We have generated an intact version of this element, SPIN(ON), which is highly active in human cells. In vitro analysis of TcBuster and SPIN(ON) shows that no proteins other than transposase are essential for recombination, a property that may contribute to the ability of SPIN to successfully invade multiple organisms. We also analyze the target site preferences of de novo insertions in the human genome of TcBuster and SPIN(ON) and compare them with the preferences of Sleeping Beauty and piggyBac, showing that each superfamily has a distinctive pattern of insertion. The high-frequency transposition of both TcBuster and SPIN(ON) suggests that these transposon systems offer powerful tools for genome engineering. Finally, we describe a Saccharomyces cerevisiae assay for TcBuster that will provide a means for isolation of hyperactive and other interesting classes of transposase mutants.

Comparative analysis of the recently discovered hAT transposon TcBuster in human cells.

BACKGROUND: Transposons are useful tools for creating transgenic organisms, insertional mutagenesis, and genome engineering. TcBuster, a novel hAT-family transposon system derived from the red flour beetle Tribolium castaneum, was shown to be highly active in previous studies in insect embryoes. METHODOLOGY/PRINCIPAL FINDINGS: We tested TcBuster for its activity in human embryonic kidney 293 (HEK-293) cells. Excision footprints obtained from HEK-293 cells contained small insertions and deletions consistent with a hAT-type repair mechanism of hairpin formation and non-homologous end-joining. Genome-wide analysis of 23,417 piggyBac, 30,303 Sleeping Beauty, and 27,985 TcBuster integrations in HEK-293 cells revealed a uniquely different integration pattern when compared to other transposon systems with regards to genomic elements. TcBuster experimental conditions were optimized to assay TcBuster activity in HEK-293 cells by colony assay selection for a neomycin-containing transposon. Increasing transposon plasmid increased the number of colonies, whereas gene transfer activity dependent on codon-optimized transposase plasmid peaked at 100 ng with decreased colonies at the highest doses of transposase DNA. Expression of the related human proteins Buster1, Buster3, and SCAND3 in HEK-293 cells did not result in genomic integration of the TcBuster transposon. TcBuster, Tol2, and piggyBac were compared directly at different ratios of transposon to transposase and found to be approximately comparable while having their own ratio preferences. CONCLUSIONS/SIGNIFICANCE: TcBuster was found to be highly active in mammalian HEK-293 cells and represents a promising tool for mammalian genome engineering.

The mosquito Aedes aegypti has a large genome size and high transposable element load but contains a low proportion of transposon-specific piRNAs.

BACKGROUND: The piRNA pathway has been shown in model organisms to be involved in silencing of transposons thereby providing genome stability. In D. melanogaster the majority of piRNAs map to these sequences. The medically important mosquito species Aedes aegypti has a large genome size, a high transposon load which includes Miniature Inverted repeat Transposable Elements (MITES) and an expansion of the piRNA biogenesis genes. Studies of transgenic lines of Ae. aegypti have indicated that introduced transposons are poorly remobilized and we sought to explore the basis of this. We wished to analyze the piRNA profile of Ae. aegypti and thereby determine if it is responsible for transposon silencing in this mosquito. RESULTS: Estimated piRNA sequence diversity was comparable between Ae. aegypti and D. melanogaster, but surprisingly only 19% of mosquito piRNAs mapped to transposons compared to 51% for D. melanogaster. Ae. aegypti piRNA clusters made up a larger percentage of the total genome than those of D. melanogaster but did not contain significantly higher percentages of transposon derived sequences than other regions of the genome. Ae. aegypti contains a number of protein coding genes that may be sources of piRNA biogenesis with two, traffic jam and maelstrom, implicated in this process in model organisms. Several genes of viral origin were also targeted by piRNAs. Examination of six mosquito libraries that had previously been transformed with transposon derived sequence revealed that new piRNA sequences had been generated to the transformed sequences, suggesting that they may have stimulated a transposon inactivation mechanism. CONCLUSIONS: Ae. aegypti has a large piRNA complement that maps to transposons but primarily gene sequences, including many viral-derived sequences. This, together the more uniform distribution of piRNA clusters throughout its genome, suggest that some aspects of the piRNA system differ between Ae. aegypti and D. melanogaster.

DNA sequence requirements for hobo transposable element transposition in Drosophila melanogaster.

We have conducted a structure and functional analysis of the hobo transposable element of Drosophila melanogaster. A minimum of 141 bp of the left (L) end and 65 bp of the right (R) end of the hobo were shown to contain sequences sufficient for transposition. Both ends of hobo contain multiple copies of the motifs GGGTG and GTGGC and we show that the frequency of hobo transposition increases as a function of the copy number of these motifs. The R end of hobo contains a unique 12 bp internal inverted repeat that is identical to the hobo terminal inverted repeats. We show that this internal inverted repeat suppresses transposition activity in a hobo element containing an intact L end and only 475 bp of the R end. In addition to establishing cis-sequences requirements for transposition, we analyzed trans-sequence effects of the hobo transposase. We show a hobo transposase lacking the first 49 amino acids catalyzed hobo transposition at a higher frequency than the full-length transposase suggesting that, similar to the related Ac transposase, residues at the amino end of the transposase reduce transposition. Finally, we compared target site sequences of hobo with those of the related Hermes element and found both transposons have strong preferences for the same insertion sites.

DNA binding activities of the Herves transposase from the mosquito Anopheles gambiae.

BACKGROUND: Determining the mechanisms by which transposable elements move within a genome increases our understanding of how they can shape genome evolution. Class 2 transposable elements transpose via a 'cut-and-paste' mechanism mediated by a transposase that binds to sites at or near the ends of the transposon. Herves is a member of the hAT superfamily of class 2 transposons and was isolated from Anopheles gambiae, a medically important mosquito species that is the major vector of malaria in sub-Saharan Africa. Herves is transpositionally active and intact copies of it are found in field populations of A gambiae. In this study we report the binding activities of the Herves transposase to the sequences at the ends of the Herves transposon and compare these to other sequences recognized by hAT transposases isolated from other organisms. RESULTS: We identified the specific DNA-binding sites of the Herves transposase. Active Herves transposase was purified using an Escherichia coli expression system and bound in a site-specific manner to the subterminal and terminal sequences of the left and right ends of the element, respectively, and also interacted with the right but not the left terminal inverted repeat. We identified a common subterminal DNA-binding motif (CG/AATTCAT) that is critical and sufficient for Herves transposase binding. CONCLUSIONS: The Herves transposase binds specifically to a short motif located at both ends of the transposon but shows differential binding with respect to the left and right terminal inverted repeats. Despite similarities in the overall structures of hAT transposases, the regions to which they bind in their respective transposons differ in sequence ensuring the specificity of these enzymes to their respective transposon. The asymmetry with which the Herves terminal inverted repeats are bound by the transposase may indicate that these differ in their interactions with the enzyme.

Phylogenetic and functional characterization of the hAT transposon superfamily.

Transposons are found in virtually all organisms and play fundamental roles in genome evolution. They can also acquire new functions in the host organism and some have been developed as incisive genetic tools for transformation and mutagenesis. The hAT transposon superfamily contains members from the plant and animal kingdoms, some of which are active when introduced into new host organisms. We have identified two new active hAT transposons, AeBuster1, from the mosquito Aedes aegypti and TcBuster from the red flour beetle Tribolium castaneum. Activity of both transposons is illustrated by excision and transposition assays performed in Drosophila melanogaster and Ae. aegypti and by in vitro strand transfer assays. These two active insect transposons are more closely related to the Buster sequences identified in humans than they are to the previously identified active hAT transposons, Ac, Tam3, Tol2, hobo, and Hermes. We therefore reexamined the structural and functional relationships of hAT and hAT-like transposase sequences extracted from genome databases and found that the hAT superfamily is divided into at least two families. This division is supported by a difference in target-site selections generated by active transposons of each family. We name these families the Ac and Buster families after the first identified transposon or transposon-like sequence in each. We find that the recently discovered SPIN transposons of mammals are located within the family of Buster elements.

Mobility properties of the Hermes transposable element in transgenic lines of Aedes aegypti.

The Hermes transposable element has been used to genetically transform a wide range of insect species, including the mosquito, Aedes aegypti, a vector of several important human pathogens. Hermes integrations into the mosquito germline are characterized by the non-canonical integration of the transposon and flanking plasmid and, once integrated, Hermes is stable in the presence of its transposase. In an effort to improve the post-integration mobility of Hermes in the germline of Ae. aegypti, a transgenic helper Mos1 construct expressing Hermes transposase under the control of a testis-specific promoter was crossed to a separate transgenic strain containing a target Hermes transposon. In less than 1% of the approximately 1,500 progeny from jumpstarter lines analyzed, evidence of putative Hermes germline remobilizations were detected. These recovered transposition events occur through an aberrant mechanism and provide insight into the non-canonical cut-and-paste transposition of Hermes in the germ line of Ae. aegypti.

Pathogenomics of Culex quinquefasciatus and meta-analysis of infection responses to diverse pathogens.

The mosquito Culex quinquefasciatus poses a substantial threat to human and veterinary health as a primary vector of West Nile virus (WNV), the filarial worm Wuchereria bancrofti, and an avian malaria parasite. Comparative phylogenomics revealed an expanded canonical C. quinquefasciatus immune gene repertoire compared with those of Aedes aegypti and Anopheles gambiae. Transcriptomic analysis of C. quinquefasciatus genes responsive to WNV, W. bancrofti, and non-native bacteria facilitated an unprecedented meta-analysis of 25 vector-pathogen interactions involving arboviruses, filarial worms, bacteria, and malaria parasites, revealing common and distinct responses to these pathogen types in three mosquito genera. Our findings provide support for the hypothesis that mosquito-borne pathogens have evolved to evade innate immune responses in three vector mosquito species of major medical importance.