Characterizing freshwater macroinvertebrates of Bangladesh using metagenetic techniques

Abstract

The degradation of freshwater ecosystems has become a global concern, in particular, the critical conditions of rivers in Bangladesh demand a monitoring programme through the assessment of bioindicator organisms. Macroinvertebrates as prominent bioindicators are widely used for assessing the health of aquatic ecosystems. Recent technological advances have enabled routine assessment with the genomic characterization of macroinvertebrates using different metagenetic techniques such as DNA barcoding for individual specimen identification, metabarcoding for multi-species identification of bulk samples and mitochondrial metagenomics for extraction of mitogenomes from mixed samples. In this thesis, I commence by generating Cytochrome Oxidase subunit (COI) barcodes for Bangladeshi freshwater macroinvertebrates belonging to the Ephemeroptera, Plecoptera, Trichoptera, Coleoptera, Hemiptera, Odonata, Diptera, Gastropoda and Bivalvia. These barcodes can be used as a DNA reference library for species identification in metabarcoding of macroinvertebrates. I also aim for exploring complete mitogenomes from selected macroinvertebrates using a mitochondrial metagenomic pipeline. I carry out phylogenetic analysis with protein-coding genes that reveals the evolutionary relationship of Bangladeshi macroinvertebrate lineages and also support deeper level identification of barcodes placing them into the phylogenetic tree (chapter 2). In chapter 3, I assess some methodological aspects of the metabarcoding pipeline required for diversity estimation from complex bulk samples of macroinvertebrates in large-scale biomonitoring programmes. These include preparation of bulk macroinvertebrate samples, optimization of the procedure of homogenization of samples required for DNA extraction, strategies for DNA pooling from these extracts, choice of robust universal primers, and viable OTU clustering for reliable diversity estimation. The results have implications for the optimization and standardization of these steps in metabarcoding of freshwater macroinvertebrates. In chapter 4, I apply the metabarcoding technique to establish the macroinvertebrate diversity and impact of various types of anthropogenic disturbances on the freshwater macroinvertebrates in highland and lowland rivers. The results document high diversity, local endemicity and pronounced responses to disturbance in largely unexplored but threatened habitats of Bangladesh. My investigations manifest the viability of metagenetic techniques for applied conservation management as a step towards building a biomonitoring system in freshwater ecosystems globally.