Kent S. Shelby

Polydnavirus-mediated suppression of insect immunity.

Polydnaviruses are symbiotic proviruses of some ichneumonid and braconid wasps that modify the physiology, growth and development of host lepidopteran larvae. Polydnavirus infection targets neuroendocrine and immune systems, altering behavior, stunting growth, and immobilizing immune responses to wasp eggs and larvae. Polydnavirus-mediated disruption of cellular and humoral immunity renders parasitized lepidopteran larvae suitable for development of wasp larvae as well as more susceptible to opportunistic infections. Evidence from the Campoletis sonorensis polydnavirus system indicates that the unique genomic organization of polydnaviruses may have evolved to amplify the synthesis of immunosuppressive viral proteins. Immunosuppressive viruses have been essential to elucidating vertebrate immunity. Polydnaviruses have similar potential to clarify insect immune responses and may also provide novel insights into the role of insect immunity in shaping polydnavirus genomes.

Polydnavirus infection inhibits translation of specific growth-associated host proteins

The wasp Campoletis sonorensis injects a polydnavirus (CsPDV) along with its egg during parasitization of Heliothis virescens larvae. CsPDV protects the wasp egg and larvae by selectively disabling the hosts cellular immune response, and by altering host physiology, growth, and development. Among the changes in host physiology brought about by CsPDV infection is a rapid, and specific decline in the translation of fat body mRNAs encoding selected major plasma proteins. Translational inhibition of the synthesis of all storage protein monomers, p82 (Riboflavin binding hexamer), and p74/p76 (arylphorin), occurs upon infection with CsPDV. Moreover, the prewandering peak of the plasma enzyme juvenile hormone esterase (JHE) was blocked by CsPDV injection. Northern blotting of fat body mRNA demonstrated that transcript levels of storage proteins were not affected by infection. Plasma titers of the iron binding proteins transferrin (p72) and ferritin (p24/26), and of the plasma juvenile hormone binding protein (p25) were not changed by CsPDV infection. That storage protein and JHE synthesis are translationally suppressed, while the synthesis of other plasma proteins continues apace, suggests that CsPDV infection may lead to translational discrimination among available mRNAs in CsPDV infected fat bodies. The effect of this translational discrimination is to shunt host resources away from larval growth and adult development, which presumably makes them available to the developing endoparasitoid.

Polydnavirus‐mediated inhibition of lysozyme gene expression and the antibacterial response

Parasitism of lepidopteran host larvae by hymenopteran parasitoids impairs the cellular immune response via expression of polydnavirus genes. Encapsulation of parasitoid eggs is thereby prevented. Parasitized insects are susceptible to opportunistic infections, suggesting that additional components of the immune system are affected. Insects normally respond to infection by inducing the synthesis of an array of antibacterial factors, including cecropins and lysozyme via a NFkappaB/lkappaB-like signal transduction pathway. To characterize the effects of PDVs on the antibacterial immune response, plasma antibacterial activities were assayed in H. virescens larvae infected with the C. sonorensis PDV. Plasma lysozyme activity in Heliothis virescens was reduced in parasitized and PDV-infected larvae after immune challenge. To examine the regulation of lysozyme after CsPDV injection, the Heliothis virescens lysozyme cDNA was cloned. In contrast to plasma lysozyme activity, the 1.1 kb lysozyme mRNA was induced in fat body and haemocytes by known elicitors. The data suggest that CsPDV, like some other viruses, regulates host cell gene expression at the level of translation. We propose that the immunodeficiencies caused by CsPDV injection are caused, in part, by the targeted translational inhibition of specific humoral immune response transcripts.

Polydnavirus infection inhibits synthesis of an insect plasma protein, arylphorin

The wasp Campoletis sonorensis injects a segmented, double-stranded DNA polydnavirus (CsPDV) along with its egg during parasitization of Heliothis virescens larvae. After parasitization, CsPDV protects the wasp egg and larva by selectively disabling the hosts cellular immune response. Other host physiological systems including growth and development are affected to the apparent benefit of the parasite. To begin the characterization of the biochemical effects and mode of action of CsPDV on host growth, the titre of a developmentally regulated insect storage protein, arylphorin, was studied. Parasitized or virus-infected insects had substantially less circulating arylphorin than control insects. Fat bodies from parasitized larvae also synthesized less arylphorin in vitro. However, Northern blots of total RNA from parasitized and non-parasitized, control insects showed that the arylphorin transcript level was unaffected by parasitization suggesting a biochemical block at the translational level. In vitro translation followed by immunoprecipitation of arylphorin indicated that the mRNA was present and translatable at equal levels in both parasitized and control insects. Injection of purified virus elicited the response observed in naturally parasitized larvae, demonstrating that the effect on arylphorin synthesis is mediated, either directly or indirectly, by polydnavirus gene product(s).

Plasma phenoloxidase of the larval tobacco budworm, Heliothis virescens, is virucidal.

Abstract Heliothis virescens larval plasma contains high levels of an antiviral activity against the budded form of the Helicoverpa zea single nucleopolyhedrovirus (HzSNPV) in vitro. Preliminary results indicated that phenoloxidase is primarily responsible for this virucidal effect. However it is known that other enzymes that generate antimicrobial reactive oxygen intermediates and reactive nitrogen intermediates are present in hemolymph that could contribute to the observed virucidal activity. To elucidate the contributions of phenoloxidase and other candidate activities to plasma innate immune response against baculovirus infection specific metabolic inhibitors were used. In vitro the general inhibitors of melanization (N-acetyl cysteine, ascorbate and glutathione), and specific inhibitors of phenoloxidase (phenylthiourea and Kojic acid), completely blocked virucidal activity up to the level seen in controls. Addition of the enzyme superoxide dismutase to plasma did not affect virucidal activity; however addition of catalase had an inhibitory effect. Inhibitors of nitric oxide synthase activity did not affect virucidal activity. Our results confirm that phenoloxidase is the predominate activity in larval plasma accounting for inactivation of HzSNPV in vitro, and that phenoloxidase-dependent H2O2 production may contribute to this virucidal activity.

Analysis of ESTs generated from immune-stimulated hemocytes of larval Heliothis virescens.

Heliothis virescens immunome components responding to baculoviral and bacterial infection were identified from expressed sequence tags (ESTs) generated from an immune-stimulated larval hemocyte cDNA library. A total of 5548 ESTs were generated comprising 448 contigs and 1114 singletons, totaling 1606 putative transcripts 1101 of which had BLAST scores, including many known orthologs from other insect species. Orthologs of known or putative immune function were identified among them melanization pathway components, proteases, antibacterial proteins, lectins, bacteria-binding proteins, ferritins, scavenger receptors, cell surface receptors, signaling pathway components, and stress response enzymes. Additionally, many enzymes of central metabolism, cytoskeletal, mitochondrial, and ribosomal components, as well as transcriptional and translational regulators were identified. The effect of bacterial and baculoviral infection upon transcript levels of three identified immunome targets from among the ESTs was quantitated using real time PCR. Scolexin-B, C-type lectin and growth-blocking peptide binding protein transcripts were significantly elevated by bacterial infection. Per os infection with the baculovirus Helicoverpa zea single nucleopolyhedrovirus however did not significantly alter transcript levels of these three genes. The ESTs reported here are the first large scale report of the H. virescens immunome responding to entomopathogens, and represent a first step to a more complete transcriptome for this pest moth.

Baculovirus induced transcripts in hemocytes from the larvae of Heliothis virescens.

Using RNA-seq digital difference expression profiling methods, we have assessed the gene expression profiles of hemocytes harvested from Heliothis virescens that were challenged with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). A reference transcriptome of hemocyte-expressed transcripts was assembled from 202 million 42-base tags by combining the sequence data of all samples, and the assembled sequences were then subject to BLASTx analysis to determine gene identities. We used the fully sequenced HzSNPV reference genome to align 477,264 Illumina sequence tags from infected hemocytes in order to document expression of HzSNPV genes at early points during infection. A comparison of expression profiles of control insects to those lethally infected with HzSNPV revealed differential expression of key cellular stress response genes and genes involved in lipid metabolism. Transcriptional regulation of specific insect hormones in baculovirus-infected insects was also altered. A number of transcripts bearing homology to retroviral elements that were detected add to a growing body of evidence for extensive invasion of errantiviruses into the insect genome. Using this method, we completed the first and most comprehensive gene expression survey of both baculoviral infection and host immune defense in lepidopteran larvae.

Characterization of the Asian Citrus Psyllid Transcriptome.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is a vector for the causative agents of Huanglongbing, which threatens citrus production worldwide. This study reports and discusses the first D. citri transcriptomes, encompassing the three main life stages of D. citri, egg, nymph and adult. The transcriptomes were annotated using Gene Ontology (GO) and insecticide-related genes within each life stage were identified to aid the development of future D. citri insecticides. Transcriptome assemblies and other sequence data are available for download at the International Asian Citrus Psyllid Genome Consortium website [http://psyllid.org/download] and at NCBI [http://www.ncbi.nlm.nih.gov/bioproject/29447].

Transcriptome of the Invasive Brown Marmorated Stink Bug, Halyomorpha halys (Stål) (Heteroptera: Pentatomidae)

Halyomorpha halys (Stål) (Heteroptera: Pentatomidae), the brown marmorated stink bug, is an invasive agricultural and nuisance pest rapidly expanding its incidence in North America. This voracious pest poses a significant threat to rural and urban agriculture, especially to specialty crops such as apples, grapes and ornamentals, as well as staple crops including soybean and corn. The object of this study was to generate transcript sequence resources for H. halys. RNA-seq libraries derived from distinct developmental stages and sexes were sequenced and assembled into 248,569 putatively unique transcripts (PUTs). PUTs were segmented into three disjoint tiers of varying reliability, with 4,794 classified as gold tier (highest quality), 16,878 as silver, and 14,357 as bronze. The gold-tier PUTs associated with 2,580 distinct non-redundant protein sequences from the NCBI NR database—1,785 of these (69%) mapped to annotated UniProtKB database proteins, from which 1,273 unique Pfam families and 459 unique Molecular Function GO terms were encountered. Of the silver tiers 6,527 PUTs associated with unique proteins, 4,193 mapped to UniProtKB (64%), from which 1,941 and 640 unique Pfam and Molecular Function GO terms were extracted. H. halys PUTs related to important life processes like immunity, endocrinology, reproduction, development, behavior, neurotransmission, neurotoxicity, olfaction, and small RNA pathways were validated through quantitative Real-Time PCR (qRT-PCR) for differential expression during distinct life stages (eggs, 2nd instar nymphs, 4th instar nymphs, female adults, male adults). PUTs similar to hypothetical proteins identified in symbiont microbes, including Pantoea and Nosema species, were more abundantly expressed in adults versus nymphs. These comprehensive H. halys transcriptomic resources can be utilized to aid development of novel control methodologies to disrupt life processes; to conduct reverse genetic screens to determine host gene function; and to design environmentally unobtrusive means to control host populations or target specific H. halys life stages, such as molecular biopesticides.

Cloning and characterization of the secreted hemocytic prophenoloxidases of Heliothis virescens

The plasma enzyme phenoloxidase plays an important role in host resistance against viral, bacterial, fungal, filarial, and parasitoid challenge. Two Heliothis virescens prophenoloxidase transcripts, HvPPO-1 and HvPPO-2, were assembled from ESTs derived from a hemocyte cDNA library. The 2,363-bp HvPPO-1 contig encoded a 696-amino acid protein. The 3,255-bp HvPPO-2 contig encoded a 684-amino acid protein. Hemocyte and fat body transcript levels of HvPPO-1 were slightly elevated by bacterial infection in 5th instar larvae; however, HvPPO-2 expression was not significantly elevated above controls by bacterial infection. Per os infection of 4th instar larvae with the baculovirus Helicoverpa zea SNPV (HzSNPV) had a mild but significant suppressive effect upon fat body and hemocytic HvPPO-1 expression when compared to expression in same-aged controls. HvPPO-2 expression levels in fat bodies and hemocytes from 4th instar larvae was not significantly altered by HzSNPV infection. HzSNPV infection of 5th instar larvae caused no significant alteration of HvPPO-1 or of HvPPO-2 expression in either fat bodies or hemocytes. Thus, even though prophenoloxidase subunits are constitutively expressed at high levels in larval H. virescens hemocytes and fat bodies, the subunit HvPPO-1 is differentially regulated by bacterial and baculoviral infection.