Identification and Characterization of a Differentially Expressed Gene (07E12) in the Infective Larvae of the Parasitic Nematode Ascaris suum.
AbstractBackground: Parasitic nematodes cause animal and human diseases of major socio-economic importance worldwide. The suppression of parasite development at particular developmental stages could provide an alternative approach for nema-tode control. In this study, Ascaris suum was used as a model system in the study of the differentially expressed genes in the infective L3 stage.Methods: The gene (07E12) was screened and identified from the subtractive cDNA library for the infective larvae of Ascaris suum using real-time quantitative PCR. Then, the full-length cDNA of 07E12 was characterized by 3′ and 5′ rapid amplification of cDNA ends (RACE). The characteristics of the gene were further analyzed using bioinformatic analyses.Results: The results showed that the gene 07E12 was differentially expressed in the third-stage larvae of A. suum and its expression level in the infective larvae was much higher than in other stages. It was shown that the gene 07E12 had 99% iden-tity with the corresponding sequences of the A. suum whole genome shotgun se-quence containing the homologous sequences with conserved sequences of Neu-ropeptide-Like Protein family member. Likewise, by performing BLASTN and BLASTP searches in the GenBank™, it was shown that this gene had 99 % identity with A. suum cre-nlp-2 protein.Conclusion: This gene 07E12 which is differentially expressed in the third-stage larvae of A. suum may encode a neuropeptide-like protein family member, a very important molecule in the process of infecting a host.
Wang M. Veterinary parasitology.3rd Ed. Bei-jing; China’s Agriculture Press: 2003. (in Chi-nese)
Inatomi Y, Murakami T, Tokunaga M, Ishiwa-ta K, Nawa Y, Uchino M. Encephalopathy caused by visceral larva migrans due to Ascaris suum. J Neurol Sci. 1999; 164(2): 195-199.
Jex AR, Liu S, Li B, et al. Ascaris suum draft genome. Nature. 2011; 479 (7374): 529-533.
Wang J, Czech B, Crunk A, Wallace A, Mitreva M, Hannon GJ, Davis RE. Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles. Genome Res. 2011; 21(9): 146214-146277.
Newton SE, Boag PR, Gasser RB. Opportuni-ties and prospects for investigating develop-mentally regulated and sex-specific genes and their expression in intestinal nematodes of hu-mans. In: Holland C Kennedy MW, Editors. World class parasites. Boston: Kluwer Aca-demic Press, 2002. p. 235-268.
Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymer-ase chain reaction assays. J Mol Endocrinol. 2000; 25(2): 169-193.
U'Ren JM, Van Ert MN, Schupp JM, Easter-day WR, Simonson TS, Okinaka RT, Pearson T, Keim P. Use of a real-time PCR TaqMan assay for rapid identification and differentiation of Burkholderia pseudomallei and Burkholderia mallei. J Clin Microbiol. 2005; 43(11): 5771-5774.
McGuigan FE, Ralston SH. Single nucleotide polymorphism detection: allelic discrimination using TaqMan. Psychiatr Genet. 2002; 12(3): 133-136.
Moal V, Textoris J, Ben Amara A, Mehraj V, Berland Y, Colson P, Mege JL. Chronic hepati-tis E infection is specifically associated with an interferon-related transcriptional program. J In-fect Dis. 2013; 207(1): 125-132.
Alasaad S, Soriguer RC, Abu-Madi M, El Behairy A, Jowers MJ, Baños PD, Píriz A, Fickel J, Zhu XQ. A TaqMan real-time PCR-based assay for the identification of Fasciola spp. Vet Parasito. 2011; 179(1-3): 266-271.
Siwińska AM, Bąska P, Daniłowicz-Luebert E, Januszkiewicz K, Długosz E, Wędrychowicz H, Cappello M, Wiśniewski M. Cloning and mo-lecular characterization of cDNAs encoding three Ancylostoma ceylanicum secreted proteins. Acta Parasitol. 2013; 58(1): 112-118.
Weksberg R, Hughes S, Moldovan L, Bassett AS, Chow EW, Squire JA. A method for accu-rate detection of genomic microdeletions using real-time quantitative PCR. BMC Genomics. 2005; 6: 180.
VanGuilder HD, Vrana KE, Freeman WM. Twenty-five years of quantitative PCR for gene expression analysis. BioTechniques. 2008; 44(5): 619–626.
Frohman MA. On beyond classic RACE (rap-id amplification of cDNA ends). PCR Methods Appl. 1994; 4(1): S40-S58.
Seufi AM, Galal FH, Hafez EE. Characteriza-tion of Multisugar-Binding C-Type Lectin (SpliLec) from a Bacterial-Challenged Cotton Leafworm, Spodoptera littoralis. PLoS One. 2012; 7(8): e42795.
Yamaguchi Y, Hasegawa Y, Honma T, Nagas-hima Y, Shiomi K. Screening and cDNA clon-ing of Kv1 potassium channel toxins in sea anemones. Mar Drugs. 2010; 8(12): 2893-2905.
Yeku O, Frohman MA. Rapid amplification of cDNA ends (RACE). Methods Mol Biol., 2011; 703: 107-122.
Huang CQ, Gasser RB, Cantacessi C, Nisbet AJ, Zhong W, Sternberg PW, Loukas A, Mul-venna J, Lin RQ, Chen N, Zhu XQ. Genomic-bioinformatic analysis of transcripts enriched in the third-stage larva of the parasitic nematode Ascaris suum. PLoS Negl Trop Dis. 2008; 2(6): e246.
Fagerholm HP, Nansen P, Roepstorff A, Frandsen F, Eriksen L. Differentiation of cu-ticular structures during the growth of the third-stage larva of Ascraris suum (Nematoda, Ascaridoidea) after emerging from the egg. J Parasitol. 2000; 86(3): 421-427.
Murrell KD, Slotved HC, Eriksen L, Bjerre-gaard J, Nansen P, Roepstorff A. Improved method for the recovery of Ascaris suum larvae from pig intestinal mucosa. J Parasitol. 1997; 83: 321-324.
Douvres FW, Tromba FG, Malakatis GM. Morphogenesis and migration of Ascaris suum larvae developing to fourth stage in swine. J Parasitol. 1969; 55: 689-712.
Huang CQ, Chen N, Zou FC, Lin RQ, Zhu XQ. Studies on methods for collecting larvae of different developmental stages of Ascaris su-um. J Trop Med. 2006; 6: 487-489. ( in Chinese)
Peng W, Yuan K, Hu M, Peng G, Zhou X, Hu N, Gasser RB. Experimental infections of pigs and mice with selected genotypes of Ascaris. Parasitol. 2006; 133: 651-657. (in Chinese)
Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔCt Method. Methods. 2001; 25: 402–408.
Li C. The ever-expanding neuropeptide gene families in the nematode Caenorhabditis elegans. Parasitol. 2005; 131: S109-S127.
Li C and Kim K. Neuropeptide. Wormbook. 2008; 1-36.
Nathoo AN, Moeller RA, Westlund BA. Hart AC. Identification of neuropeptide-like protein gene families in Caenorhabditis elegans and other species. Proc Natl Acad Sci USA. 2001; 98: 14000-14005.
Tetteh KK, Loukas A, Tripp C, Maizels RM. Identification of abundantly expressed novel and conserved genes from the infective larval stage of Toxocara canis by an expressed sequence tag strategy. Infect Immun. 1999; 67: 4771-4779.
Yew JY, Kutz KK, Dikler S, Messinger L, Li L, Stretton AO. Mass spectrometric map of neu-ropeptide expression in Ascaris suum. J Comp Neurol. 2005; 488: 396-413.
Maule AG, Geary TG, Bowman JW, Marks NJ, Blair KL, Halton DW, Shaw C, Thompson DP. Inhibitory effects of nematode FMRFamidere-lated peptides (FaRPs) on muscle strips from Ascaris suum. Invert Neurosci. 1995; 1: 255-265.
Huang CQ, Huang QC, Chen XX. Primary studies on the functions of differentially ex-pressed gene (07E12) in infective larvae of As-caris suum by RNA interference. Chin Ani Husb Vet Med. 2010; 10: 63-67 (in Chinese).