Analysis of sexual development pathways in decapod crustaceans of commercial significance

Nguyen Tran

doi:10.25907/01067

Sexual dimorphism refers to the consistent differences between males and females of the same species in physiology, morphology, behaviour, and molecular traits, including body size and shape, colouration, appendage development, behavioural patterns, reproductive organs, secondary sexual characteristics, sex-biased gene expression, and tissue specialisation. In crustaceans, the most notable aspects are sexual size dimorphism and sexually dimorphic behaviours, as these have significant applied value for improving culture systems of commercially important crustaceans. The use of sexual size dimorphism aims to produce a monosex population. In some species, males or females grow faster and are larger at harvest, making all-male or all-female populations more profitable. Sexually dimorphic behaviours are often linked to key biological activities such as mating, courtship, territorial or competitive aggression, and gathering food for offspring and partners. Therefore, a thorough understanding of the regulatory mechanisms underlying behavioural differences between males and females, including sex-biased genes and chemical communication (e.g., pheromones), will benefit the aquaculture industry. The ornate spiny lobster or tropical rock lobster (TRL), Panulirus ornatus, and the slipper lobster (SL), Thenus australiensis, are increasingly recognised as promising candidates for sustainable onshore marine aquaculture in Australia due to their high growth rates, strong market demand, and premium commercial value. In contrast, the red claw crayfish (RCC), Cherax quadricarinatus, is a commercially important freshwater aquaculture species with high survival rates under controlled conditions and tolerance of a wide range of environmental conditions. Therefore, exploring sexual dimorphism, particularly in terms of size and behavioural differences, can be strategically utilised in crustacean aquaculture to boost productivity and profits. TRL and SL exhibit sexual size dimorphism, in which males grow faster and attain a larger final size than females (TRL), whereas in SL, females are larger than males. Thus, this study investigated the mechanisms underlying sexual development in TRL and SL to aid the aquaculture industry in producing monosex populations. In the first research chapter, the study investigated the functions of the Y-linked iDmrt1 paralogue (Po-iDMY) and insulin-like androgenic gland hormone (Po-IAG) in sexual development in TRL. In a previous study of our group, 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, as it was not differentially expressed across the 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. In the following research chapter, we generated the first draft male genome assembly for SL by integrating whole-genome shotgun short-read sequencing, Diversity Arrays Technology sequencing (DArT-seq), and RNA-seq data. The assembled genome size was 1.44 Gb, with a BUSCO completeness of 93.8%, indicating high assembly quality. Gene prediction identified 85,335 protein-coding genes, of which 87.3% were BUSCO-complete, and 14,937 (18%) were functionally annotated. Using a k-mer subtraction method, we identified six male-specific contigs to investigate the sex markers. Of these, we annotated the Class II DNA transposons (Tc1-like located in Contig k141_7345028) and degraded Class I retrotransposons (LINE-1/Jerky in Contig k141_6893598), embedded within the intronic regions of CENP-E, a kinesin-7 motor protein essential for chromosome segregation. This suggests the accumulation of divergent transposable elements and retrotransposons in non-coding regions of an essential centromeric gene, potentially defining the Y chromosome region. In combination with these results, DArT-seq genotyping and reproductive transcriptome analysis provided strong evidence that SL follows an XX/XY sex-determination system, with males carrying heterozygous and Y-linked genomic regions. Primers designed from the male-specific region (Contig k141_7345028) successfully amplified a 273 bp PCR in males, achieving 100% accuracy in sex identification. These findings offer a valuable tool for early sex identification and support a sex-determination system (XY), which is crucial for developing monosex aquaculture techniques to enhance growth rates and profitability in slipper lobster farming. In the final chapter, this study reported the first case of a female intramandibular gland (FIG) connected to the antennal gland and characterised by a distinct yellow–white colouration, which exhibited a strongly female-biased dimorphism in the red claw crayfish (RCC), Cherax quadricarinatus. Histological examination revealed that the FIG is composed of densely packed glandular epithelial cells with a distinct cellular organisation, whereas the antennal gland displayed a more typical tubular gland with broader luminal spaces and epithelial cells. A comprehensive survey conducted over three years, encompassing 58 females, 33 males, and 16 juveniles (including 7 males, 6 females, and 3 intersex individuals), confirmed that the FIG was consistently present in all females and absent in all males, irrespective of moulting stage, reproductive condition, or season, highlighting a consistent morphological feature in female RCC. Transcriptomic analysis of three FIGs and three male antennal glands collected from the same anatomical region revealed that the urocanate hydratase gene (UROC1) exhibits marked sex-biased regulation. In males, the UROC1 transcript was truncated at the N-terminus, resulting in the loss of core functional domains, including the NAD-binding region, whereas females expressed the full-length transcript. Moreover, UROC1 expression was restricted, as it was approximately 10-fold higher in females than in males. Future functional studies are needed to clarify the biological role of UROC1 in females, particularly in relation to sexually dimorphic behaviours, such as egg and juvenile care and protection, and the lower levels of aggression observed in females compared with males. This finding provides an important understanding of the female-biased gland in RCC and may offer a conceptual framework for other crustacean species. In summary, our studies combined genomic, molecular, and anatomical frameworks to understand sexual dimorphism and sex determination in three commercially important crustacean species: TRL, SL and RCC. By generating draft male genomes for TRL and SL, identifying sex markers, and characterising an XX/XY sex chromosome system in both TRL and SL, this study provides critical genomic resources and practical tools for early sex identification and monosex development. Additionally, the discovery of a female-specific intramandibular gland (FIG) and the identification of UROC1 as a strongly female-biased gene in RCC reveal an unknown female-biased mechanism pathway in the antennal gland.

Analysis of sexual development pathways in decapod crustaceans of commercial significance

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