Male E.sinensis were purchased from Panjin, Liaoning province, China. No official approval was required for this study of the crab in China.

Balb/c female mice, used for antibody production, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China) and housed in an SPF animal house (constant temperature of 22–25 °C, relative humidity of 40–70%). Male rabbits, used for antibody production, were from Animal Experiment Center of Hebei University, and were provided food and water ad libitum. Experimental protocols were approved by the Institutional Animal Care and Use Committee of Hebei University. The mice and rabbits were managed per the guidelines for Animal Care and Use of Hebei University.

GM130 (ab52649), Goat anti-Rabbit IgG H&L (Alexa Fluor® 488) secondary antibody (ab150077), goat anti-mouse IgG H&L (Alexa Fluor® 594) secondary antibody (ab150116) were purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse antibodies were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

Seminal vesicles and accessory glands were obtained from mature male crabs frozen on ice for 15 min before dissection. The spilled spermatophores from seminal vesicles were washed twice using precooled (4 °C) Ca${}^{2+}$-free artificial seawater (FASW)-(475 mM NaCl, 12 mM KCl, 30 mM MgCl${}_2$$\cdot$6H${}_2$O, 20 mM Tris, pH 8.2). The liquid was discarded using a straw after spermatophores precipitated on ice. Proteins from the accessory gland were collected after homogenization and centrifugation at 4 °C, 12,000 g, and 5 min to digest the spermatophores. The crude sperm were released within 15 min; all the components were transferred to a centrifuge tube. The supernatant containing released sperm was collected after centrifugation at 4 °C, 200 g, 10 min. After washing 3 times with FASW and centrifugation at 4 °C, 850 g, 10 min, the pure sperm were obtained.

Proteins from crab sperm were extracted on ice using 8 M urea lysate (8 M urea, 3 µM total sialic acid, and 50 mM nicotinamide). Phenylmethylsulfonyl fluoride (PMSF) was added before use (final concentration 1 mM). 1 mL of precooled urea lysate was added to 50 mg sperm and incubated for 5 min on ice, then ultrasonication was conducted at 30% power, 2 s on, 3 s off, for 2 min using an ultrasonic processor (Scientz, Ningbo, China). Then, the proteins were obtained via centrifugation at 4 °C, 12,000 g for 20 min, with the sperm proteins ultimately collected from the supernatant. Proteins were stored at –80 °C after measuring their concentration with a BCA Kit ( P0012S, Beyotime, Beijing, China).

The proteins were mixed with loading buffer (SL1170-1 mL, Coolaber, Beijing, China) and denatured at 100 °C for 5 min. A total of 20 µg of each sample was separated in 15% polyacrylamide gels with 5% stacking gel for SDS-PAGE and the gels were stained for protein with Coomassie Brilliant Blue R-250 (CBB).

LC-MS/MS analyses of sperm proteins were conducted at Jingjie Company (Hangzhou, China). We summarized the steps as follows. The proteins were separated using 10% polyacrylamide gels and SDS-PAGE was finished when the protein migrated 1 cm from the stacking gel. After staining by CBB and then a destaining buffer, the gels were cut into small granules on clean glass. For digestion, the protein solution was reduced with 5 mM dithiothreitol for 30 min at 56 °C and alkylated with 11 mM iodoacetamide for 30 min at room temperature in darkness. The protein sample was then diluted by adding 100 mM triethylammonium bicarbonate (TEAB) to a urea concentration less than 2 M. Finally, trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4 h-digestion. Finally, the peptides were desalted by C18 solid phase extraction (SPE) column.

The desalted and lyophilized peptides were reconstituted with 0.1% formic acid (FA) which contains 2% acetonitrile and loaded onto a home-made reversed-phase analytical column (25-cm length, 75/100 µm i.d.). Peptides were separated with a gradient from 6% to 24% solvent B (0.1% formic acid in acetonitrile) over 70 min, 24% to 35% in 14 min, and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant flow rate of 450 nL/min on a nanoElute Ultra-high-pressure liquid chromatography (UHPLC) system (Bruker Daltonics, Bremen, Germany).

The peptides were subjected to a capillary source followed by the timsTOF Pro (Bruker Daltonics) mass spectrometry. The electrospray voltage applied was 1.60 kV. Precursors and fragments were analyzed at the TOF detector, with an MS/MS scan range from 100 to 1700 m/z. The timsTOF Pro was operated in parallel accumulation serial fragmentation (PASEF) mode. Precursors with charge state 0 to 5 were selected for fragmentation, and 10 PASEF-MS/MS scans were acquired per cycle.

The resulting MS/MS data were processed using the MaxQuant search engine (v.1.6.15.0, http://www.maxquant.org/). Tandem mass spectra were searched against the transcriptome of testis and sperm connected with downloaded data that reported data in the SwissProt database on E.sinensis. Reverse decoy databases were used to guarantee the correction rate. Trypsin/P was specified as the cleavage enzyme allowing up to 2 missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in the first search and 5 ppm in the main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as a fixed modification, and acetylation on protein N-terminal and oxidation on Met were specified as variable modifications. The false discovery rate (FDR) was adjusted to 1%.

Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/). Firstly, the identified protein ID was converted to UniProt ID and then mapped to GO IDs by the protein ID. If some identified proteins were not annotated by the UniProt-GOA database, the eggNOG-mapper (v2.0 https://github.com/eggnogdb/eggnog-mapper) was used to annotate the protein’s GO function based on the protein sequence alignment method. Then proteins were classified by GO annotation based on three categories: biological process, cellular component, and molecular function.

The cells of eukaryotic organisms are elaborately subdivided into functionally distinct membrane-bound compartments. Some major constituents of eukaryotic cells are the extracellular space, cytoplasm, nucleus, mitochondria, Golgi apparatus, endoplasmic reticulum, peroxisome, vacuoles, cytoskeleton, nucleoplasm, nucleolus, nuclear matrix and ribosomes. There, we used wolfpsort a subcellular localization prediction software to predict subcellular localization.

Testes from male crabs were dissected on ice and washed twice with Ca${}^{2+}$-free artificial seawater (Ca${}^{2+}$-FASW; containing 475 mM NaCl, 12 mM KCl, 30 mM MgCl${}_2$, 20 mM Tris, pH 8.2). RNA was extracted with Trizol and then reverse-transcribed into cDNA using a kit (RR047A, Takara, Beijing, China).

Primers of Rab2 (GenBank accession No. LOC126998668) and Rab6 (GenBank accession No. LOC127008679) were designed in our laboratory; the sequences were as follows: Rab2. F1, 5′-ATGTCCTACGCCTATTTATTCAA-3′, and R1, 5′-CTTGTTTCCAATCAGCATGAT-3′. Rab6. F2, 5′-ACAGTTCGACTGCAGCTCT-3′, R2, 5′-ACACCCACCATCGGCTGTC-3′. Primers were synthesized by Songon Biotech (Shanghai, China). The PCR steps were as follows: Rab2: 94 °C for 30 s, 62 °C for 30 s, 72 °C for 40 s, and 30 cycles. 72 °C was kept for 10 min for the last cycle. Rab6: 94 °C for 30 s, 58 °C for 30 s, 72 °C for 40 s, and 30 cycles. 72 °C was kept for 10 min for the last cycle. 1.0% agarose gels were used to check the PCR products, Rab2 was obtained at 360 bp of and Rab6 at 450 bp, as expected. To further verify the accuracy, the PCR products were sequenced by the Sanggon Company (Shanghai, China) after recovery with an Agarose Gel DNA Recovery kit (Beijing Biotechnology Co. Ltd., Beijing, China).

Pet-19T-Rab2 and Pet-19T-Rab6 were prepared with a Pet-19T cloning vector (Takara). After sequencing by the Sanggon Company (Shanghai, China), both recombined vectors were transformed into BL21 for multiplication. Stocks were stored at –80 °C. Rab2 primers were designed with BamH I (identified in the sequence by underlining) at the forward primer 5′-CGCGGATCCATGTCCTACGCCTATTTATTCAA-3′ and XhoI (underlined) at the reverse primer 5′-ACGTCACTCGAGCTTGTTTCCAATCAGCATGAT-3′. Rab6 primers were designed with NdeI (underlined) at the forward primer. 5′-ACGCGCCATATGACAGTTCGACTGCAGCTCT-3′, and XhoI (underlined) at the reverse primer 5′-ACGTCACTCGAGACACCCACCATCGGCTGTC-3′.

Pet-19T-Rab2 and Pet-19T-Rab6 were used as the template using the above primers for PCR. The products were digested with BamH I and XhoI for Rab2, and NdeI and XhoI for Rab6. Rab2 was incorporated in the Pet-28b vector while Rab6 was incorporated in the Pet-21b vector. Recombinant clones for expression are named Pet-28b-Rab2 and Pet-21b-Rab6. All were validated via sequencing by the Sangon Company (Shanghai, China).

The recombinant plasmid Pet-28b-Rab2 was transformed into electrocompetent BL-21 and selected with Luria–Bertani (LB) plates containing kanamycin (10 µg/mL). The remaining BL-21 was expanded at 37 °C for 4 h with shaking at 180 RPM with a final concentration of 0.2 mmol/L of isopropyl $\beta$-D thiogalactoside (IPTG). The culturing was ended when the OD value was read as 0.4–0.6, then centrifuged (4 °C, 1000 g, 10 min) to collect the pellets. A fragmentation buffer (100 mM NaH${}_2$PO${}_4$, 10 mM Tris–HCl, 8 M urea) was added to the strains, then fragmented using ultrasonic shearing (Ningbo, Scientz D) on ice (60% intensity, 5 s, 10 s interval, 6 min). The suspensions were centrifuged (4 °C, 12,000 g, 30 min) in 50 mL tubes to separate the supernatant and precipitant. The supernatant was purified using a nickel column and eluted with buffer (25 mM Tris 25 mL, 15 mM NaCl, 3% Midazole, pH 8.0) to collect the target protein. We used 15% SDS-PAGE to verify the purified Rab2 and Rab6.

Balb/c female mice aged 6 weeks were habituated for 1 week. 50 µg Rab2 polypeptide antigen mixed with 50 µg Freund’s complete adjuvant (lot number: F5881; Sigma, St. Louis, MO, USA) was injected into the back of the mice for the first immunization. 10 days later, a second immunization was performed by emulsifying half of the antigen polypeptide with an equal volume of Freund’s incomplete adjuvant (lot number: F5506; Sigma, St. Louis, MO, USA) using intraperitoneal injection. The health status of each animal was observed after each injection. The last two immunizations were prepared as the second injection and injected once weekly. Three days after the last immunization, blood was collected from the eye socket with a glass capillary and the serum was extracted. Anti-Rab2 serum was stored at –80 °C.

Rab6 polyclonal antibodies were prepared similarly, but by using rabbits instead of mice for more anti-serum. Two male New Zealand White rabbits (2 $\pm$ 0.3 kg) were housed in 2 pens (1.0 $\times$ 0.7 m${}^2$) individually at the Animal Experiment Center of Hebei University. A week later, each rabbit was immunized with 0.5 mg of the Rab6 polypeptide mixed with Freund’s Complete Adjuvant (lot number: F5881; Sigma, St. Louis, MO, USA) via multi-point subcutaneous injection. The last 3 immunization were performed every 10 days using Freund’s Incomplete Adjuvant (lot number: F5506; Sigma, St. Louis, MO, USA), delivered via intraperitoneal injection. Three days after the last immunization, blood was collected from the ear vein and serum was separated. Anti-Rab6 serum was stored at –80 °C.

Anti-Rab2 antiserum was collected after centrifugation (12,000 g, 10 min, 4 °C) and quantified by indirect ELISA using the above purified Rab2 as the antigens. The optimal dilution (2 µg/mL) of recombinant protein was used to coat the 96-well plate, and the samples were blocked with 100 µL 10% BSA diluted in washing buffer (0.0015 M KH${}_2$PO4, 0.008 M Na${}_2$HPO${}_4$·12 H${}_2$O, 0.14 M NaCl, 0.003 M KCl, 0.05% v/V Tween-20) for 2 h at room temperature. The primary antibody anti-Rab2 serum and pre-immune serum were diluted (1:100 to 1:102,400). A total of 100 µL goat to mouse IgG with conjugated HRP (dilution 1:1000, Solarbio, Beijing, China) was added and incubated at 37 °C for 1 h. After washing three times the reaction termination liquid (10% v/V H${}_2$SO${}_4$) was added. Anti-Rab6 antiserum was similarly detected. The difference is that the second polyclonal goat anti-rabbit IgG with conjugated HRP (dilution 1:1000, Solarbio) was used. Finally, the titer of the polyclonal antibody was measured and analyzed based on a 450 nm absorbance value using a multifunctional microplate reader (Molecular Devices, Shanghai, China).

Western Blot was used to detect the specificity of Rab2 and Rab6 polyclonal antibodies. Proteins from crab testes were extracted on ice using a radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China, cat#P0013B) with 10 µL PMSF to a final concentration of 1 mM. A total of 20 µg of the sample was loaded using 15% sodium dodecyl sulfate-polyacrylamide separation gel electrophoresis (SDS-PAGE) for detection. The gel was cut between 10–25 kD and transferred to a polyvinylidene fluoride membrane (PVDF) membrane. The membranes were incubated with Rab2 polyclonal antibodies overnight at 4 °C after blocking with 5% skim milk in TBST (tris buffered saline and 0.1% Tween-20) for 2 h at room temperature. Then, HRP-conjugated goat anti-mouse IgG (dilution 1:1000, Solarbio) after washing 3 times with TBST. Finally, enhanced chemiluminescence (ECL) reagent kit (Solarbio, China, cat#PE0010) was used for detection. Rab6 or GM130 (ab52649) was detected using the same method, the difference is that Rab6 or GM130 antibodies were used instead of Rab2, and the secondary antibodies for HRP-conjugated goat anti-rabbit IgG (dilution 1:1000, Solarbio) were used for detection. $\beta$-actin was used as the loading control for the western blot. All results were acquired and analyzed with Image Lab software (v.4.1, Bio-Rad, Hercules, CA, USA). Image J software (v.13, LOCI, University of Wisconsin, Madison, WI, USA) was used for the relative protein quantification analysis.

To observe the colocalization of Rab2 and GM130, testes from male crabs were fixed with 4% paraformaldehyde overnight at 4 °C and dehydrated using 30% (w/w) sucrose in 0.1 M PBS, pH 7.4. Frozen sections were permeabilized with 0.5% Triton X-100 at room temperature for 10 min, and blocked with 5% BSA, 10% goat serum, and 0.3 M glycine for 1 h at RT after 3 washes with PBS. Anti-Rab2 antiserum (dilution 1:500) along with GM130 (dilution 1:200, Abcam) were incubated for co-staining overnight at 4 °C. After washing 3 times for 10 min, the secondary antibodies of goat anti-rabbit Alexa Fluor® 488 (1:200, Abcam) and goat anti-mouse Alexa Fluor® 594 secondary antibodies (dilution 1:200, Abcam) were added and incubated at RT in the dark for 1 h at room temperature. After washing 2 times for 5 min with PBS, 6-diamidino-2-phenylindole (DAPI) was used for nuclear staining for 5 min following the last washing. Control sections were incubated with non-immune goat serum (Solarbio, China, cat#SL038) in place of primary antibodies under the same condition described above.

To observe the characteristics of Rab2 during different periods of spermatids, only Rab2 poly-antibodies were used (1:500) and goat anti-mouse Alexa Fluor® 594 secondary antibodies (dilution 1:200, Abcam) were incubated. Rab6 detection was conducted using the method described above. The difference is that anti-Rab6 antiserum was used (1:400) and the secondary antibodies of Alexa Fluor 488-conjugated goat anti-rabbit (dilution 1:200) were incubated at RT after washing. Both control sections were incubated without a primary antibody.

All the stained sections were mounted on slides using an anti-fade mounting medium (Southern Biotech, Cat#.0100–01). Images were acquired using an Olympus FV1000-IX81 laser scanning confocal microscope (Olympus, Tokyo, Japan).

The SDS-PAGE result of sperm proteins showed a total of 23 detectable bands (Fig. 1A). Rab proteins represent 21–25 kD and the histones are under 15 kD.

LC-MS/MS was conducted to identify the sperm proteome. Raw data produced by the timsTOF Pro (Bruker Daltonics) mass spectrometry were subsequently searched by using the MaxQuant search engine (v.1.6.15.0). Tandem mass spectra were searched with the transcriptome of testis and sperm in E.sinensis connected with SwissProt database. A total of 26,430 peptides corresponding to 1247 proteins were identified (Supplementary Table 1). As expected, the major proteins fell under either catalytic activity or binding functions. A wide range of protein classes was identified with hydrolase, oxidoreductase, nucleic acid binding, transferase, cytoskeleton protein, and enzyme modulator being the most common. The results could be divided into the biological process (BP), cell component (CC), and molecular function (MF) categories (Fig. 1B).

Importantly, all previously identified histone proteins including H1, H2A, H2B, H3, and H4 were detected [31, 32, 33, 34]. Further cell component association analysis showed that the sperm proteome contains proteins from different organelles (Fig. 1C), such as mitochondria (287), endoplasmic reticulum (18), and Golgi apparatus (2) (Supplementary Table 2). 6 cathepsin proteins including A, B, C, D, F, and L were detected in the mature sperm. In addition, 7 types of Rab proteins, including Rab1, Rab2, Rab5, Rab6, Rab11, Rab14, and Rab18 were found.

RNA quality was assessed with 1% agarose gel electrophoresis (Fig. 2A). A band was seen at approximately 360 bp as expected for Rab2 (Fig. 2B) while Rab6 was seen at 450 bp (Fig. 2C). Rab2 was cleaved from Pet-19T-Rab2 with BamH I and XhoI and sequenced after gel-purification; Rab6 was cleaved using NdeI and XhoI from Pet-19T-Rab6 and sequenced. The results of these analyses validated our Rab2 and Rab6 preparation.

Rab2 sequencing results are the same as part of the gene Rab2 sequence (total 642 nucleotides). The primers are underlined and the sequence of Rab2 (360 nucleotides) for PCR are denoted in bold.

ATGTCCTACGCCTATTTATTCAAGTACATCATCATCGGAGACACTGGTGTGGGGAAGTCATGTCTC
CTGCTACAGTTCACGGACAAGAGGTTCCAGCCAGTGCACGACCTCACCATCGGGGTGGAGTTT
GGTGCCCGCATGATCACCATTGACAACAAGCAGATCAAGCTTCAGATTTGGGACACAGCTGGC
CAAGAGGCATTCAGGTCCATCACAAGGTCATACTACCGTGGAGCAGCCGGCGCCCTTCTGGTC
TATGACATCACCAGGCGGGAAACCTTCAACCACCTCACGCAGTGGCTAGAAGATGCTCGCCAG
CACTCCAACTCCAACATGGTCATCATGCTGATTGGAAACAAGAGTGATCTTGACTCACGGCGAGA
GGTAAAGCGAGAAGAGGGCGAAGCCTTTGCGCGGGAGCACGGGCTGGTGTTCATGGAGACCTCCGC
CAAGACCGCCGCCAACGTAGAGGAAGCTTTCATCAACACTGCCAGGGAAATCTACGAGAAGATCCAG
GAGGGGGTGTTTGACGTCCACAATGAGGCTAACGGCATCAAGATTGGGCCCCAGCACTCCCCCGGAG
GAGCCGGCCTCACCAACAACCAGGGAGCCGCTGGCGGTCAAGGCGGCGGCTGCTGTTAA

Rab2 coding proteins sequence. The protein sequence of Rab2 (total 213 amino acids) is shown as follows and the selected immunogenic protein sequence of Rab2 (120 amino acids) is denoted in bold.

MSYAYLFKYIIIGDTGVGKSCLLLQFTDKRFQPVHDLTIGVEFGARMITIDNKQIKLQIWDTAGQEAF
RSITRSYYRGAAGALLVYDITRRETFNHLTQWLEDARQHSNSNMVIMLIGNKSDLDSRREVKREEGEA
FAREHGLVFMETSAKTAANVEEAFINTAREIYEKIQEGVFDVHNEANGIKIGPQHSPGGAGLTNNQGAAGG
QGGGCC

Rab6 sequencing results are the same as part of the gene Rab6 sequence (total of 639 nucleotides). The primers are underlined and the sequence of Rab6 (total 450 nucleotides) for PCR are denoted in bold.

ATGTCCATGTCGGGCGAATTTGGGAACCCGCTCCGGAAATTCAAGCTGGTCTTTCTGGGCGAGCAAAG
CGTCGGAAAGACCTCGCTGATAACAAGGTTTATGTACGACTCCTTTGACAACACATACCAGGCAACGA
TTGGCATAGACTTCCTCTCCAAAACAATGTACCTCGAGGATAGAACAGTTCGACTGCAGCTCTGGGAC
ACGGCCGGCCAGGAGAGGTTCCGTAGCCTCATCCCATCCTATATCCGAGACTCGACTGTCGCTG
TGGTTGTGTACGACATTACCAATGCCAATTCTTTCCACCAAACTTCTAAGTGGATAGACGATGTG
AGGACTGAGCGAGGCAGCGACGTAATTATCATGCTGGTTGGAAATAAGACTGATCTTTCGGACA
AGAGACAGGTTTCCACGGAGGAGGGCGAGCGCAAGGCCAAGGAACTCAACGTGATGTTCATTG
AGACGAGTGCAAAGGCAGGATACAATGTGAAACAGCTCTTCCGAAGGGTGGCCGCCGCCCTAC
CCGGTATGGAGTCCAATCCTGAGAAGGGCAAGGTTGACATGACCGAAGTTGTCCTGAGGGACT
CAGACAACACTGCAGAGATTGGGCGGACAGCCGATGGTGGGTGTGCTTGCTAG

Rab6 coding proteins sequence. The protein sequence of Rab6 is shown as follows (total 212 amino acids) and the selected immunogenic protein sequence of Rab6 (150 amino acids) is denoted in bold.

MSMSGEFGNPLRKFKLVFLGEQSVGKTSLITRFMYDSFDNTYQATIGIDFLSKTMYLEDRTVRLQLWDTA
GQERFRSLIPSYIRDSTVAVVVYDITNANSFHQTSKWIDDVRTERGSDVIIMLVGNKTDLSDKRQVSTE
EGERKAKELNVMFIETSAKAGYNVKQLFRRVAAALPGMESNPEKGKVDMTEVVLRDSDNTAEIGRT
ADGGCAC

The structures of Rab2 and Rab6 domains predicted by SWISS-MODEL showed that both two Rab proteins contain β-sheets flanked by α-helices (Fig. 3).

The results of SDS-PAGE showed that Rab2 and Rab6 were purified as expected. A band at 15 kD represents purified Rab2 and a band at 17 kD of Rab6 after Ni column purification (Fig. 4).

Quantification of Rab2 and Rab6 in the antisera using ELISA confirmed that both poly-antibodies were ready for the following experiments. Proteins extracted from the testes and sperm of E.sinensis were subjected to western blot, and the results showed a band at 22 kd for Rab2 (Fig. 5A) and a band at 26 kd for Rab6 (Fig. 5C), confirming our preparation. A band is shown at 130 kd for GM130 (Fig. 5B), validating this GM130 antibody as suitable for our research (Fig. 5D). Quantification by Image J showed that the expression of Rab2, Rab6 and GM130 in the testes was higher than in sperm (Fig. 5E–G).

In the crab testis, different stages of germ cells can be recognized according to the unique characteristics of their DAPI-stained nuclei (Fig. 6). Spermatogonia has the largest nuclei with fewer chromatin clumps compared with spermatocytes. At the spermatid stage, the chromatin becomes dense and the clumps disappear. Early spermatids have round donut-like nuclei. Middle spermatids have crescent-shaped nuclei that wrap the proacrosome. Late spermatids have cup-like nuclei that hold the acrosome. The spermatozoa in seminal vesicles have a nuclear annulus resembling a ring when observed from above.