Matthew W. Turnbull

Helicoverpa armigera (Hübner) is a major crop pest native to Europe, Asia, Australia, and Africa which has recently invaded South America and has caused billions of dollars in agricultural losses. Because of challenges in differentiating between H. armigera and Helicoverpa zea (Boddie), a closely related species native to North and South America, genetic tests have previously been developed to detect H. armigera DNA in pooled samples of moth legs. In this study, a field-based recombinase polymerase amplification (RPA) assay using a lateral flow strip and a qPCR melt curve assay were developed for specific detection of H. armigera DNA in pooled moth samples. In addition, a crude DNA extraction protocol for whole moths was developed to allow rapid preparation of DNA samples. The RPA field test was able to detect ≥ 10 pg of purified H. armigera DNA and the crude DNA of one H. armigera sample in a background of 999 H. zea equivalents. The qPCR assay was able to detect ≥ 100 fg of purified H. armigera DNA and the crude DNA of one H. armigera sample in a background of up to 99,999 H. zea equivalents. Both RPA and qPCR assays detected H. armigera in the crude DNA extracted in the field from a pool of one H. armigera moth and 999 H. zea moths. These newly developed molecular assays to detect H. armigera will contribute to large-scale surveillance programs of H. armigera.

Gap junctions are integral membrane proteins that play a role in cell-to-cell communication. They are coded by the functional genes called connexins in chordates and innexins in invertebrates. However, recently pannexins were also found in mammalian genomes, which are homologous to insect innexins. Gap junction intercellular communication (GJIC) has different functions ranging from their role in ontogenesis to the transfer of intracellular signal molecules and minimizing the adverse effects of xenobiotics by dilution and steady-state catabolism. Perturbations of these gap junctions are known to promote cancers besides, and many tumor inducers reduce the functioning of these gap junctions. Insect gap junctions play a crucial role in the development of insects and perhaps might be one of the reasons for the success of insects on terrestrial habitats. Majority of the work on innexins was done on Drosophila melanogaster Meigen, 1830 (Diptera: Drosophilidae), and its innexins regulate size of the central nervous system, embryonic gut formation, metamorphosis, and the fertility of germ lines in the reproductive system. There are several insecticides like cyclodienes, organochlorines, phenypyrazoles, synthetic pyrethroids, avermectins, milbemycins, oxadiazines, semicarbazones, meta-diamides, isoxazolines which target ion channels in the insects, but there is no evidence that supports the possible toxic effects of insecticides on insect gap junctions. In this review, we discuss the importance of insect gap junctions and how they could be a potential target for chemical pest management.

Polydnaviruses are dsDNA viruses associated with endoparasitoid wasps. Delivery of the virus during parasitization of a caterpillar and subsequent virus gene expression is required for production of an amenable environment for parasitoid offspring development. Consequently, understanding of Polydnavirus gene function provides insight into mechanisms of host susceptibility and parasitoid wasp host range. Polydnavirus genes predominantly are arranged in multimember gene families, one of which is the vinnexins, which are virus homologues of insect gap junction genes, the innexins. Previous studies of Campoletis sonorensis Ichnovirus Vinnexins using various heterologous systems have suggested the four encoded members may provide different functionality in the infected caterpillar host. Here, we expressed two of the members, vnxG and vnxQ2, using recombinant baculoviruses in susceptible host, the caterpillar Heliothis virescens. Following intrahemocoelic injections, we observed that >90% of hemocytes (blood cells) were infected, producing recombinant protein. Larvae infected with a vinnexin-recombinant baculovirus exhibited significantly reduced molting rates relative to larvae infected with a control recombinant baculovirus and mock-infected larvae. Similarly, larvae infected with vinnexin-recombinant baculoviruses were less likely to survive relative to controls and showed reduced ability to encapsulate chromatography beads in an immune assay. In most assays, the VnxG protein was associated with more severe pathology than VnxQ2. Our findings support a role for Vinnexins in CsIV and more broadly Ichnovirus pathology in infected lepidopteran hosts, particularly in disrupting multicellular developmental and immune physiology.

Polydnaviruses associated with ichneumonid parasitoid wasps (Ichnoviruses) encode large numbers of genes, often in multigene families. The Ichnovirus Vinnexin gene family, which is expressed in parasitized lepidopteran larvae, encodes homologues of Innexins, the structural components of insect gap junctions. Here, we have examined intracellular behaviours of the Campoletis sonorensis Ichnovirus (CsIV) Vinnexins, alone and in combination with a host Innexin orthologue, Innexin2 (Inx2). QRT-PCR verified that transcription of CsIV vinnexins occurs contemporaneously with inx2, implying co-occurrence of Vinnexin and Inx2 proteins. Confocal microscopy demonstrated that epitope-tagged VinnexinG (VnxG) and VinnexinQ2 (VnxQ2) exhibit similar subcellular localization as Spodoptera frugiperda Inx2 (Sf-Inx2). Surface biotinylation assays verified that all three proteins localize to the cell surface, and cytochalasin B and nocodazole that they rely on actin and microtubule cytoskeletal networks for localization. Immunomicroscopy following co-transfection of constructs indicates extensive co-localization of Vinnexins with each other and Sf-Inx2, and live-cell imaging of mCherry-labelled Inx2 supports that Vinnexins may affect Sf-Inx2 distribution in a Vinnexin-specific fashion. Our findings support that the Vinnexins may disrupt host cell physiology in a protein-specific manner through altering gap junctional intercellular channel communication, as well as indirectly by affecting multicellular junction characteristics.

1. Polydnaviruses are dsDNA viruses associated with endoparasitoid wasps. Delivery of the virus during parasitization of a caterpillar and subsequent virus gene expression is required for production of an amenable environment for parasitoid offspring development. Consequently, understanding of Polydnavirus gene function provides insight into mechanisms of host susceptibility and parasitoid wasp host range. Polydnavirus genes predominantly are arranged in multimember gene families, one of which is the vinnexins , which are virus homologues of insect gap junction genes, the innexins . Previous studies of Campoletis sonorensis Ichnovirus Vinnexins using various heterologous systems have suggested the four encoded members may provide different functionality in the infected caterpillar host. Here, we expressed two of the members, vnxG and vnxQ2 , using recombinant baculoviruses in susceptible host, the caterpillar Heliothis virescens . Following intrahemocoelic injections, we observed >90% of hemocytes (blood cells) were infected, producing recombinant protein. Larvae infected with a vinnexin -recombinant baculovirus exhibited significantly reduced molting rates relative to larvae infected with a control recombinant baculovirus and mock infected larvae. Similarly, larvae infected with vinnexin -recombinant baculoviruses were less likely to molt relative to controls, and showed reduced ability to encapsulate chromatography beads in an immune assay. In most assays, the VnxG protein was associated with more severe pathology than VnxQ2. These results, in light of previous findings, support that Polydnavirus Vinnexin gene family members may provide complementary, rather than redundant, effects. This in turn indicates a need to test gene family member functionality across infected hosts for effects to determine member contribution to host range. 2. Importance: Polydnaviruses are obligate mutualistic associates of highly speciose wasp taxa that parasitize caterpillars. Expression of Polydnavirus-encoded genes in hosts parasitized by wasps is necessary for successful parasitization, and an unusual genome structure including multiple-membered gene families is hypothesized to contribute to host manipulation. We have tested this hypothesis by in vivo expression of two members of a family of Polydnavirus homologues of Innexins, or insect gap junction proteins. Previous findings demonstrated that the two Vinnexins induce different physiological alterations in heterologous systems. Here, in host caterpillars, we observed differential alteration by the two proteins of host immune cell (hemocyte) bioelectrical physiology and the immune response of encapsulation. Not only do our data suggest a linkage between cellular bioelectricity and immunity in insects, but they support that gene family expansion has functional consequences to both Polydnavirus and host wasp success.

The corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), preferentially oviposits and feeds on ears of corn (Zea mays L.) and can be managed using transgenic hybrids that produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Concentrations of Bt proteins can vary spatially and temporally in plant tissues, creating a heterogeneous environment that can increase the risk of resistance development. We planted small-plot trials of nine Bt and non-Bt corn hybrids in South Carolina in 2016 and 2017 and investigated the development, survival, feeding injury, and feeding behavior in corn ear tissues. ELISA was used to quantify the concentrations of Cry1F and Cry2Ab2 in young silk, old silk, maternal tip tissue, kernels, and husk. Cry1F and Cry2Ab2 significantly varied with silk age and both proteins were generally highest in the silk and tip tissue. Hybrids with pyramided proteins significantly reduced feeding injury to the silk, tip, and kernel ear tissues, which was less apparent with single Bt protein hybrids. The pyramided hybrid expressing Vip3A incurred no injury to either the ear tip or kernels, and only eight 1^st instar larvae were collected in the silk of 520 sampled ears. Age of larvae significantly varied among ear tissues but not between hybrids. Depending on hybrid family, mean larval instar in the silk, tip, and kernels was 1^st or 2^nd, 3^rd, and 5^th, respectively. Instar-specific feeding penetrance into corn ears increased with age but did not differ between hybrids. We characterized the instar- and tissue-specific feeding behavior of H. zea larvae but did not detect differences in feeding behavior between Bt and non-Bt hybrids. Implications for resistance management strategies such as seed mixtures are discussed.

Insect herbivores, especially sap-feeders, are sensitive to host-plant nitrogen quantity. However, past studies present contradicting results on sap-feeder life history traits influenced by plant nitrogen supplementation. This study analyzed the bottom-up effects of below-recommended nitrogen fertilization rates (0, 0.021, 0.048, and 0.091 g N/liter) on life history and total protein and lipid contents of a significant pest species, Phenacoccus madeirensis Green (the Madeira mealybug) (Hemiptera: Pseudococcidae). Developmental durations and survivorship from egg to adulthood of male and female mealybugs were similar across nitrogen fertilization levels. Females reared on plants fertilized at 0.021, 0.048, and 0.091 g N/liter produced, respectively, 152, 142, and 67% more eggs than females reared on unfertilized plants. Finite and intrinsic rates of increase and net reproductive rates of females were similar among the nitrogen fertilization levels, whereas the generation times of females from fertilized plants were significantly shorter than those from the unfertilized plants. Lipid contents of adult females and eggs, and average adult female protein content were similar across the nitrogen treatments. Average egg protein content increased with increasing host-plant fertilization rate. These results suggest that the response of the female Madeira mealybug to nitrogen fertilization is complex and may involve trade-offs and nutrient re-allocation.

Certain parasitoid wasps are associated with Polydnaviruses, symbiotic viruses that encode virulence factors which are essential to successful parasitization by the wasp of a caterpillar host. Members of one group of Polydnaviruses, the Ichnoviruses, encode a multi-gene family known as Vinnexins. Vinnexins are homologues of insect gap junction genes, and form functional gap junctions that may affect host cell physiology. However, the role of Vinnexins in host pathology and the mechanism by which these affect their caterpillar host are largely unknown. In this article, we generated recombinant baculoviruses to express vinnexins in Spodoptera frugiperda (Sf9) cells. To measure cell physiological changes caused by Vinnexins, cells were probed with a membrane potential-sensitive probe, DiBac4(3), and a pH indicator, carboxyfluorescein diacetate (CFDA). In addition, we utilized carbenoxolone and ouabain, respectively, to probe the role of gap junctions and hemi-channels, and Na+/K+-ATPase in establishing membrane potential in studied cells. Our results indicate that Vinnexins induce cell membrane depolarization and cytoplasmic alkalization to a degree specific to each tested Vinnexin, and that neither Vinnexin hemi-channels nor Na+/K+-ATPase appear to underlie these effects directly. These results hint that members of the Vinnexin protein family may affect host bio-electrical phenomena to disrupt host cell physiology, and that the individual proteins of the family may differentially affect host physiology.

There are a limited number of model species for decapod experimental embryology. To improve our understanding of developmental pattern evolution in the Decapoda, here we describe the early embryonic development of the caridean shrimp Lysmata boggessi, from immediately after fertilization to the hatching of the zoea larva, using fluorescence microscopy and whole-mount nuclear staining with 4′,6-diamidino-2-phenylindole. Lysmata boggessi follows the standard caridean pattern of early development, with early holoblastic cleavage that will later become superficial, to form a blastoderm. We found no evidence of stereotypical cleavage and the formation of blastomere interlocking bands, which suggests there is diversity in developmental patterns within the Caridea. Gastrulation starts 37 hours after fertilization, and the embryonized nauplius is formed 2 days later. Enlarged headlobes, early retinal differentiation, and delayed pereopod development are characteristics of the post-naupliar stages in this species. To facilitate comparative studies with other crustacean species, we propose a staging method based on our findings. Lysmata boggessi is a protandric simultaneous hermaphrodite that is relatively easy to breed in captivity and amenable to laboratory experimentation in studies of embryonic development.