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By Anna Moseley

The Potentialfor Culturing

Ecsenius bicolor for the

Marine Aquarium Industry

MAppSc Aquaculture

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AimsMain 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 Pre-Hatching Indicators

3. Carry out Larvae Hatching and First-Feeding trials

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

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

Introduction: Controversies of Wild-Collection

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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

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Reduce pressures on

wild population

&

Replenish over-fished stocks

(Job 2011)

New methods

of culture can

often be

transferred

(Dhert et al. 1997).

Advance human knowledge of the biology & life history of coral reef fish

Studying Reproduction & Early Life History….

Measuring

reproductive

success can

determine safe

catch level (Pomeroy et

al. 2006)

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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).

FEW STUDIES ON BLENNIES

VA

ST

MA

JOR

ITY

SIN

CE

2000

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• 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.

Introduction: The Bicolor Blenny• Species: bicolor• Genus: Ecsenius• Family: Blenniidae• Class: Actinopterygii• Subphylum: Vertebrata• Phylum: Chordata• Kingdom: Animalia

(Springer 1988)

• Never been cultured successfully (Wickler, 1965; Job 2011)

• No publications on species regarding embryology, spawning intervals, fecundity or larval feeding

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SU

ITA

BL

E A

QU

AR

IUM

SP

EC

IES

Brightly coloured & small (10-11cm) (Randall et al. 1997; Scott 2005)

Herbivorous; do not eat ornamental corals or

invertebrates (Scott 2005)

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

MOST DESIRED

BICOLOURED (~48%)

RARE (~4%)

STRIPED

BROWN (~48%)

BLACK DOT

Springer, 1988

Misconception that colour relates to gender!

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

et al. 2003; Papanikos et al. 2004; Salze et al, 2005).

• Increased protein = promote reproduction efficiency and gonadosomatic index (Lin et al. 2007; Job

2011).

BLENDED FISHSQUID

MUSSEL PRAWN

MULTI-VITAMINSMINERALS

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Methodology: Broodstock Tanks

M.A.R.F.U Conditions

• Undercover

• Natural photoperiod

• Water temperature 27.5-29.5˚C

• Salinity at 29-27‰, pH at 8.0-8.2, NH3, NH2 at <0.02 ppm and N03 at >6ppm.

PAIR 1

PAIR 2

GROUP

800L 800L

300L

Eventual harmony achieved

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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).

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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.

DEFIES HOBBYIST MANUALS?

No apparent synchronisation(Does not follow

lunar cycles)

VERY SIMULAR

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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.

SAME SPAWN

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Methodology: Egg Counting

2) MEAN NO. PER SQUARE FOUND

1) GRID OVERLAY (PHOTOSHOP)

3) SQUARES COUNTED

(100% EGGS) 4) UNFILLED SQUARES: EGGS COUNTED

INDIVIDUALLY

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Results: Body Length vs. Fecundity

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

500

1000

1500

2000

2500

3000

3500

Mea

n N

o.

Eg

gs

± S

. E

.

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Results: Annual and Lifetime Fecundity

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)

Photoperiod = No effect on spawning intervals

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

500

1000

1500

2000

2500

3000

3500

Mea

n N

o.

Eg

gs

± S

. E

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Part 2

Embryology and Hatching Prediction

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Aquacultural Perspectives

• Document length of embryological phase (28-29 ºC)

• Morphology that signify hatching is imminent

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Egg Size Results: Embryological Life History

DAY 1

DAY 7

±150

±120

(10 randomly sampled)

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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).

Results: Embryological Life History

0h

40h30h

20h10h

25h

Page 26Newly 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).

75h

105h 125h 150h

85h

55h 65h 70h

95h

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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)

• 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 afternoon of day 7.

105h 125h 150h

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Part 3

Larvae Hatching and Pilot first-feeding 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 (Olivotto et al. 2005;

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

• 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.

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Results: Newly Hatched Larvae

• Eggs had high survival and larvae hatch rate.

• Hatched ~157 hours post-fertilization, at dusk.

• Body length: 3.15mm± 1.4mm.

• Well developed open jaw

• Pectoral fins heavily pigmented + lower jaw & tail.

•  Well developed eyes

• A functional gut

• No visible yolk sac or oil droplet

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First Feeding

• Mouth gape height was 346µm±56 (10 randomly selected larvae);

capable in ingesting rotifers (70- 360µm)

GH=Ë(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)

Bottom Jaw = 223µm±38

Top Jaw = 264µm±44

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Methodology: Larvae First Feeding Experiment

Many marine ornamentals will not thrive on rotifers; do not not meet the larvae’s 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)

ENRICHED FOR 8 HOURS

BASIC F

EED

Replicates:

Each feeding regime = 3 tanks with 50 larvae in eachStandard error found

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Methodology: Larvae First Feeding ExperimentHIGH WATER QUALITY

• Salinity 35 to 37‰

• pH 8.0 to 8.3

• NH3< 0.25, NO2< 0.05 ppm, NO3 < 20 ppm

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.

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).

• 28-29˚C

• Tank size: 8L

• Photoperiod: 24h L: 0h D. Survival and growth benefits (Olivotto et al., 2003, 2005, 2006).

• Static water.

• 100% manual water change every 24 hours 

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0 1 2 3 4 5 6 7 80

10

20

30

40

50

60

70

80

90

100

Starvation

Non-Enriched

Enriched

Results: Larvae First Feeding Experiment

ALL FED LARVAE DEAD BY DAY 8

UNFED LARVAE DEAD BY DAY 3

Mea

n M

orta

lity

(%)

± S

. E

.

Days Post-Hatch

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Results: Day 5 Larvae

GUT FULL OF ARTEMIA NAUPLII

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• Large scale culture techniques for copepod nauplii still under R&D (Stottrup & Norsker 1997; Ostrowski & Laidley 2000).

• Wild-caught zooplankton between 53 – 125µm (first feeding size) = 60-80% copepod nauplii and copepodites (Job 2011)(Moe 1997).

• Should not be attempted for research purposes; inconsistency (Ostrowski & Laidley 2000).

Discussion: Larvae First Feeding Experiment

NEXT STAGE: Copepods for Bicolor Blenny larvae?

Appear to be designed to catch fast moving, large, high energy zooplankton:

1) Large, highly developed eyes2) Very relatively large mouth gape3) Energetic foraging behaviour

• 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).

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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 priceSpawns readily in captivityHigh egg survival and hatching ratesShort spawning intervalsRobust larvae able to cope with handlingEat Artemia nauplii readily× Does not feed well on rotifers

?

Page 38

Embryology and larviculture of the Bicolor Blenny (Ecsenius bicolor)

Chaoshu ZengPamela BenjasirichaiJonathan Moorhead

Amanda Rickets

Page 39

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