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Comparative Transcriptomes Analysis of Taenia Pisiformis at Different bioRxiv preprint doi: https://doi.org/10.1101/490276; this version posted December 9, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Comparative Transcriptomes Analysis of Taenia 2 pisiformis at Different Development Stages 3 Lin Chen1†*, Jing Yu1†, Jing Xu2†, Wei Wang1, Lili Ji 1, Chengzhong Yang3, Hua Yu4 4 1 Key Lab of Meat Processing of Sichuan Province, College of Pharmacy and Biological Engineering, 5 Chengdu University, Chengdu, 610106, China; [email protected] (L.C.); [email protected](J.Y.); 6 [email protected](W.W.); [email protected](L.J.) 7 2 Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 8 611130, China; [email protected] (J.X.) 9 3 Chongqing Key Laboratory of Animal Biology, College of Life Sciences, Chongqing Normal University, 10 Chongqing, 401331, China; [email protected](C.Y.) 11 4 Sichuan EntryExit Inspection and Quarantine Breau，Chengdu，610041，China；[email protected] (H.Y.) 12 * Correspondence: [email protected]; Tel.: 086-28-84616805 13 † These authors contributed equally to this work. 14 Abstract: To understand the characteristics of the transcriptional group of Taenia pisiformis at 15 different developmental stages, and to lay the foundation for the screening of vaccine antigens and 16 drug target genes, the transcriptomes of adult and larva of T. pisiformis were assembled and 17 analyzed using bioinformatic tools. A total of 36,951 unigenes with a mean length of 950bp were 18 formed, among which 12,665, 8,188, 7,577, and 6,293 unigenes have been annotated respectively by 19 sequence similarity analysis with four databases (NR, Swiss-Prot, KOG, and KEGG). It should be 20 noted there are 5,662 unigenes that share good similarity with the four databases and get a 21 relatively perfect functional annotation. Besides, a total of 10,247 differentially expressed genes 22 were screened. To be specific, 6,910 unigenes were up-regulated in the larva stage while 3,337 23 were down-regulated in the adult stage. To sum up, this study sequenced and analyzed the 24 transcriptomes of the larval and adult stages of T. pisiformis. The results of differentially expressed 25 genes in these two stages could provide basis for functional genomics, immunology and gene 26 expression profiles of T. pisiformis. 27 Keywords: Taenia pisiformis, transcriptome, differentially expressed genes (DEGs), RNA-Seq 28 29 30 1. Introduction bioRxiv preprint doi: https://doi.org/10.1101/490276; this version posted December 9, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 2 of 16 31 Cysticercosis is one of the common parasitic diseases in rabbits, caused by the metacestode of 32 Taenia pisiformis (Cestoidea; Cyclophyllidea; Taeniidae), also known as Cysticercus pisiformis. The 33 adult T. pisiformis often parasitized the small intestine of canines and felines, such as dogs, wolves, 34 jackals, foxes, raccoon dogs and cats (Owiny, 2001; Foronda and Valladares et al., 2003; Saeed and 35 Maddox et al., 2006; Martinez and Hernandez et al., 2007; Lahmar and Sarciron et al., 2008; Bagrade 36 and Kirjusina et al., 2009; Jia and Yan et al., 2010). The larvae (Cysticercus pisiformis) often inflict 37 the liver capsule, greater omentum, mesentery and rectal serous membrane of rabbits, squirrels, 38 mice, and other rodents (Zhou and Du et al., 2008). T. pisiformis can cause serious health problems 39 and even death (Yang and Fu et al., 2012). 40 So far, there have been a lot of research on T. pisiformis, including its biological characteristics 41 (Chen and Yang et al., 2015), population genetic diversity (Yang and Ren et al., 2013), gene function 42 (Yang and Chen et al., 2013; Chen and Yang et al., 2014; 2014; Chen and Yang et al., 2016) and 43 preventative measures against it (Chen and Yang et al., 2014) . However, although the 44 transcriptome of the adult of T. pisiformis has been determined before (Yang and Fu et al., 2012), 45 there is no report on comparative transcriptomics of T. pisiformis at different developmental stages. 46 Therefore, the exploration of gene expression patterns in different developmental stages of T. 47 pisiformis will contribute to elucidating the mechanism of the infection of the host and provide an 48 important basis for the prevention and control of these tapeworms. Yet, due to the limitation of 49 materials and research methods, the infection mechanism of T. pisiformis is still unclear. In recent 50 years, high throughput sequencing technology has been widely accepted thanks to its various 51 advantages, such as large number of data, high accuracy, high sensitivity and low running cost. It 52 has been used in many parasite species such as Itch mite (He and Xu et al., 2016), Sarcoptes scabiei 53 (He and Gu et al., 2017), Dirofilaria immitis (Fu and Lan et al., 2012), Taenia ovis (Zheng, 2017), 54 Echiococcus graulosus (Ju, 2013), Taenia multiceps (Li and Zhang et al., 2017) and others (Kolev and 55 Franklin et al., 2010; Cantacessi and Young et al., 2011; Sorber and Dimon et al., 2011; 2012; Schicht 56 and Qi et al., 2014). By using this method, it is possible to understand the molecular mechanism of 57 specific biological processes and understand the overall level of gene expression at different stages 58 of the parasite. In the current research, the Illumina sequencing techniques were applied to study 59 the transcriptome of the two different developmental stages of T. pisiformis. By clarifying the gene 60 expression status in the adult and larva of T. pisiformis, this research will lay a foundation for the 61 diagnosis, prevention and treatment of Cysticercosis. 62 2. Results 63 2.1. Illumina sequencing and assembly bioRxiv preprint doi: https://doi.org/10.1101/490276; this version posted December 9, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 3 of 16 64 RNA-Seq generated approximately 20 to 26 million raw sequence reads for each of the six 65 cDNA libraries obtained from T. pisiformis (three from adult stage and three from larva stage ). The 66 average GC content was 47.25% (Table 1). All of the clean reads were assembled into a 67 transcriptome, which was used as a reference sequence for further analyses. In total, 36,951 68 unigenes were detected in all transcriptomes (Table 2). The mean length and N50 were 950bp and 69 1,998bp, respectively (Table 2). According to the statistics of length distribution, 13,759 unigenes 70 (43.50%) ≥ 500bp, and 8,975 (24.29%) ≥ 1,000bp (Figure 1, Additional file 1: Table S1). The 71 transcriptome raw reads dataset obtained has been submitted to the NCBI Short Read Archive 72 (http://www.ncbi.nlm.nih.gov/Traces/sra_sub/sub.cgi) with the accession number: SUB4234089. 73 Table1 Summary of transcriptome data for adult (Tp) and larva (Cp) of T. pisiformis Reads After Filter GC content Sample Total raw reads Length Adapter (%) Low quality (%) Reads Number (%) (%) (bp) Cp-1 27,788,994 26,124,150 (94.01%) 150 47.38% 696,628 (2.51%) 965,736 (3.48%) Cp-2 25,598,082 23,928,260 (93.48%) 150 47.44% 653,040 (2.55%) 1,013,594 (3.96%) Cp-3 22,221,546 20,382,060 (91.72%) 150 47.19% 640,246 (2.88%) 1,195,572 (5.38%) Tp-1 23,644,240 22,091,652 (93.43%) 150 46.95% 615,538 (2.6%) 932,282 (3.94%) Tp-2 25,641,834 24,071,136 (93.87%) 150 48.22% 658,922 (2.57%) 908,514 (3.54%) Tp-3 24,713,304 23,233,138 (94.01%) 150 46.30% 551,842 (2.23%) 921,362 (3.73%) Average 24,934,667 23,305,066（93.42%） 150 47.25% 636,036(2.56%)- 989,510(4.01%)- 74 75 Table 2 Filtered results of transcriptome data Genes GC Total Max length Min length Average Number percentage N50(bp) assembled (bp) (bp) length (bp) (%) bases 36,951 46.54 1,998 26,041 201 950 35,115,151 76 77 2.2 Functional annotation of unigenes 78 A total of 12,844 unigenes (34.76% of all unigenes) were annotated by the Nr (12,665, 34.28%), 79 Swiss-Prot (8,188, 22.16%), KEGG (6,293, 17.03%) and KOG (7,577, 20.51%) protein databases using bioRxiv preprint doi: https://doi.org/10.1101/490276; this version posted December 9, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 4 of 16 80 Blastx, and 5,662 unigenes were similar to the sequences of all these four databases (Figure 2). Rest 81 of the unigenes (24,107) failed to match against any sequence with E-value < 10− 5. Homologous 82 genes came from several species, with 59.01% of the unigenes having the highest homology to 83 genes from Echinococcus granulosus (7,474, 59.01%), followed by Echinococcus multilocularis (3,654, 84 28.85%), Hymenolepis microstoma (557, 4.4%), Daphnia magna (72, 0.57%), Taenia solium (67, 0.53%) 85 and other species (841, 6.64%). 86 The analysis of GO function annotation provided a functional classification and enrichment 87 analysis for differentially expressed genes (DEGs) (Li and Zhang et al., 2017). As shown in Figure 3, 88 16,269 unigenes can be classified into 25 independent KOG functional items.
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