Sayed M.S. Khalil

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.

First Transcriptome of the Testis-Vas Deferens-Male Accessory Gland and Proteome of the Spermatophore from Dermacentor variabilis (Acari: Ixodidae)

Ticks are important vectors of numerous human diseases and animal diseases. Feeding stimulates spermatogenesis, mating and insemination of male factors that trigger female reproduction. The physiology of male reproduction and its regulation of female development are essentially a black box. Several transcriptomes have catalogued expression of tick genes in the salivary glands, synganglion and midgut but no comprehensive investigation has addressed male reproduction and mating. Consequently, a new global approach using transcriptomics, proteomics, and quantitative gene expression is needed to understand male reproduction and stimulation of female reproduction. This first transcriptome to the reproductive biology of fed male ticks, Dermacentor variabilis, was obtained by 454 pyrosequencing (563,093 reads, 12,804 contigs). Gene Ontology (Biological Processes level III) recognized 3,866 transcripts in 73 different categories; spermiogenesis; spermatogenesis; peptidases, lipases and hydrolases; oxidative and environmental stress; immune defense; and protein binding. Reproduction-associated genes included serine/threonine kinase, metalloendoproteinases, ferritins, serine proteases, trypsin, cysteine proteases, serpins, a cystatin, GPCR and others. qRT-PCR showed significant upregulation from unfed versus fed adult male reproductive organs of zinc metalloprotease, astacin metalloprotease and serine protease, enzymes important in spermiogenesis and mating activity in insects, as well as a GPCR with the greatest similarity to a SIFamide receptor known to be important in regulating courtship behavior in Drosophila. Proteomics on these organs and the spermatophore by tryptic digestion/Liquid chromatography/Mass spectrometry/Mass spectrometry (LC/MS/MS) demonstrated expression of many of the same messages found by 454 sequencing, supporting their identification, and revealed differences in protein distribution in the reproductive system versus the spermatophore. We found Efα but no EF β in the transcriptome and neither of these proteins in the spermatophore. Thus, the previously described model for male regulation of female reproduction may not apply to other ticks. A new paradigm is needed to explain male stimulation of female tick reproduction.

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.

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.

Synganglion transcriptome and developmental global gene expression in adult females of the American dog tick, Dermacentor variabilis (Acari: Ixodidae)

454 Pyrosequencing was used to characterize the expressed genes from the synganglion and associated neurosecretory organs of unfed and partially fed virgin and mated replete females of the American dog tick, Dermacentor variabilis. A total of 14 881 contiguous sequences (contigs) was assembled, with an average size of 229 bp. Gene ontology terms for Level 2 biological processes were assigned to 4366 contigs. Seven acetylcholinesterases, a muscarinic acetylcholine (ACh) receptor, two nicotinic ACh receptor β‐subunits, two ACh unc‐18 regulators, two dopamine receptors, two gamma aminobutyric acid (GABA) receptors, two GABA transporters, two norepinephrine transporters and an octopamine receptor are described. Microarrays were conducted to examine global gene expression and quantitative real‐time polymerase chain reaction was used to verify expression of selected neuropeptides. Hierarchical clustering of all differentially expressed transcripts grouped part‐fed and replete ticks as being more similar in terms of differentially expressed genes with unfed ticks as the outgroup. Nine putative neuropeptides (allatostatin, bursicon‐β, preprocorazonin, glycoprotein hormone α, insulin‐like peptide, three orcokinins, preprosulphakinin) and a gonadotropin releasing hormone receptor were differentially expressed, and their developmental expression and role in reproduction was investigated. The presence of eclosion hormone, corazonin and bursicon in the synganglion, which in insects regulate behaviour and cuticle development associated with moulting, suggest that this system may be used in ticks to regulate blood feeding, cuticle expansion and development related to female reproduction; adult ticks do not moult.

Molecular characterization of the major hemelipoglycoprotein in ixodid ticks

The major hemelipoglyco‐carrier protein (CP) found throughout the development of male and female adult American dog ticks, Dermacentor variabilis (Say) was sequenced. DvCP is a single transcript coding for two protein subunits that together contain three motifs: (1) a lipoprotein n‐terminal domain that is a common attribute of proteins that bind lipids, carbohydrates and metals; (2) a domain of unknown function characteristic of proteins with several large open beta sheets; and (3) a von Willebrand factor type D domain near the carboxy‐terminus apparently important for multimerization. These motifs, which are also found in tick vitellogenin, are not shared by heme‐binding proteins studied thus far in other hematophagous insects. DvCP message was highest in fat body and salivary gland but was also found in midgut and ovary tissue. Expression was initiated by blood feeding in virgin females and not by mating, as is typical of tick vitellogenin; and the message was found in fed males at levels similar to part fed, virgin females. CP appears to be highly conserved among the Ixodida. The closest match by BlastP to DvCP is vitellogenin from Caenorhabditis elegans (AAC04423), suggesting that CP is a novel protein. The role of CP in heme sequestration, the evolution of hematophagy and host complementation are discussed.

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.

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.

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.

Enhanced activity of an insecticidal protein, trypsin modulating oostatic factor (TMOF), through conjugation with aliphatic polyethylene glycol

BACKGROUND Trypsin modulating oostatic factor (TMOF), a decapeptide (Tyr-Asp-Pro-Ala-Pro(6)) isolated from the ovaries of the adult yellow fever mosquito, Aedes aegypti, regulates trypsin biosynthesis. TMOF per os is insecticidal to larval mosquitoes and a good model for the development of technologies to enhance protein insecticide activity by reduced catabolism and/or enhanced delivery to the target. RESULTS TFA-TMOF-K (TFA = trifluoro acetyl) allowed the specific conjugation of monodispersed, aliphatic polyethylene glycol (PEG) to the amino group of lysine-producing TMOF-K-methyl(ethyleneglycol)(7)-O-propionyl (TMOF-K-PEG(7) P). The addition of lysine to TMOF reduced its per os larval mosquitocidal activity relative to the parent TMOF, but conjugation of TMOF-K with methyl(ethyleneglycol)(7)-O-propionyl increased its toxicity 5.8- and 10.1-fold above that of TMOF and TMOF-K for Ae. aegypti. Enhanced insecticidal activity was also found for larval Ae. albopictus and for neonates of Heliothis virescens and Heliocoverpa zea. Only TMOF-K was found by MS/MS in the hemolymph for H. virescens fed on TMOF-K-PEG(7) P. No TMOF, TMOF-K or PEGylated TMOF-K was detected in the hemolymph after topical applications. CONCLUSIONS This research suggests that aliphatic PEG polymers can be used as a new method for increasing the activity of insecticidal proteins.