THE UNIVERSITY OF HULL Chilled Mist as a Viable Alternative Method for Transporting Commercially Caught Crustacean and Mollusc Species Being a Thesis submitted for the degree of MSc by Research In the University of Hull by Kelan Cherry BSc (Hons.) September 2019 Acknowledgements: I would like to give a huge thank you to Dr. Katie Smyth for her invaluable support during this project, and for putting up with my constant moaning and numerous dilemmas. I’d like to extend a big thanks to Professor Mike Elliott for his help and guidance during this project, especially with writing up. I would like to extend my thanks and appreciation to everyone in the Institute of Estuarine and Coastal Studies who have all been a huge help, and have put up with my inability to use sharp objects. Eleanor Adamson and The Fishmonger’s Company have been a huge help during this project, the offers of emotional support and the financial help provided have been invaluable to me and the completion of this project, and for this I would like to say a big thank you. The people at Sutherland Game and Shellfish were a pleasure to meet and deal with, I would like to thank them for being so helpful and friendly Thank you to Pete Ekers and the people at MacDuff Shellfish for providing the whelks and Lobsters, and being a pleasure to work with I’d like to also thank John Garland and Roger Uglow for their help and insights during the experiments, especially regarding the experimental procedures used in this project Finally, the emotional support given to me by my parents, family members, and my good friend Andrew has been invaluable, and for this I would like to thank them dearly Thank you everyone! 2 Abstract: Shellfish are often transported live to markets to ensure freshness upon arrival. Traditional transport methods involve large volumes of water (1:1 animal:water, vivier) hence reducing the mass of animals that can be transported with one journey, or they are transported dry, with both methods often inducing high stress levels. To assess the viability of an alternative method, the physiological stress of three commercially important species (Buccinum undatum, Nephrops norvegicus, and Homarus gammarus) was measured over time (24h – N. norvegicus, 72h – H. gammarus, and 96h – B. undatum) within an experimental re-circulating intermittent (IM) and continuous (CM) mist environment. Haemolymph stress parameters such as L-lactate, ammonia, D- glucose, total protein, pH, and behaviour were measured every 24h to determine the condition of the animals. The responses of animals in the misted environments were compared to the traditional method of transport for each species: vivier or dry. The mist was effective at reducing levels of haemolymph ammonia in the animals compared to simulated dry transport (3 and 2.4 fold lower ammonia concentration in B. undatum and H. gammarus haemolymph; at 96 hours and 72 hours; respectively). The IM group had 8.8 times lower ammonia concentration in the reservoir water compared to the CM group at 96 hours for the B. undatum trials, suggesting that IM may be a more efficient use of water for longer journeys. In its current form, misting is not suitable for the transport of N. norvegicus, as high mortalities were recorded in both IM – 30%, CM – 10%, compared to traditional vivier – 0%, however IM reduced mortality rates compared to traditional dry transport of B. undatum (IM - 5.25%, CM – 28.07%, dry - 22.8%). The efficacy of Accutrend handheld meter for L-lactate determination in decapod crustaceans is discussed in detail within this study. This study offers a novel, easily implemented method of transport with potential for replacing traditional methods, whilst maintaining animal health. This study can be used by fishers as a base for developing more efficient, cost-effective methods of live shellfish transport. 3 Contents: 1. Introduction………………………………………………………………………………………………… ……..9 1.1. General Introduction………………………………………………………………………………………..9 1.2. Current Transport Practices………………………………………………………………………… ..11 1.2.1. Vivier System 13 1.2.1.1. Limitations for vivier system…………………………………………………… 13 1.2.2. Dry/Semi-dry system…………………………………………………………………………..15 1.2.2.1. Limitations for dry system………………………………………………………..15 1.3. Novel Transport methods – misting…………………………………………………………………16 1.4. Biology, Ecology, & Commercial Considerations of Test Organisms………………….19 1.4.1. Crustaceans…………………………………………………………………………………………19 1.4.1.1. Homarus gammarus………………………………………………………………..19 1.4.1.1.1. Biology and Distribution……………………………………………….19 1.4.1.1.2. Capture, Transport, and market……………………………………20 1.4.1.2. Nephrops norvegicus……………………………………………………………….20 1.4.1.2.1. Biology and Distribution……………………………………………....20 1.4.1.2.2. Capture, Transport, and Market……………………………………21 1.4.2. Molluscs……………………………………………………………………………………………. 21 1.4.2.1. Buccinum undatum………………………………………………………………….22 1.4.2.1.1. Biology and Distribution……………………………………………….22 1.4.2.1.2. Capture, Transport, and Market……………………………………22 1.5. Biomarkers of Stress……………………………………………………………………………………….23 1.6. Mitigation of Stress During the Transport of Organisms 25 1.7. Measuring L-lactate concentration 25 1.8. Aims, Objectives, and Hypotheses 27 2. Materials & Methods……………………………………………………………………………………………29 2.1. Animals Used………………………………………………………………………………………………….29 2.2. Laboratory Testing………………………………………………………………………………………….30 2.2.1. Misting System……………………………………………………………………………………31 2.2.2. Traditional Transport Methods……………………………………………………………32 2.2.3. Haemolymph Withdrawal……………………………………………………………………34 2.2.3.1. Haemolymph Preparation……………………………………………………….35 2.2.4. Stress Parameters……………………………………………………………………………….36 2.2.4.1. Lactate…………………………………………………………………………………….36 2.2.4.2. Ammonia…………………………………………………………………………………37 2.2.4.3. Glucose……………………………………………………………………………………40 2.2.4.4. Total Protein……………………………………………………………………………40 2.2.4.5. pH……………………………………………………………………………………………41 2.2.4.6. Behavioural Parameters…………………………………………………………..41 2.2.5. Evaluation of Accutrend Plus Lactate Meter………………………………………..45 2.3. Statistical Analysis…………………………………………………………………………………………..46 3. Results……………………………………………………………………………………………………………….…47 3.1. Buccinum undatum…………………………………………………………………………………………47 3.1.1. General Observations………………………………………………………………………….47 3.1.2. Between Transport Methods Comparisons………………………………………....48 3.1.3. PROBIT…………………………………………………………………………………………….….50 3.1.4. Within Transport Methods Comparisons……………………………………….…….50 3.1.5. Water Parameters……………………………………………………………………………….53 4 3.2. Nephrops norvegicus……………………………………………………………………………………… 55 3.2.1. General Observations………………………………………………………………………….55 3.2.2. Between Transport Methods Comparisons………………………………………….56 3.2.3. PROBIT………………………………………………………………………………………………..58 3.2.4. Within Transport Methods Comparisons……………………………………….…….59 3.2.5. Water Parameters……………………………………………………………………………….61 3.3. H. gammarus 62 3.3.1. General Observations………………………………………………………………………….62 3.3.2. Between Transport Methods Comparisons…………………………………….……64 3.3.3. PROBIT………………………………………………………………………………………………..65 3.3.4. Within Transport Methods Comparisons………………………………………….….65 3.3.5. Water Parameters……………………………………………………………………………….69 3.4. Accutrend Handheld Meter Validation…………………………………………………………….70 4. Discussion…………………………………………………………………………………………………………….73 4.1. Buccinum undatum…………………………………………………………………………………………73 4.2. Nephrops norvegicus………………………………………………………………………………………77 4.3. Homarus gammarus……………………………………………………………………………………….80 4.4. Accutrend Meter Comparison…………………………………………………………………….….85 4.5. General Discussion ……………………………………………………………………………………… 86 4.6. Critique of Study…………………………………………………………………………………………….89 4.7. Directions for Future Study…………………………………………………………………………….90 5. Conclusion……………………………………………………………………………………………………………91 6. Ethical Statement…………………………………………………………………………………………………91 References ……………………………………………………………………………………………………..92 5 List of Figures: Figure 1 – a typical vivier set-up 14 Figure 2 – a typical dry set-up 16 Figure 3 – the suggested set-up for a mist system 18 Figure 4 – possible responses to environmental perturbation 24 Figure 5 – the recirculating mist system 32 Figure 6 – a means of separating haemolymph into separate Eppendorf tubes 36 Figure 7 – a) flow injection analysis equipment used b) how the flow injection analysis equipment works 39 Figure 8 – The difference in measured stress parameters of B. undatum, between the three simulated transport treatments 49 Figure 9 – The change in haemolymph ammonia over the course of the trials for B. undatum 53 Figure 10 – The change in ammonia concentration in the reservoir for each mist method for the B. undatum trials 54 Figure 11 – Stress parameter measurements for N. norvegicus between three simulated transport treatments 57 Figure 12 – The change in haemolymph ammonia over the course of the trials for N. norvegicus 61 Figure 13 – Stress parameter measurements for H. gammarus between three simulated transport treatments 64 Figure 14 – Changes in mean haemolymph ammonia concentrations over time during each trial for H. gammarus 69 Figure 15 - The change in ammonia concentration in the reservoir for each mist method for the H. gammarus trials 70 6 Figure 16 – a) the correlation between the traditional method of L-lactate determination, and the Accutrend handheld meter for H. gammarus. b) the correlation between the traditional method of L-lactate determination, and the Accutrend handheld meter for N. norvegicus. 72 Figure 17 – hypothetical ammonia excretion