Genomic analysis of coconut rhinoceros Jeanne Jacobs

Ela Hiszczynska-Sawicka,
Mitchell Weston, Charles Hefer,

beetle and its biocontrol agent Oryctes Sean Marshall

AgResearch, Private Bag 4749,
Christchurch, New Zealand
jeanne.jacobs@agresearch.co.nz

rhinoceros nudivirus
Introduction
Coconut rhinoceros beetle (CRB, Oryctes rhinoceros) is an introduced pest
species of coconut palms in the Pacific. Oryctes rhinoceros nudivirus (OrNV) was
introduced in the 1960s as a biocontrol agent for CRB. Following a very effective
period of control there are now genetic variants of CRB that are no longer affected
by commonly used OrNV isolates. We are using different approaches to
understand this phenomenon. Here we describe our study of OrNV isolates,
originally identified in the CRB native range (Asia) to start to answer the question
do different isolates of OrNV have divergent efficacy as biocontrol agents of CRB?
The wider goal of our research project is to identify suitable biocontrol agents that
can help manage the new variants of CRB.

Aim
Develop an assay for the rapid identification of different OrNV
isolates from CRB samples based on multilocus sequence analysis. Figure 1. Position of 13 target regions on the OrNV X2B
genome (black bars). Regions selected for diagnostic analysis
with multiplex nanopore sequencing are underlined in red.
What did we do?
For initial identification of viral isolate diagnostics 13 target regions were identified Table 1. OrNV isolates from the AgResearch collection and
based on OrNV reference genomes in GenBank (AH015832.2; MN623374; the GenBank accessions of the genome sequence assemblies
MW298153; MW298154; MZ727584; NC_011588). Primers were designed for 20 obtained with nanopore sequencing (Oxford Nanopore
amplicons (0.8-1.5 kb) covering these regions (Figure 1). Technologies). Adapted from Weston et al. 2023.

Six OrNV isolates from the collection held at AgResearch were selected (Table 1). OrNV isolate GenBank accession
DNA was extracted from purified isolates. DUG42 OP831186
Sanger sequences from 20 amplicons from the 6 isolates were aligned to the PNG OP831187
OrNV_X2B genome (Figure 1). Based on the alignments, we selected 7 gene PV505 OP831188
regions with the highest variability. These were PCR amplified and barcoded with S2A OP831189
V23B OP831190
each OrNV isolate receiving a unique barcode. Multiple barcoded samples were
X2B OP831191
pooled and sequenced on a MinION R9 flowcell (ONT). SNP analysis of the
sequences successfully identified each OrNV isolate.
To test this high-throughput, rapid turnaround assay with ’real’ CRB samples, we What is next?
developed the workflow below. We now routinely use this multiplex assay for Complete a population genetic study on the diversity of
OrNV identification of CRB samples collected from field settings in the Pacific. CRB across the Pacific using Genotyping-by-
Sequencing; can we identify CRB genotypes that are
more susceptible to OrNV and/or more successful in
establishing and spreading?
Can we identify (novel) OrNV isolates that are more
successful as biocontrol agents for CRB?

Summary
A diagnostic assay for the rapid and efficient identification of
different OrNV isolates was developed.
This assay enables field monitoring of the establishment,
spread and efficacy of OrNV isolates for biocontrol within
CRB populations.
A better understanding of the OrNV isolate diversity present
within native and invasive ranges contributes towards
improving the management strategies for Pacific regions
invaded by CRB.

Weston M, C Hefer, J Jacobs, S Marshall, 2023 Complete genome sequences of six isolates of the Oryctes rhinoceros nudivirus. Microbiology Resource Announcements, doi:10.1128/mra.00126-23

Funding: This research was funded by the New Zealand Ministry of Foreign Affairs and Trade, Aid Programme ‘Pacific Response to Coconut
Rhinoceros Beetle: Biocontrol and Integrated Pest Management’