1 Author running head: Y. J. Fang et al. 2 Title running head: Sialotranscriptome of An. sinensis 3 Correspondence: Bin Chen, Institute of Entomology and Molecular Biology 4 College of Life Sciences, Chongqing Normal University 5 University Town, Chongqing 401331, China. Tel: +86 23 65918391 (office), 13638385631 6 (mobile); fax: +86 23 65910315; email: [email protected], [email protected] 7 *These authors contributed equally for the work. 8 9 ORIGINAL ARTICLE 10 Sialotranscriptome sequencing and analysis of Anopheles sinensis and 11 comparison with Psorophora albipes sialotranscriptome (Diptera: Culicidae) 12 13 Ya-Jie Fang*, Zhen-Tian Yan and Bin Chen* 14 Chongqing Key Laboratory of Vector Insects; Institute of Entomology and Molecular Biology, 15 College of Life Sciences, Chongqing Normal University, Chongqing, China. This is an Accepted Article that has been peer-reviewed and approved for publication in the Insect Science but has yet to undergo copy-editing and proof correction. Please cite this article as doi: 10.1111/1744-7917.12431. This article is protected by copyright. All rights reserved. 16 17 18 Abstract Most of adult female mosquitoes secrete saliva to facilitate blood sucking, 19 digestion and nutrition, and mosquito-borne disease prevention. The knowledge of 20 classification and characteristics of sialotranscriptome genes are still quite limited. Anopheles 21 sinensis is a major malaria vector in China and Southeast countries. In the study, the An. 22 sinensis sialotranscriptome was sequenced using Illumina sequencing technique with a total of 23 10 907 unigenes to be obtained and annotated in biological function and pathway, and 10 470 24 unigenes were mapped to An. sinensis reference genome with 70.46% genes having 25 90%–100% genome mapping through bioinformatics analysis. These mapped genes were 26 classified into four categories: housekeeping (6632 genes), secreted (1177), protein-coding 27 genes with function-unknown (2646) and transposable element (15). The housekeeping genes 28 were divided into 27 classes, and the secreted genes were divided into 11 classes and 96 29 families. The classification, characteristics and evolution of these classes/families of secreted 30 genes were further described and discussed. The comparison of the 1177 secreted genes in An. 31 sinensis in Anophelinae subfamily with 811 in Psorophora albipes in Culicinae subfamily 32 show that 6 Classes/subclasses have the gene number more than twice and 2 classes (Uniquely 33 found in anophelines, and Orphan proteins of unique standing) are unique in the former 34 compared with the later, whereas 4 Classes/subclasses are much expanded and the Uniquely 35 found in Aedes class is unique in the later. The An. sinensis sialotranscriptome sequence data is 36 the most complete in mosquitoes to date, and the analyses provide a comprehensive 37 information frame for further research of mosquito sialotranscriptome. This article is protected by copyright. All rights reserved. 38 Key words Anopheles sinensis; comparative analysis; gene classification; 39 Psorophora albipes; sialotranscriptome 40 41 Introduction 42 Anopheles sinensis is a major malaria vector in China and in eastern and southeastern Asia, 43 with wide distribution from Afghanistan, China (northern China and Taiwan), Japan, Korea, 44 northeast India, and southward to western Indonesia (Sinka et al., 2011). It also transmits the 45 filarial nematodes (Zhang et al., 1994). The adult female mosquitoes need blood feeding to 46 obtain nutrition for egg‟s maturation. Salivary gland is an important organ of mosquitoes to 47 facilitate: (1) blood feeding and digestion, (2) sugar, protein and lipid digestion, (3) reducing of 48 the parasite infection and transmission (Das et al., 2010). When mosquito sucks blood, the 49 salivary gland releases mixture, containing anti-hemostatic, blood platelet aggregation 50 inhibitor and vasodilator to obtain blood, and meanwhile, it also releases the defensin and 51 lysozyme to minimize parasite infection. The mosquito salivary glands are paired and located 52 at the anterior portion of the thorax. There are three lobes each gland, two lateral lobes and one 53 median lobe (Siriyasatien et al., 2005a). The lateral can be divided into proximal region and 54 distal region. The proximal region mainly secretes enzymes related to sugar feeding, while the 55 distal region and the median lobe secrete the compounds associated with blood-sucking, which 56 may be specific for female mosquitoes. 57 The research on transcriptome and protein of mosquito salivary gland are essential to 58 understand the mechanism of blood sucking, digestion and nutrition, and the prevention of 59 mosquito-borne diseases. Up to date, 11 species of mosquito transcriptomes of salivary glands This article is protected by copyright. All rights reserved. 60 (sialotranscriptome) have sequenced and analyzed, including Anopheles gambiae 61 (Francischetti et al., 2002), Aedes aegypti (Francischetti et al., 2002; Ribeiro et al., 2007), An. 62 stephensi (Valenzuela et al., 2003), An. darlingi (Calvo et al., 2004; Calvo et al., 2009b), Culex 63 pipiens quinquefasciatus (Ribeiro et al., 2004), Ae. albopictus (Arca et al., 2007), An. funestus 64 (Calvo et al., 2007), Toxorhynchites amboinensis (Calvo et al., 2008), Cx. tarsalis (Calvo et 65 al., 2010a), Ochlerotatus triseriatus (Calvo et al., 2010b), and Psorophora albipes (Chagas et 66 al., 2013). Comparative analyses of sialotranscriptomes in relation to blood feeding in An. 67 gambiae and in response to sugar feeding in An. stephensi have also been reported (Das et al., 68 2010; Dixit et al., 2009). However, all of these species of sialotranscriptomes, except for 69 Psorophora albipes, were sequenced based on first generation of sequencing techniques, thus 70 the transcripts sequenced were quite limited with only 281–1273 contigs. Due to partial 71 sequencing, the classifications of these species of sialotranscriptome were still preliminary. 72 The sialotranscriptome of Ps. albipes has been well sequenced based on the second generation 73 of sequencing techniques, and assembled into 43 466 contigs; however, only 3247 CDs were 74 classified (Chagas et al., 2013). Ribeiro et al. (2010) (Ribeiro et al., 2010) summarized and 75 reviewed the sialotranscriptomes of Nematocera in Diptera, in which 10 species of mosquitoes 76 were inclusive. The proteomics researches of salivary gland have mainly concentrated on the 77 comparison before and after mosquito blood sucking or Plasmodium infection (Siriyasatien et 78 al., 2005b; Cotama et al., 2013). A small amount of salivary proteins have been preliminarily 79 investigated: the blood feeding related proteins such as vasodilator (Ribeiro, 1992), apyrase 80 (Champagne et al., 1995) and anophelin (Francischetti et al., 1999), and the immunity related 81 proteins such as aedesin with antimicrobial activity (Godreuil et al., 2014), inhibitor of 82 cysteine proteases associated with Plasmodium infection (Boysen & Matuschewski, 2013). This article is protected by copyright. All rights reserved. 83 Generally, the knowledge of classification, characteristics, evolution, and function of salivary 84 gland genes are still limited. 85 Although the genome and general transcriptome in An. sinensis have been reported (Zhou 86 et al., 2014; Chen et al., 2014), the sialotranscriptome of An. sinensis is still unknown. In the 87 study, we sequenced and annotated the sialotranscriptome of An. sinensis based on next 88 generation of sequencing technique, classified the unigenes of the sialotranscriptome into 89 various categories, and compared the sialotranscriptome with Ps. albipes sialotranscriptome. 90 The An. sinensis sialotranscriptome sequence data is most complete so far, and the analyses 91 provide a comprehensive information frame for further research of the mosquito 92 sialotranscriptome. 93 94 Materials and methods 95 Insect samples, salivary gland dissection and RNA extraction 96 The laboratory colony of An. sinensis was reared in the Institute of Entomology and 97 Molecular Biology, Chongqing Normal University, China in a 12 : 12h light cycle and at 26 ± 98 1°C with 75% ± 5% relative humidity. The colony was established five years ago based on a 99 single pregnant female adult originally collected from Wuxi, Jiangsu, and confirmed to be An. 100 sinensis by genome sequencing in our institute. Female adult mosquitoes were fed with 10% 101 glucose solution and collected at the age of 3–5 days post emergence for salivary gland 102 dissection. This article is protected by copyright. All rights reserved. 103 The collected mosquitoes were frozen in a freezer to be knocked down, and then placed in a 104 Petri dish that was kept cold on ice. Salivary glands were dissected in a microplate-well with a 105 drop of sterilized 1× Phosphate Buffer Saline (1×PBS) solution using fine-tipped forceps. The 106 thorax was fixed with a pair of forceps, and the head was pull apart with another pair of forceps. 107 The salivary glands located at the anterior portion of the thorax were exposed in the process of 108 pulling-apart, peeled off with removing connected tissues, and cleaned with the 1×PBS buffer. 109 A total of 180 salivary glands were collected and dipped into 1 mL Trizol solution (Ambion, 110 USA) for RNA preparation. 111 Total RNA was separately extracted using TRIzol Reagent (Ambion, USA) following the 112 manufacturer‟s protocol. To eliminate genomic DNA, the RNA samples were treated with 113 RNase-Free DNase I