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Identification and characterization of Oryctes rhinoceros nudivirus on Oryctes rhinoceros Linnaeus
Dissertation Abstract:
The coconut rhinoceros beetle (CRB), Oryctes rhinoceros, is a destructive pest of palm trees, in Malaysia. Oryctes rhinoceros nudivirus (OrNV), a natural pathogen of CRB, was discovered in Malaysia in 1963. It has been widely used to control CRB in oil palm plantations. The peroral inoculation of larvae showed the highest mortality rate of 100%. There were 4 types of OrNV (A, B, C, and D) reported in 2011 with different efficacy against CRB. Incidence of mixed viruses in the field may happen and may affect the efficacy of OrNV. There were also reports that CRBs with different haplotypes have different tolerances to OrNV infection. To date, the latest information on the OrNV and the CRB haplotype in Malaysia is not available. Therefore, the main objectives of the study were to identify and characterize the OrNV-infected CRB larvae, examine the mitochondrial genomes of CRB, the whole genome of OrNV, and the microbiomes of gut CRB using shotgun sequencing, and elucidate the response of CRB-infected OrNV in early infection using transcriptomics sequencing. The diseased CRB larvae collected from the field were subjected to viral purification using 10-50% (w/v) sucrose gradient density. Three fractions (F5, F6, and F7) were found. The average of nucleocapsids of the fractions was 162.92 ± 38.94 nm in length and 71.61 ± 10.86 nm in width. According to the bioassays study, lethal time at 50% (LT50) of F6 and F7 of 1st generation OrNV showed shorter than lethal time at 50% (LT50) of F5. Shotgun sequencing was conducted to study the whole genome of OrNV, mitogenome and microbiomes of CRB. This study discovered CRB-G and CRB-S haplotypes. Haplotype-G was found in CRB OPl, OP2, and CTI, while Haplotype-S was found in CRB OP3 and CT2. The lengths of the mitogenomes of OPl, OP2, OP3, CTI, and CT2 are 15,315 bp, 15,475 bp, 17,142 hp, 17,275 hp, and 15,484 hp, respectively. New strains of OrNV were discovered, called OrNV strain Kluang and OrNV strain Batu Pahat, with genome lengths of 125,794 hp and 124,925 hp, respectively. Several entomopathogenic microbes were detected from the gut of the CRB, including Providencia rettgeri, Hirsutella minnesotensis, Metarhizium majus, Ophiocordyceps sinensis, Cordyceps confragosa, C. brongniartii, M. brunneum, Beauveria bassiana, and Aspergillus versicolor. Furthermore, to elucidate the response of CRB to OrNV infection, de novo transcriptomics study was conducted. The transcriptomics study revealed that the treated CRB larvae expressed genes involved in insect cuticle proteins, antimicrobial proteins, defensin, and immune proteins. The OrNV encoded up-regulated proteins, including semaphorin 1A-like, Thymidylate synthase (TS), PIF6, PIF5, and PIF6, DNA helicases, VP91, Patatin-like phospholipases, lef-4, and lef-9, and Ribonucleotide reductase. In conclusion, the OrNV strains identified in this study varied in pathogenicity; two new strains of OrNV were discovered; CRB-G and CRB-S haplotypes were first reported in Malaysia; and other entomopathogenic microbes were found in CRB gut. CRB larvae expressed genes to defend against OrNV infection; on the other hand, the OrNV also expressed proteins to invade the CRB.