Multiplex PCR improves efficiency for determining coconut rhinoceros beetle haplotype and presence of its biocontrol virus

The re-emergence of coconut rhinoceros beetle (CRB; Oryctes rhinoceros) as an invasive pest in the Pacific was unexpected after more than 30 years of successful biological control through the introduction of the biocontrol virus Oryctes rhinoceros nudivirus (OrNV) in the 1970s. OrNV was originally isolated from the beetle’s native range in Malaysia. An explanation for the recent wave of Pacific invasions by CRB emerged on discovering that most of the invasive populations belonged to a new haplotype grouping; CRB-G (Clade I). CRB-G is less affected by the commonly used biocontrol OrNV isolates. Hence, determining CRB haplotype and presence of its biocontrol virus are key steps that assist timely management decisions pertaining to outbreaks of this major palm tree pest. Until recently, three separate PCR reactions (using two different DNA dilutions), followed by a restriction digest, have been used to distinguish between invasive CRB populations and to determine presence of OrNV. This study describes a multiplex PCR targeting CRB and OrNV DNA in the same PCR, followed by a restriction digest. Method validation using gut samples collected from more than 250 CRB, representing OrNV infected and uninfected samples and key haplotypes, showed multiplex PCR virus results closely correlated with single PCR-based results. Weak positive virus bands in the multiplex PCR were followed up with a single PCR for OrNV using more dilute DNA, to rule out sample cross contamination and confirm establishment of infection. Multiplex PCR followed by restriction digest was effective at distinguishing the invasive beetle haplotype and determining the presence of OrNV. The multiplex PCR method has significantly reduced processing time and laboratory resources. We will present details of our progress and discuss the opportunity that multiplex assays present within the context of efforts to improve CRB management.