Chemoattraction in Aquatic Organisms

The Paradigm• As profit margins decline for many aquaculture

species, the need to optimize feed efficiency is paramount:

• Accomplished by: • complete consumption of the minimal amount of

nutrients required to maximize growth• minimizing amount of time between feed

application and ingestion • reducing feed input and concomitant waste

• In summary: use of feeds containing highly digestible nutrients and chemical stimulants

• Our focus: chemoattraction and feeding stimulation

Background

• Aquatics live in a complex world (chemically), aquatic organisms are capable of receiving and transmitting stimuli essential for their existence

• Chemical stimmulii are fundamental to dynamic equilibria of ecosystems: they modulate feeding behavior, migration and synchronize mating

• However: just because an aquatic organism can detect chemical stimuli does not mean it can be attracted to it

• Nor does it imply that once attracted (say, to a feed containing an attractant), will it consume it

• In essence, the feeding behavior of aquatic organisms is quite complicated

Feeding Model (Lee and Myers, 1996; Lindstedt, 1971)

Detection & Orientation Move?

No

Arrestant, stop movement

Yes

Repellant, movement away from feed

Yes

Attractant, locomotion towards feed

Ingest?

Suppressant, rejection of feed

No

Yes

Incitant, initiation of feeding

Ingest?

Deterrant, rejection of feed

No

Yes

Stimulant, continuation of feeding

Chemoreception: Finfish

• Accomplished by chemoreceptory systems (gustatation, taste, olfaction, common chemical sense) and solitary chemoreceptor cells

• Olfaction system and solitary cells also important in social behavior (communication, homing; Lamb, 2001)

• Receptor cell locations: • olfactory pouches within the snout (most fish)• taste buds within the oral cavity (e.g., catfish; Bardach and

Villars, 1974)• bottom dwellers: gustatory receptors on barbels, fins and

most of the body surface (Bardach and Villars, 1974)

Chemoreception: Crustaceans

• primarily anterior (Hindley, 1975; and Laverack, 1968), but on most appendages

• This extensive distribution related to many uses (e.g., locating feed, mates, avoiding predators, migration)

• contact receptors in flagellae of antennules used for distance chemoreception

• those in mouth parts and dactyls for food handling, gustatation and ingestion (Daniel and Derby, 1991; Derby et al., 1991a,b; Heinen, 1980)

How Introduced

• Chemicals should insure location, rapid and complete ingestion (overfeeding blows it)

• Issue: how to introduce the chemical to the animal• Most probable method of introduction: feed

ingredient• Depends on: lability of chemical, feed stability,

labor costs associated with feeding• Also: all feed ingredients are subject to leaching

(depends upon particle size, shape, texture, buoyancy)

Role of “Chemoattractants”

1) Increased initial palatability and consumption (Cordova- Murueta and Garcia-Carreno, 2002; Smith et al.,

2000; Harpaz, 1997; Lee and Meyers, 1997) • result: reduced feed input (complete

consumption) and improved overall nutrient mass transfer (feed target)

2) Reduced levels of uneaten or wasted feed • result: improved feed conversion, water

quality, both potentially enhancing growth rate (Lee and Myers, 1997)

Role of “Chemoattractants”3) Increased feed performance and growth

rate (Heinen, 1980; Papatryphon and Soares, 2001)

result: reduced leaching, excreted nitrogen, more variation possible in feed formulation

4) Reduced time in weaning from living prey to inert feeds (Roa et al., 1982)

5) Reduced dietary levels of expensive feed ingredients (Arndt et al., 1999; Adron and Mackie, 1978; Kolkovski et al., 2000;

Kubitza et al., 1997; McGoogan and Gatlin, 1997)

result: improved cost:benefit, increased byproduct usage

Chemoattractant Characteristics

• Most common physiochemical properties of feeding stimulants for fish include: non-volatile, low molecular weight (<1,000 kDaltons), nitrogen-containing, amphoteric, water soluble, heat stable, broad biological distribution (Lamb, 2001)

• These include: betaine, amino acids, nucleic acids, quaternary ammonia compounds (Carr, 1978; Conan et al., 1996; Hartati and Briggs, 1993; Smith et al., 2000)

Chemoattractant Characteristics

• Reality regards the chemicals within these sources, how they interact, rates of diffusion

• Determine what molecules elicit specific response, extract them from cheap sources, add to feed

• For finfish, previous research has shown that natural extracts were superior to chemically-formulated ones (Carr and Derby, 1986)

• Chemical-based feeding stimulation appears to be species specific (Takeda and Takii, 1992)

Evaluation of Chemoattractants

• Laminar flow choice chambers (Benfield and Aldrich, 1991, 1992)

• Y-maze choice chambers (Lee, 1992; Lee and Myers, 1996, 1997)

• Partitioned static aquariums w/feeds (Hartati and Briggs, 1993)

• Large tanks and trays (Texas Agricultural Experiment Station, Port Aransas; 2001-present)

Recent Research: Finfish

• Use of freeze-dried krill for feed training of largemouth bass (Kubitza and Lovshin, 1997)

• Use of inosine-5-monophosphate as a feed enhancer (Kubitza et al., 1997)

• Use of krill hydrolysate as a feed attractant (Kolkovski et al., 2000)

• Improved growth and performance of striped bass fed a plant feedstuff-based diet (Papatryphon and Soares, 2000)

• Feeding stimulants for young yellowtail (Hidaka et al., 2001)

• Effect of feeding stimulants on diet preference by juvenile gibel carp (Xue and Cui, 2001)

Recent Research: Shrimp• Focus has been on several levels: physiology of

detection, palatability, chemoattraction, feeding stimulation

• detection: (Coman, 1996)• palatability bioassay (Mendoza et al., 2000)• In reality, relatively few studies have addressed

chemoattraction and feeding stimulation in shrimp• Bottlenecks include: achieving equal choice

opportunity, variable physical environments, effect of dietary protein on attraction, and statistical issues

Recent Research: Shrimp

• Currently, the most common practical “chemoattractants” and feeding stimulants used in commercial shrimp production feeds include: fish meal, fish oils and squid meal)

• Others are being evaluated fish solubles, shrimp head meal, squid meal, etc.

• Normal rates of inclusion are highly variable because some “chemoattractants” are also important nutrient sources (e.g., fish meal), used for other purposes

Recent Research: Shrimp

• Cruz-Ricque et al. (1987) showed various species- and inclusion level-related growth responses to protein extracted from frozen squid: increased feed performance attributed to an “unknown growth factor” or UGF.

• Use of semi-purified ingredients (e.g., squid meal, protein hydrolysates of squid and finfish) has been shown to increase growth in L. vannamei (Cordova-Murueta and Garcia-Carreno, 2002)

Recent Research: Shrimp

• Inclusion of betaine in compounded feeds offered Macrobrachium rosenbergii increased feed consumption and growth (Harpaz, 1997; Harpaz and Steiner, 1990)

• Costero and Myers (1993) coated feeds with various naturally occurring marine compounds and offered them to Litopenaeus vannamei

• Result: improved perception and 50% shorter time to feeding

Recent Research: Shrimp

• These aforementioned bottlenecks to attraction research in shrimp were addressed by Sanchez et al. (2002)

• Objectives:• Develop a practical method to evaluate the efficacy of

chemoattractants used in feeds• Obtain reproducible results under controlled conditions• Evaluate attractability of commercial feeds, feed

ingredients

• Metric: feed preference

Recent Research: Shrimp• Used 5m diameter tanks and opposing sets of

shallow feeding trays• Used “bland” and “attractive” feeds to identify

optimum:• number of shrimp in tank• contact time (when to count shrimp on trays)• interim feeding rate (between trials)• protein level of feeds (REM: proteins can be attractive)• statistical approach to conducting research

• Feed preference was determined by percentage of shrimp in trays at predetermined time intervals

Current Research: Shrimp

• The following criteria were identified:• 50 shrimp/tank• 1h and 2h sample periods post-introduction• 2% feed rate in each tray• 2% maintenance feed rate between trials• dietary protein level of 16%

• Also: protein level of feed effected feeding stimulation

• Problem: feed ingestion was not quantified

Sanchez (2002): Review

• Recently, efforts have been undertaken to develop a consistent, quantifiable means of comparison of chemoattractants for shrimp feeds

• Static two choice chamber approach using maturation tanks at TAES (Port Aransas)

Sanchez (2002): Review

• Evaluated shrimp density in tanks, observation time, between-trial feed rate, protein source and protein level

• Results indicated an optimum density of 4.8 shrimp/m2, one or two-hr observation time, interim feed rate of 4%, protein and protein source effect, and a correlation between number of shrimp on trays and chemoattraction

Sanchez (2002): Refinement

• Surface area of trays was questioned

• Shrimp showed initial non-feeding response to white trays

• Statistics possibly not appropriate for ranking chemoattractants using a common positive control

Recent Research

• Development of a methodology to evaluate the effectiveness of one chemoattractant relative to another is an important step in reducing the amount of feed wasted in commercial aquaculture

• The benefits of such a methodology for commercial shrimp farmers could be both economic and environmental

Current Research: TAES1) Continued development of a methodology

for evaluating potential chemoattractants incorporated into shrimp feeds

2) Development of an index of relative attractability of various potential attractants;

3) Optimization of inclusion level of these compounds in laboratory and pond growth trials in order to reduce overall feed costs;

4) Relating attractability to consumption

Experimental System

• Incoming seawater from onsite storage was continuously added to each recirculating system at a rate of 3.41 L/min

Experimental System

1. intake sump

2. trickle filter

3. return sump

4. foam fractionator

5. charcoal filter

6. external standpipe

7. screened center drain

8. feed tray

Experimental System

• Cylindrical fiberglass tanks

• 3.4 m diameter.

• Water depth = 0.40 m

• Freeboard = 0.60 m

Discussion: Total Tray Surface Area

• Comparison of one tray versus two trays during feeding trials showed that not only were more shrimp attracted to the feeds at each observation time on two vs. one tray, but (with one exception each for both feeds A and B) the CV of means were substantially lower for shrimp on two trays

Discussion: Total Tray Surface Area

• Because more shrimp were found on two-tray arrangements, the potential for showing significant differences in number of shrimp on trays between experimental and control feeds was enhanced

• Lower CV for shrimp on two trays implies that separation of means between experimental and control responses was facilitated

• In both cases, sensitivity of experimental methodology was enhanced

Conclusions: Tray Surface Area

• Lower CVs would allow for greater sensitivity to minor variations in feed preference

• Future chemoattraction trials should be conducted using two feed trays for each feed, thus providing greater surface area than the single feed tray used by Sanchez (2002)

Discussion: Black vs. White Trays with Bland Feed

• At T-60 there were more shrimp on white trays than black (possible photo-tactic response)

• At T0 a sharp increase in number of shrimp attracted to white trays was observed (possibly due to agitation)

• For all observation times, shrimp were more attracted to the white trays

Discussion: Black vs. White Trays with Commercial Feed

• More shrimp were observed on white trays at T-60 as well as T0

• Lack of significant difference between the numbers of shrimp present on the black trays vs. the white trays at the remaining observation times may have been due to the attractiveness of the commercial feed

Conclusion: Black vs. White Trays

• Shrimp demonstrated a non-feeding-related attraction to the white feed trays

• To eliminate this potentially compounding behavior and, thus, eliminate any influence it may have on future chemoattraction studies, the decision was made to use black trays instead of white

Results: Trial 1 Chemoattractant Ranking

Rank1 Identifier Chemoattractant α = 0.05

1 a Intact Casein 2 a

2 b Squid Muscle Meal b

3 c Intact Casein 1 c,d

4 d Whole Squid Meal c,d,e

5 e Squid Liver Powder d,e,f,g,h,i

6 f Casein Digest e,f,g,h,i

7 g Cheese Product Intact e,f,g,h,i

8 h Whey Protein Concentrate 80% e,f,g,h,i

9 i Whole Squid Digest e,f,g,h,i

1 Rank based on pairwise comparisons of all possible choice combinations of LRS.

Results: Trial 2 Chemoattractant Ranking

Rank1 Identifier Chemoattractant α = 0.05

1 a Squid Muscle Meal a

2 b Whole Squid Meal b

3 c Squid Viscera Digest c,d,e,f,g,h

4 d Cheese Product Digest c,d,e,f,g,h

5 e Spray Dried Egg Digest c,d,e,f,g,h

6 f Whey Protein Concentrate 34% Digest c,d,e,f,g,h

7 g Spray Dried Egg Intact c,d,e,f,g,h

8 h Squid Liver Powder c,d,e,f,g,h

9 i Proprietary Attractant Mixture i

1 Rank based on pairwise comparisons of all possible choice combinations of LRS.

Results: Trial 3 Chemoattractant Ranking

Rank1 Identifier Chemoattractant α = 0.05

1 a Squid Muscle Meal a

2 b Putrescine b,c,d,e

3 c L-tyrosine b,c,d,e

4 d Taurine b,c,d,e,f,g

5 e Glycine b,c,d,e,f,g

6 f DL-lysine d,e,f,g

7 g L-arginine d,e,f,g,h

8 h Betaine-HCl g,h,i

9 i DL-histidine g,h,i

1 Rank based on pairwise comparisons of all possible choice combinations of LRS.

Discussion:

• SMM was significantly more attractive than WSM (α = 0.05) for both trials 1 and 2

• WSM was not significantly more attractive than SLP for trial 1, but was for trial 2

• SLP was not significantly more attractive than SVD in trial 2

Discussion:

• Intact Casein-2 was significantly more attractive than Intact Casein-1 (α = 0.05)

• Intact Casein- 1 was significantly more attractive than Casein Digest (α = 0.05)

Discussion:

• None of the single low-molecular weight compounds in trial 3 were as attractive as the SMM control

Conclusions:

• Developed methodology to evaluate and rank chemoattractants relative to a common control

• Different types of squid meal elicit different strengths of chemoattractant response in L. vannamei

Conclusions:

• Different types of casein elicit different strengths of chemoattractant response in L. vannamei

• Supports prior research that single low-molecular weight compounds are not as attractive as natural extracts

Implications of Chemoattraction Research

• You’d be surprised what compounds, molecules, substances could potentially serve as chemoattractants, feeding stimulants!

• If “chemoattraction” is evaluated on a molecular basis, least-cost attractants could be identified

• Subsequent incorporation into formulated feeds might not constitute an increase in per unit cost of feed (waste recycling?)

• If this compound increases consumption, lower protein level feeds could be used, reducing per unit cost

• With increased feed/protein efficiency would come a concomitant reduction in feed cost

Overall Conclusions

• Chemoattraction and feed ingestion studies are needed to ensure ingestion of expensive protein sources

• Chemoattractants will ultimately not contribute to feed cost

• In combination with improved protein/energy digestibility will come improved feed efficiency and reduced nutrient loading of receiving streams