**Applied Ecophysiology: An Integrative Form to Know How Culture Environment Modulates the Performance of Aquatic Species from an Energetic Point of View**

Carlos Rosas1, Cristina Pascual1, Maite Mascaró1, Paulina Gebauer2, Ana Farias3,4, Kurt Paschke3 and Iker Uriarte3,4 *1Unidad multidisciplinaria de Docencia e Investigación, Fac. de Ciencias, UNAM, Puerto de abrigo s/n, Sisal, Yucatán 2Centro I-Mar. Universidad de los Lagos, Puerto Montt, Chile 3Instituto de Acuicultura, Universidad Austral* 

> *de Chile, Sede Puerto Montt 4CIEN Austral 1México 2,3,4Chile*

#### **1. Introduction**

Ecological energetics as a part of ecophysiology, appears to have grown out of the Age of enlightenment and the concerns of the physiocrats, a group of economists who believed that the wealth of nations was derived solely from the value of "land agriculture" or "land development." Their theories originated in France, were most popular during the second half of the 18th century. Physiocracy is perhaps the first well-developed theory of economics (Danbom, 1979). The Age of Enlightenment (or simply the Enlightenment) is the era in Western philosophy, intellectual, scientific and cultural life, centered upon the 18th century, in which reason was advocated as the primary source for legitimacy and authority. It is also known as the Age of Reason. The enlightenment was a movement of science and reason. Ecological energetic began in the works of Podolinksy in the late 1800s, and subsequently was developed by the Soviet ecologist Stanchinskii, the Austro-American Alfred James Lotka, and American limnologists, Raymond Lindeman and George Evelyn Hutchinson. It underwent substantial development by H.T. Odum and was applied by system ecologists, and radiation ecologists to understand how the forcing factors modulate the ecosystem interactions (Weiner, 2000). Currently ecophysiology is a discipline that, in aquaculture, have been widely used to establish how the environment modulates the performance of animals in order to obtain the highest amount of biomass in the shortest possible time and cost. The use of physiological capacities of organisms to obtain biomass has been one of the basic premises of the application of ecophysiological studies to the production of aquatic organisms.

Applied Ecophysiology: An Integrative Form to Know How Culture

**3. Molluscs ecophysiology** 

resulting spat (Fig. 1).

**3.1 The case of bivalve** *Argopecten purpuratus*

Environment Modulates the Performance of Aquatic Species from an Energetic Point of View 163

This chapter will dedicated to show some examples that illustrate how applied ecophysiology have been solving some questions related with culture organisms and its consequences on production. In this chapter an integrative perspective related with immune condition is included taking into account that many of healthy problems observed on cultured animals are derived from immune problems provoked by inappropriate culture environments. Finally, some aspects related with experimental designs that must be considered in studying of applied ecophysiology are proposed. To exemplify, two species groups were formed: molluscs and crustaceans. Into the mollusks, examples with bivalves, gastropods and cephalopods are presented. Into the crustacean, examples with crabs and shrimp are used to illustrate how applied ecophysiology can improve aquaculture. In the case of fish a new encyclopaedia of fish physiology (from genome to environment) was published recently (Farrell, 2011). For that reason fish were not included in this chapter.

Bivalve filter feeders are capable of ingesting living and inert particles suspended in the water column that are responsible for the energetic input. In some species was demonstrated that detritus may contribute to the diet during periods in which the environmental offering of phytoplankton is unable to satisfy their energetic requirements. Although organic materials from discarded feed and faeces from salmon culture contributes to the diet of pectinids maintained in culture in southern Chile, phytoplankton is the main nutrient source for bivalves aquaculture (Farias & Uriarte, 2006). As pointed out, the shell growth and biochemical composition of larvae give clear indications about changes in the quality of the environment that are basic to determine how water nutrients and

phytoplankton modulates the biomass production of bivalves (Ferreiro et al*.*, 1990).

soluble protein and total carbohydrates of eggs, larvae and spat were evaluated.

Studying pectinid *A. purpuratus* several ecophysiological parameters were investigated to find the best conditions to cultivate this bivalve species. In a first step biochemical composition of larvae and spat was used to evaluate the energy metabolism and the nutritional condition of hatchery reared bivalves (Farías et al*.*, 1998). Adults from commercial long lines were stimulated to spawn using temperature shock and total lipids,

From that result was evident that lipids were the main source of energy while protein is deposited into the biomass. At the same time, an increment on carbohydrates was observed suggesting that the metabolic pathways related with transformation of lipids into glucose could be activated during larvae development and putting in evidence that quality of food used during larvae culture greatly influenced the storing of reserves and the survival of the

These results were confirmed latter when the effect of dietary protein content on biochemical composition of postlarvae, spat and gonadal development of *A*. *purpuratus* was tested (Uriarte & Farías, 1999; Farías & Uriarte, 2001). From that results it was evident that pectinid, and other bivalves production depends on the quality of the diet and the environment in which they inhabit (Fig. 2). Results on reproductive conditioning of adults showed that feed quality affect the quality of eggs depending of the species, so for *A. purpuratus* an increase in micro algal protein produce an increase in fertility and growth rate

#### **2. General aspects of applied ecophysiology**

The physiological ecologist seeks to understand the organism in relation to its environment. Defining the environment in aquaculture is relatively easy because it is, in many cases, a relatively closed system with the exception of marine cages or extensive culture. Three basic concepts are involved in applied ecophysiology (1) perception, (2) distribution on the culture environment and (3) the environment. Different animals perceive their surroundings in different ways, and to some extent, physiological and behavioural responses are dependent on perception. There is always a danger that we will superimpose our own human perceptions on other species and environments. Aquaculture is plenty of procedures that farmers have been translated from agriculture to aquaculture with not always success and in so doing fail to appreciate the interaction between organism and culture environment. The aquatic environment is completely different from the terrestrial environment and thus the perception of animals is so different from that we have. Water is more dense than the terrestrial environment provoking that organisms have developed a series of adaptations to perceive smells, spaces and physical and chemical conditions completely different from those of terrestrial animals.

Distribution on the culture environment take into account that individuals have limited tolerance ranges of temperature, salinity and other physical and chemical factors. Thus, is a truism that populations of organisms not be found in abundance beyond the tolerance regions of most individuals. Physiological tolerance and functional morphology go a long distance toward predicting where a particular organism will not occur, but they often give little indication of how well or what the organism will be doing, whether it will occur at all, within its tolerance limits. Applied to aquaculture, ecophysiology tries to establish the tolerance and resistance of species to environmental variables in order to provide the best culture conditions and how must be the environment to obtain the maximum scope for growth. Aquaculture environment is one of the topics of applied ecophysiology because the structure of the environment, interaction between individuals, water chemical and physical characteristics, type of food, etc., affect the physiology of aquatic animals enhancing or limiting animal performance, and at the end the biomass production. In this context, seed density, behavior characteristics that enhance or reduce cannibalism, turbidity, ecological characteristics of the culture systems that include meio-fauna and vegetation, are analyzed.

A physiological response represents the sum of all cellular and biochemical reactions as influenced by the environment or the animal itself. For this reason, organisms are capable of reflecting any environment condition even before the effects are observed in the population and community level. In ecological energetics an energy budget equation is defined as the sum of the energy from food ingested, which is divided into metabolizable, egested and excreted energy. This energy will varied according to the effects of different extrinsic and intrinsic factors and therefore it is important to calculate the cost of production in terms of growth when considering total aquaculture activity. Thus, the animal production (P) or growth is represented by the difference between the absorbed energy and the energy lost in respiration and excretion, taking age, sex, and body type into account. As with growth, other measures of biological productivity, such as work, egg production and body condition, will also affected by type of food or/and environmental characteristics. Thus, a satisfactory diet/environment in terms of the best efficiency will be needed in order to achieve optimal animal production.

This chapter will dedicated to show some examples that illustrate how applied ecophysiology have been solving some questions related with culture organisms and its consequences on production. In this chapter an integrative perspective related with immune condition is included taking into account that many of healthy problems observed on cultured animals are derived from immune problems provoked by inappropriate culture environments. Finally, some aspects related with experimental designs that must be considered in studying of applied ecophysiology are proposed. To exemplify, two species groups were formed: molluscs and crustaceans. Into the mollusks, examples with bivalves, gastropods and cephalopods are presented. Into the crustacean, examples with crabs and shrimp are used to illustrate how applied ecophysiology can improve aquaculture. In the case of fish a new encyclopaedia of fish physiology (from genome to environment) was published recently (Farrell, 2011). For that reason fish were not included in this chapter.

#### **3. Molluscs ecophysiology**

162 Aquaculture and the Environment - A Shared Destiny

The physiological ecologist seeks to understand the organism in relation to its environment. Defining the environment in aquaculture is relatively easy because it is, in many cases, a relatively closed system with the exception of marine cages or extensive culture. Three basic concepts are involved in applied ecophysiology (1) perception, (2) distribution on the culture environment and (3) the environment. Different animals perceive their surroundings in different ways, and to some extent, physiological and behavioural responses are dependent on perception. There is always a danger that we will superimpose our own human perceptions on other species and environments. Aquaculture is plenty of procedures that farmers have been translated from agriculture to aquaculture with not always success and in so doing fail to appreciate the interaction between organism and culture environment. The aquatic environment is completely different from the terrestrial environment and thus the perception of animals is so different from that we have. Water is more dense than the terrestrial environment provoking that organisms have developed a series of adaptations to perceive smells, spaces and physical and chemical conditions

Distribution on the culture environment take into account that individuals have limited tolerance ranges of temperature, salinity and other physical and chemical factors. Thus, is a truism that populations of organisms not be found in abundance beyond the tolerance regions of most individuals. Physiological tolerance and functional morphology go a long distance toward predicting where a particular organism will not occur, but they often give little indication of how well or what the organism will be doing, whether it will occur at all, within its tolerance limits. Applied to aquaculture, ecophysiology tries to establish the tolerance and resistance of species to environmental variables in order to provide the best culture conditions and how must be the environment to obtain the maximum scope for growth. Aquaculture environment is one of the topics of applied ecophysiology because the structure of the environment, interaction between individuals, water chemical and physical characteristics, type of food, etc., affect the physiology of aquatic animals enhancing or limiting animal performance, and at the end the biomass production. In this context, seed density, behavior characteristics that enhance or reduce cannibalism, turbidity, ecological characteristics of the culture systems that include meio-fauna and vegetation, are analyzed. A physiological response represents the sum of all cellular and biochemical reactions as influenced by the environment or the animal itself. For this reason, organisms are capable of reflecting any environment condition even before the effects are observed in the population and community level. In ecological energetics an energy budget equation is defined as the sum of the energy from food ingested, which is divided into metabolizable, egested and excreted energy. This energy will varied according to the effects of different extrinsic and intrinsic factors and therefore it is important to calculate the cost of production in terms of growth when considering total aquaculture activity. Thus, the animal production (P) or growth is represented by the difference between the absorbed energy and the energy lost in respiration and excretion, taking age, sex, and body type into account. As with growth, other measures of biological productivity, such as work, egg production and body condition, will also affected by type of food or/and environmental characteristics. Thus, a satisfactory diet/environment in terms of the best efficiency will be needed in order to

**2. General aspects of applied ecophysiology** 

completely different from those of terrestrial animals.

achieve optimal animal production.

#### **3.1 The case of bivalve** *Argopecten purpuratus*

Bivalve filter feeders are capable of ingesting living and inert particles suspended in the water column that are responsible for the energetic input. In some species was demonstrated that detritus may contribute to the diet during periods in which the environmental offering of phytoplankton is unable to satisfy their energetic requirements. Although organic materials from discarded feed and faeces from salmon culture contributes to the diet of pectinids maintained in culture in southern Chile, phytoplankton is the main nutrient source for bivalves aquaculture (Farias & Uriarte, 2006). As pointed out, the shell growth and biochemical composition of larvae give clear indications about changes in the quality of the environment that are basic to determine how water nutrients and phytoplankton modulates the biomass production of bivalves (Ferreiro et al*.*, 1990).

Studying pectinid *A. purpuratus* several ecophysiological parameters were investigated to find the best conditions to cultivate this bivalve species. In a first step biochemical composition of larvae and spat was used to evaluate the energy metabolism and the nutritional condition of hatchery reared bivalves (Farías et al*.*, 1998). Adults from commercial long lines were stimulated to spawn using temperature shock and total lipids, soluble protein and total carbohydrates of eggs, larvae and spat were evaluated.

From that result was evident that lipids were the main source of energy while protein is deposited into the biomass. At the same time, an increment on carbohydrates was observed suggesting that the metabolic pathways related with transformation of lipids into glucose could be activated during larvae development and putting in evidence that quality of food used during larvae culture greatly influenced the storing of reserves and the survival of the resulting spat (Fig. 1).

These results were confirmed latter when the effect of dietary protein content on biochemical composition of postlarvae, spat and gonadal development of *A*. *purpuratus* was tested (Uriarte & Farías, 1999; Farías & Uriarte, 2001). From that results it was evident that pectinid, and other bivalves production depends on the quality of the diet and the environment in which they inhabit (Fig. 2). Results on reproductive conditioning of adults showed that feed quality affect the quality of eggs depending of the species, so for *A. purpuratus* an increase in micro algal protein produce an increase in fertility and growth rate

Applied Ecophysiology: An Integrative Form to Know How Culture

**3.2 The case of gastropod** *Haliotis fulgens* **and** *H. rufescens*

(Farías & Uriarte, 2001).

(Lovatelli et al*.*, 2007).

balanced foods.

(Farías et al*.*, 2003).

different sized green abalone *H. fulgens*.

Environment Modulates the Performance of Aquatic Species from an Energetic Point of View 165

of larvae while in *Crassotrea gigas* non effect in fecundity was observed but eggs showed the highest lipid reserves and larvae showed highest survival. The scope for growth and reproductive conditioning of *A. purpuratus* is the product of the combined effect of high clearance rate and absorption efficiency that animals showed when fed high protein levels, indicating that phytoplankton abundance and composition will determine culture success

The cost of the feeding depends as much on the processes pre- as post-ingestion and its modelling allows predicting, quantitatively, the growth of the bivalves based on simple but robust relationships between the conducts of feeding and the quality and amount of the food available in the environment. Although the importance of food quality and environment have been acknowledge, variability of the environmental quality and food remains as the principal concern between mussel and bivalve farmers, that depends on the natural food and environmental quality to maintain long line marine bivalves production

From the 130 species Halliotidae reported worldwide, only 14 species have been commercially exploited, either from fishery or through aquaculture. Since the commercial overfishing has severely depleted most populations of abalone, their aquaculture is replacing great part of the demand of these species. For abalone or any other cultured species, feed composition and ingestion rate are between most important factors to consider to predicting biomass production. In this sense, the study of *H. fulgens* have been used as an example to understand how the type of diet modulates the use and destination of the ingested energy, in this particular case, in attempt to optimize the use of macro algae or

Energy budget is a basic tool to evaluate the effect of type of food or other factors on physiological condition of aquatic animals. Using this model and considering different sizes of organisms, it is possible to obtain enough information to estimate their energy needs, and in a particular condition, predict the energetic costs associated with a particular environmental condition or type of food. In *H. fulgens* the energetic balance of animals between 0.1 to 2.5g was obtained in animals maintained in optimal conditions (Table 1)

Dry body weight (g) DBW vs Total lenght (mm) L DBW = a Lb 0.00001 3.08 0.98 Wet body weight (g) WBW vs Live weight (g) LW WBW = a LW-b 0.15 0.63 0.99 Dry body weight (g) DBW vs Live weight (g) LW DBW = a LW+b 0.02 0.11 0.98

Ingestion rate (mg day-1) I vs dry body weight (g) DBW I = a DBWb 24.3 0.6 0.69 Absorption rate (mg day-1) Ab vs dry body weight (g) DBW Ab = a DBWb 21.01 0.6 0.69 Respiration rate ml O2 day-1 R vs dry body weight (g) DBW R = a DBWb 12.01 12 0.69 Nitrogen excretion (μmol N-NH4 day-1) U vs dry body weight (g) DBW U = a DBWb 43.6 0.85 0.63 Table 1. Relationship between morphometric characteristics and physiological responses of

Relationship Type of model a b r2

Fig. 1. Biochemical composition changes during larvae and postlarvae development of *A. purpuratus*. Arrow indicates metamorphosis.

Fig. 2. Nutrient flows and biochemical pathways of planktonic larvae, spat and broodstock of *A. purpuratus* under culture condition. High quality eggs from healthy broodstock favors the use of lipids as a source of energy and synthesis of glycogen, while protein are used to protein deposition . After metamorphosis, carbohydrates and lipids are used mainly as a source of energy for maintenance and to accumulate protein via biomass production of spat. Food quality, measured as protein content, and environmental quality (Temperature, salinity, dissolved oxygen, turbidity etc.) determine the growth rate of bivalves, being the clearance rate and energy absorption key of culture success.

Soluble Protein Total Lipids Total Carbohydrates

0 1 3 12 16 19 38 73

**Age, days**

Fig. 1. Biochemical composition changes during larvae and postlarvae development of *A.* 

Fig. 2. Nutrient flows and biochemical pathways of planktonic larvae, spat and broodstock of *A. purpuratus* under culture condition. High quality eggs from healthy broodstock favors the use of lipids as a source of energy and synthesis of glycogen, while protein are used to protein deposition . After metamorphosis, carbohydrates and lipids are used mainly as a source of energy for maintenance and to accumulate protein via biomass production of spat. Food quality, measured as protein content, and environmental quality (Temperature, salinity, dissolved oxygen, turbidity etc.) determine the growth rate of bivalves, being the

clearance rate and energy absorption key of culture success.

*purpuratus*. Arrow indicates metamorphosis.

**% of Ash free dry weight**

of larvae while in *Crassotrea gigas* non effect in fecundity was observed but eggs showed the highest lipid reserves and larvae showed highest survival. The scope for growth and reproductive conditioning of *A. purpuratus* is the product of the combined effect of high clearance rate and absorption efficiency that animals showed when fed high protein levels, indicating that phytoplankton abundance and composition will determine culture success (Farías & Uriarte, 2001).

The cost of the feeding depends as much on the processes pre- as post-ingestion and its modelling allows predicting, quantitatively, the growth of the bivalves based on simple but robust relationships between the conducts of feeding and the quality and amount of the food available in the environment. Although the importance of food quality and environment have been acknowledge, variability of the environmental quality and food remains as the principal concern between mussel and bivalve farmers, that depends on the natural food and environmental quality to maintain long line marine bivalves production (Lovatelli et al*.*, 2007).

#### **3.2 The case of gastropod** *Haliotis fulgens* **and** *H. rufescens*

From the 130 species Halliotidae reported worldwide, only 14 species have been commercially exploited, either from fishery or through aquaculture. Since the commercial overfishing has severely depleted most populations of abalone, their aquaculture is replacing great part of the demand of these species. For abalone or any other cultured species, feed composition and ingestion rate are between most important factors to consider to predicting biomass production. In this sense, the study of *H. fulgens* have been used as an example to understand how the type of diet modulates the use and destination of the ingested energy, in this particular case, in attempt to optimize the use of macro algae or balanced foods.

Energy budget is a basic tool to evaluate the effect of type of food or other factors on physiological condition of aquatic animals. Using this model and considering different sizes of organisms, it is possible to obtain enough information to estimate their energy needs, and in a particular condition, predict the energetic costs associated with a particular environmental condition or type of food. In *H. fulgens* the energetic balance of animals between 0.1 to 2.5g was obtained in animals maintained in optimal conditions (Table 1) (Farías et al*.*, 2003).


Table 1. Relationship between morphometric characteristics and physiological responses of different sized green abalone *H. fulgens*.

Applied Ecophysiology: An Integrative Form to Know How Culture

growth abalone culture.

*vulgaris*

Environment Modulates the Performance of Aquatic Species from an Energetic Point of View 167

in high quantities that could be demanded to produce biomass could be a limit for the

Of cephalopods, octopuses are considered economically interesting species for aquaculture. Landings in this area have been steadily decreasing since the 90's, which has led to increase the demand for octopuses and thus the technological-scientific efforts to rear them. Rearing is defined as the development of juveniles from an egg or paralarva with the ability to reach a second stage (Boletzky, 2003). Many cephalopods have been subject of several studies in captivity intended to investigate behavioural aspects (Hanlon & Wolterding, 1989; Hochner et al., 2006), used as models in neurophysiology studies (Flores, 1983; Wollesen et al., 2009), in predator-prey relationships (Villanueva, 1993; Scheel, 2002; Smith, 2003) or to provide live specimens for aquariums (Summer & McMahon, 1970; Bradley, 1974; Anderson & Wood, 2001). In the last years, many studies related with octopus and sepia culture have been putting in evidence the increased interest in their culture (for reviews see) (Sykes et al., 2006; Iglesias et al., 2007; Uriarte et al., 2011). Octopus are semelparous, that means that females spawn once in their life and die soon after the eggs hatch (Hanlon et al., 1991).The life span of the most of cephalopods species is short varying from about 6 months in small species to between one to three years in larger ones (Villanueva & Norman, 2008). Recent works show the very fast and high rates of food consumption, food conversion and growth of octopus. Most of our knowledge of octopus energy budget comes from studies of few species

**3.3 The case of cephalopods** *Octopus maya***,** *Enteroctopus megalocyathus* **and** *O.* 

potentially due to their fisheries value and their potential as culture species.

(Rrout) expressed as joules per day (Fig. 3).

*Octopus vulgaris* is by far the most studied between octopus species. At the date there are many investigations related with some aspects of energy budget. To exemplify the use of ecophysiological studies on octopus culture there are several studies in which octopuses were fed squid and tested at different experimental temperatures (Katsanevakis et al*.*, 2005; Miliou et al*.*, 2005). Katsanevakis et al. (2005) tested the effects of temperature and body weight on respiratory metabolism of juveniles of *O. vulgaris.* Using data obtained on that study a surface plot was constructed using a quadratic model for routine metabolism

That model was constructed using an interval of octopus living weight between 0.1 to 1kg and a sea water temperature between 13 to 28°C. With this information we can now calculate how much energy will be metabolized if octopuses of 0.1kg are cultivated at 15°C and in consequence how much food should be used to cover that energetic demand: Using the model:

Rrout = 19403.7-[15.3 (0.1kg)] – [2040.6(15°C)] – [0.003 (0.1kg) 2] + [2.4 (0.1kg x 15°C)] + [52.2 (15°C)2] = 541.2 joules day-1 To validate that model, we re-calculated the Rrout value obtained with last equation but using the living weight of octopuses used in other studies where Rrout was also measured. To do that we used weight values from García-Garrido et al. (2011) how made respirometric measurements of *O. vulgaris* (850g) fed squid at 15°C. The Rrout found by authors in that study was 671 joules day-1, close to that obtained if the quadratic model is applied (554.4 joules day-1). That concordance suggests that the quadratic model calculated now gave

comparable data with other experimentally obtained (García-Garrido et al*.*, 2011).

With these equations it is possible to calculate how much of energy abalones needs per mg of biomass, how much of energy could be obtained, as a scope for growth per joule of ingested food (Table 2) and at the end the quantity of food that is needed to produce 1g of living abalone.


Table 2. Energetic balance obtained on optimal culture conditions of *H. fulgens*. Values obtained from equations derived from table 1. Circle indicates the proportion (%) of ingested energy (100%) that is channeled to each physiological response.

To make the calculations it is necessary to consider that 1g living abalone have 0.13g of dry weight biomass DBW (Table 1), that food have 18000 joules g-1 (Farias et al., 2003) and that scope for growth obtained was around 207 joules day-1 mg-1 DBW (Table 2). Following the next steps:

1. Joules day-1 needed to produce 1g living abalone =

( ) ( ) 1 1 130 mg DBW of abalone contained into 1g living weight x 207 joules day mg DBW − −

> 1 1 1 mg DBW 35,100 joules day g LW − − =

2. If 1g of food have a content of 18,000 joules g-1, but only 45% of it (8,100 joules g-1) are really converted in biomass, then the quantity of food required to produce 1g of living abalone will be:

Food to produce 1g of living abalone = 35.1 kjoules day-1 g-1/8.1 kjoules g-1 = 4.3g or 4.3kg of food for 1kg of abalone. With these models it is possible to help farmers to calculate not only biomass production levels but cost of that biomass production.

In a more generalized application of energetic balance results it is possible estimate the food needed to produce other abalone species as *H. rufescens* (Hernández et al*.*, 2009). In that case formulated diet with 4.15 kjoules g-1 wet weight were compared with two macro algae with 2.7(*Porphyra columbiana*) and 0.92(*Macrocystis pyrifera*) kjoules g-1 wet weight energy contents, respectively. At the light of that energy contents and assuming the same conversion efficiency of *H. fulgens* (45%) it is possible to observe that to produce 1kg of abalone with macro algae will be necessary to use 4.15kg of formulated food, 28.9kg of *P. columbiana* and 84 kg *M. pyrifera*, both given as a fresh food. Results of the *H. rufescens*  assay showed that abalones fed with macro algae grew 120% and 40% higher than observed in animals fed formulated diet suggesting that macro algae could be used to cultivate abalones instead of formulated diet. However natural production of macro algae

With these equations it is possible to calculate how much of energy abalones needs per mg of biomass, how much of energy could be obtained, as a scope for growth per joule of ingested food (Table 2) and at the end the quantity of food that is needed to produce 1g of

> joules day-1 mg-1

Table 2. Energetic balance obtained on optimal culture conditions of *H. fulgens*. Values obtained from equations derived from table 1. Circle indicates the proportion (%) of

To make the calculations it is necessary to consider that 1g living abalone have 0.13g of dry weight biomass DBW (Table 1), that food have 18000 joules g-1 (Farias et al., 2003) and that scope for growth obtained was around 207 joules day-1 mg-1 DBW (Table 2). Following the

( ) ( ) 1 1

2. If 1g of food have a content of 18,000 joules g-1, but only 45% of it (8,100 joules g-1) are really converted in biomass, then the quantity of food required to produce 1g of living

Food to produce 1g of living abalone = 35.1 kjoules day-1 g-1/8.1 kjoules g-1 = 4.3g or 4.3kg of food for 1kg of abalone. With these models it is possible to help farmers to calculate not only

In a more generalized application of energetic balance results it is possible estimate the food needed to produce other abalone species as *H. rufescens* (Hernández et al*.*, 2009). In that case formulated diet with 4.15 kjoules g-1 wet weight were compared with two macro algae with 2.7(*Porphyra columbiana*) and 0.92(*Macrocystis pyrifera*) kjoules g-1 wet weight energy contents, respectively. At the light of that energy contents and assuming the same conversion efficiency of *H. fulgens* (45%) it is possible to observe that to produce 1kg of abalone with macro algae will be necessary to use 4.15kg of formulated food, 28.9kg of *P. columbiana* and 84 kg *M. pyrifera*, both given as a fresh food. Results of the *H. rufescens*  assay showed that abalones fed with macro algae grew 120% and 40% higher than observed in animals fed formulated diet suggesting that macro algae could be used to cultivate abalones instead of formulated diet. However natural production of macro algae

130 mg DBW of abalone contained into 1g living weight x 207 joules day mg DBW 1 mg DBW 35,100 joules day g LW

− − =

1 1

ingested energy (100%) that is channeled to each physiological response.

14 4

% H U R SFG

45

38

− −

Physiological response % Ingestion rate I 457 100 Feces H 62 14

Nitrogen excretion rate U 16 4 Respiration rate R 172 38 Scope for growth SFG 207 45

1. Joules day-1 needed to produce 1g living abalone =

biomass production levels but cost of that biomass production.

Absorption rate Ab 395

living abalone.

next steps:

abalone will be:

in high quantities that could be demanded to produce biomass could be a limit for the growth abalone culture.

#### **3.3 The case of cephalopods** *Octopus maya***,** *Enteroctopus megalocyathus* **and** *O. vulgaris*

Of cephalopods, octopuses are considered economically interesting species for aquaculture. Landings in this area have been steadily decreasing since the 90's, which has led to increase the demand for octopuses and thus the technological-scientific efforts to rear them. Rearing is defined as the development of juveniles from an egg or paralarva with the ability to reach a second stage (Boletzky, 2003). Many cephalopods have been subject of several studies in captivity intended to investigate behavioural aspects (Hanlon & Wolterding, 1989; Hochner et al., 2006), used as models in neurophysiology studies (Flores, 1983; Wollesen et al., 2009), in predator-prey relationships (Villanueva, 1993; Scheel, 2002; Smith, 2003) or to provide live specimens for aquariums (Summer & McMahon, 1970; Bradley, 1974; Anderson & Wood, 2001). In the last years, many studies related with octopus and sepia culture have been putting in evidence the increased interest in their culture (for reviews see) (Sykes et al., 2006; Iglesias et al., 2007; Uriarte et al., 2011). Octopus are semelparous, that means that females spawn once in their life and die soon after the eggs hatch (Hanlon et al., 1991).The life span of the most of cephalopods species is short varying from about 6 months in small species to between one to three years in larger ones (Villanueva & Norman, 2008). Recent works show the very fast and high rates of food consumption, food conversion and growth of octopus. Most of our knowledge of octopus energy budget comes from studies of few species potentially due to their fisheries value and their potential as culture species.

*Octopus vulgaris* is by far the most studied between octopus species. At the date there are many investigations related with some aspects of energy budget. To exemplify the use of ecophysiological studies on octopus culture there are several studies in which octopuses were fed squid and tested at different experimental temperatures (Katsanevakis et al*.*, 2005; Miliou et al*.*, 2005). Katsanevakis et al. (2005) tested the effects of temperature and body weight on respiratory metabolism of juveniles of *O. vulgaris.* Using data obtained on that study a surface plot was constructed using a quadratic model for routine metabolism (Rrout) expressed as joules per day (Fig. 3).

That model was constructed using an interval of octopus living weight between 0.1 to 1kg and a sea water temperature between 13 to 28°C. With this information we can now calculate how much energy will be metabolized if octopuses of 0.1kg are cultivated at 15°C and in consequence how much food should be used to cover that energetic demand: Using the model:

Rrout = 19403.7-[15.3 (0.1kg)] – [2040.6(15°C)] – [0.003 (0.1kg) 2] + [2.4 (0.1kg x 15°C)] + [52.2 (15°C)2] = 541.2 joules day-1

To validate that model, we re-calculated the Rrout value obtained with last equation but using the living weight of octopuses used in other studies where Rrout was also measured. To do that we used weight values from García-Garrido et al. (2011) how made respirometric measurements of *O. vulgaris* (850g) fed squid at 15°C. The Rrout found by authors in that study was 671 joules day-1, close to that obtained if the quadratic model is applied (554.4 joules day-1). That concordance suggests that the quadratic model calculated now gave comparable data with other experimentally obtained (García-Garrido et al*.*, 2011).

Applied Ecophysiology: An Integrative Form to Know How Culture

0.9272lnW + 0.1138T°C – 0.0026T°C2) (Miliou et al., 2005)

**I, Cold: 522 Tropical: 358**

Food

**T E M P E R A T U R E**

considered at the time of culture.

**H, Cold: 146 Tropical: 64**

**U, Cold: 2.05**

Fig. 4. The energy balance between species inhabiting cold (*E. megalociathus)* and tropical environments *(O. maya)*, showed that the scope for growth (P) of *E. megalocyathus* was 1.5 times higher than that of *O. maya*. In that study was demonstrated that cold-water cephalopod species could be more efficient than tropical species, a fact that should be

That study shows also that each octopod species is adapted to use ingested energy in a different way as a consequence of the specific forces in each particular ecosystem: *O. maya*

**Tropical: 7.18**

R, Cold: 9.07 Tropical: 37.6

Cold: *Enteroctopus megalociathus* Tropical: *Octopus maya*

Environment Modulates the Performance of Aquatic Species from an Energetic Point of View 169

Living weight,g Temperature, °C g day-1 % living weight 15 4.8 2.4 20 5.4 2.7 25 5.3 2.7 15 9.2 2.3 10.3 2.6 10.1 2.5 15 13.4 2.2 15.0 2.5 14.7 2.5 15 17.4 2.2 19.6 2.4 19.2 2.4 15 21.5 2.1 24.0 2.4 23.7 2.4

Table 3. Relationship between living weight (g) and temperature (°C) on feeding rate of *Octopus vulgaris* fed squid. Data obtained from the equation: Feeding rate g day-1 = -4.4608 +

Feeding rate

Fig. 3. Surface plot derived from a quadratic model to describe the relationship between Respiratory metabolism (joules day-1) (Rrout = Routine metabolism; Rsda = Specific dynamic action metabolism; Rtot = Total metabolism) calculated for animals between 0.1 to 1kg living weight. (Data from Kasanevakis et al., 2005)

But, not always the models obtained give results applied at all mainly because the experimental procedures are not always are made in comparable conditions and depends on the form in which a determined factor affect the physiological response of animals. For example, using a model proposed by (Miliou et al*.*, 2005) to evaluate the combined effect of temperature and living weight on feeding rate of *O. vulgaris* (Feeding rate g day-1 = -4.4608 + 0.9272lnW + 0.1138T°C – 0.0026T°C2) we calculated the feeding rate (g day-1) for octopuses fed squid with different living (W) weight and exposed at different temperatures (Table 3).

In their research, Miliou et al. (2005) feed octopuses with squid at a rate of 3% living weight day-1. When the equation is applied the proportion of food consumed by octopuses resulted closed to that food rate (Table 3) suggesting that the data obtained with equation increased with temperature and living weight in a constant form (Table 3), producing a constant feeding rate. Using newly the study of García-Garrido et al., (2011), where octopuses were fed squid at a ratio of 10% living weight day-1 we observed that the model cannot predict the real feeding rate obtained by authors, that in this case was 8% living weight, because for octopuses feeding rate, and in consequence the energy derived of food, is modulated by the quantity of food that is offered, and certainly 3% could not be an ad libitum ratio.

Other species of octopus have been studied in attempt to quantify the physiological condition of octopus in similar conditions; a study was done comparing energetic balance of two species: *Enteroctopus megalociathus* (living at 10°C) and *O. maya* (living at 28°C) (Farias et al., 2009). The scope for growth of both species was calculated as: P = I – (R + H + U), and values of P of 522 and 358 joules day-1 g-1 were obtained of *E. megalociathus* and *O. maya,*  respectively (Fig. 4).

0 200 400 600 800 1000 1200 Living weight, g

Fig. 3. Surface plot derived from a quadratic model to describe the relationship between Respiratory metabolism (joules day-1) (Rrout = Routine metabolism; Rsda = Specific

dynamic action metabolism; Rtot = Total metabolism) calculated for animals between 0.1 to

But, not always the models obtained give results applied at all mainly because the experimental procedures are not always are made in comparable conditions and depends on the form in which a determined factor affect the physiological response of animals. For example, using a model proposed by (Miliou et al*.*, 2005) to evaluate the combined effect of temperature and living weight on feeding rate of *O. vulgaris* (Feeding rate g day-1 = -4.4608 + 0.9272lnW + 0.1138T°C – 0.0026T°C2) we calculated the feeding rate (g day-1) for octopuses fed squid with different living (W) weight and exposed at different temperatures (Table 3). In their research, Miliou et al. (2005) feed octopuses with squid at a rate of 3% living weight day-1. When the equation is applied the proportion of food consumed by octopuses resulted closed to that food rate (Table 3) suggesting that the data obtained with equation increased with temperature and living weight in a constant form (Table 3), producing a constant feeding rate. Using newly the study of García-Garrido et al., (2011), where octopuses were fed squid at a ratio of 10% living weight day-1 we observed that the model cannot predict the real feeding rate obtained by authors, that in this case was 8% living weight, because for octopuses feeding rate, and in consequence the energy derived of food, is modulated by the

quantity of food that is offered, and certainly 3% could not be an ad libitum ratio.

Other species of octopus have been studied in attempt to quantify the physiological condition of octopus in similar conditions; a study was done comparing energetic balance of two species: *Enteroctopus megalociathus* (living at 10°C) and *O. maya* (living at 28°C) (Farias et al., 2009). The scope for growth of both species was calculated as: P = I – (R + H + U), and values of P of 522 and 358 joules day-1 g-1 were obtained of *E. megalociathus* and *O. maya,* 

+2.4WT°C+52.2T°C2

Rrut (Joules day-1) = 19403.7-15.3W-2040.6T°C-0.003W 2

respectively (Fig. 4).

1kg living weight. (Data from Kasanevakis et al., 2005)

Temperature, °C


Table 3. Relationship between living weight (g) and temperature (°C) on feeding rate of *Octopus vulgaris* fed squid. Data obtained from the equation: Feeding rate g day-1 = -4.4608 + 0.9272lnW + 0.1138T°C – 0.0026T°C2) (Miliou et al., 2005)

Fig. 4. The energy balance between species inhabiting cold (*E. megalociathus)* and tropical environments *(O. maya)*, showed that the scope for growth (P) of *E. megalocyathus* was 1.5 times higher than that of *O. maya*. In that study was demonstrated that cold-water cephalopod species could be more efficient than tropical species, a fact that should be considered at the time of culture.

That study shows also that each octopod species is adapted to use ingested energy in a different way as a consequence of the specific forces in each particular ecosystem: *O. maya*

Applied Ecophysiology: An Integrative Form to Know How Culture

salinities from 32-35 UPS and 9-26 UPS, respectively.

low salinities obtaining good survival and growth.

**4.1.1 Growth and survival** 

metamorphic moult (PL20).

Environment Modulates the Performance of Aquatic Species from an Energetic Point of View 171

Salinity affects the distribution of a variety of estuarine and marine organisms. Some marine species, such as shrimps, have life cycles that include an estuarine phase. Since the 1950´s, there have been reports on congregations of juvenile shrimps at low salinities, whilst adults reproduced under strict marine conditions. It has also been observed that larval phases inhabit oceanic waters, where they develop and moult into postlarvae. Through certain chemo tactile mechanisms and water currents, postlarvae are capable of moving towards estuaries and coastal lagoons, where they recruit as early juveniles. It is in these habitats, with strong salinity variations, where some shrimp species are capable of colonizing more diluted environments than others. For example, studies carried out in the 1960´s by Mac Farland & Lee, (1963) showed that juvenile *L. setiferus* are better adapted to tolerate low salinities than juvenile *Farfantepenaeus aztecus.* Similarly, Mair, (1980) observed that amongst *L. stylirostris, F. californiensis,* and *L. vannamei*, the latter is the species that best tolerates highly diluted environments in estuaries and costal lagoons, and is therefore capable of inhabiting areas restricted to the other two species. This ecophysiological trait allows *L. vannamei* to reduce ecological pressures of competition for space and food, both strong limiting resources in these habitats. In an experimental study, Mair, (1980) observed that *L. vannamei* postlarvae and early juveniles placed in an experimental salinity gradient preferred salinities from 3-6 UPS, whereas *L. stylirostris* and *F. californiensis* preferred

The wide tolerance of *L. vannamei* to salinity has also been observed under culture conditions, both experimentally and within production facilities. Ogle et al*.* (1992) examined the effects of salinity on growth in *L. vannamei* postlarvae under different culture conditions. In that study the authors observed that tolerance to salinity is independent of postlarval (PL) age, showing that both PL8 and PL22 had better survival and growth at salinities ranging from 16 to 32 UPS. In that same study, it was demonstrated that tolerance to salinity increases as temperature declines from 30-16°C, since the highest survival and growth of postlarvae was obtained at salinities of 8 and 16 UPS at that temperature. Postlarvae can be acclimated to low salinities for production purposes. Recent studies have shown that a rate of salinity decline of 25% per hour is appropriate for postlarvae (PL10 to 4 UPS) and early juveniles (PL 20 to 1 UPS), demonstrating that this species is well adapted to highly diluted environments (McGraw et al*.*, 2002). From these studies it is possible to conclude that *L. vannamei* postlarvae can tolerate broad salinity ranges, although shrimp between PL1 and PL10 are more sensible to low salinities that juveniles with more than 20 days after the last

In this study juvenile *L. vannamei* (initial mean weight 2.26g) were cultivated at 2, 4 and 8 UPS during 70 days in a semi-closed recirculating experimental system. Density was kept constant at 28 juveniles per m2 in all salinity treatments. Results showed high survival rates in all treatments (98.7-100%). Mean final weight varied slightly between 19.0 -19.28g, whereas weekly growth rates varied between 1.67-1.7g. No significant differences in growth or survival amongst treatments were found, suggesting that *L. vannamei* can be cultivated at

Bray et al*.*, (1994) reported that juvenile *L. vannamei* positive to IHHN virus showed better growth exposed during 35 days at salinities of 5 and 15 UPS than at 25, 35 and 49 UPS. A


loses more energy through ammonia excretion than *E. megalocyathus*, suggesting different physiological mechanisms for using the ingested protein (Table 4).

Table 4. Equation parameters (Y = a Wb) of oxygen consumption, ammonia excretion and ingestion rate of *Enteroctopus megalociathus* and *O. maya*. (Data from Farías et al., 2009).

#### **4. Ecophysiology of crustacean**

Between crustacean shrimp, crab and lobster has been deeply studied from an ecophysiological point of view. Crustaceans live in a wide variety of environments including fresh water, salty water and sea water. Crustaceans can be finding in holes in earth living with a little bit of humidity or in the crown of palms, several meters above the ground. Crustacean aquaculture has been growing in the last decades being the shrimp culture the most important activity. The world production of crustaceans was 5 million tons in 2008 being shrimp culture 73.3% of the total production (FAO, 2011). Today the white leg shrimp *Litopenaeus vannamei* is the most important species for aquaculture. *Litopenaeus vannamei* was introduced in China where its cultivation shifted to that of *Penaeus monodon.*  Ecophysiological studies of shrimp have been important to define culture environments mainly when this species is mostly cultivated in salty water, where joint with diluted environment, animals are exposed to high temperatures, wide oxygen variations and nitrogen waste products.

#### **4.1 Effect of salinity on shrimp physiological condition**

White shrimp, *Litopenaeus vannamei*, the most cultivated shrimp species in America, grows well at salinities ranging from freshwater to hyper saline waters. Although *L. vannamei*  tolerates broad salinity ranges, values for optimal growth have not yet been determined, and studies up to date show contradictory results. Whilst studies conducted under laboratory conditions have shown that maximal growth occurs between 5 and 15 UPS (Bray et al*.(*1994); Boyd, 1995; Rosas et al*.*, 2001a), other studies have reported maximal growth at salinities above 35 UPS (Ponce-Palafox et al*.*, 1997; Decamp et al*.*, 2003). Recent studies have demonstrated a close relationship between nutrition and environmental characteristics at which shrimp are cultivated. This is because dietary levels of carbohydrates, lipids and proteins largely determine the shrimps capacity to respond to changes in the ionic composition of culture water.

#### **4.1.1 Growth and survival**

170 Aquaculture and the Environment - A Shared Destiny

loses more energy through ammonia excretion than *E. megalocyathus*, suggesting different

*Enteroctopus megalociathus* 0.2 0.71 0.82 *Octopus maya* 0.93 0.69 0.93

*Enteroctopus megalociathus* 0.32 0.37 0.53 *Octopus maya* 0.75 0.43 0.76

*Enteroctopus megalociathus* 0.19 0.67 0.95 *Octopus maya* 0.07 0.82 0.95 Table 4. Equation parameters (Y = a Wb) of oxygen consumption, ammonia excretion and ingestion rate of *Enteroctopus megalociathus* and *O. maya*. (Data from Farías et al., 2009).

Between crustacean shrimp, crab and lobster has been deeply studied from an ecophysiological point of view. Crustaceans live in a wide variety of environments including fresh water, salty water and sea water. Crustaceans can be finding in holes in earth living with a little bit of humidity or in the crown of palms, several meters above the ground. Crustacean aquaculture has been growing in the last decades being the shrimp culture the most important activity. The world production of crustaceans was 5 million tons in 2008 being shrimp culture 73.3% of the total production (FAO, 2011). Today the white leg shrimp *Litopenaeus vannamei* is the most important species for aquaculture. *Litopenaeus vannamei* was introduced in China where its cultivation shifted to that of *Penaeus monodon.*  Ecophysiological studies of shrimp have been important to define culture environments mainly when this species is mostly cultivated in salty water, where joint with diluted environment, animals are exposed to high temperatures, wide oxygen variations and

White shrimp, *Litopenaeus vannamei*, the most cultivated shrimp species in America, grows well at salinities ranging from freshwater to hyper saline waters. Although *L. vannamei*  tolerates broad salinity ranges, values for optimal growth have not yet been determined, and studies up to date show contradictory results. Whilst studies conducted under laboratory conditions have shown that maximal growth occurs between 5 and 15 UPS (Bray et al*.(*1994); Boyd, 1995; Rosas et al*.*, 2001a), other studies have reported maximal growth at salinities above 35 UPS (Ponce-Palafox et al*.*, 1997; Decamp et al*.*, 2003). Recent studies have demonstrated a close relationship between nutrition and environmental characteristics at which shrimp are cultivated. This is because dietary levels of carbohydrates, lipids and proteins largely determine the shrimps capacity to respond to changes in the ionic

a bR2

physiological mechanisms for using the ingested protein (Table 4).

Oxygen consumption, mgO2 h-1 animal-1

Ammonia excretion, mgN-NH3 h-1 animal-1

Ingestion rate, g day-1 animal-1

**4.1 Effect of salinity on shrimp physiological condition** 

**4. Ecophysiology of crustacean** 

nitrogen waste products.

composition of culture water.

Salinity affects the distribution of a variety of estuarine and marine organisms. Some marine species, such as shrimps, have life cycles that include an estuarine phase. Since the 1950´s, there have been reports on congregations of juvenile shrimps at low salinities, whilst adults reproduced under strict marine conditions. It has also been observed that larval phases inhabit oceanic waters, where they develop and moult into postlarvae. Through certain chemo tactile mechanisms and water currents, postlarvae are capable of moving towards estuaries and coastal lagoons, where they recruit as early juveniles. It is in these habitats, with strong salinity variations, where some shrimp species are capable of colonizing more diluted environments than others. For example, studies carried out in the 1960´s by Mac Farland & Lee, (1963) showed that juvenile *L. setiferus* are better adapted to tolerate low salinities than juvenile *Farfantepenaeus aztecus.* Similarly, Mair, (1980) observed that amongst *L. stylirostris, F. californiensis,* and *L. vannamei*, the latter is the species that best tolerates highly diluted environments in estuaries and costal lagoons, and is therefore capable of inhabiting areas restricted to the other two species. This ecophysiological trait allows *L. vannamei* to reduce ecological pressures of competition for space and food, both strong limiting resources in these habitats. In an experimental study, Mair, (1980) observed that *L. vannamei* postlarvae and early juveniles placed in an experimental salinity gradient preferred salinities from 3-6 UPS, whereas *L. stylirostris* and *F. californiensis* preferred salinities from 32-35 UPS and 9-26 UPS, respectively.

The wide tolerance of *L. vannamei* to salinity has also been observed under culture conditions, both experimentally and within production facilities. Ogle et al*.* (1992) examined the effects of salinity on growth in *L. vannamei* postlarvae under different culture conditions. In that study the authors observed that tolerance to salinity is independent of postlarval (PL) age, showing that both PL8 and PL22 had better survival and growth at salinities ranging from 16 to 32 UPS. In that same study, it was demonstrated that tolerance to salinity increases as temperature declines from 30-16°C, since the highest survival and growth of postlarvae was obtained at salinities of 8 and 16 UPS at that temperature. Postlarvae can be acclimated to low salinities for production purposes. Recent studies have shown that a rate of salinity decline of 25% per hour is appropriate for postlarvae (PL10 to 4 UPS) and early juveniles (PL 20 to 1 UPS), demonstrating that this species is well adapted to highly diluted environments (McGraw et al*.*, 2002). From these studies it is possible to conclude that *L. vannamei* postlarvae can tolerate broad salinity ranges, although shrimp between PL1 and PL10 are more sensible to low salinities that juveniles with more than 20 days after the last metamorphic moult (PL20).

In this study juvenile *L. vannamei* (initial mean weight 2.26g) were cultivated at 2, 4 and 8 UPS during 70 days in a semi-closed recirculating experimental system. Density was kept constant at 28 juveniles per m2 in all salinity treatments. Results showed high survival rates in all treatments (98.7-100%). Mean final weight varied slightly between 19.0 -19.28g, whereas weekly growth rates varied between 1.67-1.7g. No significant differences in growth or survival amongst treatments were found, suggesting that *L. vannamei* can be cultivated at low salinities obtaining good survival and growth.

Bray et al*.*, (1994) reported that juvenile *L. vannamei* positive to IHHN virus showed better growth exposed during 35 days at salinities of 5 and 15 UPS than at 25, 35 and 49 UPS. A

Applied Ecophysiology: An Integrative Form to Know How Culture

nutritional state of shrimp maintained at high and low salinities (Fig. 5).

the center of penaeid shrimp nutrition.

It can be seen that CHO (carbohydrates) enter as starch and are transformed into glucose and glycogen. Laboratory studies have shown that starch breakup by amylase has a saturation point, indicating that shrimp capacity to breakup starch increases to a maximum level. At CHO dietary concentrations higher than 35%, the processing rate of amylase stays constant even with increasing CHO levels Rosas et al*.*, 2000). Because in diluted environments amylase is less efficient, the saturation point of amylase has a larger effect under low than high salinities, (Rosas et al., 2001a). Another important aspect is the saturation of glycogen in the digestive gland (digestive gland) of shrimp fed with diets containing excessive CHO. Results of laboratory studies have shown that shrimp capacity to store glycogen is limited, and the digestive gland is rapidly saturated with levels of dietary inclusion higher than 30 %. When shrimp are kept at low salinities, this saturation has a larger effect, because it prevents adequate absorption of amino acids used to maintain internal osmotic pressure and cell volume Rosas et al., (2001a). These results have been interpreted taking into account that shrimp basically use CHO to form chitin, which in turn is the principal component of the exoskeleton. A glucose molecule and one ammonia radical product of the amino acid degradation form chitin. The combination of these products emphasizes the fact that chitin formation depends on CHO metabolism, which constitutes

The adaptations of shrimp cultivated in hypersaline environments have not yet been studied in depth. Studies conducted in our laboratory have shown that at high salinity the effects of dietary proteins and CHO operate in a different way than when at low salinity Pascual et al*.*, (2003); Pascual et al*.*, (2004a). From our results, it can be concluded that at high salinity

**4.1.2 Relationship between salinity and food** 

Environment Modulates the Performance of Aquatic Species from an Energetic Point of View 173

Salinity is factor modulating the physiological state of shrimp. In costal environments where juveniles inhabit, estuaries and lagoons offer salinity conditions that fluctuate markedly both with tidal and seasonal variations. Penaeid shrimp have adapted to these changes taking maximum profit of food richness and protection found in these nursing areas. In order to survive and grow at the highest rates, shrimp have evolved through diverse physiological mechanisms that allow compensation for the marked changes in salinity. A decrease in water salinity results in a massive entry of water into the tissues by simple diffusion. In order to avoid extreme dilution and the consequent increment in cell volume, shrimp respond by: i) Reducing the concentration of ions dissolved in the cytoplasm. ii) Transporting amino acids from the cells to the blood, and then to the digestive gland. iii) Changing cell permeability at the gills. In crustaceans, ions transport has been extensively studied. Studies of Na, K and Cl transport as a result of exposure to diluted environments have shown that these ions play an active role in the reduction of cell volume by helping to keep osmotic pressure constant. However, because ions are small molecules with low osmotic power this mechanism is unsuccessful when shrimp are kept for long periods in diluted environments. A second mechanism has been described in which shrimp use the amino acid pool free in the cytoplasm to regulate cell volume. Amino acids are the principal component of proteins, and are abundant within shrimp cells. When a salinity change occurs, amino acids are transported towards the hemolymph where they are re-absorbed by the digestive gland. There, amino acids are stored as hemocyanin, as peptides for growth, or as proteins for the immune system. A recent study by Rosas et al*.*,(2002) includes a summary of the effects of dietary and protein and carbohydrate levels on the physiological and

weekly growth of 2g was obtained at low salinities 5 and 15 UPS. Mean final weights of shrimp were 12g (5 UPS), 12.2g (15 UPS), 10.8g (25 UPS), 11.1g (35 UPS) y 9,8g (49 UPS). No significant differences in mean final weight were found amongst low salinity treatments (5 and 15 UPS), but these were both significantly higher than that obtained at 49 UPS. Survival values were statistically similar amongst all treatments. A second group of juveniles with mean initial weights of 2.2g were infected with the IHHN virus, and maintained at 25 and 49 UPS. Their growth and survival was then compared with those of shrimp of the first group kept at those salinities. Results showed that growth of infected shrimp was similar to that obtained in the previous trial. In addition, there was a significant interaction between shrimp group and salinity, indicating that the group infected with IHHN grew less at high salinities. Mean final weights in the second trial were 10.3g (25 UPS) and 8.5g (49 UPS). Survival was also statistically similar for infected shrimp kept under the two salinity treatments.

In a more recent study Ponce-Palafox et al., (1997) reported that optimum growth and survival for juvenile *L. vannamei* were between 33 and 40 UPS and 28 and 30°C. Besides growth and survival of *L. vannamei* postlarvae were measured at 20, 25, 30, and 35ºC and salinities of 20, 30, 35, 40 and 50 UPS. Groups of 30 individuals with three replicates were used in each temperature and salinity combination. Results clearly showed that juveniles of this specie survive best at 20 and 30ºC and at salinities higher than 20 UPS. Shrimp growth was highest at temperatures between 25 and 35ºC, with small differences amongst salinity treatments. Rosas et al*.*,(2001b) observed that better growth of juvenile *L. vannamei* can be attained when shrimp are kept at 15 UPS than at salinities of 35 UPS or more. Although much research on juvenile *L. vannamei* culture has been conducted, there is yet no decisive demonstration of the optimal salinity range. Whilst some researchers report that salinities lower than 25 UPS promote growth, others confirm that salinities higher than 35 UPS result best for this species. Combinations of salinity and temperature, as well as the type and quality of food appear to be determining factors to establish optimum salinity levels. Other shrimp species have also grown best at estuarine salinities

Chen et al*.*, (1992) observed that *P. chinensis* kept at 25-31°C showed best growth and survival at salinities between 10 and 20 UPS. In addition (Staples & Heales, 1991) report best survival and growth in *P. merguiensis* kept under a combination of 20°C and 20 UPS. Rosas et al*.*, (1999) showed that juvenile *L. vannamei* are energetically more efficient when exposed at salinities of 15 UPS than those lower (10 y 5 UPS) or higher (20, 25 y 30 UPS) than 15 UPS. In another study Brito et al., (2000) showed that juvenile *F. brasiliensis* have low tolerance to salinities lower than 25 UPS, below which shrimp stop growing and die. The authors explain that postlarvae and juveniles of this species occur in the southern Gulf of Mexico and Caribbean Sea, where they are rarely exposed to salinities lower than these. Rosas et al., (1999) found that salinity affects shrimp growth and survival differentially depending on postlarval age. These authors observed that optimal salinity for PL 10 lies between 30 and 35 UPS, whereas optimal salinity for PL 20 and older lies between 5 and 15 UPS. Optimal salinity ranges have been identified for other shrimp species. In general terms, species of the genus *Litopenaeus* are more tolerant to low salinities than those of the genus *Penaeus* or *Farfantepenaeus*. The former have optimal salinity ranges below 25 UPS, whilst the latter have optimal ranges at salinities above 30 UPS (Brito et al*.*, 2000).

#### **4.1.2 Relationship between salinity and food**

172 Aquaculture and the Environment - A Shared Destiny

weekly growth of 2g was obtained at low salinities 5 and 15 UPS. Mean final weights of shrimp were 12g (5 UPS), 12.2g (15 UPS), 10.8g (25 UPS), 11.1g (35 UPS) y 9,8g (49 UPS). No significant differences in mean final weight were found amongst low salinity treatments (5 and 15 UPS), but these were both significantly higher than that obtained at 49 UPS. Survival values were statistically similar amongst all treatments. A second group of juveniles with mean initial weights of 2.2g were infected with the IHHN virus, and maintained at 25 and 49 UPS. Their growth and survival was then compared with those of shrimp of the first group kept at those salinities. Results showed that growth of infected shrimp was similar to that obtained in the previous trial. In addition, there was a significant interaction between shrimp group and salinity, indicating that the group infected with IHHN grew less at high salinities. Mean final weights in the second trial were 10.3g (25 UPS) and 8.5g (49 UPS). Survival was also statistically similar for infected shrimp kept under the two salinity

In a more recent study Ponce-Palafox et al., (1997) reported that optimum growth and survival for juvenile *L. vannamei* were between 33 and 40 UPS and 28 and 30°C. Besides growth and survival of *L. vannamei* postlarvae were measured at 20, 25, 30, and 35ºC and salinities of 20, 30, 35, 40 and 50 UPS. Groups of 30 individuals with three replicates were used in each temperature and salinity combination. Results clearly showed that juveniles of this specie survive best at 20 and 30ºC and at salinities higher than 20 UPS. Shrimp growth was highest at temperatures between 25 and 35ºC, with small differences amongst salinity treatments. Rosas et al*.*,(2001b) observed that better growth of juvenile *L. vannamei* can be attained when shrimp are kept at 15 UPS than at salinities of 35 UPS or more. Although much research on juvenile *L. vannamei* culture has been conducted, there is yet no decisive demonstration of the optimal salinity range. Whilst some researchers report that salinities lower than 25 UPS promote growth, others confirm that salinities higher than 35 UPS result best for this species. Combinations of salinity and temperature, as well as the type and quality of food appear to be determining factors to establish optimum salinity levels. Other

Chen et al*.*, (1992) observed that *P. chinensis* kept at 25-31°C showed best growth and survival at salinities between 10 and 20 UPS. In addition (Staples & Heales, 1991) report best survival and growth in *P. merguiensis* kept under a combination of 20°C and 20 UPS. Rosas et al*.*, (1999) showed that juvenile *L. vannamei* are energetically more efficient when exposed at salinities of 15 UPS than those lower (10 y 5 UPS) or higher (20, 25 y 30 UPS) than 15 UPS. In another study Brito et al., (2000) showed that juvenile *F. brasiliensis* have low tolerance to salinities lower than 25 UPS, below which shrimp stop growing and die. The authors explain that postlarvae and juveniles of this species occur in the southern Gulf of Mexico and Caribbean Sea, where they are rarely exposed to salinities lower than these. Rosas et al., (1999) found that salinity affects shrimp growth and survival differentially depending on postlarval age. These authors observed that optimal salinity for PL 10 lies between 30 and 35 UPS, whereas optimal salinity for PL 20 and older lies between 5 and 15 UPS. Optimal salinity ranges have been identified for other shrimp species. In general terms, species of the genus *Litopenaeus* are more tolerant to low salinities than those of the genus *Penaeus* or *Farfantepenaeus*. The former have optimal salinity ranges below 25 UPS, whilst the latter have optimal ranges at salinities above 30

shrimp species have also grown best at estuarine salinities

treatments.

UPS (Brito et al*.*, 2000).

Salinity is factor modulating the physiological state of shrimp. In costal environments where juveniles inhabit, estuaries and lagoons offer salinity conditions that fluctuate markedly both with tidal and seasonal variations. Penaeid shrimp have adapted to these changes taking maximum profit of food richness and protection found in these nursing areas. In order to survive and grow at the highest rates, shrimp have evolved through diverse physiological mechanisms that allow compensation for the marked changes in salinity. A decrease in water salinity results in a massive entry of water into the tissues by simple diffusion. In order to avoid extreme dilution and the consequent increment in cell volume, shrimp respond by: i) Reducing the concentration of ions dissolved in the cytoplasm. ii) Transporting amino acids from the cells to the blood, and then to the digestive gland. iii) Changing cell permeability at the gills. In crustaceans, ions transport has been extensively studied. Studies of Na, K and Cl transport as a result of exposure to diluted environments have shown that these ions play an active role in the reduction of cell volume by helping to keep osmotic pressure constant. However, because ions are small molecules with low osmotic power this mechanism is unsuccessful when shrimp are kept for long periods in diluted environments. A second mechanism has been described in which shrimp use the amino acid pool free in the cytoplasm to regulate cell volume. Amino acids are the principal component of proteins, and are abundant within shrimp cells. When a salinity change occurs, amino acids are transported towards the hemolymph where they are re-absorbed by the digestive gland. There, amino acids are stored as hemocyanin, as peptides for growth, or as proteins for the immune system. A recent study by Rosas et al*.*,(2002) includes a summary of the effects of dietary and protein and carbohydrate levels on the physiological and nutritional state of shrimp maintained at high and low salinities (Fig. 5).

It can be seen that CHO (carbohydrates) enter as starch and are transformed into glucose and glycogen. Laboratory studies have shown that starch breakup by amylase has a saturation point, indicating that shrimp capacity to breakup starch increases to a maximum level. At CHO dietary concentrations higher than 35%, the processing rate of amylase stays constant even with increasing CHO levels Rosas et al*.*, 2000). Because in diluted environments amylase is less efficient, the saturation point of amylase has a larger effect under low than high salinities, (Rosas et al., 2001a). Another important aspect is the saturation of glycogen in the digestive gland (digestive gland) of shrimp fed with diets containing excessive CHO. Results of laboratory studies have shown that shrimp capacity to store glycogen is limited, and the digestive gland is rapidly saturated with levels of dietary inclusion higher than 30 %. When shrimp are kept at low salinities, this saturation has a larger effect, because it prevents adequate absorption of amino acids used to maintain internal osmotic pressure and cell volume Rosas et al., (2001a). These results have been interpreted taking into account that shrimp basically use CHO to form chitin, which in turn is the principal component of the exoskeleton. A glucose molecule and one ammonia radical product of the amino acid degradation form chitin. The combination of these products emphasizes the fact that chitin formation depends on CHO metabolism, which constitutes the center of penaeid shrimp nutrition.

The adaptations of shrimp cultivated in hypersaline environments have not yet been studied in depth. Studies conducted in our laboratory have shown that at high salinity the effects of dietary proteins and CHO operate in a different way than when at low salinity Pascual et al*.*, (2003); Pascual et al*.*, (2004a). From our results, it can be concluded that at high salinity

Applied Ecophysiology: An Integrative Form to Know How Culture

**4.2 Physiological condition and immune system of shrimp** 

this species.

Destoumieux et al*.*, (2000).

capacities Pascual et al., (2004).

Environment Modulates the Performance of Aquatic Species from an Energetic Point of View 175

were statistically similar. This is probably due lipids in the digestive gland being used during the first 3 hours of fasting, allowing for animals to have sufficient energetic reserves to tackle the salinity change Rosas et al*.*, (1995). This study concluded that movement of lipids to satisfy the energetic demand of osmoregulation could be operating in shrimp of

The shrimp immune system has a solid protein base and hemocyanin plays an important role in its function. Recent studies have demonstrated that in addition to its multifunctional role (oxygen transporter, storage protein, carotenoids carrier, osmolite, ecdysone transporter) hemocyanin has a fungistatic Destoumieux et al*.*,(2001) and prophenol oxydase-like function Adachi et a*l.*, (2003). Proteins are also involved in recognizing foreign glucans through lipopolysaccharide binding protein (LPSBP) and β glucan binding protein (BGBP) Destoumieux et al*.*, (2000); Vargas-Albores & Yepiz-Plascencia, (2000). A clotting protein (with the change of fibrinogens to fibrin) is involved in engulfing foreign invading organisms and prevents blood loss upon wounding Hall et al*.*, (1999); Montaño-Pérez et al., (1999). Defense reactions in shrimp are often accompanied by melanization. Prophenoloxidase (ProPO) activating system, mediated by hemocytes, is a zymogen of phenoloxidase (PO) enzyme that catalyzes both o-hydroxylation of monophenols and oxidation of phenols to quinones leading to synthesis of melanin Sritunyalucksana & Söderhall, (2000). Conversion of ProPO to PO occurs through a serine protease called prophenoloxidase-activating enzyme (ppA) regulated by another protein, a-2 macroglobulin, a trypsin inhibitor Perazzolo & Barracco, (1997). The innate immune response of shrimp also relies upon a production, in hemocytes, of antimicrobial peptides called peneidins that are active against a large range of pathogens essentially directed against Gram-positive bacteria via a strain-specific inhibition mechanism

 In order to reach effects of dietary protein level on energetic balance we probe two protein levels in a range of optimal reported levels of 15% and 40% (equivalent to 15 and 40g DP/kg body weight/day [g DP/kg BWd]) and one extremely low (5% equivalent to 5g DP/kg BWd) were used to feed juveniles for 50 days. Dietary protein level enhanced ingestion rate in shrimp fed 5g DP/kg BWd compared to shrimp fed 40g DP/ kg BWd, however, daily growth coefficient (DGC,%) of *L. vannamei* juveniles was high in shrimp fed 40g DP/kg BWd. An inverse relation between wastes (H+U) and dietary protein level was observed indicating that shrimp lose 81% of ingested energy when fed 5g DP/kg BWd and only 5.6% when fed 40g DP/kg BWd. A higher assimilation and production efficiency (P/As) was obtained when shrimp were fed 40g DP/kg BWd, than obtained in shrimp fed 15 or 5g DP/kg BWd. An increase in Oxy hemocyanin was observed with increasing dietary protein levels indicating that shrimp accumulated protein as hemocyanin. A reduction of hemocytes occurred when shrimp were fed sub-optimal dietary protein; same patron was observed in the respiratory burst. The compensatory mechanism used by *L. vannamei* to respond nutritional stress, sub-optimal dietary protein level (5 and 15g DP/kg BWd) induced not only a severe reduction in growth rate and assimilation efficiency but also in immune

In an attempt to know how the protein level modulates catabolism and its effects on the immune response, we studied juvenile *L. vannamei* that had been starved for varying period

Fig. 5. Diagram showing a summary of the relationship between dietary proteins and carbohydrates and the metabolic pathways involved in the uptake of ingested nutrients.

shrimp may require lower protein levels than those they require at low salinity. This is because at concentrated environments ammonia produced by the degradation of dietary amino acids may be toxic. At low salinity degraded amino acids are transformed to ammonia and used for ion exchange, whilst at concentrated environments ammonia molecules are trapped in the blood and cannot be released as rapidly and efficient. Notwithstanding the high tolerance of crustaceans to high ammonia in blood Schmitt & Uglow, (1997), levels above 12 mg l-1 could inhibit growth, delay moult and reduce breathing capacity of shrimp Danford et al*.*(2001). High levels such as those can easily be attained when the diet contains more than 35% proteins and shrimp are kept at salinities higher than 40 UPS Rosas et al., (2000)

The effect of salinity on lipid metabolism in has neither been studied in depth. In a recent study the effect of salinity on shrimp survival, the activity of the Na/K bomb and lipids was examined Palacios et al*.*, (2004). In that study 20 day old fed and unfed (3 and 24 h fasting) postlarvae were compared to demonstrate the effect of energy reserves on survival and osmoregulatory mechanisms. Activity of the Na+-K+ ATPase was 5 times higher in posterior gills than in anterior ones, a response associated with the osmoregulatory function of this organ. In addition, a non-significant increase in anhydrase activity was observed in postlarvae exposed for 20 days to 10 UPS. This is explained by the role of this enzyme in the hydration of CO2 produced in respiration. Under low salinity conditions, the internal media dilutes and the output of Na + decreases. Na+ in then exchanged for H+ and Cl- for HCO3, provided by CO2 through the carbonic anhydrase. In that same study total lipids in digestive gland were significantly lower in fasting than in fed PL20, whereas triacyl glycerids were statistically similar. This is probably due lipids in the digestive gland being used during the first 3 hours of fasting, allowing for animals to have sufficient energetic reserves to tackle the salinity change Rosas et al*.*, (1995). This study concluded that movement of lipids to satisfy the energetic demand of osmoregulation could be operating in shrimp of this species.

#### **4.2 Physiological condition and immune system of shrimp**

174 Aquaculture and the Environment - A Shared Destiny

**E P I D E R M I S C h y t i n S y n t h e s i s**

**Energy**

**Glycogen**

**Glucose**

**+ NH4+**

**Glucosamine**

**Energy Glycogen**

**Glucosamine to epidermis Glutamine to gill**

> **Energy + NH4+ NH3**

**NH4+ - NH3**

Fig. 5. Diagram showing a summary of the relationship between dietary proteins and carbohydrates and the metabolic pathways involved in the uptake of ingested nutrients.

shrimp may require lower protein levels than those they require at low salinity. This is because at concentrated environments ammonia produced by the degradation of dietary amino acids may be toxic. At low salinity degraded amino acids are transformed to ammonia and used for ion exchange, whilst at concentrated environments ammonia molecules are trapped in the blood and cannot be released as rapidly and efficient. Notwithstanding the high tolerance of crustaceans to high ammonia in blood Schmitt & Uglow, (1997), levels above 12 mg l-1 could inhibit growth, delay moult and reduce breathing capacity of shrimp Danford et al*.*(2001). High levels such as those can easily be attained when the diet contains more than 35% proteins and shrimp are kept at salinities

The effect of salinity on lipid metabolism in has neither been studied in depth. In a recent study the effect of salinity on shrimp survival, the activity of the Na/K bomb and lipids was examined Palacios et al*.*, (2004). In that study 20 day old fed and unfed (3 and 24 h fasting) postlarvae were compared to demonstrate the effect of energy reserves on survival and osmoregulatory mechanisms. Activity of the Na+-K+ ATPase was 5 times higher in posterior gills than in anterior ones, a response associated with the osmoregulatory function of this organ. In addition, a non-significant increase in anhydrase activity was observed in postlarvae exposed for 20 days to 10 UPS. This is explained by the role of this enzyme in the hydration of CO2 produced in respiration. Under low salinity conditions, the internal media dilutes and the output of Na + decreases. Na+ in then exchanged for H+ and Cl- for HCO3, provided by CO2 through the carbonic anhydrase. In that same study total lipids in digestive gland were significantly lower in fasting than in fed PL20, whereas triacyl glycerids

**Glucose 6 P**

**Hemocyanin**

Immune Response

**Peptides + AA**

**Blood**

**Blood Free AA Pool Free AA Pool**

**Muscle**

**Piruvate**

**Protein synthesis + NH4+**

**Growth**

**Osmotic Regulation**

**Digestive Gland**

**Glucose + NH4+**

**Piruvate + NH4+**

**Dietary** 

**Gills**

**Blood**

**Glutamine to gill**

**Glutamate + NH4**

higher than 40 UPS Rosas et al., (2000)

**Protein Peptides**

Dietary CHO **Starch Glucose 6 P**

**Peptides + AA**

**Free AA Pool**

The shrimp immune system has a solid protein base and hemocyanin plays an important role in its function. Recent studies have demonstrated that in addition to its multifunctional role (oxygen transporter, storage protein, carotenoids carrier, osmolite, ecdysone transporter) hemocyanin has a fungistatic Destoumieux et al*.*,(2001) and prophenol oxydase-like function Adachi et a*l.*, (2003). Proteins are also involved in recognizing foreign glucans through lipopolysaccharide binding protein (LPSBP) and β glucan binding protein (BGBP) Destoumieux et al*.*, (2000); Vargas-Albores & Yepiz-Plascencia, (2000). A clotting protein (with the change of fibrinogens to fibrin) is involved in engulfing foreign invading organisms and prevents blood loss upon wounding Hall et al*.*, (1999); Montaño-Pérez et al., (1999). Defense reactions in shrimp are often accompanied by melanization. Prophenoloxidase (ProPO) activating system, mediated by hemocytes, is a zymogen of phenoloxidase (PO) enzyme that catalyzes both o-hydroxylation of monophenols and oxidation of phenols to quinones leading to synthesis of melanin Sritunyalucksana & Söderhall, (2000). Conversion of ProPO to PO occurs through a serine protease called prophenoloxidase-activating enzyme (ppA) regulated by another protein, a-2 macroglobulin, a trypsin inhibitor Perazzolo & Barracco, (1997). The innate immune response of shrimp also relies upon a production, in hemocytes, of antimicrobial peptides called peneidins that are active against a large range of pathogens essentially directed against Gram-positive bacteria via a strain-specific inhibition mechanism Destoumieux et al*.*, (2000).

 In order to reach effects of dietary protein level on energetic balance we probe two protein levels in a range of optimal reported levels of 15% and 40% (equivalent to 15 and 40g DP/kg body weight/day [g DP/kg BWd]) and one extremely low (5% equivalent to 5g DP/kg BWd) were used to feed juveniles for 50 days. Dietary protein level enhanced ingestion rate in shrimp fed 5g DP/kg BWd compared to shrimp fed 40g DP/ kg BWd, however, daily growth coefficient (DGC,%) of *L. vannamei* juveniles was high in shrimp fed 40g DP/kg BWd. An inverse relation between wastes (H+U) and dietary protein level was observed indicating that shrimp lose 81% of ingested energy when fed 5g DP/kg BWd and only 5.6% when fed 40g DP/kg BWd. A higher assimilation and production efficiency (P/As) was obtained when shrimp were fed 40g DP/kg BWd, than obtained in shrimp fed 15 or 5g DP/kg BWd. An increase in Oxy hemocyanin was observed with increasing dietary protein levels indicating that shrimp accumulated protein as hemocyanin. A reduction of hemocytes occurred when shrimp were fed sub-optimal dietary protein; same patron was observed in the respiratory burst. The compensatory mechanism used by *L. vannamei* to respond nutritional stress, sub-optimal dietary protein level (5 and 15g DP/kg BWd) induced not only a severe reduction in growth rate and assimilation efficiency but also in immune capacities Pascual et al., (2004).

In an attempt to know how the protein level modulates catabolism and its effects on the immune response, we studied juvenile *L. vannamei* that had been starved for varying period

Applied Ecophysiology: An Integrative Form to Know How Culture

Lipids Fatty acids

pyruvate

**enzyms**

glycogen chitin

**glycogen**

**Hemocyanin** energy **Protein synthesis**

**Growth Neurotransmitters**

(**FAA**), which can be used to generate muscle tissue.

**Lectins**

**GL**

amilase **++ CHO --**

**Wild shrimp**

**+**

glucose

**+**

alpha-

A-CoA

**CAG**

**CK CR**

GC

glucose 6F

**IMMUNE RESPONSE**

Fig. 6. Conceptual model: protein metabolism and homeostasis. The scheme includes the main organs and systems of juvenile shrimp: digestive gland, **GD**, gills, **G**; muscle, **M**; cuticle, **C**; nervous system, **SN**, hematopoietic tissue, **HT** and hemolymph, **H**. Genetic variability is the basis of the model as it affects the flow of energy from food chemistry to physiological processes. *Litopenaeus vannamei* juveniles grown in closed loop and sorted by size (F7, F25) have a limited capacity to respond to carbohydrate diets (CHO) demonstrating

high protein dependence in their metabolism. The degradation of proteins generates peptides and amino acids (**aa**) that are oxidized or used to synthesize **glycogen**. The **aa** can also be used for the synthesis of digestive and metabolic enzymes, and immune proteins (such as prophenoloxidase, **ProPO**, lectins, **L**, lipoprotein recognition, **BGBP**, clothing protein, **CP**, antimicrobial peptides, **AP**; of regulation of the immune response; pacifastine and α2-macroglobulin, and hemocyanin, **H**, which seems to play a role in the immune response of shrimp, in addition to functioning as a storage protein in the hemolymph. Amino acids can also be transported to the hemolymph to form the pool of free amino acids

direction, for example in conditions of nutritional stress, catabolism of glucogenic amino acids is the basis for the gluconeogenic route for CHO can be used as energy substrate. The cycle of fatty acids (**CAG**) is connected to the glycolytic pathway through **A-CoA**, a high concentration of **ATP** or **NADH** promotes the synthesis of fatty acids from the A-CoA. The ß-oxidation of lipids from food or reserves represents the reverse direction to generate

Regulation of osmotic pressure is mainly associated with the Na-K ATPase, ion exchange, the catabolism of aa and the activity of glutamate dehydrogenase (**GDH**), which controls

**AAF**

**DG**

**Metabolic rate Moult**

**Protein**

+ NH4

**Peptides**

**Farmed,**

**aa**

**HT**

**HT**

**C**

metabolic energy.

**pathogens**

**SN**

C O M P E N S A T I O N

I M M U N I T Y

Environment Modulates the Performance of Aquatic Species from an Energetic Point of View 177

Cholesterol

Reserves

**DIGESTIVE CAPACITY**

**aa**

**AAF**

**LP**

**ßGBP**

**CP**

**Hemocytes**

**2-M pacifastine**

**Free amino acids catabolism** 

**OxyHC**

glucose 6F

**OxyHC**

**H**

**HC**

**proFO**

**PA**

**GROWTH**

**M**

+ NH4

**CR**

**aa**

**aa**

peptides

**aa**

**pyruvate**

**glycogen**

A-CoA

**CK** GC

**OSMORREGULATION**

**G**

H+ NH4 + NH3

+ NH4

Na+

GDH

glutarate

O2

**AAF**

CO2

**HC**

**LP** Fatty acids

O2

**GL**

Cholesterol

**CAG**

H2O

H2O iones **pathogens**

**O2 CO2**

Na+

**K+**

after being conditioned on diet containing either maintenance or optimal dietary protein levels Pascual et al*.*,( 2006). Juvenile shrimp were fed for 21 days on diets containing 5% and 40% dietary protein. Hemolymph metabolites (glucose, cholesterol, protein, acylglycerols, and lactate), hemocyanin, osmoregulatory capacity, digestive gland glycogen and lipids, and immune conditions (hemocytes concentration, phenoloxidase activity, respiratory burst: basal and activated) were evaluated and considered as initial condition. After that time, shrimp were starved for 21 days. A reduction in all physiological and immunological indicators was observed with starvation. The results demonstrate that shrimp are well adapted to tolerate food deprivation for some time but that this tolerance is closely related to its previous nutritional condition. In the case of shrimp fed 40% DPL, wet weight, nutritional and immune condition was significantly affected after 14 days of starvation. In shrimp previously fed 5% DP, tolerance to starving condition was limited to only a few days (7 days) as a result of low reserves of circulatory and mussel proteins. All these results demonstrate that dietary protein levels can governor the immune condition of shrimp through the management reserves metabolism.

Domestication is other important aspect to obtain better results during culture; however, artificial selection has important implication on physiological adaptations. Pascual et al., (2004) studied wild and seventh-generation cultivated shrimp to determine how size-based selection could alter the nutritional and immunological conditions of *Litopenaeus vannamei*. Wild juveniles and a sample of seventh-generation cultured shrimp were acclimated under identical conditions. During 55 days, shrimp were fed a high (HCHO: 44%) or a low (LCHO: 3%) carbohydrate diet. Wild shrimp showed a direct relation between dietary CHO and lactate, protein and hemocyte levels indicating that dietary CHO was used for protein synthesis via transamination pathways. In seventh-generation cultured shrimp these parameters were inversely proportional to dietary CHO level, indicating the capacity to synthesize protein from dietary CHO was repressed in cultured shrimp. Farmed shrimp showed a limited capacity to respond to LCHO diets demonstrating high protein dependence in their metabolism and immune response. These results demonstrate that during size-based breeding programs other metabolic process than CHO catabolism can be selected. The incapacity of shrimp to use dietary CHO could limit protein reduction of diets and limit the efforts of the shrimp industry to be ecologically and environmentally profitable.

Considering the results arising from a series of studies, Arena et al*.*, (2003); Pascual et al*.*, (2004 a and b), developed a conceptual model about how dietary components modulate the fate of energy intake, the nutritional status and immune system of juvenile *L. vannamei* (Fig. 6). The environment and genetic variability are the basis of the model, since as mentioned; the interaction between these elements affects the digestive capacity, the flow of energy, protein synthesis, osmoregulation, disease resistance and the degree homeostatic control.

Degradation of starch leads to glucose uptake, it is transported directly to the hemolymph or metabolized through glycolysis (**GL**); subsequently pyruvate through acetyl-coenzyme-A (**A-CoA**) can enter the Krebs cycle (**KC**) and continue with the respiratory chain (**RC**) to obtain energy (**ATP**). The digestive capacity of wild shrimp revealed the importance of taking advantage of dietary CHO, which can be associated with the various points of metabolic regulation of the glycolytic pathway. Nine of the ten essential amino acids can be synthesized from glucose, where glutamate provides the amino group. In the opposite

after being conditioned on diet containing either maintenance or optimal dietary protein levels Pascual et al*.*,( 2006). Juvenile shrimp were fed for 21 days on diets containing 5% and 40% dietary protein. Hemolymph metabolites (glucose, cholesterol, protein, acylglycerols, and lactate), hemocyanin, osmoregulatory capacity, digestive gland glycogen and lipids, and immune conditions (hemocytes concentration, phenoloxidase activity, respiratory burst: basal and activated) were evaluated and considered as initial condition. After that time, shrimp were starved for 21 days. A reduction in all physiological and immunological indicators was observed with starvation. The results demonstrate that shrimp are well adapted to tolerate food deprivation for some time but that this tolerance is closely related to its previous nutritional condition. In the case of shrimp fed 40% DPL, wet weight, nutritional and immune condition was significantly affected after 14 days of starvation. In shrimp previously fed 5% DP, tolerance to starving condition was limited to only a few days (7 days) as a result of low reserves of circulatory and mussel proteins. All these results demonstrate that dietary protein levels can governor the immune condition of shrimp

Domestication is other important aspect to obtain better results during culture; however, artificial selection has important implication on physiological adaptations. Pascual et al., (2004) studied wild and seventh-generation cultivated shrimp to determine how size-based selection could alter the nutritional and immunological conditions of *Litopenaeus vannamei*. Wild juveniles and a sample of seventh-generation cultured shrimp were acclimated under identical conditions. During 55 days, shrimp were fed a high (HCHO: 44%) or a low (LCHO: 3%) carbohydrate diet. Wild shrimp showed a direct relation between dietary CHO and lactate, protein and hemocyte levels indicating that dietary CHO was used for protein synthesis via transamination pathways. In seventh-generation cultured shrimp these parameters were inversely proportional to dietary CHO level, indicating the capacity to synthesize protein from dietary CHO was repressed in cultured shrimp. Farmed shrimp showed a limited capacity to respond to LCHO diets demonstrating high protein dependence in their metabolism and immune response. These results demonstrate that during size-based breeding programs other metabolic process than CHO catabolism can be selected. The incapacity of shrimp to use dietary CHO could limit protein reduction of diets and limit the efforts of the shrimp industry to be ecologically and environmentally

Considering the results arising from a series of studies, Arena et al*.*, (2003); Pascual et al*.*, (2004 a and b), developed a conceptual model about how dietary components modulate the fate of energy intake, the nutritional status and immune system of juvenile *L. vannamei* (Fig. 6). The environment and genetic variability are the basis of the model, since as mentioned; the interaction between these elements affects the digestive capacity, the flow of energy, protein

Degradation of starch leads to glucose uptake, it is transported directly to the hemolymph or metabolized through glycolysis (**GL**); subsequently pyruvate through acetyl-coenzyme-A (**A-CoA**) can enter the Krebs cycle (**KC**) and continue with the respiratory chain (**RC**) to obtain energy (**ATP**). The digestive capacity of wild shrimp revealed the importance of taking advantage of dietary CHO, which can be associated with the various points of metabolic regulation of the glycolytic pathway. Nine of the ten essential amino acids can be synthesized from glucose, where glutamate provides the amino group. In the opposite

synthesis, osmoregulation, disease resistance and the degree homeostatic control.

through the management reserves metabolism.

profitable.

Fig. 6. Conceptual model: protein metabolism and homeostasis. The scheme includes the main organs and systems of juvenile shrimp: digestive gland, **GD**, gills, **G**; muscle, **M**; cuticle, **C**; nervous system, **SN**, hematopoietic tissue, **HT** and hemolymph, **H**. Genetic variability is the basis of the model as it affects the flow of energy from food chemistry to physiological processes. *Litopenaeus vannamei* juveniles grown in closed loop and sorted by size (F7, F25) have a limited capacity to respond to carbohydrate diets (CHO) demonstrating high protein dependence in their metabolism. The degradation of proteins generates peptides and amino acids (**aa**) that are oxidized or used to synthesize **glycogen**. The **aa** can also be used for the synthesis of digestive and metabolic enzymes, and immune proteins (such as prophenoloxidase, **ProPO**, lectins, **L**, lipoprotein recognition, **BGBP**, clothing protein, **CP**, antimicrobial peptides, **AP**; of regulation of the immune response; pacifastine and α2-macroglobulin, and hemocyanin, **H**, which seems to play a role in the immune response of shrimp, in addition to functioning as a storage protein in the hemolymph. Amino acids can also be transported to the hemolymph to form the pool of free amino acids (**FAA**), which can be used to generate muscle tissue.

direction, for example in conditions of nutritional stress, catabolism of glucogenic amino acids is the basis for the gluconeogenic route for CHO can be used as energy substrate. The cycle of fatty acids (**CAG**) is connected to the glycolytic pathway through **A-CoA**, a high concentration of **ATP** or **NADH** promotes the synthesis of fatty acids from the A-CoA. The ß-oxidation of lipids from food or reserves represents the reverse direction to generate metabolic energy.

Regulation of osmotic pressure is mainly associated with the Na-K ATPase, ion exchange, the catabolism of aa and the activity of glutamate dehydrogenase (**GDH**), which controls

Applied Ecophysiology: An Integrative Form to Know How Culture

0 0.5 1 1.5 2 2.5

O:N

Respiration rate (J\*ind-1\*day-1)

a)

c)

9 12 15 Temperature (ºC)

9 12 15 Temperature (ºC)

ZI ZII ZIII Megalopa

ZI ZII ZIII Megalopa

Protein

Fig. 7. Temperature effect on a) the respiration rate; b) the excretion rate (J\*ind-1\*day-1); c) O:N ratio; d) the cumulative energy losses (J) during larval development of *Lithodes santolla*.

Protein-Lipid Lipid

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08

Excretion rate (J\*ind-1\*day-1)

Cumulative energy losses

d)

(J)

b)

9 12 15 Temperature (ºC)

9 12 15 Temperature (ºC)

ZI ZII ZIII Megalopa

ZI ZII ZIII Megalopa Total

Environment Modulates the Performance of Aquatic Species from an Energetic Point of View 179

Our results showed that the development time fluctuate between 55 and 42 days approximately, at 9 and 15ºC, respectively. The sustained even increasing respiration rate throughout the zoeal stages is interpreted as a confirmation of lecithotrophic development, otherwise a drop in metabolic rate in starved larvae were expected (Fig. 7a). Megalopa showed at lower temperatures a decreasing respiration rate attributed to a behavioral change in the swimming activity, while at higher temperature the metabolic rate increased (Fig. 7a). A similar pattern was observed for the nitrogen excretion rate (Fig. 7b). The atomic proportion of consumed oxygen and excreted nitrogen (O:N) is considered as an estimator of the metabolized substratum; values between 3 and 16 would indicate protein metabolism, an O:N ratio between 50 and 60 corresponds to similar proportion of lipid and protein catabolism and higher values than 60 represents lipid metabolism Mayzaud & Conover, (1988). *L. santolla* larvae reared at 12ºC, in general showed a combined protein and lipid catabolism, but, at 9 and 15ºC the Zoea I showed an evident lipid metabolism (Fig. 7c). Respiration rate, independent to temperature, contributed principally on the energy losses, ranging between 93 and 96% at 9 and 15ºC. Although the shortest development time was at 15ºC, low survival suggests unsuitable rearing conditions. Indeed, the highest energy losses expressed as excretion and respiration rate (Figs. 7 a and b) occurred at 15ºC. Multiplying the energy losses of respiration and excretion rates by the respective developmental time, the cumulative energy losses were estimated. Similar energy losses during Zoea I and II were observed for the different temperatures, while an evident impact of higher temperature on the megalopa stage was observed (Fig 7d). The results suggest that elevated temperature ≥15ºC could generate a mismatch on the larval metabolism of *L. santolla* due the increasing energy losses and the restriction in the limited stored energy in the form of yolk remaining

the addition or removal of ammonia. Glutamate cycle (**GC**) is represented by a circle attached to KC. The amino group can be transferred to glutamine, which is less toxic than NH4, so is involved in nitrogen excretion through the gills. The amino group can also be coupled to a carbohydrate to the formation of glucosamine for chitin formation. Therefore, growth and immunity of juveniles of *L. vannamei* are linked to domestication and protein metabolism.

The hematopoietic tissue and hemocytes are represented in the model under a deep interaction with the GD and the nervous system, since nutritional status and some hormones affect the rate of proliferation of hemocytes and thus the state of the immune response, because in the hemocytes are synthesized many of the immune effectors. On the other hand, the concentration of hemocytes in the epidermis during some stages of moult may be related to the release of glycogen for the synthesis of chitin, which is consistent with the ability of hemocytes to synthesize glycogen and store huge amounts (proportionately greater than content in the digestive gland). Also, the high amount of amino acids within the hemocytes could point to a possible release into the hemolymph associated with osmotic regulation and/ or energy demand Claybrook, (1983). Since the metabolic functions of hemocytes have not been demonstrated, in the model are marked with a dashed arrow.

#### **4.3 Effect of temperature on energy losses during southern king crab larval development**

Temperature is one of the most important environmental factors affecting every aspect of an organism's physiology, from the basic structures of the macromolecules that are responsible for catalysis and information processing to the rates at which chemical reactions occur Hochachka & Somero, (2002). Thus, energy acquisition, losses, allocation to maintenance and growth in any organism is affected by temperature. In heterotrophic organisms, the ingested food provides the energy input which is balanced with the main sources of energy losses i.e. bio deposition or faeces rejection, metabolism quantifiable by the oxygen consumption rate, and in less extent nitrogen excretion. The reminder energy is canalized to growth and/or reproduction. In a lecithotrophic (i.e. food independent) condition, the principal energy losses are related to respiration and nitrogen excretion.

The lithodids, commonly known as king crabs or stone crabs, inhabits high latitude cold waters of both hemispheres. Several species of king crabs represent valuable fisheries but commercial exploitation and environmental factors has dramatically declined the landings. In the southernmost part of South America (Chile and Argentina) *Lithodes santolla* (Molina), the southern king crab, is commercially exploited and severe fishing restrictions are necessary to protect the populations. Both circumstances increased the interest and expectative about the development of cultivation technologies for the southern king crab.

In order to establish larval rearing conditions, ecophysiological studies on *L. santolla* larvae have been carried out to define culture temperature based in the energy losses.

Early life-history stages tolerate low temperatures and their larval development is fully independent of food (lecithotrophic) Anger et al., (2004); Calcagno et al., (2004); Kattner et al., (2003); Lovrich et al., (2003); Thatje et al., (2003).

the addition or removal of ammonia. Glutamate cycle (**GC**) is represented by a circle attached to KC. The amino group can be transferred to glutamine, which is less toxic than NH4, so is involved in nitrogen excretion through the gills. The amino group can also be coupled to a carbohydrate to the formation of glucosamine for chitin formation. Therefore, growth and immunity of juveniles of *L. vannamei* are linked to domestication

The hematopoietic tissue and hemocytes are represented in the model under a deep interaction with the GD and the nervous system, since nutritional status and some hormones affect the rate of proliferation of hemocytes and thus the state of the immune response, because in the hemocytes are synthesized many of the immune effectors. On the other hand, the concentration of hemocytes in the epidermis during some stages of moult may be related to the release of glycogen for the synthesis of chitin, which is consistent with the ability of hemocytes to synthesize glycogen and store huge amounts (proportionately greater than content in the digestive gland). Also, the high amount of amino acids within the hemocytes could point to a possible release into the hemolymph associated with osmotic regulation and/ or energy demand Claybrook, (1983). Since the metabolic functions of hemocytes have not been demonstrated, in the model are marked

**4.3 Effect of temperature on energy losses during southern king crab larval** 

principal energy losses are related to respiration and nitrogen excretion.

Temperature is one of the most important environmental factors affecting every aspect of an organism's physiology, from the basic structures of the macromolecules that are responsible for catalysis and information processing to the rates at which chemical reactions occur Hochachka & Somero, (2002). Thus, energy acquisition, losses, allocation to maintenance and growth in any organism is affected by temperature. In heterotrophic organisms, the ingested food provides the energy input which is balanced with the main sources of energy losses i.e. bio deposition or faeces rejection, metabolism quantifiable by the oxygen consumption rate, and in less extent nitrogen excretion. The reminder energy is canalized to growth and/or reproduction. In a lecithotrophic (i.e. food independent) condition, the

The lithodids, commonly known as king crabs or stone crabs, inhabits high latitude cold waters of both hemispheres. Several species of king crabs represent valuable fisheries but commercial exploitation and environmental factors has dramatically declined the landings. In the southernmost part of South America (Chile and Argentina) *Lithodes santolla* (Molina), the southern king crab, is commercially exploited and severe fishing restrictions are necessary to protect the populations. Both circumstances increased the interest and expectative about the development of cultivation technologies for the

In order to establish larval rearing conditions, ecophysiological studies on *L. santolla* larvae

Early life-history stages tolerate low temperatures and their larval development is fully independent of food (lecithotrophic) Anger et al., (2004); Calcagno et al., (2004); Kattner et

have been carried out to define culture temperature based in the energy losses.

al., (2003); Lovrich et al., (2003); Thatje et al., (2003).

and protein metabolism.

with a dashed arrow.

southern king crab.

**development** 

Our results showed that the development time fluctuate between 55 and 42 days approximately, at 9 and 15ºC, respectively. The sustained even increasing respiration rate throughout the zoeal stages is interpreted as a confirmation of lecithotrophic development, otherwise a drop in metabolic rate in starved larvae were expected (Fig. 7a). Megalopa showed at lower temperatures a decreasing respiration rate attributed to a behavioral change in the swimming activity, while at higher temperature the metabolic rate increased (Fig. 7a). A similar pattern was observed for the nitrogen excretion rate (Fig. 7b). The atomic proportion of consumed oxygen and excreted nitrogen (O:N) is considered as an estimator of the metabolized substratum; values between 3 and 16 would indicate protein metabolism, an O:N ratio between 50 and 60 corresponds to similar proportion of lipid and protein catabolism and higher values than 60 represents lipid metabolism Mayzaud & Conover, (1988). *L. santolla* larvae reared at 12ºC, in general showed a combined protein and lipid catabolism, but, at 9 and 15ºC the Zoea I showed an evident lipid metabolism (Fig. 7c). Respiration rate, independent to temperature, contributed principally on the energy losses, ranging between 93 and 96% at 9 and 15ºC. Although the shortest development time was at 15ºC, low survival suggests unsuitable rearing conditions. Indeed, the highest energy losses expressed as excretion and respiration rate (Figs. 7 a and b) occurred at 15ºC. Multiplying the energy losses of respiration and excretion rates by the respective developmental time, the cumulative energy losses were estimated. Similar energy losses during Zoea I and II were observed for the different temperatures, while an evident impact of higher temperature on the megalopa stage was observed (Fig 7d). The results suggest that elevated temperature ≥15ºC could generate a mismatch on the larval metabolism of *L. santolla* due the increasing energy losses and the restriction in the limited stored energy in the form of yolk remaining

Fig. 7. Temperature effect on a) the respiration rate; b) the excretion rate (J\*ind-1\*day-1); c) O:N ratio; d) the cumulative energy losses (J) during larval development of *Lithodes santolla*.

Applied Ecophysiology: An Integrative Form to Know How Culture

Environment Modulates the Performance of Aquatic Species from an Energetic Point of View 181

Fig. 8. Effect of dissolved oxygen (DO) on different physiological responses of *L. santolla*  juveniles. Pc: critical point of aerobic metabolism shift; LDH: Lactate dehydrogenase; Prot: protein concentration; Oxy-Hc: oxygenated hemocyanin; Glyc: glycogen; Chol: cholesterol;

concentration. These responses to reduced DO were complemented with anaerobic metabolism evidenced by the increase in the activity of gill-LDH and a consequent increase of Lactate concentration. A reduction in nutritional compounds in the hepatopancreas was interpreted as a consequence of a notorious reduction of food ingestion, and as an additional consequence of hypoxic conditions (the ingestion was affected severely and showed a linear relationship to dissolved oxygen (DO) between 2 and 13 kPa: 0.59 and 1.92 mg Dry weight h-1 ind-1, respectively). Severe chronic hypoxia (1 mgO2\*l-1 or 2 kPa for 10 days) led to an unsustainable reduction in the aerobic and anaerobic activity, where a reduction of the gill-

AG: acylglycerids; Gluc: glucose.

from the embryonic development. For cultivation purposes physiological data suggest rearing temperatures below 15°C and special caution to reducing larval stress and wasteful energy losses.

After the increase in the atmospheric oxygen availability 2000 million of years ago, most animals were selected for a more efficient energy acquiring mechanism using oxygen as the last electron acceptor. While anaerobic pathways persist, aerobic ATP yield increase as much as 15 to 18-folds (depending on the metabolic pathway) the efficiency in the process of gaining energy Wieser, (1986). Hence, oxygen has a main role in obtaining energy for the cells, therefore for the organism. Oxygen decrease (hypoxia, both environmental and cellular; hypoxic generally refers to low oxygen conditions that are physiologically stressful, Levin, (2003)) has many effects in different levels, from population to individual and cellular, affecting the capability to obtain energy, which affects also the organism's activity and their growth performances. One of the most important mechanisms to deal with hypoxia is to improve the use of energy available, by means of reducing the metabolic rate. In fact, organisms consider well adapted to hypoxia events show a lower ATP consumption, to balance the reduction in the capability to generate ATP. During hypoxia, the ion pump slightly decreases, but the most important is the low in protein synthesis and degradation Hochachka & Somero, (2002). The reduction in protein synthesis result in an efficient energy saving mechanism and, at the same time, an effective mechanism to face hypoxic events, but for aquaculture purposes, such mechanisms activated by low dissolved oxygen (DO) results in a drop in the ingestion rate, animal activity and finally a marked reduction or even ceasing in growth. In marine animals, adaptation to low oxygen availability includes also modifying the acid-base balance in the hemolymph Martinez et al., (1998), hemocyanin binding capacity, oxyhemocyanin protein relationship, hemolymph osmolality, and ion concentrations as well as hemocyanin synthesis Johnson & Uglow, (1985); Charmantier et al., 1994; Chen & Kou, (1998); Taylor & Anstiss, (1999) Environmental parameters like high pH and low dissolved oxygen (DO) have been reported to cause reduction in hemocyte counts Cheng & Chen, (2000).

#### **4.4 Dissolved oxygen as key factor in the bioenergetics of southern king crab juveniles**

Crustacean aquaculture is widespread in tropical areas where higher water temperatures reduces oxygen solubility and accelerates the decomposition of organic material by bacteria and other oxygen consuming microorganisms both conducting to a decreasing in DO levels Rosas et al., (1998, 1999). For cold water organisms the relationship between DO and temperature described by Pörtner (2005) by the Metabolic Cold Adaptation hypothesis implies that the thermal tolerance of the animal is affected by the supply of oxygen to their mitochondria.

In an attempt to establishing the impact of culture environmental factors on the physiology of southern king crab *Lithodes santolla* with a final goal to develop cultivation techniques, the effect of dissolved oxygen (DO) on different physiological responses of *L. santolla* juveniles is analysed. Paschke et al. (2010) showed physiological responses in juveniles of *L. santolla*  exposed 10 days to reduced Oxygen availability treatments (Fig. 8).

The juveniles regulate their metabolism until 4-9 kPa (ca. 4-6 mg O2\*l-1), maintaining their oxygen consumption rates, hemolymphatic protein levels, as well as the oxyhemocyanin

from the embryonic development. For cultivation purposes physiological data suggest rearing temperatures below 15°C and special caution to reducing larval stress and wasteful

After the increase in the atmospheric oxygen availability 2000 million of years ago, most animals were selected for a more efficient energy acquiring mechanism using oxygen as the last electron acceptor. While anaerobic pathways persist, aerobic ATP yield increase as much as 15 to 18-folds (depending on the metabolic pathway) the efficiency in the process of gaining energy Wieser, (1986). Hence, oxygen has a main role in obtaining energy for the cells, therefore for the organism. Oxygen decrease (hypoxia, both environmental and cellular; hypoxic generally refers to low oxygen conditions that are physiologically stressful, Levin, (2003)) has many effects in different levels, from population to individual and cellular, affecting the capability to obtain energy, which affects also the organism's activity and their growth performances. One of the most important mechanisms to deal with hypoxia is to improve the use of energy available, by means of reducing the metabolic rate. In fact, organisms consider well adapted to hypoxia events show a lower ATP consumption, to balance the reduction in the capability to generate ATP. During hypoxia, the ion pump slightly decreases, but the most important is the low in protein synthesis and degradation Hochachka & Somero, (2002). The reduction in protein synthesis result in an efficient energy saving mechanism and, at the same time, an effective mechanism to face hypoxic events, but for aquaculture purposes, such mechanisms activated by low dissolved oxygen (DO) results in a drop in the ingestion rate, animal activity and finally a marked reduction or even ceasing in growth. In marine animals, adaptation to low oxygen availability includes also modifying the acid-base balance in the hemolymph Martinez et al., (1998), hemocyanin binding capacity, oxyhemocyanin protein relationship, hemolymph osmolality, and ion concentrations as well as hemocyanin synthesis Johnson & Uglow, (1985); Charmantier et al., 1994; Chen & Kou, (1998); Taylor & Anstiss, (1999) Environmental parameters like high pH and low dissolved oxygen (DO) have been reported to cause reduction in hemocyte counts Cheng & Chen, (2000).

**4.4 Dissolved oxygen as key factor in the bioenergetics of southern king crab** 

Crustacean aquaculture is widespread in tropical areas where higher water temperatures reduces oxygen solubility and accelerates the decomposition of organic material by bacteria and other oxygen consuming microorganisms both conducting to a decreasing in DO levels Rosas et al., (1998, 1999). For cold water organisms the relationship between DO and temperature described by Pörtner (2005) by the Metabolic Cold Adaptation hypothesis implies that the thermal tolerance of the animal is affected by the supply of oxygen to their

In an attempt to establishing the impact of culture environmental factors on the physiology of southern king crab *Lithodes santolla* with a final goal to develop cultivation techniques, the effect of dissolved oxygen (DO) on different physiological responses of *L. santolla* juveniles is analysed. Paschke et al. (2010) showed physiological responses in juveniles of *L. santolla* 

The juveniles regulate their metabolism until 4-9 kPa (ca. 4-6 mg O2\*l-1), maintaining their oxygen consumption rates, hemolymphatic protein levels, as well as the oxyhemocyanin

exposed 10 days to reduced Oxygen availability treatments (Fig. 8).

energy losses.

**juveniles** 

mitochondria.

Fig. 8. Effect of dissolved oxygen (DO) on different physiological responses of *L. santolla*  juveniles. Pc: critical point of aerobic metabolism shift; LDH: Lactate dehydrogenase; Prot: protein concentration; Oxy-Hc: oxygenated hemocyanin; Glyc: glycogen; Chol: cholesterol; AG: acylglycerids; Gluc: glucose.

concentration. These responses to reduced DO were complemented with anaerobic metabolism evidenced by the increase in the activity of gill-LDH and a consequent increase of Lactate concentration. A reduction in nutritional compounds in the hepatopancreas was interpreted as a consequence of a notorious reduction of food ingestion, and as an additional consequence of hypoxic conditions (the ingestion was affected severely and showed a linear relationship to dissolved oxygen (DO) between 2 and 13 kPa: 0.59 and 1.92 mg Dry weight h-1 ind-1, respectively). Severe chronic hypoxia (1 mgO2\*l-1 or 2 kPa for 10 days) led to an unsustainable reduction in the aerobic and anaerobic activity, where a reduction of the gill-

Applied Ecophysiology: An Integrative Form to Know How Culture

and *p* values that cannot be used for prediction purposes.

over time.

Environment Modulates the Performance of Aquatic Species from an Energetic Point of View 183

requirement of regression models, namely that each data point be independent from others Zar, (1999); Zuur et al*.*, (2007). Finally, size-at-age variation generally increases with age, resulting in strong heterogeneity of variance in different ages, another impediment for ordinary regression analysis Zuur et al*.*, (2007). These characteristics violate important regression assumptions, thus producing unreliable models, i.e. models with dubious *F*, *X2*

A methodological approach is fitting a regression model to the weight of each individual at a known fixed time (*t*2) against its weight at an earlier point in time (*t*1). This method ensures linearity of the *X–Y* function and independence of data points (as long as they are individually labelled). The resulting linear equation describes a type of relative growth, and its slope represents the proportionality of the difference between two individuals at *t*<sup>2</sup> relative to the difference between them at *t*1. In turn, the line's intercept represents the final weight reached by the smallest individual in the dataset. In this context, comparing the slopes of different lines gives information on how much inter-individual weight differences change over experimental time. Concomitantly, comparing different line intercepts informs on individual growth rates: lines with different intercepts indicate different growth rates, because two animals with the same initial weight reach different final weights within the same period. Although this approach allows indirect corroboration of whether individuals with different final size have different growth rates, it does not permit estimation (mean value ± standard error) of those parameters in the equation that describe individual growth

Generalized linear mixed models (GLMM) are a statistical tool that can complement growth analyses, because they allow modelling of the large variability in individual size observed within a culture population. GLMM can be applied to non-normal data in which random effects are present (Zuur et al., 2009). By incorporating components that modify the structure of variance, mixed models yield more-reliable estimators of model coefficients. In addition, through certain variance and correlation structures, mixed models may produce new parameters that estimate size-at-age variability and the time elapsed for two size measures to be statistically unrelated. GLMM procedures include the validation of models by visual inspection of residuals Montgomery & Peck, (1992); Draper & Smith, (1998. ),

Cephalopod growth has some remarkable characteristics: (i) growth rates are among the highest in metazoans (the highest in invertebrate metazoans, higher than those of fish and similar to those of mammals) Calow, (1987); (ii) it lacks an asymptotic growth phase Moltschaniwskyj, (2004); (iii) it is highly plastic owing to its strong dependence on abiotic and biotic factors, mainly temperature Pecl et al*.*, (2004), the amount and quality of food André et al*.*, (2008), and sexual maturation Semmens et al., (2004); (iv) it follows a biphasic pattern (as it often does in captivity), consisting of an initial rapid exponential phase followed by a second phase, where growth slows down progressively (André et al*.*, 2009);

and (v) it is highly variable intra-specifically Pecl et al., (2004); Leporati et al., (2007).

Size-at-age variability has been attributed to the lack of a strong association between age and size of these soft bodied invertebrates, and is well documented both in wild and in culture populations Leporati et al*.*, (2007); Leporati et al*.*, (2008). This great variability, patent even under controlled temperature and food conditions, has been associated with

thereby assuring that regression assumptions are adequately met.

LDH activity and Lactate concentration as well as oxygen consumption coincide with a significant reduction in hemolymphatic hemocytes and finally a survival reduction to 70%.

In accordance with these results and applying Ecophysiology to the development of cultivation techniques, the rearing systems were modified to ensure the adequate Oxygen supply in the first millimeter over the bottom surface, the place where juveniles of the southern king crab *Lithodes santolla* feed, moult and growth.

#### **5. Modelling individual growth: Size-at-age variability in cephalopods and mixed models tools**

Both individual and population growth is central to aquaculture practice and research, and modelling growth has been an important aim in methodological and theoretical studies Moltschaniwskyj, (2004); Vigliola & Meekan, (2009). Models have been extensively developed and applied to understand population growth (e.g. Malthussian models, logistic models, Leslie matrix, etc.), and those for individual growth have followed closely (von Bertalanffy model). Methods for measuring and modelling individual growth can be classified as either indirect or direct Semmens et al*.*,(2004). Indirect methods are those using modal progression analysis based on length-frequency data obtained from animals caught in the wild. These have been used to predict length-at-age in individuals of a variety of both vertebrate and invertebrate fisheries Solis-Ramírez, (1997); Cortez et al*.*, (1999) Direct methods, such as mark and recapture using a variety of tags, have been used to follow the growth of juvenile and adult stages in the wild Sousa Reis & Fernándes, (2002); Cortez et al*.*, (1999).

From an ecophysiological point of view, however, more useful are direct methods examining the growth of individuals whose age and specific culture conditions are known. The growth of an individual is the result of a series of energy transformations that begin with food ingested, together with the balance between the uses and destinations of the energy contained in that food Lucas, (1993); Rosas et al*.*, (2007). Thus, the growth curve represents the manner in which the physiological and energy demands are expressed over time O'Dor & Wellls, (1987); Pauly, (1998) at differing levels of biological organization, i.e. body size, organs, tissue, and cells Moltschaniwskyj, (2004). In the context of energy balance, the ultimate need to measure and compare the way in which individuals grow becomes clear: individual growth models can be used not only to predict size at age, hence biomass productions at certain points in time, but can also serve to estimate growth curve parameters that can later be compared amongst different culture conditions.

The mathematical tool generally used to obtain growth curve parameters is least square fitting, which can be used both for linear and non-linear models. The linear least-squares problem occurs in statistical regression analysis where it has a closed-form solution. The non-linear problem has no closed-form solution and is usually solved by iterative refinement, where chi-square (*X2*) and probability (p) values are obtained and evaluated at each iteration.

Analyses of individual growth have commonly involved fitting linear regression models of weight against time. However, such an approach involves difficulties because the weight vs. time relationship is rarely linear and, when it is, it is only for very short and specific periods. Moreover, repeated measurement of the same individuals violates an indispensible

LDH activity and Lactate concentration as well as oxygen consumption coincide with a significant reduction in hemolymphatic hemocytes and finally a survival reduction to 70%. In accordance with these results and applying Ecophysiology to the development of cultivation techniques, the rearing systems were modified to ensure the adequate Oxygen supply in the first millimeter over the bottom surface, the place where juveniles of the

**5. Modelling individual growth: Size-at-age variability in cephalopods and** 

Both individual and population growth is central to aquaculture practice and research, and modelling growth has been an important aim in methodological and theoretical studies Moltschaniwskyj, (2004); Vigliola & Meekan, (2009). Models have been extensively developed and applied to understand population growth (e.g. Malthussian models, logistic models, Leslie matrix, etc.), and those for individual growth have followed closely (von Bertalanffy model). Methods for measuring and modelling individual growth can be classified as either indirect or direct Semmens et al*.*,(2004). Indirect methods are those using modal progression analysis based on length-frequency data obtained from animals caught in the wild. These have been used to predict length-at-age in individuals of a variety of both vertebrate and invertebrate fisheries Solis-Ramírez, (1997); Cortez et al*.*, (1999) Direct methods, such as mark and recapture using a variety of tags, have been used to follow the growth of juvenile and adult stages in the wild Sousa Reis & Fernándes, (2002); Cortez et al*.*,

From an ecophysiological point of view, however, more useful are direct methods examining the growth of individuals whose age and specific culture conditions are known. The growth of an individual is the result of a series of energy transformations that begin with food ingested, together with the balance between the uses and destinations of the energy contained in that food Lucas, (1993); Rosas et al*.*, (2007). Thus, the growth curve represents the manner in which the physiological and energy demands are expressed over time O'Dor & Wellls, (1987); Pauly, (1998) at differing levels of biological organization, i.e. body size, organs, tissue, and cells Moltschaniwskyj, (2004). In the context of energy balance, the ultimate need to measure and compare the way in which individuals grow becomes clear: individual growth models can be used not only to predict size at age, hence biomass productions at certain points in time, but can also serve to estimate growth curve

The mathematical tool generally used to obtain growth curve parameters is least square fitting, which can be used both for linear and non-linear models. The linear least-squares problem occurs in statistical regression analysis where it has a closed-form solution. The non-linear problem has no closed-form solution and is usually solved by iterative refinement, where chi-square (*X2*) and probability (p) values are obtained and evaluated at

Analyses of individual growth have commonly involved fitting linear regression models of weight against time. However, such an approach involves difficulties because the weight vs. time relationship is rarely linear and, when it is, it is only for very short and specific periods. Moreover, repeated measurement of the same individuals violates an indispensible

parameters that can later be compared amongst different culture conditions.

southern king crab *Lithodes santolla* feed, moult and growth.

**mixed models tools** 

(1999).

each iteration.

requirement of regression models, namely that each data point be independent from others Zar, (1999); Zuur et al*.*, (2007). Finally, size-at-age variation generally increases with age, resulting in strong heterogeneity of variance in different ages, another impediment for ordinary regression analysis Zuur et al*.*, (2007). These characteristics violate important regression assumptions, thus producing unreliable models, i.e. models with dubious *F*, *X2* and *p* values that cannot be used for prediction purposes.

A methodological approach is fitting a regression model to the weight of each individual at a known fixed time (*t*2) against its weight at an earlier point in time (*t*1). This method ensures linearity of the *X–Y* function and independence of data points (as long as they are individually labelled). The resulting linear equation describes a type of relative growth, and its slope represents the proportionality of the difference between two individuals at *t*<sup>2</sup> relative to the difference between them at *t*1. In turn, the line's intercept represents the final weight reached by the smallest individual in the dataset. In this context, comparing the slopes of different lines gives information on how much inter-individual weight differences change over experimental time. Concomitantly, comparing different line intercepts informs on individual growth rates: lines with different intercepts indicate different growth rates, because two animals with the same initial weight reach different final weights within the same period. Although this approach allows indirect corroboration of whether individuals with different final size have different growth rates, it does not permit estimation (mean value ± standard error) of those parameters in the equation that describe individual growth over time.

Generalized linear mixed models (GLMM) are a statistical tool that can complement growth analyses, because they allow modelling of the large variability in individual size observed within a culture population. GLMM can be applied to non-normal data in which random effects are present (Zuur et al., 2009). By incorporating components that modify the structure of variance, mixed models yield more-reliable estimators of model coefficients. In addition, through certain variance and correlation structures, mixed models may produce new parameters that estimate size-at-age variability and the time elapsed for two size measures to be statistically unrelated. GLMM procedures include the validation of models by visual inspection of residuals Montgomery & Peck, (1992); Draper & Smith, (1998. ), thereby assuring that regression assumptions are adequately met.

Cephalopod growth has some remarkable characteristics: (i) growth rates are among the highest in metazoans (the highest in invertebrate metazoans, higher than those of fish and similar to those of mammals) Calow, (1987); (ii) it lacks an asymptotic growth phase Moltschaniwskyj, (2004); (iii) it is highly plastic owing to its strong dependence on abiotic and biotic factors, mainly temperature Pecl et al*.*, (2004), the amount and quality of food André et al*.*, (2008), and sexual maturation Semmens et al., (2004); (iv) it follows a biphasic pattern (as it often does in captivity), consisting of an initial rapid exponential phase followed by a second phase, where growth slows down progressively (André et al*.*, 2009); and (v) it is highly variable intra-specifically Pecl et al., (2004); Leporati et al., (2007).

Size-at-age variability has been attributed to the lack of a strong association between age and size of these soft bodied invertebrates, and is well documented both in wild and in culture populations Leporati et al*.*, (2007); Leporati et al*.*, (2008). This great variability, patent even under controlled temperature and food conditions, has been associated with

Applied Ecophysiology: An Integrative Form to Know How Culture

σ 0.209

AIC 251.4

was estimated to be 0.20 (Table 5).

transformed values; b *p* < 0.001.

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

predicted values, respectively.

W (g)

Environment Modulates the Performance of Aquatic Species from an Energetic Point of View 185

and (ii) it made the cyclic residual pattern disappear. The estimated range parameter associated with this correlation structure was 74.3 (Table 5), representing the interval (in days) necessary to avoid correlation between two consecutive weight measurements of the same animal. Heterogeneity of variances was identified based on an increase in weight variation with age and was accounted for by including a variance structure of the type:

**εi ~ N(0, σ2 • |agei|2δ)**  Including this term significantly improved the model (L-ratio = 70.00; *p* < 0.001; AIC = 251.4). Finally, visual inspection of residuals revealed uniform dispersion and no evident patterns, and σ (a constant representing the increase in residual weight variation with age)

**Parameter Value Significance test**  α -2.182 ± 0.22 a t = -100.92 b β 0.030 ± 0.001 t = 48.60 b

δ 0.204 L.ratio = 70.00 b Range 74.3 L.ratio = 114.20 b

Table 5. Parameters of the exponential growth model: α = intercept; β = slope; σ = residual standard error; δ = variance structure parameter; range: correlation structure parameter defined by the variogram. Significance values of statistics used are also shown; a log-

W = 0.013 *e* 0.03 (age)

0 15 30 45 60 75 90 105 Age (d)

Fig. 9. Exponential relationship of weight and age (to age 105 d) in individually housed juvenile *Octopus maya* kept at 27 ± 1°C. Data points and continuous line are observed and

initial size Leporati et al., (2007), which in turn is highly variable both within and between broods. High coefficients of variation (>20%) in initial weight have been observed in several octopod species (*Octopus bimaculoides*, Forsythe & Van Heukelem, (1987); *Octopus diguetti*, DeRusha et al*.*, (1989); *Octopus ocellatus*, Segawa & Nomoto, (2002). Intra-brood variation, however, could not be related to either the mother´s weight or day of hatching Briceño-Jacques et al*.*, (2010). Results suggest that growth rate does not depend on initial size in *O. maya* Briceño et al., (2010), but small differences in size during early life can be amplified and accumulated in time Vigliola & Meekan, (2009). This means that inter-individual variation in initial size has to be considered in experimental designs and data analyses aimed at understanding cephalopod growth.

*Octopus maya* is endemic to the Yucatan Peninsula Voss & Solis Ramirez, (1966), and its culture has received considerable attention Martinez et al*.*, (2011); Uriarte et al*.*, (2011). This species provides an interesting biological model to test hypotheses on heterogeneity in growth amongst cultured siblings, and serves to illustrate problems that arise in growth analysis and possible solutions to them.

Juveniles of *O. maya* from a single female were used. The eggs were held at 28°C (± 1°C) in an artificial incubator (without maternal care) until hatching. The weight of a total of 84 juveniles, that hatched over the course of 8 d, was recorded 24 h after hatching (W1 ± 0.01g), and then again on days 15, 45, 75 and 105. Octopuses were housed individually in 300 ml containers connected to a recirculation system in which water temperature was kept constant at 27 ± 1 oC. Octopuses were fed live adult brine shrimp (*Artemia salina*) and pieces of blue crab (*Callinectes sapidus*) meat *ad libitum*. Overall mortality (at day 105) was 60.7%.

To obtain a curve of weight gain as a function of time, a linear model was adjusted to the relationship between the natural logarithm of W1, W15, W45, W75, and W105 weights of all individuals and their corresponding ages:

$$\mathbf{\color{red}{Y\_{ij}}} = \mathbf{\color{red}{a}} + \mathbf{\color{red}{p\_l}} \,\mathbf{\color{red}{X\_i}} + \mathbf{\color{red}{e\_{ij}}}$$

Previous regression analysis, graphic representations of the data were explored to (i) identify extreme points (point graphs); (ii) assess normality (histograms and percentile graphs); and (iii) verify linear relationships *X–Y* graphs; Zuur et al*.*, (2007). The regression was fitted with a generalized least square (GLS) procedure through restricted maximum likelihood (REML) and incorporated correlation and variance structures, using GLMM to ensure that homocedasticity and independence requisites were met (Fig. 9). Models featuring optimal correlation and variance structures were selected by considering values of the Akaike information criterion (AIC) and hypothesis tests based on *F* and likelihood ratio (L ratio) values. Once the significance of regression parameters were established through *F*, L ratio and *t*-tests, the model was validated by visual inspection of residuals Montgomery & Peck, (1992). We used the parameter δ as an estimator of the tendency of weight variances to increase with age.

Regression parameters differed significantly from 0 (Table 5). Interdependence of data over time (because we weighed animals repeatedly) resulted in a cyclic residual pattern, so we incorporated an autocorrelation structure (spherical spatial structure) in the random-effects term of the model (εi). Following Pinheiro & Bates, (2000), we kept this structure in the model, because (i) AIC values indicated that using it improved the model (AIC = 319.45),

initial size Leporati et al., (2007), which in turn is highly variable both within and between broods. High coefficients of variation (>20%) in initial weight have been observed in several octopod species (*Octopus bimaculoides*, Forsythe & Van Heukelem, (1987); *Octopus diguetti*, DeRusha et al*.*, (1989); *Octopus ocellatus*, Segawa & Nomoto, (2002). Intra-brood variation, however, could not be related to either the mother´s weight or day of hatching Briceño-Jacques et al*.*, (2010). Results suggest that growth rate does not depend on initial size in *O. maya* Briceño et al., (2010), but small differences in size during early life can be amplified and accumulated in time Vigliola & Meekan, (2009). This means that inter-individual variation in initial size has to be considered in experimental designs and data analyses

*Octopus maya* is endemic to the Yucatan Peninsula Voss & Solis Ramirez, (1966), and its culture has received considerable attention Martinez et al*.*, (2011); Uriarte et al*.*, (2011). This species provides an interesting biological model to test hypotheses on heterogeneity in growth amongst cultured siblings, and serves to illustrate problems that arise in growth

Juveniles of *O. maya* from a single female were used. The eggs were held at 28°C (± 1°C) in an artificial incubator (without maternal care) until hatching. The weight of a total of 84 juveniles, that hatched over the course of 8 d, was recorded 24 h after hatching (W1 ± 0.01g), and then again on days 15, 45, 75 and 105. Octopuses were housed individually in 300 ml containers connected to a recirculation system in which water temperature was kept constant at 27 ± 1 oC. Octopuses were fed live adult brine shrimp (*Artemia salina*) and pieces of blue crab (*Callinectes sapidus*) meat *ad libitum*. Overall mortality (at day 105) was 60.7%.

To obtain a curve of weight gain as a function of time, a linear model was adjusted to the relationship between the natural logarithm of W1, W15, W45, W75, and W105 weights of all

**Yij = α + β1 Xi + eij** Previous regression analysis, graphic representations of the data were explored to (i) identify extreme points (point graphs); (ii) assess normality (histograms and percentile graphs); and (iii) verify linear relationships *X–Y* graphs; Zuur et al*.*, (2007). The regression was fitted with a generalized least square (GLS) procedure through restricted maximum likelihood (REML) and incorporated correlation and variance structures, using GLMM to ensure that homocedasticity and independence requisites were met (Fig. 9). Models featuring optimal correlation and variance structures were selected by considering values of the Akaike information criterion (AIC) and hypothesis tests based on *F* and likelihood ratio (L ratio) values. Once the significance of regression parameters were established through *F*, L ratio and *t*-tests, the model was validated by visual inspection of residuals Montgomery &

Regression parameters differed significantly from 0 (Table 5). Interdependence of data over time (because we weighed animals repeatedly) resulted in a cyclic residual pattern, so we incorporated an autocorrelation structure (spherical spatial structure) in the random-effects term of the model (εi). Following Pinheiro & Bates, (2000), we kept this structure in the model, because (i) AIC values indicated that using it improved the model (AIC = 319.45),

as an estimator of the tendency of weight variances to

δ

aimed at understanding cephalopod growth.

analysis and possible solutions to them.

individuals and their corresponding ages:

Peck, (1992). We used the parameter

increase with age.

and (ii) it made the cyclic residual pattern disappear. The estimated range parameter associated with this correlation structure was 74.3 (Table 5), representing the interval (in days) necessary to avoid correlation between two consecutive weight measurements of the same animal. Heterogeneity of variances was identified based on an increase in weight variation with age and was accounted for by including a variance structure of the type:

$$
\mathfrak{e}\_{\mathbf{i}} \sim \mathbf{N}(\mathbf{0}, \sigma^2 \mathbf{\bullet} \mid \mathbf{a} \mathbf{e}\_{\mathbf{i}} \mid \mathbf{a} \mathbf{e}\_{\mathbf{i}} \mid \mathbf{a})
$$

Including this term significantly improved the model (L-ratio = 70.00; *p* < 0.001; AIC = 251.4). Finally, visual inspection of residuals revealed uniform dispersion and no evident patterns, and σ (a constant representing the increase in residual weight variation with age) was estimated to be 0.20 (Table 5).


Table 5. Parameters of the exponential growth model: α = intercept; β = slope; σ = residual standard error; δ = variance structure parameter; range: correlation structure parameter defined by the variogram. Significance values of statistics used are also shown; a logtransformed values; b *p* < 0.001.

Fig. 9. Exponential relationship of weight and age (to age 105 d) in individually housed juvenile *Octopus maya* kept at 27 ± 1°C. Data points and continuous line are observed and predicted values, respectively.

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The GLMM model included the use of a variance and a correlation structure that enhanced the model fit (the best AIC value was obtained: 251.4), and successfully explained octopus weight as a function of age. Because the date of hatching was registered for each individual, "age" is fixed in the model (i.e. it has no associated error), a feature in models fitted by least squares that is not usually met. According to this model, *O. maya* juveniles grow at a rate of 0.03 g/day, a rate similar to that obtained for other *O. maya* cultures (unpublished data). The δ parameter estimated in 0.204 is the exponent Given the correlation and variance structures included in the current model, and the parameters associated with them (*δ*, range), the procedures followed to adjust the GLMM model detailed here allow a high level of precision in predicting octopus weight from known age, thus, making the estimates of growth rate more reliable than other growth models.

#### **6. Conclusions**

During aquaculture technological development of species it is common that farmers or researchers start the culture only putting some animals in ponds, floating cages, or any other closed environment giving attention on productive parameters such as growth and survival. It is also common that, at the beginning of culture, animals are feed with the local available food in attempt to evaluate if in such conditions animals can be raised. However, this approach does not allow getting enough information for a sustainable development of culturing technology. In this sense, ecophysiology can help to evaluate how environmental conditions modulate several responses of cultured organisms, including the scope for growth, one of the key parameters involved in the energy balance equation. This model could be used as a base to make economical inferences or to modify culture methods. As showed in the present work, independently of the cultured species, the bulk of papers dedicated to demonstrate the effects of many environmental factors on physiological condition of animals validate applied ecophysiology as a useful method for the development and improvement of cultivation technology of aquatic organisms.

#### **7. Acknowledgements**

The present chapter is the result of many research made with the financial support of FONDEF D02i1163, D05i10217, Fondecyt 1110637, FONDEF D09I1153 in Chile and CONACYT through their financial support to make the sabbatical year of CR and Papiit IN 290327 to CR in Mexico.

#### **8. References**


The GLMM model included the use of a variance and a correlation structure that enhanced the model fit (the best AIC value was obtained: 251.4), and successfully explained octopus weight as a function of age. Because the date of hatching was registered for each individual, "age" is fixed in the model (i.e. it has no associated error), a feature in models fitted by least squares that is not usually met. According to this model, *O. maya* juveniles grow at a rate of 0.03 g/day, a rate similar to that obtained for other *O. maya* cultures (unpublished data). The δ parameter estimated in 0.204 is the exponent Given the correlation and variance structures included in the current model, and the parameters associated with them (*δ*, range), the procedures followed to adjust the GLMM model detailed here allow a high level of precision in predicting octopus weight from known age, thus, making the estimates of

During aquaculture technological development of species it is common that farmers or researchers start the culture only putting some animals in ponds, floating cages, or any other closed environment giving attention on productive parameters such as growth and survival. It is also common that, at the beginning of culture, animals are feed with the local available food in attempt to evaluate if in such conditions animals can be raised. However, this approach does not allow getting enough information for a sustainable development of culturing technology. In this sense, ecophysiology can help to evaluate how environmental conditions modulate several responses of cultured organisms, including the scope for growth, one of the key parameters involved in the energy balance equation. This model could be used as a base to make economical inferences or to modify culture methods. As showed in the present work, independently of the cultured species, the bulk of papers dedicated to demonstrate the effects of many environmental factors on physiological condition of animals validate applied ecophysiology as a useful method for the

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**10** 

*USA* 

**Metals in Crawfish** 

*Lake Charles, Louisiana* 

Joseph Sneddon\* and Joel C. Richert

*Department of Chemistry, McNeese State University,* 

Much of the information in the first nine sections of this chapter was derived and condensed from the Louisiana Crawfish Production Manual (McClain et al, 2007). The harvesting of crawfish (or crayfish) for human consumption has become an important industry in several areas throughout the world. In the United States, crawfish are common in Louisiana (located in the southern United States on the Gulf of Mexico) and throughout the southeastern states. Crawfish (McClain, 2005) are well suited for habitats that have seasonal flooding and drying, especially when the dry periods occur in the summer and fall. Periods of flooding allow the crawfish to feed, grow and mature. Dry periods help aeration of sediments, reduce the abundance of predators and allow for establishment of vegetation, which serves as cover and food resources when flooded. Crawfish are common in parts of northeastern Mexico, can be found extensively in southeastern Asia and are grown commercially in China. In the 1970s, crawfish were introduced into Portugal and Spain and thrived in the rivers and estuaries to the extent that commercial harvesting now contributes significantly to the food supply in these regions (Alcorlo et al, 2006) (Maranhao et al, 1999). In Louisiana there are numerous varieties of crawfish but the two dominant species are the red swamp crawfish (*Procamburas clarkii*) and the white river crawfish (*Procamburas zonangulas*). Both species dominate harvests in natural habitats and on crawfish farms, therefore, unless otherwise

noted these two species are the species of crawfish to which the authors refer.

In Louisiana, crawfish has been a part of the human diet for centuries but the commercial sales of crawfish began only in the late 1800s. As the demand for crawfish outgrew the harvest from the wild, the crawfish aquaculture industry was born. Early crawfish aquaculture consisted only of re-flooding rice fields after the harvest to produce food for the farm family. Excess production from these fields was sold to nearby consumers. Over time the demand and supply of crawfish continued to grow and by the 1960s commercial processing of crawfish began. The peeling and packaging of crawfish tail meat allowed for the establishment of regional markets. Commercial processing required a steady supply of crawfish to meet demand. This increased demand was greater than could be supplied by harvests in natural habitats and lead to a great increase crawfish aquaculture in Louisiana. In the United States there are few commercial producers of crawfish outside of Louisiana.

**1. Introduction 1.1 Overview** 

**1.2 History** 

