USE OF MTCOI SEQUENCES FOR THE IDENTIFICATION OF SUBTERRANEAN TERMITE SPECIES FROM PUNJAB, PAKISTAN Prof . Farkhanda Manzoor Lahore College for Women University, Lahore, Pakistan.

AIM OF STUDY Collection and identification of worker and soldier caste termites. Sequencing of mitochondrial genes. DNA barcoding Study of nucleotide sequence alignments and phylogenetic analysis

SUBTERRANEAN TERMITES Termites are little insects that are termed along with the ants. The scientific name for Termites is ISOPTERA. Generally feed on dead plant material : wood, leaf litter, soil or animal dung/poo and break down dead plant or animal matter About 10% of the approximate amount of 4,000 different species are pests that can cause serious structural damage to buildings, crops or forests. Termites are sometimes called "white ants" even though they are not really related to them. In Pakistan some 53 species have been identified.

TERMITES

SUBTERRANEAN TERMITES A severe economic timber pest of Pakistan. They build distinctive tunnels, often referred to as "mud tubes”. These are the most destructive termite species. The hard, saw-toothed jaws of termites work like shears and are able to bite off extremely small fragments of wood, one piece at a time. Over time, they can collapse a building entirely, meaning possible financial ruin for a homeowner

LIFE CYCLE

DNA BARCODING  Molecular markers are used to study the genetic polymorphism. mtCOI gene is the most widely applied DNA region for the determination of the genetic structure . On the basis mtCOI gene; different biotypes can be identified Used to study molecular character for species recognition and discrimination.

MATERIALS AND METHODS

MORPHOLOGICAL IDENTIFICATION OF TERMITES Termites were collected and identified on the basis of Total head length (THL) • Length of head to the side base of mandibles (LHSBM) • Width of head to the side-base of mandibles (WHSBM) • Maximum width of head (MWH) • Length of mandible (LM) • Distance of tooth from the tip of mandible • Pronotum length • Pronotum width • Postmentum length • Postmentum width

DNA EXTRACTION DNA extractions was isolated by using Erlandson et al. (2003) method. PRIMERS Primers for amplification of (COI-3 AND COI-5) were deigned

LepF2_t1 (TGTAAAACGACGGCCAGTAATCATAARGATATYGG) LepR1 (TAAACTTCTGGATGTCCAAAAAATCA) & C1-J 2195 (TTGATTYTTTGGTCATCCAGAAGT) TL2-N-3014 (TCCAATGCACTAATCTGCCATATTA)

MTCOI (COI-3 AND COI-5) AMPLIFICATION AND SEQUENCING Two fragments of mtCOI (COI-3 AND COI-5) were PCR amplified from each termite. PCR conditions were; 1 minute at 94°C, 40 seconds at 94°C ( 5 cycles) 40 seconds at 45°C 1 minute at 72°C

Nucleotide sequence alignments and phylogenetic analysis 40 seconds at 94°C (35 cycles) 40 seconds at 51°C 1 minute at 72°C 5 minutes at 72°C for final extension. Sequencing DNA barcoding Nucleotide sequence alignments and phylogenetic analysis

RESULTS

MORPHOLOGICAL IDENTIFICATION OF TERMITES Twelve termite species from two families were identified based on morphological features of soldier caste. Termitidae (Odontotermes obesus ) Odontotermes lokanandi Odontotermes gurdaspurensis Odontotermes sp.,

Microtermes obesi Microtermes unicolor Microtermes mycophagus) Rhinotermitidae (Heterotermes indicola, Coptotermes heimi, Angulitermes dehraensis, Microcerotermes longignathus, Coptotermes travian)

TABLE??

Figure 1. DNA-barcode based evolutionary relationships of nine termite species collected from various locations of Punjab.

Figure 2. COI sequence based evolutionary relationships of twelve termite species .

Figure 3. Identification of worker caste by determination of evolutionary relationships with identified soldier caste specimens .

CONCLUSION Both the DNA barcodes and COI-3ʹ sequences discriminated the analyzed termites to the species level. The cluster analysis showed species level relationships between different termite species. Disparity Index Test of species based on barcode data showed significant differences among majority of the species pairs.

Nucleotide similarity analysis of the species showed that none of the species had a similarity of more than 98% and in most cases similarity was less than 89%. Analysis of COI-3ʹ sequences of seven termite species when performed along with species sequences reported in the GenBank placed the species of each genus together in a single cluster.