The Endosymbiotic Wolbachia and Host COI Gene Enables to Distinguish Between Two Invasive Palm Pests; Coconut Leaf Beetle, Brontispa longissima and Hispid Leaf Beetle, Octodonta nipae

Abstract

To elucidate taxonomic eminence of identical pest species is essential for many ecological and conservation studies. Without proficient skills, accurate molecular identification and characterization are laborious and time-consuming. The coconut leaf beetle, Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae), is biologically and morphologically identical to hispid leaf beetle, Octodonta nipae (Maulik) (Coleoptera: Chrysomelidae), and is known as the most harming nuisances of palm cultivation worldwide. The present examination was to establish Wolbachia genotyping analysis along with host cytochrome oxidase subunit I (COI) gene for accurate identification between these individuals of the same family (Chrysomelidae). Here, we have cloned and sequenced a gene coding Wolbachia surface protein (wsp) and COI gene regions amplified from both species by polymerase chain reaction. The nucleotide sequences were directly determined (≈600 bp for wsp and ≈804 bp for COI) and aligned using the multiple alignment algorithms in the ESPript3 package and the MEGA5 program. Comparative sequence analysis indicated that the representative of wsp and COI sequences from these two beetles were highly variable. To ensure this bacterial variation, multilocus sequence typing (MLST) of bacterial genes was conducted, and the results vindicated the same trend of variations. Furthermore, the phylogenetic analysis also indicates that B. longissima and O. nipae being the two different species harbors two distinct Wolbachia Hertig and Burt (Rickettsiales: Anaplamataceae) supergroups B and A, respectively. The present outcomes quickly discriminate between these two species. Considering its simplicity and cost-effectiveness, it can be used as a diagnostic tool for discriminating such invasive species particularly B. longissima and O. nipae which has overlapping morphologic characters.

Fig. 1.

Various life stages (egg to adult) of Brontispa longissima and Octodonta nipae. Life cycle durations (in days) information of B. longissima were presented in detail from Takasu et al. (2010) and information of O. nipae are presented from Hou et al. (2014a).

Fig. 2.

MSA of host COI gene regions of ≈804 bp amplified from Brontispa longissima and Octodonta nipae. Shaded nucleotides indicate the sequence homology, while no shaded indicates the heterogeneity between the both species.

Fig. 3.

MSA of (A)Wolbachia outer surface protein (wsp) of ≈600 bp and (B) concatenated MLST loci of five conserved regions (2073 or 20179 bp) isolated from Brontispa longissima and Octodonta nipae. Shaded nucleotides indicate the sequence homology, while no color indicates the heterogeneity between the both species.

Fig. 3.

MSA of (A)Wolbachia outer surface protein (wsp) of ≈600 bp and (B) concatenated MLST loci of five conserved regions (2073 or 20179 bp) isolated from Brontispa longissima and Octodonta nipae. Shaded nucleotides indicate the sequence homology, while no color indicates the heterogeneity between the both species.

Fig. 4.

Phylogenetic placement of Wolbachia strains isolated from Brontispa longissima and Octodonta nipae by ML inference phylogeny using MEGA (v. 5.05). (A) ML tree is constructed based on 2 (one from each beetle) Wolbachia outer surface protein (wsp) sequences (≈600 bp) with 20 Wolbachia strains from various arthropods belong to A, and B Wolbachia supergroups were assembled and aligned together for phylogenetic analysis. (B) ML tree based on the 2 (one from each beetle) concatenated MLST loci (2073 or 2079 bp) with 13 closely related sequence type (ST) retrieved from MLST database (http://pubmlst.org/wolbachia/). Sequence from this study is highlighted in bold, while alphabetic letters (A, B, H, F, and D) indicate different Wolbachia supergroups.