THE EFFECT OF DIET ON DMSP ACCUMULATION AND TASTE IN SOUTH AFRICAN ABALONE ( Haliotis midae ) Deborah Robertson-Andersson 1 ; A. J. Smit 2 ; S. Peall 3 and Bolton J. J 1 1 Botany Department, U CT 2 School of Biological and Conservation Sciences, UKZN 3 Hearshaw and Kinnes Analytical Laboratory, UCT,

What do DMSP and abalone taste have to do with each other? Abalone farming and the importance of macroalgal feeds The effect of macroalgae on abalone growth The problem DMSP Our studies Work in progress and the future

Abalone farming and the importance of macroalgal feeds

The effect of macroalgae on abalone growth

The problem

DMSP

Our studies

Work in progress and the future

Integrated abalone-macroalgal cultivation number of benefits: Increased growth rates, Improved vigor and stress resistance (Naidoo et al ., in press)

number of benefits:

Increased growth rates,

Improved vigor

and stress resistance

Benefits of a mixed algal diet KELP ONLY DIET MIXED ALGAL DIET

However….. Two farms which feed Ulva and Gracilaria exclusively experienced that their abalone developed an ‘off’ taste and sulphur-like smell after the canning process

Two farms which feed Ulva and Gracilaria exclusively experienced that their abalone developed an ‘off’ taste and sulphur-like smell after the canning process

Abalone Mariculture Operations in South Africa Abalone Mariculture Operations in South Africa Durban Port Elizabeth Cape Town I&J Abalone Atlantic Fishing Sea Plant Products Roman Bay Marine Growers HIK Farm Abagold Aquafarm Development Avuca Abalone Fish Bay Mariculture Jacobsbaai Sea Products West Coast Abalone Port Nolloth Sea Farms Wild Coast Abalone Abaseed

Questions… What is the cause of the problem? What can be done about it?

What is the cause of the problem?

What can be done about it?

Preliminary investigations Methionine, cysteine? No… Hill et al . (2000; 2004) mentioned a similar problem in tridacnid clams and implicated DMS (produced from DMSP)… Do abalone receive DMSP in their diet? 16.8 15.7 18.5 Methionine (mg.g -1 protein) 28.1 Gracilaria 13.3 Ulva 29.1 Ecklonia Cysteine (mg.g -1 protein)

Methionine, cysteine? No…

Hill et al . (2000; 2004) mentioned a similar problem in tridacnid clams and implicated DMS (produced from DMSP)…

Do abalone receive DMSP in their diet?

Preliminary investigations Do abalone receive DMSP from their feed? Van Alstyne et al . (2003) - Ulva… yes! 16.8 15.7 18.5 Methionine (mg.g -1 protein) 1.6 - 48.6 28.1 Gracilaria 4101 - 7961 13.3 Ulva 0.5 - 46.1 29.1 Ecklonia DMSP ( μg.g -1 fresh mass ) Cysteine (mg.g -1 protein)

Do abalone receive DMSP from their feed?

Van Alstyne et al . (2003) - Ulva… yes!

Background: DMSP and DMS in algae Some algal species contain high concentrations of intercellular DMSP Produced in response to freezing temperatures and fluctuating salinity hypothesised function as osmolyte DMSP is oxidised in the presence of OH - , O 3- or H 2 O 2 reaction with reactive oxygen species implies role as antioxidant Enzymatic cleavage of DMSP leads to DMS and acrylate

Some algal species contain high concentrations of intercellular DMSP

Produced in response to freezing temperatures and fluctuating salinity

hypothesised function as osmolyte

DMSP is oxidised in the presence of OH - , O 3- or H 2 O 2

reaction with reactive oxygen species implies role as antioxidant

Enzymatic cleavage of DMSP leads to DMS and acrylate

DMSP and DMS Produced by algal or bacterial DMSP lyase Thermal decomposition of DMSP Highly volatile Distinct taste and odor Noticeable at 0.03 - 0.045 μg.ml -1 in water Produced by algae Accumulated by grazers Taste - and odorless Do abalone contain DMSP?

Produced by algal or bacterial DMSP lyase

Thermal decomposition of DMSP

Highly volatile

Distinct taste and odor

Noticeable at 0.03 - 0.045 μg.ml -1 in water

Produced by algae

Accumulated by grazers

Taste - and odorless

If they contain DMSP, where is it accumulated? Top of foot Epipodial frill

DMSP values from various tissue types in mixed diet cultivated abalone diet (25% Ulva; 1 year) Preliminary investigations In Tridacnidae, siphonal mantle not abductor muscle had high concentrations (Hill et al . 2000) Oysters and mussles have highest concentrations in abductor muscle (Iida et al . 1986) TF F BF AM Top of foot Epipodial frill Bottom of foot Abductor muscle

DMSP values from various tissue types in mixed diet cultivated abalone diet (25% Ulva; 1 year)

In Tridacnidae, siphonal mantle not abductor muscle had high concentrations (Hill et al . 2000)

Oysters and mussles have highest concentrations in abductor muscle (Iida et al . 1986)

Abductor muscle preparation Step 1 Step 4 Step 3 Step 2

DMSP diet experiment Values for fish, crustaceans and other molluscs usually below 1800 μg.g-1 (Hill et al . 2000) Previous recorded highest DMSP accumulation in tridacnids with zooxanthellae (Hill et al . 2004) Abalone about 25 % higher

Values for fish, crustaceans and other molluscs usually below 1800 μg.g-1 (Hill et al . 2000)

Previous recorded highest DMSP accumulation in tridacnids with zooxanthellae (Hill et al . 2004)

Abalone about 25 % higher

DMS content in canned abalone DMS in cans up to 44 μg.ml -1 Taste threshold of DMS in water about 1000 times lower

DMS in cans up to 44 μg.ml -1

Taste threshold of DMS in water about 1000 times lower

Depuration experiment Variation is consistent with that found in bivalve mollusks (Hill et al . 1995)

Variation is consistent with that found in bivalve mollusks (Hill et al . 1995)

Wild abalone Effect of DMS content on taste/odour of the product (quality control)? Important taste consequences in seafoods (Peavy & Riley, 1993; 1994) which limit cultivation e.g. tridacnids Excessive DMS in cod is commercially problematic, but very low concentrations of DMS are part of the “valued flavor” of some clams and oysters (Ackman & Hingley, 1968; Iida & Toukuaga, 1986; Brooke et al . 1968; Hill et al . 2000) > 1 000 g 250 – 750 g 100 -200 g 10 -20 g Small Medium Large Smallest

Effect of DMS content on taste/odour of the product (quality control)?

Important taste consequences in seafoods (Peavy & Riley, 1993; 1994) which limit cultivation e.g. tridacnids

Excessive DMS in cod is commercially problematic, but very low concentrations of DMS are part of the “valued flavor” of some clams and oysters (Ackman & Hingley, 1968; Iida & Toukuaga, 1986; Brooke et al . 1968; Hill et al . 2000)

Continuing studies… The effect of DMSP on abalone physiology Partitioning of DMSP – are there tissue properties that specifically act to exclude DMSP? May affect enzyme function (Karsten et al . 1996) therefore have specialised enzymes developed to function in a high DMSP environment (Hill et al . 2004)?

The effect of DMSP on abalone physiology

Partitioning of DMSP – are there tissue properties that specifically act to exclude DMSP?

May affect enzyme function (Karsten et al . 1996) therefore have specialised enzymes developed to function in a high DMSP environment (Hill et al . 2004)?

The Future Physiology of DMSP production by macroalgae Can the DMSP production in Ulva be reduced by altering the cultivation conditions ? Evaluate the role of DMSP as antioxidant in algae by studying its photophysiology (stress to photosystems)

Physiology of DMSP production by macroalgae

Can the DMSP production in Ulva be reduced by altering the cultivation conditions ?

Evaluate the role of DMSP as antioxidant in algae by studying its photophysiology (stress to photosystems)

THANK YOU ACKNOWLEDGEMENTS I would like to extend special thanks to the following people and organizations without whose help this project would be impossible: Swedish and South African Collaborative Program I & J Mariculture farm JSP Mariculture farm Wild Coast Abalone Abagold N R F J SP

Preliminary investigations A small experiment was instigated to see if DMS was detectable in abalone. 1 – 2 g of tissue was removed from 5 abalone fed a mixed (kelp, Ulva and Gracilaria ) diet. Samples were placed in 20 ml of Methanol Measurement of DMS: Quantitative conversion of DMSP to DMS through alkali hydrolysis DMS analysis via gas chromatography

A small experiment was instigated to see if DMS was detectable in abalone.

1 – 2 g of tissue was removed from 5 abalone fed a mixed (kelp, Ulva and Gracilaria ) diet.

Samples were placed in 20 ml of Methanol

Measurement of DMS:

Quantitative conversion of DMSP to DMS through alkali hydrolysis

DMS analysis via gas chromatography

DMSP and DMS Ever notice the salty sea smell when you're out on or near the ocean? Trace concentrations of a volatile sulphur compound, Dimethylsulfide (DMS) , were discovered in the Earth's atmosphere about three decades ago. The gas was also found to occur in ocean surface waters . Some phytoplankton synthesize the molecule dimethylsulfoniopropionate (DMSP) DMS is released by algal cells following death and ‘sloppy feeding’ by zooplankton. DMS a naturally produced biogenic gas essential for the Earth's biogeochemical cycles.

Ever notice the salty sea smell when you're out on or near the ocean?

Trace concentrations of a volatile sulphur compound, Dimethylsulfide (DMS) , were discovered in the Earth's atmosphere about three decades ago.

The gas was also found to occur in ocean surface waters .

Some phytoplankton synthesize the molecule dimethylsulfoniopropionate (DMSP)

DMS is released by algal cells following death and ‘sloppy feeding’ by zooplankton.

DMS a naturally produced biogenic gas essential for the Earth's biogeochemical cycles.

DMSP and DMS in herbivores Assumptions: (To be confirmed by taste tests and future studies) DMSP is not produced by animals and may accumulate following feeding DMSP is proportionately converted to DMS (Hill et al . 1995) DMS causes the taste change (Hill et al . 1995, 2000, 2004) DMSP loss is through excretion (e.g. planktonic copepods (Hill et al. 1993) or mucus loss (corals, giant clams - Hill et al. 1995, 2004). No DMS in live abalone (Hill et al . 1995) Post mortem DMS content is due to: DMS production from DMSP resulting from heat treatment during canning Bacterial activity : DMSP lyase Abalone do not have DMSP-lyase (true for other invertebrates - Hill et al . 2004)

Assumptions: (To be confirmed by taste tests and future studies)

DMSP is not produced by animals and may accumulate following feeding

DMSP is proportionately converted to DMS (Hill et al . 1995)

DMS causes the taste change (Hill et al . 1995, 2000, 2004)

DMSP loss is through excretion (e.g. planktonic copepods (Hill et al. 1993) or mucus loss (corals, giant clams - Hill et al. 1995, 2004).

No DMS in live abalone (Hill et al . 1995)

Post mortem DMS content is due to:

DMS production from DMSP resulting from heat treatment during canning

Bacterial activity : DMSP lyase

Abalone do not have DMSP-lyase (true for other invertebrates - Hill et al . 2004)

The Future DMSP physiology Important taste consequences in seafoods (Peavy and Riley 1993, 1994) which limit cultivation e.g. Giant clams. Values in cod and other finfish in the range 0.2 – 1 u mol.g -1 are considered commercially problematic, BUT very low concentrations of DMS are part of the “valued flavor” of some clams and oysters ( Ackman & Hingley, 1968; Iida & Toukuaga, 1986; Brooke et al . 1968; Hill et al . 2000) Abalone physiology Partitioning of DMSP – have tissue evolved properties that specifically act to exclude DMSP Can affect enzyme function (Karsten et al . 1996) therefore have specialized enzymes formed to function in a high DMSP environment (Hill et al . 2004). Does it play role in ameliorating tissue oxygen stress (Sundra et al . 2002).

DMSP physiology

Important taste consequences in seafoods (Peavy and Riley 1993, 1994) which limit cultivation e.g. Giant clams.

Values in cod and other finfish in the range 0.2 – 1 u mol.g -1 are considered commercially problematic, BUT very low concentrations of DMS are part of the “valued flavor” of some clams and oysters ( Ackman & Hingley, 1968; Iida & Toukuaga, 1986; Brooke et al . 1968; Hill et al . 2000)

Abalone physiology

Partitioning of DMSP – have tissue evolved properties that specifically act to exclude DMSP

Can affect enzyme function (Karsten et al . 1996) therefore have specialized enzymes formed to function in a high DMSP environment (Hill et al . 2004).

Does it play role in ameliorating tissue oxygen stress (Sundra et al . 2002).