13th International Research Conference General Sir John Kotelawala Defence University Basic and Applied Sciences Sessions Paper ID: 204 Identification of Wolbachia like endosymbiont DNA in Setaria digitata genome and phylogenetic analysis of filarial nematodes MSA Kothalawala1#, N Rashanthy1, TS Mugunamalwaththa1, WAS Darshika1, GLY Lakmali1, RS Dassanayake1, YINS Gunawardene2, NV Chandrasekharan1, Prashanth Suravajhala3, Kasun de Zoysa4 1Department of Chemistry, Faculty of Science, University of Colombo, Sri Lanka. 2Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka. 3Department of Biotechnology and Bioinformatics, Birla Institute of Scientific Research, Statue Circle, Jaipur 302001, RJ, India 4Department of Communication and Media Technologies, University of Colombo School of Computing, Sri Lanka. #For correspondence; <[email protected]> Abstract - Setaria digitata is a Wolbachia-free BLAST2GO analysis was able to identify 6055 filarial parasite that causes cerebrospinal annotations and 95 metabolic pathways nematodiasis in non-permissive hosts such as within the S. digitata genome. Based on goats and sheep, leading to substantial FASTA36 and phylogenetic analyses, it can be economic losses in animal husbandry. concluded that ancestors of S. digitata were Therefore, there arises a considerable need for colonized with Wolbachia in the distant past, the development of new interventions to and suspected gene transfer may have brought disease control and eradication of this filarial Wolbachia DNA into the S. digitata nuclear parasite. Owing to the limited knowledge on S. genome prior to endosymbiont loss. digitata genome, it's host-parasite Keywords - Setaria digitata, Wolbachia, NGS relationship and the potential impact of the Wolbachia endosymbiont in filarial Introduction nematodes, this research was focused on the S. digitata is an ivory-color slimy filarial generation of the draft genome of S. digitata; parasitic-worm with a coiled tapering tail. It is identification of Wolbachia like endosymbiont classified under class Secernentea, order DNA in S. digitata genome; phylogenetic Spirurida and family Setariidae. S. digitata analysis as well as functional annotation and resides in the peritoneal cavity of grazing metabolic pathway analysis of the genome. A hoofed animals (Shiny et al., 2011; Shin et al., draft genome of 78.8 Mbp size with a GC% of 2002) and cause cerebrospinal nematodiasis, 31.45% was generated for the S. digitata a neuropathological disorder that causes worms collected from the peritoneal cavity of dysfunction of the central nervous system. It slaughtered cattle, using NGS Illumina leads to lumbar paralysis and eventual death platform. FASTA36 sequence similarity of non-permissive domesticated hosts such as analysis was able to identify homologous goats, sheep and horses, which cause sequences of coxA and gatB Wolbachia MLST substantial economic loss in animal genes within the S. digitata genome, while husbandry in Asia and the Far East. However, phylogenetic analysis using Geneious Prime infection of natural hosts such as cattle and Software revealed that S. digitata is more buffalos by the S. digitata cause mild disease closely related to the filarial nematodes with conditions such as mild fibrinous peritonitis Wolbachia endosymbiont than Wolbachia- and not considered as parasitic in their natural free filarial nematodes. Furthermore, hosts. Human infections are also reported 15 13th International Research Conference General Sir John Kotelawala Defence University Basic and Applied Sciences Sessions causing allergic reactions, eye lesions, potential impact of the endosymbiont and to abscesses, enlarged lymph nodes and lung gain insight into the host-parasite inflammation (Taylor and Hoerauf, 1999; relationship. Gunawardene and Dassanayake, 2015; Sundar S. digitata also considered as a model and Souza, 2015) organism for human lymphatic filariasis Most of the filarial nematodes mutually (HLF), due to their close resemblance to associate with Wolbachia and without Wuchereria bancrofti, the primary causative Wolbachia, such filarial nematodes cannot agent of HLF, in morphology, histology and survive (Werren et al., 2008). The reason is antigenic properties (Gunawardene and that the genome of Wolbachia carries the Dassanayake, 2015). Hence, generation of a genes required for the metabolism of heam, draft genome of S. digitata and complete riboflavin, FAD, glutathione and nucleotides functional analysis and metabolic pathway whereas its filarial host does not (Fenn et al., reconstructions of S. digitata will have a huge 2006). However, S. digitata does not harbor impact on the development of novel drugs Wolbachia endosymbiont, but undergo and/or vaccines for human filariasis and other biosynthetic pathways of heam, riboflavin and filarial diseases. nucleotides. The genes of those pathways Methodology should be present in its genome itself (Voronin et al., 2015). There are two possible Adult worm samples of S. digitata were explanations for the independent survival of S. collected from the peritoneal cavity of cattle digitata. It can be hypothesized that either the (Bos taurus) from the western province. S. ancestors of S. digitata were colonized with digitata worms were washed thoroughly in Wolbachia in the distant past and horizontal PBS and preserved in 80% ethanol at −20˚C gene transfer (HGT) may have brought before analysis. Genomic DNA of adult S. Wolbachia DNA into the nuclear genome of S. digitata worms was extracted by DNA Micro digitata prior to endosymbiont loss, or the kit, QIAGEN. Confirmation of extracted S. endosymbiotic relationship between digitata DNA was done by carrying out a series Wolbachia and its filarial host is dispensable. of PCRs using S. digitata ARV1 specific Studies showed that Wolbachia free filarial primers. nematode Loa loa evolved own DNA After performing quality control (QC), the sequences to code heam and riboflavin passed sample was proceeded with the Next biosynthesis pathways. For some pathways, Generation Sequencing (NGS) library they have gained partial gene sequences from construction. Sequencing libraries were Wolbachia, and this indicates the horizontal constructed from the extracted DNA using the gene transfer within Loa loa and Wolbachia at TruSeq™ DNA PCR-Free Kit. Purified libraries some point during the evolution (Desjardins et were loaded into an Illumina HiSeq4000 for al., 2013). Therefore, independent survival of paired-end sequencing. Sequenced data (base S. digitata also can be explained by horizontal call files) were converted to raw FASTQ files gene transfer of Wolbachia gene fragments to using the sequencer soon after the initial the S. digitata genome and/or evolution of sequencing. The reads were filtered before their own DNA sequences to code those assembly such that for a pair of PE reads each pathways (Mcnulty et al., 2010). Therefore, read should have more than 90 % of bases identification of Wolbachia like endosymbiont with base quality greater than or equal to Q20. DNA in S. digitata genome and phylogenetic Generated 150bp reads were analyzed for K- analysis of S. digitata and other filarial mers using JELLYFISH. Once the optimum k- nematodes, are important to understand the 16 13th International Research Conference General Sir John Kotelawala Defence University Basic and Applied Sciences Sessions mer size is identified, a de novo draft assembly 31.77% and a Q20 value of 99.18%. A draft was built using SOAPdenovo2. genome of 78,774,594 bases belongs to a total of 2,075 contigs was generated after the In order to identify Wolbachia like assembly. The percentage of guanine-cytosine endosymbiont DNA in S. digitata genome, base pairs (GC%) in the assembled draft FASTA file containing 2075 S. digitata contigs genome is 31.45%. In a typical random library, was blasted against several Wolbachia specific it is expected to see a roughly normal reference sequences such as Wolbachia distribution of GC content where the central surface protein (WSP), Wolbachia-specific 16S peak corresponds to the overall GC content. An rRNA and Wolbachia MLST genes (coxA, gatB, unusually shaped distribution could indicate a fbpA, ftsZ, and hcpA) using FASTA36 program. contaminated library or some other kinds of a Later, using Wolbachia marker sequences biased subset. Here, the obtained histogram (gatB, coxA, ftsZ, fbpA, hcpA, WSP and 16S showed a normal distribution and therefore, it rRNA), BLASTx was performed against S. can conclude that there is no contamination. digitata, Loa loa, Brugia malayi, W. bancrofti, Five Wolbachia marker gene sequences were Onchocerca volvulus and Wolbachia selected for identification of Wolbachia like endosymbiont of B. malayi (wBm) protein endosymbiont DNA in S. digitata genome sequences available on the NCBI non- because of their presence in the Rickettsiales redundant (NR) protein sequence database. order. These marker genes have a single copy, Resulted best hit for each organism for each a wide spatial distribution and a strong marker gene was recorded, and the aligned stabilizing selection within the Wolbachia region was obtained. Those aligned sequences genome. Homologous sequences were found of each organism for each marker gene were only for coxA and gatB. For coxA, homologous used to generate a phylogenetic tree using sequence was found in the S. digitata Geneious Prime software. Firstly, sequences contig_633 and for gatB, homologous were loaded into the program and alignment sequences