Samples Collection and Nucleic acid Preparation
The experimental specimens of adult C. quadricarinatus (body weight, 55±10 g; body length 12.5±2 cm) were obtained from the Balidian breeding base of Zhejiang Institute of Freshwater Fisheries (Huzhou, Zhejiang Province). Various tissues (testes, ovaries, heart, muscle, gill, intestine and hepatopancreas) were dissected on an ice bath, immediately frozen in liquid nitrogen, and stored at -80℃ until used for RNA extraction. Embryo samples at different developmental stages from the offsprings of the overwintered gravid crayfish (fertilized eggs, cleavage and blastula, gastrula, nauplius, eye pigments forming, and prehatching) were collected and preserved in liquid nitrogen for RNA extraction. Description of C. quadricarinatus embryonic development was based on a previously published method . Ovaries at different periods (Stage I to VI) were sampled based on the biological process and histological characteristics.
Total RNA was extracted from tissues and embryos of C. quadricarinatus using Trizol reagent (Total RNA Extractor Kit, Sangon Biotech), and the RNA quality was assessed by electrophoresis on 1% agarose gel. Total RNA isolated from all samples was reverse transcribed using HiFiScript cDNA first-strand synthesis kit (Cwbio, Beijing) according to the manufacturer's procedures for next reverse transcription-PCR (RT-PCR) or Real-time quantitative PCR (qRT-PCR), respectively. Animal experiments were approved by the Committee of Laboratory Animal Experimentation at Zhejiang Institute of Freshwater Fisheries, Huzhou, China.
1.1 Full-length dsx cDNA Sequence Amplification
Firstly, downloaded the dsx coding sequence from known species, such as Drosophila melanogaster (Genbank accession No. NM_169202), Bombyx mori (Genbank accession No. NM_001043406) and Fenneropenaeus chinensis (Genbank accession No. JQ965255), and searched of homulogous fragment sequence based on RNA-seq data from C. quadricarinatus (data not published). As a result, an EST sequence containing a DM domain was found. On this basis, gene-specific primers (Table 1) were designed to amplify the full-length cDNA sequence using the SMARTerTM RACE Amplification Kit (Clontech, USA) following the manufacturer’s protocol. The amplified PCR products of 3’/5’ RACE reactions were subjected to clone into the pMD18-T vector for sequencing, respectively. Finally, the full-length dsx cDNA was assembled from the sequenced results of RACEs by DNAMAN 5.0 software.
The nucleotide sequence and deduced amino acid sequence of dsx were analyzed using Jellyfish software (3.3.1). The secondary structure and basic physicochemical parameters of Dsx protein were predicted using the online software InterProScan (http://www.ebi.ac.uk/interpro/scan.html) and ExPASy (https://web.expasy.org/compute_ pi/), respectively. The conserved amino acid sequence of Dsx DM-domain was aligned with that of DM-domain from other species using DNAMAN, and a phylogenetic tree was constructed by the neighbor-joining method using MEGA 5.0 software. The reliability of the branching was tested using bootstrap re-sampling (with 500 pseudo replicates).
The cDNA from different tissues, embryos and ovary developmental stages synthesized by reverse transcription were used as template, and qRT-PCR was performed for detection transcript levels of dsx gene using SYBR Premix Ex Taq kit (TaKaRa, Japan). The primers used for qRT-PCR was shown in Table 1, and 18S-rRNA (Genbank accession No. AF235966) was used as a normalizing gene. SYBR Green based qRT-PCR was performed using LightCycler® 480 System (Roche, Switzerland), and set the program of 95°C for 10 s followed 40 cycles of 95°C for 5 s and 60°C for 20 s. Each test and their endogenous control were performed in triplicate. Gene expression data was analyzed using the 2-ΔΔCT method  and the histogram using GraphPad Prism 5 software was obtained.
Tissues Section Preparation and In situ Hybridization
Testes and ovaries from adult C. quadricarinatus were carefully dissected and fixed in 4% paraformaldehyde overnight at 4℃, and then embedded in paraffin for cross-sections. A target fragment was amplified with probe primers (Table 1) and cloned into pbluescriptSKII plasmid. Dig-labeled probe was transcribed in vitro and synthesized according to the instructions of the Roche DIG RNA Labeling Mix. For in situ hybridization, the general procedures consisted of 4% paraformaldehyde fixation, proteinase K digestion, room temperature prehybridization, and followed by hybridization with Dig-labeled probe at 55℃ for 12-16 h, and the antibody was eluted after blocking solution treatment. Finally, the hybridization signals were visualized by microscopy and recorded with chromogenic solution NBT/BCIP.
dsRNA Preparation and In-vivo Injection
Gene specific primers with T7 promoter sequence (Table 1) were designed to amplify a 400 bp cDNA fragment of dsx gene followed the PCR program described below: 94℃ for 4 min; 35 cycles of 94℃ for 30 s, 60℃ for 30 s and 72℃ for 30 s; followed by one cycle of 72℃ for 10 min. PCR product was purified and cloned into pUC57 vector, and the EGFP DNA fragment with T7 promoter sites was synthesized by the GenScript Company. Linearized vectors with EcoR I digestion and EGFP fragment were purified and MEGAscript RNAi Kit (Thermo Scientific, USA) was used in dsRNA synthesys according to the manufacturer’s instructions.
For a short silencing experiment, 5 μg/g body dsRNA was injected into a single undifferentiated crayfish with an average weight of o.2 g and average body length of 2 cm by microsyringe (0–50 μL), which manufactured by Ningbo zhenhai sanai instrument factory. Cephalothoraxes of dsRNA-injected individuals were collected at two weeks after injection with dsEGFP or dsDsx, respectively. qRT-PCR was performed to investigate the expression level of Cqdsx and Cq-IAG.
Statistical analyses were performed using SPSS software version 13.0. Data were expressed as mean ± SD (n=3) and statistical significance was determined by one-way ANOVA. Significance was set at P < 0.05.