Anna Moseley Dissertation

7/29/2019 Anna Moseley Dissertation

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The Potential for Culturing the Bicolor Blenny (Ecsenius

bicolor) for the Marine Aquarium Industry

Minor Project by Anna Moseley (12494288)

MAppSc Aquaculture

AQ5023

Supervisor: Chaoshu Zeng

March 2012

Word Count: 7900

Abstract

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The bicolor blenny (Ecsenius bicolor) adult possesses many characteristics that make it suitable for

culture for the marine aquarium industry. However this species, or indeed any species from theEcsenius

genus, has never been reported to have been cultured successfully. In fact prior to this study, no literature

existed on important aspects of reproductive biology, such as spawning intervals and fecundity, as well as

embryology and larvae first-feeding for this species, all of which are crucial for any captive breeding efforts;

this study investigated these factors. Results showed that of four successfully paired females ofE. bicolorin

this study all spawned readily after 1-3 months after being introduced to the system and all spawning occurred

consistently at dawn in a variety of differently sized PVC tubes. The mean spawning interval was usually

short (4 days), but ranged from 2 to 7 days. The largest female produced the highest mean number off eggs

per spawn (3223). Newly aid oocytes where approximately spherical and measured ~1500150m in diameter

at the widest point. They reduced in diameter to 1220120m immediately before hatching, which occurred

approximately 157 h post-fertilization (PF), at dusk. Newly hatched larvae had a mean body length of

3.150.3mm, and an open jaw. Mouth gape height (34615m) was sufficient for consuming rotifers and

indeed, predatory behaviour and rotifer ingestion was observed in healthy larvae within 1 days post-hatch

(DPH). A first feeding trial (3 replicates per regime, 29C, 8L, static green water, rotifer density 20/mL)

showed that newly hatched larvae left unfed reached 100% mortality after 3 DPH. In contrast, larvae fed on

rotifers enriched withNannochloropsis sp. (InstaAlgae Nanno 3600) and a high protein enriched algae

(AlgaMac Protien Plus) survival longer and achieved 100% mortality on 8 DPH. Observation under a

microscope revealed that the vast majority of larvae failed to ingest sufficient rotifers for survival, suggesting

that they do not provoke a sufficient predatory response inE. bicolorlarvae. However, good ingestion of

Artemia nauplii was also observed on larvae at 5 DPH. First-feeding trials using copepod nauplii is advised as

the next step in devising a working culture protocol for this species.

Contents

1. Introduction

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2. Materials and Methods

2.1 Broodstock Management and Conditioning

2.2 Spawning Times and Spawning Intervals

2.3 Fecundity

2.4 Embryological Life History

2.5 Hatching Technique

2.6 Larval Measurements

2.7 First-Feeding Experiment

3. Results

3.1 Reproductive and Territorial Behaviour

3.2 Spawning Intervals

3.3 Fecundity


3.5 Larvae Observations

3.6 First Feeding Experiment4. Discussion


4.2 Fecundity and Spawning Intervals

4.3 Embryology and Hatching Indicators

4.4 Larvae First Feeding

5. Conclusions

6. References

Introduction

Marine ornamentals make up around 10% of the global exports of ornamental fish (UNEP-WCMC

2008) and there has been exponential growth (Larkin 2003) in the marine aquarium industry in recent years

(US $278 million in 2005; FAO 1996-2005). Approximately 90-98% of marine ornamentals are wild-caught

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(Job 2011; AIMS 2011) and over 90% of these are collected from coral reefs (Sugiyama et al. 2004).

Increasing public and personal and marine aquarium ownership is creating an ever increasing demand for

specimens (Green 2003; Balboa 2003; Hall & Warmolts 2003), which encourages unsustainable and harmful

fishing techniques used by collectors (Wood 2001; Bunting et al. 2003). As a consequence, the marine

aquarium trade is increasingly being referred to as one of the major contributing factors responsible for the

current global decline in coral reef biodiversity (Cesaret al. 1997), and is being criticized more frequently by

conservation groups (Tlusty 2002; Green 2003). Often key fish species that maintain the health of the reef

ecosystem are objects of these commercial fisheries (Green 2003). Development of captive breeding methods

for marine ornamental species targeted by the aquarium trade could help meet the demands for specimens and

consequentially take the pressure off wild-caught species (Pomeroy et al. 2006), and could even be used to

replenish over-fished stocks through the release of juveniles (ranching) (Tlusty 2002). Ornamental

aquaculture also leads to the development of new methods of breeding, larval rearing and feeding which can

often be transferred to other species (Dhert et al. 1997). Alongside the obvious significance for aquacultural

endeavours and advancing human knowledge of the biology of ornamental coral reef fish; studying

reproduction and early life history of these species is also vital if they are to be managed correctly. For

example, measuring the levels of reproductive output and success can help provide important information for

determining the safe catch level (Miller & Kendall 2009).

Only around 100 marine ornamental fishes of the ~1000 species (Wabnitz et al. 2003) involved in the

trade have been reported to have been successfully cultured, and the vast majority of these are on a hobbyist

or research scale (Job 2011). Only 30-35% of the 100 species are in commercial production (Job 2011). Most

culture information is generated by commercial ventures and is consequentially of a proprietary nature and not

publically available (Job 2011). Therefore, the development of marine ornamental aquaculture is currently

stunted by a lack of published culturing protocols for most species in the trade. Furthermore, most of the

scientific research into addressing the challenges of culturing marine ornamentals has only occurred after the

year 2000. As a result of this, the vast majority of marine ornamental aquaculture remains poorly understood

(Job 2011).

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Species of Blenniidae form one of the top ten most highly traded marine ornamental fish families (Job

2011), and the adult form of the bicolor blenny (Ecsenius bicolor) has many attributes that make it a suitable

species for the marine aquarium trade, and a good choice for responsible marine aquarium owners (Livengood

& Chapman 2005). Positive traits include its herbivorous nature, meaning that they do not eat ornamental

corals or invertebrates (Scott 2005). Furthermore, they do not put high pressure on the aquariums filtration

system, adapt well to small aquariums, are known to thrive on a large variety of marine based foodstuffs, and

are generally considered to be hardy (Scott 2005)E. bicoloris also non-aggressive towards other aquarium

species, with the exception of their own kind and simular species (Scott 2005; Skomal 2007). It is one of the

more brightly pigmented species ofEcsenius (Springer 1988), and this combined with its relatively small

maximum size of 10-11cm (Scott 2005; Randall et al. 1997) means thatE. bicoloris already a very popular

aquarium species that is recommended in many hobbyist marine aquarium-keeping guides (Paletta 2001;

Scott 2005; Skomal 2007; Fenner & Wittenrich 2008).

Although the larval dispersal distances are not known forE. bicolor, its wide distribution and

relatively abundant nature (Springer 1988) suggests that there is great potential for broodstock to be collected

from a wide range of locations, in a sustainable way. The natural range ofE. bicolor(See Fig. 1) includes the

Maldives, the central Indian Ocean, westward to Samoa, the tropical Pacific, and from the southern Ryukyu

Islands in the north to the Capricorn islands at the southern limit of the Great Barrier Reef (Springer 1988).

Despite the relatively common abundance ofE. bicolor(Springer 1988) and its consequent high availability

for purchase (Job 2011), prices for individual adults are still relatively high (up to US$45 at online stores).

Furthermore, tank-bred marine ornamentals are in high demand from avid hobbyists and generally command

prices 25% higher than the wild-caught equivalent (Job 2011). It is likely that the increased awareness of coral

reef degradation, to which wild-collection is a contributor, demand for tank-bred specimens will remain high

(Job 2011).

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Figure 1. Map published in Springer (1988) showing the distribution of the Bicolor Group. (L in symbol forEcsenius bicolorindicates acceptable distribution record basedon literature).

E. bicoloradults exhibit three distinct colour variations. The bicolored pattern is common; individuals

possess a uniformly dark blue head, with a contrastingly yellow/orange pale caudal fin (Fig. 2a)(Losey 1972;

Springer 1988; Chapman & Schultz 1952; Springer & Smith-Vaniz 1972). The uniformed brown coloration

(Fig. 2b) is described as being uniformly dark from head to tail with a dark pigment extending as a triangle

into the caudal fin (Springer 1988; Springer & Smith-Vaniz 1972). A striped pattern also occurs; both the

head and dorsal proportion of the body are dark with a darker stripe on the body side (Fig. 2c), which is the

least common colour variety (Springer 1988; Chapman & Schultz 1952). In a specimen collection described

by Springer (1988), only 1 or 2 individuals were observed to have the striped pattern, whereas more than 25

possessed the uniformed or bicolored colour pattern in the same collection.

Figure 2. Photos published in Springer (1988) showing the three distinct colour variations of adultE. bicolor; (a)Bicolored, (b)Uniform& (c)Striped.6

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Although the basic adult biology, ecology and behavioural traits ofE. bicolorwere studied, mainly in

the field during the 1950s, 60s and 70s (Wickler 1965; Losey 1972; Chapman & Schultz 1952; Springer &

Smith-Vaniz 1972), no literature exists on the important aspects of reproductive biology, such as spawning

intervals and fecundity, as well as embryology and larvae first-feeding for this species. To date, there are no

reports thatE. bicolorlarvae, or any other species from theEcsenius genus, have ever been cultured

successfully (Job 2011). Therefore, the main aim of this project is to determine the potential for culturing

E.bicolorfor the marine aquarium industry. This will be done in three ways. Firstly by investigating the

spawning behaviour, spawning intervals, and fecundity for this species. Secondly, by recording the

embryological life history and pre-hatching indicators of the oocytes. Finally, larvae hatching and first-

feeding trials will be carried out. Once a working culture protocol for this species has been devised, it is most

likely it can be transferred to other species of theEcsenius genus, none of which have been successfully

cultured to date.

E.bicoloradults are known to spawn (iteroparous) readily in captivity (Wickler 1965). Other species

of Blenniidae are known to lay their eggs on a number of artificial and natural substrates including PVC pipe,ceramic tiles and live rock (Olivotto et al. 2005; Wittenrich et al. 2007; Moorhead & Zeng 2011). E. bicolor

adults will most likely require plenty of structures for spawning as wild specimens live in crevices on coral

reefs (Randall et al. 1997). Literature on other marine demersal spawners reveals that spawns should remain

in paternal care until hatching as removing them earlier is dramatically detrimental to hatching efficiency

(Olivotto et al. 2005; Wittenrich et al. 2007; Moorhead & Zeng 2011). Aeration is typically provided to flow

across demersal eggs, which has proved to improve egg hatching efficiency when they are away from paternal

care (Olivotto et al. 2005; Wittenrich et al. 2007; Moorhead & Zeng 2011). Wickler (1965) recounts the

breeding, hatching and a failed attempt to rear newly hatched larvae ofE. bicolorin captivity; the newly

hatched larvae failed to first-feed (Wickler 1965). However, the rotifer was not commercially available at that

time, as culture techniques were still in the research and development phase (Theilacker & McMaster 1971;

Ostrowski & Laidley 2000). Blenniidae larvae are normally raised in green water systems (Moorhead &

Zeng 2011), as is typical with marine finfish larvae that experience a pelagic phase (Job et al. 1997; Palmeret

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al. 2007). This technique is used in order to dissipate light and maintain the nutritional value of zooplankton

(Job et al. 1997; Olivotto et al. 2008, Moorhead & Zeng 2011). Also, greater turbidity is believed to improve

feeding efficiency in marine ornamental finish (larvae react more readily to prey that is moving notably and

prey encounter rates are increased) (Job 2011; Mackenzie & Kiorboe 1995). Furthermore, recent studies show

survival and growth benefits for many species when using a 24L: 0D photoperiod; including the yellow-tailed

damsel fish (Chrysiptera parasema), the cleaner goby (Gobiosoma evelynae), the sunrise dottyback

(Pseudochromis flavivertex), and the lemon peel angelfish (C. flavissimus) (Olivotto et al., 2003, 2005, 2006).

2. Material and Methods

2.1 Broodstock Management and Conditioning

The broodstock tanks were located undercover and experienced a natural photoperiod. Water

temperature was maintained by a recirculating system at 27.5-29.5C, salinity at 29-27,pH at 8.0-8.2, NH3

and NH2 at 6ppm. AdultE.bicolorwere obtained from a commercial supplier and

introduced into the broodstock tanks in a variety of combinations until two spawning heterosexual pairs were

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eventually established in broodstock tanks where they were the only residentE. bicolor. One pair (Female #1)

was housed in a 1000L tank and the other (Female #2) in a 300L tank. The remaining two spawning females

(Female #3 & #4) and two males occupied a single 1000L tank (Group Tank). All data in this study was

gathered from these four spawning females.

In order to bring theE.bicoloradults to breeding condition they were fed twice daily with a varied a

diet as possible. An enriched high protein and lipid home-made gelatine-bound wet feed was used, consisting

of blended fish, squid, mussel and prawn with added multi-vitamins and mineral supplements in order to

maximise egg and larvae quality (Papanikos et al. 2004; Salze et al, 2005) and gonosomatic index (Lin et al.

2007).

2.2 Spawning Times and Spawning Intervals

In this study, white PVC pipe shelters of various sizes and shapes were provided for spawning in

order to optimise the likelihood ofE.bicoloradults deeming a hide suitable for spawning (Moorhead & Zeng

2011). Spawning tubes included 50mm and 25mm open pipes and 50mm capped pipes withal single 25mm

reduced entrance. In order to determine the spawning intervals for this species under captive conditions, all

PVC pipes in each of the three broodstock tanks were checked daily for fresh oocyte deposits over a 62 day

period (September to December, 2011) and the findings recorded.

In order to determine the time of day when spawning occurs, frequented spawning pipes used by the

females were checked for fresh spawns on an hourly basis over a 24 hour period. This procedure was repeated

on 3 separate instances to improve the reliability of the findings.

2.3 Fecundity

In order to determine the fecundity of each of the four breeding femaleE.bicolor, the PVC spawning

tubes were lined with a clear plastic sheet. Once an egg deposit was made, this lining was extracted and

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photographed with a digital camera (HP Photosmart R818) and analysed using photo software (Adobe

Photoshop CS3 version 10.0). Each digital image was overlaid with a grid and the mean number of oocytes

per grid square was calculated, and the total number of eggs was estimated thusly. In order to determine

fecundity, a total of three egg deposits were counted for each female and the mean calculated. In the Group

tank containing two females, the issue of determining which spawn belonged to which female was overcome

by observing which female was visibly pregnant prior to a spawn. The body length of each female was

measured and compared to their fecundity to see if there was a correlation.


The embryological life history ofE.bicolorwas recorded photographically using a light microscope

and a digital camera (Olympus DB25). Photos were enhanced and analysed using photo software (Adobe

Photoshop CS3 version 10.0). Photographs were taken at 0h post-fertilization (PF), 10h, 20h, 25h, 30h, 40h,

55h, 65h, 70h, 75h, 85h, 95h, 105h, 125h and 150h. All oocytes remained in paternal care until required in

order to ensure normal growth and development. Egg diameter and heart rate (where existent) were measured

at multiple stages for 10 randomly selected eggs. During these measurements the salinity remained constant at

29-27, as did the temperature at 27.5-29.5C.

2.5 Hatching Technique

E. bicoloroocytes were left in paternal car until hatching. Based on embryological development

described in the present study; on the evening (at dusk) of the expected hatching the entire PVC spawning

tube was removed from the broodstock tank and the exterior of the pipe scrubbed clean to maintain water

quality inside the hatching tank. It was then placed in a 20L dark coloured hatching tank; water was of the

same temperature and water quality parameters as that of the broodstock tanks (27.5-29.5C, salinity at 29-

27,pH at 8.0-8.2, NH3 and NH2 at 6ppm). Aeration was provided to flow across the

eggs at an approximate flow rate of 1L/min (Wittenrich et al. 2007; Moorhead & Zeng 2011).The tank was

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then covered to eliminate all light (simulate dusk). Once hatching was complete healthy larvae displaying

normal swimming behaviour, were counted and transferred gently to larvae rearing tanks as required.

2.6 Larval Measurements

Upon hatching, the mouth gape of 10 randomly selected larvae was measured from three separate

batches. They were initially preserved with 10% buffered formalin. The following formula was used to

determine mouth gape height: GH = UJL 2+ LJL 2, where UJL and LJL stand for the upper and lower jaw

lengths respectively (Moorhead & Zeng 2011). Gape height was recorded as the distance between the left and

right postero-ventral tips of the articular bones of the jaw (Kiorboe et al. 1985). The standard length was also

measured from 10 randomly selected larvae immediately after hatching and at 5 days post-hatch (DPH). A

typical larval specimen from both of these samples was photographed under a dissecting microscope fitted

with a digital camera (Olympus DB25).


Live zooplanktons were provided immediately after hatching. Due to their current commercial

availability in comparison to other zooplankton of first-feeding size, (Tamaru et al. 2003) rotifers (Brachionus

rotundifornis andB. plicatilis) were used as a test first-feed zooplankton forE.bicolorlarvae. A rotifer

enrichment experiment was chosen due to pilot studies revealing thatE. bicolorlarvae stalk their prey in an

energetic but cautious fashion, approaching the target from multiple angles for a considerable length of time

before attempting a strike, which is energetically costly and suggested that they require a highly nutritious diet

to remain active. Growth and survival benefits were anticipated by undertaking high protein rotifer

enrichment. Alongside highly unsaturated fatty-acid enrichment (Avella et al. 2007), rotifer protein

enrichment is frequently required for marine ornamental finfish culture. For example, non-enrichment of live

rotifers for yellow-tailed damselfish larvae Chrysiptera parasema resulted in 100% mortality within 48 hours

(Job 2011). Similar results have been found withP. flaviverex when using the high-protein enrichment

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product Algamac ProtienPlus (Olivotto et al. 2003; 2006). This particular high-protein enrichment

product was chosen for this experiment based on the remarkably positive results observed for the previously

mentioned study.

Three first-feeding regimes, i.e. 1) No zooplankton; 2) Feeding newly hatched larvae with non-

enriched rotifers fed on InstantAlgae Nanno 3600 at 20/mL and withArtemia nauplii added at 1/mL from

5 DPH; and 3) Feeding newly hatched larvae with enriched rotifers fed on Algamac ProtienPlus at 20/mL

and withArtemia nauplii added at 1/mL from 5 DPH, were tested to determine their effect on survival of

larvae ofE. bicolor. For each treatment, 3 replicate tanks were used with 50 healthy newly hatched larvae

introduced within a few hours after hatching to each circular 10L larvalrearing tank containing 8L of staticseawater. A 100% water change was carried out every 24h in the morning; surviving larvae were gently

transferred with a 50ml beaker into to a new tank containing the same zooplankton density and water quality

parameters as the previous tank. During which time the remaining healthy larvae in each tank were counted.

Moribund larvae were discarded. Water temperature was maintained at 28-29C, salinity 35 to 37, pH 8.0 to

8.3, NH3< 0.25, NO2< 0.05 ppm, NO3 < 20 ppm. Throughout all culture experiments, the photoperiod was

maintained at 24 L: 0 D.

All rotifers used for the experiment were initially cultured using a commercially available algal paste

ofNannochloropsis sp.(InstantAlgae Nanno 3600). For the enrichment treatment, rotifers were first

harvested from the culture tank containingNannochloropsis sp. and enriched using a high protein algae

powder for 8h according to the product guidelines (Algamac Protien Plus).Artemia nauplii (INVE

technologies, Thailand LTD; GSL) were hatched daily and added fed to larvae without enrichment.

In all larval culture tanks green water was used;Nannochloropsissp.(InstantAlgae Nanno

3600)was added to the water until the bottom of the larvae rearing tank could not be seen. Continuous

aeration was provided. On 0-2 DPH aeration was provided at a lower rate of ~50mL/min to minimise

mechanical damage (Olivotto et al. 2006). This was increased to ~100mL/min on days 3 to 8.

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3. Results


Eighteen individuals were introduced into three 300L and two 1000L broodstock tanks until

combinations were found where no aggressive territorial behaviour was observed. It was found to be

impossible to keep more than 2 adults in a single 300L tank and no more than 4 adults in the 1000L without

aggressive territorial behaviour taking place. Between 1-3 months following their introduction, and the

harmonious relations being established, four females began spawning. Simply grouping a male and female

together did not necessarily result in spawning. Meanwhile, individuals did not have to be of the opposite sex

to live harmoniously. The females spawned in both the large (1000L) and small (300L) tanks. As is typical

with species of the genus Blenniidae (Kunz 2004), the males were observed to guard and care of the eggs

(oxygenate, remove debris and diseased/unfertile eggs). Although spawning was observed in all of the sizes

and shape of PVC pipe shelters, the majority (74.5%) of egg deposits (41 out of 55 recorded spawns) were

made in capped 50mm PVC pipe shelters with a single narrow entry hole (25mm).

3.2 Spawning Intervals

Spawning intervals were recorded for a 62 day period between September and December, 2011. The

change in season, photoperiod and lunar cycle over this time appeared to have no effect on spawning intervals

(See Fig. 3). The individual females spawning times did not align with other females; proving that this

species does not coordinate their spawning times with lunar cycles under captive conditions.

Of the single pairs, the number of spawns made by Female #1 and Female #2 over the 62-day period

was very simular. Female #1 spawned 14 times and Female #2 spawned 15 times. The mean spawning

interval for both females was the same (4 days); although the standard deviation for Female #1 and #2 was

different at 15h and 32h respectively. For example, the shortest spawning interval for Female #1 was 2

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days, whereas for Female #2 it was 3 days. Similarly, the longest spawning interval for Female #1 was 7 days,

whereas for Female #2 it was only 5 days.

Figure 3. Spawning intervals for twoE.bicolorfemales (F#1 & F#2) and the day on which fresh spawns were found in the group tankcontaining two males and two females (F#3 & F#4).

In the Group tank (Female #3, #4 and 2 males), fresh spawns were found much more frequently that

in the tanks containing only one female. The mean time between fresh spawns being discovered was every 2-3

days (60h 21). Out of the 62 days, fresh spawns present on 24 of these days. In this tank, it was not possible

to accurately determine which spawn belonged to which of the two females over the full 62 day period. Both

females were observed to become pregnant and all eggs were spawned in the PVC pipe inhabited by the same

male. The other male failed to take place in the reproductive process.

3.3 Fecundity

Individual spawns were laid on the substrate directly next to the previous spawn. Fresh spawns could

easily be distinguished as sufficient time had past between spawns for the former one to become noticeably

more pigmented. The largest female (Female #4; 9.1cm) produced the highest mean number of eggs in a

single spawn (3223 556 eggs), whilst the smallest female (Female #2; 6.4cm) produced the lowest mean

fecundity (2300 986 eggs). Female #4 had significant higher mean fecundity than all other females (p0.05) (See Fig. 4).

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Figure 4. Mean no. of eggs produced per spawn of the four femaleE.bicolorrelated to their total length.


Freshly spawned eggs

The newly laid lithophilic eggs were 1200150m at the widest point and possessed a

transparent chorion. The yolk sphere (YS) could be viewed through the shell; it was pale yellow in colour.

The egg envelope was spherical in shape with a flattened surface where they were attached to the substrate in

a near-geometric consistency. The egg envelope was attached to the substrate with fibrils, which were

restricted to the animal pole. These fibrils made up an adhesive disc; seemingly at the site of the micropyle.

Cleavage Phase

By 1h (Fig. 5a) after being exposed to saltwater the pervitilline space (PV) had formed. Initially there

was one single large oil globule (OG), at the animal pole, with numerous others that were tiny in comparison;

this large globule became fragmented soon after fertilization and began to migrate towards the vegetal pole.

The cleavage stage (2 cells) occurred approximately 1.5h post fertilization (PF) with approximately 0.5 h

intervals between cell divisions thereafter. The cytoplasmic divisions were meroblastic in nature and

displayed great regularity. The first divisions created a one-layer blastodisc (B) (meridional cleavages) and

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this was followed by horizontal cleavages which yielded a bilayer disc. The cleavage stage was complete by

5h PF (the blastomers rearranged into two layers). As the eggs were attached to the substrate at the animal

pole, therefore embryo development began between the yolk sphere and the substrate making it impossible to

clearly photograph the cleavage phase.

Blastula Phase

By 10h PF (Fig. 5b) the blastoderm (BD) and periblast (P) had formed at the animal pole and covered

approximately 50% of the yolk sac. At this time the volume of the perivitelline space had increased

substantially; this coincided with a contraction of the yolk sphere. The oil globules were numerous and of

various sizes, and located at the vegetal pole.

Gastrula Phase

By 20 h PF (Fig. 5c) the blastoderm covered around 90% of the yolk sphere and epiboly was

underway (periblast and outermost envelope layer (EVL) spread over the yolk sac). At this time epiboly had

advanced to a stage where involation of the blastodisc (BD) could be observed along the whole germ ring

(GR) of the expanding blastodisc. At this time the oil globules had consolidated into a single structure. By 25h

PF (Fig. 5d) the dorsal lip (DL) could be observed; here involation appeared more extensive, most likely as it

was the site of the future embryonic axis (Kunz 2004).

Embryo Phase

By 30h PF (Fig. 5e), closure of the germ ring and formation of the yolk sac (YS) has taken place; the

periblast, envelope layer, and yolk syncytial layer (YSL) surrounded the yolk sphere completely. This

signified the beginning of the embryo phase and the end of the gastrula phase (Kunz 2004). The tail bud (TB)

could be observed as a mass of undifferentiated cells. By this time the first migratory melanophores (Mm)

had developed on the blastoderm covering the yolk sac.

Neurula Phase

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By 40h PF (Fig. 5f), the tail bud had given rise to the tail (T) and posterior trunk somite formation

was complete. Eye buds (E) and lens (L) formation was also visible for the first time. Migratory melanophores

were larger at this time and more numerous as the blastopore continued to advance over the surface of the

yolk sac. By 55h PF (Fig. 5g) the embryo had grown a beak-like structure forming at the anterior of the

embryo (cephalization). The brain continued to form at the end of the embryonic body and eye pigmentation

was visible for the first time.

Turn-Over Phase

By 65h PF (Fig. 5h), the turn-over phase was complete; the embryo has completely turned over so

that the yet-to-be -formed jaw (J) pointed upwards, away from the substrate (vegetal pole). At this point the

embryos length was approximately that of the eggshell and the posterior part of the body had begun to detach

from the yolk sac, so that the tailed moved freely. The heart rate was 6515 beats per minute (bpm). Blood

was without pigmentation at this time.

By 70h PF (Fig. 5i), the yolk sac had detached more extensively from the posterior of the embryo

and had also begun to detach anteriorly from the central body. Formation of the jaw was underway and

became visible, at this time, as a hole in the head. At 75h PF (Fig. 5j), the embryo had grown in size and the

yolk sac had diminished and detached anteriorly from the central body to such an extent that the atrium and

ventricle of the heart (H) could be viewed working in tandem (the pericardium was fully developed). The

heart rate was 13512 bpm. Pale pigmented blood cells could be observed at this point. The eye appeared to

be fully pigmented and the telescopic lens had begun to form.

At 85h PF (Fig. 5k), as the embryo continued to grow and size and the yolk sac continued to

diminish, migratory melanophores had begun to move to the body. They appeared as body melanophores

(Mb) for the first time at the base of the tail of the embryo. By 95h PF (Fig. 5l) trunk movements could be

observed and xanthophores had formed on the ventral side of the head. Blood could also be observed pumping

inside the gills. At this point the heart rate was 156 24bpm and the blood had darkened from a pink to red.

By 105h PF (Fig. 5m), alongside trunk movements, eye movement could also be observed at this stage,

signifying that their development was complete. The iris (I) now appears blue/silver and reflective. At 125h

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PF (Fig. 5n), the jaw was fully formed and begins to move. Body melanophores appeared at the opening of the

jaw and migratory melanophores were no longer present on the yolk sac. At this stage the digestive tract

could be observed alongside the bright green gall bladder (GB). Little difference could be noticed regarding

the embryos morphology between 125h and 150h PF (Fig. 5o), with the exception of continued diminishment

of the yolk sac and oil droplet. Immediately before hatching the yolk sac and oil globule had become so

diminished that they could no longer be observed clearly. At this time the oocytes measured 1500120m in

diameter at the widest point; a decrease in size from when they were freshly laid (1200150m).

Figure 5. Newly deposited oocyte (a) ofEcsenius bicolor, showing oil globule (OG), chorion (C), vitelline membrane, perivitelline space (PV), andyolk sphere (YS). Blastula and Gastrula embryonic development (b-e), showing the blastodisc (B), blastoderm (BD), dorsal lip (DL), yolk syncytiallayer (YSL), periblast (P), envelope layer (EVL), tail bud (TB) and germ ring (GR). The Neurula phase (f-g), showing the eye (E), migratorymelanophore (Mm), and somites. The turnover phase (h-o), showing melanophore in the body (Mb), lens (L), iris (I), the cornea (C), tail (T), jaw (J),heart (H) and gall bladder (GB).

3.5 Larvae Observations

Larvae hatched approximately 157h PF, at dusk. Upon hatching the majority of larvae remained on

the bottom and displayed erratic swimming patterns, after a few hours however most swam in an evenly

dispersed pattern within the water column. Larvae were robust in nature as they could withstand transferral

between tanks and striking the tank wall seemingly without incidence.

The mean body length of newly hatched larvae was 3.150.3mm. The pectoral fins were heavily

pigmented. Pigmentation was also observed on the head, lower jaw and tail.Newly hatched larvae possessed

well developed eyes, a finfold, a functional gut and no visible yolk sac or oil droplet upon hatching. First

feeding did not occur immediately upon hatching (dusk) even when artificial light and prey was provided.

However predatory behaviour was observed in healthy larvae by the natural dawn time (less than 1 DPH). The

jaw was well developed upon hatching and the vast majority possessed open mouths. Mouth gape height was

18

(j) 75h

(m) 105h (n) 125h (o) 150h

(k) 85h

(g) 55h (h) 65h (I) 70h

(l) 95h

(a) 0h

(f) 40h(e) 30h

(c) 20h(b) 10h

(d) 25h


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34656m; they were therefore capable in ingesting most rotifers which range in size from 70 to 360m

(Theilacker & McMaster 1971). Individuals stalked their prey in a very energetic but cautious fashion;

approaching the target from multiple angles for a considerable length of time before attempting a strike.

Larvae at 5 DPH, alongside increased pigmentation, had undergone antero-posterior elongation of the

head and overall had increased in length to 3.720.4mm. At this time the largest individuals in the sample had

sufficient mouth gape to ingestArtemia nauplii.E.bicolorlarvae exhibited a strong predatory response

towards the nauplii and so could easily be distinguished in the gut (See Fig. 6).Artemia nauplii used in this

study had a mean length of 491125m at the widest point. Mean mouth gape height at 5 DPH was

38596m.

Figure 6.E. bicolorlarvae at 5 DPH measuring 4.2mm withArtemia nauplii visible in the gut.

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E.bicolorlarvae that were not fed achieved 100% mortality on 3 DPH (See Fig. 7), whereas fed larvae

had a survival rate on 3 DPH of 25.36.1% and 34.010.0% for rotifers enriched withNannochloropsis sp.

(InstantAlgae Nanno 3600) and Algamac ProtienPlus respectively. Larvae fed on both of these rotifer

feeding treatments did not reach total mortality until 8 DPH. There was a significant difference on 2 & 3 DPH

(p


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Figure 7. The mean survival(%) of newly hatched larvae ofE.bicolorlarvae under 3 different feeding regiems. 1) starvation 2) larvaefed rotifers cultured on microalgae nanochloropsis (InstantAlgae Nanno 3600) +Artemia nauplii. 3) Larvae fed rotifers enrichedwith Algamac ProtienPlus andArtemia nauplii.

4. Discussion


Firstly, when acquiring adults for this study, a widely held misconception regardingE. bicolour

gender determination was revealed. Ornamental suppliers believe that colour variation is an indicator of

gender, i.e. the uniform brown variations are females and the bicoloured variations are males. This

misconception initially led to unsuccessful parings ofE. bicolorin some cases and time and efforts being

wasted. In fact, there is no correlation between colour form and gender; rather it is the length and number of

caudal fin elements which make this species sexually dimorphic (Springer 1988). Furthermore, this

misconception led to an initial delay in obtaining the correct broodstock as the individuals with the uniform

colour form (mistaken as females) were found to be much more difficult to source since they are not as

popular as bicoloured form in the aquarium trade.

During this study, the females were shown to spawn in both the large (1000L) and small (300L) tanks,

therefore this species seems to be able to adapt well to relatively small broodstock tanks as well as various

sizes and shapes of spawning shelters (spawning was observed in all types of PVC pipe shelters). This is a

major plus for a candidate species for aquaculture as it means lower requirements for space and facilities and

therefore, costs for maintaining broodstock. However, the majority (74.5%) of egg deposits were made in

capped 50mm PVC pipe shelters with a single narrow entry hole (25mm), which suggests a preference for a

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more confined space. During future aquacultural endeavours into culturing this species, providing a hide of

this type might help in improving reproductive efficiency.

As is consistent with most coral reef fish species, it was necessary to keepE.bicolorbroodstock at a

low density (Hoff 1996; Tucker 1998; Tlusty 2002) due to their territorial behaviour. The observations from

the present study show that when attempting to pair adults, it was not possible to contain more than 2E.

bicolorin a single 300L tank, nor was it possible to contain more than 4 in the 1000L tank. It is constantly

stated in hobbyist manuals for aquarium keeping (Scott 2005) thatE. bicolorshould not be kept in groups, as

they are highly territorial towards individuals of their own species. This study raises some interesting

questions regarding the densities needed to trigger the territorial behaviour ofE. bicolor. It is general practicewith this species that only one pair should be contained in a tank no smaller than 75 L in order to prevent

aggressive territorial behaviour (Scott 2005). In this study broodstock were provided with tanks much larger

than 75 L and although in the smaller tanks (300L) only two individuals could coexists without aggressive

territorial behaviour being observed, it would seem if the tank is large enough and sufficient hides are present;

this species can exist in numbers greater than a single pair (in this study four individuals remained healthy in a

1000L tank). It is therefore possible that more than one pair ofE. bicolorbroodstock might be kept together

and with other fish in large tanks to minimums the costs, considering that this species is not aggressive

towards other species and they are benthic in nature.

Another very interesting result observed in this study is the proof that not only canE. bicolorbe kept

groups in larger tanks, but a harem breeding structure can also be established. All spawns deposited by

Female #3 and #4 living in the group tank were fertilized, often on the same night, by the same male and in

the same hide. The other male in the tank failed in participating in the reproduction process. Therefore from

an aquacultural perspective, as females have been shown to willing to share a male, egg production in larger

tanks can be maximised in the future by attempting to keep multiple females with a single male.

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4.2 Fecundity and Spawning Intervals

The results of this experiment show thatE. bicolorhave a relatively low fecundity per spawn in

comparison to other species with a simular demersal spawning technique (Kunz 2004; Olivotto et al. 2005;

Yasir & Qin 2007; Wittenrich et al. 2007). However, unlike many reef fishes who only spawn once or a few

times during breeding season,E. bicolorspawns regularly and with very short intervals (every 4 days on

average during the observation period of this study). Furthermore, it appeared that under a controlled

temperature (27.5-29.5C), the spawning fecundities and intervals were not affected by seasonality,

photoperiod or lunar cycle, despite the broodstock tanks being located outside. Due to the time limitations of

this project there is no hard evidence that the species spawns consistently all year round. However it is very

likely based on the observation that spawning was ongoing for more than 6 months. This is a major advantage

from an aquacultural perspective as eggs can be obtained reliably and extremely frequently.

It is typical with most teleost fish (Thresher 1984) for the number of eggs produced by a female to

vary depending upon her age and size, and that this is true withE.bicolor; the largest female had the greatest

mean fecundity. When aquaculture is undertaken for this species, it might be advantageous to obtain large

broodstock. Furthermore, given that this species can live for 10 years in captivity, it clearly has the capability

of producing large amount of eggs over its lifetime.

4.3 Embryology and Hatching Indicators

As is consistent with the majority of bottom-dwelling territorial fishes (Kunz 2004);E. bicoloreggs

were demersal. The method of attaching the egg envelope with an adhesive disk at the animal pole to the

substrate was found to be similar to another species of Blenniidae;Blennius fluviatilis (Wickler 1957). The

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pale colouring of the yolk contained withinE. bicoloroocytes revealed that it contained very few carotenoids

in comparison to other demersal spawners (Kunz 2004; Yasir & Qin 2007). Therefore, as carotenoids are the

mechanism for egg taking up oxygen from the surrounding water (Kunz 2004),E. bicolormost likely restricts

depositing its eggs to areas with relatively high oxygen saturation.

Overall the embryonic development time was relatively long in comparison to other demersal

spawning species (Yasir & Qin 2007) therefore paternal care is likely to be more crucial for this species.

Indeed, accurately predicting the hatching time was paramount as it was found during this project that moving

the eggs away from paternal care too early is extremely detrimental to the hatching success; this actually

initially led to several unsuccessful hatching attempts during the present study. In many deposit spawnerspecies, such as the clownfish, the pigmentation of the eye happens immediately before hatching and this can

be seen with the naked eye through the egg shell (Yasir & Qin 2007; Miller & Kendall 2009). However forE.

bicolor, this appears to not be a valid method of predicting hatching time, as when observing the eggs using

only the human eye there appears to be little difference regarding pigmentation of eggs from day 5 PF

onwards, until hatching (at 27.5-29.5C, salinity at 29-27). From an aquacultural perspective, this could

cause a problem regarding the miss-timing of hatchings and lead to egg mortality. The detailed description of

embryonic development ofE. bicolorby the present study provided crucial information on accurate predicting

of hatching time for the species. It revealed the key subtle signs to look for in order to determine imminent

hatching; Firstly, when the yolk sac and oil globule are fully depleted; this occurs in the afternoon of day 7

PF. Secondly; late on day 6 PF the reflective blue-silver iris starts to become visible to the naked eye through

the egg shell, and by day 7 PF this becomes much more pronounced. This is the most obvious indicator for

determining imminent hatching without a microscope.

4.4 Larvae First Feeding

Based on embryonic developmental observations in this study, newly hatchedE. bicolorlarvae have

been proved to possess almost zero yolk reserve upon hatching. Which suggests thatE. bicolorshould be

provided with food immediately upon hatching, as starvation will occur quickly otherwise. Indeed, in the first-

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feeding experiment only 26% of unfedE.bicolorlarvae survived to 2 DPH, and all of them died within 3

DPH.

The first-feeding stage is commonly a bottleneck in the culture of many marine ornamental finfish

larvae (Job 2011). This is due to many newly hatched tropical marine finfish larvae possessing a very narrow

mouth gape (Holt 2003; Moorhead & Zeng 2011). However results from this study have revealed that

E.bicolorlarvae have a sufficient mouth gape height (34615m) upon hatching to feed on smaller sized

rotifers (70 to 360m) and indeed some rotifers were observed in the gut of a fewE.bicolorlarvae, so it is

clear that they were not too large.

In many cases marine ornamentals will not thrive on conventional rotifers andArtemia feeding

regimes as they do not meet the larvaes nutritional requirements (Tamaru et al. 1995; Doi et al. 1997;

Ostrowski & Laidley 2000). This seems unlikely to be the reason why fedE. bicolorlarvae failed to survive

past 7 DPH as the results of this study showed that high-protein rotifer enrichment had no effect on larvae

survival (larvae fed on the two different rotifer diets both died within the same time period). Although it is

clear that some rotifers were being ingested, as fed larvae survived 5 days longer than unfed ones, the results

of the first-feeding experiment suggest that rotifers do not elicit a strong enough predatory response for the

larvae to obtain sufficient nutrition. Simular results have been found with other marine ornamental species

(Job 2011); it has been reported that various species of angelfish (Pomacanthus spp.) refuse to eat rotifers

despite having a sufficient mouth gape (Ostrowski & Laidley 2000). It is believed that the slow whirling

swimming pattern of rotifers does not provoke a strong predatory response; rather the fast stop-start

swimming pattern of copepod nauplii is required (Young 1994; Moe 1997). Furthermore, larvae ofE. bicolor

were observed to be strong swimmers shortly after hatching and were very active most of time. Individuals

stalked their prey in an energetic but cautious fashion, approaching the target from multiple angles for a

considerable length of time before attempting a strike; all of which is likely to be very energetically costly and

most likely goes some way to explaining the eventual starvation and mortality of the fed larvae. These factors

combined (the energetic nature of the larvae and poor predatory response towards rotifers) suggest that this

species may have a preference for fast-moving, highly nutritious zooplankton, such as copepod nauplii.

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The next stage of culture experimentations for this species should revolve around using alternative

preys such as copepod nauplii. However, large scale culture techniques for copepod nauplii are still under

research and development (Stottrup & Norsker 1997; Ostrowski & Laidley 2000). Wild-caught zooplankton

between 53125m (first feeding size for marine ornamentals) is made up of approximately 60-80% copepod

nauplii and copepodites (Job 2011). Indeed, wild zooplankton has been used to successfully culture some

marine ornamental finfish larvae which have a poor predatory response to rotifers (Moe 1997). However,

using wild zooplankton should not be attempted for research purposes due to the inherent inconsistency of

supply (Ostrowski & Laidley 2000).Other first-feeding size plankton including oyster trocophores, tintinnids,

diatoms and phytoplankton have been used as first-feed prey for grouper larvae (Epinephelus spp.), which are

similar in terms of their complexity to culture as marine ornamental larvae (Riley & Holt 1995; Watanabe et

al.1996; Ostrowski & Laidley 2000).

Once a sufficient mouth gape had been obtained by the larvae at 5 DPH,E.bicolorappeared to have a

strong predatory response towardsArtemia nauplii; in that they fed until the gut was clearly bloated with this

zooplankton. The reason why surviving larvae at 5 DPH continued to die despite feeding well onArtemia

nauplii cannot be reliably determined from the results of this project. However, the most likely explanation is

that the survivors were already moribund at 5 DPH, having failed to ingest sufficient calories and micro-

nutrients to carry out metamorphosis prior toArtemia nauplii feeding. Once the first-feeding (0-4 DPH)

stumbling block has been overcome, it is likely thatE. bicolorlarvae will be able to thrive, perhaps with

nutritional enrichment, onArtemia nauplii, due to the strong predatory response that was observed.

Conclusion

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In answer to question of the potential for culturingE.bicolorfor the marine aquarium industry, it can

be concluded that it is a good species for further study. This conclusion is justified when considering the

adults suitability for personal aquariums; they are non-aggressive towards other species, adapt well to

commercial foodstuffs and are considered to be a hardy species by hobbyists. They command a relatively

high retail price, there is good potential for broodstock to be collected in a sustainably way. Furthermore,

females can be organised into harem breeding structures to maximise egg production. Many of the stumbling

blocks that are frequently encountered when attempting to culture marine ornamentals are not an issue forE.

bicolor. For example, they spawn readily in captivity and in a short time frame after establishment, on a

variety of easily obtainable substrates. Furthermore, they have relatively short spawning intervals meaning

that eggs can be obtained regularly and predictably, a major plus for aquaculture. The larvae themselves are

robust in nature and able to cope with the stress of handling. However, based on the results of this study, the

most likely cause for larvae mortality was starvation due to insufficient rotifer ingestion as a result of them not

provoking a sufficient predatory response. The next stage in devising a successful culture protocol for this

species should be to change the first-feeding zooplankton (copepod nauplii are recommended by the findings

of this study). Once this first-feeding stumbling block has been overcome, it is likely thatE. bicolorlarvae

will be able to thrive, perhaps with nutritional enrichment, onArtemia nauplii, as they expressed a strong

predatory response towards them.

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