Ellie Watts

Cryopreservation of Greenshell™ mussel (Perna canaliculus) trochophore larvae ☆

The Greenshell™ mussel (Perna canaliculus) is the main shellfish species farmed in New Zealand. The aim of this study was to evaluate the effects of cryoprotectant concentration, loading and unloading strategy as well as freezing and thawing method in order to develop a protocol for cryopreservation of trochophore larvae (16-20 h old). Toxicity tests showed that levels of 10-15% ethylene glycol (EG) were not toxic to larvae and could be loaded and unloaded in a single step. Through cryopreservation experiments, we designed a cryopreservation protocol that enabled 40-60% of trochophores to develop to D-larvae when normalized to controls. The protocol involved: holding at 0 °C for 5 min, then cooling at 1 °C min⁻¹ to -10 °C, holding for a further 5 min, then cooling at 0.5 °C min⁻¹ to -35 °C followed by a 5 min hold and then plunging into liquid nitrogen. A final larval rearing experiment of 18 days was conducted to assess the ability of these frozen larvae to develop further. Results showed that only 2.8% of the frozen trochophores were able to develop to competent pediveligers.

SETTLEMENT OF HALIOTIS AUSTRALIS LARVAE: ROLE OF CUES AND ORIENTATION OF THE SUBSTRATUM

ABSTRACT Haliotis australis is a medium-size New Zealand abalone with aquaculture potential. H. australis can be readily ripened and spawned, but larval settlement has proved difficult. The current study addressed aspects of larval settlement (i.e., attachment and metamorphosis) in laboratory experiments and small-scale hatchery trials. Competence to metamorphose was first seen weakly in larvae at 8 days old at 14.5°C, but larval metamorphosis rates increased progressively up to at least 12 days of age. In laboratory experiments, larvae showed a dose-dependent response to dissolved &ggr;-aminobutyric acid (GABA), with settlement peaking at 1 µM (57% metamorphosis 4 days after settlement induction) and declining at higher and lower doses. Across a range of cues, metamorphosis was highest on crustose coralline algae (97 ± 4% metamorphosis after 4 days), intermediate on 1 µM GABA (55 ± 14%), and low on diatom films (<20%). GABA combined additively with diatom film to give strong metamorphosis (81 ± 6%). Metamorphosis induction by a diatom film was doubled if the film was pregrazed by juvenile H. australis (76% vs. 35%, P < 0.0001), but the grazing effect appeared to be species specific, because pregrazing by Haliotis iris had no effect (41% vs. 35%, P = 0.720). The species-specific response to trail mucus appears to preclude GABA as being the main inducer of metamorphosis in this case. On hatchery plates, settlement on pregrazed biofilms was approximately 4-fold higher when the plates were horizontal rather than vertical (41 % vs. 10% metamorphosis, P = 0.003). Settlement on ungrazed horizontal plates remained low even if those plates had been held in water with adult conspecifics, but without contacting the abalone. We conclude that tanks offering sloped or horizontal surfaces will be more effective than vertical plates for settlement of H. australis, and that pregrazed biofilms and/or GABA are promising settlement cues.

Identifying Thermally-Stressed Adult Green-Lipped Mussels (Perna canaliculus Gmelin, 1791) via Metabolomic Profiling*

Abstract: The green-lipped mussel (Perna canaliculus Gmelin, 1791) is an economically and ecologically important marine species within New Zealand, yet the ability of adult mussels to cope with acute temperature change remains unknown. To address this, we sought to characterize the thermotolerance capacity of P. canaliculus adults and, using metabolomics, identify any metabolic biomarkers of thermal stress in this species. To achieve this, mussels were exposed to a 3 h acute temperature challenge using temperatures of 20 °C (Ambient), 25 °C, 29 °C, 31 °C, 33 °C, and 35 °C. No mortality was observed in mussels exposed to 31 °C or less, even after 30 days recovery. However, mussels exposed to 33 and 35 °C exhibited 100% mortality within 48 h of the challenge. Gill tissues were harvested from mussels for metabolomic analysis of thermal stress biomarkers via Gas Chromatography-Mass Spectrometry (GC-MS). Discriminant Function Analysis (DFA) of 52 metabolites identified eight key biomarkers indicative of thermal stress in this species (i.e., cis-11-Eicosenoic acid, Palmitic acid, Proline, GABA, Aspartic acid, Fumaric acid, beta-Alanine and Asparagine). These metabolites were entirely consistent in their ability to classify the exposure temperature that mussels experienced, indicating that the discriminatory capacity of these biomarkers was strong. Therefore, our results reveal that mortality in thermally-stressed adult P. canaliculus seems assured once temperatures reach 33 °C. Additionally, metabolite biomarkers can discriminate those mussels exhibiting varying levels of thermal stress; thus, metabolomics offers a new tool for physiologists seeking to gain greater mechanistic understanding of the effects of acute thermal stresses on invertebrate species.

Physiological Indicators of Stress and Morbidity in Commercially Handled Abalone Haliotis iris

ABSTRACT Abalone are enigmatic, displaying subtle responses to stress, including a delayed onset of morbidity, and death after severe stress. The present study therefore sought to further elucidate the nature of stress responses in the blackfoot abalone Haliotis iris and develop practical indices to quantify stress and forecast survival. Abalone were challenged with air exposure (16°C, 4–120 h). Physiological responses were assessed at three organizational levels: whole organism (heart rate), extracellular (hemolymph metal concentrations), and intracellular (hemocyte lysosome stability). Hemolymph sodium:potassium ratio showed a strong negative correlation with emersion time (R2 = 0.82) and a reasonable capacity to predict subsequent survival (R2 = 0.50); a [Na]:[K] of 23 was representative of an undisturbed abalone, whereas values less than 15 typically indicated morbidity. Hemocytes tended to lyse before lysosome destabilization could be quantified by neutral red loss; the stability assay therefore failed to yield meaningful data. Resting heart rate declined from 19.4 min-1 (undisturbed) to a minimum of 1 min-1 recorded after 48 h emersion; however, the correlation between heart rate and emersion time or subsequent survival was weak. Hemolymph sodium:potassium ratio warrants further investigation as a practical and sensitive forensic tool to quantify stress, but will require complementary approaches to robustly determine the survival tipping point.

Investigation of early mussel (Perna canaliculus) development using histology, SEM imaging, immunochemistry and confocal microscopy

ABSTRACT A comprehensive study, incorporating histology, light microscopy, scanning electron microscopy, immunochemistry and confocal microscopy, was performed to investigate embryogenesis and larval development of the New Zealand Greenshell™ mussel, Perna canaliculus. Detailed observations with this multi-technique approach revealed a gastrula stage at 18 hours post-fertilization, with the appearance of a blastopore, apical sense organ and enclosing vegetal pole. Early D-stage larvae showed limited alimentary organogenesis and clear initiation of a developing nervous system. Shell morphology of D-larvae was characterized by a flat, hinged, pitted–punctate prodissoconch I shell, followed closely by commarginal growth lines within the prodissoconch II shell. Early umbo larvae had a protruding functioning velum, and well-developed posterior adductor and velar retractor muscles. Significant progression in neuronal development occurred just before the umbo stage with noticeable paired cerebral, pedal and visceral ganglia. Shell morphology was characterized by further prodissoconch II secretion with a more rounded umbonate appearance. During the transition through the pediveliger stage, rapid development of the gill rudiment, eye spot and functioning foot was observed with ongoing neuronal development. The first appearance of the dissoconch shell layer took place during this transition, at which point the nervous system was highly distinct with innervations extending throughout muscle regions and between ganglia. This study provides the first comprehensive documentation of the developmental stages of P. canaliculus larvae from fertilization to settlement. The study highlights the advantages of using a combination of techniques to understand larval development and provides crucial information to identify larval performance during larval rearing.

Practical Fertilization Procedure and Embryonic Development of the New Zealand Geoduck Clam (Panopea Zelandica)

Cultivation of the geoduck Panopea zelandica (Quoy & Gaimard, 1835) requires knowledge on embryonic development to produce spat in hatcheries. This study investigated the development of P. zelandica embryos at 15°C and 35 ppt and the optimal sperm:egg ratios for fertilization under hatchery conditions. Panopea zelandica broodstock were induced to spawn by serotonin injection. Sperm and eggs were collected and fertilization was conducted at sperm:egg ratios of: 50:1, 100:1, 500:1, 1000:1 and 10,000:1 over 40 min. The optimal sperm:egg ratio was Panopea zelandica eggs (~80 μm diameter) developed the first and second polar bodies within 15–20 and 50–55 min post-fertilization, respectively. The blastula appeared at ~8 hpf, including the X R and X L cells and the presumptive shell field depression. Gastrulation occurred at 12–18 hpf with organic material apparent at the shell field depression. The mid-stage trochophore, which appeared at around 35 hpf had an apical plate with an apical tuft. The shell field spread to form the periostracum, which expanded and folded into right and left segments covering the late trochophore. The early D-stage veliger appeared at 45 hpf with the soft body being enclosed by two valves and the appearance of the velum. These observations will serve as the basis for future analyses of P. zelandica embryogenesis and for optimization of commercial production of D-veliger larvae.