122 Cryopreservation effects on larval development of the New Zealand greenshell™ mussel, Perna canaliculus

Abstract

Effects of cryopreservation on development of trochophore (18 h post-fertilisation) and D-stage larvae (48 h post-fertilisation) of the New Zealand greenshell™ mussel, Perna canaliculus were evaluated over a 21 day rearing period. Examination of development was undertaken through histology, scanning electron microscopy (SEM), confocal microscopy, light microscopy, and visual observations. Larvae were cryopreserved in 10% ethylene glycol and 0.4 M trehalose which had been made up at twice the final concentration using Milli-Q water. Larvae diluted 1:1 with cryoprotectant solution were loaded into 0.5 mL straws and frozen in a controlled rate freezer that was programmed to hold at 0 °C for 5 min then cool at 1 °C min−1 to −10 °C, hold for 5 min then cool at 0.5 °C min−1 to −35 °C before being plunged into liquid nitrogen. Straws were held for 1–2 h in liquid nitrogen then thawed in a 28 °C water bath. Thawed larvae were then reared under standard hatchery conditions and compared with larvae which were not cryopreserved (controls). Results showed that there were significant differences in survivability, shell length, and feeding consumption between controls and cryopreserved treatments, but no comparable differences among the two larval stages cryopreserved after the 21-day development period. At 18 days post-fertilisation, ∼23% of control larvae had progressed to competent pediveliger, while <1% of thawed larvae survived and were unable to develop to competent pediveliger or post-larva. Settlement was achieved in ∼9% of controls at 21 days post fertilisation with most individuals developing eye spots. Differences in neurogenesis between cryopreserved trochophore larvae and controls were observed, while cryopreserved D-stage larvae did not differ greatly to controls. Characterisation of shell morphology revealed abnormalities to both cryopreserved stages, with thawed trochophore larvae showing the greatest shell abnormalities. Organogenesis was delayed in the cryopreserved treatments and no cryopreserved larvae developed an eye spot. However, controls successfully made the transition to settlement. This delay in organogenesis and overall development was indicative of cryo-injuries sustained at a cellular level. Overall, results indicated that D-stage larvae have a higher resilience to cryopreservation. This research emphasises the need to assess the effects of cryopreservation not just immediately post-thawing but over longer time frames. Further on-going research aims to optimise cryopreservation protocols to improve the long-term viability of cryopreserved Perna canaliculus D-stage larvae. Source of funding: This work was funded by the New Zealand Ministry of Business Innovation and Employment (CAWX0802) and by the Auckland University of Technology. Conflict of interest: None declared. serean.adams@cawthron.org.nz