Detection and Characterisation of Endosymbiont Wolbachia (Rickettsiales: Anaplasmataceae) in Elaeidobius kamerunicus (Coleoptera: Curculionoidea), Pollinating Agent of Oil Palm, and Its Relationships between Populations

Main Article Content

Mohd Nur Azad Rushidi
Muhammad Luqman Hakim Azhari
Salmah Yaakop
Izfa Riza Hazmi


Elaeidobius kamerunicus is the most efficient pollinator of oil palm. Wolbachia is an endosymbiotic bacteria associated with E. kamerunicus that has a potential to affect the fecundity and fitness of the E. kamerunicus. Despite their importance, no studies have been conducted to investigate its prevalence in E. kamerunicus. The objectives of this study were to detect and characterize Wolbachia in E. kamerunicus and determine the phylogenetic relationship of Wolbachia strains that infect E. kamerunicus by using three genetic markers namely Filamenting temperature-sensitive mutant Z (ftsZ), Chaperonin folding protein (groEL), and Citrate Synthase Coding Gene (gltA). DNA was extracted from 210 individuals of E. kamerunicus and the Wolbachia infections were detected using the wsp marker. The infected samples (n = 25, 11.9%) were then sequenced using ftsZ, gltA and groEL markers for strain characterisation. In this study, a combination of four markers was used to construct the phylogeny of Wolbachia. Similar topologies were shown in all trees; Neighbour-Joining (NJ), Maximum Parsimony (MP), and Bayesian Inference (BI), which showed the mixing of individuals that harbor Wolbachia between populations. Interestingly, Wolbachia on E. kamerunicus was claded together with the species Drosophila simulans under supergroup B. This is the first report of Wolbachia infecting E. kamerunicus which is very valuable and significant as one of the parameters to evaluate the quality of the E. kamerunicus population for sustaining its function as a great pollinator for oil palm.

Article Details

How to Cite
Detection and Characterisation of Endosymbiont Wolbachia (Rickettsiales: Anaplasmataceae) in Elaeidobius kamerunicus (Coleoptera: Curculionoidea), Pollinating Agent of Oil Palm, and Its Relationships between Populations . (2023). Tropical Life Sciences Research, 34(3), 95–111.
Original Article


Arai S, Tabara K, Yamamoto N, Fujita H, Itagaki A, Kon M, Satoh H, Araki K, Tanaka- Taya K, Takada N, Yoshikawa Y, Ishihara C, Okabe N and Oishi K. (2013). Molecular phylogenetic analysis of Orientia tsutsugamushi based on the groES and groEL genes. Vector-Borne and Zoonotic Diseases 13(11): 825–829.

Ashraf H J, Akutse K S, Mukhtar I, Aguila L C R, Qasim M, Wang W, Bamisile B S and Wang L. (2021). Genetic diversity of Tamarixia radiata populations and their associated endosymbiont Wolbachia species from China. Agronomy 11(10): 2018.

Badrulisham A S, Kageyama D, Halim M, Aman-Zuki A, Masri M M, Ahmad S N, Md-Zain B M and Yaakop S. (2021). New insights into the phylogeography of the oil palm pest, Metisa plana towards its management control. Journal of Oil Palm Research 34(3): 427–438.

Bourtzis K, Nirgianaki A, Markakis G and Savakis C. (1996). Wolbachia infection and cytoplasmic incompatibility in Drosophila species. Genetics 144(3): 1063–1073.

Casiraghi M, Bordenstein S R, Baldo L, Lo N, Beninati T, Wernegreen J J, Werren J H and Bandi C. (2005). Phylogeny of Wolbachia pipientis gltA groEL and fisZ gene sequences: Clustering of arthropod and nematode symbionts in the F supergroup and evidence for further diversity in the Wolbachia tree. Microbiology 151(12): 4015–4022.

Chang Y H, Shangkuan Y H, Lin H C and Liu H W. (2003). PCR assay of the groEL gene for detection and differentiation of Bacillus cereus group cells. Applied and Environmental Microbiology 69(8): 4502–4510.

Daud S N S M and Ghani I A. (2016). Population density of oil palm pollinator weevil Elaeidobius kamerunicus based on seasonal effect and age of oil palm. AIP Conference Proceedings 1784: 060051.

Dedeine F, Vavre F, Shoemaker D D and Boulétreau M. (2004). Intra?individual coexistence of a Wolbachia strain required for host oogenesis with two strains inducing cytoplasmic incompatibility in the wasp Asobara tabida. Evolution 58(10): 2167– 2174.

Duplouy A, Couchoux C, Hanski I and van Nouhuys S. (2015). Wolbachia infection in a natural parasitoid wasp population. PLoS One 10(8): 1–16.

Fenollar F, La Scola B, Inokuma H, Dumler J S, Taylor M J and Raoult D. (2003). Culture and phenotypic characterization of a Wolbachia pipientis isolate. Journal of Clinical Microbiology 41(12): 5434–5441.

Fialho R F and Stevens L. (2000). Male-killing Wolbachia in a flour beetle. Proceedings of the Royal Society B: Biological Sciences 267(1451): 1469–1474.

Huigens M E, De Almeida R P, Boons P A H, Luck R F and Stouthamer R. (2004). Natural interspecific and intraspecific horizontal transfer of parthenogenesis-inducing Wolbachia in Trichogramma wasps. Proceedings of the Royal Society B: Biological Sciences, 271(1538): 509–515.

Kageyama D, Nishimura G, Hoshizaki S and Ishikawa Y. (2002). Feminizing Wolbachia in an insect, Ostrinia furnacalis (Lepidoptera: Crambidae). Heredity 88(6): 444–449.

Kageyama D, Narita S and Watanabe M. (2012). Insect sex determination manipulated by their endosymbionts: Incidences, mechanisms and implications. Insects 3(1): 161–199.

Kapli P, Yang Z and Telford M J. (2020). Phylogenetic tree building in the genomic age. Nature Reviews Genetics 21(7): 428–444.

Kovanen S M, Kivistö R I, Rossi M, Schott T, Kärkkäinen U M, Tuuminen T, Uksila J, Rautelin H and Hänninen M L. (2014). Multilocus sequence typing (MLST) and whole-genome MLST of campylobacter jejuni isolates from human infections in three districts during a seasonal peak in Finland. Journal of Clinical Microbiology 52(12): 4147–4154.

Kumar S, Tamura K and Nei M. (2004). MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings in Bioinformatics 5:150–163.

Li S J, Ahmed M Z, Lv N, Shi P Q, Wang X M, Huang J L and Qiu B L. (2017). Plant mediated horizontal transmission of Wolbachia between whiteflies. ISME Journal 11(4): 1019–1028.

Lin G N, Zhang C and Xu D. (2011). Polytomy identification in microbial phylogenetic reconstruction. BMC Systems Biology 5(Suppl. 3): S2.

Meléndez M R and Ponce W P. (2016). Pollination in the oil palms Elaeis guineensis, E. oleifera and their hybrids (OxG), in tropical America. Pesquisa Agropecuária Tropical 46(1): 102–110.

Mohammed M A, Aman Zuki A, Yusof S, Md Zain B M and Yaakop S. (2017). Prevalence and evolutionary history of endosymbiont Wolbachia (Rickettsiales: Anaplasmataceae) in parasitoids (Hymenoptera: Braconidae) associated with Bactrocera fruit flies (Diptera: Tephritidae) Infesting carambola. Entomological Science 20(1): 382– 395.

Mohammed M A, Aman Zuki A, Yusof S, Othman N W, Md Zain B M and Yaakop S. (2018). Classification of endosymbiont Wolbachia (Rickettsiales: Anaplasmataceae) in Opiine wasps (Hymenoptera: Braconidae). AIP Conference Proceedings 1940(1): 020039.

Moslim R and Kamarudin N. (2016). Comparative traits of pollinating weevils and factors affecting its population in Malaysia. In: Task Force Oil Palm Pollinating Weevil and Fruit Set. Bangi, Selangor: Malaysian Palm Oil Board.

Muhammad Luqman H A, Dzulhelmi M N, Idris A B and Izfa Riza H. (2017). The potential natural predators for oil palm pollinating weevil Elaeidobius kamerunicus Faust 1878 (Coleoptera: Curculionidae) in Malaysia. Serangga 22(2): 239–252.

Narita S, Kageyama D, Nomura M and Fukatsu T. (2007). Unexpected mechanism of symbiont-induced reversal of insect sex: Feminizing Wolbachia continuously acts on the butterfly Eurema hecabe during larval development. Applied and Environmental Microbiology 73(13): 4332–4341.

Parvizi P, Bordbar A and Najafzadeh N. (2013). Detection of Wolbachia pipientis, including a new strain containing the wsp gene, in two sister species of Paraphlebotomus sandflies, potential vectors of zoonotic cutaneous leishmaniasis. Memorias Do Instituto Oswaldo Cruz 108(4): 414–420.

Poinar G O Jr, Jackson T A, Bell N L and Wahid M B. (2002). Elaeolenchus parthenonema n. g., n. sp. (Nematoda: Sphaerularioidea: Anandranematidae n. fam.) parasitic in the palm-pollinating weevil Elaeidobius kamerunicus Faust, with a phylogenetic synopsis of the Sphaerularioidea Lubbock, 1861. Systematic Parasitology 52(3): 219–225.

Prasetyo A E, Purba W O and Susanto A. (2014). Elaeidobius kamerunicus: Application of hatch and carry technique for increasing oil palm fruit set. Journal of Oil Palm Research 26(3): 195–202.

Ren W, Wei H, Yang Y, Shao S, Wu H, Chen X and Yang Z. (2020). Molecular detection and phylogenetic analyses of Wolbachia in natural populations of nine galling Aphid species. Scientific Reports 10(1): 1–10.

Ros V I D, Fleming V M, Feil E J and Breeuwer J A. (2009). How diverse is the genus Wolbachia? Multiple-gene sequencing reveals a putatively new Wolbachia supergroup recovered from spider mites (Acari: Tetranychidae). Applied and Environmental Microbiology 75(4): 1036–1043.

Ronquist F, Teslenko M, van der Mark P, Ayres D L, Darling A, Höhna S, Larget B, Liu L, Suchard M A and Huelsenbeck J P. (2012). MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematics Biology 61(3): 539–542.

Schulenburg J H G V D, Hurst G D D, Huigens T M E, van Meer M M M, Jiggins F M and Majerus M E N. (2000). Molecular evolution and phylogenetic utility of Wolbachia ftsZ and wsp gene sequences with special reference to the origin of male-killing. Molecular Biology and Evolution 17: 584–600.

Schuler H, Kern P, Arthofer W, Vogt H, Fischer M, Stauffer C, and Riegler M. (2016). Wolbachia in parasitoids attacking native European and introduced eastern cherry fruit flies in Europe. Environmental Entomology 45(6): 1424–1431.

Siswanto and Soetopo D. (2020). Population of oil palm pollinator insect (Elaeidobius kamerunicus Faust.) at PTP Nusantara VIII Cisalak Baru, Rangkasbitung-Banten. IOP Conference Series: Earth and Environmental Science 418: 012045. https://

Sugimoto T N and Ishikawa Y. (2012). A male-killing Wolbachia carries a feminizing factor and is associated with degradation of the sex-determining system of its host. Biology Letters 8(3): 412–415.

Susko E. (2016). Support measures, phylogenetic tree. Encyclopedia of Evolutionary Biology 4(1): 256–260.

Swofford D. L. (2002). PAUP*. Phylogenetic Analysis Using Parsimony (* and other methods) (version 4.0b10). Sinauer Associates.

Teo T M. (2015). Effectiveness of the oil palm pollinating weevil, Elaeidobius kamerunicus, in Malaysia. UTAR Agriculture Science Journal 1(4): 40–43.

Turelli M and Hoffman A A. (1991). Rapid spread of an inherited incompatibility factor in California Drosophila. Nature 353: 440–442.

Wan Mohd Nor W N A, Othman N W, Yaakop S and Md Nor N S. (2018). Morphology and histology of reproductive organ and first screening of Wolbachia in the ovary of red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae). Serangga 23(2):183–193.

Weeks A R, Turelli M, Harcombe W R, Reynolds K T and Hoffmann A A. (2007). From parasite to mutualist: Rapid evolution of Wolbachia in natural populations of Drosophila. PloS Biology 5(5): 997–1005.

Yue J, Yan Z, Bai C, Chen Z, Lin W and Jiao F. (2015). Pollination activity of Elaeidobius kamerunicus (Coleoptera: Curculionoidea) on oil palm on Hainan Island. Florida Entomologist 98(2): 499–505.

Zhou W, Rousset F and O’Neill S. (1998). Phylogeny and PCR–based classification of Wolbachia strains using wsp gene sequences. Proceedings of the Royal Society of London B: Biological Sciences 265(1395): 509–515.