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.

A study was conducted to determine the effects of incubation salinity and temperature . and post-hatching temperature on salinity tolerance of Pacific herring (Clupea pallasi Valenciennes) larvae. Calorimetry experiments were also conducted on herring larvae that were incubated and reared in various combinations of salinity and temperature. In the salinity tolerance test, herring eggs were artificially fertilized in three salinities (13, 21 and 29°/ooS) at 6°C. Newly fertilized eggs were incubated in the same salinities as those in which they were fertilized at two levels of incubation temperature (6 and 12°C), making six incubation groups. During the peak of the hatching period, groups of larvae from each incubation group were exposed to a series of test salinities, ranging from 20 to 55°/ooS at three levels of post-hatching temperatures (6, 9 and 12°C). Salinity tolerance was measured by determining the mortality of larvae occurring in each test salinity over a period of 72 hours. Two approaches were employed: 1) time-mortality, and 2) dosage-mortality. The former provided estimates of the effective time to 50% mortality (ET₅₀), the latter, the effective dosage (salinity) to 50% mortality (ED₅₀). The ET₅₀ or ED₅₀ values were used as bases for comparison of the responses of larvae to incubation temperature and salinity, and post-hatching temperature. The results indicate that both incubation salinity and temperature exert a significant influence on the salinity tolerance of larvae. Eggs incubated in the highest salinity (29°/ooS) and lower temperature (6°C) produced larvae most tolerant to higher post-hatching salinities. Post-hatching temperature also showed a possible influence, although the trends seen were not statistically significant. Salinity tolerance of larvae hatching from the 6°C incubation temperature appeared to be maximized at post-hatching temperatures between 6 and 9°C; whereas, those larvae from the 12°C incubation temperature showed maximum salinity tolerance at 9 to 12°C post-hatching temperatures. The response. of. larvae to salinity may be a result of an irreversible non-genetic adaptation of the embryo to salinity and temperature during incubation. In the calorimetry experiments, samples of herring larvae were combusted in a microbomb calorimeter to determine caloric values. These larvae were hatched and reared in the same six combinations of incubation salinities and temperatures as those used in the main experiment. Larvae were sampled at day 0 (peak of hatching), 3, 6 and 9. Standard lengths, dry weights, ash-free dry weights and ash content of the larval samples were also taken. There was a considerable variability in the data for calorimetry of the herring larva. In general, larvae from all treatments, at hatching, averaged 6,586 cal/g ash-free dry weight and had a mean ash value of 8.25%, obtained by direct ashing. Mean standard length of larvae at the peak of hatching (day 0) ranged from 7.67 mm (12°C, 29°/ooS) to 9.93 mm (6°C, 13°/ooS). There was a trend towards larger larvae (greater standard length) at lower temperatures in lower salinities in all ages tested. Data on dry weights show a linear decrease in larval (body and yolk) weight from day 0 to day 9. Mean dry weight of larva at the peak of hatching ranged from 0.142 mg (6°C, 13°/OOS) to 0.162 mg (12°C, 21°/ooS; 12°C, 29°/ooS). At day 0, the largest larvae (in terms of dry weight) were found at higher temperatures in higher salinities, while at day 9 (past yolk-sac stage), the largest larvae were found at lower temperatures in lower salinities. The same trend was observed in terms of ash-free dry weight. / Science, Faculty of / Zoology, Department of / Graduate

Larval fish distributions were determined in the lower Winfield Pool, Kanawha River, West 85, Virginia, using a 0.5-m plankton net and a 1-m² dropbox. Five habitats were sampled with the plankton net, 3 habitats with the dropbox. The 5 deep water water habitats, greater than 1.5-m in depth, sampled by the plankton net included surface tows in Bill's Creek backwater, main channel border upstream and downstream of Little Guano backwater, and Little Guano Creek backwater, where deep tows (1.5 m deep) were also taken. The 3 shallow water habitats, less than 1 m in depth, sampled by the dropbox included open water over silt substrate, open water over a sand substrate, and emergent vegetation. <i>Lepomis</i> species, emerald shiners (<i>Notropis atherinoides</i>), and gizzard shad (<i>Dorosoma cepedianum</i>) were the dominant taxa. The emerald shiner taxa could also have included some larvae of <i>Notropis</i> species which are also present in the Kanawha River but whose larvae have not been described. Overall, the mean total larval density did not differ between the backwater or the main channel borders but the species associated with each habitat differed greatly. The <i>Lepomis</i> larvae were found predominantly in backwater areas. These areas provide suitable spawning sites for many centrarchids of this river. Upon leaving the nest, the <i>Lepomis</i> larvae moved into the deeper open water areas within the backwater. After reaching the juvenile stage, these same larvae returned to the shallow water habitats where they inhabited vegetated areas. Emerald shiner larvae, while present in both backwater and main channel habitats, were most abundant in the main channel borders. This is probably a result of their parent's pelagic spawning strategy. In all habitats, emerald shiner larvae predominated in the upper 1 m of water. Upon becoming larger, the emerald shiner larvae appeared in the backwaters. This increase in numbers could be due to movement, differential mortality, or higher growth rates. Other cyprinids (excluding emerald shiners and carp) were equally abundant in both backwater and main channel areas. These other cyprinid larvae were also distributed equally, Gizzard shad larvae were found predominantly in the main channel borders. Presumably, these higher densities were the result of main channel spawning. The gizzard shad larvae present in the backwater areas were distributed evenly throughout the water column. Overall, the backwaters were important for the nest-building species found in the river and also for the larger larvae of the pelagic species, and thus acts as a nursery area for these species. Therefore, the backwaters do seem to be important for the fishery of the Kanawha River. / Master of Science

published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy

The larval fish assemblage in the shallow, nearshore region of a proposed marine protected area (MPA) in eastern Algoa Bay, temperate South Africa was investigated. Current velocities and direction and the swimming abilities of late-stage larvae were further assessed to determine potential larval movement to and from the MPA. In total, 6045 larval fishes were collected along two depth contours (~5m and ~15m) in the shallow nearshore of eastern Algoa Bay using stepped-oblique bongo net tows, twice per season for two years (March 2005 – January 2007). These larvae represented 32 families and 78 species. The Gobiidae, Cynoglossidae, Clupeidae, Engraulidae and Sparidae were the dominant fish families. Catches varied significantly between seasons peaking in spring with a mean of 64 larvae/100m3. Preflexion stage larvae dominated catches (75%). All developmental stages of Diplodus capensis, Engraulis capensis, Heteromycteris capensis, Sardinops sagax and Pomadasys species were found in the study area. It appears that these species use the shallow nearshore as a nursery area. Analysis of 12 months (May 2006 – May 2007) data from a bottom-moored Acoustic Doppler Current Profiler within the study area showed that offshore south eastward (39%) and onshore north westward currents (33%) dominated. The south westward current (15%) and north eastward current (12%) occurred less frequently. Current velocity decreased with depth in the nearshore, with a mean velocity of ~29 cms⁻¹ recorded at a depth of 4 m and a mean velocity of ~11 cms⁻¹ recorded at a depth of 14 m. Understanding the dispersal and movement of marine fish larvae in coastal habitats requires knowledge of active swimming abilities. The critical speed and endurance swimming of late stage larvae of two common inshore species occurring in the study area, Diplodus capensis and Sarpa salpa (Family Sparidae), were measured in a laboratory swimming chamber. The mean U-crit value for D. capensis (18.6 cms⁻¹) was similar to that of S. salpa (18.0 cms⁻¹), whereas mean endurance (km swum) was greater in S. salpa (8.4 km) than D. capensis (5.9 km). These swimming abilities exceed the average current velocities observed in the shallow nearshore providing larvae with the ability to greatly alter their passive dispersal trajectories and ultimately influence their distribution in the nearshore.

Larval development and reproduction of 19 species of Central Amazonian fish (five cichlids, two siluriforms, one osteoglossiform and 11 characiforms) were studied over two years. Most species spawn during the flooding season. The cichlids, the siluriforms and two species of characiform are nest spawners, reproducing in the littoral areas of the floodplain. The osteoglossiform is a mouth-brooder. The remaining characiforms spawn in the river channels and show no parental care. Two main strategies explain 90% of the variability of reproductive traits found among the 19 species. The first strategy is used by riverine spawners (characiforms). They have high fecundity , high to very high reproductive expenditure (calories per spawn per wet weight of female) and spawn once a year during a short season. Their eggs vary in size from 0.06 to 0.3 mg and have intermediate to high calorific value. The second strategy is used by the cichlids and two species of characiforms. They have low fecundity, low reproductive expenditure, long spawning season, multiple spawnings per season and some of them show parental care. Their eggs vary in size from 0.4 to 1.2 mg and have an intermediate calorific content. The other three species show distinct combinations of reproductive traits, but have as common feature a high reproductive expenditure, a short annual spawning season and parental care. Patterns of larval development are correlated with egg size and adult spawning sites. Egg size explained most of variability of larval body size at hatching, pectoral fin bud, eye pigmentation, jaw formation, swim bladder inflation, onset of swimming, first feeding and maximum size attained with exclusively endogenous feeding. The pattern of blood circulation of the larvae was correlated with the spawning sites. Larvae of riverine spawners are small, utilize yolk efficiently and are relatively resistant to starvation. Newly hatched larvae of riverine spawners seem to be very sensitive to physico-chemical conditions of the floodplain lakes, but by the first feeding stage they develop some resistance to the low availability of oxygen. Larvae of littoral spawners are large, utilize yolk less efficiently, and seem to be resistant to low concentrations of oxygen. The resistance of larvae to oxygen deficiency is correlated with the development of the larval respiratory system. It is suggested that egg size of riverine spawners was selected to optimize the distance of the dispersal of the larvae in a range of floodplain lakes. Conversely, egg size of floodplain spawners seemed to be selected to optimize larval survival in the spawning lake. The results are further discussed in relation to life history models.

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Common snook (Centropomus undecimalis) is a relatively new species for aquaculture; considered as a recreational species and not commercial. The aim of this study was to develop common snook larval rearing techniques for stock enhancement. Common snook culture has two main bottlenecks, broodstock management and larval culture. High mortality during the first 6 days is the main limitation for successful larval survival. Broodstock management of common snook is still developing and the only source of common snook eggs is from wild broodstock. Securing a regular supply during the natural spawning was essential to reach the main objective. Finding the optimal spawning sites, as well as optimal spawning time was achieved. Results showed Terra Ceia, Longboat and Cayo Costa to be the best sites for wild broodstock collection. The onset of spawning was triggered by a rise in water temperature. During the 4 years of this study spawning started at the end of May and finished in September. Total capture results and egg quality results, such as fertilization, hatching rate and lipid analysis, indicated June and July as the peak months during the spawning season. Common snook follow a lunar spawning cycle. Results showed that one to three days after the new and full moon were the peak spawning periods and therefore the best days to capture wild stock. Common snook egg lipid composition fits the general marine fish fatty acid composition with saturated fatty acids predominating. On the other hand, the omega 3, omega 6 (n-3/n-6) ratio was lower than the typical marine fish and arachidonic acid values were significantly higher than other marine species. This egg fatty acid profile will be helpful in the future to compare it with captive spawned eggs for egg quality purposes. Description of the common snook embryonic and larval development for the first 14 days was carried out. This has strengthened knowledge for this species’ development, and should provide a helpful tool to identify common snook embryos and larvae in the wild. Novel improvements to existing common snook larval culture protocols were implemented. Larval lipid analysis throughout development, and high mortality around day 6 post hatching, suggested that common snook larvae were dying of starvation. Gross morphological development and ultra-structure findings in the digestive and eye system development during the first three days indicated that day 2 post hatching larvae were capable of capturing and digesting food. Additionally, larval nutritional improvements were made, increasing the larval survival. The most significant ones were: finding a smaller and more nutritional prey (SS type rotifers and copepods), finding an optimal stocking and feeding density and the importance that green water technique has on larval survival. Overall, larval success was improved from a zero percent survival during the first 14 days to a 2% survival rate.

No description available.

Ichthyoplankton dynamics in the permanently open Kowie Estuary, in the warm temperate region of South Africa was investigated. The composition, abundance, distribution and seasonality of larval fishes were studied over a two year period between 2004 and 2006. Additionally, tidal exchange of ichthyoplankton and the use of frontal zones in the mouth region of the estuary were also explored between 2008 and 2009. Temporal and spatial trends in occurrence of larval fishes within the estuary and associated marina were obtained from data collected seasonally using boat-based plankton netting at 14 sampling stations along the length of the estuary. A total of 11 128 larval fishes were collected, representing 23 families and 38 taxa. Clupeidae and Gobiidae were the dominant fish families, contributing 47.0 % and 24.7 % respectively to the total catch. Estuarine resident species dominated the overall catch (91 %). A notable absence of older stage larvae and early juveniles characterised the artificial channels of the marina and estuary mouth region. This was attributed to the absence of a shallow, marginal water habitat typical of successful estuarine nursery areas. Tidal exchange of larval fishes was investigated in the Kowie Estuary using a new technique. A set of drifting light traps were set repetitively on the ebb and flood tide every second night for two consecutive 14 day periods during the peak estuarine recruitment period. A total of 553 larval fishes were caught during the study, representing nine families and 26 species. Blenniidae and Clupeidae dominated the catches. Family and species occurrence changed with tide state. Species richness (d) and diversity (H’) varied with tide and was highest on flood tides. Estuary-dependent species, such as Omobranchus woodii were more dominant on flood tides, whilst larvae of marine-spawned species, such as Sardinops sagax, were dominant on the ebb tide. Light trap catches yielded a different composition in terms of development stage / size and species, when compared to towed net studies. Towed plankton nets were again used to study the shear fronts that characterise the mouth region of the canalised Kowie Estuary. It was hypothesised that the convergence zone of the front provides feeding opportunities for ichthyoplankton. Ichthyoplankton, zooplankton and phytoplankton was collected from within and immediately outside of the convergence zone during frontal conditions. Species specific distribution trends emerged from this study. Postflexion larvae and early juvenile stages of the estuary dependent Mugilidae were only present in the foam line of the convergence zones. Some known predators of ichthyoplankton were also present in slightly higher numbers in the convergence zone (isopods) while other predators such as mysid shrimps, chaetognaths and cnidarians were more abundant out of the convergence zone. It appears that these zones may provide access to temporary food patches in the lower estuary but will be accompanied by a trade-off with increased isopod predators. The Kowie Estuary is a heavily impacted system and lack of adequate freshwater supply; artificial channelling and pollution ultimately have a negative impact of the success of this system as a nursery area. Rehabilitation of marginal areas in the lower estuary and marina is suggested as remedial action to re-establish the integrity of the nursery function this system could offer.