Role of CYP9E2 and a long non-coding RNA gene in resistance to a spinosad insecticide in the Colorado potato beetle, Leptinotarsa decemlineata.

Spinosads are insecticides used to control insect pests, especially in organic farming where limited tools for pest management exist. However, resistance has developed to spinosads in economically important pests, including Colorado potato beetle (CPB), Leptinotarsa decemlineata. In this study, we used bioassays to determine spinosad sensitivity of two field populations of CPB, one from an organic farm exposed exclusively to spinosad and one from a conventional farm exposed to a variety of insecticides, and a reference insecticide naïve population. We found the field populations exhibited significant levels of resistance compared with the sensitive population. Then, we compared transcriptome profiles between the two field populations to identify genes associated primarily with spinosad resistance and found a cytochrome P450, CYP9E2, and a long non-coding RNA gene, lncRNA-2, were upregulated in the exclusively spinosad-exposed population. Knock-down of these two genes simultaneously in beetles of the spinosad-exposed population using RNA interference (RNAi) resulted in a significant increase in mortality when gene knock-down was followed by spinosad exposure, whereas single knock-downs of each gene produced smaller effects. In addition, knock-down of the lncRNA-2 gene individually resulted in significant reduction in CYP9E2 transcripts. Finally, in silico analysis using an RNA-RNA interaction tool revealed that CYP9E2 mRNA contains multiple binding sites for the lncRNA-2 transcript. Our results imply that CYP9E2 and lncRNA-2 jointly contribute to spinosad resistance in CPB, and lncRNA-2 is involved in regulation of CYP9E2 expression. These results provide evidence that metabolic resistance, driven by overexpression of CYP and lncRNA genes, contributes to spinosad resistance in CPB.

Plastid Transformation of Micro-Tom Tomato with a Hemipteran Double-Stranded RNA Results in RNA Interference in Multiple Insect Species.

Plant-mediated RNA interference (RNAi) holds great promise for insect pest control, as plants can be transformed to produce double-stranded RNA (dsRNA) to selectively down-regulate insect genes essential for survival. For optimum potency, dsRNA can be produced in plant plastids, enabling the accumulation of unprocessed dsRNAs. However, the relative effectiveness of this strategy in inducing an RNAi response in insects using different feeding mechanisms is understudied. To investigate this, we first tested an in vitro-synthesized 189 bp dsRNA matching a highly conserved region of the v-ATPaseA gene from cotton mealybug (Phenacoccus solenopsis) on three insect species from two different orders that use leaf-chewing, lacerate-and-flush, or sap-sucking mechanisms to feed, and showed that the dsRNA significantly down-regulated the target gene. We then developed transplastomic Micro-tom tomato plants to produce the dsRNA in plant plastids and showed that the dsRNA is produced in leaf, flower, green fruit, red fruit, and roots, with the highest dsRNA levels found in the leaf. The plastid-produced dsRNA induced a significant gene down-regulation in insects using leaf-chewing and lacerate-and-flush feeding mechanisms, while sap-sucking insects were unaffected. Our results suggest that plastid-produced dsRNA can be used to control leaf-chewing and lacerate-and-flush feeding insects, but may not be useful for sap-sucking insects.

Transplastomic Tomato Plants Expressing Insect-Specific Double-Stranded RNAs: A Protocol Based on Biolistic Transformation.

Expressing insecticidal double-stranded RNA (dsRNA) molecules in plant plastids is a novel approach for in planta production of dsRNA that has enormous potential for developing improved plant-mediated RNA interference (RNAi) strategies for insect pest control. In this chapter, we describe the design of a transformation vector containing an expression cassette which can be used to stably transform plastids of tomato plants for production and accumulation of dsRNA . Such dsRNA can trigger the mechanisms of RNAi in pest insects and selectively suppress the expression of target genes, resulting in lethality. We also describe a protocol for detection of full-length dsRNA molecules in plastids using an RT-PCR-based method.

The ABCB Multidrug Resistance Proteins Do Not Contribute to Ivermectin Detoxification in the Colorado Potato Beetle, Leptinotarsa decemlineata (Say).

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a significant agricultural pest that has developed resistance to many insecticides that are used to control it. Investigating the mechanisms of insecticide detoxification in this pest is important for ensuring its continued control, since they may be contributors to such resistance. Multidrug resistance (MDR) genes that code for the ABCB transmembrane efflux transporters are one potential source of insecticide detoxification activity that have not been thoroughly examined in L. decemlineata. In this study, we annotated the ABCB genes found in the L. decemlineata genome and then characterized the expression profiles across midgut, nerve, and Malpighian tubule tissues of the three full transporters identified. To investigate if these genes are involved in defense against the macrocyclic lactone insecticide ivermectin in this insect, each gene was silenced using RNA interference or MDR protein activity was inhibited using a chemical inhibitor, verapamil, before challenging the insects with a dose of ivermectin. Survival of the insects did not significantly change due to gene silencing or protein inhibition, suggesting that MDR transporters do not significantly contribute to defense against ivermectin in L. decemlineata.

RNA Interference in the Tobacco Hornworm, Manduca sexta, Using Plastid-Encoded Long Double-Stranded RNA.

RNA interference (RNAi) is a promising method for controlling pest insects by silencing the expression of vital insect genes to interfere with development and physiology; however, certain insect Orders are resistant to this process. In this study, we set out to test the ability of in planta-expressed dsRNA synthesized within the plastids to silence gene expression in an insect recalcitrant to RNAi, the lepidopteran species, Manduca sexta (tobacco hornworm). Using the Manduca vacuolar-type H+ ATPase subunit A (v-ATPaseA) gene as the target, we first evaluated RNAi efficiency of two dsRNA products of different lengths by directly feeding the in vitro-synthesized dsRNAs to M. sexta larvae. We found that a long dsRNA of 2222 bp was the most effective in inducing lethality and silencing the v-ATPaseA gene, when delivered orally in a water droplet. We further transformed the plastid genome of the M. sexta host plant, Nicotiana tabacum, to produce this long dsRNA in its plastids and performed bioassays with M. sexta larvae on the transplastomic plants. In the tested insects, the plastid-derived dsRNA had no effect on larval survival and no statistically significant effect on expression of the v-ATPaseA gene was observed. Comparison of the absolute quantities of the dsRNA present in transplastomic leaf tissue for v-ATPaseA and a control gene, GFP, of a shorter size, revealed a lower concentration for the long dsRNA product compared to the short control product. We suggest that stability and length of the dsRNA may have influenced the quantities produced in the plastids, resulting in inefficient RNAi in the tested insects. Our results imply that many factors dictate the effectiveness of in planta RNAi, including a likely trade-off effect as increasing the dsRNA product length may be countered by a reduction in the amount of dsRNA produced and accumulated in the plastids.

Transcriptomic analysis of the Malpighian tubules of Trichoplusia ni: Clues to mechanisms for switching from ion secretion to ion reabsorption in the distal ileac plexus.

Excretion of metabolic wastes and toxins in insect Malpighian tubules (MTs) is coupled to secretion of ions and fluid. Larval lepidopterans demonstrate a complex and regionalized MT morphology, and recent studies of larvae of the cabbage looper, Trichoplusia ni, have revealed several unusual aspects of ion transport in the MTs. Firstly, cations are reabsorbed via secondary cells (SCs) in T. ni, whereas in most insects SCs secrete ions. Secondly, SCs are coupled to neighbouring principal cells (PCs) via gap junctions to enable such ion reabsorption. Thirdly, PCs in the SC-containing distal ileac plexus (DIP) region of the tubule reverse from cation secretion to reabsorption in response to dietary ion loading. Lastly, antidiuresis is observed in response to a kinin neuropeptide, which targets both PCs and SCs, whereas in most insects kinins are diuretics that act exclusively via SCs. Recent studies have generated a basic model of ion transport in the DIP of the larval T. ni. RNAseq was used to elucidate previously uncharacterised aspects of ion transport and endocrine regulation in the DIP, with the aim of painting a composite picture of ion transport and identifying putative regulatory mechanisms of ion transport reversal in this tissue. Results indicated an overall expression of 9103 transcripts in the DIP, 993 and 382 of which were differentially expressed in the DIP of larvae fed high-K+ and high-Na+ diets respectively. Differentially expressed transcripts include ion-motive ATPases, ion channels and co-transporters, aquaporins, nutrient and xenobiotic transporters, cell adhesion and junction components, and endocrine receptors. Notably, several transcripts for voltage-gated ion channels and cell volume regulation-associated products were detected in the DIP and differentially expressed in larvae fed ion-rich diet. The study provides insights into the transport of solutes (sugars, amino acids, xenobiotics, phosphate and inorganic ions) by the DIP of lepidopterans. Our data suggest that this region of the MT in lepidopterans (as previously reported) transports cations, fluid, and xenobiotics/toxic metals. Besides this, the DIP expresses genes coding for the machinery involved in Na+- and H+-dependent reabsorption of solutes, chloride transport, and base recovery. Additionally, many of the transcripts expressed by the DIP a capacity of this region to respond to, process, and sometimes produce, neuropeptides, steroid hormones and neurotransmitters. Lastly, the DIP appears to possess an arsenal of septate junction components, differential expression of which may indicate junctional restructuring in the DIP of ion-loaded larvae.

The Effect of Diet on Midgut and Resulting Changes in Infectiousness of AcMNPV Baculovirus in the Cabbage Looper, Trichoplusia ni.

Insecticide resistance has been reported in many important agricultural pests, and alternative management methods are required. Baculoviruses qualify as an effective, yet environmentally benign, biocontrol agent but their efficacy against generalist herbivores may be influenced by diet. However, few studies have investigated the tritrophic interactions of plant, pest, and pathogen from both a gene expression and physiological perspective. Here we use microscopy and transcriptomics to examine how diet affects the structure of peritrophic matrix (PM) in Trichoplusia ni larvae and consequently their susceptibility to the baculovirus, AcMNPV. Larvae raised on potato leaves had lower transcript levels for chitinase and chitin deacetylase genes, and possessed a thicker and more multi-layered PM than those raised on cabbage or artificial diet, which could contribute to their significantly lower susceptibility to the baculovirus. The consequences of these changes underline the importance of considering dietary influences on pathogen susceptibility in pest management strategies.

Overexpression of a cytochrome P450 and a UDP-glycosyltransferase is associated with imidacloprid resistance in the Colorado potato beetle, Leptinotarsa decemlineata.

Current control of insect pests relies on chemical insecticides, however, insecticide resistance development by pests is a growing concern in pest management. The main mechanisms for insecticide resistance typically involve elevated activity of detoxifying enzymes and xenobiotic transporters that break-down and excrete insecticide molecules. In this study, we investigated the molecular mechanisms of imidacloprid resistance in the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), an insect pest notorious for its capacity to develop insecticide resistance rapidly. We compared the transcriptome profiles of imidacloprid-resistant and sensitive beetle strains and identified 102 differentially expressed transcripts encoding detoxifying enzymes and xenobiotic transporters. Of these, 74 were up-regulated and 28 were down-regulated in the resistant strain. We then used RNA interference to knock down the transcript levels of seven up-regulated genes in the resistant beetles. Ingestion of double-stranded RNA successfully knocked down the expression of the genes for three cytochrome P450s (CYP6BQ15, CYP4Q3 and CYP4Q7), one ATP binding cassette (ABC) transporter (ABC-G), one esterase (EST1), and two UDP-glycosyltransferases (UGT1 and UGT2). Further, we demonstrated that silencing of CYP4Q3 and UGT2 significantly increased susceptibility of resistant beetles to imidacloprid, indicating that overexpression of these two genes contributes to imidacloprid resistance in this resistant strain.

Octopamine receptor gene expression in three lepidopteran species of insect.

The invertebrate octopaminergic system affects many diverse processes and represents the counterpart to the vertebrate adrenergic/noradrenergic system with the classes of octopamine receptor (OAR) being homologous to those of vertebrate adrenergic receptors. However, there is still little information on the OARs present in different insect species, and the levels and distribution of these receptors throughout the body. cDNAs sharing high similarity with known insect OARs were cloned in three lepidopteran species: the cabbage looper, Trichoplusia ni; the true armyworm, Pseudaletia unipuncta; and the cabbage white, Pieris rapae. Seven major larval tissues and one adult tissue were examined in T. ni using quantitative real-time PCR to determine the relative expression levels of each receptor transcript across different tissues, as well as of all receptor transcripts within individual tissues. A subset of these tissues was also examined in P. unipuncta and P. rapae. All receptor transcripts were expressed in the nervous system of all three species, however, the distribution of the different receptor types varied between species. In all tissues, the OARbeta2 transcript was the most highly expressed, except in the Malpighian tubules where OARbeta1 was highest, and the midgut where there was no significant difference in receptor transcript levels.

Multidrug resistance protein gene expression in Trichoplusia ni caterpillars.

Many insect species exhibit pesticide-resistant phenotypes. One of the mechanisms capable of contributing to resistance is the overexpression of multidrug resistance (MDR) transporter proteins. Here we describe the cloning of three genes encoding MDR proteins from Trichoplusia ni: trnMDR1, trnMDR2 and trnMDR3. Real-time quantitative PCR (qPCR) detected trnMDR mRNA in the whole nervous system, midgut and Malpighian tubules of final instar T. ni caterpillars. To test whether these genes are upregulated in response to chemical challenge in this insect, qPCR was used to compare trnMDR mRNA levels in unchallenged insects with those of insects fed the synthetic pyrethroid, deltamethrin. Only limited increases were detected in a single gene, trnMDR2, which is the most weakly expressed of the three MDR genes, suggesting that increased multidrug resistance of this type is not a significant part of the response to deltamethrin exposure.

Expression of multidrug resistance proteins is localized principally to the Malpighian tubules in larvae of the cabbage looper moth, Trichoplusia ni.

The multidrug resistance proteins (MRPs) serve a number of important roles in development, physiological homeostasis and metabolic resistance. In insects, they may also contribute to resistance against xenobiotics including insecticides and plant secondary metabolites. To investigate their contribution to xenobiotic resistance, we have examined the tissue distribution of gene and protein expression of the multidrug resistance proteins TrnMRP1 and TrnMRP4 of the lepidopteran insect, Trichoplusia ni. Using quantitative PCR and immunohistochemistry, we have identified high expression levels of both transporters in the Malpighian tubules relative to levels in other major tissues of the body, where they probably contribute to excretion of metabolic wastes or ingested xenobiotics. We have specifically located TrnMRP protein expression in a subpopulation of Malpighian tubule secondary cells. Expression of TrnMRP1 was also detected both at a high level in specific cortical neurons of larval ganglia and at a lower level throughout the cortex, where it may act in signaling or protective functions, respectively. In contrast, expression of TrnMRP4 was low to absent in larval ganglia, with the exception of single cells in the central connective. We discuss the potential implications of this TrnMRP activity on insect development and metabolic resistance.

Autographa californica multiple nucleopolyhedrovirus ac142, a core gene that is essential for BV production and ODV envelopment.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac142 is a baculovirus core gene and encodes a protein previously shown to associate with occlusion-derived virus (ODV). To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac142 deletion virus (AcBAC(ac142KO-PH-GFP)). Fluorescence and light microscopy revealed that AcBAC(ac142KO-PH-GFP) exhibits a single-cell infection phenotype. Titration assays and Western blot confirmed that AcBAC(ac142KO-PH-GFP) is unable to produce budded virus (BV). However, viral DNA replication is unaffected and the development of occlusion bodies in AcBAC(ac142KO-PH-GFP)-transfected cells evidenced progression to very late phases of the viral infection. Western blot analysis showed that AC142 is expressed in the cytoplasm and nucleus throughout infection and that it is a structural component of BV and ODV which localizes to nucleocapsids. Electron microscopy indicates that ac142 is required for nucleocapsid envelopment to form ODV and their subsequent occlusion, a fundamental process to all baculoviruses.

Functional comparison of full-length and N-terminal-truncated octopamine transporters from Lepidoptera.

We have cloned two new lepidopteran octopamine transporters (OATs), members of the solute-linked carrier family 6 (SLC6) of nutrient transporters, from the CNS of the European corn borer Ostrinia nubilalis and the cabbage white Pieris rapae. Comparison of these sequences with the previously cloned OAT from the cabbage looper Trichoplusia ni showed that the T. ni OAT sequence previously reported was truncated by 74 amino acids at the N-terminus. The cytoplasmic N-termini deduced here are considerably longer than the N-termini of other monoamine transporters in the SLC6 family and contain many more high-probability serine- and threonine-phosphorylation sites. Monoamine uptake and competitive inhibition studies on baculovirus-infected Sf9 cells expressing these three cloned OATs indicate that they are able to transport tyramine, octopamine and dopamine with high affinity (K(m) and K(i) range, 0.4 microM-2.7 microM) and capacity ((3)H-dopamine uptake by TrnOAT, 2.5 pmol/well/min). We aimed to examine the role of the N-terminus of OAT by comparing the properties of the full-length T. ni OAT with those of the previously reported N-truncated version. Results for the new full-length T. ni OAT showed no difference in the protein's affinity for octopamine or dopamine, although at low levels of viral infection it did show slightly higher transport activity ((3)H-dopamine uptake by truncated TrnOAT, 1.5 pmol/well/min). Treatment of Sf9 cells expressing full-length or truncated TrnOAT with a variety of protein kinase activators and inhibitors, however, did not change transporter activity. Neither an intact N-terminus, nor apparently a particular phosphorylation state of this extended N-terminus, is required for OAT to transport monoamines.

Ancestry of neuronal monoamine transporters in the Metazoa.

Selective Na(+)-dependent re-uptake of biogenic monoamines at mammalian nerve synapses is accomplished by three types of solute-linked carrier family 6 (SLC6) membrane transporter with high affinity for serotonin (SERTs), dopamine (DATs) and norepinephrine (NETs). An additional SLC6 monoamine transporter (OAT), is responsible for the selective uptake of the phenolamines octopamine and tyramine by insect neurons. We have characterized a similar high-affinity phenoloamine transporter expressed in the CNS of the earthworm Lumbricus terrestris. Phylogenetic analysis of its protein sequence clusters it with both arthropod phenolamine and chordate catecholamine transporters. To clarify the relationships among metazoan monoamine transporters we identified representatives in the major branches of metazoan evolution by polymerase chain reaction (PCR)-amplifying conserved cDNA fragments from isolated nervous tissue and by analyzing available genomic data. Analysis of conserved motifs in the sequence data suggest that the presumed common ancestor of modern-day Bilateria expressed at least three functionally distinct monoamine transporters in its nervous system: a SERT currently found throughout bilaterian phyla, a DAT now restricted in distribution to protostome invertebrates and echinoderms and a third monoamine transporter (MAT), widely represented in contemporary Bilateria, that is selective for catecholamines and/or phenolamines. Chordate DATs, NETs, epinephrine transporters (ETs) and arthropod and annelid OATs all belong to the MAT clade. Contemporary invertebrate and chordate DATs belong to different SLC6 clades. Furthermore, the genes for dopamine and norepinephrine transporters of vertebrates are paralogous, apparently having arisen through duplication of an invertebrate MAT gene after the loss of an invertebrate-type DAT gene in a basal protochordate.

Molecular cloning and functional characterization of a neuronal choline transporter from Trichoplusia ni.

A cDNA encoding a high-affinity Na(+)-dependent choline transporter (TrnCHT) was isolated from the CNS of the cabbage looper Trichoplusia ni using an RT-PCR-based approach. The deduced amino acid sequence of the CHT cDNA predicts a 594 amino acid protein of 64.74 kDa prior to glycosylation. TrnCHT has 80%, 79%, 76%, and 58% amino acid identity to putative CHTs from Anopheles gambiae, Drosophila melanogaster and Apis mellifera, and a cloned CHT from Limulus polyphemus, respectively. In situ hybridization of TrnCHT cRNA in whole-mount preparations of caterpillar CNS revealed that TrnCHT mRNA is expressed by hundreds of presumably cholinergic neurons present in both the brain and cortex of all segmental ganglia. Na(+)-dependent [(3)H]-choline uptake was induced in Sf9 cells in vitro following infection with a TrnCHT-expressing recombinant baculovirus. Virally induced [(3)H]-choline uptake was found to approximately equal the endogenous rate of choline uptake in insect cells, seen either after infection with a control virus or in TrnCHT-infected cells exposed to [(3)H]-choline in the absence of Na(+). The Na(+)-dependent component of [(3)H]-choline uptake by TrnCHT-infected cells was saturable with a K(m) for choline transport of 8.4 microM. Several compounds reported to be potent blockers of [(3)H]-choline uptake by cloned vertebrate choline transporters proved to be relatively weak inhibitors of choline uptake by Sf9 cells expressing TrnCHT. Hemicholinium-3 (K(i)=4.1 microM) and two oxoquinuclidium analogues of choline, quireston-A (K(i) approximately 10 microM) and quireston (K(i) approximately 100 microM) inhibited 50% of control uptake only at micromolar concentrations. The endogenous low-affinity Na(+)-independent uptake of [(3)H]-choline was also inhibited by high micromolar concentrations of hemicholinium-3.

Complete comparative genomic analysis of two field isolates of Mamestra configurata nucleopolyhedrovirus-A.

A second genotype of Mamestra configurata nucleopolyhedrovirus-A (MacoNPV-A), variant 90/4 (v90/4), was identified due to its altered restriction endonuclease profile and reduced virulence for the host insect, M. configurata, relative to the archetypal genotype, MacoNPV-A variant 90/2 (v90/2). To investigate the genetic differences between these two variants, the genome of v90/4 was sequenced completely. The MacoNPV-A v90/4 genome is 153 656 bp in size, 1404 bp smaller than the v90/2 genome. Sequence alignment showed that there was 99.5 % nucleotide sequence identity between the genomes of v90/4 and v90/2. However, the v90/4 genome has 521 point mutations and numerous deletions and insertions when compared to the genome of v90/2. Gene content and organization in the genome of v90/4 is identical to that in v90/2, except for an additional bro gene that is found in the v90/2 genome. The region between hr1 and orf31 shows the greatest divergence between the two genomes. This region contains three bro genes, which are among the most variable baculovirus genes. These results, together with other published data, suggest that bro genes may influence baculovirus genome diversity and may be involved in recombination between baculovirus genomes. Many ambiguous residues found in the v90/4 sequence also reveal the presence of 214 sequence polymorphisms. Sequence analysis of cloned HindIII fragments of the original MacoNPV field isolate that the 90/4 variant was derived from indicates that v90/4 is an authentic variant and may represent approximately 25 % of the genotypes in the field isolate. These results provide evidence of extensive sequence variation among the individual genomes comprising a natural baculovirus outbreak in a continuous host population.

Functionally distinct dopamine and octopamine transporters in the CNS of the cabbage looper moth.

A cDNA was cloned from the cabbage looper Trichoplusia ni based on similarity to other cloned dopamine transporters (DATs). The total nucleotide sequence is 3.8 kb in length and contains an open reading frame for a protein of 612 amino acids. The predicted moth DAT protein (TrnDAT) has greatest amino acid sequence identity with Drosophila melanogasterDAT (73%) and Caenorhabditis elegansDAT (51%). TrnDAT shares only 45% amino acid sequence identity with an octopamine transporter (TrnOAT) cloned recently from this moth. The functional properties of TrnDAT and TrnOAT were compared through transient heterologous expression in Sf9 cells. Both transporters have similar transport affinities for DA (Km 2.43 and 2.16 micro m, respectively). However, the competitive substrates octopamine and tyramine are more potent blockers of [3H]dopamine (DA) uptake by TrnOAT than by TrnDAT. D-Amphetamine is a strong inhibitor and l-norepinephrine a weak inhibitor of both transporters. TrnDAT-mediated DA uptake is approximately 100-fold more sensitive to selective blockers of vertebrate transporters of dopamine and norepinephrine, such as nisoxetine, nomifensine and dibenzazepine antidepressants, than TrnOAT-mediated DA uptake. TrnOAT is 10-fold less sensitive to cocaine than TrnDAT. None of the 15 monoamine uptake blockers tested was TrnOAT-selective. In situ hybridization shows that TrnDAT and TrnOAT transcripts are expressed by different sets of neurons in caterpillar brain and ventral nerve cord. These results show that the caterpillar CNS contains both a phenolamine transporter and a catecholamine transporter whereas in the three invertebrates whose genomes have been completely sequenced only a dopamine-selective transporter is found.

Characterization of Mamestra configurata nucleopolyhedrovirus enhancin and its functional analysis via expression in an Autographa californica M nucleopolyhedrovirus recombinant.

Enhancin genes have been identified in a number of baculoviruses and enhancin proteins are characterized by their ability to enhance the oral infectivity of heterologous baculoviruses in various lepidopteran insects. Here, we describe the putative enhancin gene from Mamestra configurata nucleopolyhedrovirus (MacoNPV), only the second NPV in which an enhancin-like ORF has been identified. The putative enhancin gene from MacoNPV has a typical baculovirus late promoter (ATAAG) 15 bp upstream from the ATG codon. The enhancin ORF encodes an 847 amino acid protein with a predicted molecular mass of 98 kDa and contains a conserved zinc-binding domain (HEIAH) common to metalloproteases. The MacoNPV enhancin shows approximately 20 % amino acid identity with other baculovirus enhancins. An Autographa californica M nucleopolyhedrovirus (AcMNPV) recombinant, AcMNPV-enMP2, expressing the MacoNPV enhancin gene under control of its native promoter was developed and characterized. Northern blot analysis showed expression of enhancin from 24 through 72 h post-infection. In 2nd-instar Trichoplusia ni larvae, the LD50 of the AcMNPV-enMP2 recombinant was 2.8 polyhedral inclusion bodies (PIB) per larva, 4.4 times lower than that of AcMNPV E2 wild-type virus (12.4 PIB per larva). At biologically equivalent doses, i.e. LD90, the survival time 50 % (ST50) of AcMNPV-enMP2 recombinant and AcMNPV E2 wild-type viruses were not significantly different.

Vasa expression and germ-cell specification in the spider mite Tetranychus urticae.

The specification of germ cells is an important process during the development of all animals. Expression of an evolutionarily conserved gene such as vasa can be used as a marker for germ cell fate. We have isolated a vasa-related gene from the two-spotted spider mite (Tetranychus urticae) and used it to examine the segregation of germ cells in this animal. In spider mites, vasa expression first appears in a group of cells that do not join the initial blastoderm surface. Instead, these cells remain in the interior of the blastoderm and then migrate to posterior regions of the embryo, where they form a cluster that appears in regions of the embryo consistent with the gonads. The expression pattern of this spider mite vasa homologue implies a novel process acts to specify germ cells in this species and that the specification of germ cells is an evolutionarily labile process.

Identification and genomic analysis of a second species of nucleopolyhedrovirus isolated from Mamestra configurata.

MacoNPV-96B is a nucleopolyhedrovirus isolated from naturally infected Mamestra configurata (Lepidoptera: Noctuidae) larvae. It was initially identified due to its completely different restriction endonuclease profile relative to the previously sequenced Mamestra configurata virus MacoNPV-90/2 (Q. Li, C. Donly, L. Li, L. G. Willis, D. A. Theilmann, and M. Erlandson, 2002, Virology 294, 106-121). The MacoNPV-96B host range and virulence were also found to differ significantly from those of the previous isolate. To further understand the complex of viruses infecting M. configurata, the genome of MacoNPV-96B was completely sequenced and analyzed in comparison with the genome of MacoNPV-90/2 and other sequenced baculoviruses. MacoNPV-96B consists of 158,482 bp, and 168 open reading frames (ORFs) of 150 nucleotides or longer with minimal overlap have been identified. The genome of MacoNPV-96B is 3422 bp larger than MacoNPV-90/2 and although gene arrangement is virtually identical, there are 9 ORFs unique to MacoNPV-96B and 10 unique to MacoNPV-90/2. bro genes were found to be associated with nonhomologous regions, suggesting that bro genes may facilitate recombination between genomes. A major difference in the gene content between the two viruses is a 5.4-kb insert in MacoNPV-96B, which is highly homologous to a cluster of Xestia c-nigrum granulovirus (XecnGV) ORFs, suggesting recent recombination events between these two viruses. Nucleotide sequence and amino acid sequence identity between the common ORFs of MacoNPV-96B and MacoNPV-90/2 average 87 and 90%, respectively. The sequence data suggest that MacoNPV-96B and MacoNPV-90/2 are closely related but have diverged and evolved into two separate species. This is the first study to identify highly related but separately evolving viruses in the same insect host and geographic location. A new Identity-GeneParityPlot analysis was developed to perform a comparison of two viral genomes in gene content and arrangement as well as homology level of individual ORFs.