The Australian red claw crayfish Cherax quadricarinatus, an emerging species within the freshwater aquaculture trade, is not only an ideal species for commercial production due to its high fecundity, fast growth, and physiological robustness but also notoriously invasive. Investigating the reproductive axis of this species has been of great interest to farmers, geneticists, and conservationists alike for many decades; however, aside from the characterisation of the key masculinising insulin-like androgenic gland hormone (IAG) produced by the male-specific androgenic gland (AG), little remains known about this system and the downstream signalling cascade involved. This investigation used RNA interference to silence IAG in adult intersex C. quadricarinatus (Cq-IAG), known to be functionally male but genotypically female, successfully inducing sexual redifferentiation in all individuals. To investigate the downstream effects of Cq-IAG knockdown, a comprehensive transcriptomic library was constructed, comprised of three tissues within the male reproductive axis. Several factors known to be involved in the IAG signal transduction pathway, including a receptor, binding factor, and additional insulin-like peptide, were found to not be differentially expressed in response to Cq-IAG silencing, suggesting that the phenotypic changes observed may have occurred through post-transcriptional modifications. Many downstream factors displayed differential expression on a transcriptomic level, most notably related to stress, cell repair, apoptosis, and cell proliferation. These results suggest that IAG is required for sperm maturation, with necrosis of arrested tissue occurring in its absence. These results and the construction of a transcriptomic library for this species will inform future research involving reproductive pathways as well as biotechnological developments in this commercially and ecologically significant species.

In the face of rising global demand and unsustainable production methods, cultivated crustacean meat (CCM) is proposed as an alternative means to produce delicious lobster, shrimp, and crab products. Cultivated meat requires starting stem cells that may vary in terms of potency and the propensity to proliferate or differentiate into myogenic (muscle-related) tissues. Recognizing that regenerating limbs are a non-lethal source of tissue and may harbor relevant stem cells, we selected those of the crayfish Cherax quadricarinatus as our model. To investigate stem cell activity, we conducted RNA-Seq analysis across six stages of claw regeneration (four pre-molt and two post-molt stages), along with histology and real-time quantitative PCR (qPCR). Our results showed that while genes related to energy production, muscle hypertrophy, and exoskeletal cuticle synthesis dominated the post-molt stages, growth factor receptors (FGFR, EGFR, TGFR, and BMPR) and those related to stem cell proliferation and potency (Cyclins, CDKs, Wnts, C-Myc, Klf4, Sox2, PCNA, and p53) were upregulated before the molt. Pre-molt upregulation in several genes occurred in two growth peaks; Stages 2 and 4. We therefore propose that pre-molt limb regeneration tissues, particularly those in the larger Stage 4, present a prolific and non-lethal source of stem cells for CCM development.

RNA interference (RNAi)-based biotechnology has been previously implemented in decapod crustaceans. Unlike traditional RNAi methodologies that investigate single gene silencing, we employed a multigene silencing approach in decapods based on chimeric double-stranded RNA (dsRNA) molecules coined ‘gene blocks’. Two dsRNA constructs, each targeting three genes of the crustacean hyperglycaemic hormone (CHH) superfamily of neuropeptides, were produced: Type II construct targeting Cq-Molt-inhibiting hormone 1 (MIH1), Cq-MIH-like 1 (MIHL1), and Cq-MIHL2 isoforms and Type I construct targeting Cq-ion transport peptide (Cq-ITP; a putative hybrid of CHH and MIH) and Cq-CHH and Cq-CHH-like (CHHL) isoforms. Both constructs were injected into juvenile redclaw crayfish, Cherax quadricarinatus, to determine the effects of multigene knockdown on molting and developmental processes. A 20-Hydroxyecdysone (20E) enzyme-linked immunosorbent assay (ELISA) and glucose assay were used to determine the effects of RNAi on molting and hemolymph glycemic activities, respectively. Multigene silencing reduced the intermolt interval by 23%. Statistically significant elevated 20E was recorded in treated intermolt individuals, consistent with the reduced intermolt interval as well as unique and abnormal phenotypes related to the molting process, which indicates a shift in 20E-induced cascade. There was no effect of RNAi treatment on hemolymph glucose level or molt increment. Through multigene silencing and subsequent annotation of gene networks, gene blocks may provide a tailored approach to investigate complex polygenic traits with RNAi in a more efficient and scalable manner.

Understanding the mechanisms of sexual development would pave the way for producing mono-sex populations to aid the aquaculture industry. This study investigates the functions of the Y-linked iDmrt1 paralogue (Po-iDMY) and insulin-like androgenic gland hormone (Po-IAG) in the process of sexual development in the tropical rock lobster, Panulirus ornatus (TRL). Previously, we identified that Po-iDMY, a male-specific heterogametic (Y-linked) paralogue of the autosomal Po-iDmrt1 found in TRL, is a second sex-linked iDmrt gene identified in invertebrates. Using 5′ and 3′ rapid amplification of cDNA ends and data from a draft male genome (with an assembly genome size of approximately 2.446 Gbp and 87% BUSCO completeness), we obtained the full-length Po-iDMY gene (encoding a protein of 312 amino acids). A 411 bp male-specific sequence located at the 3′ untranslated region of Po-iDMY mRNA was used as a sex marker, which was reported for the first time in our draft genome. However, Po-iDMY is not a master sex-determining factor since it was not expressed across developmental stages of embryos, juveniles and adults. Instead, we silenced Po-IAG at an early juvenile stage, generating two potential neo-females, implying that sexual manipulation could be a promising technique in TRL.

Panulirus ornatus, the ornate spiny lobster, is a stenohaline weak hyper-osmoregulator, yet its osmoregulatory response to salinity stress remains poorly understood. This study investigated six osmoregulatory genes—Na⁺/K⁺-ATPase (nka), V-type H⁺-ATPase (vhe), Na⁺/HCO₃⁻ exchanger (nbc), Na⁺/K⁺/2Cl⁻ co-transporter (nkcc), Na⁺/H⁺ exchanger (nhe), and carbonic anhydrase (ca)—in juvenile gills exposed to 25 ppt, 34 ppt (control), and 40 ppt salinities during acute (48 h) and chronic (>38 d) phases. Transcriptome analysis revealed that all genes were unresponsive following either 25 ppt or 40 ppt salinity acute exposure. However, nkcc showed a tendency toward for upregulation under 25 ppt salinity during acute exposure. Additionally, glutathione S-transferase and putative ferrous reductase 1 were upregulated under 25 ppt salinity, suggesting increased metabolic demand. In contrast, glutathione peroxidase and an ammonia transporter were upregulated in 40 ppt salinity, indicating protein catabolism. Quantitative PCR confirmed nkcc- and nka upregulation under chronic 25 ppt salinity. Vhe, nbc, nhe and ca showed no response, and 40 ppt salinity did not affect the six target genes. These findings suggest P. ornatus relies on nkcc- and nka-mediated ion transport and lacks mechanisms to tolerate high salinity, resulting in reduced growth and survival. These findings define optimal salinity range for aquaculture (25–34 ppt), highlighting the need to avoid high-salinity stress in lobster water quality management