John P. Burand

RNAi: future in insect management.

RNA interference is a post- transcriptional, gene regulation mechanism found in virtually all plants and animals including insects. The demonstration of RNAi in insects and its successful use as a tool in the study of functional genomics opened the door to the development of a variety of novel, environmentally sound approaches for insect pest management. Here the current understanding of the biogenesis of the two RNAi classes in insects is reviewed. These are microRNAs (miRNAs) and short interfering RNAs (siRNAs). Several other key approaches in RNAi -based for insect control, as well as for the prevention of diseases in insects are also reviewed. The problems and prospects for the future use of RNAi in insects are presented.

Virus transmission in gypsy moths is not a simple mass action process

We used the nuclear polyhedrosis virus (LdNPV) of the gypsy moth, Ly- mantria dispar (Lepidoptera: Lymantriidae), to test one of the basic assumptions of most models of disease dynamics, that the rate of horizontal transmission is directly proportional to the product of the densities of healthy larvae and virus. To do this we made measurements of virus transmission, using small-scale experiments in bags on red oak (Quercus rubra) foliage and field data on naturally occurring populations from a previous study. We observed a decline in the transmission constant as the densities of both healthy larvae and pathogen increased, indicating that the rate of disease transmission is not directly proportional to the product of these variables.

Two viruses that cause salivary gland hypertrophy in Glossina pallidipes and Musca domestica are related and form a distinct phylogenetic clade.

Glossina pallidipes and Musca domestica salivary gland hypertrophy viruses (GpSGHV and MdSGHV) replicate in the nucleus of salivary gland cells causing distinct tissue hypertrophy and reduction of host fertility. They share general characteristics with the non-occluded insect nudiviruses, such as being insect-pathogenic, having enveloped, rod-shaped virions, and large circular double-stranded DNA genomes. MdSGHV measures 65x550 nm and contains a 124 279 bp genome (approximately 44 mol% G+C content) that codes for 108 putative open reading frames (ORFs). GpSGHV, measuring 50x1000 nm, contains a 190 032 bp genome (28 mol% G+C content) with 160 putative ORFs. Comparative genomic analysis demonstrates that 37 MdSGHV ORFs have homology to 42 GpSGHV ORFs, as some MdSGHV ORFs have homology to two different GpSGHV ORFs. Nine genes with known functions (dnapol, ts, pif-1, pif-2, pif-3, mmp, p74, odv-e66 and helicase-2), a homologue of the conserved baculovirus gene Ac81 and at least 13 virion proteins are present in both SGHVs. The amino acid identity ranged from 19 to 39 % among ORFs. An (A/T/G)TAAG motif, similar to the baculovirus late promoter motif, was enriched 100 bp upstream of the ORF transcription initiation sites of both viruses. Six and seven putative microRNA sequences were found in MdSGHV and GpSGHV genomes, respectively. There was genome. Collinearity between the two SGHVs, but not between the SGHVs and the nudiviruses. Phylogenetic analysis of conserved genes clustered both SGHVs in a single clade separated from the nudiviruses and baculoviruses. Although MdSGHV and GpSGHV are different viruses, their pathology, host range and genome composition indicate that they are related.

Effect of nuclear polyhedrosis virus infection on the development and pupation of gypsy moth larvae

Abstract The effects of Lymantria dispar nuclear polyhedrosis virus infection on gypsy moth larval development with respect to size, molting, and pupation were examined. Virus-infected noenate larvae inoculated by droplet feeding were significantly smaller than uninfected control larvae. In later instars, virus infection was found to slow weight gain, delay the onset of apolysis, inhibit molting, and block pupation. The magnitude of these effects increased with an increase in virus dose. These observed consequences may be the result of nutritional or hormonal interactions between virus and host and may have an effect on epidemiological studies on virus-infected insects in the field.

The effect of baculovirus infection on ecdysteroid titer in gypsy moth larvae (Lymantria dispar)

Abstract Insect baculovirus carries a gene referred to as egt. This gene encodes an enzyme known as ecdysteroid UDP-glycosyl transferase which catalyzes the sugar conjugation of ecdysteroids. Using a gypsy moth embryonic cell line EGT activity of Lymantria dispar nuclear polyhedrosis virus was identified. The effect of egt gene expression following virus infection in gypsy moth larvae was studied by determining the ecdysteroid titer in hemolymph. A radioimmunoassay (RIA) was used to determine the ecdysteroid titer and TLC coupled with RIA was used to estimate levels of conjugated and unconjugated ecdysteroids. Both the level and pattern of hemolymph ecdysteroid titer in virus-infected larvae are different from uninfected control insects. The hemolymph ecdysteroid titer of virus-infected insects never rise above basal level during larval to pupal molting. However, during fourth to fifth larval molting, the hemolymph of virus-infected larvae shows higher levels of ecdysteroids than controls. In these insects the proportion of the sugar conjugated form of ecdysone was found to increase as virus replication proceeded.

The effect of gypsy moth metamorphosis on the development of nuclear polyhedrosis virus infection

Abstract The development of nuclear polyhedrosis virus (NPV) infection in gypsy moth ( Lymantria dispar ) was studied before, during, and after host metamorphosis, and in larvae and pupae in the subsequent generation, to determine whether NPV ingested by late instars can replicate in host tissues through metamorphosis and whether it can be vertically transmitted to progeny. Individuals that survived sublethal dosages of NPV did not differ from undosed insects in pupal weight, fecundity, larval and pupal weight of progeny, or response of progeny to NPV challenge. No evidence of NPV infection or of abnormal histology was found in adult tissues examined by light microscopy and no virus was detected on the surface of eggs produced by NPV-treated moths. No NPV-caused mortality was recorded among undosed progeny of dosed or undosed parents. The progeny of dosed parents were neither more resistant nor more susceptible to LdMNPV than were progeny of undosed parents and lethal times did not differ between groups. Examination of larval, pupal, and adult tissues by DNA hybridization revealed that insects in which NPV DNA was detected died prior to adult eclosion. NPV was not detected in any hosts surviving to the adult stage. These results suggest that survivors of sublethal dosages of NPV avoid infection and are therefore incapable of vertically transmitting infectious virus to progeny.

Presence and prevalence of viruses in local and migratory honeybees (Apis mellifera) in Massachusetts.

ABSTRACT Migratory and local bees in Massachusetts were analyzed for seven viruses. Three were detected: black queen cell virus (BQCV), deformed wing virus (DWV), and sacbrood virus (SBV). DWV was most common, followed closely by BQCV and then by SBV. BQCV and SBV were present at significantly higher rates in the migratory bees assayed, bringing into question the impact that these bees have on the health of local bee populations.

Nudivirus genomics: Diversity and classification

Nudiviruses represent a diverse group of arthropod specific, rod-shaped and dsDNA viruses. Due to similarities in pathology and morphology to members of the family Baculoviridae, they have been previously classified as the so-called “non-occluded” baculoviruses. However, presently they are taxonomically orphaned and are not assigned to any virus family because of the lack of genetic relatedness to Baculoviridae,. Here, we report on recent progress in the genomic analysis of Heliothis zea nudivirus 1 (HzNV-1), Oryctes rhinoceros nudivirus (OrNV), Gryllus bimaculatus nudivirus (GbNV) and Heliotis zea nudivirus 2 (HzNV-2). Gene content comparison and phylogenetic analyses indicated that the viruses share 15 core genes with baculoviruses and form a monophyletic sister group to them. Consequences of the genetic relationship are discussed for the classification of nudiviruses.

The use of polymerase chain reaction and shortwave UV irradiation to detect baculovirus DNA on the surface of gypsy moth eggs.

Polymerase chain reaction (PCR) technology was employed to detect baculovirus DNA sequences from viral occlusion bodies (OB) contaminating the surface of gypsy moth eggs. The level of sensitivity of the technique was as low as 5 viral genome copies and DNA from 1 OB equivalent. Thirty minutes of shortwave UV irradiation of eggs contaminated with 8.4 x 10(4) OBs prevented amplification of viral DNA sequences from OBs on the egg surface. These methods are important for providing a better understanding of gypsy moth virus epizootiology as well as for the examination of insect eggs for the persistence of baculovirus gene sequences inside the egg or on the egg surface. In addition, these methods can be easily modified for monitoring the persistence of genetically engineered baculoviruses in insect populations as well as the fate of genes that these viruses might carry.

Baculovirus replication alters hormone-regulated host development.

The baculovirus Lymantria dispar nuclear polyhedrosis virus interferes with insect larval development by altering the hosts hormonal system. The level of haemolymph ecdysteroids, the insect moulting hormone, was found to be higher in virus-infected larvae than in uninfected controls. This was consistently observed in both fourth instars and day 5-infected fifth instars. The rate of hormone synthesis was examined by in vitro incubation of the prothoracic gland. Gland activity in virus-infected larvae was higher than controls and continued until the late stages of virus infection, even during the time that controls had ceased to secrete ecdysone after moulting. During virus replication, the prothoracic gland was observed to maintain morphological and ultrastructural characteristics indicative of ecdysone biosynthetic activities. Therefore, it is likely that the insects are no longer under the control of the normal hormonal system after virus infection. It is felt that the alteration of hormone titre and the rate of ecdysone synthesis is the result of the activity of ecdysteroid UDP-glucosyl transferase (EGT), a virus-encoded enzyme which is thought to inactivate ecdysteroids by sugar conjugation.