KEET-Larva Ikan.pdf

The development of a larval feeding regimen for dusky kob,
Argyrosomus japonicus, with a specific focus on the effect of
weaning period on larval development and survival
Submitted in fulfilment of the requirements for the degree of
MASTER OF SCIENCE
At
RHODES UNIVERSITY
by
Thomas Keet
September 2018

Abstract
One of the biggest limiting factors in marine finfish aquaculture is the low survival rate of
early-stage larvae. Most mortalities can be ascribed to the poor nutritional value of live feeds,
sibling cannibalism, and various stressors that result in swim bladder hyperinflation and/or
starvation during the larval stage.
Research results vary on the best timing for the introduction of artificial feed for good survival
and growth rate in dusky kob larvae. The main objective of this experiment was to improve
survival and growth rate. The experiment focused on a new feeding regime that sought to wean
larvae onto an artificial diet earlier than the current Argyrosomus japonicus standard (weaning
commenced at 16 days after hatch (DAH) versus 20 days after hatch), based on findings and
recommendations made by Musson & Kaiser (2014). Three trials were conducted, each with
five replicates of the two treatments, namely the new feeding regime and the standard feeding
regime in a fully randomised design. Samples from each tank were collected every two days
for the duration of the trial. Morphometric measurements (standard length; body depth; eye
diameter) obtained from these sample larvae were used to compare growth rates between
treatments. The ratio of BD:SL was used to assess larval condition throughout each trial. Tank
survival rates were calculated on the last day of each trial.
The study indicated that in mean water temperatures ranging from 24.3 – 25.2 °C, dusky kob
larvae can be weaned onto an artificial pellet diet from 16 - 21 DAH without any negative
effects on growth, condition and survival. Results from the highest mean temperatures of Trial
2 show a better mean condition in the treatment group during the weaning period (p < 0.05). In
Trial 3, with its lower mean water temperatures of 23.2 °C, larvae in both treatments showed
stunted absolute growth rates of all biometrics when compared to results from the higher mean
temperatures of Trials 1 and 2. During the first 6 days of Trial 3 larvae were in relatively poor
condition, BD:SL ≤ 0.30. During this same period in Trials 1 and 2, mean BD:SL ≥ 0.31,
suggesting that a BD:SL ratio of ≤ 0.30 in non-weaned dusky kob larvae is an indicator of a
degree of starvation. A future study on the morphology and histology of the larval
gastrointestinal tract, specifically the liver and intestines, and how this early weaning regime
affects their ontogeny under differing temperature conditions this needed to investigate the
validity of these initial data on dusky kob larvae condition.

ii
Contents
Abstract................................................................................................................................................... ii
Tables and Figures................................................................................................................................. iv
Acknowledgements............................................................................................................................... vii
Chapter 1: General Introduction and Literature Review.........................................................................1
Global Aquaculture Trends.................................................................................................................1
South African fisheries and aquaculture .........................................................................................1
Dusky kob (Argyrosomus japonicus)..................................................................................................3
Viability as a candidate for aquaculture..............................................................................................3
Commercial rearing of Argyrosomus japonicus larvae.......................................................................4
Larval husbandry and feeding.............................................................................................................5
The beginning of exogenous feeding..............................................................................................6
Cannibalism ....................................................................................................................................6
Larval feeding.................................................................................................................................6
Ontogenetic development ...................................................................................................................7
Gastrointestinal tract development..................................................................................................8
Eye development.............................................................................................................................9
Larval rearing feeding strategies.......................................................................................................10
Larval food: Form and composition..............................................................................................10
Larval mouth gape ........................................................................................................................10
Live feeds......................................................................................................................................11
A comparison of South African and Australian research recommendations ....................................13
A. japonicus sub populations ........................................................................................................13
Larval rearing comparison ............................................................................................................15
Objectives .........................................................................................................................................16
Chapter 2: Materials and Methods........................................................................................................17
Source of experimental fish ..............................................................................................................17
System Design ..................................................................................................................................18
Experimental Design.........................................................................................................................22
Rotifers..............................................................................................................................................22
Brine shrimp (Artemia spp) ..............................................................................................................23
Artificial pellet feed..........................................................................................................................24
Feeding regimes (Treatments) ..........................................................................................................24
Sampling and data collection............................................................................................................29

iii
Data Analysis....................................................................................................................................31
Chapter 3: Trial 1 Results .....................................................................................................................32
Temperature and water quality .........................................................................................................32
Development of larval morphology..................................................................................................32
Survival rate......................................................................................................................................37
Discussion.........................................................................................................................................38
Larval development and growth ...................................................................................................38
Larval survival ..............................................................................................................................38
Development of larval morphometrics .............................................................................................39
Survival rate......................................................................................................................................45
Discussion.........................................................................................................................................46
Larval survival ..............................................................................................................................46
Development of larval morphometrics .............................................................................................47
Survival rate......................................................................................................................................52
Discussion.........................................................................................................................................53
Larval survival ..............................................................................................................................53
Chapter 6: General Discussion..............................................................................................................54
Challenges.........................................................................................................................................55
Review of methods ...........................................................................................................................55
Larval growth and survival ...............................................................................................................58
Larval condition................................................................................................................................62
Observations .....................................................................................................................................63
Conclusions.......................................................................................................................................63
References.............................................................................................................................................65

iv
Tables and Figures
Table 1.1: Water quality variables reported from two dusky kob larval rearing trials.
Table 2.1: Water quality variable measured throughout each trial, the frequency of each
measurement, the instrument used, and the optimal range.
Table 2.2: Number of Artemia ml-1
added to each treatment from 10 to 25 days after
hatching (DAH).
Table 2.3: A daily breakdown of the total dry weight (DW) of the different feed types fed
to the two treatments.
Table 3.1: Pooled water quality data from Trial 1.
Table 6.1 The pooled mean absolute growth rate (mm d-1
) of all biometrics; and the pooled
survival rates (%) in each Trial.
Figure 2.1: Experimental tank setup at Mtunzini Fish Farm Pty Ltd, 2014-2015.
Figure 2.2: An image of a live Brachionus plicatilis, a euryhaline rotifer and a commercially
important live feed for fish larvae in the aquaculture industry.
Figure 2.3: A schematic of the different feeds introduced at different stages during the trial
period in the C treatment.
period in the T treatment.
Figure 2.5: A 23 DAH dusky kob larvae indicating where measurements of total length
(TL); notochord length (NL) / standard length (SL); body depth (BD); eye
diameter (ED) were taken.
Figure 3.1: The development of mean SL (mm) of larvae fed two feeding regimens
(Treatments C & T) over time (DAH) in Trial 1.

v
Figure 3.2: Frequency histograms of SL from the control and treatment, C and T
respectively, on the last day of sampling in Trial 1 (28 DAH).
Figure 3.3: The development of mean BD (mm) of larvae fed two feeding regimens
Figure 3.4: Frequency histograms of BD from the control and treatment, C and T
Figure 3.5: The development of mean ED (mm) of larvae fed two feeding regimens
Figure 3.6: Frequency histograms of ED from the control and treatment, C and T
Figure 3.7: Mean BD:SL (mm) of larvae fed different feeding regimens (Treatments C &
T) over time (DAH) in Trial 1.
Figure 3.8: Regression analysis of BD as a function of SL for each treatment in Trial 1.
Figure 3.9: Box & Whisker plot of the mean survival rates of each treatment group on the
final day of the Trial 1 (28 DAH).
T) over time (DAH) in Trial 2

vi
T) over time (DAH) in Trial 3.
Figure 6.1: A scatterplot and linear model of pooled larval standard lengths in each trial.
Figure 6.2: Evidence of Type II cannibalism at 31 DAH in Trial 3.

vii
Acknowledgements
This research project was a THRIPS funded initiative, for which I’m thankful to the NRF for the
financial support.
The experiments were carried out at Zini Fish Farms (formerly Mtunzini Fish Farm) near the town of
Mtunzini on the northern coast of KZN. Without the generous access to the hatchery facility I was
afforded, as well as all the support from the farm staff, I never could have completed all three trials. To
Gavin Carter, thank you for giving me the opportunity to come and work at the farm. To the farm staff,
thank you for all your encouragement and help in the hatchery. And to Neil Stallard, thank you for
everything you have taught me, and continue to teach me, about all things fishy.
I would like to thank my supervisors, Dr Horst Kaiser and Dr Tom Shipton, for their encouragement
and patience over the years. Dr Shipton, thank you for being such a hospitable host during my visits to
Grahamstown. Dr Kaiser, thank you for your guidance and patience during both the field trials and the
write-up phases of this research project.
Lastly, I’d like to thank my family. I wouldn’t be where I am today without my parents, and I wouldn’t
be the person I am today without my siblings. Thanks for putting up with me. A special mention of
gratitude must go to my sister, Emma, and her editing skills.

Chapter 1: General Introduction and Literature Review
Global Aquaculture Trends
Aquaculture is understood to mean the farming of aquatic organisms, including fish, molluscs,
crustaceans and aquatic plants. Farming implies some form of intervention in the rearing
process to enhance production, such as regular stocking, feeding or protection from predators.
Farming also implies individual or corporate ownership of the stock being cultivated (FAO,
1988).
According to data released by the Food and Agriculture Organisation of the United Nations in
their 2016 report, The State of World Fisheries and Aquaculture (FAO, 2016), aquaculture is
the fastest growing food production sector globally. It now provides more than half of all the
fish consumed by humans. This contribution to total fish consumption is projected to increase
over the next decade as wild capture fisheries stagnate and begin to decline (FAO, 2016). Wild
capture fisheries source fish primarily in the oceans, although some also occur in lakes and
rivers, and areas that support populations of commercially valuable aquatic species.
Worldwide growth in aquaculture production has increased due to the increase in demand for
food fish. The term ‘food fish’ refers to any species of fish used as a food source by humans.
Factors such as human population growth, a substantial increase in “demand from an emerging
global middle class” (FAO, 2016), and the now well-publicised health benefits of eating fish
have aided this growth. Production of teleost fish from major industrialised producers, i.e.
USA, Spain, France, Italy, Japan, and the Republic of Korea, however, has declined in recent
years. A major reason for this is the availability of fish imported from developing countries
where production costs are lower (FAO, 2016).
Aquaculture in Africa contributes only 0.15% towards the total gross domestic product (GDP)
of all African countries, while marine and inland fisheries contribute 1.11% towards total GDP
(FAO, 2016). South Africa is a minor player in the African aquaculture sector. Instead it relies
heavily on wild capture fisheries (FAO, 2016).
South African fisheries and aquaculture
South African fisheries depend on natural but highly variable fish stocks. This variation is a
result of both natural causes and human exploitation. South Africa has long benefited from
abundant fish resources along its coastline. A number of inshore fisheries are now overfished,

2
both legally and illegally. According to the Department of Agriculture, Forestry and Fisheries
(DAFF), 68% of all commercial line-fish stocks have collapsed and a further 15% are
overexploited (Department of Agriculture, 2014). This will inevitably lead to a reduction in
wild capture supply, stimulating the development of a marine aquaculture sector, or mariculture
sector. Geographically, the country does not have many features conducive to aquaculture.
South Africa is surrounded by volatile and destructive seas, and has very few sheltered bays,
coves or fjords. The farming of filter-feeding bivalves such as oysters and mussels requires
nutrient-rich waters in sheltered water bodies such as inland bays, estuaries and lagoons.
Saldanha Bay, on the west coast of South Africa, is one of the few sites that fit these
requirements. Cage culture of marine finfish species in the open ocean is risky and expensive.
An attempt to introduce salmon cage farming in the Western Cape failed due to persistent
damage along the high-energy coastline (Moehl et al., 2004).
In monetary value the South African commercial mariculture industry is dominated by the
culture of the South African abalone, Haliotis midae. By taking advantage of a collapsed wild
capture fishery and a strong demand from countries in Southeast Asia, this mollusc is now
successfully farmed in land-based recirculating systems in several places along the western,
southern and south-eastern coastline of South Africa.
Marine finfish aquaculture is still in its developmental phase. The dusky kob, Argyrosomus
japonicus has been flagged as an important candidate species for aquaculture in South Africa
(Hinrichsen, 2008). This species is highly sought after as a food fish (Ballagh et al., 2008), it
has a high tolerance to varying water quality conditions (Whitfield, 1999) and a fairly good
growth rate (Griffiths, 1996).

3
Dusky kob (Argyrosomus japonicus)
Dusky kob is a carnivorous, euryhaline finfish of the family Sciaenidae. This family includes
croakers and drums. Sciaenids are mostly demersal fish and are found in fresh, estuarine and
coastal marine waters in subtropical to temperate regions of the Atlantic, Indian and Pacific
oceans (Trewavas, 1977). The family contains approximately 70 genera and about 270 species
worldwide (Nelson, 1994; Watson et al., 2003). Griffiths & Heemstra (1995) reported that A.
japonicus has been known by at least 13 other synonyms and, until 1995, it was misidentified
as A. hololepidotus in some areas, notably Australia and South Africa (Lin, 1940; Trewavas,
1977; Griffiths & Heemstra, 1995).
A. japonicus is a popular line fish found along the southern and eastern coastline of southern
Africa, as well as the coastlines of Australia, Hong Kong, Pakistan and Japan (Fielder &
Bardsley, 1999). It has mildly flavoured white flesh, making it very popular with both
commercial and recreational line fisheries. This, together with its desirable life-history traits
(fast growth and late sexual maturity) and high tolerance to varying water quality conditions
(Whitfield, 1999), indicate its potential as an aquaculture species (Griffiths, 1996).
Griffiths (1996) suggested that the species’ longevity (maximum estimated age of 42 years)
and large size at sexual maturity (50% of males and female mature at 92 and 107 cm,
respectively) evolved in conjunction with a low natural mortality rate, making it susceptible to
stock depletion and recruitment due to overfishing. This hypothesis is backed up by data from
DAFF (2014), which illustrate a steady decline in stock size of kob, whereby silver kob (A.
inodorus) and dusky kob (A. japonicus) have been lumped into the same category caught
between 2000 (547 t) and 2012 (221 t). Dusky kob wild stocks were last assessed in 1997 by
per-recruit analysis and were categorised as ‘collapsed’ or < 25% of unexploited level
(Griffiths, 1997). The decline in natural stock size, similar to that seen in many popular marine
line fish species worldwide, provides additional incentive for the development of commercial
aquaculture.
Viability as a candidate for aquaculture
Marine finfish aquaculture is in its developmental phase in South Africa, but the importance of
the culture of dusky kob (Argyrosomus japonicus) is expected to increase greatly (Hinrichsen,
2008). Dusky kob have several traits that make it an attractive candidate species for fish
farming. Since the 1990s, several applied studies on rearing protocols for dusky kob larvae

4
have been conducted. A large portion of this research was conducted in Australia, where A.
japonicus occurs naturally.
A. japonicus has a wide global distribution. In Australia, it is highly sought-after by recreational
fishers (Kailola et al., 1993; Henry & Lyle, 2003). It is commonly referred to as mulloway.
Farming of the species, mostly in sea cages and earthen ponds, has been practiced for a longer
time in Australia in comparison with South Africa, but the same rearing protocols are utilised
in both countries. However, the geographical isolation and different environmental conditions
may result in phenotypic plasticity (Scheiner, 1993). Phenotypic plasticity is “the ability of a
single genotype to produce more than one alternative form of morphology, physiological state,
and/or behaviour in response to environmental conditions” (West-Eberhard, 1998). In
aquaculture, polymorphisms in fish are thought to be phenotypically plastic responses to their
rearing environment (Meyer, 1987), meaning that differing rearing environments can illicit
differing morphological development responses from the same species.
Wild-caught broodstock are used to produce most of the seed for commercial grow-out
operations (Musson & Kaiser, 2014; Hunter, 2015) in South Africa, with the first generation
(F1) of captive-bred broodstock in South Africa producing seed for the first time in 2014 under
the supervision of Andre Bok at Pure Ocean Pty Ltd.’s facility in East London. The species’
relatively late onset of sexual maturity, an advantage when viewing its commercial viability in
grow-out operations in isolation, is a hindrance for seed supply. Sexually mature specimens
are relatively large (> 100 cm total length) and difficult to capture without harming the animal.
Captive-bred specimens (F1 progeny) selected for desirable traits only reach sexual maturity
after a minimum of 5–6 years, a long-term investment for a start-up venture. Thus, dusky kob
broodstock in South Africa are scarce. As a result, maximising the efficiency of larval rearing
with the currently limited supply of broodstock is essential for the success of the industry.
Commercial rearing of Argyrosomus japonicus larvae
Research to determine the feasibility of farming the species began in Australia in 2008 (Guy &
Cowden, 2012; Guy & Nottingham, 2014), which lead to an emergent industry. A key
constraint of marine aquaculture globally is the low availability and high cost of juveniles for
grow-out operations (Guy & Cowden, 2015). This is recognised as one of the main bottlenecks
to commercial expansion for new aquaculture species (Schwartz et al., 2009). To illustrate the
high cost of dusky kob fingerling production, Allan (2008) reported a price of AU$ 1.05 per
35–mm kob fingerling in NSW, Australia. Comparatively, Barramundi (Lates calcarifer) is an

5
established commercial aquaculture species in Australia and cost approximately AU$ 0.48 -
0.60 per 40–mm fingerling in 2015 (pers. comm. David Borgelt, hatchery manager at Jungle
Creek Aquaculture Pty Ltd).
Guy & Cowden (2015) studied the feasibility of modifying and adapting unused prawn
hatcheries for temperate marine fish culture in New South Wales, Australia, and reported on
production costs for the rearing of two fingerling size classes, i.e., 40 mm total length (TL), 1
g fish, and 100 mm TL, 12 g fish. Producing 630 000 1-g fingerlings cost an average of AU$
0.104 per fish, while producing 150 000 12-g fingerlings cost an average of AU$ 0.281 per
fish.
The production of dusky kob for commercial operations in South Africa has been centred in
the East London Industrial Development Zone (ELIDZ), specifically at Oceanwise Pty Ltd.
and Pure Ocean East London Pty Ltd. (POEL). The cost per 0.35-0.5 g fingerling in 2015 was
R2.60 – 3.00 (Andre Bok, chief executive at POEL), which equates to AU$ 0.280 – 0.323 per
fingerling. Although cheaper than that of the Australian-produced fingerlings, this price acts as
a disincentive to potential grow-out operations. This cost is further increased by transport
mortality (Guy & Cowden, 2015).
Larval husbandry and feeding
While the main reasons for high costs of juveniles in any pioneering animal production industry
can normally be attributed to initial capital outlays for research and development (R & D) and
a lack of economy of scale, marine finfish hatcheries have the added complexity of needing to
produce stable quantities of high-quality live feed (Støttrup & McEvoy, 2008) for the early
stages of larval development. Greater efficiency in production protocols is thus needed, and
this can be attained by species-specific applied research (Guy & Cowden, 2015). Studies on
the environmental conditions for the rearing of A. japonicus included research on the effect of
salinity on juvenile fish growth (Fielder & Bardsley, 1999), photoperiod and feeding interval
(Ballagh et al., 2008), temperature (Collett et al., 2008) and the effect of stocking density on
food conversion ratio and survival (Collett, 2007). Husbandry plays an important role in larval
rearing success, and a major contributing factor within this context is larval nutrition.
Optimisation of larval rearing feeding protocols has the potential to reduce the impact of two
major mortality events experienced in marine finfish hatcheries. These occur at the start of
exogenous feeding and as a result of sibling cannibalism.

6
The beginning of exogenous feeding
The first is mortality that occurs during yolk sac absorption and the beginning of exogenous
feeding, from 1 – 5 DAH (Musson & Kaiser, 2014). A large portion of the mortality associated
with this period can be attributed to sensitive physiological events at this early stage as dusky
kob larvae inflate their swim bladder by gulping air from the water surface. Any oily
contaminant accumulation on the water surface will hinder this process (Fielder & Heasman,
2011). Losses due to swim bladder malformation are typically in the order of 5 – 10% of all
marine finfish fingerlings produced but can reach as much as 50% in some cases (Woolley &
Qin, 2010). Surface skimmers are employed during this period to reduce the impact of this
issue. The speed at which enriched rotifers lose their nutritional value, i.e. within 6 hours at
30°C (Fielder & Heasman, 2011), and the undeveloped larval gastrointestinal tract’s (GIT)
inability to efficiently absorb nutrients play a role in this event (Musson & Kaiser, 2014).
Enrichment products such as frozen algal pastes and powders have been used as a reliable
alternative to live algal cultures in rotifer production, but “further work is required on the
effectiveness of these frozen concentrates, before recommending the wide scale use of them”
(Guy & Cowden, 2015).
Cannibalism
The second cause of mortality is cannibalism, which tends to coincide with the weaning period
or metamorphosis. Hecht & Appelbaum (1988) introduced the terms Type I and Type II
cannibalism. Type I, or partial cannibalism, where biting of the abdomen or caudal fin occurs,
often results in bacterial infections. This is later partly replaced by Type II or complete
cannibalism when size variation between siblings increases and total prey ingestion ‘head-first’
becomes possible because of a sufficiently large gape width in some of the larger individuals
(Hecht & Appelbaum, 1988; Baras & Jobling, 2002; Kestemont et al., 2003). A considerable
portion of this size variation within commercially reared A. japonicus cohorts can be attributed
to genetic variation due to the undomesticated nature of the commercial populations of
broodstock in South Africa (Hunter, 2015). Nutritional and environmental factors also play an
important role. Providing larvae with a feeding regime that reduces early developmental size
variation could limit the impact of this mortality event.
Larval feeding
Studies with a focus on ideal feed types and the optimal feeding regime for dusky kob larvae
have been conducted in Australia. Ballagh et al. (2010) conducted three experiments. In the
first, three Artemia ration sizes (0, 50, 100% of a standard Artemia ration) and two timings of

7
artificial pellet feed introduction (14 and 23 days after hatching) were tested for their effect on
larval weight, total length (TL) development and survival. The second and third experiments
were short-duration prey preference experiments to determine the optimal larval TL for first
introduction of two feed types, Artemia nauplii and a pelleted artificial feed.
Results from the first experiment (Ballagh et al., 2010) indicated that no interactions existed
between the two main factors i.e. Artemia ration and timing of diet change for larval weight or
TL for all sampling times. There was also no effect on larval weight and TL for the timings of
artificial pellet introduction. There was, however, a significant difference in weight and TL
between Artemia-starved larvae (i.e. 0% Artemia ration size) and larvae fed the 50 and 100%
ration size, but no difference between the 50 and 100% Artemia ration size treatments. There
was a significant statistical interaction between time of pellet introduction and Artemia ration
size for larval survival. The two treatments with a 50% Artemia ration, but differing pellet
introduction time, i.e. 14 and 23 days after hatching (DAH), had survival rates of 13.7 ± 4.1%
and 1.0 ± 0.6%, respectively. This interaction suggests that a 50% Artemia ration was
insufficient to achieve suitable survival rates without the earlier introduction of artificial feed.
The second experiment determined that the larvae selected Artemia nauplii equally to rotifers
at a TL of 5.2 ± 0.5 mm. The third trial showed that larvae selected pellets equally to Artemia
at a TL of 10.6 ± 1.8 mm.
As fish are poikilotherms, their rate of development is determined by the ambient water
temperature. For this reason, larval size, as opposed to age, is more suitable for estimating the
onset of metamorphosis (Fukuhara, 1991). This has been demonstrated for many species
(Policansky, 1983). Weaning and metamorphosis also tend to coincide in the marine larval
rearing process. Different recommendations relating to the optimal larval size for weaning
dusky kob in South Africa and Australia (Ballagh et al., 2010; Musson & Kaiser, 2014) could
be due to the two populations’ differing responses to environmental aspects (feeding, tank
environment, etc.). Alternatively, differences between locations where research has taken
place, even when standardised methods and experimental conditions are used, could have an
unexplained effect on results (Wahlsten et al., 2003).
Ontogenetic development
Fish larvae are tiny self-supporting vertebrates that need to undergo significant histological and
morphological change and development during their early life stages to be able to avoid
predation and to find and digest food (Osse et al., 1997). Larvae, during their early

8
developmental stages, lack full functionality of many physiological structures, with the
gastrointestinal tract (GIT) being a major example. They therefore have different feed
requirements to those of juvenile and adult fish (Kestemont et al., 1995).
Gastrointestinal tract development
GIT development of farmed fish species has been studied and described (Elbal et al., 2004;
Gisbert et al., 2004; Micale et al., 2006; Suzer et al., 2013; Solovyev et al., 2016), but a study
of the morphology and histology of the GIT development in A. japonicus, spawned on a South
African farm, has only recently been published (Musson & Kaiser, 2014). More knowledge of
the developmental process is needed to determine when to wean fish larvae from live food onto
an artificial diet (Cahu & Infante, 2001). Teleost digestive system development can be divided
into three stages; the eleutheroembryonic stage, the exogenous feeding stage, and the functional
digestive tract stage (Buddington & Christofferson, 1985; Boulhic & Gabaudan 1992; Bisbal
& Bengtson 1995).
The eleutheroembryonic stage occurs during the period from hatching to the completion of
endogenous feeding. The gut is straight and undifferentiated with ciliated cells (Kjørsvik et al.
1991). The larvae rely solely on an endogenous source of nutrition, namely the remaining yolk
sac post-hatch.
The exogenous feeding stage is characterised by pinocytosis and intracellular digestion and
nutrient absorption (Watanabe, 1982) and the appearance of supranuclear vacuoles in the
hindgut, allowing larvae to digest proteins and absorb nutrients in the absence of a functional
stomach (Govoni et al., 1986). This stage ends before the formation of gastric glands, while
digestive capabilities remain insufficient for the effective assimilation of an artificial diet
(Buddington & Christofferson, 1985). During this phase, live feeds are introduced into the
larval rearing tanks.
The functional digestive tract stage begins with the appearance of gastric glands and pyloric
caecae. Gastric glands are tubular branched glands in the mucosa of the fundus of the stomach.
These glands contain parietal cells that secrete hydrochloric acid and zymogenic cells that
produce pepsin, a protease enzyme. Gastric glands increase digestive efficiency by providing
extra-cellular protein digestion followed by membrane transport, replacing the less efficient
processes of pinocytosis and intracellular digestion (Govoni et al., 1986). Pyloric caecae are
finger-like projections that branch off from the junction between the stomach and the large
intestine. The brush border membranes of each caecum contain hydrolytic enzymes and, aided

9
by the large surface area provided by the organ’s structure, are a site where the digestion of
sugars and amino acids occurs. The presence of gastric glands and pyloric caecae are associated
with the ability of marine larval finfish to digest complex proteins and sugars. At this
developmental stage, clownfish, Amphiprion percula, were ready to accept an artificial diet
(Gordon & Hecht, 2002).
Musson & Kaiser (2014) conducted a study of the histological development of the GIT of dusky
kob larvae during the first 30 days post-hatch. Recommendations were made regarding when
larvae had a mature GIT that would allow them to digest and assimilate lipids and complex
proteins. This was done by randomly sampling 50 larvae daily from 15 separate spawning
events over two spawning seasons, for 30 days. Average temperature for the trials was 21.0 ±
1.42 °C and larvae had a total length of 18.9 ± 3.56 mm at 30 DAH. Degree days (DD), a
cumulative summation of average daily temperatures over the trial period, were utilised to
measure the effect of varying temperatures between trials on growth rate, ranged from 608 to
672 DD at 30 DAH. Larvae commenced exogenous feeding at 4 DAH (110 DD), 22 DD after
the presence of a functional mouth. Differentiation of the alimentary canal into the
buccopharynx, oesophagus, intestine and visible hindgut was completed by 88 DD. This
coincided with the appearance of supranuclear vacuoles in the hindgut. Supranuclear vacuoles
suggest the presence of pinocytosis and intracellular protein metabolism (Watanabe, 1982),
facilitating the absorption of proteins from the GIT. Gastric glands were visible by 224 DD (9-
11 DAH), while fully formed pyloric caeca with associated goblet cells were observed at 364
DD. By 338 DD, the differentiation of the digestive system was complete, and it was fully
functional by 466 DD.
Eye development
Fish feeding behaviour is controlled by mechanical, chemical and optical stimuli that are
received and processed by the brain based on input from the respective sensory organs, such
as the lateral line, taste buds and the eye.
Vision is considered to be the main sensory system in fish larvae, since it is required for
orientation, feeding, learning and avoidance of predators (Hubbs & Blaxter, 1986; Schreck et
al., 1997). A study of the eye ontogeny of meagre larvae (Argyrosomus regius), a
Mediterranean fish of the same genus as dusky kob, showed that larvae had well-developed
scotopic sensitivity by 17 DAH (TL 8.14 ± 1.64 mm) (Papadakis et al., 2018). Studying the
eye development or the relative size of the eye in larvae reared under different feeding

10
conditions could provide insight into whether dusky kob larvae have fully developed eyes
around the time of weaning in each treatment.
Larval rearing feeding strategies
The first major landmark in larval development post hatching is the change from endogenous
to exogenous feeding. This transition is a critical first step in A. japonicus larval development
due to high mortality occurring between 1 and 5 DAH (Musson & Kaiser, 2014). Once
endogenous feeding is complete, the availability of sufficient food in a size suitable throughout
their development is essential for high rearing success of any marine finfish species on a
commercial scale (Appelbaum & Uland 1979; Holmefjord, 1993; Jähnichen & Kohlmann,
1999).
Larval food: Form and composition
Although many attempts have been made to find suitable alternatives to live food, the feed
supply for marine finfish larvae in commercial aquaculture relies heavily on the culture of live
food organisms during the early stages of larval development, as opposed to using an artificial
diet. The GIT of larvae transitioning from endogenous to exogenous food is rudimentary and
lacks the ability to efficiently digest and absorb many nutrients. Detailed investigations into
the use of artificial microdiets containing pre-digested and soluble proteins in marine larval
rearing trials have been conducted by Kvale et al. (2002) and Tonheim et al. (2007). Findings
from these studies suggest that these diets had limited success. The appearance of taste buds at
a relatively early stage of development in most teleost species, 4 DAH or 110 DD in A.
japonicus (Musson & Kaiser, 2014), suggests that larvae are capable of evaluating the
palatability of prey items from a very early stage.
Larval mouth gape
Larval mouth gape size at this early stage is another factor determining the success of feeding.
Gape size is considered to reflect the maximum size of prey a larva can swallow. This has been
shown in a number of fish species, including red snapper, Lutjanus argentimaculatus (Doi et
al., 1997). Therefore, all food items presented during rearing need to be smaller than the
average gape size. These limitations are species-dependent with a paucity of data for dusky
kob.
Brown et al. (2003) measured first-feeding Atlantic cod (Gadus morhua) at 4.3 mm TL with a
gape width of approximately 160 µm. Larvae were fed small (192 X 150 µm) and large (242
X 181 µm) rotifers under different conditions. First-feeding cod seemed not to ingest the large

11
rotifers until day 8, and preferred the small rotifers until day 20, when the larvae were 5.7 mm
long with a gape width of about 290 µm. Larvae were able to ingest rotifers longer than their
gape, presumably because rotifer width was less than the larval gape and/or because the rotifers
are deformable.
First-feeding red drum, Sciaenops ocellatus, consumed relatively large prey as long as prey
width was lower than larval gape of approximately 220 µm (Krebs & Turingan,
2003). Hamasaki et al. (2009) demonstrated that Amberjack (Seriola spp.), a particularly
small-mouthed fish, prefer 140-µm rotifers at first feeding.
The mouth of dusky kob larvae opened by 65 DD (Musson & Kaiser, 2014) and a rudimentary
oesophagus appeared by 80 DD or 3 DAH. No data on dusky kob larvae gape size could be
found in a review of the literature.
Live feeds
Live feeds are crucial to the larva’s digestive tract development. Amino acids from digested
proteins found in the GIT are freely available in live feeds and support larval digestion in
marine fish larvae (Rønnestad et al., 1999). Fish zymogens, enzymatically inactive precursors
of proteolytic enzymes found in the pancreas, are activated by invertebrate enzymes
(Dabrowski, 1991; Abi-Ayad & Kestemont, 1994; Mischke & Morris, 1998). The gut contents
of live feed act as kick-starters to the gut function of the larvae, as they contain digestive
enzymes and partially digested molecules from the feeding activity of the live food.
Zooplankton of a suitable size should be present in sufficient quantities in the culture tank so
as to be easily available to larvae that are ready to commence exogenous feeding. Care should
be taken to not overload the culture tank with too much live feed as this can reduce water
quality, especially if live feed does not survive for very long under larval tank conditions.
Artemia nauplii, for example, do not survive for long in freshwater and those that are not
consumed within the first hour after feeding reduce water quality as they decompose (Paulet,
2003).
Live feed can be added to larval culture tanks in two ways. Either a sterile culture of rotifers in
a suitable density is prepared in the larval rearing tank, or concentrated rotifers are added to
the larval tank at the appropriate time from a rotifer culture tank in which rotifers are
continuously produced. Quantities are then increased as the demand by the larvae increases.
The most common first live feed organisms cultured in commercial marine finfish hatcheries
are rotifers, predominantly Brachionus plicatilis and B. rotundiformis, (Lubzens et al. 2001)

12
and brine shrimp (Artemia spp.). Together, they are the most widely used live prey in
aquaculture (Conceição et al., 2010).
Rotifers are the best choice as a first feed in marine finfish hatcheries because of their small
body size, 70–350 µm, depending on the strain and age (Conceição et al., 2010), relatively low
mortality rate, and ease to rear in high-density cultures (Yoshimatsu & Hossain, 2014). They
are easily detected and captured by young larvae due to their swimming movements in the
water column, and they are highly digestible (Conceição et al., 2010).
Rotifer production requires labour and capital input for infrastructure, and, under most
circumstances, the use of enrichment additives such as algal pastes, fish oil emulsions and
probiotics. Sterile live cultures of different microalgae can also be maintained and used to
bolster enrichment. Whenever microalgae are used as a direct food source or as an indirect food
source, in the production of rotifers, brine shrimp or copepods, growth of the animals is usually
superior when a mixture of several microalgae species is used (Becker, 2007). Rotifers are non-
selective feeders and consume particles of a suitable size suspended in the water column.
Rotifers act as nutrition delivery vessels to larvae, providing them with enzymes, bacteria and
feed, a process known as bioencapsulation.
There are three methods of rotifer culture typically used in hatcheries (Yoshimatsu & Hossain,
2014). These methods are batch cultures, continuous cultures, and semi-continuous cultures.
Batch cultures entail inoculating a culture tank at a certain density and then harvesting the
entire contents of the tank after a period of time, while continuous and semi-continuous cultures
entail regular to semi-regular harvesting of a portion of the culture, and then replacing the
harvest volume with fresh or filtered culture medium.
Brine shrimp (predominantly Artemia salina) are used as a follow-up live feed to rotifers. The
cost and quality of brine shrimp can fluctuate as the supply is dependent on both the worldwide
aquaculture demand as well as the weather patterns affecting the primary harvest areas
(Sorgeloos et al., 2001; Callan et al., 2003). In most circumstances, brine shrimp culture costs
constitute a substantial percentage of hatchery feed costs (Ballagh et al., 2010). Thus, reducing
the amount of brine shrimp needed for rearing can reduce production costs. Decapsulation and
enrichment of brine shrimp is crucial. The decapsulation process removes the hard outer layer
of the cyst, making the hatching process less energy-demanding for the nauplii. In many cases,
when this is done, the nauplii do not hatch as the embryo is being fed without the shell. This
has the highest nutritional value as hatching reduces the calorie content. The composition of

13
decapsulated brine shrimp cysts is globally the same as that of newly hatched nauplii, with
about 50 – 57% protein, 13 – 14% lipid, 6 – 7% carbohydrate and 5 – 9% ash, but their dry
weight (DW) and energy content is on average 30 – 40% higher than that of instar I nauplii
(Conceição et al., 2010). Enrichment techniques and products have been developed in order to
overcome essential fatty acid deficiencies and are generally quite effective in boosting the brine
shrimp levels of eicosapentaenoic acid (EPA) and arachidonic acid (ARA) (Conceição et al.,
2010).
Enriching entails feeding post-hatch nauplii a paste or powder or emulsion solution containing
nutrients (mostly lipids, fatty acids and vitamins) essential to marine finfish larval development
(Van Stappen, 1996; Fernandez, 2001). All enrichment products, of which there are many
different brands and formulations, contain unsaturated fatty acids in varying proportions.
“Feeding of recently hatched Artemia nauplii to mulloway larvae is discouraged as nutrient
deficiencies have been found to promote high mortality in pre-metamorphic larvae.” (Fielder
& Heasman, 2011). The amino acid profile of brine shrimp protein has been shown to be
unbalanced for several larval species (Conceição et al., 1998; Conceição et al. 2003; Aragaão
et al. 2004; Ruiz et al. 2008). Enrichment is costly. Reducing the amount of brine shrimp used
can be approached by either reducing the daily per-larva number of brine shrimp fed or weaning
larvae onto an artificial diet at an earlier age.
To replace the live food component in larval diets, it is necessary to find a diet that is water
stable and can be accepted, ingested, digested and assimilated at rates comparable to that found
for live feeds (Jones et al., 1993). There are many commercially available artificial diets with
varying quality and price levels. Examples for marine finfish larvae diets are: Golden Pearl®,
Otohime®, INVE Proton®, and INVE NRD 4/6 crumble. Fielder & Heasman (2011)
recommend a particle size of between 200 – 400 µm at first introduction of artificial feed.
A comparison of South African and Australian research recommendations
A. japonicus sub populations
Comparative genetic studies of local populations of A. japonicus that occur around Australia
(western, southern and eastern coastlines) and South Africa show evidence of divergent
genotypes (Farmer, 2008; Silberschneider & Gray, 2008).
Farmer (2008) compared the mitochondrial DNA (mtDNA) of A. japonicus collected in
different regions of Western Australia, as well as corresponding, albeit restricted, data on
specimens caught on the east coast of Australia and southern Africa. There were significant

14
differences in genetic composition. These comparisons indicated a relatively recent divergence
between the Australian sub-populations, but a much longer period of isolation with regard to
the South African population, indicating that the Australian and southern African populations
could represent different species (Farmer, 2008).
Silberschneider & Gray (2008) identified that A. japonicus in south-eastern Australia had
similar growth rates, but matured at smaller lengths and younger ages, compared to the
populations in Western Australia and South Africa. They found that 50% of wild-caught A.
japonicus males in New South Wales (NSW), eastern Australia, were sexually mature at 51 cm
total length (TL), while 50% of females were mature at approximately 68 cm TL. This is lower
than the estimates reported by Griffiths (1996) for the South African population.
Thus, due to geographical separation and environmental pressures exerted on each population,
there could be differences in the morphological development between species or populations.
This could have implications for larval rearing protocols currently being used by the South
African industry, as some aspects in the procedure have been taken from studies conducted in
Australia.
The objective of this applied research project was to test methods of weaning larvae onto an
artificial diet. By comparing findings from this study to the results from the research conducted
in Australia larval rearing methods and their differences between experiments will be
discussed.
Although, specific research on effective dusky kob larvae feeding techniques in the two above-
mentioned countries is difficult to compare due to a lack of common variables in the
recommendations proposed at the end of studies, an attempt at comparing two, one from each
country and both relevant to this research, is described and shown below (Table 1.1).

15
Larval rearing comparison
Table2.1: Water quality variables reported from two dusky kob larval rearing trials.
pH DO (mg
L-¹)
Temperature
(°C)
Salinity
(g L-¹)
Total Ammonia
(mg L-¹)
Reference
8.2 6.7 ± 0.3 23.3 ± 0.8 35.3 ± 0.1 0.1 ± 0.0 Ballagh et al.
(2010). AUS
8.00 7.3 ± 0.9 21.0 ± 1.42 Not
available
< 1.0 Musson & Kaiser
(2014). RSA
Ballagh et al. (2010) recommend weaning periods that coincide with 5.2 ± 0.5 mm TL for
Artemia and 10.6 ± 1.8 mm TL for artificial pellet diets. This recommendation was based on
behavioural observation and gut contents analysis.
Musson & Kaiser (2014) proposed that weaning onto a suitable artificial diet should commence
at 16 DAH. This recommendation is based on a morphological and histological study of the
GIT development of dusky kob larvae. The growth curve of dusky kob larvae, constructed
using TL measurements taken daily as a function of DD was represented by the regression
model (Musson & Kaiser, 2014):
y = 3.37 – 0.0099 x + 0.00006 x²
where y = TL (mm), and x = DD
(r² = 0.987; F2, 47 = 861, p < 0.0001, for all model coefficients).
DD is a common variable that can be calculated from each of the respective recommendations
to compare them. Using the minimum and maximum TL measurements recommended by
Ballagh et al. (2010) for optimal time of weaning onto an artificial pellet diet in the regression
model (Musson & Kaiser 2014) for larval growth, their recommendation equates to the period
of 394 – 479 DD.

16
The suggestion by Musson & Kaiser (2014) for the optimal time of weaning onto an artificial
diet equates to 315 – 360 DD, at a TL range of 6.2 – 7.6 mm, as opposed to the TL range of
8.8 – 12.4 mm suggested by Ballagh et al. (2010). The average temperature in the South African
study (Musson & Kaiser 2014) of 21.0 ± 1.42 o
C was lower than that used by Ballagh et al.
(2010) of 23.3 ± 0.8 o
C. Therefore, larvae in the former study should have experienced
relatively slower growth and development (Table 1.1). These differences in research methods
and results support the need for further investigation.
Objectives
The objective of this applied research project was to test methods of weaning larvae onto an
artificial diet. By comparing findings from this study to the results from the research conducted
in Australia, larval rearing methods and their differences between experiments will be
discussed.
Using histological and morphological data on the development of the dusky kob larvae’s GIT
(Musson & Kaiser, 2014), it was hypothesised that a dusky kob larva’s GIT is physiologically
capable of digesting and absorbing complex proteins and fats at an earlier stage than suggested
by Ballagh et al. (2010). Therefore, weaning of larvae onto an artificial pellet diet may be
possible at an earlier stage, reducing live feed culture costs and labour. The study was designed
to test whether earlier weaning had an effect on growth, condition, and survival rate of dusky
kob larvae. The benefits of weaning larvae at an earlier stage also include:
i) minimising weaning difficulty due to larvae becoming accustomed to Artemia, as
discussed by Canavate & Fernandez-Diaz (1999),
ii) reducing labour and Artemia enrichment costs,
iii) and addressing cannibalism in the rearing of A. japonicus larvae. Incidences of
cannibalism tend to peak during the time of weaning larvae onto artificial feed,
approximately 20 DAH (Musson & Kaiser, 2014). Weaning at an earlier stage of
development may help to reduce the effect of cannibalism. The younger the larvae, the
less extreme the size variation within the cohort, thus survival during the larval rearing
period could be influenced positively by an earlier inclusion and acceptance of artificial
feed into the larval diet.

17
Chapter 2: Materials and Methods
Source of experimental fish
Due to a scarcity of broodstock in South Africa, fertilised eggs were sourced from several
places throughout the country. Dusky kob broodstock at Mtunzini Fish Farm (Pty) Ltd (MFF)
were not in suitable spawning condition during the initial stages of the research. Some fish
were recently-caught wild specimens, some had been transported from East London only one
month prior to the beginning of the research, and some had been housed in temporary porta-
pool tanks during construction of the permanent broodstock holding tanks.
For these reasons, larvae for Trial 1 were sourced from broodstock held by Pure Ocean
Aquaculture Pty Ltd in East London. Larvae for Trial 2 were sourced from broodstock held by
the Department of Agriculture, Forestry and Fisheries (DAFF) at their research facility in Cape
Town. For Trial 3 larvae were obtained from broodstock held at MFF.
Using photoperiod and temperature manipulation, the captive specimens were exposed to
conditions that emulate their natural spawning conditions. Gonad development was tracked by
taking scheduled sample biopsies of developing oocytes. This was done by anaesthetising the
fish in their holding tanks using a bath treatment of the synthetic clove-oil derivative Aqui-S®
at 5 – 10 mlL-1
. Each sedated fish was corralled into a sling and flipped over to expose its
ventral side. A catheter was gently inserted into the reproductive tract of the cloaca to the point
where the end of the catheter had likely penetrated the gonads. This was dependent on the size
of the fish. A syringe was attached to the end of the catheter. Gently withdrawing the plunger,
while slowly retracting the catheter, a few oocytes could be siphoned up.
The diameter of the oocytes was measured using a grid under a dissecting microscope. Fish
with the majority of eggs of a diameter > 450 µm (Musson & Kaiser, 2014) were hormonally
induced by intramuscular injection with either an LHRH analogue or the commercially
available product AquaSpawn®. The suggested concentration administered for each of these
products was 70 µg kg-1
and 0.5 ml kg-1
, respectively.
Spawning occurred 24 – 48 hours after induction, during the night. This was in accordance
with the spawning latency period of 34 hours at 22°C, recorded by Fielder & Heasman (2011).
Fertilised eggs floated, while unfertilised eggs sank to the bottom of the tank.
Eggs supplied for Trials 1 and 2 were collected, counted, incubated and hatched prior to
transportation to Mtunzini, from East London and Cape Town, respectively. To reduce

18
bacterial growth in the transport containers, egg casings and unhatched eggs were removed
from the containers. Egg counting was done by concentrating all eggs into a container of known
volume, then gently mixing the concentrate to attain an even egg distribution. A 10-ml test tube
was then used to scoop a sample from the surface of the container. That sample was transferred
to a petri dish, where eggs were manually counted. This was repeated five times, and the
average number of eggs from the five samples was used to estimate the total number of eggs.
Hatched larvae were concentrated into 20-L plastic bags, filled half with sterilised seawater
and inflated with pure oxygen. The closed plastic bags were placed in polystyrene cooler boxes,
along with an ice pack, and the boxes were sealed so that no light could enter. The boxes were
then air-freighted to King Shaka International Airport in Durban, where they were collected
and transported to MFF by motor vehicle. Upon arrival, the larvae were slowly acclimatised to
the conditions in the hatchery. The transport boxes were cracked open initially, which exposed
the larvae to a small amount of light. After ten minutes, the sealed bags were placed into the
prepared rearing tanks so that the temperature equalised between the water in the bag and the
tank water. Once temperatures were similar, the bags were opened and left floating on the tank
surface. Small amounts of tank water were added to each bag, allowing the larvae to become
gradually exposed to the chemical characteristics of the tank water.
System Design
Ten 400-L circular tanks with cone-shaped bottoms were configured in two lines of five tanks
(Figure 2.1) in a section of the MFF hatchery. This part was separate from the rest of the
hatchery with its own water supply, oxygen supply and lighting system. The water supply
system was configured as a flow-through setup that could supply either seawater from a pump
station situated on the Mtunzini beach, or estuarine water from the Umlalazi River. Water from
both sources was mechanically filtered through a 10- μm mesh size screen filter.
The estuarine water was pumped to the hatchery from a screen-filtered submersible pump
situated in a fertilised one-hectare pond. Samples viewed under a light microscope (Nikon
Eclipse E100) contained several phytoplankton genera. Identification was done by comparing
samples to slides and descriptions by Hoff & Snell (2008). The predominant genus in the pond
samples was Nannochloropsis. Other genera included Isochrysis, Nannochloris, and
Chlamydomonas.

19
Figure 2.1: Experimental tank setup at Mtunzini Fish Farm Pty Ltd, 2014-2015. Photo credit:
Thomas Keet, 2014.
To maintain good water quality, 20 % of the water was exchanged daily from 6 DAH by
siphoning 80 L of tank water out through a 50- μm mesh screen, and then slowly replacing it
with seawater that had been filtered through a 10- μm mesh. Tank surfaces were brushed and
the water was swirled every second day, starting around 8 DAH. Suspended solids settled at
the bottom of the cone. This build-up was then purged, by opening the ball-valves attached to
the bottom of each tank. A bucket with a 100-μm mesh screen bottom, placed in a larger
container, was positioned below the ball-valve prior to purging. Larvae accidently siphoned
out during the purge could then be caught and returned to the rearing tank. From 10 DAH
onwards, a slow continuous water exchange was initiated, starting at 4 L hour-1
, or a 10 % daily
turn-over rate, which was increased as larvae developed. Water exited the tanks by means of a
50-μm mesh screen strainer attached to the top of the central standpipe (the photograph in
Figure 2.1 was taken prior to their installation).
The lighting system consisted of four sets of Osram® L 58W/640 ‘Cool White’ fluorescent
tube lights. Each line of five tanks had two sets of lights suspended approximately 1 m above
the water surface. This light source produced 4600 lumens of luminous flux, which, in an area
of 25 m², produces a maximum illuminance of 184 lux. Initially (< 6 DAH) light intensity was
reduced manually using a dimmer, producing a medium to low intensity of approximately 100
lux as recommended by Ballagh et al. (2008) and Fielder & Heasman (2011). The latter

20
suggested a light intensity in green culture rearing tanks of 225 - 400 lux, starting with the
lights dimmed to the lower intensity and increasing after swim bladder inflation had occurred
(3 – 6 DAH).
Photoperiod was controlled by an automatic timer switch. It was initially set at 12L:12D, until
swim bladder inflation occurred (3 – 6 DAH). After this, the light : dark regime was changed
to 14L:10D to promote larval growth. Ballagh et al. (2010) used a 12L:12D regime for the
duration of the larval rearing process of dusky kob. Fielder & Heasman (2011) proposed using
12L:12D initially until all larvae had inflated their swim bladders. The light period was then
increased to 18 hours. This was done to promote larval growth. This tactic is most effective in
fish species that are strict diurnal feeders, such as certain members of the Sparidae family
farmed in the Mediterranean, for example, Sparus aurata (Tandler & Helps, 1985) and Pagrus
major (Biswas et al., 2006).
Each tank was equipped with an oxygen diffuser, perforated piping attached to an air-blower
line (used predominantly for water mixing), a banjo screen filter (50 μm ) attached to a central
stand pipe for water exchange, and an aquarium heater. Surface skimmers designed to remove
oil from the water surface were used in each tank for the first week post hatching. This was
done during the period of swim bladder inflation, when larvae gulp air from the water surface.
Fielder & Heasman (2011) reported swim bladder inflation in dusky kob larvae at 3 – 4 DAH.

21
Table 2.1: Water quality variable measured throughout each trial, the frequency of each
measurement, the instrument used, and the optimal range according to the literature (Ballagh
et al., 2010; Fielder & Heasman, 2011; Musson & Kaiser, 2014).
Measurement
Frequency of
measurement
Instrument used Target range
Dissolved Oxygen
(mg /L)
Temperature (°C)
Twice daily
7:30 AM / 16:00
PM
Handy Polaris® 2
Meter
>6.00 mg L-1
O2
22.0 - 25.0 °C
pH
Salinity (ppt) Every second day
Hanna® pH probe
Refractometer
7.6 - 8.2
12.5 - 30 ppt
Total Ammonia
Nitrogen (NH3 &
NH4
+
)
Twice a week
Hanna®
spectrophotometer
<1.00 mg L-1
A stocking density (SD) of 25 fish L-1
, as recommended by Musson & Kaiser (2014), was used
for all trials.
Ten thousand one-day-old larvae were stocked into each experimental tank at the beginning of
Trials 1 and 2. Larvae used in Trial 3 were housed in a single 11000-L tank until 10 DAH, after
which they were transferred into the experimental tanks, prior to the beginning of the
experiment. The reason for this change in method was that Trial 3 was conducted during the
winter months of 2015. Diurnal fluctuations in water temperature in the relatively small 400-L
experimental tanks were more severe than those measured for the larger tanks. Aquarium
heaters had a limited effect on negating these fluctuations in the experimental tanks, but a slow
continuous water exchange of 5 – 10 % of tank volume hour-1
was more effective. Continuous

22
water exchange only began after 10 DAH in the first two trials, so the larvae for Trial 3 were
moved into the experimental tanks to coincide with the beginning of water exchange.
Experimental Design
For each of the three trials, five tanks were randomly selected and assigned to a treatment (T),
while the remaining five were control tanks (C), thus this was a completely randomised design.
Tanks were reassigned for each trial.
Rotifers
Rotifers, Brachionus plicatilis (Figure 2.2), were cultured in the experimental tanks prior to the
beginning of the trial to provide sufficient feed for the first few days of exogenous feeding (3
– 6 DAH). These cultures were seeded from stock cultures held in glass aquaria in a
temperature-controlled laboratory. The stock cultures were fed a mixture of yeast,
Saccharomyces cerevisiae, and treacle sugar daily. Larval rearing tanks were seeded two weeks
prior to a spawning event and a combination of the enrichment product Ori-One® and live
algae harvested from fertilised earthen ponds was added. Pond water quality was tested prior
to introduction of larvae into the tanks. The live algae were filtered through a 10- μm mesh
screen prior to being added to the experimental tank. Rotifer numbers were reduced thereafter
from 20 ml-1
to 5 ml-1
by 16 DAH (Fielder & Heasman, 2011). Enriched and concentrated
cultures from supplementary culture tanks were added twice daily at 09:00 and 16:00 to keep
the desired rotifer densities in the rearing tanks.
Figure 2.2: An image of a live Brachionus plicatilis, a euryhaline rotifer and a commercially
important live feed for fish larvae in the aquaculture industry. Rotifers of this species have a
lorica length of 100 - 340 μm. Photo credit: Thomas Keet, 2015.

23
Brine shrimp (Artemia spp)
Artemia cysts were obtained from Brine Shrimp Direct Inc., Utah, United States of America.
The required quantity of cysts was weighed (Table 2.3), re-hydrated and de-capsulated
(Sorgeloos et al. 1977) before being stocked into conical hatching tanks with strong aeration
emanating from the bottom of the cone. Cysts were hydrated in soft fresh water for 60 – 90
minutes at 67 g L-1
. The suspension was then diluted with an equal volume of commercial
hypochlorite (HTH®) resulting in the cysts being exposed to a 2.12 % solution of the active
ingredient. The suspension was cooled with ice packs prior to the introduction of the
hypochlorite, to limit the sharp elevation in temperature caused by the exothermic reaction
between hypochlorite and the chorion of the cysts. Sorgeloos et al. (1977) suggested that > 40
°C will be lethal to the cysts.
At a water temperature of 28 ± 1.5 °C (mean ± standard deviation), 85 – 90% of the de-
capsulated cysts hatched. Aeration in the cones was then switched off, allowing the unhatched
cysts to sink to the bottom of the cone. Once settled, the cysts were purged using a ball-valve
tap at the bottom of the cone. After purging, the newly hatched nauplii were concentrated and
rinsed with clean seawater over a 50-μm mesh. Nauplii to be used for the enrichment procedure
were stocked into aerated conical enrichment tanks, while those that were being stocked
straight into the larval tanks were concentrated into a 20-L bucket and distributed to the
experimental tanks. Table 2.2 shows the daily schedule of Artemia concentrations for the
treatments.
Only Instar I nauplii were introduced into larval tanks from 10 DAH until 13 DAH. From 14 –
16 DAH, the nauplii added to each tank were a 1:1 ratio of Instar I nauplii and enriched Instar
V nauplii. After 16 DAH, only enriched Instar V nauplii were fed.
Live feed from previous feedings was not removed from the tanks prior to the introduction of
new live feed. This is normally done because Artemia nauplii tend to lose their nutritional value
post hatching. However, as the algal culture maintained in the experimental tanks acted a
source of enrichment to the nauplii, this was not considered necessary. Dead Artemia settled to
the bottom of the cone and were siphoned out daily.

24
Artificial pellet feed
The artificial pellet feed used during the early weaning period was Golden Pearl® (GP) from
Brine Shrimp Direct Inc., Utah, USA. The ingredients were marine fish, krill (23%), fish roe,
soy lecithin, yeast autolysate, micro-algae, fish gelatine, squid meal, hydrogenated vegetable
fat, vitamins and minerals and antioxidants, with proximate analysis of protein, 55%; lipids,
15%; ash, 12%; moisture, 8%; Vitamin C, 2550 mg L-1
; Vitamin E, 425 mg L-1
; EPA,10 mg
g-1
; DHA, 12 mg g-1
.
Two pellet sizes were used. Initially the 200 – 300 μm larval diet was distributed into each tank
four times day-1
. Two days after first feeding of GP 200-300, the Active Sphere GP 300 – 500
μm larval diet was introduced. This was done by splitting the daily artificial feed ration for
each tank into half, i.e., 50% GP 200 - 300 and 50% GP 300 – 500. A further two days after
this, only GP 300 – 500 was fed.
Feeding regimes (Treatments)
Figures 2.3 and 2.4 represent schematics of the feed type introduced into the experimental
tanks, and their timing of introduction.
period in the C treatment.
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Rotifers
Artemia
Pellet Diet
C Treatment (Control Feeding Regime)
Days after Hacthing (DAH)

25
period in the T treatment.
This research aimed to test the effects of weaning dusky kob larvae onto an artificial pellet diet
four days earlier than the current industry standard (in both South Africa and Australia) on the
morphological development and survival of the larvae. The currently utilised feeding regimen,
i.e., C (Figure 2.4), began weaning at 20 DAH. The new feeding regimen, T (Figure 2.5), began
weaning at 16 DAH.
Using the initial live prey stocking density (SD) suggested by Ballagh et al. (2010), Artemia
nauplii were first stocked into all tanks at a density of 0.2 ml-1
at 10 DAH. This number was
then doubled each day until 16 DAH in treatments C and T. The amount of Artemia added to
the treatment T was then reduced by approximately 20% each day until 21 DAH, when Artemia
was fed in the treatment tanks for the last time. In the control treatment, C, the number of
Artemia added each day was kept the same until 20 DAH, when weaning onto the artificial
pellet diet began. The amount of Artemia was then halved each day until 25 DAH, when
Artemia was fed for the last time (Table 2.2).
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Rotifers
Artemia
Pellet Diet
T Treatment (Novel Feeding Regime)
Days after Hatching (DAH)

26
Table 2.2: Number of Artemia ml-1
added to each treatment from 10 to 25 days after hatching
(DAH).
DAH Artemia (number of Artemia.ml-1)
C T
10 0.2 0.2
11 0.4 0.4
12 0.8 0.8
13 1.6 1.6
14 3.2 3.2
15 6.4 6.4
16 12.8 10.3
17 12.8 7.7
18 12.8 5.1
19 12.8 2.6
20 12.8 1.6
21 6.4 0.8
22 3.2
23 1.6
24 0.8
25 0.4

27
To keep treatment and control comparable, the total dry weight of all food items added to each
tank was the same, irrespective of which treatment a tank belonged to. To achieve this, it was
assumed that economy-grade Artemia cysts hatched in the ideal conditions produced an
average of 195 000 hatched nauplii g-1
(Brine Shrimp Direct Inc., 2010). Using this conversion
factor, the dry weight of each concentration (Table 2.2) was estimated using the formula:
When a difference in dry weight between the daily amounts of Artemia added to the two
treatments started at 16 DAH (Table 2.3), this difference was used as the starting dry weight
of Golden Pearl (GP) pellet feed added to each treatment tank. As the difference between the
two Artemia rations fed in each treatment group increased, so too did the amount of artificial
pellet feed to the treatment T, so as to keep the total dry weight of all feed types fed across the
two treatments equal. From 20 DAH onwards, when weaning onto pellet feed started in the
control, the sums of the dry weights of both Artemia and pellet feed fed to each treatment were
equal.
DW =
ASD×TV
195000
,
Where DW = dry weight (g) of Artemia nauplii added to each tank; ASD = daily Artemia
stocking density assigned to each tank (Table 2.2), and TV = tank volume (ml).

28
Table 2.3: A daily breakdown of the total dry weight (DW) of the different feed types fed to
the two treatments.
DAH Artemia (g) Pellet feed (g)
C T C T
10 0.4 0.4
11 0.8 0.8
12 1.6 1.6
13 3.3 3.3
14 6.6 6.6
15 13.1 13.1
16 26.3 21.1 5.2
17 26.3 15.8 10.5
18 26.3 10.5 15.8
19 26.3 5.3 21
20 21.1 3.3 5.2 23.3
21 13.1 1.6 13.5 25
22 6.6 20 26.6
23 3.3 25.4 28.7
24 1.6 29.1 30.7
25 0.8 32 32.8
26 35 35

29
Sampling and data collection
A small aquarium net was used to catch larvae. The net was swept at varying speeds, depending
on the age of the larvae at the time, from one side of the tank, downwards towards the bottom
of the cone, upwards towards the opposite side of the tank, and then circled around on the water
surface, to end up at the starting point. As the larvae developed, their swimming abilities
improved, allowing them to dodge a slow-moving net sweep. Therefore, as the larvae grew,
the net sweeps had to become more vigorous to catch sufficient larvae for sampling. Samples
of five larvae were either randomly selected from the net thereafter or netting continued until
five larvae were obtained from each tank. Samples were then placed in a labelled vial
containing a 10% formalin physiological saline solution (pH 7.2) to euthanise and preserve the
larvae.
Shrinkage caused by the immersion of larvae in a fixative solution was considered. Varying
shrinkage rates have been reported for both marine and freshwater larvae, with Gomez et al.
(2014) reporting that shrinkage rate mostly depended on the species studied, and the
concentrations or combinations of the preservation media used. Degree of shrinkage is
primarily dependent on the osmotic strength of the fixative solution and un-buffered 4%
formalin in freshwater gave the most accurate length measurements (Tucker & Chester, 1984).
In this experiment, physiological saline solution was used to minimise the osmotic pressure
exerted on the larvae.
Samples were taken from all replicates of the two treatments every two days to measure the
development of the three biometrics standard length (SL), body depth (BD), and eye diameter
(ED) (Figure 2.6).
Absolute growth rate (AGR) was calculated by dividing the difference between larval SL at
the start (SL1) and the end (SL2) of the trial by the number of days or degree days (DD) in the
trial.
AGR =
𝑆𝐿2−𝑆𝐿1
𝑛𝑜.𝑜𝑓 𝑑𝑎𝑦𝑠 𝑜𝑟 𝐷𝐷

30
Survival rate was determined at the end of each trial by counting the number of larvae left in
each tank. The samples collected throughout each trial were excluded from the denominator
used in the survival rate calculation:
Survival rate =
n1
n0−TTS
× 100 ;
where n1 is the number of larvae in the tank at the end of the trial, n0 is the total number of
larvae stocked into the tank, and TTS is the total number of larvae taken from the tank for
sampling purposes throughout the trial.
Figure 2.5: A 23 DAH dusky kob larvae indicating where measurements of total length (TL);
notochord length (NL) / standard length (SL); body depth (BD); eye diameter (ED) were taken.
Upon completion of three trials, preserved larval samples were transported to the Department
of Ichthyology and Fisheries Sciences (DIFS) at Rhodes University, Grahamstown, South
Africa. Larvae were individually photographed with a Leica dissection microscope that had a
mounted digital camera. The magnification on the camera was set at a standard level and a
pixel-to-millimetre conversion factor was obtained from the associated Leica digital camera
software. ImageJ, an open-source Java image processing program that was first developed by
Wayne Rasband in 1997 for use in the scientific community (Schneider et al., 2012), was used
to measure SL, BD, and ED, using pixels as the unit of measurement (Figure 2.6). The reason
for using NL/SL, as opposed to TL, for this part of the data collection was due to the fact that
the caudal fins of a number of the preserved samples were damaged or difficult to make out on
NL or SL
ED
BD

31
the digital photographs. These measurements were used in the statistical analysis of data from
all three trials.
Data Analysis
Accurate biometric measurements from each trial were obtained using the digital imaging and
measuring method described above. These values were used in the construction of growth
curves, i.e. the change of length or body mass or biometric measurements as a function of time.
Statistical analyses were conducted using the Statistica 13 software package. Residuals were
tested for normality (Kolmogorov - Smirnov test) and equality of variance (Levene’s test).
Sphericity testing was done to validate the use of analysis of variance with repeated measures
(ANOVA RM). If no testing violations occurred, ANOVA RM was conducted on the mean
standard length (SL), body depth (BD), eye diameter (ED) and condition (BD:SL) of each
treatment. Confidence intervals of 95% and mean ± SE were used to describe all biometrics,
while most physicochemical parameters were described by mean ± SD, unless specifically
indicated otherwise.
The ratio of BD:SL was used as an indicator of larval condition (Smith et al., 2005). Regression
analyses of SL vs. BD was conducted for each trial. The slopes of treatments regression models
were compared using the General Linear Model (ANCOVA) to test for significant differences
in slope between treatments.
ANOVA RM was the preferred data analysis method as it accounts for within-subjects
variance. In addition, measurements of larvae taken from each tank were averaged before
analysis, thus preventing bias due to pseudoreplication, which resulted in the tank being the
experimental unit. The null hypothesis was that there was no significant interaction between
treatment and time.
If no significant difference between treatments was found, data from the treatments were
combined to develop a model to estimate values of dependent data as a function of time.
A student’s t-test was conducted to test for differences in mean survival rate on the last day of
sampling for each trial. Where necessary, a t-test for unequal variances was used thereby
avoiding the need for non-parametric tests.

32
Chapter 3: Trial 1 Results
Temperature and water quality
There was no significant difference in mean temperature (p = 0.537), dissolved oxygen, DO,
(p = 0.111) or pH (p = 0.580, student’s t-test) between treatments, allowing for the pooling of
water quality data (Table 3.1). Salinity ranged between 28 – 30 ppt for the duration of the trial
and total ammonia nitrogen (TAN) did not exceeded 1.1 mg L-1
in any of the experimental
tanks.
Table 3.1: Pooled water quality data from Trial 1. Data, except for pH, are mean ± standard
deviation of the mean.
Temperature (°C) DO (mg L-¹) pH (mean & range)
24.3 ± 0.6 6.8 ± 1.5 7.88 (7.47 – 8.7)
Development of larval morphology
Both feeding regimens were successful in rearing dusky kob. Fish grew from a mean standard
length (SL) of 3.98 to 18.30 mm over 18 days (438 DD), thus increasing their starting length
by 14.32 mm (Figure 3.1). Both treatments showed a similar change in SL over time (ANOVA
RM F9, 72 = 0.727; p = 0.683). The absolute growth rate (AGR) of SL was 0.80 mm d-1
or 0.03
mm DD-1
for the duration of the trial. On the last day of sampling (28 DAH) the mean SL in
the C and T treatments were 19.67 ± 1.27 and 16.93 ± 1.27 (mm ± SE), respectively (Figure
3.2).
Growth of mean body depth (BD) and mean eye diameter (ED) followed the same trend as SL.
Both treatments showed similar changes in BD (ANOVA RM F9, 72 = 0.656 p = 0.745) over
the trial period (Figure 3.3) and on the last day of sampling (28 DAH) the mean BD in the C
and T treatments were 6.38 ± 0.45 and 5.50 ± 0.45 (mm ± SE), respectively (Figure 3.4). Both
treatments also showed similar changes in ED, Figure 3.5, (ANOVA RM F9, 72 = 1.231; p =
0.290) over the trial period and on the last day of sampling the mean ED in the C and T
treatments were 1.771 ± 0.09 and 1.534 ± 0.09 (mm ± SE), respectively (Figure 3.6).
BD increased from 1.26 to 5.94 mm, and ED increased from 0.43 to 1.65 mm over 18 days
(438 DD). Linear growth in BD was 0.26 mm d-1
or 0.01 mm DD-1
and it was 0.06 mm d-1
or
0.003 mm DD-1
for ED.

33
Laval condition (BD:SL) in each treatment showed a similar change (ANOVA RM F9, 72 =
0.49; p = 0.88) over time (Figures 3.7; 3.8) suggesting that the earlier weaning period did not
result in temporary starvation in treatment T.
Figure 3.1: The development of mean SL (mm) of larvae fed two feeding regimens (Treatments
C & T) over time (DAH) in trial 1. Vertical bars represent 95% confidence intervals of the
mean.
On the first day (10 DAH) of sampling, the Coefficient of Variance (CV) for SL in the C and
T groups was 20.36% and 11.77% respectively. On the last day of sampling (28 DAH), the SL
CV’s for the C and T groups were 26.17% and 12.67% respectively.

34
Treatment=C
12 14 16 18 20 22 24 26 28 30 32
Standard length (mm)
0
1
2
3
4
No.
of
obs.
Treatment=T
12 13 14 15 16 17 18 19 20
Standard legth (mm)
0
1
2
3
4
5
No.
of
obs.
Figure 3.2: Frequency histograms of SL from the control and treatment, C and T respectively,
on the last day of sampling in Trial 1 (28 DAH), with the line representing expected normal
distribution.
(Treatments C & T) over time (DAH) in Trial 1. Vertical bars represent 95% confidence
intervals of the mean.

35
Treatment=C
4 5 6 7 8 9 10 11
Body depth (mm)
0
1
2
3
4
5
6
7
No.
of
obs.
Treatment=T
4.0 4.5 5.0 5.5 6.0 6.5 7.0
Body depth (mm)
0
1
2
3
4
5
6
7
No.
of
obs.
Figure 3.4: Frequency histograms of BD from the control and treatment, C and T respectively,
distribution.
Figure 3.5: The development of mean ED (mm) of larvae fed two feeding regimens (Treatments
C & T) over time (DAH) in Trial 1. Vertical bars represent 95% confidence intervals of the
mean.

36
Treatment=C
1.2 1.4 1.6 1.8 2.0 2.2 2.4
Eye diamter (mm)
0
1
2
3
No.
of
obs.
Treatment=T
1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9
Eye diameter (mm)
0
1
2
3
4
5
No.
of
obs.
Figure 3.6: Frequency histograms of ED from the control and treatment, C and T respectively,
distribution.
Figure 3.7: Mean BD:SL (mm) of larvae fed different feeding regimens (Treatments C & T)
over time (DAH) in Trial 1. Vertical bars represent 95% confidence intervals of the mean.
Fish in the two treatments showed no change in condition over time. Analysis of the slopes of
the regression models of BD vs. SL (Figure 3.8) using the general linear model indicated that
there was no significant difference in the slopes between treatments in Trial 1 (F1, 347 = 2.02; p

37
= 0.156). On the first day (10 DAH) of sampling, the Coefficient of Variance (CV) for BD:SL
in the C and T groups was 8.53% and 5.47% respectively. On the last day of sampling (28
DAH), the BD:SL CV’s for the C and T groups were 4.23% and 4.3% respectively.
Treatment=C
0 5 10 15 20 25 30 35
SL
0
2
4
6
8
10
12
BD
SL:BD: y = 0.1398 + 0.3118*x; r = 0.9877, p = 0.0000;
r2
= 0.9756
Treatment=T
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
SL
0
1
2
3
4
5
6
7
8
9
10
BD
SL:BD: y = 0.0729 + 0.3184*x; r = 0.9939, p = 0.0000;
r2
= 0.9878
Survival rate
Mean survival rates of the C and T treatments were 2.55 and 3.06 %, respectively. There was
no significant difference between treatments (t = 0.404; df = 8; p = 0.697, student’s t-test,
Figure 3.9).
T C
Treatment
1.00%
2.00%
3.00%
4.00%
5.00%
Survival
rate
Mean
Mean±SE
Mean±1.96*SE
Figure 3.9: Box & Whisker plot of the mean survival rates of each treatment group on the final
day of the Trial 1 (28 DAH).

38
Discussion
Larval development and growth
No significant differences between treatments in any of the measured morphometrics were
found during this trial. The water temperature in Trial 1 averaged 24.3 ± 0.6 °C (mean ±
standard deviation) and resulted in an SL AGR that was higher than the TL AGR reported by
Musson & Kaiser (2014), i.e. 0.80 mm d-1
versus 0.53 ± 0.19 mm d-1
. Larvae in both treatments
had larger mean SL-values at 28 DAH (C: 19.67 ± 1.27 ; T: 16.93 ± 1.27) than the results from
an experiment conducted at a lower temperature of 23.3 ± 0.8 °C (mean ± SEM) by Ballagh et
al. (2010). Larvae from two treatment groups similar to C and T in Trial 1, had TL values of
14.2 ± 0.2 and 13.7 ± 0.4 mm, respectively, at 29 DAH.
These results indicating faster growth are corroborated by research by Bernatzeder & Britz
(2007) on the temperature preferences of juvenile dusky kob (with an average weight of 23.7
g fish-1
) in South Africa, which suggested a preferred range of 25 – 26.4 °C. Collett et al. (2008)
also found that juvenile dusky kob showed the fastest specific growth rate (2.05% day-1
) and
optimal food conversion ratio (0.72 kg gain-1
) at a temperatures of 25.3 °C and 21.7 °C,
respectively.
BD:SL ranged from 0.30 to 0.36 and was similar between treatments throughout the trial
period, indicating that the earlier weaning period had no effect on larval condition, and that
larval condition did not change with age.
Larval survival
Survival rate was not significantly different between treatments. Both treatments experienced
a low survival rate due to high levels of cannibalism. Type I cannibalism, or antagonistic
behaviour, was observed in larvae from 16 – 20 DAH in both treatments. Type II cannibalism
was observed from 20 DAH.
Size variation was evident in all tanks, and Figure 3.2 illustrates the size distribution of larvae
sampled on the last day of the trial from each treatment. The larvae from C had a wider size
range (14 –32 mm SL) than T (13 – 20 mm SL), but this did not result in a lower survival rate
in C, as predicted by Kestemont et al. (2003). The bimodal distribution displayed in Figure 3.3
indicated two size groupings of larvae in the T treatment on the last day of sampling, one larger,
and one smaller.

39
Chapter 4: Trial 2 Results
Temperature and water quality
There was no significant difference in mean temperature (p = 0.101, student’s t-test), dissolved
oxygen concentration (p = 0.128) or pH (p = 0.586) between treatments, allowing for the
pooling of water quality data (Table 4.1). Salinity ranged between 27 – 29 ppt for the duration
of the trial and TAN did not exceed 1.0 mg L-1
in any of the experimental tanks.
Table 4.1: Pooled water quality data from Trial 2. Data, with the exception of pH, are mean
± standard deviation of the mean.
Temperature (°C) DO (mg L-¹) pH (mean & range)
25.2 ± 0.6 6.9 ± 1.7 7.94 (7.45 – 8.2)
Development of larval morphometrics
Both feeding regimens were successful in rearing dusky kob. Fish grew from a mean standard
length (SL) of 4.36 to 23.92 mm over 18 days (454 DD), thus increasing their starting length
by 19.56 mm (Figure 4.1). Both treatments showed a similar change in SL over time (ANOVA
RM F9, 72 = 0.4226; p = 0.9189). The AGR of SL was 1.09 mm d-1
or 0.04 mm DD-1
for the
duration of the trial. On the last day of sampling (28 DAH) the mean SL in the C and T
treatment groups were 24.23 ± 2.17 and 23.60 ± 2.17 (mm ± SE), respectively (Figure 4.2).
Growth of mean body depth (BD) and mean eye diameter (ED) followed the same trend as SL.
Both treatments showed similar changes in BD (ANOVA RM F9, 72 = 0.499; p = 0.870) over
the trial period (Figure 4.3) and on the last day of sampling (28 DAH) the mean BD in the C
and T treatment groups were 7.49 ± 0.65 and 7.58 ± 0.65 (mm ± SE), respectively (Figure 4.4).
Both treatments also showed similar changes in ED (ANOVA RM F9, 72 = 0.284; p = 0.977)
over the trial period (Figure 4.5) and on the last day of sampling (28 DAH) the mean ED in the
C and T treatment groups were 2.00 ± 0.15 and 1.98 ± 0.15 (mm ± SE), respectively (Figure
4.6).
BD increased from 1.41 to 7.54 mm, and ED increased from 0.47 to 1.99 mm over 18 days
(454 DD). The AGR of BD was 0.34 mm d-1
or 0.01 mm DD-1
and it was 0.08 mm d-1
or 0.003
mm DD-1
for ED.

40
Larval condition (BD:SL) differed in the way it changed over time between treatments
(ANOVA RM F9, 72 = 2.16; p = 0.035) (Figures 4.7; 4.8).
10 12 14 16 18 20 22 24 26 28
DAH
0
5
10
15
20
25
30
Standard
length
(mm)
Treatment C
Treatment T
Figure 4.1: The development of mean SL (mm) of larvae fed different feeding regimens
intervals of the mean.
On the first day (10 DAH) of sampling, the Coefficient of Variance (CV) for SL in the C and
T groups was 18.91% and 20.11% respectively. On the last day of sampling (28 DAH), the SL
CV’s for the C and T groups were 16.01% and 26.12% respectively.

41
Treatment=C
16 18 20 22 24 26 28 30 32
0
1
2
3
4
5
No.
of
obs
. Treatment=T
10 15 20 25 30 35 40
0
1
2
3
4
5
6
7
8
9
10
No.
of
obs.
Figure 4.2: Frequency histograms of SL from the control and treatment, C and T respectively,
distribution.
10 12 14 16 18 20 22 24 26 28
DAH
0
1
2
3
4
5
6
7
8
9
Body
depth
(mm)
Treatment C
Treatment T
intervals around the means.

42
Treatment=C
5 6 7 8 9 10 11
Body depth (mm)
0
1
2
3
4
5
6
7
8
9
No.
of
obs. Treatment=T
4 5 6 7 8 9 10 11 12
Body depth (mm)
0
1
2
3
4
5
6
7
No.
of
obs.
Figure 4.4: Histograms of BD data from each treatment group on the last day of sampling in
Trial 2 (28 DAH).
10 12 14 16 18 20 22 24 26 28
DAH
0.0
0.5
1.0
1.5
2.0
2.5
Eye
diameter
(mm)
Treatment C
Treatment T
Figure 4.5: The development of mean ED (mm) of larvae fed two feeding regimens (Treatments
C & T) over time (DAH) in Trial 2. Vertical bars represent 95% confidence intervals around
the means.

43
Treatment=C
1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8
Eye diameter (mm)
0
1
2
3
4
5
6
7
8
9
No.
of
obs. Treatment=T
1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8
Eye diameter (mm)
0
1
2
3
4
5
6
7
No.
of
obs.
Figure 4.6: Histograms of ED data from each treatment group on the last day of sampling in
Trial 2 (28 DAH).
10 12 14 16 18 20 22 24 26 28
DAH
0.29
0.30
0.31
0.32
0.33
0.34
0.35
0.36
0.37
Values
Treatment C
Treatment T
Figure 4.7: The development of mean condition of larvae fed two feeding regimens
intervals around the means.
Fish in the two treatments showed significant changes in condition over time. An analysis of
the slopes of the regression models of BD vs. SL using the general linear model (Figures 4.8)

44
indicated that there was a significant difference in slopes of the models between treatments (F1,
358 = 0.851; p = 0.003). On the first day (10 DAH) of sampling, the Coefficient of Variance
(CV) for BD:SL in the C and T groups was 6.66% and 5.58% respectively. On the last day of
sampling (28 DAH), the BD:SL CV’s for the C and T groups were 6.24% and 5.49%
respectively.
Treatment=C
2
0 5 10 15 20 25 30 35
SL
0
2
4
6
8
10
12
BD
SL:BD: y = 0.2247 + 0.2997*x; r = 0.9906, p = 0.0000;
r2
= 0.9813
Treatment=T
2
0 5 10 15 20 25 30 35 40
SL
0
2
4
6
8
10
12
14
BD
SL:BD: y = 0.1588 + 0.3133*x; r = 0.9908, p = 0.0000;
r2
= 0.9816
Figure 4.8: Regression analysis of BD vs SL for each treatment in Trial 2 (C: y = 0.2247 +
0.2997*x; T: y = 0.1588 + 0.3133*x)
The T curve had a steeper gradient than C (0.3133 vs. 0.2997), which could either indicate
temporary starvation in the earlier stages of the trial, or an accelerated growth rate for larvae in
the T treatment.

45
Survival rate
Mean survival rates (Figure 4.9) of the C and T treatments, were 11.82 and 10.34 %,
respectively. Student’s t-test indicated no significant difference in mean survival rate between
treatments (t = 0.387; df = 8; p = 0.709).
T C
Treatment
4.00%
6.00%
8.00%
10.00%
12.00%
14.00%
16.00%
18.00%
Survival
rate
Mean
Mean±SE
Mean±1.96*SE
Figure 4.9: Box & Whisker plot of the mean survival rates of each treatment group on the final
day of the Trial 2 (28 DAH).

46
Discussion
Larval development and growth
No significant differences between treatments in the change of SL, BD, or ED were found
during Trial 2, but the change in BD:SL (larval condition) between treatments over time did
differ, illustrated by Figure 4.8. The water temperatures experienced in Trial 2 were 25.2 ± 0.6
°C (mean ± standard deviation) and resulted in an SL AGR that was higher than the TL AGR
reported by Musson & Kaiser (2014), namely 1.09 mm d-1
versus 0.53 ± 0.19 mm d-1
.
The change in BD:SL differed between treatments during the weaning period. This is illustrated
by the differing gradients of each treatments’ linear regression curve for BD vs SL, and by
Figure 4.7, which shows a change in development between the two treatments curves during
the period 18 DAH and 22 DAH. This suggests that after 18 DAH, larvae weaned from 16
DAH were in better condition than those weaned from 20 DAH. Alternatively, it could indicate
a degree of starvation in the control group (Smith et al., 2005).
Larval survival
Survival rate was not significantly different between treatments in Trial 2. Survival was higher,
and the mean water temperature was 0.9 degrees Celsius warmer than in Trial 1, which suggests
a possible positive interaction between temperature and survival. Average temperatures in Trial
1 were 24.3 ± 0.6 compared to 25.2 ± 0.6 °C in Trial 2.
Type I cannibalism, or antagonistic behaviour, was observed in larvae from 16 – 20 DAH in
both treatments. Type II cannibalism was observed from 20 DAH. Type II was more frequently
observed during this trial, compared to Trial 1. The higher average temperatures may have
resulted in faster larval development in Trial 2, resulting in an earlier onset of Type II
cannibalism. This observation did not impact overall survival rates; therefore, it was deemed
insignificant.
Growing fish larvae at temperatures in the upper limits of their thermal preference range can
be both beneficial and detrimental to their growth and survival. Beneficial in that the grow and
develop at a faster rate and therefore be able to consume larger prey earlier than counterparts
grown at lower temperatures. Detrimental in that the water quality in the larval rearing tank
could be adversely affected by the conditions, as a result of lower DO levels and increased
microbial growth rates in the warmer water.

KEET-Larva Ikan.pdf

Recommended

Recommended

More Related Content

Similar to KEET-Larva Ikan.pdf

Similar to KEET-Larva Ikan.pdf (20)

Recently uploaded

Recently uploaded (20)

KEET-Larva Ikan.pdf