Molecular Alliance of Lymantria dispar Multiple Nucleopolyhedrovirus and a Short Unmodified Antisense Oligonucleotide of Its Anti-Apoptotic IAP-3 Gene: A Novel Approach for Gypsy Moth Control

Abstract

Baculovirus IAP (inhibitor-of-apoptosis) genes originated by capture of host genes. Unmodified short antisense DNA oligonucleotides (oligoDNAs) from baculovirus IAP genes can down-regulate specific gene expression profiles in both baculovirus-free and baculovirus-infected insects. In this study, gypsy moth (Lymantria dispar) larvae infected with multiple nucleopolyhedrovirus (LdMNPV), and LdMNPV-free larvae, were treated with oligoDNA antisense to the RING (really interesting new gene) domain of the LdMNPV IAP-3 gene. The results with respect to insect mortality, biomass accumulation, histological studies, RT-PCR, and analysis of DNA apoptotic fragmentation suggest that oligoRING induced increased apoptotic processes in both LdMNPV-free and LdMNPV-infected insect cells, but were more pronounced in the latter. These data open up possibilities for promising new routes of insect pest control using antisense phosphodiester DNA oligonucleotides.

Keywords: DNA insecticides; RING (really interesting new gene); baculovirus infection; gypsy moth Lymantria dispar; insect pest management; multiple nucleopolyhedrovirus IAP (inhibitor-of-apoptosis) genes.

Figure 1

Alignment of the sequenced DNA fragments of gypsy moth and nucleopolyhedrovirus (LdMNPV) IAP-3 gene fragments performed using ClustalW 2.0.3; identities found by simple comparison of the sequences are marked with *.

Figure 2

Expression of gypsy moth IAP-Z and IAP-1 genes after treatment with oligoDNAs. (a) Treatment of LdMNPV-free larvae with oligoRING (RING column) leads to significant down-regulation of host IAP-Z gene expression on the 14th day. Values represent means and standard errors of mRNA expression for 3 replicates relative to the water-treated control group. The significant difference between the oligoRING group and the water-treated control group is indicated by * when p < 0.05; (b) Treatment of LdMNPV-free gypsy moth larvae with oligoRING (RING column) leads to markedly decreased expression of the host IAP-1 gene on the 14th day. Values represent means and standard errors of mRNA expression for 3 replicates relative to the control water-treated control group. The significant difference between the oligoRING group and the water-treated control group is indicated by ^ when p < 0.12.

Figure 3

Light microscopy of the gypsy moth larvae histological slides. (a) Control sample (water-treated control): 1—outer layers; 2—intestinal epithelium; 3—esophagus; 4—pigment cells and their septum; 5—mandible; 6—maxilla; (b) OligoRING-treated larvae showing pronounced apoptotic destruction of tissues: 1—outer layers; 2—pigment cells with destroyed septum; 3—separate clusters of epithelial cells of the digestive tube; 4—destroyed mouth apparatus; 5—heart; 6—anal section of the digestive tube; 7—normal cell; 8—apoptotic signatures (pyknosis, karyorrhexis, and involution of cells); 9—vacuolated cell. Magnification: ×40 for (a), (b) and ×80 for the zoomed fragment of (b).

Figure 4

The oligoRING’s effect on the mortality and biomass accumulation of LdMNPV-infected gypsy moth larvae. (a) Non-cumulative curve of mortality of LdMNPV-infected larvae after treatment with oligoDNAs. The significant difference is indicated by * for p < 0.05. Each group (water-treated control, oligoCpG, and oligoRING) used 20 to 25 larvae per replicate. Each experiment was conducted in triplicate; (b) The mean body mass of LdMNPV-infected larvae after treatment with water, oligoCpG, or oligoRING is shown in mg. SE (standard errors) are given for three replicates. Significant difference: ** for p < 0.01; *** for p < 0.001.

Figure 5

Expression of gypsy moth IAP-1 gene and joint expression of baculovirus IAP-3 and host IAP-Z genes after treatment with oligoDNAs. (a) Treatment of LdMNPV-infected larvae with oligoRING leads to significant down-regulation of total expression of hIAP-Z and vIAP-3 genes on the 6th day. Values represent means and standard errors of mRNA expression for 3 replicates relative to the water-treated control group. The significant difference between oligoRING group vs. water control is indicated by ** when p < 0.01; (b) Treatment of LdMNPV-infected gypsy moth larvae with the oligoRING leads to a significant decrease in expression of the host IAP-1 gene on the 14th day. Values represent means and standard errors of mRNA expression for 3 replicates relative to the water-treated control group. The significant difference between oligoRING group vs. the water-treated control group is indicated by *** when p < 0.001.

Figure 6

Electrophoretic separation of DNA extracted from tissues of live LdMNPV-infected gypsy moth larvae (1% agarose gels). The positions of DNA markers are indicated on the left. Lanes: 1,2—water control; 3,4—oligoCpG treatment; 5,6—oligoRING treatment. Signs: FD—apoptotic fragmented DNA (around 180 and 360 bp); HF—higher fraction of undergraded genomic DNA. Data in each agarose lane represent DNA from three live larvae from every group in the experiment.