Doublesex is essential for masculinization but not feminization in Lygus hesperus.

In most holometabolous insects, sex differentiation occurs via a hierarchical cascade of transcription factors, with doublesex (dsx) regulating genes that control sex-specific traits. Although less is known in hemimetabolous insects, early evidence suggests that substantial differences exist from more evolutionarily advanced insects. Here, we identified and characterized dsx in Lygus hesperus (western tarnished plant bug), a hemipteran pest of many agricultural crops in western North America. The full-length transcript for L. hesperus dsx (Lhdsx) and several variants encode proteins with conserved DNA binding and oligomerization domains. Transcript profiling revealed that Lhdsx is ubiquitously expressed, likely undergoes alternative pre-mRNA splicing, and, unlike several model insects, is sex-biased rather than sex-specific. Embryonic RNA interference (RNAi) of Lhdsx only impacted sex development in adult males, which lacked both internal reproductive organs and external genitalia. No discernible impacts on adult female development or reproductivity were observed. RNAi knockdown of Lhdsx in nymphs likewise only affected adult males, which lacked the characteristic dimorphic coloration but had dramatically elevated vitellogenin transcripts. Gene knockout of Lhdsx by CRISPR/Cas9 editing yielded only females in G0 and strongly biased heterozygous G1 offspring to females with the few surviving males showing severely impaired genital development. These results indicate that L. hesperus male development requires Lhdsx, whereas female development proceeds via a basal pathway that functions independently of dsx. A fundamental understanding of sex differentiation in L. hesperus could be important for future gene-based management strategies of this important agricultural pest.

ß-tubulin functions in spermatogenesis in Lygus hesperus Knight.

Lygus hesperus Knight is an important insect pest of crops across western North America, with field management heavily reliant on the use of chemical insecticides. Because of the evolution of resistance to these insecticides, effective and environmentally benign pest management strategies are needed. Traditional sterile insect technique (SIT) has been successfully employed to manage or eradicate some insect pests but involves introducing irradiated insects with random mutations into field populations. New genetically-driven SIT techniques are a safer alternative, causing fixed mutations that manipulate individual genes in target pests to produce sterile individuals for release. Here, we identified seven ß-tubulin coding genes from L. hesperus and show that Lhßtub2 is critical in male sperm production and fertility. Lhßtub2 is expressed primarily in the male testes and targeting of this gene by RNA interference or gene editing leads to male sterility.

Sindbis virus is suppressed in the yellow fever mosquito Aedes aegypti by ATG-6/Beclin-1 mediated activation of autophagy.

Autophagy is a critical modulator of pathogen invasion response in vertebrates and invertebrates. However, how it affects mosquito-borne viral pathogens that significantly burden public health remains underexplored. To address this gap, we use a genetic approach to activate macroautophagy/autophagy in the yellow fever mosquito (Aedes aegypti), infected with a recombinant Sindbis virus (SINV) expressing an autophagy activator. We first demonstrate a 17-amino acid peptide derived from the Ae. aegypti autophagy-related protein 6 (ATG-6/beclin-1-like protein) is sufficient to induce autophagy in C6/36 mosquito cells, as marked by lipidation of ATG-8 and puncta formation. Next, we engineered a recombinant SINV expressing this bioactive beclin-1-like peptide and used it to infect and induce autophagy in adult mosquitoes. We find that modulation of autophagy using this recombinant SINV negatively regulated production of infectious viruses. The results from this study improve our understanding of the role of autophagy in arboviruses in invertebrate hosts and also highlight the potential for the autophagy pathway to be exploited for arboviral control.

Chromosome-scale genome assembly of the pink bollworm, Pectinophora gossypiella, a global pest of cotton.

The pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), is a major global pest of cotton. Current management practices include chemical insecticides, cultural strategies, sterile insect releases, and transgenic cotton producing crystalline (Cry) protein toxins of the bacterium Bacillus thuringiensis (Bt). These strategies have contributed to the eradication of P. gossypiella from the cotton-growing areas of the United States and northern Mexico. However, this pest has evolved resistance to Bt cotton in Asia, where it remains a critical pest, and the benefits of using transgenic Bt crops have been lost. A complete annotated reference genome is needed to improve global Bt resistance management of the pink bollworm. We generated the first chromosome-level genome assembly for pink bollworm from a Bt-susceptible laboratory strain (APHIS-S) using PacBio continuous long reads for contig generation, Illumina Hi-C for scaffolding, and Illumina whole-genome re-sequencing for error correction. The pseudo-haploid assembly consists of 29 autosomes and the Z sex chromosome. The assembly exceeds the minimum Earth BioGenome Project quality standards, has a low error rate, is highly contiguous at both the contig and scaffold levels (L/N50 of 18/8.26 MB and 14/16.44 MB, respectively), and is complete, with 98.6% of lepidopteran single-copy orthologs represented without duplication. The genome was annotated with 50% repeat content and 14,107 protein-coding genes, further assigned to 41,666 functional annotations. This assembly represents the first publicly available complete annotated genome of pink bollworm and will serve as the foundation for advancing molecular genetics of this important pest species.

RNAi-Mediated Manipulation of Cuticle Coloration Genes in Lygus hesperus Knight (Hemiptera: Miridae).

Cuticle coloration in insects is a consequence of the accumulation of pigments in a species-specific pattern. Numerous genes are involved in regulating the underlying processes of melanization and sclerotization, and their manipulation can be used to create externally visible markers of successful gene editing. To clarify the roles for many of these genes and examine their suitability as phenotypic markers in Lygus hesperus Knight (western tarnished plant bug), transcriptomic data were screened for sequences exhibiting homology with the Drosophila melanogaster proteins. Complete open reading frames encoding putative homologs for six genes (aaNAT, black, ebony, pale, tan, and yellow) were identified, with two variants for black. Sequence and phylogenetic analyses supported preliminary annotations as cuticle pigmentation genes. In accord with observable difference in color patterning, expression varied for each gene by developmental stage, adult age, body part, and sex. Knockdown by injection of dsRNA for each gene produced varied effects in adults, ranging from the non-detectable (black 1, yellow), to moderate decreases (pale, tan) and increases (black 2, ebony) in darkness, to extreme melanization (aaNAT). Based solely on its expression profile and highly visible phenotype, aaNAT appears to be the best marker for tracking transgenic L. hesperus.

Characterization of Mayaro virus (strain BeAn343102) biology in vertebrate and invertebrate cellular backgrounds.

Mayaro virus (MAYV) is an emerging New World alphavirus (genus Alphavirus, family Togaviridae) that causes acute multiphasic febrile illness, skin rash, polyarthritis and occasional severe clinical phenotypes. The virus lifecycle alternates between invertebrate and vertebrate hosts. Here we characterize the replication features, cell entry, lifecycle and virus-related cell pathology of MAYV using vertebrate and invertebrate in vitro models. Electron-dense clathrin-coated pits in infected cells and reduced viral production in the presence of dynasore, ammonium chloride and bafilomycin indicate that viral entry occurs through pH-dependent endocytosis. Increase in FITC-dextran uptake (an indicator of macropinocytosis) in MAYV-infected cells, and dose-dependent infection inhibition by 5-(N-ethyl-N-isopropyl) amiloride (a macropinocytosis inhibitor), indicated that macropinocytosis is an additional entry mechanism of MAYV in vertebrate cells. Acutely infected vertebrate and invertebrate cells formed cytoplasmic or membrane-associated extracytoplasmic replication complexes. Mosquito cells showed modified hybrid cytoplasmic vesicles that supported virus replication, nucleocapsid production and maturation. Mature virus particles were released from cells by both exocytosis and budding from the cell membrane. MAYV replication was cytopathic and associated with induction of apoptosis by the intrinsic pathway, and later by the extrinsic pathway in infected vertebrate cells. Given that MAYV is expanding its geographical existence as a potential public health problem, this study lays the foundation for biological understanding that will be valuable for therapeutic and preventive interventions.

Knockout of ABC transporter gene ABCA2 confers resistance to Bt toxin Cry2Ab in Helicoverpa zea.

Evolution of pest resistance reduces the benefits of widely cultivated genetically engineered crops that produce insecticidal proteins derived from Bacillus thuringiensis (Bt). Better understanding of the genetic basis of pest resistance to Bt crops is needed to monitor, manage, and counter resistance. Previous work shows that in several lepidopterans, resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2. The results here show that mutations introduced by CRISPR/Cas9 gene editing in the Helicoverpa zea (corn earworm or bollworm) gene encoding ABCA2 (HzABCA2) can cause resistance to Cry2Ab. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains. A multiple concentration bioassay with one of these strains revealed it had > 200-fold resistance to Cry2Ab relative to its parental susceptible strain. All Cry2Ab-resistant individuals tested had disruptive mutations in HzABCA2. We identified five disruptive mutations in HzABCA2 gDNA. The most common mutation was a 4-bp deletion in the expected Cas9 guide RNA target site. The results here indicate that HzABCA2 is a leading candidate for monitoring Cry2Ab resistance in field populations of H. zea.

Cas9-mediated gene editing in the black-legged tick, Ixodes scapularis, by embryo injection and ReMOT Control.

Despite their capacity to acquire and pass on an array of debilitating pathogens, research on ticks has lagged behind other arthropod vectors, such as mosquitoes, largely because of challenges in applying available genetic and molecular tools. CRISPR-Cas9 is transforming non-model organism research; however, successful gene editing has not yet been reported in ticks. Technical challenges for injecting tick embryos to attempt gene editing have further slowed research progress. Currently, no embryo injection protocol exists for any chelicerate species, including ticks. Herein, we report a successful embryo injection protocol for the black-legged tick, Ixodes scapularis, and the use of this protocol for genome editing with CRISPR-Cas9. We also demonstrate that the ReMOT Control technique could be successfully used to generate genome mutations outside Insecta. Our results provide innovative tools to the tick research community that are essential for advancing our understanding of the molecular mechanisms governing pathogen transmission by tick vectors and the underlying biology of host-vector-pathogen interactions.

CRISPR-mediated knockout of cardinal and cinnabar eye pigmentation genes in the western tarnished plant bug.

The western tarnished plant bug, Lygus hesperus, is a key hemipteran pest of numerous agricultural, horticultural, and industrial crops in the western United States and Mexico. A lack of genetic tools in L. hesperus hinders progress in functional genomics and in developing innovative pest control methods such as gene drive. Here, using RNA interference (RNAi) against cardinal (LhCd), cinnabar (LhCn), and white (LhW), we showed that knockdown of LhW was lethal to developing embryos, while knockdown of LhCd or LhCn produced bright red eye phenotypes, in contrast to wild-type brown eyes. We further used CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) genome editing to generate germline knockouts of both LhCd (Card) and LhCn (Cinn), producing separate strains of L. hesperus characterized by mutant eye phenotypes. Although the cardinal knockout strain Card exhibited a gradual darkening of the eyes to brown typical of the wild-type line later in nymphal development, we observed bright red eyes throughout all life stages in the cinnabar knockout strain Cinn, making it a viable marker for tracking gene editing in L. hesperus. These results provide evidence that CRISPR/Cas9 gene editing functions in L. hesperus and that eye pigmentation genes are useful for tracking the successful genetic manipulation of this insect.

Examination of Rickettsial Host Range for Shuttle Vectors Based on dnaA and parA Genes from the pRM Plasmid of Rickettsia monacensis.

The genus Rickettsia encompasses a diverse group of obligate intracellular bacteria that are highly virulent disease agents of mankind as well as symbionts of arthropods. Native plasmids of Rickettsia amblyommatis (AaR/SC) have been used as models to construct shuttle vectors for genetic manipulation of several Rickettsia species. Here, we report on the isolation of the complete plasmid (pRM658B) from Rickettsia monacensis IrR/Munich mutant Rmona658B and the construction of shuttle vectors based on pRM. To identify regions essential for replication, we made vectors containing the dnaA and parA genes of pRM with various portions of the region surrounding these genes and a selection reporter cassette conferring resistance to spectinomycin and expression of green fluorescent protein. Rickettsia amblyommatis (AaR/SC), R. monacensis (IrR/Munich), Rickettsia bellii (RML 369-C), Rickettsia parkeri (Tate's Hell), and Rickettsia montanensis (M5/6) were successfully transformed with shuttle vectors containing pRM parA and dnaA. PCR assays targeting pRM regions not included in the vectors revealed that native pRM was retained in R. monacensis transformants. Determination of native pRM copy number using a plasmid-carried gene (RM_p5) in comparison to chromosomally carried gltA indicated reduced copy numbers in R. monacensis transformants. In transformed R. monacensis strains, native pRM and shuttle vectors with homologous parA and dnaA formed native plasmid-shuttle vector complexes. These studies provide insight on the maintenance of plasmids and shuttle vectors in rickettsiae. IMPORTANCERickettsia spp. are found in a diverse array of organisms, from ticks, mites, and fleas to leeches and insects. Many are not pathogenic, but others, such as Rickettsia rickettsii and Rickettsia prowazeckii, can cause severe illness or death. Plasmids are found in a large percentage of nonpathogenic rickettsiae, but not in species that cause severe disease. Studying these plasmids can reveal their role in the biology of these bacteria, as well as the molecular mechanism whereby they are maintained and replicate in rickettsiae. Here, we describe a new series of shuttle plasmids for the transformation of rickettsiae based on parA and dnaA sequences of plasmid pRM from Rickettsia monacensis. These shuttle vectors support transformation of diverse rickettsiae, including the native host of pRM, and are useful for investigating genetic determinants that govern rickettsial virulence or their ability to function as symbionts.

Growth Dynamics and Antibiotic Elimination of Symbiotic Rickettsia buchneri in the Tick Ixodes scapularis (Acari: Ixodidae).

Rickettsia buchneri is the principal symbiotic bacterium of the medically significant tick Ixodes scapularis This species has been detected primarily in the ovaries of adult female ticks and is vertically transmitted, but its tissue tropism in other life stages and function with regard to tick physiology is unknown. In order to determine the function of R. buchneri, it may be necessary to produce ticks free from this symbiont. We quantified the growth dynamics of R. buchneri naturally occurring in I. scapularis ticks throughout their life cycle and compared it with bacterial growth in ticks in which symbiont numbers were experimentally reduced or eliminated. To eliminate the bacteria, we exposed ticks to antibiotics through injection and artificial membrane feeding. Both injection and membrane feeding of the antibiotic ciprofloxacin were effective at eliminating R. buchneri from most offspring of exposed females. Because of its effectiveness and ease of use, we have determined that injection of ciprofloxacin into engorged female ticks is an efficient means of clearing R. buchneri from the majority of progeny.IMPORTANCE This paper describes the growth of symbiotic Rickettsia buchneri within Ixodes scapularis through the life cycle of the tick and provides methods to eliminate R. buchneri from I. scapularis ticks.

CRISPR-Cas9-Based Genome Editing in the Silverleaf Whitefly (Bemisia tabaci).

Bemisia tabaci cryptic species Middle East-Asia Minor I (MEAM1) is a serious agricultural polyphagous insect pest and vector of numerous plant viruses, causing major worldwide economic losses. B. tabaci control is limited by lack of robust gene editing tools. Gene editing is difficult in B. tabaci due to small embryos that are technically challenging to inject and which have high mortality post injection. We developed a CRISPR-Cas9 gene editing protocol based on injection of vitellogenic adult females rather than embryos ("ReMOT Control"). We identified an ovary-targeting peptide ligand ("BtKV") that, when fused to Cas9 and injected into adult females, transduced the ribonucleoprotein complex to the germline, resulting in efficient, heritable editing of the offspring genome. In contrast to embryo injection, adult injection is easy and does not require specialized equipment. Development of easy-to-use gene editing protocols for B. tabaci will allow researchers to apply the power of reverse genetic approaches to this species and will lead to novel control methods for this devastating pest insect.

c-Src regulates cargo transit via the Golgi in pancreatic acinar cells.

The exocrine pancreatic acinar cell is unique for its rapid protein synthesis and packaging in zymogen granules (ZGs). However, while crucial to the pathogenesis of pancreatitis, the signaling involved in the transit of proteins via the Golgi is poorly understood in these cells. Noting the evidence of c-Src in regulating transit of cargo via the Golgi in other systems, we explored this in acinar cells. Stimulation of ZG formation with dexamethasone activated Src and increased the Golgi area in acinar cells. c-Src localized to the microsomes of acinar cells on immunofluorescence and subcellular fractionation. While other Src family members had no effect on the Golgi markers P115 and GM130, active c-Src increased the Golgi area these stained, extending them into the ER. Src inhibition reduced amylase staining outside the Golgi and increased it in a stack like Golgi morphology. In vivo pharmacologic inhibition or acinar specific genetic deletion of c-Src reduced ZG number and staining of amylase in ZGs along with increasing amylase retention in the microsomal fraction. Morphologically this was associated with smaller Golgi stacks, and dilation of the endoplasmic reticulum. Therefore the role c-Src regulated Golgi function, ZG formation and microsomal zymogen transit in acinar cells needs to be explored in pancreatitis.

Fluorescent Protein Expressing Rickettsia buchneri and Rickettsia peacockii for Tracking Symbiont-Tick Cell Interactions.

Rickettsiae of indeterminate pathogenicity are widely associated with ticks. The presence of these endosymbionts can confound a One Health approach to combatting tick-borne diseases. Genomic analyses of symbiotic rickettsiae have revealed that they harbor mutations in gene coding for proteins involved in rickettsial pathogenicity and motility. We have isolated and characterized two rickettsial symbionts-Rickettsia peacockii and R. buchneri-both from ticks using tick cell cultures. To better track these enigmatic rickettsiae in ticks and at the tick-mammal interface we transformed the rickettsiae to express fluorescent proteins using shuttle vectors based on rickettsial plasmids or a transposition system driving insertional mutagenesis. Fluorescent protein expressing R. buchneri and R. peacockii will enable us to elucidate their interactions with tick and mammalian cells, and track their location and movement within individual cells, vector ticks, and host animals.

Transcriptional Analysis of the Conjugal Transfer Genes of Rickettsia bellii RML 369-C.

Rickettsia bellii is an obligate intracellular bacterium that is one of the few rickettsiae that encode a complete set of conjugative transfer (tra) genes involved in bacterial conjugation and has been shown to exhibit pili-like structures. The reductive genomes of rickettsiae beg the question whether the tra genes are nonfunctional or functioning to enhance the genetic plasticity and biology of rickettsiae. We characterized the transcriptional dynamics of R. bellii tra genes in comparison to genes transcribed stably and above the background level to understand when and at what levels the tra genes are active or whether the tra genes are degenerative. We determined that the best reference genes, out of 10 tested, were methionyl tRNA ligase (metG) or a combination of metG and ribonucleoside diphosphate reductase 2 subunit beta (nrdF), using statistical algorithms from two different programs: Normfinder and BestKeeper. To validate the use of metG with other rickettsial genes exhibiting variable transcriptional patterns we examined its use with sca2 and rickA, genes involved in actin based motility. Both were shown to be up-regulated at different times of replication in Vero cells, showing variable and stable transcription levels of rickA and sca2, respectively. traATi was up-regulated at 72 hours post inoculation in the tick cell line ISE6, but showed no apparent changes in the monkey cell line Vero and mouse cell line L929. The transcription of tra genes was positively correlated with one another and up-regulated from 12 to 72 hours post inoculation (HPI) when compared to RBE_0422 (an inactivated transposase-derivative found within the tra cluster). Thus, the up-regulation of the tra genes indicated that the integrity and activity of each gene were intact and may facilitate the search for the optimal conditions necessary to demonstrate conjugation in rickettsiae.

Rickettsia buchneri sp. nov., a rickettsial endosymbiont of the blacklegged tick Ixodes scapularis.

We obtained a rickettsial isolate from the ovaries of the blacklegged tick, Ixodes scapularis. The isolate (ISO7(T)) was grown in the Ixodes ricinus embryonic cell line IRE11. We characterized the isolate by transmission electron microscopy and gene sequencing. Phylogenetic analysis of 11 housekeeping genes demonstrated that the isolate fulfils the criteria to be classified as a representative of a novel rickettsial species closely related to 'Rickettsia monacensis'. These rickettsiae form a clade separate from other species of rickettsiae. Gene sequences indicated that several genes important in rickettsial motility, invasiveness and temperature adaptation were mutated (e.g. sca2, rickA, hsp22, pldA and htrA). We propose the name Rickettsia buchneri sp. nov. for this bacterium that infects the ovaries of the tick I. scapularis to acknowledge the pioneering contributions of Professor Paul Buchner (1886-1978) to research on bacterial symbionts. The type strain of R. buchneri sp. nov. is strain ISO-7(T) ( = DSM 29016(T) = ATCC VR-1814(T)).

Development of shuttle vectors for transformation of diverse Rickettsia species.

Plasmids have been identified in most species of Rickettsia examined, with some species maintaining multiple different plasmids. Three distinct plasmids were demonstrated in Rickettsia amblyommii AaR/SC by Southern analysis using plasmid specific probes. Copy numbers of pRAM18, pRAM23 and pRAM32 per chromosome in AaR/SC were estimated by real-time PCR to be 2.0, 1.9 and 1.3 respectively. Cloning and sequencing of R. amblyommii AaR/SC plasmids provided an opportunity to develop shuttle vectors for transformation of rickettsiae. A selection cassette encoding rifampin resistance and a fluorescent marker was inserted into pRAM18 yielding a 27.6 kbp recombinant plasmid, pRAM18/Rif/GFPuv. Electroporation of Rickettsia parkeri and Rickettsia bellii with pRAM18/Rif/GFPuv yielded GFPuv-expressing rickettsiae within 2 weeks. Smaller vectors, pRAM18dRG, pRAM18dRGA and pRAM32dRGA each bearing the same selection cassette, were made by moving the parA and dnaA-like genes from pRAM18 or pRAM32 into a vector backbone. R. bellii maintained the highest numbers of pRAM18dRGA (13.3 - 28.1 copies), and R. parkeri, Rickettsia monacensis and Rickettsia montanensis contained 9.9, 5.5 and 7.5 copies respectively. The same species transformed with pRAM32dRGA maintained 2.6, 2.5, 3.2 and 3.6 copies. pRM, the plasmid native to R. monacensis, was still present in shuttle vector transformed R. monacensis at a level similar to that found in wild type R. monacensis after 15 subcultures. Stable transformation of diverse rickettsiae was achieved with a shuttle vector system based on R. amblyommii plasmids pRAM18 and pRAM32, providing a new research tool that will greatly facilitate genetic and biological studies of rickettsiae.