Delivery Methods for RNAi in Mosquito Larvae.

Mosquito-transmitted diseases pose a threat for a great portion of the world population. Chemical insecticides are the main tool for mosquito control. Heavy dependence on chemicals created several problems such as resistance development in many mosquito species, environmental effects, and human health issues. Other tools for mosquito control were developed and used in some parts of the world. Ribonucleic acid interference (RNAi) is a reverse genetic mechanism that was recently introduced as a new tool for pest control. Regarding mosquito, RNAi was used to study gene function and to discover genes that can be used as targets for control purposes. Several delivery methods are used to induce RNAi in mosquito larvae. Some methods such as injection and soaking are used routinely in RNAi research but have no application in the field. Other methods such as nanoparticles and microbes have some characteristics that make them good candidates for field application. In this report, we will focus on delivery methods for RNAi in mosquito larvae and will give examples for each method.

RNAi-mediated mortality of Culex quinquefasciatus using two delivery methods of potential field application.

With increasing reports of resistance to traditional insecticides, there is a need for innovative ways for mosquito control. RNAi is a sequence-specific molecular biology technique for gene silencing through degradation of mRNA and prevention of protein translation. Some genes are essential for insect life and their silencing can lead to insect morbidity and/or mortality. Searching for lethal genes in Culex quinquefasciatus, we found dynamin, ROP, HMGR and JHAMT to be lethal targets for RNAi in initial screening through larval soaking in dsRNA solution. Two delivery methods, chitosan nanoparticles and genetically modified yeast cells, were used in this study and proved effective in inducing high larval mortality and low adult emergence. Adult emergence after chitosan nanoparticles/dsRNA treatment was 12.67% ± 1.76 (HMGR), 17.33% ± 1.76 (dynamin), 18.67% ± 0.67 (ROP), and 35.33% ± 0.67 (JHAMT). Genetically modified yeast increased mortalities as adult emergence was 8.33% ± 1.67 (HMGR), 13.33% ± 3.33 (dynamin), and 10% ± 2.89 (JHAMT and ROP). Chitosan nanoparticles retained 75% of its biological activity whereas yeast cells retained >95% of their activities after 7 days of incubation in water. In conclusion, our results showed that these four genes are good targets for C. quinquefasciatus control using RNAi packaged in either chitosan nanoparticles or genetically modified yeast cells.

RNAi-Mediated Screening of Selected Target Genes Against Culex quinquefasciatus (Diptera: Culicidae).

Culex quinquefasciatus, a member of the Culex pipiens complex, is widespread in Saudi Arabia and other parts of the world. It is a vector for lymphatic filariasis, Rift Valley fever, and West Nile virus. Studies have shown the deleterious effect of RNA interference (RNAi)-mediated knockdown of various lethal genes in model and agricultural pest insects. RNAi was proposed as a tool for mosquito control with a focus on Aedes aegypti and Anopheles gambiae. In this study, we examined the effect of RNAi of selected target genes on both larval mortality and adult emergence of Cx. quinquefasciatus through two delivery methods: soaking and nanoparticles. Ten candidate genes were selected for RNAi based on their known lethal effect in other insects. Disruption of three genes, chitin synthase-1, inhibitor of apoptosis 1, and vacuolar adenosine triphosphatase, resulted in the highest mortality among the selected genes using the two treatment methods. Silencing the other seven genes resulted in a medium to low mortality in both assays. These three genes are also active against a wide range of insects and could be used for RNAi-based mosquito control in the future.

Assessment and an updated list of the mosquitoes of Saudi Arabia.

BACKGROUND: Mosquito-borne pathogens are important causes of diseases in the Kingdom of Saudi Arabia. Knowledge of the mosquito fauna is needed for the appropriate control of the vectors that transmit the pathogens and prevent the diseases they cause. An important first step is to have an up-to-date list of the species known to be present in the country. Original occurrence records were obtained from published literature and critically scrutinized to compile a list of the mosquito species that occur within the borders of the Kingdom. RESULTS: Fifty-one species have been recorded in the Kingdom; however, the occurrence of two of these species is unlikely. Thus, the mosquito fauna of the Kingdom comprises 49 species that include 18 anophelines and 31 culicines. Published records are provided for each species. Problematic records based on misidentifications and inappropriate sources are discussed and annotated for clarity. CONCLUSION: Integrated morphological and molecular methods of identification are needed to refine the list of species and accurately document their distributions in the Kingdom.

Hormonal regulation of metamorphosis and reproduction in ticks.

The presence of a "status quo" hormone like JH has not been found in ticks. The most advanced understanding of tick endocrinology is associated with female reproduction, where the sequence of the first messages for storage proteins (vitellogenin (Vg) and carrier protein), the Vg receptor, and male peptidic pheromones were recently reported. The current consensus model suggests that ecdysteroids from the epidermis regulated by a putative peptidic ecdysiotrophic hormone from the synganlion initiates the expression of the Vg messages in fat body and midgut. Vg protein, secreted into the hemolymph, requires an ovary Vg receptor to be absorbed by oocytes. Male pheromones transferred into the female genital tract during mating initiate blood feeding to repletion and vitellogenesis. The work so far on tick endocrinology is limited by the paucity of identified hormones and the small number of studies on a few tick models. The role of storage proteins in the evolution of hematophagy is discussed.

Sequence and the developmental and tissue-specific regulation of the first complete vitellogenin messenger RNA from ticks responsible for heme sequestration.

The first full-length mRNA for vitellogenin (Vg) from ticks was sequenced. This also represents the first complete sequence of Vg from the Chelicerata and of a heme binding Vg. The Vg cDNA from the American dog tick, Dermacentor variabilis was 5744nt in length (GenBank Accession number AY885250), which coded for a protein of 1843 aa with a calculated molecular weight of 208 kD. This protein had an 18 aa signal sequence, a single RXXR cleavage signal that would generate two subunits (49.5 and 157K in molecular weight) and lipoprotein N-terminal and carboxy von Willebrand factor type D domains. Tryptic digest MS analysis of vitellin protein confirmed the function of the cDNA as the tick yolk protein. Apparently, vitellin in D. variabilis is oligomeric (possibly dimeric) and is comprised of a mixture of the uncleaved monomer and subunits that were predicted from the single RXXR cleavage signal. The highly conserved GL/ICG motif close to the C-terminus in insect Vg genes was different in the tick Vg message, i.e., GLCS. This variant was also present in a partial sequence of Vg from Boophilus microplus. Phylogenic analysis showed that the full length Vg cDNA from D. variabilis and the partial cDNA from B. microplus were distinct from insects and Crustacea. The Vg message was not found in whole body RNA from unfed or fed males or in unfed and partially fed (virgin) females as determined by Northern blotting. The message was found in replete (mated) pre-ovipositional females, increased to higher levels in ovipositing females and was absent after egg laying was complete. The endocrine regulation of the Vg mRNA is discussed. The tissue sources of the Vg message are both the gut and fat body. Tryptic digest MS fingerprinting suggests that a second Vg mRNA might be present in the American dog tick, which needs further study.

Characterization and RNAi-mediated knockdown of Chitin Synthase A in the potato tuber moth, Phthorimaea operculella.

Chitin is a major component of insect exoskeleton, tracheal system and gut where it is synthesized by chitin synthase (CHS) enzymes. In this paper, we report the isolation and RNAi of chitin synthase A (PhoCHSA) from the potato tuber moth Phthorimaea operculella. The full-length cDNA of PhoCHSA is 5,627 bp with 4,689 bp open reading frame coding for 1,563 amino acids. Structural analysis of conceptual amino acid translation showed three distinct regions found in all known insect CHS proteins; N-terminus region having 9 transmembrane helices, middle catalytic region containing several conserved domains identified in insect CHS enzymes, and C-terminus region containing seven transmembrane spans. Phylogenetic analysis showed that PhoCHSA protein clustered with CHSA enzymes identified from insects from different insect orders. RNAi targeting three different regions of the gene showed different efficacy against potato tuber moth larvae and dsRNA targeting the 5' region has the highest efficacy. Results were verified by qRT-PCR which showed that dsRNA targeting the 5' region caused the highest reduction in PhoCHSA mRNA level. Our results show the importance of selecting the RNAi target region and that chitin synthase A can be a suitable RNAi target for the potato tuber moth control.

Role of juvenile hormone esterase and epoxide hydrolase in reproduction of the cotton bollworm, Helicoverpa zea.

The role of juvenile hormone (JH) esterase (JHE) and epoxide hydrolase (EH) in reproduction of the cotton bollworm, Helicoverpa zea, was investigated. Peak emergence of male and female bollworm adults occurred early in the scotophase. Female adults were added to males in a 1:2 ratio, respectively, at the beginning of the first photophase after emergence (d0). The highest oviposition rates for mated females were noted on d 2-4. The in vitro JH III esterase and JH III EH activity was measured in whole body homogenates of virgin and mated females from d0 to d8 post-emergence. Maximal JHE activity for virgin females occurred on d2 (1.09+/-0.14(+/-1 SEM) nmol of JH III degraded/min/mg protein), which was approximately twice that of mated females on the same day. The same results were observed for EH where the activity peaked on d2 at 0.053+/-0.003 as compared to 0.033+/-0.003 nmol of JH III degraded/min/mg protein, respectively. By d4, both JHE and JH EH activities declined significantly in virgin and mated females and were the same through d7. The developmental changes and effects of mating on JH degradation were similar when measured per insect. The highest levels of JHE and JH EH activity/min/mg protein in d2 virgin and mated females was found in ovaries followed by the carcass and then haemolymph; no EH activity was found in haemolymph as expected. For ovary, the JHE and JH EH activity was highest in virgin compared to mated females. The role of both enzymes in the regulation of reproduction is discussed.

Genomic insights into the Ixodes scapularis tick vector of Lyme disease.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick-host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host 'questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.

In vivo role of 20-hydroxyecdysone in the regulation of the vitellogenin mRNA and egg development in the American dog tick, Dermacentor variabilis (Say).

Injection of the hormone 20-hydroxyecdysone (20-E) into partially fed (virgin) female adults of the American dog tick, Dermacentor variabilis, while they are attached and feeding on the rabbit host, initiated the expression of the vitellogenin (Vg) gene, and Vg protein secretion and uptake by the ovary. The induction of egg production by 20-E in this bioassay was dose dependent in the range of 1-50 times the concentration normally found in a replete, vitellogenic female. Ticks examined 4 d after the 50 x treatment were still attached to the host, had numerous enlarged vitellin-filled (brown) oocytes in their ovaries, but had not engorged to repletion. The ovaries reached weights similar to those found in untreated, replete (mated) females (pre-oviposition) while solvent-injected controls demonstrated no increase in oocyte size or increase in ovary weight. An increase in the levels of a putative Vg protein was observed in hemolymph samples collected 1, 2 and 3d post-20-E injection but was not observed in the corresponding solvent controls as determined by native PAGE. Analysis of the ecdysteroid-induced protein by tryptic digestion-mass fingerprinting and BLASTP found that the putative Vg had the strongest match to GP80 (U49934), the partial sequence for the vitellogenin protein from Boophilus microplus. A partial Vg cDNA was cloned and sequenced from replete females of D. variabilis with a high similarity to GP80. Using this message as a probe, Northern blots conducted with RNA collected from partially fed, virgin females 1, 2 and 3d post-20-E injection showed upregulation of the Vg mRNA on all 3 days. Controls injected with solvent only showed no Vg mRNA. Injections with juvenile hormone III did not stimulate Vg expression, oocyte growth or full engorgement. These studies indicate that ecdysteroids and not JH can initiate expression of the Vg gene, Vg protein synthesis and release into hemolymph, and Vg uptake into developing oocytes under bioassay conditions mimicking normal feeding on the host.

Molecular traces of alternative social organization in a termite genome.

Although eusociality evolved independently within several orders of insects, research into the molecular underpinnings of the transition towards social complexity has been confined primarily to Hymenoptera (for example, ants and bees). Here we sequence the genome and stage-specific transcriptomes of the dampwood termite Zootermopsis nevadensis (Blattodea) and compare them with similar data for eusocial Hymenoptera, to better identify commonalities and differences in achieving this significant transition. We show an expansion of genes related to male fertility, with upregulated gene expression in male reproductive individuals reflecting the profound differences in mating biology relative to the Hymenoptera. For several chemoreceptor families, we show divergent numbers of genes, which may correspond to the more claustral lifestyle of these termites. We also show similarities in the number and expression of genes related to caste determination mechanisms. Finally, patterns of DNA methylation and alternative splicing support a hypothesized epigenetic regulation of caste differentiation.

New approach for the study of mite reproduction: The first transcriptome analysis of a mite, Phytoseiulus persimilis (Acari: Phytoseiidae).

Many species of mites and ticks are of agricultural and medical importance. Much can be learned from the study of transcriptomes of acarines which can generate DNA-sequence information of potential target genes for the control of acarine pests. High throughput transcriptome sequencing can also yield sequences of genes critical during physiological processes poorly understood in acarines, i.e., the regulation of female reproduction in mites. The predatory mite, Phytoseiulus persimilis, was selected to conduct a transcriptome analysis using 454 pyrosequencing. The objective of this project was to obtain DNA-sequence information of expressed genes from P. persimilis with special interest in sequences corresponding to vitellogenin (Vg) and the vitellogenin receptor (VgR). These genes are critical to the understanding of vitellogenesis, and they will facilitate the study of the regulation of mite female reproduction. A total of 12,556 contiguous sequences (contigs) were assembled with an average size of 935bp. From these sequences, the putative translated peptides of 11 contigs were similar in amino acid sequences to other arthropod Vgs, while 6 were similar to VgRs. We selected some of these sequences to conduct stage-specific expression studies to further determine their function.

Full-length sequence, regulation and developmental studies of a second vitellogenin gene from the American dog tick, Dermacentor variabilis.

Vitellogenin (Vg) is the precursor of vitellin (Vn) which is the major yolk protein in eggs. In a previous report, we isolated and characterized the first Vg message from the American dog tick Dermacentor variabilis. In the current study, we describe a second Vg gene from the same tick. The Vg2 cDNA is 5956 nucleotides with a 5775 nt open reading frame coding for 1925 amino acids. The conceptual amino acid translation contains a 16-residues putative signal peptide, N-terminal lipid binding domain and C-terminal von Willebrand factor type D domain present in all known Vgs. Moreover, the amino acid sequence shows a typical GLCG domain and several RXXR cleavage sites present in most isolated Vgs. Tryptic digest-mass fingerprinting of Vg and Vn recognized 11 fragments that exist in the amino acid translation of DvVg2 cDNA. Injection of virgin females with 20 hydroxyecdysone induced DvVg2 expression, vitellogenesis and oviposition. Using RT-PCR, DvVg2 expression was detected only in tick females after mating and feeding to repletion. Northern blot analysis showed that DvVg2 is expressed in fat body and gut cells of vitellogenic females but not in the ovary. DvVg2 expression was not detected in adult fed or unfed males. The characteristics that distinguish Vg from other similar tick storage proteins like the carrier protein, CP (another hemelipoglycoprotein) are discussed.

Neuropeptide signaling sequences identified by pyrosequencing of the American dog tick synganglion transcriptome during blood feeding and reproduction.

Ticks are important vectors of numerous pathogens that impact human and animal health. The tick central nervous system represents an understudied area in tick biology and no tick synganglion-specific transcriptome has been described to date. Here we characterize whole or partial cDNA sequences of fourteen putative neuropeptides (allatostatin, insulin-like peptide, ion-transport peptide, sulfakinin, bursicon alpha/beta, eclosion hormone, glycoprotein hormone alpha/beta, corazonin, four orcokinins) and five neuropeptide receptors (gonadotropin receptor, leucokinin-like receptor, sulfakinin receptor, calcitonin receptor, pyrokinin receptor) translated from cDNA synthesized from the synganglion of unfed, partially fed and replete female American dog ticks, Dermacentor variabilis. Their homology to the same neuropeptides in other taxa is discussed. Many of these neuropeptides such as an allatostatin, insulin-like peptide, eclosion hormone, bursicon alpha and beta and glycoprotein hormone alpha and beta have not been previously described in the Chelicerata. An insulin-receptor substrate protein was also found indicating that an insulin signaling network is present in ticks. A putative type-2 proprotein processing convertase was also sequenced that may be involved in cleavage at monobasic and dibasic endoproteolytic cleavage sites in prohormones. The possible physiological role of the proteins discovered in adult tick blood feeding and reproduction will be discussed.

Characterization of vitellin protein in the twospotted spider mite, Tetranychus urticae (Acari: Tetranychidae).

In mites, vitellogenin synthesis, regulation and uptake by the oocytes as vitellin remain practically unknown. Although a partial sequence of the gene is now available, no previous studies have been conducted that describe the native vitellin protein in mites. The objective of this study was to characterize vitellin in the twospotted spider mite, Tetranychus urticae. The native twospotted spider mite vitellin migrated as a single major band with a molecular weight of 476+/-14.5 kDa as compared to 590+/-25.5 kDa for vitellin from the American dog tick, Dermacentor variabilis. However, isoelectric focusing analysis of native spider mite vitellin showed five bands with pI values slightly acidic to neutral (pH 5.8, 6.2, 6.7, 7.0 and 7.2), as is the case for insect and tick vitellins. Reducing conditions (SDS-PAGE) also revealed multiple subunits ranging from 290.9 to 3.6 kDa and was similar to that found in D. variabilis. Spider mite vitellin weakly bound lipids and carbohydrates compared to the tick. Unlike D. variabilis, the spider mite egg yolk protein does not bind heme. The significance of non-heme binding in mites is discussed.

Male engorgement factor: Role in stimulating engorgement to repletion in the ixodid tick, Dermacentor variabilis.

Mating in ticks results in profound physiological changes that eventually results in egg production. In the American dog tick, Dermacentor variabilis, mating causes partially blood-fed female ticks to commence rapid engorgement to repletion and eventual detachment from the host and egg laying. The peptidic male pheromone (engorgement factor alpha/beta) transferred to the female during mating is known only from a single tick species, Amblyomma hebraeum, and was shown to consist of two peptides produced in the testis/vas deferens (TVD) and not in the male accessory gland (MAG). In the current study, we obtained 2704bp of sequence data for efalpha from D. variabilis, of 7kb as determined by Northern blot, and show that it is also present in the Southern cattle tick, Rhipicephalus microplus and the deer tick, Ixodes scapularis. Analysis of the male gonad transcriptome by pyrosequencing produced 563,093 reads of which 636 matched with efalpha; none matched with efbeta. No evidence of efbeta orthologs could be found in any publicly available database including the I. scapularis genome. Silencing efalpha in male ticks failed to significantly reduce the engorgement weight of females compared to controls. Injection of sephadex beads, replete female synganglia, fed male MAG, fed male TVD, or replete female vagina/seminal receptacle (VA/SR), separately, failed to initiate feeding to repletion like that found in normally mated females. However, a small percentage of females injected with VA/SR that fed beyond the arbitrary weight for repletion of 300mg, produced brown eggs (an indication of vitellogenin uptake by the oocytes). The greatest effect was observed in female ticks injected with a suspension of MAG and TVD combined; 50% fed to repletion and all of these dropped off from the host and laid brown eggs. The effect was abolished if the aqueous fraction of the MAG/TVD homogenate only was injected suggesting that EF in ticks is a non-secreted membrane-bound or intracellular protein. Overall, these data suggest that EFalpha in D. variabilis is not an engorgement factor.

Heme-binding storage proteins in the Chelicerata.

Lipoglycoproteins in the Chelicerata that bind and store heme appear to represent a unique evolutionary strategy to both mitigate the toxicity of heme and utilize the molecule as a prosthetic group. Knowledge of heme-binding storage proteins in these organisms is in its infancy and much of what is known is from studies with vitellogenins (Vg) and more recently the main hemolymph storage protein in ixodid ticks characterized as a hemelipoglyco-carrier protein (CP). Data have also been reported from another arachnid, the black widow spider, Latrodectus mirabilis, and seem to suggest that the heme-binding capability of these large multimeric proteins is not a phenomenon found only in the Acari. CP appears to be most closely related to Vg in ticks in terms of primary structure but post-translational processing is different. Tick CP and L. mirabilis high-density lipoprotein 1 (HDL1) are similar in that they consist of two subunits of approximate molecular masses of 90 and 100 kDa, are found in the hemolymph as the dominant protein, and bind lipids, carbohydrates and cholesterol. CP binds heme which may also be the case for HDL1 since the protein was found to contain a brown pigment when analyzed by native polyacrylamide gel electrophoresis. Vgs in ticks are composed of multiple subunits and are the precursor of the yolk protein, vitellin. The phylogeny of these proteins, regulation of gene expression and putative functions of binding and storing heme throughout reproduction, blood-feeding and development are discussed. Comparisons with non-chelicerate arthropods are made in order to highlight the mechanisms and putative functions of heme-binding storage proteins and their possible critical function in the evolution of hematophagy.