Quantification of lipid droplets in hatchery reared veliger larvae of the green-lipped mussel, Perna canaliculus

Domestication efforts and selective breeding in bivalve aquaculture would greatly benefit from reliable screening biomarkers in the early life stages of development (e.g., larvae). In this study the lipids remaining at the completion of embryo development in selectively-bred/hatchery reared green-lipped mussel, Perna canaliculus, were targeted and quantified using two approaches: confocal laser scanning microscopy and image analysis (CLSM/IA) on Oil Red-O stained larva (i.e., lipid droplet number and volume distribution), and Iatroscan thin layer chromatography/flame ionization detection (TLC/FID) (i.e., energetic and structural lipid classes content). Four groups of newly formed pre-feeding veliger larvae, produced from two genetically distinct families (FA, FB) that completed embryo development at either low (L, 17 degrees C) or high (H, 21 degrees C) temperatures, were evaluated. The FB produced offspring better equipped to deal with higher temperatures during embryonic development, with greater survival, higher normal development, and larger larvae than FA when developing at 21 degrees C. In terms of lipids content, CLSM/IA showed that each individual larva had overall between 83 and 157 lipid droplets (ranging in volume from 2.5 to 294 & mu;m3). The TLC/FID estimation of mean total lipid content ranged from 4.1 to 6.9 ng larva-1 (29-39% structural lipid and 62-69% energetic lipid). Both lipid quantification methods were successful in detecting differences among larval groups with different genetic backgrounds, as well as providing insight into the ability to cope with changes in the growing environment when relying on maternal energy sources (triacylglycerol-TAG identified as the main source of energy for early development). Stronger differences between the two families were identified using CLSM/IA than with TLC/FID (i.e., with FA having overall more and larger lipid droplets). However, TLC/FID was able to distinguish FA larvae reared at high temperatures from the other groups (due to lower levels of TAG, 46% of total lipids, versus 52-56% in the other groups), therefore being more sensitive to the pooled population response compared to the individual lipid droplet quantification in CLSM/IA. The application of these tools, when fully developed, could be extended to controlled commercial operations to diagnose and predict larval performance, enabling more directed early screening and informing production and breeding decisions.

Automated summary

This study aimed to identify reliable screening biomarkers in the early life stages of selectively-bred/hatchery reared green-lipped mussel through lipid quantification using confocal laser scanning microscopy and image analysis, as well as Iatroscan thin layer chromatography/flame ionization detection. The results showed that both methods were successful in detecting differences among larval groups with different genetic backgrounds and growing environments, providing important insights for early screening.