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Bacterially produced dsRNA targeting SePGRP-LB reduces population fitness of Spodoptera exigua (Lepidoptera: Noctuidae) and increases its susceptibility to SeMNPV.

The beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), is an important agricultural pest, and S. exigua multiple nucleopolyhedrovirus (SeMNPV) is a host-specific biological control agent. However, baculovirus efficacy can be limited by host antiviral responses. S. exigua peptidoglycan recognition protein LB (SePGRP-LB) has been identified as an antiviral immune factor, suggesting that its suppression may increase larval susceptibility to SeMNPV. In this study, bacterially produced double-stranded RNA targeting SePGRP-LB (bac-dsPGRP-LB) was orally delivered to larvae to induce RNA interference. Feeding bac-dsPGRP-LB reduced SePGRP-LB transcript levels by 24.0% to 65.7% over 7 d. SePGRP-LB knockdown prolonged fifth-instar larval development, reduced female pupal weight, shortened male adult longevity and the oviposition period, and decreased fecundity by approximately 51%. Life table analysis further showed significant reductions in the intrinsic rate of increase (r), finite rate of increase (λ), and net reproductive rate (R0) following bac-dsPGRP-LB treatment. During SeMNPV infection, co-feeding with bac-dsPGRP-LB significantly suppressed SePGRP-LB expression, increased the SeMNPV genomic load, and reduced larval survival compared with the SeMNPV + bac-dsGFP treatment. These findings identify SePGRP-LB as a promising RNAi target for simultaneously reducing S. exigua fitness and enhancing its susceptibility to SeMNPV under laboratory conditions.

SePGRP-LB

Baculovirus (MNPV) genomic variants: characterization of Spodoptera exempta MNPV DNAs and comparison with other Autographa californica MNPV DNAs.

A nuclear polyhedrosis virus (NPV) strain from Spodoptera exempta (SeMNPV-25 baculovirus) is a restriction endonuclease DNA map variant similar to Autographa californica NPV (AcMNPV baculovirus). Fourteen restriction endonuclease variants were identified and isolated from a SeMNPV baculovirus stock with 12 of the variants found at low frequency (less than 3%). The DNA from each variant was compared to the prototype SeMNPV-25 for insertions, deletions and new restriction sites. Regions of variation were defined on the prototype SeMNPV-25 genome, and the nature of the variation within these regions was determined. These data are discussed and compared with the existing data on other variants of AcMNPV. A comparison of the physical maps revealed that all the SeMNPV variants were different from those reported for AcMNPV. Although the SeMNPV variants were distinctive, they were clearly genomic AcMNPV variants. The regions of the baculovirus genomic variation were identified, and three separate mechanisms are suggested for their generation. Five regions (hr1 to hr5) were associated with intragenic homologous viral sequences, five regions (vI to vIII) may be associated with the insertion of DNA sequences of cellular origin, and two regions (pI and pII) were associated with mutations resulting in the addition or loss of a PstI site. Physical maps were generated for SeMNPV variant regions vI, hr2, vII and vIII.

Animals

Conserved homologous regions between two baculovirus DNAs.

Regions of homology on the physical maps of Spodoptera exempta multiple-nucleocapsid nuclear polyhedrosis virus (SeMNPV-25), an Autographa californica MNPV genomic variant, and S. frugiperda (SfMNPV-2) baculovirus DNAs were identified by reciprocal DNA-DNA blot hybridization under conditions of an effective temperature of Tm -25 degrees C. In addition, cloned fragments of the viral genome which contained the homologous regions were used in hybridization experiments to confirm, refine and correlate the regions of the two physical maps. Five homologous regions conserved between the two physical maps were identified. When the stringency of the hybridization was increased (Tm -20 degrees C), only two of the original regions were identified by blot hybridization. One of the two regions contained the polyhedrin gene, and the other region was not associated with any known viral function. The five regions did not overlap with the intragenic homologous sequence (hr1 to hr5) regions on the SeMNPV-25 map or with restriction endonuclease variant (vI to vIV) regions on the SeMNPV-25 and SfMNPV-2 maps. The degree of similarity in the genomic organization of these two baculoviruses is discussed.

Animals

Nucleotide sequence and transcriptional analysis of the polyhedrin gene of Spodoptera exigua nuclear polyhedrosis virus.

The nucleotide sequence of a 1.1 kbp fragment of the multiple nucleocapsid nuclear polyhedrosis virus (MNPV) of Spodoptera exigua (Se) containing the polyhedrin gene was determined. An open reading frame (ORF) of 738 nucleotides (nt) was detected. This ORF encoded a protein of 246 amino acids with a predicted M(r) of 29K. The nucleotide and amino acid sequences were compared with the sequences of eight other NPV polyhedrins. The SeMNPV polyhedrin protein was most closely related to S. frugiperda MNPV polyhedrin with differences in only five amino acids, and most distantly related to the Lymantria dispar MNPV polyhedrin. The size of the mRNA was approximately 1,000 nt, as determined by Northern blot analysis. Using primer extension assays and S1 nuclease mapping the transcriptional start and stop sites of the polyhedrin mRNA were located. The 5' regulatory sequence appeared to be 44 nt in length with the mRNA start site predominantly at the first A of the TAAG consensus start sequence. Two degenerate poly(A) signals were found immediately downstream of the translational stop signal. The transcriptional stop was located approximately 230 nt downstream from the translational stop signal, in an AT-rich sequence that appears to be common to all baculovirus polyhedrin genes. The SeMNPV polyhedrin mRNA does not appear to be polyadenylated.

Amino Acid Sequence