Genomic-Bioinformatic Analysis of Transcripts Enriched in the Third-Stage Larva of the Parasitic Nematode Ascaris suum Cui-Qin Huang 1,2 , Robin B. Gasser 3 *, Cinzia Cantacessi 3 , Alasdair J. Nisbet 4 , Weiwei Zhong 5 , Paul W. Sternberg 5 , Alex Loukas 6 , Jason Mulvenna 6 , Rui-Qing Lin 1 , Ning Chen 1 , Xing-Quan Zhu 1 * 1 Laboratory of Parasitology, College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong Province, People’s Republic of China, 2 College of Life Sciences, Longyan University, Fujian Province, People’s Republic of China, 3 Department of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia, 4 Parasitology Division, Moredun Research Institute, Penicuik, United Kingdom, 5 Biology Division, California Institute of Technology, Pasadena, California, United States of America, 6 Helminth Biology Laboratory, Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, Brisbane, Queensland, Australia Abstract Differential transcription in Ascaris suum was investigated using a genomic-bioinformatic approach. A cDNA archive enriched for molecules in the infective third-stage larva (L3) of A. suum was constructed by suppressive-subtractive hybridization (SSH), and a subset of cDNAs from 3075 clones subjected to microarray analysis using cDNA probes derived from RNA from different developmental stages of A. suum. The cDNAs (n = 498) shown by microarray analysis to be enriched in the L3 were sequenced and subjected to bioinformatic analyses using a semi-automated pipeline (ESTExplorer). Using gene ontology (GO), 235 of these molecules were assigned to ‘biological process’ (n = 68), ‘cellular component’ (n = 50), or ‘molecular function’ (n = 117). Of the 91 clusters assembled, 56 molecules (61.5%) had homologues/orthologues in the free- living nematodes Caenorhabditis elegans and C. briggsae and/or other organisms, whereas 35 (38.5%) had no significant similarity to any sequences available in current gene databases. Transcripts encoding protein kinases, protein phosphatases (and their precursors), and enolases were abundantly represented in the L3 of A. suum, as were molecules involved in cellular processes, such as ubiquitination and proteasome function, gene transcription, protein–protein interactions, and function. In silico analyses inferred the C. elegans orthologues/homologues (n = 50) to be involved in apoptosis and insulin signaling (2%), ATP synthesis (2%), carbon metabolism (6%), fatty acid biosynthesis (2%), gap junction (2%), glucose metabolism (6%), or porphyrin metabolism (2%), although 34 (68%) of them could not be mapped to a specific metabolic pathway. Small numbers of these 50 molecules were predicted to be secreted (10%), anchored (2%), and/or transmembrane (12%) proteins. Functionally, 17 (34%) of them were predicted to be associated with (non-wild-type) RNAi phenotypes in C. elegans, the majority being embryonic lethality (Emb) (13 types; 58.8%), larval arrest (Lva) (23.5%) and larval lethality (Lvl) (47%). A genetic interaction network was predicted for these 17 C. elegans orthologues, revealing highly significant interactions for nine molecules associated with embryonic and larval development (66.9%), information storage and processing (5.1%), cellular processing and signaling (15.2%), metabolism (6.1%), and unknown function (6.7%). The potential roles of these molecules in development are discussed in relation to the known roles of their homologues/orthologues in C. elegans and some other nematodes. The results of the present study provide a basis for future functional genomic studies to elucidate molecular aspects governing larval developmental processes in A. suum and/or the transition to parasitism. Citation: Huang C-Q, Gasser RB, Cantacessi C, Nisbet AJ, Zhong W, et al. (2008) Genomic-Bioinformatic Analysis of Transcripts Enriched in the Third-Stage Larva of the Parasitic Nematode Ascaris suum. PLoS Negl Trop Dis 2(6): e246. doi:10.1371/journal.pntd.0000246 Editor: Michael Cappello, Yale Child Health Research Center, United States of America Received January 8, 2008; Accepted May 13, 2008; Published June 18, 2008 Copyright: ß 2008 Huang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Natural Science Foundation of China (grant no. 36071578), International Foundation for Science (grant no. B/ 4018-1), the Scientific and Technological Programme of Guangdong Province (grant no. 2004B50201020) (XQZ), the Australian Research Council (RBG and AL), the Howard Hughes Medical Institute (PWS), and the National Health and Medical Research Council of Australia (AL and JM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: robinbg@unimelb.edu.au (RBG); xingquanzh@scau.edu.cn (XQZ) Introduction Parasitic nematodes are of major socio-economic importance in animals. For example, hundreds of millions of people are infected with geohelminths (soil-transmitted worms), such as blood-feeding hookworms Ancylostoma duodenale and/or Necator americanus, Trichuris trichiura and Ascaris spp. [1], causing serious adverse effects on human health, particularly in children. Similarly, parasitic nematodes of livestock, such as pigs, also cause substantial economic losses due to subclinical and clinical diseases, with billions of dollars spent annually on the treatment and control of gastro-intestinal nematodes. In addition to the socioeconomic impact that these parasites have, there is potential for the emergence of resistance in them against all of the main classes of (nematocidal) compounds used to treat the diseases they cause [2–5]. Therefore, there is a significant need to work toward www.plosntds.org 1 June 2008 | Volume 2 | Issue 6 | e246