Identification of candidate genes of male sexual development from androgenic gland in 1 Macrobrachium nipponense through performing long-reads and next generation transcriptome 2 sequencing after eyestalk ablation

18 The eyestalk of crustacean species contained many neurosecretory structures, affected the 19 process of reproduction, molting, metabolism of glucose and other function in crustaceans. In 20 this study, we aimed to selected important metabolic pathways and candidate genes involved 21 in the male sexual development through performing the long-reads and next generation 22 transcriptome sequencing of androgenic gland after the ablation of eyestalk from 23 Macrobrachium nipponense . qPCR analysis revealed that the mRNA expression of Mn-IAG 24 was significantly increased after ablation both of the single-side (SS) and double-side (DS) eyestalk, compared with that of control group (CG). The long-reads transcriptome generated 26 49,480 non-redundant transcripts. A total of 1,319, 2,092 and 4,351 differentially expressed 27 genes (DEGs) were identified between CG vs SS, SS vs DS and CG vs DS, respectively, 28 indicating the ablation of double-side eyestalk has more important regulatory roles than that of 29 single-side ablation on male sexual development, which was consistent with that of qPCR 30 analysis. Cell cycle, Cellular senescence, Oxidative phosphorylation, 31 Glycolysis/Gluconeogenesis and Steroid hormone biosynthesis were the main enriched 32 metabolic pathways in all of these three comparisons, and the important genes from these 33 metabolic pathways were also selected. The qPCR verifications of 10 GEDs were as the same 34 as that of RNA-seq. The qPCR and RNAi analysis of Hydroxysteroid dehydrogenase like 1 35 (HSDL1) revealed that HSDL1 has the positive regulatory effected on testis development. This 36 study provided valuable evidences on male sexual development in M . nipponense , promoting 37 the studies on male sexual development in other crustacean as well. 38


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The oriental river prawn, Macrobrachium nipponense (Crustacea; Decapoda; Palaemonidae),  In addition, the rapid development of testis in the reproductive season is another main problem, 47 restricted the sustainable development of M. nipponense. The previous studies revealed that 48 the testis of a new born M. nipponense can reach sexual maturity within 40 days after hatching 49 [5]. Thus, inbreeding will be happened between the new born prawns. Inbreeding will lead to 50 the decrease of the ability of resistance to adversity in their offspring, the small scale of market 51 prawn, and the degradation of germplasm resources. Therefore, it is urgently needed to fully 52 understand the male sexual differentiation and development mechanism, with the aims of 53 establishment of the technique to produce all male progeny on a commercial scale, and to 54 regulate the process of testis development in M. nipponense. 55 Androgenic gland is a special tissue in crustacean species. It has been proven to play

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The expression analysis of Mn-IAG after eyestalk ablation 90 The mRNA expression of Mn-IAG were measured in three groups, including CP, SS and DS 91 ( Figure 1). The mRNA expression of Mn-IAG were increased with time of eyestalk ablation in 92 SS group and DS group. The mRNA expressions of Mn-IAG were about 5-folder higher at day 93 4 and day 7 than that of day 1 in both SS and DS group (P < 0.01). However, the Mn-IAG 94 expression was only slightly higher at day 7 than that of day 4 in both SS and DS group, and 95 showed no significant difference (P> 0.05). The Mn-IAG expression in the DS group was 96 almost 2-folder higher than SS group at the same day, and showed significant difference (P < 97 0.05).

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Long-read transcriptome 99 A total of 22.83 GB clean data was generated in the long-reads transcriptome. A total of 160,496 100 high-quality transcripts were obtained with a mean length of 2,230 bp. Finally, 49,480 non-101 redundant transcripts were identified in the long-reads transcriptome. All of the non-redundant 102 transcripts were compared with the non-redundant protein database and nucleotide sequences 103 in NCBI, in order to identify their putative functions. A total of 37,355(74.94%) unigenes were 104 annotated in Nr database. The other unannotated transcripts represent novel genes whose 105 functions need further investigation.

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GO and COG analysis aimed to provide a structured vocabulary to describe gene products.

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A total of 19,673 (39.76 %) unigenes were assigned to GO database, comprised of 52 functional 108 groups ( Figure 2). The number of unigenes in each functional group ranged from 1 to 10,057. The differentially expressed genes were identified, using the criterion of > 2.0 as up-regulatory 117 genes and < 0.5 as down-regulatory genes, and P-value < 0.05. A total of 1,319 differentially 118 expressed genes (DEGs) were identified between CG and SS, including 713 up-regulated genes 119 and 606 down-regulated genes. A total of 2,092 DEGs were identified between SS and DS, 120 including 1,036 up-regulated genes and 1,056 down-regulated genes. A total of 4,351 DEGs 121 were found between CG and DS, including 2163 up-regulatory genes and 2188 down-122 regulatory genes. KEGG analysis revealed that Cell cycle, Cellular senescence, Oxidative phosphorylation, Glycolysis/Gluconeogenesis and Steroid hormone biosynthesis were the 124 main enriched metabolic pathways in all of these three comparisons.

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A total of 15 DEGs were selected from these enriched metabolic pathways, which were 126 listed in Table 3. These genes were differentially expressed in at least two of these three  with that of DS prawns (p < 0.05). The mRNA expression of SDHB and HSDL1 between the prawns of SS and DS showed no significant difference (p > 0.05), whereas showed significant 148 difference with that of CG prawns (p < 0.05).

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The previous study has been reported that the Mn-HSDL1 mRNA showed the highest 151 expression level in the hepatopancreas, followed by the testis, which showed significant 152 difference with other tested tissues (p < 0.05). The expression in the hepatopancreas and testis   The decrease reached to 96% and 90% at day 7 and 14, respectively, compared with that in 167 control group (Figure 6-A). 168 The expressions of Mn-IAG were also measured in the cDNA template from the same 169 prawns ( Figure 6-B). According to the qPCR analysis, the expression of Mn-IAG at day 1 in 170 control group was slightly higher than that of day 7 and day 14, while it generally remained 171 stable. In RNAi group, the expressions of Mn-IAG were significantly decreased at day 7 and day 14 after the injection of Mn-HSDL1 dsRNA. The expression decreased about 61% and 54% 173 at day 7 and 14, respectively, compared with that in control group.

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ATP is an unstable high-energy compound that is the most direct energy source in 242 organisms. It is widely acknowledged that ATP is essential for the activities in an orgamisn,  It is widely acknowledged that steroid hormones play essential roles in sexual development.

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It is generally divided into five main classes, including glucocorticoids, mineralocorticoids,      Table 2. EIF was used as reference gene in this study [75]. Quantitative data were expressed as mean ± SD. Statistical differences were estimated by one-