Developing intensive culture techniques for the tropical copepod Parvocalanus crassirostris as a live feed for aquaculture

Copepods are the most common metazoans in marine environment. The number of copepod species identified has reached about 11,500 species, composed of free living pelagic, benthic and symbiotic copepods. Copepods are the most important secondary producers in marine environments and are the natural prey for larvae of most fishes. During the past decades, there has been increasing interest in the development of mass culture techniques for copepods as live food for aquaculture. Unlike traditional live feeds such as rotifers, copepods are rich with essential fatty acids, free amino acids and other essential micronutrients and their nutritional profiles meet the requirements of fish larvae. Moreover, the small size of copepod nauplii is vital for the first feeding larvae of fish species with small mouth gape sizes, such as groupers and snappers. Copepods also stimulate strong feeding responses in fish larvae through their distinctive swimming patterns. Despite multiple advantages of copepods as larval food, utilisation of copepods in aquaculture hatcheries is still sporadic, which is largely attributed to difficulties in their culture and, in particular, their low culture productivity. While recent improvements in intensive cultivation techniques for some copepod species had been achieved, sustainable and feasible mass culture methods for most species, particularly planktonic species, remain elusive. This thesis focused on filling the knowledge gaps by investigating improvements on intensive culture techniques, including several novel approaches, for calanoid copepods. The primary aim of this thesis was to develop intensive culture methods to overcome the current constraints hindering a reliable mass production of calanoid copepods, specifically improving culture productivity for the tropical calanoid Parvocalanus crassirostris.\ud \ud The first chapter of this thesis reviews the current knowledge related to the advantages of copepods as larval feeds compared to traditional live feeds and discusses the current constraints and major hurdles in copepod cultivation. The subsequent experimental chapters can be grouped into three parts. The first part (Chapter 2 and 3) investigates the optimal algal diets for the cultivation of P. crassirostris and exploits the potential use of commercial algal pastes as an alternative diet. The second part (Chapter 4) focuses on identifying optimal culture density for P. crassirostris. Finally, the third part (Chapter 5 and 6) evaluates the efficacy of selective breeding and longterm cultivation (domestication) on improving culture productivity of P. crassirostris.\ud \ud One of the major issues in the advancement of copepod mass cultivation is their species-specific dietary requirements. Generally, calanoid copepods feed on live microalgae. However suitable microalgal species for each copepod species needs to be determined, in addition, whether microalgae should be fed singly or in combination. Chapter 2 evaluated 9 different algal diets, including diet combinations and algal pastes, for the cultivation of P. crassirostris. The key parameters investigated were egg production, egg hatching success, survival and development of nauplii and copepodite stages, population growth and sex ratio of P. crassirostris. The results showed that the combination of two algae species, the Tahitian strain of Isochrysis sp. (T-Iso) and the diatom Chaetoceros muelleri, gave the overall best results. Meanwhile, a healthy culture could also be maintained using T-Iso only. Although, the use of commercial algae pastes is popular for other live feeds, yet, the results of Chapter 2 demonstrated that commercial algal pastes, including T-Iso pastes, have failed to support P. crassirostris culture. Moreover, culturing copepods using commercial pastes has been relatively unsuccessful in previous reports, with no clear explanation as to what may be the main cause of that failure. Therefore, chapter 3 investigated the potential causes that led to the failure of commercial algae pastes in supporting productive copepod culture. A series of experiments were designed to test 3 hypotheses: I) Food availability was low in the water column of algae paste treatments due to the settlement of dead algal cells on the bottom; II) Adherence of the highly condensed cells of microalgal pastes to the swimming appendages and mouthparts of P. crassirostris significantly reduced their feeding and swimming efficiency; and III) the reproductive capacity and survival of P. crassirostris were negatively impacted due to lower digestibility of the pastes as well as lower levels of essential nutrient and/or other bio-active contents in dead algal cells. The results showed significantly high adherence to the mouthparts and swimming appendages of P. crassirostris, which was the primary reason behind the failure of algal pastes as a diet for copepod cultivation. Additionally, the lower digestibility of algal pastes is likely to contribute to that failure. The results suggest that the current method of producing commercial algal pastes are not suitable for calanoid copepod cultivation, which was demonstrated by the high adherence and low digestibility by copepods as compared to live algae. Therefore, new manufacture methods for algal paste production need to be developed to provide acceptable alternatives to live algae for calanoid copepod cultivation.\ud \ud Despite increased research in recent years aimed at enhancing copepod culture techniques, the inability to achieve high culture density is still a major bottleneck in copepod culture, particularly for calanoid copepods. Sustainable culture densities were reported to be approximately two adults ml⁻¹, however, such a density would require a large volume of culture water to produce an adequate number for feeding fish larvae. P. crassirostris had been observed to sustain higher densities than those reported previously. Therefore, the aim of chapter 4 was to evaluate the effects of initial stocking density on a range of biological parameters affecting the culture productivity of the calanoid copepod P. crassirostris. Five initial stocking densities of 1000, 3000, 5000, 7000 and 9000 adults L⁻¹ were evaluated. The results demonstrated that P. crassirostris is a highly promising species as live prey for aquaculture hatcheries, and it can be stocked at a high initial density of 5000 adults L⁻¹ without negatively impacting its culture productivity. Such high culture density is substantially higher than any other calanoid species documented so far.\ud \ud Improving the reproductive capacity of calanoid copepods through optimizing diets and environmental conditions has their limitations. Therefore, chapter 5 and 6 explores the genetic improvements that could be made through selective breeding and domestication as new approaches in improving copepod cultivation. While selective breeding as a successful strategy to enhance the life history traits of aquaculture species had been well documented, it has never been tested for calanoid copepods. Chapter 5 was, hence, designed to determine if selective breeding is an effective way to improve the reproductive productivity of P. crassirostris. Using a family selection program combined with a circular mating strategy, P. crassirostris was cultured for five generations. Females were selected for their high reproductive capacity, and a control chosen by random selection was maintained alongside. The select line exhibited a positive response for selection with a significant increase in total egg production of 24.5% over the female lifespan as compared to the control line. The results provided the first clear evidence of the effectiveness and high potential of selective breeding in enhancing the reproductive capacity of calanoid copepods. Moreover, selective breeding can be employed as an effective approach for the improvement of other life history traits of copepods to enhance their culture productivity as well as other traits improving their quality as larval live food