Romano, Nicholas (2010) Investigating the survival, growth and osmoregulatory responses of blue swimmer crab, Portunus pelagicus, early juveniles to salinity and ammonia challenge. PhD thesis, James Cook University.

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Abstract

The blue swimmer crab, Portunus pelagicus, is a portunid crab native throughout the Indo-Pacific region and their harvests support important commercial fisheries as well as being an emerging aquaculture species. However, basic information on this species relevant to fisheries management and aquaculture production is limited. Two of the most important abiotic factors that can affect the productivity and physiology of aquatic crustaceans include salinity and ammonia-N. Salinities fluctuate widely in open aquaculture systems or estuaries and the ability of aquatic animals to thrive in such conditions is highly species-specific, depending on their osmoregulatory abilities. Ammonia-N, on the other hand, is the first nutrient in the nitrification cycle and, compared to nitrite and nitrate, is often the most toxic to crustaceans leading to reduced growth, physiological disruption or death. Accumulation of ammonia-N in closed aquaculture systems is ubiquitous concern, while in nature, crustaceans can experience elevated levels when burying for prolonged periods or in ecosystems receiving excessive anthropogenic discharge. Although salinity and ammonia-N may appear unrelated, the coping processes to salinity stress and elevated ammonia-N exposure are both closely linked via the highly important gill enzyme Na⁺/K⁺-ATPase activity. Furthermore, since these two factors may simutaneously challenge aquatic animals in either aquaculture systems or nature, the main focus of this thesis will investigate the effects of salinity and ammonia-N, alone and in combination, on the survival, growth, osmoregulation and coping responses of P. pelagicus juveniles. In addition, the last chapter will examine whether dietary highly unsaturated fatty acids (HUFA) and phospholipids (PL) supplemenation can improve the survival, growth and haemolymph ion maintenance of P. pelagicus juveniles at osmotically stressful conditions.

Chapter 2 was designed to investigate salinity levels of 5, 10, 15, 20, 25, 30, 35, 40 and 45 ‰ on the survival and growth of early P. pelagicus juveniles over 45 days and at the end of the experiment the haemolymph osmolality was measured to determine their osmoregulatory abilities. Results showed salinities of ≤ 15 ‰ and 45 ‰ significantly reduced survival while salinities of ≤ 15 ‰ and ≥ 40 ‰ significantly reduced the growth of P. pelagicus juveniles, indicating that this species is highly sensitive to salinities outside an optimal range of 20 - 35 ‰. Based on the haemolymph osmolality, the reduced performance of the crabs at these salinities may be attributed to their relatively weak osmoregulatory abilities.

In Chapter 3, the acute ammonia-N tolerance of P. pelagicus through juvenile development was determined and, to explain potential ammonia-N toxicity differences, gill histopathological changes were observed. Crabs at juvenile stages 1, 3, 5 and 7 were exposed to 0, 10, 20, 40, 60, 80 and 100 mg l⁻¹ ammonia-N and at 12-h intervals mortalities were measured for 96-h and then fixed to examine any histopathological gill damage. Results showed that P. pelagicus juveniles are highly tolerant to ammonia-N and this ability which increased through juvenile development possibly due to less ammonia-N induced gill damage at older juvenile stages.

Since ammonia-N tolerance was linked with gill damage, Chapter 4 was performed to determine any correlation between gill damage and coping responses, if gill damage is reversible and potential causes for the high ammonia-N tolerance of P. pelagicus juveniles. Crabs exposed to sub-lethal ammonia-N levels of 20 and 40 mg l⁻¹ were measured for haemolymph osmolality, Na⁺, K⁺, Ca2⁺, pH and ammonia-N levels, total haemocyte counts (THC) and then fixed for gill histological examination at hourly intervals for 48-h. Following 48-h, crabs were transferred to pristine seawater and again measured for these parameters at hourly intervals for 96-h. Ammonia-N had no significant effect on haemolymph osmolality, ion or pH, while haemolymph ammonia-N levels remained substantially lower than the test media. Further, ammonia-N exposure quickly caused gill damage along with adaptive/healing responses including significanlty higher THC and haemocytes within the gill lamallae. Upon transfer to pristine seawater, signs of gill healing became apparent and by 96-h, the gills were almost completley healed likely facilitated by haemocyte increases. Such findings likely explain the high ammonia-N tolerance of P. pelagicus, although this response has not yet been confirmed with other species.

These findings suggest that osmoregulation of P. pelagicus was undisrupted by ammonia-N and that haemolymph ammonia-N regulation may have been accomplished via ammonia-N excretion. To confirm this, Chapter 5 was set up to expose P. pelagicus juveniles to 0, 20, 40, 60, 80, 100 and 120 mg l⁻¹ ammonia-N at salinities of 15, 30 and 45 ‰ for 96-h and then measured for haemolymph osmolality, Na⁺, K⁺, Ca2+ and ammonia-N levels, ammonia-N excretion and gill Na⁺/K⁺-ATPase activity. Low salinities of 15 ‰ significantly increased posterior gill Na⁺/K⁺-ATPase activity, compared to 30 and 45 ‰, while anterior gill Na⁺/K⁺-ATPase activity was unaffected by salinity. However, anterior gill Na⁺/K⁺-ATPase activity significantly increased with increasing ammonia-N, whereas for the posterior gills, this result only occurred at 30 ‰. Furthermore, ammonia-N excretion and haemolymph ammonia-N levels significantly increased and decreased, respectively at 15 ‰, compared to those at 45 ‰. This likely explains the general pattern of increased ammonia-N toxicity at decreasing salinities to various crustaceans and, furthermore, ammonia-N excretion is unlikely to be the sole mechanism for haemolymph ammonia-N regulation.

During ammonia-N excretion, NH₄⁺ can substitute for K⁺ via Na⁺/K⁺-ATPase activity and elevated levels of both these ions has been demonstrated to increase Na⁺/K⁺-ATPase activity on dissected crustacean gills. While this suggests that ammonia-N excretion and osmoregulation would remain undisrupted in living crustaceans, this has yet to be proven. Therefore, Chapter 6 was set up to expose P. pelagicus juveniles to 0, 20, 40, 60, 80 and 100 mg l⁻¹ ammonia-N at low (4.25 mM), normal (8.50 mM) and high (12.75 mM) K⁺ levels of seawater for 96-h. After 96-h, haemolymph osmolality, Na⁺, K⁺, Ca2⁺ and ammonia-N levels, ammonia-N excretion, gill Na⁺/K⁺-ATPase activity and gill histopathological changes of the crabs were measured. In the presence of ammonia-N, both low and high K⁺ significantly reduced both ammonia-N excretion and gill Na⁺/K⁺-ATPase while haemolymph ammonia-N levels of the crabs significantly increased. An in vitro experiment was then performed, at different ammonia-N and K⁺ combinations, to compare gill Na⁺/K⁺-ATPase activity responses with the in vivo experiment. The in vitro experiment revealed no significant K⁺, ammonia-N or interactive effect on gill Na⁺/K⁺-ATPase activity. Greater histological gill damage and/or adverse physiogical consequences to living crabs may explain this discrepency and therefore in vitro investigations may be needed to test their applicability on living animals.

Finally, in Chapter 7, seperate experiments were set up to determine whether dietary highly unsaturated fatty acids (HUFA) and phospholipids (PL) can improve the survival, growth and haemolymph ion maintenance of early P. pelagicus juveniles at salinities of 14, 30 and 42 ‰. Results showed that increased dietary HUFA significantly improved growth and haemolymph ion maintenance of P. pelagicus juveniles at both 14 and 42 ‰, to become comparable with those cultured at 30 ‰. In contrast, dietary PL supplementation had no effect on overcoming the negative growth effects of high salinity and no significant effect on haemolymph ions. However, increased dietary PL supplementation significantly improved survival rates, particularly at 14 and 42 ‰. These findings demonstrate that the productivity of P. pelagicus juveniles can be significantly improved through both increased dietary HUFA and PL supplementation when subjected to osmotically stressful conditions for prolonged periods.

Item ID:	29822
Item Type:	Thesis (PhD)
Keywords:	blue swimmer crab; Portunus pelagicus; osmoregulation; amonia levels; diet; aquaculture industry
Date Deposited:	18 Oct 2013 04:47
FoR Codes:	06 BIOLOGICAL SCIENCES > 0608 Zoology > 060808 Invertebrate Biology @ 50% 06 BIOLOGICAL SCIENCES > 0606 Physiology > 060604 Comparative Physiology @ 50%
SEO Codes:	83 ANIMAL PRODUCTION AND ANIMAL PRIMARY PRODUCTS > 8301 Fisheries - Aquaculture > 830101 Aquaculture Crustaceans (excl. Rock Lobster and Prawns) @ 100%
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