The Potential for Culturing Ecsenius bicolor for the Marine Aquarium Industry

By Anna Moseley MAppSc Aquaculture

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Aims
Main aim: Investigate the potential of culturing the Bicolor Blenny for the aquarium industry.
1. Investigate Reproductive Behaviour, Spawning and Fecundity under captive conditions 2. Record the Embryological life history and PreHatching Indicators 3. Carry out Larvae Hatching and First-Feeding trials

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Introduction: Controversies of Wild-Collection

Exponential growth in Marine Aquarium industry (US $278 million in 2005)
(FAO 1996-2005).

90-98% = wild-caught specimens (Job 2011; AIMS 2011)

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Insensitive shipping methods Lack of sufficient husbandry

(Wabnitz 2003; Green 2003)

Destructive fishing: Target rarer species (Pomeroy et al. 2006) Use of cyanide Target key herbivores (Green 2003; Tlusty
2002).

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100 successfully cultured; vast majority on a hobbyist or research scale

(Job 2011).

Only 1/3 are in commercial production Page 5

Reduce pressures on wild population & Studying Reproduction & Early Life History. Replenish overfished stocks
(Job 2011)

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Current Literature
Commercial Culture Protocols: Stunted by a lack of published culturing protocols for most species (Job 2011).

Proprietary nature (Job 2011).

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Introduction: The Bicolor Blenny

Species: bicolor Genus: Ecsenius Family: Blenniidae Class: Actinopterygii Subphylum: Vertebrata Phylum: Chordata Kingdom: Animalia

Natural range; Indo-Pacific Wide distribution & relatively abundant Potential for broodstock to be collected from a wide range of locations, in a sustainable way. Spawns in captivity
(Wickler 1965).

Sexually dimorphic.

Never been cultured successfully (Wickler, 1965; Job 2011) No publications on species regarding embryology, spawning intervals, fecundity or larval feeding

(Springer 1988)

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Brightly coloured & small (1011cm) (Randall et al. 1997; Scott 2005)

Herbivorous; do not eat ornamental corals or invertebrates

(Scott 2005)

SUITABLE AQUARIUM SPECIES

Thrive on commercial foodstuffs

(Scott 2005)

Adapt well to small aquariums

(Scott 2005)

Clean; low pressure on filtration

(Scott 2005)

Non-aggressive; exception of own kind

(Scott 2005; Skomal 2007)

Considered to be hardy
(Scott 2005)

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 Still relatively high; US$45 (max) at online stores (e.g.

SaltWaterFish, VividAquariums, Magento, and CreateAReef).

Many other species do not acclimatize well.

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Colour Variations

Springer, 1988

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Part 1 Broodstock Conditioning, Spawning and Fecundity

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Methodology: Broodstock Conditioning

Fed daily on enriched high protein and lipid home-made gelatine-bound wet diet High in lipids (HUFAs); Maximise reproductive efficiency and egg/larvae quality
et al. 2003; Papanikos et al. 2004; Salze et al, 2005).

(Emata et al. 2000; Wittenrich et al. 2007; Lin et al. 2007; Murugan et al. 2009; Mazorra

Increased protein = promote reproduction efficiency and gonadosomatic index

(Lin et al. 2007; Job 2011).

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Methodology: Broodstock Tanks

M.A.R.F.U Conditions

Undercover
Natural photoperiod Water temperature 27.5-29.5C Salinity at 29-27, pH at 8.0-8.2, NH3, NH2 at <0.02 ppm and N03 at >6ppm.

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Methodology: Spawning times and intervals

Structures for territory & shelter; live in crevices on coral reefs (Wickler 1965, Randall et al. 1997).
Blennies known to spawn on PVC pipe, ceramic tiles and live rock (Olivotto et al. 2005; Wittenrich et al.
2007; Moorhead & Zeng 2011).

Spawning tubes: 50mm & 25mm open PVC pipes, & 50mm capped pipes with a single 25mm entrance. Spawning intervals (in pairs): Checked daily over a 62 day period. Spawning times: pipes checked hourly over 24 hours (3 replicates). Page 15

Results: Spawning Behaviour

Females spawned in both the large and small tanks (adapt well)
First time; 1-3 months. (Once successful pairing established) Males guard and care for the eggs (oxygenate, remove debris and diseased/unfertile eggs) Spawn in all of the types of artificial shelters. Preference. Majority in capped PVC pipe shelters with a single narrow entry hole. Spawning occurred within the first hour after DAWN.

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Results: Spawning Intervals

Pair 1
14 spawns during a 62 day period Average spawning interval = 96h(31) Shortest interval = 48 hours Longest interval = 168 hours

Pair 2
15 spawns during a 62 day period Average spawning interval = 96h (15) Shortest interval = 72 hours Longest interval = 120 hours

Group
Average time between egg acquisition = 60h (21) Out of 62 days, fresh eggs were laid on 24 of them. Spawns from both females laid on the same night (observed) but indistinguishable from one another.

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Methodology: Fecundity
Laid on the clear plastic substrate Easily be distinguished due to pigmentation Replicates: Three spawns (replicates) counted for each of the 4 females. Group tank: Females observed for heavy pregnancy the day before spawns were laid to determine which female laid the eggs. Page 18

Methodology: Egg Counting

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Results: Body Length vs. Fecundity

4000

3500

3000 Mean No. Eggs S. E.

2500

2000

1500

1000

500

0 Female 4 (9.4cm) Female 3 (8.2cm) Female 1 (8.7cm) Female 2 (6.4cm)

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Results: Annual and Lifetime Fecundity

Photoperiod = No effect on spawning intervals

Mean No. Eggs S. E

Batch Fecundity Female 4 (max)= 3223 ( 557) Female 2 (min)= 2300 ( 985) Annual fecundity (85 spawns) Female 1= ~273,995 eggs/year Female 2= ~195,500 eggs/year Lifetime Fecundity ~10 Years (in captivity) Able to produce millions Cannot accurately predict (fecundity changes with size)

4000 3500 3000 2500 2000 1500 1000 500 0 Female 4 (9.4cm) Female 3 (8.2cm) Female 1 (8.7cm) Female 2 (6.4cm)

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Part 2 Embryology and Hatching Prediction

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Aquacultural Perspectives
Document length of embryological phase (28-29 C) Morphology that signify hatching is imminent

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Results: Embryological Life History

Egg Size
(10 randomly sampled)

120

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Results: Embryological Life History

Newly deposited oocyte (A) of Ecsenius bicolor, showing cytoplasm (Cy), oil globule (OG), chorion (C), and yolk sac (YS). Blastula and Gastrula embryonic development (B-E), showing the blastodisc (B), blastoderm (BD), dorsal lip (DL), yolk syncytial layer (YSL), periblast (P), envelope layer (EVL), deep cell (DP), germ ring (GR). The Neurula phase (F-G), showing the eye (E), migratory melanophore (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).

0h

10h

20h

25h

30h

40h

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55h

65h

70h

75h

85h

95h

105h

125h

150h

Newly deposited oocyte (A) of Ecsenius bicolor, showing cytoplasm (Cy), oil globule (OG), chorion (C), and yolk sac (YS). Blastula and Gastrula embryonic development (B-E), showing the blastodisc (B), blastoderm (BD), dorsal lip (DL), yolk syncytial layer (YSL), periblast (P), envelope layer (EVL), deep cell (DP), germ ring (GR). The Neurula phase (F-G), showing the eye (E), migratory melanophore (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).

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Discussion: Embryological Life History

TIMING IS CRITICAL; moving the eggs away from paternal care too early = extremely detrimental to the hatching efficiency. (Hatch after ~157h) Human eye; appears to be little difference from day 5, 6 and 7 (early eye pigmentation)

105h

125h

150h

Late on day 6, the reflective blue-silver iris becomes visible to the naked eye.

When viewed under a microscope; few morphological changes between day 6 and 7.
The key sign; yolk sac and oil globule are fully depleted. Occurs in the Page 27 afternoon of day 7.

Part 3 Larvae Hatching and Pilot firstfeeding trials

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Methodology: Hatching
Eggs remained in paternal care until hatching
(Olivotto et al. 2005; Wittenrich et al. 2007; Moorhead & Zeng 2011).

Dusk- on expected hatching, PVC tube was removed Scrubbed clean 20L hatching tank (water quality parameters=
broodstock tanks)

Aeration across the eggs at 1L/min

Wittenrich et al. 2007; Moorhead & Zeng 2011).

(Olivotto et al. 2005;

Without aeration eggs suffer from high mortality

(Wittenrich et al. 2007).

Tank covered to eliminate light (dusk) Larvae (normal swimming behaviour) were counted and transferred to rearing tanks. Page 29

Results: Newly Hatched Larvae

Eggs had high survival and larvae hatch rate.

Pectoral fins heavily pigmented + lower jaw & tail. Well developed eyes A functional gut

Hatched ~157 hours postfertilization, at dusk. Body length: 3.15mm 1.4mm.

No visible yolk sac or oil Well developed open jaw droplet

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First Feeding
Mouth gape height was 346m56 (10 randomly selected larvae); capable in ingesting rotifers (70- 360m)

Top Jaw = 264m44

Bottom Jaw = 223m38

GH=(UJL2+LJL2)
(Wittenrich et al., 2007)

Predatory behaviour was observed in healthy larvae by the natural dawn time (less than 1 DPH). However, in most larvae rotifers could not be observed in the gut despite high rotifer density (20 per mL)

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Methodology: Larvae First Feeding Experiment

Many marine ornamentals will not thrive on rotifers; do not not meet the larvaes nutritional requirements (Tamaru et al.
1995; Doi et al. 1997; Ostrowski & Laidley 2000; Olivotto et al. 2006; Olivotto et al. 2008).

Three separate feeding regimes:

1) Starvation (do they eat?)
2) Non-enriched rotifers @ ~20 per mL (day 1-7) + Artemia spp. Nauplii @ 1 per mL (day 5-7) 3) Enriched rotifers @ ~20 per mL (day 1-7) + Artemia spp. Nauplii @ 1 per mL (day 5-7)

Replicates:

Each feeding regime = 3 tanks with 50 larvae in each Standard error found
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Methodology: Larvae First Feeding Experiment

HIGH WATER QUALITY
Salinity 35 to 37 pH 8.0 to 8.3 NH3< 0.25, NO2< 0.05 ppm, NO3 < 20 ppm

28-29C Tank size: 8L Photoperiod: 24h L: 0h D. Survival and growth benets

(Olivotto et al., 2003, 2005, 2006).

AEARATION
Continuous aeration (improve dissolved oxygen content & feeding efficiency) (Mackenzie & Kiorboe 1995; Job 2011).

Days 0-2 aeration ~50mL/min to minimise mechanical damage (Olivotto et al. 2006).
Increased to ~100mL/min on days 3 to 7.

Static water. 100% manual water change every 24 hours

GREEN WATER
Nanocholoropsis: Dissipate light & maintain the nutritional value of rotifer (Job et al. 1997; Olivotto et al. 2008, Moorhead & Zeng 2011) . Reduce phenomena of larvae being attracted to wall (feeding efficiency & mechanical damage (Job 2011).

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Results: Larvae First Feeding Experiment

100 90

Mean Mortality (%) S. E.

80

70

60 Starvation 50 Non-Enriched Enriched 40

30

20

10

0 0 1 2 3 4 5 6 7 8

Days Post-Hatch Page 34

Results: Day 5 Larvae

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Discussion: Larvae First Feeding Experiment

Most angelfish larvae refuse to eat rotifers despite having sufficient mouth gape (Ostrowski & Laidley 2000). Whirling of rotifers; no predatory response. Stop-start pattern of copepod nauplii is required
(Young 1994; Moe 1997).

Large NEXT STAGE: scale Copepods culture techniques for Bicolor for copepod Blenny nauplii larvae? still under R&D (Stottrup &
Norsker 1997; Ostrowski & Laidley 2000).

Appear to be designed to catch fast moving, large, high energy zooplankton: Wild-caught zooplankton between 53 125m (first feeding size) = 6080% copepod nauplii and copepodites (Job 2011)(Moe 1997). 1) Large, highly developed eyes 2) Very relatively large mouth gape 3) foraging behaviour Energetic Should not be attempted for research purposes; inconsistency (Ostrowski & Laidley
2000).

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Conclusions
What is the Potential of Culturing Ecsenius bicolor for the Marine Aquarium Industry?

Good subject for further study. Adults suitable for aquarium + good retail price Spawns readily in captivity High egg survival and hatching rates Short spawning intervals Robust larvae able to cope with handling Eat Artemia nauplii readily Does not feed well on rotifers
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Embryology and larviculture of the Bicolor Blenny (Ecsenius bicolor)

Chaoshu Zeng Pamela Benjasirichai Jonathan Moorhead Amanda Rickets

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