Newly Found Dmrt1B Gene Tied to Testis Development in Oriental River Prawns

A gene that may help steer male sexual development in the oriental river prawn has been isolated and described for the first time. Because the gene’s protein sequence and evolutionary position place it closest to the Dmrt1B gene of the giant freshwater prawn Macrobrachium rosenbergii, the team named it MniDmrt1B.

The oriental river prawn is an economically significant species in China, where production reached 224,413 tonnes in 2022 with an output value of 2.8 billion US dollars. One biological quirk makes the animal particularly interesting to aquaculture: males grow faster than females and reach a commercial size roughly two to two and a half times larger. That difference gives sex-determining genes and their regulatory networks direct commercial relevance, since understanding how sex is set could eventually inform production strategies. Sex determination in crustaceans, however, is a complex process shaped by multiple mechanisms and a network of genes rather than a single switch, which is why each new component of that network attracts attention.

The Dmrt proteins carry a distinctive zinc-finger DNA-binding motif called the DM domain, short for doublesex and Mab-3. This motif is conserved from invertebrates to humans, and several DMRT1 paralogs and their orthologs are known to regulate reproductive functions including gonad differentiation, gametogenesis and sex determination. Before this study, only one Dmrt gene had been reported in M. nipponense: a DM-domain gene called DMRT11E, isolated and characterized by Wang and colleagues in 2019. Whether other members of the protein family also participate in the molecular control of sex development in this species remained an open question, and the new work was designed to address that gap.

To find the gene, the researchers mined transcriptome libraries prepared from the ovary of female prawns and the testis of males, using a transcript annotated as Dmrt1B as their starting point. From adult testis tissue they extracted total RNA and applied rapid amplification of cDNA ends, or RACE, to clone the full-length sequence. The resulting cDNA is 3,812 base pairs long, containing a 438-base-pair untranslated region at the 5′ end, a 1,862-base-pair untranslated region at the 3′ end, and a 1,512-base-pair open reading frame that encodes a protein of 503 amino acids. The presence of a termination codon and a polyadenylation signal in the 3′-RACE product confirmed that the cloned sequence was complete. The underlying transcriptomic data were deposited under NCBI accession number PRJNA9878611.

Sequence comparisons showed that the predicted MniDmrt1B protein is most similar to its counterpart in M. rosenbergii, with 93.81 percent identity, followed by the black tiger prawn Penaeus monodon at 82.35 percent, the swimming crab Portunus trituberculatus at 80.56 percent, the Pacific white shrimp Penaeus vannamei at 80.28 percent, Penaeus chinensis at 73.30 percent and the red swamp crayfish Procambarus clarkii at 67.17 percent. Bioinformatic predictions placed the molecular weight of the protein at 55,224.39 with an isoelectric point of 9.19. InterProScan identified a DM DNA-binding domain spanning amino acids 115 to 168, and alignment of this domain with other Dmrt proteins highlighted conserved residues, including the zinc-binding sites that define the motif’s structure. A phylogenetic tree built with the neighbor-joining method and 500 bootstrap replicates placed the new gene on the same evolutionary branch as the Dmrt1B subfamily of M. rosenbergii, supporting its name.

The most striking expression result came from adult tissues. Quantitative real-time PCR on nine males and nine females, testing the second pereiopod, hepatopancreas, eyestalk, muscle, ovary and testis, showed that MniDmrt1B mRNA is present in all tested tissues of both sexes. In males, however, expression was far from uniform: the testis showed the highest level, significantly exceeding every other tissue at a threshold of p less than 0.05. In females, no significant difference in expression was detected among the tissues examined. This male-biased, testis-dominant pattern echoes findings for other Dmrt genes in related species, such as MroDMRT11E in the giant freshwater prawn, and parallels a crab study in which suppressing an EsDmrt-like gene by RNA interference reduced testicular size and impaired spermatogenesis.

Developmental time-course data reinforced that impression. The researchers hatched larvae from berried females, sampled ten larvae at 20, 25, 30 and 35 days post-hatching, and later identified the sex of each individually marked animal so that expression could be matched to sex. In males, MniDmrt1B expression stayed consistently high throughout development and rose with age, peaking at 35 days post-hatching at a level significantly higher than at 20 and 25 days. In females, expression showed no significant change across the same period. The gene was thus expressed at markedly higher levels in males than in females throughout the larval window measured, a sexually dimorphic pattern consistent with a role in male development, particularly testis development.

To place MniDmrt1B in context with known players in prawn sex differentiation, the team measured it alongside four sex-regulated genes: the insulin-like androgenic gland hormone Mn-IAG, the transcription factor MnDmrt11E, the forkhead box protein L2-like gene MnFoxl2 and the Sox-family gene MnSoxE, at 15, 20, 25, 30 and 35 days post-hatching. The four comparison genes followed a shared trajectory, rising and then falling, with peaks at 30 days post-hatching significantly higher than at other time points. MniDmrt1B instead increased steadily at every measured point, also reaching its highest value at 30 days post-hatching, significantly above its levels at 15, 20 and 25 days. That timing matters because previous work by Jin and colleagues identified the sex-sensitive period in this species as running from post-larval day 10 to post-larval day 22, a window encompassing the formation of the testis, ovary and androgenic gland. The authors interpret the concurrence of peak MniDmrt1B expression with this differentiation-sensitive phase, together with the gene’s similarity in behavior to established sex-related genes, as strong indication that MniDmrt1B probably plays an important role in testis development and sex differentiation. They note, however, that gonadal differentiation is a complex regulatory network in which genes occupy different positions and activation timings, which may explain why MniDmrt1B’s expression pattern differs from that of its neighbors.

The study also uncovered an environmental link. Because earlier work by the same group showed that lower temperature can induce masculinization in M. nipponense, the researchers reared larvae at either 26 plus or minus 0.5 degrees Celsius or 31 plus or minus 0.5 degrees Celsius for roughly 60 days and then measured MniDmrt1B in the testes of males from each treatment. Expression was significantly higher in males reared at the low temperature than at the high one. Temperature-dependent regulation of dmrt1 has been documented in fish and reptiles with temperature-dependent sex determination, generally with more dmrt1 expression at temperatures that promote male development, and the prawn result fits that pattern. The authors hypothesize that temperature, like sex hormones, may influence Dmrt gene expression and thereby affect sex differentiation in this species. They conclude that high temperature acts as a negative regulator of MniDmrt1B expression.

Important caveats frame these findings. The evidence for MniDmrt1B’s function is correlational: it rests on expression patterns, sequence similarity and phylogenetic placement, not on direct loss- or gain-of-function experiments, so the proposed role in testis development and sex differentiation remains a well-supported hypothesis rather than a demonstrated mechanism. The expression analyses used pooled biological replicates of three animals each, and the temperature experiment sampled testes from males at a single endpoint after about two months of culture. Even so, the identification of a second Dmrt-family gene in M. nipponense adds a concrete piece to the puzzle of crustacean sex determination and offers aquaculture researchers a candidate target for future functional studies in a species where the growth advantage of males makes the biology of sex a matter of direct economic interest.

Subject of Research: Identification and expression characterization of the Dmrt1B sex-development gene in the oriental river prawn Macrobrachium nipponense

Article Title: Identification and characterization of the Dmrt1B gene in the oriental river prawn, Macrobrachium nipponense

Article References: Jiang, G., Xue, Y., Arifuzzaman, A., & Huang, X. (2024). Identification and characterization of the Dmrt1B gene in the oriental river prawn, Macrobrachium nipponense. Development Genes and Evolution, 234(1), 21-32. https://doi.org/10.1007/s00427-024-00715-3

Image Credits: AI Generated

DOI: 10.1007/s00427-024-00715-3

Keywords: Macrobrachium nipponense, Dmrt1B, DM domain, sex determination, testis development, crustacean genetics, qRT-PCR, gene cloning, phylogenetics, temperature-dependent expression, aquaculture, Development Genes and Evolution