Nguyen Dinh Quang, Duy (2017) Developing hatchery culture of the tropical sea cucumber, Holothuria scabra, using micro-algae concentrates. PhD thesis, James Cook University.

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Abstract

Major bottlenecks to further development and expansion of hatchery-based production of tropical sea cucumbers include our lack of knowledge of their nutritional requirements and appropriate foods for larval stages. Live micro-algae have been used as the main source of food for hatchery culture. However, mass culture of adequate volumes of high quality cultured micro-algae is both labour and resource demanding and is often inappropriate for small-scale hatcheries in developing countries that generally lack the required resources and technical capacity. Alternative potential food sources for larvae include highly-concentrated marine micro-algae that is now available commercially. This study assessed the potential of commercially available micro-algae concentrates as a replacement for live cultured microalgae during hatchery culture of sandfish, Holothuria scabra. It examined ingestion and digestion of micro-algae concentrates by larval sandfish (Chapter 3), the nutritional value of micro-algae concentrates for larval sandfish during hatchery culture (Chapter 4), the relationships between hyaline spheres formation, larval food composition and subsequent settlement success (Chapter 5), and the ingestion, digestion and nutritional value of live microalgae and micro-algae concentrates for newly settled sandfish juveniles (Chapter 6).

Ingestion and digestion of two live (TISO and Chaetoceros muelleri) and six concentrated micro-algae products (Instant Algae®, Reed Mariculture Inc., Campbell, CA, USA, 95008) by sandfish auricularia larvae of different ages were assessed using epifluorescence microscopy. The commercial micro-algae products were purchased from an Australian distributor. They were: (1) mono-cultured Isochrysis sp. (Haptophyceae) (Isochrysis 1800®); (2) mono-cultured Pavlova sp. (Haptophyceae) (Pavlova 1800®); (3) mono-cultured Tetraselmis sp. (Chlorophycophyceae) (Tetraselmis 3600®); (4) mono–cultured Thalassiosira weissflogii (Bacillariophyceae) (TW 1200®); (5) mono-cultured Thalassiosira pseudonana (Bacillariophyceae) (3H 1800®); and (6) a mix of four concentrated single micro-algae species: Isochrysis sp., Pavlova sp. Thalassiosira pseudonana and Tetraselmis sp. (Shellfish Diet 1800®). This is the first study to report the use of epifluorescence microscopy with larval echinoderms and experiments were conducted using 2, 6 and 10 day old auricularia larvae. Seven of the eight micro-algae tested were ingested and digested by larvae with digestion occurring more rapidly in older larvae. C. muelleri was rapidly digested by 6-day and 10-day old larvae but results indicate that C. muelleri is unsuitable as a food for 2-day old sandfish larvae. TISO was well ingested by sandfish larvae in both live and concentrated forms, and live TISO was the most suitable of the micro-algae tested in terms of ingestion and digestibility. All commercially available micro-algae concentrates tested were readily ingested and digested by H. scabra larvae with the exception of Thalassiosira pseudonana (3H 1800®) which was not ingested by larvae of any of the three ages tested. Results show potential for using microalgae concentrates as alternatives to live micro-algae in hatchery culture of sandfish. The three most digestible of the ingested Instant Algae® products were used in a subsequent experiment to assess the relative nutritional values of digestible micro-algae, as a basis for optimising a diet for hatchery culture of sandfish, and to provide information on the nutritional requirements of sandfish larvae.

Three Instant Algae® (Reed Mariculture Inc., Campbell, CA, USA, 95008) products: (1) mono-cultured Isochrysis sp. (Haptophyceae) (Isochrysis 1800®); (2) mono-cultured Pavlova sp. (Haptophyceae) (Pavlova 1800®); and (3) mono-cultured Thalassiosira weissflogii (Bacillariophyceae) (TW 1200®) were used to feed sandfish larvae both singly and in ternary combination to assess their nutritional efficacy. Two-day auriculariae were held at a starting density of 0.3 mL⁻¹ and were initially fed a daily ration equivalent to the dry weight of 10,000 cells mL⁻¹ of Isochrysis 1800®. This ration was increased by the dry weight equivalent of 1000 cells mL⁻¹ of Isochrysis 1800® per day as larval development proceeded. Post-settled larvae fed TW 1200® were significantly larger than those fed the ternary diet, Isochrysis 1800® or Pavlova 1800®. There were significant differences in the mean (±SE) survival of auriculariae and post-settled larvae between treatments and survival to settlement was significantly higher (P < 0.05) for larvae fed TW 1200® (13.7 ± 0.7%) alone. Laval development, competency and survival were significantly correlated with dietary levels of total protein, lipid and nitrogenfree extract (NFE, equivalent to carbohydrate), and with total polyunsaturated fatty acid (PUFA) content of the diets, and the levels of some specific fatty acids (FA). The proportion of late auriculariae with hyaline spheres (day 13), numbers of competent doliolariae (day 15) and the total length of post-settled larvae (day 21) were all positively correlated with dietary NFE and palmitic acid (16:0) contents, as well as dietary EPA:DHA ratio. This study is the first comprehensive assessment of the nutritional value of micro-algae concentrates for sandfish larvae based on their nutrient compositions. Results confirm the feasibility of using commercially available micro-algae concentrates as a sole food source for hatchery culture of sandfish, and are the first to report successful hatchery culture of sandfish larvae without using live micro-algae. All micro-algae concentrates used in this study proved nutritious for sandfish larvae and supported normal growth and development and relatively high survival, through settlement.

The following experiment investigated the influence of diet composition on hyaline sphere (HS) development in auricularia larvae of sandfish, and the subsequent relationships between the presence and size of HS and competency through settlement and early juvenile performance. Two-day old larvae were fed one of three commercially available micro-algae diets that varied in their nutrient compositions: (1) Isochrysis sp. (Haptophyceae); (2) Pavlova sp. (Haptophyceae); and (3) Thalassiosira weissflogii (Bacillariophyceae) or a ternary combination of the three. There were positive significant correlations between HS development in late auriculariae on days 10, 11, 12 and 13 post-fertilisation, and the proportion of competent doliolariae on day 15, post-settlement size (day 21) and post-settlement survival (day 25). The dietary components that most strongly influenced these relationships were carbohydrates (as NFE) and the polyunsaturated fatty acids arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The results confirm a strong relationship between HS formation in late auriculariae of sandfish and subsequent larval competency through settlement. As such, the presence and size of HS is a reliable indicator of subsequent performance for sandfish. Given that HS development was influenced by the nutrients available to sandfish auriculariae, there is clear opportunity for development of more appropriate diets for hatchery culture of this species that will improve HS formation and larval performance supporting improved hatchery production.

Information on the nutritional requirements and preferred diets of sea cucumber juveniles is extremely limited and this has hindered development of hatchery culture methods. The final experiment in this study assessed ingestion, cell wall digestion and relative nutritional values of two live micro-algae (Isochrysis aff. galbana (TISO) and Chaetoceros muelleri) and six concentrated micro-algae products (Instant Algae®, Reed Mariculture Inc.) for early juveniles of sandfish, Holothuria scabra. The six Instant Algae® products were: (1) mono-cultured Isochrysis sp. (Haptophyceae) (Isochrysis 1800®); (2) mono-cultured Pavlova sp. (Haptophyceae) (Pavlova 1800®); (3) mono-cultured Tetraselmis sp. (Chlorophycophyceae) (Tetraselmis 3600®); (4) mono–cultured Thalassiosira weissflogii (Bacillariophyceae) (TW 1200®); (5) mono-cultured Thalassiosira pseudonana (Bacillariophyceae) (3H 1800®); and (6) a mixture of four micro-algae species: Isochrysis sp., Pavlova sp. Thalassiosira pseudonana and Tetraselmis sp. (Shellfish Diet 1800®). Seven of the eight micro-algae tested were ingested by juveniles with the exception of live TISO. Faeces excretion times varied between ingested diets that passed through the juvenile gut in less than 1 h. The cell walls of five of the eight micro-algae tested were partially or mostly digested (Chaetoceros muelleri, TW1200®, Palova 1800®, Isochrysis 1800® and Shellfish 1800®), while the cell walls of Tetraselmis 3600® and 3H 1800® remained intact. Juvenile growth rates were significantly different between diet treatments over the duration of a 14-day growth trial. Mean (±SE) length of early juveniles at the end of the growth trial was highest for those fed live C. muelleri (4.10 ± 0.03mm) followed by TW 1200® (3.49 ± 0.05mm). Juvenile survival did not differ significantly between diet treatments and was highest for those fed C. muelleri (79.33 ± 6.11%) followed by TW1200® (78.33 ± 1.20%). Pearson's correlation tests were used to identify key correlations between the levels of specific nutrients and juvenile performance (growth and survival). A significant positive correlation between growth and dietary protein content (P < 0.05), and a highly significant positive correlation between growth and dietary EPA: DHA ratio (P < 0.01) provide new information to inform diet selection for juvenile sandfish that will help improve juvenile performance and support improved hatchery production.

Hatchery culture of sandfish larvae and early juveniles using micro-algae concentrates, without the use of live micro-algae, is a significant output from this study, and results support development of cheaper, simpler hatchery rearing protocols for this species. This study also determined detailed nutritional compositional data for the commercial micro-algae products used, and on this basis, it was possible to deduce new information relating to key nutrients for sea cucumber larvae. The results provide a basis for fine-tuning diets used in hatchery culture of sandfish, and other sea cucumbers, to better address their nutritional requirements. Such a development is likely to result in improved quality of hatchery produced juveniles as well as improved hatchery production.

Item ID:	51795
Item Type:	Thesis (PhD)
Keywords:	digestion, early juveniles, epifluorescence microscopy, hatchery culture, Holothuria scabra, hyaline spheres, ingestion, larvae, larval competence, live microalgae, microalgae concentrates, micro-algae, nutrition, sandfish
Related URLs:	https://researchonline.jcu.edu.au/43082/ https://researchonline.jcu.edu.au/49154/ https://researchonline.jcu.edu.au/49166/ https://researchonline.jcu.edu.au/51799/
Additional Information:	For this thesis, Duy Nguyen Dinh Quang received the Dean's Award for Excellence 2018. Publications arising from this thesis are available from the Related URLs field. The publications are: Chapter 3: Nguyen Dinh Quang, Duy, Pirozzi, Igor, and Southgate, Paul C. (2015) Ingestion and digestion of live microalgae and microalgae concentrates by sandfish, Holothuria scabra, larvae. Aquaculture, 44. pp. 256-261. Chapter 4: Nguyen Dinh Quang, Duy, Francis, David S., Pirozzi, Igor, and Southgate, Paul C. (2016) Use of micro-algae concentrates for hatchery culture of sandfish, Holothuria scabra. Aquaculture, 464. pp. 145-152. Chapter 5: Duy, Nguyen Dinh Quang, Francis, David S., and Southgate, Paul C. (2016) Development of hyaline spheres in late auriculariae of sandfish, Holothuria scabra: is it a reliable indicator of subsequent performance? Aquaculture, 465. pp. 144-151. Chapter 6: Duy, Nguyen Dinh Quang, Francis, David S., and Southgate, Paul C. (2017) The nutritional value of live and concentrated micro-algae for early juveniles of sandfish, Holothuria scabra. Aquaculture, 473. pp. 97-104.
Date Deposited:	18 Dec 2017 21:50
FoR Codes:	07 AGRICULTURAL AND VETERINARY SCIENCES > 0704 Fisheries Sciences > 070401 Aquaculture @ 40% 07 AGRICULTURAL AND VETERINARY SCIENCES > 0702 Animal Production > 070204 Animal Nutrition @ 20% 06 BIOLOGICAL SCIENCES > 0608 Zoology > 060808 Invertebrate Biology @ 40%
SEO Codes:	83 ANIMAL PRODUCTION AND ANIMAL PRIMARY PRODUCTS > 8301 Fisheries - Aquaculture > 830199 Fisheries - Aquaculture not elsewhere classified @ 100%
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