2024 06 24 600346v1 Full

bioRxiv preprint doi: https://doi.org/10.1101/2024.06.24.600346; this version posted June 27, 2024.
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1 High-resolution chromosome-level genome provides molecular insights into
2 adaptive evolution in crabs

3 Yin Zhang1,2,3,#, Ye Yuan1,2,3,#, Mengqian Zhang1,2,3,#, Xiaoyan Yu1,2,3, Bixun Qiu1,2,3, Fangchun
4 Wu1,2,3, Douglas R. Tocher1, Jiajia Zhang1,2,3, Shaopan Ye1,2,3, Wenxiao Cui1,2,3, Jonathan Y. S.
5 Leung1,2,3, Mhd Ikhwanuddin2,3,4, Waqas Waqas1,2,3, Tariq Dildar1,2,3, Hongyu Ma1,2,3,*
6
1
7 Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University,
8 Shantou, China
2
9 International Joint Research Center for the Development and Utilization of Important
10 Mariculture Varieties Surrounding the South China Sea Region, Shantou University, Shantou,
11 China
3
12 STU-UMT Joint Shellfish Research Laboratory, Shantou University, Shantou, China
4
13 Higher Institute Centre of Excellence (HICoE), Institute of Tropical Aquaculture and
14 Fisheries, Universiti Malaysia Terengganu, Kuala Nerus, Terengganu, Malaysia

#
15 These authors contributed equally to this work.
16
*
17 Correspondence: Prof. Hongyu Ma (Email: mahy@stu.edu.cn)
18 Guangdong Provincial Key Laboratory of Marine Biotechnology
19 Shantou University
20 243 Daxue Road, Shantou 515063, China
21 Tel: +86-754-86503471
22
23
1
bioRxiv preprint doi: https://doi.org/10.1101/2024.06.24.600346; this version posted June 27, 2024. The copyright holder for this preprint
24 Abstract
25 Crabs thrive in diverse ecosystems, from coral reefs to hydrothermal vents and terrestrial
26 habitats. Here, we report a comprehensive genomic analysis of the mud crab using ultralong
27 sequencing technologies, achieving a high-quality chromosome-level assembly. The refined
28 1.21 Gb genome, with an impressive contig N50 of 11.45 Mb, offers a valuable genomic
29 resource. Gene family analysis shows expansion in development-related pathways and
30 contraction in metabolic pathways, indicating niche adaptations. Notably, Investigation into
31 Hox gene regulation sheds light on their role in pleopod development, with the Abd-A gene
32 identified as a linchpin. Posttranscriptional regulation involving novel-miR1317 negatively
33 regulates Abd-A levels. Furthermore, the fru gene's potential role in ovarian development and
34 the identification of novel-miRNA-35 as a regulator of Spfru2 add complexity to gene
35 regulatory networks. Comparative functional analysis across Decapoda species reveals
36 neofunctionalization of the elovl6 gene in the synthesis of long-chain polyunsaturated fatty
37 acids (LC-PUFA), suggesting its importance in environmental adaptation. These findings
38 contribute significantly to our understanding of crab adaptability and evolutionary dynamics,
39 offering a robust foundation for future investigations.
40 Key words: whole genome; crab; early development; ovary maturation; LC-PUFA
41
2
42 Introduction
43 Evolutionary adaptation involves organisms adjusting to their environment to increase survival
44 chances, resulting in diverse modifications including changes to body plans, development, and
45 growth patterns. These adaptations are particularly evident among arthropods, specifically
46 Decapoda crustaceans, which include economically valuable species such as shrimp, crayfish,
47 lobsters, and crabs. Decapods occupy distinct ecological niches and employ diverse adaptive
48 strategies, with shrimp navigating near the seafloor and crabs inhabiting burrows or crevices
49 within muddy substrates during day. True crabs belong to the Decapoda infraorder Brachyura
50 and can be traced back to the early mid-Jurassic period (Schweitzer and Feldmann 2010; Ma
51 et al, 2019). The success of brachyuran crabs suggests that acquiring a crab-like morphology
52 may have served as a pivotal innovation and, thus, they are an ideal group for studying trends
53 in biodiversity over time (Morrison et al, 2002; Wolfe et al, 2021). Therefore, crabs provide an
54 illuminating model system for investigating these foundations within Decapoda.
55 The majority of animals have intricate life cycles, which include larval and adult stages with
56 distinct morphologies and ecological niches. In Decapoda, particularly in crabs, the
57 morphological diversity is intriguing (Wolfe et al, 2019; Wolfe et al, 2021). Metamorphosis
58 enables crabs to develop unique body shapes, and to transition from an oceanic planktonic to a
59 benthic niche (Keiler et al, 2017). Crab larvae undergo metamorphosis from a laterally
60 compressed zoea to a dorsoventrally compressed megalopa with crab-like features,
61 recapitulating the morphological evolution of Decapoda. However, the evolutionary and
62 genetic mechanisms underpinning crab metamorphosis remain inadequately understood
63 (McNamara and Faria 2012; Bracken-Grissom et al, 2013; Scholtz 2014), despite indications
64 of a correlation between body form and ecology (Luque et al, 2019). Concurrently,
65 comprehending reproductive strategies is pivotal for elucidating evolutionary history, given the
66 contribution these strategies make to enhancing parental fitness (Stearns 1992). While
67 mammals exemplify the apogee of animal evolution with complex reproductive behaviors and
68 sophisticated physiological mechanisms (Gong et al, 2023), in fish, gametes mature in the
69 gonad before mating occurs (Turner et al, 2022). Similarly, shrimps mate after gonadal
3
70 maturation (Zhang et al, 2019), whereas crabs mate after females undergo reproductive molting
71 when their ovaries are immature, thus triggering ovary maturation.
72 In the domains of reproduction, metamorphosis, growth, and life history, fatty acids,
73 specifically long-chain polyunsaturated fatty acids (LC-PUFA), fulfill essential functions
74 (Tocher 2010; Ting et al, 2020; Závorka et al, 2023). The allocation of capacities in animals
75 entails a delicate equilibrium between acquiring diverse fatty acids from diet and synthesizing
76 LC-PUFA through multiple pathways regulated by fatty acid desaturase (fads) and elongase
77 (elovl) genes (Twining et al, 2021; Castro et al, 2016). Initially, it was postulated that marine
78 animals had limited ability to synthesize LC-PUFA due to the high availability of these fatty
79 acids in marine ecosystems (Kabeya et al, 2018). However, recent reports indicate that
80 euryhaline herbivorous fish and some invertebrates can also produce LC-PUFA endogenously
81 (Monroig et al, 2018; Xie et al, 2021). Interestingly, the genes encoding different types of Elovl
82 are more diverse in invertebrates than vertebrates (Kabeya et al, 2018; Monroig et al, 2022;
83 Ramos-Llorens et al, 2023), although this diversity remains poorly understood.
84 The utilization of genome sequencing is crucial for understanding organism evolution and
85 identifying genes involved in animal adaptations (Zhang et al, 2019; Yim et al, 2014; Gutekunst
86 et al, 2018). Despite the availability of several genome maps for shrimps and crabs (Zhang et
87 al, 2019; Song et al, 2016; Tang et al, 2021; Tang et al, 2020; Zhao et al, 2021; Cui et al, 2021;
88 Chebbi et al, 2019), the current genomic data for Decapoda are insufficient to facilitate
89 comprehensive studies on development, reproduction, and nutrient acquisition within this
90 ancient, successful lineage. Attaining a high-quality genome is imperative for establishing an
91 experimental model to understand the molecular mechanisms underlying adaptive evolution in
92 crabs. The current study presents a comprehensive analysis of the genome of the green mud
93 crab Scylla paramamosain, providing a robust model system to investigate the molecular
94 foundations of adaptive evolution within Decapoda.
95
96 Results
97 Genome sequencing, assembly and annotation
98 A total of 98.60 Gb pass reads were obtained using Nanopore ultralong-read sequencing. After
4
99 quality control, genome assembly and correction, the S. paramamosain (Fig. 1A) genome had
100 a size of 1.21 Gb with a contig N50 of 11.45 Mb. In total, 421 contigs with an average length
101 of 11,740,790 bp were obtained and further assembled into 233 scaffolds with a scaffold N50
102 of 23.61 Mb (Table 1), which was longer than those of other crustaceans (Zhang et al, 2019;
103 Gutekunst et al, 2018; Song et al, 2016; Tang et al, 2021; Cui et al, 2021; Chebbi et al, 2019)
104 (Table S1). The N50 of the sequences obtained here is approximately five times that achieved
105 in the previous genome assembly (N50 < 200 kb) (Zhao et al, 2021), indicating a significant
106 improvement compared to the previous S. paramamosain genome. Overall, 95.26% of the
107 1,013 Arthropoda Benchmarking Universal Single-Copy Orthologs (BUSCOs) were identified
108 in the assembled genome by searching against Arthropoda_odb 10. The contigs were further
109 anchored to 49 pseudochromosomes with a total length of 1,106,215,978 bp, representing 99.47%
110 of the genome (Fig. 1B). Chromosome 01 (chr01) is the largest, with a length of 42.18 Mb,
111 whereas chr49 has the shortest length (5.67 Mb). In addition, chr06 has a strongly sex-linked
112 signal using genome-wide association studies (GWAS) (Fig. 1C and Fig. S1), which have been
113 conducted to identify genes associated with various economically relevant traits, including sex,
114 growth, disease resistance and environmental adaptation, in shrimp (Zhang et al, 2019; Lyu et
115 al, 2021). Previously, we identified 13 sex-specific SNPs (Waiho et al, 2019) located on chr06
116 in S. paramamosain, which indicated that chr06 is the sex chromosome of green mud crab. We
117 observed a peak of Ks ranging from 0 to 1 and a summit at 0.2, representing a potential ancient
118 whole-genome duplication (WGD) (Fig. 1D).
119 The assembly of the genome can be affected by various factors, such as individual
120 heterozygosity, repetitive sequences, and GC bias (Zhang et al, 2012; Li et al, 2017). In addition,
121 long reads can span complex or repetitive regions with a single continuous read, thus
122 eliminating ambiguity in the positions or size, contributing to a more complete genome
123 assembly (Jain et al, 2018). Herein, we performed ultralong-read sequencing and detected
124 587.19 Mb of repetitive sequences, accounting for 58.97% of the genome, which is much
125 higher than that in Chinese mitten crab Eriocheir sinensis (Cui et al, 2021) and Pacific white
126 shrimp Litopenaeus vannamei (Zhang et al, 2019), but lower than that in other crustaceans for
127 which genome data are available (Gutekunst et al, 2018; Zhao et al, 2021). The genome
5
128 contains 206,695 simple sequence repeats (SSRs), representing 0.25% of the total genome.
129 Dinucleotide repeats were the dominant type of SSRs, accounting for 58.85% of the total SSRs.
130 Moreover, there were 345,610 tandem repeats (TRs) with a length of 24,183,525 bp,
131 representing 2.00% of the whole genome, which is much lower than that in Chinese mitten crab
132 (Cui et al, 2021). As a predominant force driving genome expansion and evolution (Bire and
133 Rouleux-Bonnin 2012; Elliott and Gregory 2015), transposable elements (TEs), with a
134 proportion of 48.50% of the S. paramamosain genome, contain 17.40% long interspersed
135 elements (LINEs), 8.22% long terminal repeats (LTRs), 22.05% DNA transposons, 0.33%
136 rolling circle (RC) and 0.35% miniature inverted-repeat transposable elements (MITEs) (Fig.
137 S2 and Table S2).
138 In S. paramamosain, 33,662 protein-coding genes were predicted with an average gene
139 length of 12,535.06 bp, CDS length of 1,208.52 bp, exon length of 225.29 bp, and intron length
140 of 2,595.33 bp (Table S3 and Fig. S3). Compared with Chinese mitten crab E. sinensis (Cui et
141 al, 2021) and marbled crayfish Procambarus virginalis (Gutekunst et al, 2018), we found that
142 S. paramamosain had relatively more coding genes. The average CDS length of green mud
143 crab protein-coding genes was similar to that of Chinese mitten crab E. sinensis (Song et al,
144 2016) and shrimp L. vannamei (Zhang et al, 2019), but longer than that of swimming crab
145 Portunus trituberculatus (Tang et al, 2020) (Table S1). Comparisons of gene number and
146 protein length indicated superior quality in the green mud crab gene models compared to those
147 of the Chinese mitten crab and swimming crab, suggesting useful resources for further
148 comparative studies and a more comprehensive understanding of genomic characteristics in
149 green mud crab. In total, 92.31% of the genes were annotated to public databases, including
150 SwissProt (14,738 genes), InterPro (28,920 genes), Kyoto Encyclopedia of Genes and
151 Genomes (KEGG; 11,128 genes), Gene Ontology (GO; 12,328 genes), EuKaryotic
152 Orthologous Groups (KOG; 12,381 genes) and Non-Redundant Protein Sequence Database (Nr,
153 20,927 genes) (Fig. S4). We also annotated noncoding RNAs, including 449 miRNAs, 1,055
154 rRNAs, 36 snRNAs, and 3,153 tRNAs (Table S4).
155 Gene family expansion, contraction and genome evolution
156 Most expansion events are associated with the adaptive evolution of specific phenotypes. To
6
157 conduct gene family expansion and contraction events in S. paramamosain, gene family
158 clustering analysis was conducted on 14 species, including five other crustacean species (P.
159 trituberculatus, E. sinensis, Penaeus vannamei, Homarus americanus, and P. monodon), six
160 insect species (Drosophila melanogaster, Bombyx mori, Eisenia andrei, Lepeophtheirus
161 salmonis, Stegodyphus mimosarum, and Strigamia maritima), and two vertebrate species
162 (Danio rerio and Homo sapiens). The results of the gene family analysis were categorized and
163 statistically analyzed for each category. S. paramamosain had 297 single-copy genes, 4,775
164 unique genes, and 11,097 unclustered genes. Furthermore, the unclustered and unique genes
165 were combined to yield a total of 15,872 species-specific genes (Fig. 1E and Fig. S5). These
166 species-specific genes were significantly enriched in aminoacyl-tRNA biosynthesis, TNF
167 signaling pathway, RNA degradation, ubiquitin mediated proteolysis, longevity regulating
168 pathway, circadian entrainment, autophagy–animal, neurotrophin signaling pathway,
169 adrenergic signaling in cardiomyocytes, MAPK signaling pathway, NF-kappa B signaling
170 pathway, mTOR signaling pathway and c-type lectin receptor signaling pathway (Fig. S6),
171 most of which are related to the development and lifestyle of the green mud crab. Phylogenetic
172 analysis based on 297 single-copy orthologous genes suggested that crabs diverged from
173 shrimp (represented by L. vannamei) ~481.54 million years ago (Mya), that marine crabs
174 diverged from freshwater crabs (E. sinensis) ~280.48 Mya, and that S. paramamosain diverged
175 from P. trituberculatus ~141.1 Mya (Fig. S7).
176 A total of 12,274 gene families were identified through family clustering, with 725 being
177 specific to S. paramamosain. Of the 33,662 genes, 22,565 genes could be classified into distinct
178 gene families, with an average of 1.84 genes per family. As variations in gene copy number
179 might support adaptive evolution, we examined the expansion and contraction of gene families
180 in the S. paramamosain genome to explore the potential mechanisms underlying the
181 adaptability of crabs. The number of expanded gene families in S. paramamosain was 1,545,
182 while the number of contracted families was 2,671 (Fig. 2). Among them, the expanded genes
183 were significantly enriched in development-related pathways such as betalain biosynthesis,
184 ubiquitin-mediated proteolysis, circadian entrainment, neurotrophin signaling pathway,
185 adrenergic signaling in cardiomyocytes, longevity regulating pathway, MAPK signaling
7
186 pathway and autophagy–animal. Interestingly, when comparing shrimps and crabs, the
187 expanded gene families in S. paramamosain were also significantly associated with these
188 pathways (Table S5), suggesting that the specific developmental pattern of green mud crabs
189 may be attributed to the expansion of these gene families. On the other hand, the contracted
190 gene families were mainly annotated to nutritional metabolism, including carbon metabolism,
191 propanoate metabolism, glyoxylate and dicarboxylate metabolism, microbial metabolism in
192 diverse environments, biosynthesis of secondary metabolites, carbon fixation pathways in
193 prokaryotes, methane metabolism, pyruvate metabolism, and glycolysis/gluconeogenesis (Fig.
194 S8), indicating that there have been alterations in nutrient intake during the process of evolution.
195 Therefore, the aforementioned species-specific and expanded gene families may have been
196 important to S. paramamosain lineage-specific environmental adaptations, enabling the crab
197 to occupy diverse niches in a myriad of stressful conditions.
198 The function and regulatory mechanisms of Hox genes in the development of pleopods
199 Crustaceans present the most impressive diversity in body plan among arthropods (Deutsch
200 and Mouchel-Vielh 2003). Hox genes encode homeodomain-containing transcription factors
201 that play crucial roles in directing tissue differentiation and morphological development
202 throughout all the principal axes of an embryo (Yan et al, 2019). Here, we found that the S.
203 paramamosain genome contained all ten canonical Hox gene clusters (lab, pb, Hox3, Dfd, Scr,
204 ftz, Antp, Ubx, Abd-A, and Abd-B) (Fig. 3 A, B), which were found in the arthropod ancestor
205 (Grenier et al, 1997; Hughes and Kaufman 2002). The Hox gene clusters were located on the
206 same chromosome (Fig. 3A), similar to the spatial collinearity in most bilaterians
207 (Uengwetwanit et al, 2021). However, most Hox gene sequences contain breaks because of
208 low-quality sequencing data, preventing the verification of the transcribed direction of the Hox
209 gene cluster (Kim et al, 2018). Herein, the continuous location of the Hox gene cluster strongly
210 supported the high integrity of our genome assembly of S. paramamosain. However, the
211 expression of the Hox genes did not show whole-cluster temporal collinearity (Fig. 3C), similar
212 to vertebrates, but exhibited subcluster-level temporal collinearity, similar to Chinese mitten
213 crab E. sinensis (Cui et al, 2021), shrimp P. monodon (Uengwetwanit et al, 2021), scallop
214 Patinopecten yessoensis (Wang et al, 2017), oyster Crassostrea gigas (Zhang et al, 2012) and
8
215 sea squirt Ciona intestinalis (Ikuta et al, 2004).

216 Green mud crab larval includes stages from zoea I to megalopa, with zoea I has sessile eyes;
217 zoea II has stalked eyes; zoea III has six segments on abdomen; zoea IV has the presence of
218 pleopod buds and zoea V has the presence of setae on pleopod well developed and also the
219 presence of setae. During the early stages of development in S. paramamosain, SpUbx, SpAntp,
220 and SpAbd-A exhibited similar expression patterns that were upregulated during zoea stages III
221 and V (Fig. 3C), suggesting their potential involvement in regulating zoeal development
222 process. Additionally, there was a significant decrease in the expression levels of both SpUbx
223 and SpAntp within the abdomen of zoea stage III to V individuals (Fig. 3D), while there was
224 an increase in SpAbd-A (Fig. 4A). Given that green mud crab zoea stages III to V are critical
225 periods for abdominal and maxillary development, it can be speculated that these three genes
226 primarily function on the abdomen and maxillae of zoea larvae. SpAbd-A gene expression was
227 significantly higher in the abdomen (Fig. 4A); however, at the protein level, it was higher in
228 the cephalothorax (Fig. 4B). Furthermore, there was a gradual increase followed by a decrease
229 in SpAbd-A protein expression in the abdomen of zoea I to zoea V, with a peak observed in the
230 zoea stage III (Fig. 4B). The SpAbd-A protein was primarily expressed in maxillopods,
231 pereiopods, and abdominal segments in the zoea (Fig. 4C). Interference with the expression
232 level of SpAbd-A during the zoea stage IV resulted in the absence of pleopods in larvae (Fig.
233 4D), highlighting its crucial role in initiating and developing pleopods in S. paramamosain
234 larvae. In crustaceans, Abd-A establishes segmental identity and boundaries; without Abd-A,
235 present abdominal segments may develop thoracic-like limbs (Martin et al, 2016). The loss of
236 Abd-A has also been associated with a lack of an abdomen in lineage Cirripedia (Mouchel-
237 Vielh et al, 1998). Considering that morphological changes are mainly driven by alterations in
238 gene expression patterns, it can be speculated that abdominal degeneration in crabs may be
239 linked to low levels of Abd-A during brachyurization metamorphosis.
240 Posttranscriptional regulation also fine-tunes Abd-A levels, wherein microRNAs such as
241 miR-10 target Abd-A transcripts for degradation (Miura et al, 2011). The potential target
242 miRNAs of SpAbd-A were identified through transcriptome data analysis of S. paramamosain
243 larvae and RNAhybrid: novel-miR1317 was found to be one of them (Fig. 4E). A negative
9
244 relationship between the expression level of novel-miR1317 and SpAbd-A was detected (Fig.
245 4F), verifying that novel-miR1317 negatively regulated the expression of the Abd-A gene,
246 possibly facilitating the development of pleopods. In most insect species, miR-iab-4 exerts a
247 negative regulatory effect on Abd-A and Ubx (Mouchel-Vielh et al, 1998), while miR-iab-8
248 regulates Abd-A and Abd-B (Garaulet and Lai 2015). In both Chinese mitten crab and
249 swimming crab genomes, miR-993, miR-10, miR-iab-4, and miR-iab-8 were identified within
250 the intergenic region of Hox gene clusters (Cui et al, 2021). Herein, the current study has
251 demonstrated the significant role of SpAbd-A in the development of abdominal pleopods in S.
252 paramamosain larvae. Its expression is negatively regulated by novel-miR1317 (Fig. 4G).
253 These findings provide a theoretical foundation for further understanding the mechanisms of
254 pleopod development of S. paramamosain larvae.
255 Gene regulation of ovarian development
256 Reproductive molting is crucial for the reproductive process of crustaceans. After reproductive
257 molting, female crabs mate with males, and the ovaries begin to develop rapidly (Li et al, 2022;
258 Liu et al, 2020; Long et al, 2020; Wang et al, 2020). Numerous studies have demonstrated that
259 the fruitless (fru) gene plays a role in the sex determination pathway in fly D. melanogaster
260 (Manoli et al, 2005; Cachero et al, 2010). Meanwhile, we obtained the transcript fragments of
261 fruitles2 (Spfru2) according to the gonadal transcriptome data of green mud crab (Yang et al,
262 2017). Additionally, we found that the expression levels of Spfru2 in the gonad, thoracic
263 ganglion, gill and muscle of females were significantly higher than those in males (Fig. S9A).
264 Moreover, the expression level of Spfru2 was the highest in ovarian stage III of the five
265 different developmental stages (Fig. 5A). As the ovary increased in volume during stage III,
266 the internal germ cells began to mature and were predominantly stage 3 oocytes (Wu et al,
267 2020). Additionally, Spfru2 was mainly localized in the cytoplasm of oocytes, and, in males, it
268 was detected mainly in the epithelia of seminiferous tubules and spermatids (Fig. 5B). This
269 implies that it may function in ovarian maturation in green mud crab.
270 The expression level of Spfru2 was higher at day three post mating (Fig. S9B). In crabs, after
271 the reproductive molt, females serve as the catalyst for the mating process and trigger ovarian
272 development, including oogenesis (Yang et al, 2022). Oogenesis represents a metabolically
10
273 demanding reproductive process that encompasses distinct phases. The subsequent stages of
274 oogenesis involve primary and secondary vitellogenesis (Tsukimura 2001), which entail
275 significant enlargement of oocyte diameter along with yolk protein accumulation within
276 maturing oocytes. Subsequent to ovulation and release from the ovaries, eggs undergo
277 fertilization through fusion with sperm residing within female spermathecae for varying
278 durations ranging from one to several months (Tsukimura 2001). This observation further
279 highlights the variable period required for sperm-egg maturation throughout the reproductive
280 cycle of green mud crabs. The in vitro tissue culture experiments demonstrated a significant
281 decrease in Spfru2 expression at 8 h (Fig. S9C), indicating the effectiveness of RNA
282 interference. Additionally, compared to the positive and negative controls, the treatment group
283 that underwent fru2 gene interference exhibited incomplete and fragmented egg cell
284 morphology at 8 h (Fig. 5C). These findings suggest that the fru2 gene also play a pivotal role
285 in maintaining egg cell morphology, thereby influencing female gonadal development and
286 maturation.
287 Furthermore, we found the expression of novel-miR35 and Spfru2 in different gonad
288 development stages displayed opposite expression patterns in both female and male crabs (Fig.
289 S9). Within 48 h after injection of miRNA mimic/inhibitor, the expression levels of novel-
290 miR35 and Spfru2 had opposite variant trends in the gonads (Fig. 5D). We further validated the
291 interaction between novel-miR35 and Spfru2 using in vitro green fluorescent reporter assays.
292 Both the novel-miR35 mimic and pre–novel-miR35 plasmid effectively reduced fluorescence
293 intensity when co-transfected with the WT 3′‐UTR reporter plasmid into Drosophila S2 cells,
294 but this effect was largely restored for the co-transfected plasmid containing the mutant type
295 (MT) 3′‐UTR (Fig. 5E, F). These findings suggest that Spfru2 might be a target of novel-miR35
296 in green mud crab.
297 Neo-functionalization of the elovl6 gene in the LC-PUFA synthesis pathway
298 The LC-PUFA are defined as fatty acids with ≥ 20 carbons and ≥ 3 double bonds and,
299 depending upon the position of the last double bond relative to the methyl end, can be
300 categorized into n-6 and n-3 series LC-PUFA, and are critical nutrients for animal reproduction
301 and metamorphosis (Xie et al, 2021). Among them, n-3 LC-PUFA, especially eicosapentaenoic
11
302 acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), contribute to organism health
303 via multiple pathways and play crucial roles in cell membrane lipid functions,
304 neurodevelopment, and immune responses, and have well-known beneficial effects in
305 mitigating several diseases (Xie et al, 2021). Generally, LC-PUFA can be enriched in body
306 tissues either by dietary acquisition or, in some animal species, through endogenous
307 biosynthesis (Xie et al, 2021). The fatty acid elongase, Elovl6, as a crucial enzyme in the
308 endogenous synthesis of fatty acids, participates in fatty acid metabolism, energy balance,
309 insulin resistance, and metabolic diseases (Matsuzaka et al, 2007; Shimano 2012). To
310 illuminate the neofunctionalization of the elovl6 gene and its involvement in LC-PUFA
311 biosynthesis in green mud crab, we first analyzed the molecular characteristics of the Spelovl6
312 gene. The variable promoter of the elovl6 gene in S. paramamosain resulted in the generation
313 of three transcripts (elovl6a, elovl6b, and elovl6c) through splicing (Fig. 6A). Elovl6a, Elovl6b,
314 and Elovl6c exhibited the typical structural features of Elovl protein family members (Fig. 6B).
315 During embryonic development, the expression levels of the elovl6 genes showed an overall
316 increasing trend, reaching highest levels in the prehatching period, and decreasing significantly
317 in the hatching period (Fig. S10 A, B, C). The expression levels of elovl6 genes in the larval
318 stages showed a generally stable trend, but there was a significant decline in the megalopa stage
319 (Fig. S10 D, E, F).
320 Furthermore, S. paramamosain Elovl6a, Elovl6b, and Elovl6c were functionally
321 characterized through a yeast heterologous expression system (Table S5). Upon addition of
322 16:0 fatty acid substrate, the Elovl6-transformed yeast exhibited the production of elongation
323 products consisting of saturated fatty acids (SFA) ranging from C18 to C22. Similarly, incubation
324 with 18:1n-9 resulted in the generation of several elongation products of monounsaturated fatty
325 acids (MUFA). Endogenous 18:1n-9 was also elongated to form 20:1n-9 and subsequently
326 further elongated to produce 22:1n-9. Regarding PUFA substrates, all C18 PUFA substrates
327 tested were significantly converted to their respective C20 products, with elongation observed
328 for 18:3n-3, 18:4n-3, 18:2n-6, and 18:3n-6 to their corresponding C20 PUFA. Notably, S.
329 paramamosain Elovl6 exhibited a higher affinity toward n-3 PUFA than n-6 PUFA when
330 utilizing C18 PUFA substrates. However, neither of the C20 LC-PUFA, arachidonic acid (ARA;
12
331 20:4n-6) or eicosapentaenoic acid (EPA; 20:5n-3), underwent elongation and so 22:4n-6 and
332 22:5n-3, respectively, were not generated. Additionally, a significant decrease in the expression
333 level of the elovl6 gene was observed following 18 h and 36 h interference in isolated
334 hepatopancreas of green mud crab (Fig. S10G), resulting in a notable reduction in 16:0, 16:1n-
335 7, 18:1n-9, 18:2n-6, 18:3n-3, 20:5n-3, and 22:6n-3. This further validated the capability of
336 Elovl6 for elongation of C18 PUFA to C20 PUFA (Fig. S10H). Thus, the capacity of Elovl6 of
337 S. paramamosain for elongation of C18 PUFA, in addition to SFA and MUFA, was confirmed.
338 In contrast, Elovl6 proteins in mammals (Matsuzaka et al, 2007; Junjvlieke et al, 2019) and
339 teleosts (Li et al, 2020; Wang et al, 2020) are only involved in the elongation of SFA and
340 MUFA as they have only been demonstrated to elongate 16:0 and 16:1 to 18:0 and 18:1,
341 respectively, with other potential activities of elovl6 gene products not yet reported.
342 The aforementioned results indicate that Elovl6 may undergo neofunctionalization in the
343 Decapoda species S. paramamosain. Decapod crabs inhabit a diverse range of environments,
344 including seawater (e.g., P. trituberculatus), freshwater (e.g., E. sinensis), and brackish water
345 (e.g., S. paramamosain). It was reported that the multifunctionalization of existing genes and
346 neo-functionalization of duplicated genes have occurred in catadromous and freshwater teleost
347 species such as flatfish family Achiridae (Matsushita et al, 2020) and freshwater three-spined
348 stickleback (Ishikawa et al, 2021). To investigate the influence of the environment on the
349 evolutionary adaptation of key enzymes of LC-PUFA synthesis in Decapoda crab species, three
350 transcripts of the E. sinensis and P. trituberculatus elovl6 genes were functionally characterized.
351 The findings demonstrated that the Ptelovl6 and Eselovl6 genes exhibited comparable fatty
352 acid elongation activities to that of Spelovl6 (Fig. 6C), implying the potential
353 neofunctionalization of Elovl6 in decapod crabs. The phylogenetic analysis of elovl6 in E.
354 sinensis, P. trituberculatus, and S. paramamosain showed the evolutionary difference between
355 elovl6 and elovl2/4/5/8 (Fig. 6D), which indicated that neo-functionalization of the elovl6 gene
356 in the LC-PUFA synthesis pathway in crustaceans occurred.
357
358 Discussion
359 Crabs represent a pivotal model system for exploring the molecular underpinnings of adaptive
13
360 evolution within Decapoda. Here, we present a comprehensive analysis of the green mud crab
361 S. paramamosain genome using Nanopore ultralong-read sequencing, achieving remarkable
362 contig N50 and scaffold N50 values that substantially surpass previous assemblies (Zhang et
363 al, 2019; Song et al, 2016; Tang et al, 2021; Tang et al, 2020; Zhao et al, 2021; Cui et al, 2021;
364 Chebbi et al, 2019). By anchoring contigs to 49 pseudochromosomes, we attained a more
365 precise depiction of the genome architecture, with Chr06 revealing a prominent sex-linked
366 signal, offering foundational insights into sex determination mechanisms in decapod
367 crustaceans. Furthermore, the detection of potential ancient whole-genome duplication events
368 suggests a dynamic evolutionary history underlying the genomic organization of S.
369 paramamosain.
370 The highly repetitive nature of crustacean genomes presents significant challenges in
371 maintaining genome integrity (Zhang et al, 2019; Polinski et al, 2021). Our investigation into
372 genome assembly emphasized the impact of ultralong-read sequencing in resolving repetitive
373 sequences, culminating in the identification of 58.97% of the genome as repetitive. Repetitive
374 sequences and TEs significantly contribute to genome expansion and evolution in S.
375 paramamosain, with a higher proportion observed compared to several other crustacean
376 species (Zhang et al, 2019; Song et al, 2016; Tang et al, 2021; Tang et al, 2020; Cui et al, 2021;
377 Chebbi et al, 2019). The identification of numerous SSRs and TRs underscores the importance
378 of these elements in shaping the genomic landscape of S. paramamosain. Analysis of SSRs,
379 TRs, and TEs provided insights into genome expansion and evolution. S. paramamosain
380 exhibits 33,662 protein-coding genes, highlighting gene richness compared to other
381 crustaceans (Zhang et al, 2019; Song et al, 2016; Tang et al, 2021; Tang et al, 2020; Zhao et al,
382 2021; Cui et al, 2021; Chebbi et al, 2019). The annotation of these genes to various databases
383 enhances our understanding of green mud crab genomics. Gene family analysis revealed
384 species-specific genes enriched in pathways related to development, indicating potential
385 adaptations. Phylogenetic analysis positioned green mud crabs among crustaceans,
386 highlighting their evolutionary divergence.
387 The investigation of Hox genes in S. paramamosain elucidated their role in directing tissue
388 differentiation and morphological development during larval stages, particularly in the
14
389 development of pleopods. The expression patterns of Hox genes during different larval stages
390 suggest their involvement in the regulation of key developmental processes, with potential
391 implications for understanding the evolution of crustacean body plans. We investigated the
392 regulatory mechanisms of Hox genes in pleopod development, demonstrating the crucial role
393 of Abd-A in abdominal limb development. Posttranscriptional regulation involving novel-
394 miR1317 fine-tunes Abd-A levels, unveiling a nuanced layer of gene regulation.
395 Additionally, our exploration of the fru gene in ovarian development underscored its
396 potential role in female gonadal development and maturation. The identification of novel-
397 miR35 as a potential regulator of Spfru2 adds complexity to the gene regulatory network
398 governing reproductive processes. The divergent expression patterns of fru in male and female
399 crabs, alongside its localization in gonadal tissues, highlight its potential involvement in
400 ovarian maturation and gonadal development. Furthermore, the identification of novel
401 microRNA targeting fru underscores the intricate posttranscriptional regulatory mechanisms
402 governing reproductive processes in crustaceans.
403 The neofunctionalization of the elovl6 gene in the long-chain polyunsaturated fatty acid (LC-
404 PUFA) synthesis pathway represents a key adaptation in S. paramamosain. Here, we elucidate
405 the neo-functionalization of the elovl6 gene in the synthesis of LC-PUFA, shedding light on its
406 multiple transcripts and tissue-specific expression. Comparative analysis with S.
407 paramamosain, P. trituberculatus, and E. sinensis suggests evolutionary divergence in LC-
408 PUFA biosynthesis pathways, potentially driven by environmental adaptation.
409 Overall, our findings shed light on various aspects of crab biology, including genome
410 sequencing, assembly, and annotation, as well as gene family expansion, contraction, and
411 regulatory mechanisms governing crucial developmental processes such as metamorphosis,
412 reproductive strategies, and fatty acid metabolism. The present findings significantly advance
413 our understanding of the green mud crab genome, providing a valuable resource for future
414 studies in comparative genomics, evolutionary biology, and functional genomics in crustaceans.
415 Future studies leveraging this genomic resource will further elucidate the genetic basis of key
416 adaptive traits and inform conservation and management strategies for economically important
417 crab species.
15
418
419 Methods and materials

420 Sample preparation and genomic DNA isolation
421 Genomic DNA for sequencing was extracted from the testis of a wild adult male green mud
422 crab S. paramamosain with stage III testis, which was caught off the coast of Shantou, China.
423 A Grandomics Genomic BAC-long DNA Kit was used to isolate ONT ultralong DNA
424 according to the manufacturer's guidelines. The total DNA quantity and quality were evaluated
425 using a NanoDrop One UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA)
426 and a Qubit 3.0 Fluorometer (Invitrogen Life Technologies, Carlsbad, CA). Large DNA
427 fragments were obtained through gel cutting with the Blue Pippin system (Sage Science,
428 Beverly, MA). Qualification of DNA involved visual inspection for foreign matter, assessment
429 of degradation and size via 0.75 % agarose gel electrophoresis, check on purity (OD260/280
430 between 1.8-2.0; OD260/230 between 2.0-2.2) using Nanodrop, and precise quantification with
431 Qubit 3.0 Fluorometer (Invitrogen, USA).
432 Library construction and sequencing
433 Approximately 8-10 µg of genomic DNA (> 50 kb) was selected using the SageHLS HMW
434 library system (Sage Science, Beverley MA, USA) and processed with the Ligation Sequencing
435 1D Kit (Oxford Nanopore Technologies, Shanghai, China) following the manufacturer's
436 instructions. Library construction and sequencing were conducted on the PromethION (Oxford
437 Nanopore Technologies) at the Genome Center of Grandomics (Wuhan, China). After quality
438 inspection, large DNA fragments were recovered using the BluePippin automatic nucleic acid
439 recovery instrument. Terminal repair, A-tailing, and ligation were performed using the LSK109
440 connection kit. Qubit was employed to assess the constructed DNA library precisely. The
441 library was loaded into a flow cell, transferred to the PromethION sequencer, and subjected to
442 real-time single-molecule sequencing. In the ONT sequencing platform, base calling, the
443 conversion of nanopore-generated signals to base sequences (Wick et al, 2019), was executed
444 using the Guppy toolkit (Oxford Nanopore Technologies). Pass reads with a mean
445 qscore_template value greater than or equal to 7 were obtained and directly used for subsequent
446 assembly (https://github.com/nanoporetech/taiyaki).
16
447 Genome assembly, evaluation and correction

448 Filtered reads post quality control were employed for a pure three-generation assembly using
449 NextDenovo software (reads_cutoff:1k, seed_cutoff:28k)
450 (https://github.com/Nextomics/NextDenovo.git). The NextCorrect module was employed to
451 correct the original data, yielding a consistency sequence (CNS sequence) after 13 Gb of error
452 correction. De novo assembly using the NextGraph module produced a preliminary assembly
453 of the genome. ONT three-generation data and Pb HiFi three-generation data were utilized with
454 Nextpolish software (https://github.com/Nextomics/NextPolish.git) for genome correction.
455 The corrected genome (Polish Genome) was obtained after three rounds of correction for both
456 ONT and Pb HiFi data. Bwa mem default parameters were used to compare next-generation
457 data to the genome, and Pilon was iteratively calibrated three times to derive the final genomic
458 sequence. GC depth analysis and BUSCO prediction (https://busco.ezlab.org/) were used to
459 assess genome quality and completeness.
460 Chromosome anchoring by Hi-C sequencing
461 To anchor hybrid scaffolds onto chromosomes, genomic DNA for Hi-C library construction
462 was extracted from green mud crab testes. Cell samples from the tissue used for genomic DNA
463 sequencing were employed for Hi-C library preparation. The process involved cross-linking
464 cells with formaldehyde, lysing cells, resuspending nuclei, and subsequent steps leading to
465 proximity ligation. After overnight ligation, cross-linking was reversed, and chromatin DNA
466 manipulations were performed. DNA purification and shearing to 400 bp lengths were followed
467 by Hi-C library preparation using the NEBNext Ultra II DNA library Prep Kit for Illumina.
468 Sequencing on the Illumina NovaSeq/MGI-2000 platform completed the Hi-C procedure.
469 Briefly, cell samples were fixed with formaldehyde and subjected to lysis and extraction for
470 sample quality assessment. After passing the quality test, the Hi-C fragment preparation
471 process involved chromatin digestion, biotin labeling, end ligation, DNA purification, and
472 library construction. The library was sequenced on the MGI-2000 platform, and data were
473 processed to extract high-quality reads. The analysis included filtering for adapters, removing
474 low-quality reads, and eliminating reads with an N content exceeding 5. Reads were aligned
475 using Bowtie2 (Langmead and Salzberg 2012), and contig clustering was performed using
17
476 LACHESIS software (Joshua and Burton 2013).

477 Gene annotation
478 Tandem repeats were annotated using GMATA
479 (https://sourceforge.net/projects/gmata/?source=navbar) and Tandem Repeats Finder (TRF)
480 (http://tandem.bu.edu/trf/trf.html), identifying simple repeat sequences (SSRs) and all tandem
481 repeat elements. Transposable elements (TEs) were identified through an ab initio and
482 homology-based approach, with RepeatMasker (https://github.com/rmhubley/RepeatMasker)
483 used for searching known and novel TEs. Gene prediction employed three methods: GeMoMa
484 (http://www.jstacs.de/index.php/GeMoMa) for homolog prediction, PASA
485 (https://github.com/PASApipeline/PASApipeline) for RNAseq-based prediction, and Augustus
486 (https://github.com/Gaius-Augustus/Augustus) for de novo prediction. EVidenceModeler
487 (EVM) (http://evidencemodeler.github.io/) integrated gene sets, which underwent further
488 filtering for transposons and erroneous genes. UTRs and alternative splicing regions were
489 determined using PASA based on RNA-seq assemblies. Functional annotation involved
490 comparisons with public databases, including SwissProt, NR, KEGG, KOG, and Gene
491 Ontology. InterProScan identified putative domains and GO terms. BLASTp
492 (https://blast.ncbi.nlm.nih.gov/Blast.cgi) against public protein databases was used to assess
493 gene function information. The noncoding RNA (ncRNA) prediction entailed using tRNAscan-
494 SE (http://lowelab.ucsc.edu/tRNAscan-SE/) and Infernal cmscan (http://eddylab.org/infernal/)
495 for tRNAs and other noncoding RNAs. BUSCO was employed for gene prediction evaluation,
496 aligning annotated protein sequences to evolution-specific BUSCO databases.
497 Evolutionary analysis
498 The evolutionary analysis entailed the examination of gene families, construction of
499 phylogenetic trees, estimation of divergence times, exploration of gene expansion and
500 contraction phenomena, identification of orthologous genes, scrutiny of positively selected
501 genes, and investigation of whole-genome duplications. To ascertain homologous relationships
502 between S. paramamosain and other animal species, protein sequences were acquired and
503 aligned using OrthMCL (https://orthomcl.org/orthomcl/). Initially, protein sets were gathered
504 from 14 sequenced animal species, and the longest transcripts for each gene were selected,
18
505 excluding miscoded and prematurely terminated genes. Subsequently, pairwise alignment of
506 these extracted protein sequences was conducted to identify conserved orthologs, employing
507 Blastp with an E-value threshold of ≤ 1 × 10-5. Further identification of orthologous
508 intergenome gene pairs, paralogous intragenome gene pairs, and single-copy gene pairs was
509 achieved using OrthMCL. Species-specific genes, encompassing S. paramamosain-specific
510 unique genes and unclustered genes, were extracted. Functional annotation and enrichment
511 tests of species-specific genes were performed utilizing information from homologs in the
512 online Gene Ontology (http://www.geneontology.org/) and KEGG (Kyoto Encyclopedia of
513 Genes and Genomes) (https://www.genome.jp/kegg/) databases.
514 Building upon the orthologous gene sets identified with OrthMCL, molecular phylogenetic
515 analysis was executed using shared single-copy genes. Coding sequences were extracted from
516 single-copy families, followed by multiple alignment of each ortholog group using MAFFT
517 (https://mafft.cbrc.jp/alignment/software/). Gblocks were applied to eliminate poorly aligned
518 sequences, and the GTRGAMMA substitution model of RAxML (https://cme.h-
519 its.org/exelixis/web/software/raxml/hands_on.html) was employed for phylogenetic tree
520 construction with 1000 bootstrap replicates. The resulting tree file was visualized using
521 Figtree/MEGA (http://tree.bio.ed.ac.uk/software/figtree/). The RelTime tool
522 (https://www.megasoftware.net/) of MEGA-CC was then utilized to compute mean
523 substitution rates along each branch and estimate species divergence times, with three fossil
524 calibration times obtained from the TimeTree (http://www.timetree.org/) database serving as
525 temporal controls, including the divergence times of S. paramamosain.
526 The detection of significant expansions or contractions in specific gene families, often
527 indicative of adaptive divergence in closely related species, was carried out based on
528 OrthoMCL results. CAFE (https://github.com/hahnlab/CAFE), employing a birth and death
529 process to model gene gain and loss over a phylogeny, was used for this purpose. Furthermore,
530 in accordance with the neutral theory of molecular evolution, the ratio of the nonsynonymous
531 substitution rate (Ka) to the synonymous substitution rate (Ks) of protein-coding genes was
532 calculated. The average Ka/Ks values were determined, and a branch-site likelihood ratio test
533 using Codeml (http://abacus.gene.ucl.ac.uk/software/) from the PAML package was conducted
19
534 to identify positively selected genes within the S. paramamosain lineage. Genes with a p value
535 < 0.05 under the branch-site model were considered positively selected.
536 Whole-genome duplication events in the green mud crab genome were investigated using
537 four-fold synonymous third-codon transversion (4DTv) and synonymous substitution rate (Ks)
538 estimation. Initial steps involved extracting protein sequences and conducting all-vs.-all
539 paralog analysis through self-Blastp in these plants. After filtering by identity and coverage,
540 the Blastp results underwent analysis with MCScanX (Wang et al, 2012), and the respective
541 collinear blocks were identified. Subsequently, Ks and 4DTV were calculated for the syntenic
542 block gene pairs using KaKs-Calculator (https://sourceforge.net/projects/kakscalculator2/),
543 and potential WGD events in each genome were evaluated based on their Ks and 4DTv
544 distribution.
545 RNA-seq sample collection and sequencing
546 A total of 1 μg of high-quality RNA per experimental group (including different developmental
547 stages, different tissues, and gonads from different mating periods of S. paramamosain) was
548 allocated for RNA sample preparations. Sequencing libraries were constructed using the
549 NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA), following the
550 manufacturer's guidelines. Subsequently, each sample was labeled with index codes for
551 sequence characterization. In brief, mRNA was isolated from total RNA using poly T oligomer
552 magnetic beads. NEBNext First-Strand Synthesis Reaction Buffer (5X) was employed,
553 utilizing bivalent cations for high-temperature cleavage. First-strand cDNA was synthesized
554 with random hexamer primers and M-MuLV Reverse Transcriptase. Subsequently, the second
555 strand of cDNA was synthesized using DNA Polymerase I and RNase H, and any remaining
556 overhangs were converted to blunt ends by exonuclease/polymerase. Following adenylation of
557 the 3' end of DNA fragments, the NEBNext adaptor with a hairpin loop structure was ligated
558 for hybridization. Library fragments, preferentially selecting 240 bp cDNA fragments, were
559 purified using the AMPure XP system (Beckman Coulter, Beverly, USA). The USER enzyme
560 (NEB, USA) was introduced to the cDNA of the selected size and adaptor, and PCR was
561 conducted with Phusion High-fidelity DNA polymerase after 15 min at 37 ℃ and 5 min at
562 95 ℃. Universal PCR primers and index (X) primers were utilized for PCR. Finally, the PCR
20
563 products were purified using the AMPure XP system, and the library quality was assessed using
564 the Agilent Bioanalyzer 2100 system. Following the manufacturer's recommendations, the
565 TruSeq PE Cluster Kit v4-cBot -HS (Illumina) was employed to cluster the index-coded
566 samples on the cBot Cluster Generation System. After clustering, library preparations were
567 sequenced on the Illumina platform to generate paired-end reads.
568 Microinjection and RNA interference
569 Primer design for the amplification of the Abd-A, fru2 and elovl6 gene fragments and
570 subsequent dsRNA synthesis was based on the gene sequences. Specific primers were designed
571 incorporating protective bases and T7 promoter sequences at the 5' end of both the positive and
572 negative primers to facilitate dsRNA synthesis. The plasmid containing the Abd-A and elovl6
573 gene sequence fragments served as the template for PCR amplification, and the resulting
574 product underwent purification. The PCR product, now equipped with the T7 promoter, was
575 utilized as the template for the synthesis of dsRNA, a crucial step for gene interference through
576 in vitro transcription. Subsequently, zoeal stage IV of the green mud crab were positioned on a
577 custom-made agarose gel plate. Injection of dsRNA and the injection indicator mixture was
578 carried out at the cuticle and abdominal space of the larvae using a microinjector. As the larvae
579 progressed to the zoea V stage, the development of the abdomen and limbs in the juveniles was
580 meticulously observed through electron microscopy.
581 Hepatopancreas treatment in vitro
582 Crabs were anesthetized on ice for 10 min, followed by sterilization in 75 % ethanol for 5 min.
583 Hepatopancreas tissues were subsequently dissected and first infiltrated with phosphate
584 buffered saline (PBS) containing 1 % penicillin and streptomycin for 30 min, then washed 8
585 times for 5 min each time using PBS solution as above. Next, hepatopancreas tissues were
586 chopped by scissors into fragments of approximately 20 mg. The fragments were then
587 precultured at room temperature (25 ℃) in a 24-well-plate with 150 µL of Leibovitz L15
588 medium (containing 1% penicillin and streptomycin). The 24-well culture plates were
589 supplemented with dsRNA-Elovl6 and dsRNA-EGFP (control group) and placed in a 28 ℃
590 incubator for cultivation. After 18 h, 24 h, and 36 h of culture, tissue fragments were collected
591 from each treatment for total RNA extraction and subsequent qRT-PCR analysis, with
21
592 corresponding parallel samples of hepatopancreas tissue also collected for fatty acid analysis.
593 Sequence and phylogenetic analysis
594 To provide important clues for predicting functions or evolution of the elovl6 genes, multiple
595 sequence alignments were performed with the DNAMAN software (v6.0.3.99, Lynnon
596 Corporation, USA). The phylogenetic tree mapped using the neighbor-Joining method with
597 MEGA software (v11.0.13, Arizona State University, USA), on the basis of deduced amino
598 acid sequences of Elovls from S. paramamosain and representative mammals, teleosts, and all
599 functionally characterized or uncharacterized Elovl from crustacean species. Confidence in the
600 resulting phylogenetic tree branch topology was measured by bootstrapping through 1000
601 replications.
602 Quantitative real time PCR (qRT-PCR)
603 Using the miRcute miRNA Isolation Kit, miRNAs were isolated from distinct developmental
604 stages and tissues of S. paramamosain. Subsequently, the commercially accessible miRcute
605 Enhanced miRNA cDNA First Strand Synthesis Kit was deployed for the reverse transcription
606 process. Finally, the miRcute Enhanced miRNA Fluorescence Quantitative Detection Kit was
607 applied in the qPCR analysis to elucidate the expression dynamics of both miRNAs and the
608 genes. The total RNA extraction was extracted using the RNAiso Plus kit (Takara Co. Ltd.,
609 Japan). Preceding the quantitative real-time polymerase chain reaction (qRT-PCR), the RNA
610 samples underwent treatment with RQ1 RNase-Free DNase (Takara Co. Ltd.) to eradicate
611 genomic DNA contamination. Subsequently, cDNA synthesis was conducted using 500 ng of
612 DNase-treated RNA and the Talent qPCR Premix (SYBR Green) kit (TIANGEN Biotech Co.,
613 Ltd., Beijing), in accordance with the manufacturer's introductions. The qPCR primers were
614 meticulously designed using Primer 6.0 software (Table S6). The qRT-PCR reactions were
615 performed employing a Mini Option real-time detector (Roche LightCycle@480). Each
616 reaction mixture comprised 10 µL of Talent qPCR Premix (2×), 0.6 µL of PCR forward primer
617 (10 µM), 0.6 µL of PCR reverse primer (10 µM), 2.0 µL of RT reaction solution containing
618 cDNA at an amount of 20 ng, and 6.8 µL of RNase-free water. The amplification protocol
619 involved an initial denaturation step at 95 °C for 3 min, followed by 40 cycles of denaturation
620 at 95 °C for 5 s, annealing at 60 °C for 10 s, and extension at 72 °C for 15 s. A subsequent
22
621 melting curve analysis ensued by sustaining the reaction at 72 °C for 6 s, followed by a concise
622 denaturation step at 95 °C for 5 s. For verification of correct amplicon sizes, all products were
623 initially resolved through agarose gel electrophoresis. Normalization of gene expression levels
624 to the reference gene (18s rRNA) was conducted. The gene expression levels were calculated
625 using the optimized comparative Ct method (2-ΔΔCt).
626 Protein expression detection using Western blotting
627 Protein extraction from cephalothorax and abdomen samples of green mud crab larvae was
628 performed using a cryogenic protein lysis solution following flash freezing with liquid nitrogen.
629 Subsequently, total protein was extracted from each sample. After quantifying the protein
630 concentration using the BCA protein assay kit (Sangon Biotech Co. Ltd., Shanghai, China), an
631 equal number of proteins were loaded for SDS-PAGE electrophoresis. Following separation
632 based on molecular weight, the proteins were transferred onto a PVDF membrane from the
633 polyacrylamide gel and subsequently probed with specific antibodies. Subsequent steps
634 included incubation with HRP-conjugated secondary antibodies and detection using an ECL
635 hypersensitive chemiluminescence kit (Sangon Biotech).
636 Fluorescence in situ hybridization (FISH)
637 The FISH analysis was performed on S. paramamosain gonads, which were fixed in 4 %
638 paraformaldehyde prepared with DEPC-treated water two hours prior to sectioning at a
639 thickness of 10-12 µm using a cryostat set at -20 to -25 °C. Subsequently, no more than three
640 sections per slide were thaw-mounted onto charged Superfrost Plus slides. The fru2 probe,
641 modified with FITC fluorescence at the 3′ end, was synthesized commercially (Sangon
642 Biotech). The prehybridization and hybridization procedures followed the method described
643 by Pinaud et al (2008). Initially, a prehybridization solution diluted with high-grade formamide
644 in a 1:1 ratio was added to each slide and coverslip section (50 µL per sample). Then, the slides
645 were placed in a humid chamber for 30 min at room temperature before removing the coverslips
646 in a 2× SSC solution. After prehybridization, the probe was added to the hybridization buffer
647 at a concentration of 1 ng/µL. Twenty-five mL hybridization mix was applied to each slide and
648 coverslip section before placing them in a metal slide holder immersed in a mineral oil bath
649 maintained at 65 °C for 14-16 h. Following hybridization, the slide holder was removed from
23
650 the mineral oil bath and slides were rinsed twice (30 s each) in chloroform followed by
651 immersion in a fresh batch of 2× SSC solution without coverslips for washing at room
652 temperature for 1 h; subsequently washed again in an SSC solution containing 50 % formamide
653 at 65 °C for 1 h; finally washed twice (each time lasting 30 min) in ten-fold diluted (0.1×) SSC
654 solution while maintaining the temperature at 65 °C throughout these washes. The signals
655 resulting from hybridization on each slide were ultimately detected using fluorescence
656 microscopy.
657 Yeast expression and fatty acid detection
658 PCR amplification of open reading frames (ORFs) corresponding to Eselovl6, Ptelovl6, and
659 Spelovl6 was performed using a high-fidelity DNA polymerase, KOD-Plus-Neo (Toyobo,
660 Japan), and cDNA templates. Specific primers that incorporated BamHI (GGATCC) and EcoRI
661 (GAATTC) restriction sites were employed in accordance with the manufacturer's instructions.
662 The primers designed for ORF cloning, with their respective restriction sites, are detailed in
663 Table S6. For Eselovl6 and Ptelovl6, the amplification process involved an initial denaturation
664 step at 96 °C for 3 min, followed by 23 cycles of denaturation at 95 °C for 30 s, annealing at
665 58 °C for 15 s, and extension at 72 °C for 20 s, culminating in a final extension at 72 °C for 1
666 min. The PCR conditions for Spelovl6 included an initial denaturing step at 94 °C for 2 min,
667 followed by 35 cycles of denaturation at 98 °C for 30 s, annealing at 94 °C for 30 s, extension
668 at 68 °C for 30 s, and a final extension at 72 °C for 7 min.
669 Subsequently, the resulting DNA fragments were purified using the TIAN quick mini
670 purification kit (Tiangen Biotech). After purification, the fragments were digested with the
671 corresponding BamHI and EcoRI restriction endonucleases (Thermo Scientific, USA) and
672 ligated into a similarly restricted pYES2 yeast expression vector (Invitrogen, USA) using the
673 DNA Ligation Kit Mighty Mix (Takara, Japan). The resulting plasmid constructs, namely,
674 pYEselovl6, pYPtelovl6, and pYSpelovl6, were then introduced into E. coli DH5α competent
675 cells (Takara, Japan), which were subsequently screened for the presence of recombinants via
676 PCR.
677 The transformation and selection of yeast with recombinant plasmids, as well as yeast culture,
678 followed modified protocols based on a previous study (Monroig et al, 2012). In summary,
24
679 purified plasmids, inserted with either one of the elovl6 ORFs or no insert, were used to
680 transform yeast (Saccharomyces cerevisiae strain INVSc1) competent cells through the
681 polyethylene glycol/lithium acetate (PEG/LiAc) method. The transformed yeast containing the
682 corresponding recombinants were selected on S. cerevisiae minimal solid media plates without
683 uracil (SC-Ura, containing 2 % glucose and 2 % yeast agar) and cultured at 30 °C for 3 d.
684 Single colonies were then picked and transferred into liquid SC-Ura medium with 2 % glucose,
685 followed by further growth in a shaking incubator at 30 °C under 250 rpm for 2 d. Yeast
686 genomic DNA was extracted from the bacterial solution using a yeast genomic DNA rapid
687 extraction kit (Solarbio, Beijing, China), and the presence of the resulting plasmid constructs
688 in S. cerevisiae was identified and confirmed by PCR screening.
689 For heterologous expression, successfully transformed yeast was initially cultured for 24 h
690 in SC-Ura broth containing 2 % raffinose at 30 °C and 250 rpm. Cultured S. cerevisiae cells
691 were then diluted to an OD600 of 0.4 in SC-Ura broth and allowed to grow until the OD600
692 reached 1. The suspensions were centrifuged to obtain precipitated yeast, which was then
693 resuspended in SC-Ura broth (containing 2 % galactose) at an OD600 of 0.4. Next, a mixture
694 of the suspensions, 10 % tergitol-type (NP-40, Beyotime Biotechnology, Shanghai, China), and
695 corresponding fatty acid substrates substrates (including 16:0, 18:1n-9, 18:2n-6, 18:3n-3,
696 18:3n-6, 18:4n-3, 20:4n-6, and 20:5n-3) at final concentrations of 0.50 mM for C16, 0.50 mM
697 for C18, and 0.75 mM for C20 were added to the centrifuge tubes. The tubes were placed into a
698 shaker and incubated at 30 °C for 2 d at 250 rpm. Subsequently, the yeast was collected via
699 centrifugation, washed with Hanks’s balanced salt solution (Solarbio), and processed for fatty
700 acid analysis. This experiment was replicated using three separate recombinant colonies for
701 each recombinant yeast strain.
702 Fatty acid analysis by GC-MS
703 The yeast obtained above underwent a 48-h freeze-drying process and was subsequently
704 ground into powder. Approximately 200 mg of yeast powder was then thawed at 4 °C and
705 placed in a 12-mL screw-top glass tube with a Teflon-sealed lid and 0.25 mg/mL methylation
706 solution, which was composed of 99 mL methanol, 1 mL sulfuric acid, and 0.025 g butylated
707 hydroxy toluene as antioxidant, was added to the tube. After vortexing for 2 min and ultrasonic
25
708 disruption for 30 min, the samples were incubated in a water bath at 80 °C for 4 h. After cooling,
709 1 mL of n-hexane was added to the mixture and shaken vigorously for 1 min, and 1 mL of
710 ultrapure water then added to facilitate layer separation. The resulting supernatant was then
711 filtered through a nylon syringe filter with a 0.22 µm ultrafiltration membrane (SCAA-104,
712 ANPEL, China) and collected into a clean ampoule. The fatty acid methyl ester (FAME)
713 solution was concentrated under a stream of nitrogen gas in a Termovap sample concentrator
714 and the FAME resuspended in 600 µL of n-hexane and stored at -20 °C until analysis. The
715 FAME samples were separated and analyzed by gas chromatography‒mass spectrometry (GC‒
716 MS) using an Agilent 7890B-5977A GC-MS (Agilent Technologies, Santa Clara, CA, USA)
717 equipped with a fused-silica ultra inert capillary column (DB-WAX, 30 m × 250 µm internal
718 diameter, film thickness 0.25 µm; Agilent J & W Scientific, CA, USA). The oven temperature
719 was initially increased from 100 ℃ to 200 ℃ at a rate of 10 ℃/min, with a hold time of 5 min
720 at 200 ℃. Subsequently, the temperature was increased to 230 ℃ at 2 ℃/min with a hold time
721 of 10 min at 230 ℃, followed by a final ramp from 230 to 240 at 10 ℃/min. The injection,
722 interface, and ion source temperatures were adjusted to 250, 240, and 230 °C, respectively.
723 High-purity helium (99.999 %) served as the carrier gas with a constant flow rate of 1 mL/min.
724 A 0.5 µL sample was injected at a 1:20 split ratio by an autosampler. The collision energy was
725 set at 70 eV, and mass spectra data were acquired in full scan mode (scanning range 40–500
726 m/z). Fatty acids were identified through mass spectrometry, referencing a commercially
727 available standard library (National Institute of Standards and Technology Mass Spectral
728 Library 2011) and the relative retention times of standards. The elongation conversion
729 efficiencies of the respective added fatty acid substrates were determined by calculating the
730 proportion of exogenously added fatty acids (FAs) to the elongated FA products, given by the
731 following equation.
𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎
732 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 (%) = × 100
𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 + 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎
733 Data availability
734 The whole genome sequences are deposited at DDBJ/ENA/GenBank under the accession
735 JAYKKS000000000. The version described in this paper is version JAYKKS010000000. Raw
736 sequencing data are deposited in NCBI under BioProject PRJNA1059155.
26
737
738 Acknowledgements
739 This study was supported by the National Key Research & Development Program of China
740 (2018YFD0900201), the National Natural Science Foundation of China (42076133, 42306126,
741 42306125), the National Plan for the Special Support for Top-notch Talents
742 (ZUTINGZI201548), the Leading Talent Project of Special Support Plan of Guangdong
743 Province (2019TX05N067), the Guangdong Natural Science Foundation (2022A1515110488,
744 2022A1515111151), and the STU Scientific Research Foundation for Talents (NTF21016,
745 NTF21020). We express our gratitude to Dr. Noah Esmaeili for his valuable input in enhancing
746 the linguistic quality of the manuscript.
747
748 Author contributions

749 Hongyu Ma provided the funding of the research and conceptualized the whole experiment.
750 Yin Zhang, Ye Yuan, Mengqian Zhang, Xiaoyan Yu, Bixun Qiu and Fangchun Wu collected
751 the specimens. Yin Zhang, Ye Yuan, Xiaoyan Yu, Bixun Qiu, Fangchun Wu, Jiajia Zhang and
752 Mengqian Zhang performed the experiment. Yin Zhang, Ye Yuan and Mengqian Zhang
753 analyzed the data and prepared the required figures. Yin Zhang, Ye Yuan and Mengqian Zhang
754 wrote the manuscript. Shaopan Ye and Yin Zhang upload the genome data. Hongyu Ma,
755 Shaopan Ye, Wenxiao Cui, Jonathan Y. S. Leung, Mhd Ikhwanuddin, Tariq Dildar, Waqas
756 Waqas and Douglas R. Tocher revised the manuscript and contributed to additional discussion.
757 All authors read and approved the final manuscript.
758
759 Additional information

760 Supplementary information is available for this paper.
761 Correspondence and requests for materials should be addressed to Hongyu Ma.
762
763 Competing interests

764 The authors declare that they have no competing interests.
765
27
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975
35
976 Table 1. Summary of Scylla paramamosain genome assembly

Gap
Contig Length Contig Scaffold Length Scaffold Gap
Stat Type Length
(bp) Number (bp) Number Number
(bp)
N50 11,448,802 37 23,607,973 20 100 113
N60 9,208,292 49 21,816,234 25 100 135
N70 6,865,899 64 19,336,055 31 100 158
N80 3,900,000 87 15,570,585 38 100 180
N90 1,000,000 151 9,316,856 47 100 203
Longest 30,957,417 1 42,181,690 1 100 225
Total 1,210,737,920 458 1,210,760,420 233 22,500 225
Length>=1kb 1,210,737,920 458 1,210,760,420 233 0 0
Length>=2kb 1,210,737,920 458 1,210,760,420 233 0 0
Length>=5kb 1,210,737,920 458 1,210,760,420 233 0 0
977
36
978 Figures legends

979 Fig. 1. Schematic representation of the genomic characteristics of mud crab Scylla
980 paramamosain
981 (A) Appearance of S. paramamosain. (B) Schematic representation of the genomic
982 characteristics of S. paramamosain. From the outer to inner, Track 1: The 49 chromosomes of
983 S. paramamosain; Track 2: Scaffolds anchored to each chromosome; Track 3: Protein-coding
984 genes; Track 4: Distribution of gene density across the genome; Track 5: Distribution of GC
985 content in the genome; Track 6: Distribution of 5 significantly expanded gene families in the
986 genome; Track 7: Distribution of SSRs in the genome; Track 8: Distribution of transposable
987 elements in the genome; Track 9: Schematic presentation of major interchromosomal
988 relationships in the mud crab. (C) Sex linked region in the chromosomes of S. paramamosain.
989 (D) Whole genome duplicates event in 12 species including S. paramamosain, Portunus
990 trituberculatus, Eriocheir sinensis, Penaeus vannamei, Homarus americanus, Penaeus
991 monodon, Drosophila melanogaster, Bombyx mori, Eisenia andrei, Stegodyphus mimosarum,
992 Strigamia maritima, Danio rerio. (E) Single-copy orthologs are defined as orthologs that were
993 present as a single-copy gene in all 14 species (S. paramamosain, P. trituberculatus, E. sinensis,
994 P. vannamei, H. americanus, P. monodon, D. melanogaster, B. mori, E. andrei, Lepeophtheirus
995 salmonis, S. mimosarum, S. maritima, D. rerio, and Homo sapiens). Multiple-copy orthologs
996 represent the gene groups present in all species with a gene number of > 1 in at least one species.
997 Species-specific paralogs represent genes uniquely present in only one species. Other types of
998 orthologs represent the gene groups that are absent in some species and not species-specific
999 paralogs.
1000 Fig. 2. Phylogenetic tree and gene family expansion/contraction of Scylla paramamosain.
1001 Phylogenetic tree inferred from 338 single-copy orthologs among 14 selected species (S.
1002 paramamosain, Portunus trituberculatus, Eriocheir sinensis, Penaeus vannamei, Homarus
1003 americanus, Penaeus monodon, Drosophila melanogaster, Bombyx mori, Eisenia andrei,
1004 Lepeophtheirus salmonis, Stegodyphus mimosarum, Strigamia maritima, Danio rerio, and
1005 Homo sapiens). Numbers of expanded gene families are marked in green, and numbers of
1006 contracted gene families are marked in red. The number below the MRCA (most recent
37
1007 common ancestor) represents the total number of orthologs from OrthoMCL analysis used as
1008 input for CAFE expansion/contraction analysis.
1009 Fig. 3. The conserved hox gene clusters and their expressions in Scylla paramamosain
1010 (A) Location and transcript direction in the genome of S. paramamosain Hox genes. (B)
1011 Distribution and relationships of Hox gene cluster in S. paramamosain, Bombyx mori (Bmo),
1012 Drosophila melanogaster (Dme), Eriocheir sinensis (Esi), Homarus americanus (Ham),
1013 Parhyale hawaiensis (Pha), Penaeus monodon (Pmo), Penaeus vannamei (Pva), and Portunus
1014 trituberculatus (Ptr). (C) Temporal expression of S. paramamosain Hox cluster genes. E:
1015 embryo, Z1: zoea I, Z3: zoea III, Z5: zoea V, M: megalopa stage, and C1: crablet I. (D)
1016 Expressions of Ubx and Antp in early different developmental stages of S. paramamosain. (E)
1017 The expression locations of Ubx, Antp and Abd-A in S. paramamosain larvae.
1018 Fig. 4. The function and regulation of Abd-A gene in Scylla paramamosain larvae
1019 (A) Expressions of Abd-A in different stages of S. paramamosain larvae. (B) Expression of
1020 SpAbd-A protein in the abdomen of S. paramamosain larvae. (C) Expression locations of
1021 SpAbd-A protein in S. paramamosain larvae. (D) Phenotype changes of S. paramamosain at
1022 Zoeal stage V after RNAi at Zoeal stage IV. (E) Prediction binding sites of Abd-A and novel-
1023 miR1317. (F) Negative expressions patterns between novel-miR1317 and Abd-A in S.
1024 paramamosain larvae. (G) The S2 cells were co-transfected with WT Abd-A 3´-UTR, and the
1025 mutated-type of Abd-A 3´-UTR (MT), together with novel-miR1317 mimics or negative
1026 control mimic. The data were expressed as the relative fluorescence intensity. Z1-5: zoea I-V,
1027 M: megalopa stage, C1: crablet I.
1028 Fig. 5. The expression and regulation of fru2 genes in gonads of Scylla paramamosain
1029 (A) Relative expression of Spfru2 in different developmental stages of the gonads. O: ovary; T:
1030 testis; O1-O5: stage OⅠ to stage OⅤ; T1-T3: stage TⅠ to stage TⅢ. Bars with different lowercase
1031 letters indicate significant difference (p < 0.05). (B) Distribution of Spfru2 in ovary (i, ii) and
1032 testis (iii, iv) of S. paramamosain. N: nucleus; Oo: oocyte; ET: epithelia; SP: spermatid. Scale
1033 bars: 100 μm. (C) Phenotype changes of the ovary tissue after the interference of fru2
1034 expression. Yg: yolk granule; Va: Vacuole. (D) Relative expression of novel-miR35 and Spfru2
1035 of the ovary after overexpression and silencing of *novel-miR35. i: Relative expression of
38
1036 novel-miR35 in the ovary after overexpression of novel-miR35. ii: Relative expression of
1037 Spfru2 in the ovary after overexpression of novel-miR35. iii: Relative expression of novel-
1038 miRNA-35 in the ovary after silencing of novel--miR35. iv: Relative expression of Spfru2 in
1039 the ovary after silencing of novel-miR35. Bars with different lowercase letters indicate
1040 significant difference (p < 0.05). (E) The S2 cells were co-transfected with WT fru2 3´-UTR,
1041 and the mutated-type of fru2 3´-UTR (MT), together with novel-miR35 mimics or negative
1042 control mimic. The data were expressed as the relative fluorescence intensity.
1043 Fig. 6. Neo-functionalization of the elovl6 gene in the LC-PUFA synthesis pathway in
1044 crustacean species
1045 (A) Schematic diagram of the alternative splicing in elovl6. elovl6-DNA, the DNA sequence
1046 of elovl6; elovl6a, the elovl6 transcript containing the first and second introns; elovl6b, the
1047 elovl6 transcript containing the first intron; elovl6c, the elovl6 transcript of introns normal
1048 splicing. Black boxes represent exons. Gray boxes represent introns. (B) Alignment of the
1049 amino acid sequences of elovl6 from crustacean species, including Scylla olivacea
1050 (QEV88898.1), Gecarcoidea lalandii (QKG32709.1), Macrobrachium nipponense
1051 (ANM86278.1), and Penaeus vannamei (AKJ77890.1). Identical amino acid residues were
1052 indicated by dark green, and similar residues were indicated by blue and orange. The conserved
1053 HXXHH histidine motif was in the box, and highly conserved motifs (KXXEXXDT,
1054 NXXXHXXMYSYY, KXXKXX, and TXXQXXQ) were framed in a black box. (C)
1055 Phylogenetic tree comparing S. paramamosain Elovl6 with elongase proteins from other
1056 organisms. The tree was constructed using the neighbour-Joining method by MEGA 11.0.13.
1057 The horizontal branch length is proportional to amino acid substitution rate per site. The
1058 numbers represent the frequencies with which the tree topology presented was replicated after
1059 1000 iterations. (D) Comparison of Elovl6 extended carbon chain conversion efficiency in E.
1060 sinensis, P. trituberculatus, and S. paramamosain.
1061
39
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bioRxiv preprint doi: https://doi.org/10.1101/2024.06.24.600346; this version posted June 27, 2024.

The copyright holder for this preprint

1 High-resolution chromosome-level genome provides molecular insights into

2 adaptive evolution in crabs

14 Fisheries, Universiti Malaysia Terengganu, Kuala Nerus, Terengganu, Malaysia

18 Guangdong Provincial Key Laboratory of Marine Biotechnology

20 243 Daxue Road, Shantou 515063, China

215 sea squirt Ciona intestinalis (Ikuta et al, 2004).

419 Methods and materials

447 Genome assembly, evaluation and correction

476 LACHESIS software (Joshua and Burton 2013).

748 Author contributions

759 Additional information

763 Competing interests

976 Table 1. Summary of Scylla paramamosain genome assembly

978 Figures legends

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