Nutritional aspects of three species of marine fish larvae, herring Clupea harengus L., plaice Pleuronectes platessa L. and turbot, Scophthalmus maximus L. were examined. Larvae were fed three different diets all of which were derived from rotifers Brachionus plicatilis (O.F. Mtlller). These rotifers had been mass cultivated on baker's yeast and then enriched with baker's yeast, or with one of two unicellular algae, Isochrysis galbana Park or Nannochloropsis (Nannochloris) oculata (Droop) Hibberd. The biochemical changes that these enrichments conferred on the rotifer were examined. There was little difference in proximate and amino acid compositions. Dry weights, and calorific contents of the rotifers fluctuated according to the type, temperature and duration of enrichment, with the yeast enriched rotifers tending to weigh less (200-320 ng) than their algal counterparts (250-370ng). Major differences were found in the total fatty acid profiles of the rotifers. Yeast-enriched rotifers had no 18:3n-3 or 20:5n-3 and only trace amounts of 22:6n-3 fatty acids. The Nanrinochioropsis-enriched rotifers had substantial amounts of n-3 fatty acids though only of 20:5n-3 (11-14%), while rotifers enriched with Isochrysis had only trace amounts of 20:5n-3 (2-4%). All three species of fish responded similarly to the yeast enriched rotifer diet. Their growth, in terms of length and weight, was minimal and they never developed any fin ray elements. Both flatfishes developed a looped gut in the short growth duration on this diet and some turbot (20%) inflated their swim bladder. This retardation of growth was attributed to the lack of long chain highly unsaturated n-3 fatty acids (n-3 HUFA) in the diet. During one month feeding trials it was shown that herring and plaice were able to exploit both algal-enriched rotifer diets equally well. Turbot, on the other hand grew better, over 14 days, on rotifers enriched with lsochrysis compared with Nannochioropsis. The reaction of the different species was linked to the fatty acid profiles of the diet. Hence, turbot require a dietary input of 22:6n-3 while herring and plaice thrived equally well on diets containing only 20:n-3. Juvenile plaice consistently exhibited improved pigmentation when Isochrysis compared to Nannochloropsis were used as the rotifer enrichment. The effect was attributed to specific nutrients particular to algal chloroplasts, which need to be present in the diet of the larvae up to metamorphosis. Assessment of daily rations, employing the rotifer enriched with Isochrysis diet only, showed herring to reduce their food intake from 46 to 19% body dry weight/d between days 10-21 post hatch while turbot consumed between 34-169% body dry weight/d with no apparent age related effects. The gross growth efficiencies (Ki, Brett & Groves, 1979) for both species were seemingly unaffected by age related processes and fluctuated between 15-50% in turbot and 20-61% in herring according to fluctuations in daily food intake. Ingestion is further described for both species with a linear model that related food intake to larval weight. The weight-specific ingestion was found to be a constant 13% and 43% of body dry weight in herring and turbot respectively. The weight-specific ingestion and specific growth rate estimates were combined with other data available from the literature, revealing that larvae tend to consume food (in dry weight) at a level equivalent to approximately twice their daily dry weight increase.

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The study of larvae, their behaviour & physiology has gathered momentum in recent years due to the development of improved techniques of rearing during the past decade. Many marine larvae have been successfully reared in the laboratory (Shelbourne, 1964; Schumann, 1967; Blaxter, 1968,1969; Houde & Palko, 1970; Futch & Detwyler, 1970; Lasker, Feder, Theilacker & May, 1970). Fish larvae, in particular those of most marine species form an important tool for research in that at hatching they only possess the rudiments of most organs. Thus they are ideal material for ontogenetical studies. Although the respiratory mechanisms of adult fish have been the subject of a great deal of investigation from the point of view of gas exchange (see Randall 1970) gill structure (Hughes, 1966; Hughes & Grimstone, 1965; Newstead, 1967; to name a few) and dimensions (see Muir, 1969) gill ventilation and perfusion (see Shelton, 1970) circulatory systems (see Randall, 1970) bioenergetics, (see Brett, 1970) the respiratory systems of larvae have not been investigated in any great detail. Apart from a few studies on oxygen uptake (see Blaxter, 1969) and Harder (1954) on the development of branchial elements, no detailed study of the development of respiratory mechanisms have been made in marine fish larvae. The purpose of this study was to investigate the development of respiration in two species of marine larvae viz. the herring (Clupea harengus L.) and the plaice (Pleuronectes plates sa L.) These two species are well separated taxonomically and both adults and young have very different life histories. Herring lay demersal eggs, the plaice pelagic ones. The yolk-sac larvae of both species are planktonic, later feeding on diatoms and copepod nauplii and much later copepods. Adult herring are pelagic, living in mid water as juveniles and moving into deeper water with age, ranging from offshore to about 200m. They perform migrations partly caused by the distribution and density of food organisms. In contrast to this plaice at metamorphosis show an interesting asymmetry in that one eye migrates over the head and comes to lie against its opposite number. At this stage pelagic life ceases and the young fish assumes a bottom-living existence. Other features associated with asymmetry are secondary to the migration of the eye and follow on from the adoption of the benthic mode of life. They range from the shoreline when young to lOOOm feeding on bottom living organisms. During development the mechanism of respiration changes from a cutaneous one to gill respiration typical of the adult form. There is apparently no respiratory pigment in the early stages but the blood becomes pink at metamorphosis. The problem was approached from a morphological and a physiological viewpoint. The main parts of the study are as follows. (1) The survival times in water of low oxygen concentrations. (2) The oxygen uptake at normal oxygen concentrations. (3) The oxygen uptake at low oxygen concentrations. (4) Measurement of the body surface area as well as the gill area available for respiration. (5) The appearance of haemoglobin and its quantitative measurement.

Manipulation or control of the microbiological aquaculture environment has been identified as an important focus area for future hatchery development. Subsequently, alternatives to obtain control of the microbiological environment are being sought of which the field of probiotics appears highly promising. Probiotics are usually selected based on various in vitro characteristics, however, the methods used differ and are sometimes unsuccessful due to poor experimentation. The aim of this work is to contribute towards the development of a protocol for the in vitro screening of bacterial candidate probiotics for marine fish larviculture. To reduce the number of candidate probiotics to be tested in vivo, various in vitro experiments need to be conducted, each screening for a particular mode of action – antagonism towards pathogen through production of antimicrobial compounds, growth and attachment to fish intestinal mucus, and the production of other beneficial compounds such as vitamins, fatty-acids and digestive enzymes. A total of 108 bacteria species were isolated from the digestive tract of the adult common clownfish, Amphiprion percula to screen for potential probiotics to be used in clownfish larval rearing. The antagonistic compounds assay identified twelve isolates which showed antagonism towards two or more aquatic pathogens. This was followed by an in vitro test that involved growing the organisms in fish intestinal mucus and modeling their growth parameters. A ranking index (RI) was developed using the lag period (λ) and doubling time (td) of the organism, where (1over λ x td) x 100. Five candidate probionts (AP1-AP5) with varied growth parameters were used for further in vitro experiments. The attachment to mucus assay introduced a novel tool for quantifying competition for attachment sites between candidate probionts and pathogens on mucus. Candidate probiont Pseudoalteromonas AP5 reduced the attachment ability of Vibrio alginolyticus when added before the pathogen and partially out-competed the pathogen for attachment sites when added second. In vitro screening for the production of beneficial compounds tested the candidate probiotics’ ability to produce digestive enzymes - trypsin, lipase and alkaline phosphatase as well as carotenoids and vitamin C. Candidate probiont Pseudoalteromonas AP5 produced high levels of the enzymes (98.2, 34.1 and 91.3 mU product liberated.ml⁻¹, respectively) and contained carotenoids while Kocuria AP4 contained carotenoids but produced low quantities of enzymes (7.8, 0 and 59 mU product liberated.ml⁻¹, respectively). None of the candidate probiotics produced vitamin C. To eliminate potential pathogenic or toxic candidate probionts, Artemia nauplii were exposed to each candidate probiont and the percentage Artemia mortality after 24-hours was determined. Candidate probiont AP2 caused high mortality of Artemia nauplii (98.4%) and was excluded from further studies. Identification of candidate probionts AP3-AP5 was performed using 16S-rDNA molecular techniques and the bacteria were assigned the names Bacillus AP3, Kocuria AP4 and Pseudoalteromonas AP5, respectively. Two methods of larval probiont delivery were tested – attachment to Artemia, and in-water delivery. Attachment to Artemia was high for both Kocuria AP4 and Pseudoalteromonas AP5 (7.2 x10³ and 2.7x10⁴ bacteria.nauplius⁻¹, respectively) while the in-water viability experiment showed that Kocuria AP4 comprised 23.9% of the total culturable water microflora after 24 hours while Pseudoalteromonas AP5 contributed 100%. To validate the findings from the in vitro experiments, in vivo trials using clownfish larvae were performed. Of the four candidate probiotics tested, only Kocuria AP4 showed potential to increase larval survival. In vitro tests produced a better understanding of the possible mode of action and strategies of competition between bacteria, however, the number of criteria in which a candidate probiont is successful in vitro may not be the best predictor for its effectiveness in vivo. Commercial studies that reduce between-treatment variation are required to test predictions about the most suitable probiont or combinations thereof.