Aquaculture of stalked barnacles (Pollicipes pollicipes) Sofia Cota Franco A thesis submitted to Newcastle University in candidature for the Degree of Doctor of Philosophy School of Marine Science and Technology July 2014 Abstract The stalked barnacle, Pollicipes pollicipes, is considered a delicacy on the Iberian Peninsula and has a high market value. Despite being a dangerous activity, increased collection efforts and associated stock shortage have raised awareness of the need for effective conservation and stock management policies. Accordingly, aquaculture has received interest as an alternative to supply the market and for re-stocking programmes. However, knowledge on the aquaculture requirement of this species and applicable production cycles is limited. Research challenges span the entire P. pollicipes life cycle, from adult reproduction to larval settlement. Though adults have been kept in culture, the conditions required for broodstock reproduction and larval release remain poorly studied and larvae have been routinely extracted from wild-collected adults and reared to cyprids. Optimization of larval culture is essential for the production of high-quality larvae and avoidance of high mortality. Furthermore, cyprid settlement on artificial substrata presents a bottleneck to production, with settlement occurring mostly on conspecific adults. The conditions that mediate settlement on preferential substrata have yet to be established. Though juvenile behaviour and growth in the wild have been the subject of ecological studies, research on culture conditions is limited and the influence of environmental factors is poorly understood. In the present work, the effect of environmental conditions on the behaviour and development of P. pollicipes was tested throughout the life cycle to identify optimal culture conditions and assess potential for larger-scale culture. Research focused on broodstock reproductive conditioning, larval culture, larval settlement and juvenile culture and behaviour. Broodstock reproductive conditioning was investigated by monitoring larval production and nauplius quality from adults reared under different temperature regimes. P. pollicipes larvae were also cultured under different conditions of temperature, food quality, photoperiod and salinity, and monitored for growth and survival. Larval attachment and metamorphosis on adults were tested for cyprids of different age and batch, and according to environmental factors such as temperature, salinity, hydrodynamics and light conditions. Optimal conditions were then used for investigating settlement on substrata in culture, and natural and artificial structures were i tested for settlement in the wild. Juvenile feeding behaviour and performance in culture were investigated in relation to hydrodynamics, temperature, food quality and quantity. Results indicated that P. pollicipes conditioning requires 28 days after acclimatization, when subjected to increasing temperatures (from 16 ᵒC to 24 ᵒC), as found during the breeding season. This could be achieved either by steady increase, or increase with daily temperature oscillations. Larvae were released daily and peak releases (≥ 10000 – 30000 nauplii per tank; ≈122 adults per tank) occurred 1 – 2 times during the conditioning period. Higher fecundity was recorded for broodstock reared under steady temperature increase, while lamella maturation and larval production were higher at oscillating temperatures. No differences in larval quality were recorded among temperature regimes. Larval growth and survival were improved by feeding daily with Tetraselmis chuii/Skeletonema costatum or Isochrysis galbana/S. costatum mixed diets, temperatures of 15 – 20 ᵒC, 24:0 L/D photoperiod and salinity of 20 – 40 psu. Under these conditions, development to the cyprid was achieved within 16 days (20 ᵒC), with 20 – 30 % survival. Larval settlement on the adult was higher for older cyprids (6 days), at 20 ᵒC, with water circulation, light and salinity of 30 – 40 psu to a maximum of 30 – 35 % settlement. Metamorphosis was affected by temperature and cyprid age, with a maximum of 70 – 80 % larvae metamorphosing within a week. Settlement occurred predominantly on the adults, mainly on the capitulum (≈ 60 % of settled larvae), and was greatly reduced on artificial substrata tested in the laboratory (< 3 %) and, in this experiment, was not recorded in the wild. Settlement on natural substrata (e.g. Chthamalus sp., Corallina sp., rocks) was higher (< 17 % per surface) and in the wild appeared to be related to future survival potential on the substrata. On another occasion, settlement was observed on marine epoxy in the wild, used as a fixative for structures, being comparable to that on adults (2 recruits per cm2). Juvenile feeding varied with the degree of laboratory conditioning, as unconditioned individuals only responded to water speeds above 23 cm s-1 in comparison to above 6 cm s-1 for conditioned animals. Those barnacles conditioned in the laboratory prior to testing also showed more frequent prey captures, although live nauplii of Artemia sp. promoted the highest feeding rates for both groups. Alterations to juvenile morphology were observed in culture, though reversible after transfer to the wild. Growth and survival were optimized in cultures not subjected to daily tides, at 16:8 L/D ii photoperiod, fed daily with excess Artemia sp., under which conditions laboratory specimens achieved growth values comparable to the wild. Due to the limited previous knowledge on P. pollicipes rearing, the present study aimed at identifying the conditions required to the culture of this species, over the various production phases. It also aimed at building on ecological studies, towards the understanding of the factors that mediate habitat selection and distribution. The present work has established the initial basis for adult reproduction in culture and provided the first reference values on broodstock performance in captivity. Future studies should focus on investigating the use of different diets and other environmental factors for P. pollicipes broodstock conditioning, in order to maximize yields. The use of recirculating systems for larval culture should be investigated, using as a basis the knowledge acquired on environmental conditions and further looking at improving feeding and husbandry protocols. Current reference values on larval growth and survival can be used as a base for future culture optimization. The present work investigated some of the factors affecting larval settlement and has given important clues over key factors mediating settlement and potential substrata to be used for recruitment. However, settlement remains the largest limitation to culture and efforts should build on the current work to target the testing of settlement substrata that afford larval protection post-settlement, as well as the use of chemical attractants to induce settlement. The development of field trials should be a priority as well as further assessing the performance of laboratory-cultured barnacles transferred to the wild. The investigation on the effect of environmental factors on larval and juvenile behaviour provides new data on P. pollicipes behaviour in response to factors such as hydrodynamics, towards the understanding of P. pollicipes distribution in the wild, and the impact of larval choice in substrata selection. The acquired knowledge on juvenile performance under culture can be used to direct future work on P. pollicipes production and tackle expected pitfalls (e.g. changes in morphology). Studies on juvenile growth should be directed at the assessment of economical viability of commercial scale culture, considering both culture options in captivity and in the natural environment. iii Acknowledgments I would like to express my sincere gratitude to the many people that made this project a reality, for they have been essential. From the guidance, to the technical support, friendship and teamwork, it would have not been possible without them. I would like to thank my supervisors, Prof. Tony Clare, Dr. Nick Aldred and Prof. Teresa Cruz, for their invaluable support throughout this project. For all the guidance, inspiration and contribution, from the high-times, to when the deadline was on the same day – thank you! It has been a pleasure and an honour to work with you. Thank you to the colleagues and staff at the School of Marine Science and Technology (Newcastle University), Laboratório de Ciências do Mar (CIEMAR, Universidade de Évora) and Centro de Ciências do Mar (CCMAR, Universidade do Algarve). Your support has been invaluable and your company has made my days brighter. I would like to further express my debt to Antonio P. Cribeiro, and others that with little bits and pieces of their time and vision have made this project better. A special note of appreciation to my examiners, for their words of wisdom, striking questions and most fascinating discussion. To my family and closest friends, who have made my life richer, my most heartfelt thank you. You are irreplaceable and I cannot ever repay you. You have made my journey remarkable and have inspired me to always reach further. A special thank you to my sister and my boyfriend - you are my everything. Finally, this research would not have been possible without the financial support from the Fundação para a Ciência e Tecnologia, research grant SFRH/BD/63998/2009, and from ASSEMBLE, grant no. 227799. What started from a simple idea and a few questions, soon became an overreaching