Panulirus ornatus (Ornate spiny lobster)

Ventura et al. 2020, Multi-Tissue Transcriptome Analysis Identifies Key Sexual Development-Related Genes of the Ornate Spiny Lobster (Panulirus ornatus) https://doi.org/10.3390/genes11101150

Six mature ornate spiny lobsters (P. ornatus; three males and three females, 1.37–1.83 kg) were purchased from wild-caught stocks captured in the Torres Strait in 2019 and reared at the University of the Sunshine Coast for at least one month prior to dissections. Six immature P. ornatus individuals (three males and three females, 300–350 g) derived from cultured stocks supplied by the Institutefor Marine and Antarctic Studies (IMAS) aquaculture facility, Tasmania. Lobsters were reared as previously described [53]. Multiple tissues were dissected from 12 P. ornatus individuals, and samples were snap-frozen using liquid nitrogen and stored at −80 ◦C until use. RNA was then extracted from up to 100 mg from each tissue using RNAZol (MRC, Melbourne, VIC, Australia), as previously described [54]. Total RNA was isolated from the testes (n = 6), ovaries (n = 6), and hepatopancreas (n = 11) of all individuals (with the omission of one immature male hepatopancreas). In addition, total RNA was extracted from the eyestalk, brain, thoracic ganglia, antennal gland, stomach, intestine, epidermal tissue, fat tissue, anterior gills, posterior gills, heart, and tail muscle of one mature P. ornatus male and one mature female (n = 2 per tissue). Two replicates from each sample were uploaded to represent this dataset on CB.

P. ornatus is one of over 80 spiny lobster species found around the globe, and is found on shallow reefs in the tropical Indopacific. During larval development, spiny lobsters undergo two distinct, transparent larval phases which are unique to the Achelata - the first is the flat, leaf-shaped phyllosoma and the second is the nektonic, shrimp-like puerulus. One notable hallmark of phyllosoma metamorphosis is the withdrawal of the hepatopancreas (known as gut-retraction) from the cephalic shield, a process which continues for 1–3 days prior to the metamorphic molt. Since phyllosoma larvae of this stage reach a size of 20-40 mm, gut retraction can be traced by eye with relative ease, providing a visible index of metamorphic progression. This unique trait, together with a relatively predictable intermolt period (approximately 10 days in P. ornatus), makes spiny lobsters a potentially useful model for studying crustacean molting and larval development.

The samples were sent to Novogene (Beijing) for paired-end sequencing in Illumina HiSeq™ 2500 (PE150). RNA libraries were constructed by oligo(dT) mRNA enrichment, followed by random fragmentation and cDNA synthesis with random hexamers. This was followed by second-strand synthesis by nick translation and purification with AMPure XP beads. Raw reads were trimmed to remove adapter contamination and reads with high uncertainty (N > 10%) or low base quality were discarded. Due to the absence of a reference genome, clean reads were de novo assembled using Trinity software with hierarchical clustering by Corset. Transcript expression quantitation was calculated by RSEM, producing raw read counts which were then used to calculate FPKM with the software DESeq2.