Essentiality of dietary cholesterol and its interactions with phospholipid in juvenile slipper lobster (Thenus australiensis).

This study was conducted to verify the essentiality of dietary cholesterol for early juvenile slipper lobster, Thenus australiensis (initial weight 4.50 ± 0.72 g, mean ± SD, CV = 0.16), and to explore the potential for interactions between dietary cholesterol and phospholipid. An 8-week experiment was conducted using six experimental feeds containing three supplemental cholesterol concentrations (0, 0.2 and 0.4% dry matter) at two supplemental phospholipid concentrations (0% and 1.0% dry matter). Dietary cholesterol concentrations of ≥ 0.2% resulted in up to threefold greater weight gain compared to 0% dietary cholesterol, but without any significant main or interactive dietary phospholipid effect. An interaction was observed for lobster survival with lowest survival (46%) recorded for combined 0% cholesterol and 0% phospholipid compared to every other treatment (71-100%). However, all surviving lobsters at 0% dietary cholesterol, regardless of dietary phospholipid level, were in poor nutritional condition. Apparent feed intake (AFI) was significantly higher at dietary cholesterol ≥ 0.2% but was lower for each corresponding dietary cholesterol level at 1% dietary phospholipid. This implied that the feed conversion ratio was improved with supplemental phospholipid. In conclusion, this study confirms the essential nature of dietary cholesterol and that dietary phospholipid can provide additional benefits.

Physiological status and nutritional condition of cultured juvenile Thenus australiensis over the moult cycle.

The moult cycle is arguably the most critical aspect of crustacean biology and is associated with dramatic changes in behaviour, physiology and condition. Here we describe the first detailed investigation of the combined changes in morphology, physiological status and nutritional condition over the moult cycle of juvenile T. australiensis. Haemolymph refractive index (measured as Brix) was evaluated as a non-destructive method for predicting physiological status and nutritional condition. Post-moult, inter-moult and pre-moult stages were identifiable by microscopic examination of the pleopod distal tips, though differentiation of the pre-moult substages was not possible using this technique. Monitoring of ecdysial suture lines on the exoskeleton gill chambers was found to be highly useful for visually determining progression through the pre-moult stage and predicting the timing of ecdysis. A classical pattern of inter-moult growth was observed where size and wet weight remained relatively uniform over the moult cycle while highly significant changes in whole-body composition were simultaneously observed over time. Growth was most evident by changes in dry weight and dry matter content which more than doubled by the onset of pre-moult. Changes in dry matter content were generally mirrored by Brix and whole-body crude protein, total lipid and gross energy content. Brix strongly correlated with dry matter content and significant correlations were also found for all other primary measures of nutritional condition. Typical moult-related changes were also found for circulating ecdysteroids. This study clearly demonstrates pronounced cyclical changes in physiology and condition of juvenile T. australiensis over a typical moult cycle. The significant correlations between haemolymph and whole-body composition validates Brix as a practical and non-destructive method to objectively assess physiological status, nutritional condition and quality in T. australiensis and further demonstrates its potential for individual crustacean assessment in experimental research and practical commercial applications.

The role of the kidney in compensating the alkaline tide, electrolyte load, and fluid balance disturbance associated with feeding in the freshwater rainbow trout, Oncorhynchus mykiss.

The effect in freshwater rainbow trout of digesting a commercial pellet meal on the renal handling of water, ions and acid-base equivalents was investigated through urine collection over a 48 h period following meal ingestion. The glomerular filtration rate (GFR) and urine flow rate (UFR) were reduced in fed fish between 12 and 24h following the meal, likely reflecting a loss of endogenous water across the gastric epithelium as a result of ingesting dry, ion-rich food pellets. The kidney was also responsible for the excretion of some excess dietary Ca(2+), and, to a much lesser extent, Na(+) and Cl(-), while the urinary excretion of K(+) was unaffected. The most dramatic effect of feeding was the elevation of renal Mg(2+) excretion, with the kidney transitioning from net Mg(2+) reabsorption to net Mg(2+) secretion during digestion. The renal handling of dietary ions accounted for 3-27% of the total ions absorbed from the diet, indicating that a majority of the ions are excreted extra-renally or incorporated into growth. However this does highlight the underestimation of renal ion handling when using unfed fish models. The metabolic alkalosis created by digestion (the alkaline tide) resulted in an increase in urine pH as well as a transition from net acidic equivalent excretion in the urine to net basic equivalent excretion. This was due to a decrease in the titratable acidity minus bicarbonate component of urine as well as a decrease in ammonia secretion. Additionally, the experimental separation of the urinary component of acid-base excretion from that of the gills highlighted the substantially larger contribution of the latter. During the alkaline tide, renal excretion accounted for approximately 5% of the total basic equivalent excretion to the external water.

Flow cytometric characterization of freshwater crayfish hemocytes for the examination of physiological status in wild and captive animals.

Enumeration of invertebrate hemocytes is a potentially powerful tool for the determination of physiological effects of extrinsic stressors, such as hypoxia, disease, and toxicant exposure. A detailed flow cytometric method of broad application was developed for the objective characterization and enumeration of the hemocytes of New Zealand freshwater crayfish Paranephrops planifrons for the purpose of physiological health assessment. Hemocyte populations were isolated by flow cytometric sorting based on differential light scatter properties followed by morphological characterization via light microscopy and software image analysis. Cells were identified as hyaline, semigranular, and granular hemocytes based on established invertebrate hemocyte classification. A characteristic decrease in nuclear size, an increase in granularity between the hyaline and granular cells, and the eccentric location of nuclei in granular cells were also observed. The granulocyte subpopulations were observed to possess varying degrees of granularity. The developed methodology was used to perform total and differential hemocyte counts from three lake populations and between wild and captive crayfish specimens. Differences in total and differential hemocyte counts were not observed among the wild populations. However, specimens held in captivity for 14 d exhibited a significant 63% reduction in total hemocyte count, whereas the relative hemocyte proportions remained the same. These results demonstrate the utility of this method for the investigation of subacute stressor effects in selected decapod crustaceans.

Carotenoid depletion and ovarian dysfunction in common bully exposed to a New Zealand pulp mill effluent.

In a previous cumulative impacts assessments of fish populations along the Waikato River, New Zealand, reduced ovary pigmentation was observed in the common bully (Gobiomorphus cotidianus) with an associated reduction in recruitment in the pulp mill-impacted Kopakorahi Arm of Lake Maraetai compared to an upstream reference site. Carotenoid pigments are typically responsible for the yellow to red colouration of fish eggs and a number of functions for these pigments such as pro-vitamins and antioxidants have been proposed. The purpose of the current study was first to quantify the observed pigment reduction and then to establish possible consequences of carotenoid deficiency. Significantly lower levels of all measured carotenoids were observed in the ovaries of fish from below the mill outfall compared to the reference site. Using in vitro incubations of ovarian follicles, it was also found that these fish had a significantly reduced ability to produce the sex steroids testosterone and estradiol. This study demonstrates changes in ovarian carotenoids associated with pulp mill effluent exposure. Significantly reduced biosynthetic capacity of ovarian follicles demonstrates reproductive endocrine disruption potential associated with exposure to this particular effluent. These results may provide an important link between reproductive dysfunction, oxidative stress and food chain modifications in pulp mill-impacted systems.

Altered physiology of rainbow trout in response to modified energy intake combined with pulp and paper effluent exposure.

Two experiments using rainbow trout (Oncorhynchus mykiss) were conducted to examine the combined effects of energy intake as manipulated by ration and pulp and paper mill effluent exposure over either one, or two consecutive reproductive cycles. This study demonstrated that the level of energy intake affected the full range of measured parameters from energy allocation to somatic growth and the gonadal development, steroid production and hematology. Increasing ration level expectedly increased growth, condition, liver and gonad size. Female trout in the higher ration treatments produced more follicles and had larger eggs, investing the same relative proportion of total energy into ovarian development. Sex steroid levels and hematological parameters were also positively influenced by increasing ration level in males and females. By far, the most dramatic impact of reduced ration on reproduction was to substantially reduce the frequency of sexually maturing fish. The effects of effluent exposure were not as marked as those linked to ration level and typically did not manifest unless fish were exposed through two consecutive reproductive cycles. The physiological effects of pulp and paper effluent exposure observed in these experiments were not consistent between the two experiments conducted herein, nor were they consistent with previously observed impacts in similar experiments with this effluent. Effluent exposure over one reproductive cycle did not impact physiological parameters in trout. However, when effluent exposure was maintained over two reproductive cycles, a new pattern of effluent response emerged including increased condition factor in both sexes, a decrease in the potential ability of the blood of females to transport oxygen, and increased sex steroids and reproductive investment in males. Effluent was also observed to cause reduced growth in male trout over two years. The effects of ration on gonad and liver size were far more obvious and consistent when a longer exposure was employed, thus, it appears to take more than one full year for energy intake changes to be reflected in those particular physiological endpoints.

Responses of shortfin eel (Anguilla australis) exposed in situ to pulp and paper effluent.

The responses of shortfin eel (Anguilla australis) to discharges from two pulp and paper mills, municipal wastewater, and a geothermal power plant wastewater were examined. Eels were caged at 3 sites along the Tarawera River, North Island, New Zealand, to explore effects of a 3-wk exposure down a contamination gradient (Ref --> D1 --> D2). Most of the observed effects were seen in eels caged at the furthest downstream site (D2), below all the discharge areas. General hematology in eels was unaffected, as measures did not differ markedly at the two downstream sites compared with the reference site. At D2, eels were significantly lighter per unit length (reduced condition factor), although liver and spleen size (LSI and SSI) were unaffected. Significantly elevated circulating sex steroid concentrations (testosterone and estradiol) were measured in D2 eels and increasing sex steroid levels at both sites downstream of the reference site were observed. Significant ethoxyresorufin O-deethylase (EROD) activity induction was seen in D2 eels and bile chemistry showed significant accumulation of pyrene and retene equivalents. However, significantly greater concentrations of total resin acids were found in the bile of eels from the intermediate site (D1), between the two pulp and paper mills. The higher polycyclic aromatic hydrocarbon (PAH) equivalents found in the bile of D2 eels suggest that resin acid neutrals, particularly retene, are responsible for some of the effects observed in eels at the furthest downstream exposure site. Levels of pulp and paper mill extractives in sediment, including the PAH retene, support this conclusion.

Combined effects of pulp and paper effluent, dehydroabietic acid, and hypoxia on swimming performance, metabolism, and hematology of rainbow trout.

Experiments were conducted to examine the effects of a thermomechanical (TMP)/bleached kraft pulp and paper mill effluent (BKME), dehydroabietic acid (DHAA), hypoxia, and combinations of hypoxia and effluent on juvenile rainbow trout. In the first two experiments, trout were exposed for 4 weeks to 0%, 10%, 30%, and 70% TMP/BKME or 0, 35, 110, and 250 microgL(-1) DHAA, respectively. Endpoints of those dose-response studies included critical swimming speed, oxygen consumption, and hematology. Reduced swimming performance was found for fish exposed to 70% TMP/BKME. Moderate increases in mean cell hemoglobin concentration at 70% TMP/BKME and blood glucose at 30% and 70% TMP/BKME were also seen. The opposite trend for glucose was found for DHAA-exposed fish, where a slight decrease in glucose was seen at 110 and 250 microgL(-1) DHAA. The third experiment examined the effects of 15% v/v TMP/BKME exposure at 2.5 and 5.0 mgL(-1) dissolved oxygen (DO) for 4 weeks. This experiment found no effect of low DO on swimming ability. An interactive effect between DO and effluent exposure was seen only on hematocrit, where effluent caused an increase in hematocrit at 5 mgL(-1) and a decrease at 2.5 mgL(-1) DO. Effluent exposure in this experiment resulted in a greater number of smaller red blood cells. The current study demonstrated physiological effects in rainbow trout exposed to varying concentrations (15-70% v/v) of a TMP/BKME and no substantial effects of DHAA exposure. With the exception of the reduced swimming performance in fish exposed to TMP/BKME, the observed effects are considered relatively small in magnitude but are occurring at concentrations of effluent that occur in the receiving environment.