Molecular and Morphometric Analyses of Coptotermes spp. (Blattodea: Rhinotermitidae) with a Description of Novel Microsatellite Markers

Norsyarizan, Jamil (2018) Molecular and Morphometric Analyses of Coptotermes spp. (Blattodea: Rhinotermitidae) with a Description of Novel Microsatellite Markers. Masters thesis, Universiti Malaysia Sarawak(UNIMAS).

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Abstract

Coptotermes genus, a subterranean termite from the family Rhinotermitidae, is among one of the most destructive pests in Malaysia as it can cause more than 90% damage in buildings and structures. An accurate Coptotermes spp. identification is essential for proper pest control and management. However, this is hampered by difficulty in distinguishing the within and between population variations of the species under this genus. The relationship of the Coptotermes spp. were identified by using three mitochondrial DNA sequences of 12S, 16S and cytochrome oxidase subunit II (COII) and were supported by morphometric measurement data. A total of 193 individuals consist of four different species of Coptotermes (C. curvignathus, C. sepangensis, C. kalshoveni and C. travians) were used in a morphometric analyses. The species grouping evaluated from morphometric result showed many overlapping morphological characters between C. sepangensis and C. kalshoveni and both species had difficulties in being differentiated based on Discriminant Function Analyses (DFA). The morphological ambiguities between C. sepangensis and C. kalshoveni and the relationships of Coptotermes spp. were then validated by using mitochondrial DNA sequences (12S, 16S and COII gene). The phylogenetic relationship of the five putative Coptotermes spp. (C. curvignathus Holmgren 1913, C. travians Haviland 1898, C. sepangensis Krishna 1956, C. kalshoveni Kemner 1934, C. gestroi Wasmann 1896) were used and the available sequences for these species also were included in the analyses. “Neighbour Joining (NJ)”, “Maximum Parsimony (MP)” and “Maximum Likelihood (ML)” were constructed for each genes. The phylogenetic trees resulted four major clades: I (C. curvignathus), II (C. gestroi), III (mixture of C. sepangensis and C. kalshoveni) and IV (C. travians). The genetic distance by “Kimura 2-parameter” model between C. sepangensis and C. kalshoveni was 0% to 3.2% in COII, 0% to 4.2% in 16S and 0% to 2.2% in 12S gene. v The discriminant function analyses (DFA) results were corresponded well with molecular phylogenetic tree constructed for C. sepangensis and C. kalshoveni. It can be proposed that C. sepangensis and C. kalshoveni might be possible synonyms based on morphometric and molecular data. A mitochondrial DNA sequences used in the current analyses showed limited genetic variation at inter- and intra- colonial level of Coptotermes spp.. Therefore, a more sensitive DNA markers such as polymorphic genetic marker is required to elucidate the details of colony organization and colony breeding structure of an important urban pest termites for identifying the origin for groups of foragers. The rubber termite, Coptotermes curvignathus is a common subterranean termite which often causes structural damages to the built environment. The incidence of this species infestation in Malaysia occurred in the early 1900’s and was reported to infest on rubber trees, Hevea brasilliensis. Consequently, this xylophagous insect is considered, by far to be a destructive pest to buildings in urban dwelling as well as a major agricultural pest especially to oil palm plantations in its native range in Southeast Asia. Therefore, we isolated and characterize novel polymorphic microsatellite markers from the genome of C. curvignathus in order to understand their cryptic population genetic structure and breeding strategies in oil palm plantation that would further enhance our knowledge on the infestation dynamics of this pest species. A modest volume of 454 next generation pyrosequencing technique generated 47,462 reads whereby 1996 (4.2%) of the reads contain microsatellites with di-, tri- and tetra- nucleotide repeat motifs. Sixty primer pairs were randomly selected for preliminary test of polymorphism across five individual of C. curvignathus from distinct geographic sample locations collected within the Sarawak region in Malaysia. Ten of 30 primer sets tested were found to be polymorphic with 4-15 alleles per locus and were subsequently assigned into four multiplex groups for future population genetic studies. Observed and expected heterozygosities ranged between 0.19 to 0.86 and 0.44 to 0.92, respectively. No linkage disequilibrium was found between any pair of loci and all loci do not deviate from Hardy-Weinberg equilibrium. The high degree of polymorphism among these 10 microsatellite loci will be useful as a sensitive tool to investigate the colony and population genetic structure of C. curvignathus in this region. To further validate the current findings, more extensive sample collection from peninsular Malaysia and Borneo region should be done and includes type of specimens (if available) with the original descriptions to provide evidence for a more robust phylogenetic positions of each species. Morphological identification based on the soldier’s caste among Coptotermes spp. can be improved by comparing with the reproductive caste, i.e. alates, king, queen. The advances in termite taxonomy when combined with conventional methods and molecular tools are important to validate many species names as real biological taxa. An accurate species identification can have important implications for control practices to avoid duplicative testing of termite management strategies which were thought to be a different species in different geographical areas. Therefore, it will save times, resources and finances for pest management strategies. Meanwhile, a more sensitive microsatellite markers will enable understanding of termite colony social organization and their breeding system. Results from this study are hopefully applied for future identification and in turn important for effective pest management.

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