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Crustaceans have aquatic, terrestrial and semi terrestrial representatives and so have had to adapt to the huge physical and chemical diversity of these environments. Brackish, estuarine and intertidal environments are probably among the most stressful aquatic habitats, and the establishment of crustaceans in these environments implies highly adapted physiological features (Péqueux 1995). The stronger the adaptive response, the more likely the detection of stress- induced change in that response (Gould and Greig 1983). The species used in this study are found in a fully marine, sublittoral and therefore normally stable environment, however crustaceans are very sensitive to changes in water quality, chemistry, light and temperature. It is important that these balances are maintained in order for them to survive in the aquarium or outside their natural environment. Each species has its own tolerances to levels of applied stressors such as temperature, oxygen saturation, pH and toxins. Cancer pagurus and Carcinus maenas have a much higher tolerance to these than Necora puber (Bernasconi 2006). Environmental stressors of this nature may rapidly become lethal to some species and can negatively affect their quality and hence the market price. Intertidal species generally have a greater range of environmental tolerances than subtidal species. When an environmental stressor such as salinity moves outside the limits of homeostasis the organism adapts its metabolism to cope and this increase or decrease in metabolic processes is what can eventually lead to death, or sublethal impacts decreasing the marketability. Sublethal effects may impact the long term sustainability of populations (and therefore the commercial market) by affecting growth and reproduction.

Increases in oxygen consumption as salinity is reduced (or increased maybe) may be due to increased energy needed to actively pump ions needed to maintain homeostasis. It is uncertain whether animals with higher respiration rates would be able to survive in these unfavourable conditions for any length of time and may need to migrate into areas of more ambient salinity to survive. This will be one of the factors addressed in this thesis.

1.6.1 Cardioventilatory activity

Blood circulation in decapod crustaceans is achieved by rhythmic contractions of a single- chambered, neurogenic heart. Heart rates between 15-150 beats per minute (bpm) are not unusual in individuals however rates are normally at levels between these extremes (Cumberlidge and

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Uglow 1977a). Heart rate is typically elevated in response to physical and environmental stress and this ability to increase respiration rate is probably a factor in the ability of crustaceans to escape from predators or unfavourable conditions. Variability in cardiac activity has been shown to be an indicator of the Darwinian fitness of decapods (Depledge and Lundebye 1996), and therefore their physical ability to cope with changing environmental gradients and high variability in any measured behaviour or parameter may itself be an indicator of stress as animals do not necessarily respond in a uniform way to environmental changes.

Branchial irrigation in decapod crustaceans is maintained by the pumping action of the scaphognathites. The scaphognathites are enlarged blade shaped exopodites of the second maxillae, one in each of the left and right branchial chambers. They are capable of both synchronous and independent activity (Cumberlidge and Uglow 1977a). In crustaceans the scaphognathites pump water over the gills in a forward direction in most species including the lobsters, but this is interrupted at irregular intervals by reversed beats which propel water backward over the gills (Wilkens and McMahon 1972). The scaphognathite in Homarus americanus moves as a rigid blade which does not flex but instead effects water propulsion by changing its angle of attack during each half beat (Wilkens and McMahon 1972). Change in the cardioventilatory activity of crustaceans is used as a sublethal indicator of the impact of environmental change (Ansell 1973; Cumberlidge and Uglow 1977a; Walters and Uglow 1981).

1.6.2 Autotomy

This is the ability to regrow lost limbs and is an adaptation that is shared by all the crustaceans included in this study. Under stressful conditions, a limb can be shed and a new one is grown in replacement, e.g. due to injury, or to aid escape from a predator. The limb is lost at a fracture plane at its base which is covered in a thin membrane which has a hole in the centre through which blood passes into the rest of the limb, but on fracture this hole is quickly sealed by a blood clot. In a premoult animal, the new limb grows out of the old stump and is soft and encased in a membrane, only becoming hard when the animal moults. The new limb will eventually attain a size that is slightly smaller than the previous one (Spence 1989). If an animal is already experiencing environmental stress in the form of changes to salinity, pollution etc, it might have an impact on the ability of the animal to complete this process successfully, if the fracture plane is not closed quickly

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pathogens could enter the body. In addition if energy is going into maintaining homeostasis under environmental stress, there will be less available for growth of a new limb at the next moult, if the onset of the moult is not delayed by the stressor. As the main feeding appendages of the lobster are the subchelate walking legs, loss of the crushing and/or cutting claw (which are used mainly for defence and opening hard food) does not mean starvation (Ingram 1985). However in crabs the chelae are used for feeding and loss of one or both can lead to malnutrition and eventually death.

1.6.3 Haemolymph O

affinity and anaerobic metabolism in crustaceans

Hypersaline conditions have already been shown to affect mobility in both H. gammarus and C. pagurus (Macdonald and Elliott 2005). If this reduction of mobility also involves the scaphognathites (gill bailers), it could mean that even in a fully oxygenated media, the crustacean could face an internal hypoxia as it cannot draw enough water across the gills for respiration, thus affecting the acid base balance of the haemolymph. This hypothesis is investigated in Chapter 6.

Crustaceans are ectothermic, gaining most of their heat from their environment. For most ectothermic animals, the colder the environment the less active they are, both in terms of behavioural activity and internal processes such as metabolism and respiration. In crustaceans metabolic rates are determined largely by external temperatures. Within lethal limits the higher the temperature the higher the metabolic rate and the more rapidly animals will become distressed by external stressors such as a low oxygen supply (Wyman et al. 1985), water borne pollutants, or environmental salinity change (i.e. hypersalinity). Stress of this kind often induces anaerobic respiration which results in the production of lactic acid and the build up of lactate in the blood and tissues as a shortage of oxygen means the tissues respire inefficiently. Lactate is a toxic substance which, if high concentrations persist, will ultimately kill the animal, but before that will reduce its survival and its condition so as to make it a less attractive commercial product (Wyman et al. 1985). Therefore the raised temperature of hypersaline discharges is a concern with regards to the metabolic processes of affected species. Lactate can also accumulate under conditions other than aerial exposure, such as during exercise. L-Lactate is the primary metabolic acid product produced

by decapod crustaceans and exercise induced acidosis is caused almost totally by L-Lactate

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1.6.4 Nitrogen metabolism in crustaceans

Ammonia is the primary nitrogenous waste excreted by aquatic crustaceans and is excreted constantly via the gills by a passive diffusion into the water in the branchial chamber. Here the water in is continuously renewed, so protecting the animal from the toxic effects of high ammonia levels (Durand et al. 1999). Other important waste products are urea and uric acid. Some minor excretory products of nitrogen metabolism are guanine, trimethylamine oxide (TMAO), creatine, creatinine and amino acids. Increased ammonia production may indicate a higher rate of protein metabolism (Emerson 1969) and elevated ammonia levels in H. americanus in low salinity media have been shown to disrupt ionoregulatory functions (Young-Lai et al. 1991). So it is important that crustaceans have an effective ammonia excretion or detoxification system in order to iono- osmoregulate efficiently and respond to changing environmental conditions (i.e. salinity).

Quantifying the changes in ammonia in crustaceans in response to hyper/hypo salinity, shows the initiation of changes in the physiology in response to environmental change and is considered as indicative of a sublethal response to stress in aquatic organisms. Therefore assessing ammonia production in crustaceans in response to changing environmental variables (i.e. salinity) may be a way of assessing not only their tolerance levels, but also the degree of stress imposed.

For a comprehensive review of the effects of ammonia on the body (though not just applicable to crustacea) see Wright (1995), but briefly summarised they are:

• Modification of the properties of the blood brain barrier and disruption of cerebral blood flow.

• NH4+ can directly substitute itself for K+ in nerve conduction.

• Interfere with transport of amino acids and impede the process of amino acid excretion • Cause mutations in astrocytes and neurons (nervous system/brain tissues).

• Alter carbohydrate and fat metabolism • Alter ATP levels in the brain and other tissues

Many aquatic species also can be uricotelic (they can produce uric acid as well as ammonia). They can switch to an alternative biochemical pathway so that they produce alternate forms of waste to ammonia in higher amounts. This is an energetically expensive option but other forms of waste

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such as urea and uric acid are less toxic to the organism if they build up in the body (Uglow and Williams 2001).

Hence it is important to establish how nitrogen metabolism, especially ammonia production and excretion is affected by changing environmental salinity in order to determine the potential sublethal impacts that may occur in salinity stressed species.

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