Comparison of stress induced by manual restraint and immobilisation in the estuarine crocodile, Crocodylus porosus

This study compared the stress induced in captive estuarine crocodiles, Crocodylus porosus, by two different handling methods: manual restraint (noosing with ropes) and immobilization by electro-stunning. To stun, a short charge (approx. 6 s) at 110 V was delivered to the back of the necks of C. porosus using a custom-built device, which immobilized the animals for 5-10 min. Immobilized and restrained animals were measured and sexed, and the condition of the skin assessed. Blood samples were taken from some animals immediately after restraint or immobilization. Other animals were returned to their pens to recover for periods of 30 min, 1, 4, 12, 24 or 48 hours after which they were stunned and blood samples taken. Individual animals (mean body length 1.96 m, N=99) were bled only once. Haematocrit and haemoglobin concentrations were measured and plasma samples were analysed for corticosterone, glucose and lactate levels. Following restraint, there were significant increases in haematocrit, haemoglobin, glucose, lactate and corticosterone concentrations in C. porosus. For restrained animals, recovery to baseline levels occurred after approximately 8 hours. The stress response of stunned animals was significantly reduced compared to manually captured and restrained crocodiles. Both groups showed a significant increase in haematocrit, haemoglobin concentration and lactate levels, however the magnitude of change was significantly reduced, and recovery was faster in stunned animals. No increase in either glucose or corticosterone levels occurred with immobilisation. The results imply that immobilization by electro-stunning is much less stressful. (C) 2003 Wiley-Liss, Inc.

Hypertension in Pagothenia borchgrevinki caused by X-cell disease

Approximately 15% of a population of the cryopelagic nototheniid fish Pagothenia borchgrevinki, found constantly swimming immediately beneath the annual fast ice, in McMudro Sound. Ross Sea, Antarctica, was affected by X-cell gill disease. This disease affected blood flow through the gill lamellae, and this in turn affected oxygen uptake. Exercise caused increases in heart rate and ventral aortic blood pressure. Heart rate increased from 15.1 +/- 1.55 to 23.1 +/- 0.93 beats min(-1) in healthy fish, with a similar increase from 15.1 +/- 1.55 to 23.1 +/- 0.93 beats min(-1) in healthy fish, with a similar increase (to 24.6 +/- 0.26 beats min(-1)) in X-cell-affected animals. In healthy fish, pressures rose with exercise (from 2.72 +/- 0.11 to 3.75 +/- 0.19 kPa) and then rapidly returned to resting levels during recovery. In X-cell fish pressures rose during exercise, but then continued to rise, to reach a high of 4.18 +/- 0.13 kPa, close to the predicted maximum pressure able to be generated by these hearts. Recovery was rapid in healthy fish, but was prolonged in diseased animals. As they are constantly swimming, there is the potential that X-cell-affected fish suffer from chronic hypertension. (C) 2003 The Fisheries Society of the British Isles.

Prostaglandins are important in thermoregulation of a reptile (Pogona vitticeps)

The effectiveness of behavioural thermoregulation in reptiles is amplified by cardiovascular responses, particularly by differential rates of heart beat in response to heating and cooling (heart-rate hysteresis). Heart-rate hysteresis is ecologically important in most lineages of ectothermic reptile' and we demonstrate that heart-rate hysteresis in the lizard Pogona vitticeps is mediated by prostaglandins. In a control treatment (administration of saline), heart rates during heating were significantly faster than during cooling at any given body temperature. When cyclooxygenase 1 and 2 enzymes were inhibited, heart rates during heating were not significantly different from those during cooling. Administration of agonists showed that thromboxane B-2 did not have a significant effect on heart rate, but prostacyclin and prostaglandin F-2alpha caused a significant increase (3.5 and 13.6 beats min(-1), respectively) in heart rate compared with control treatments. We speculate that heart-rate hysteresis evolved as a thermoregulatory mechanism that may ultimately be controlled by neurally induced stimulation of nitric oxide production, or maybe via photolytically induced production of vitamin D.

Scaling of rectal gland mass in the European lesser-spotted dogfish

In the European lesser-spotted dogfish Scyliorhinus canicula, rectal gland mass in mg (M-Rg) followed the allometric relationship: M-Rg = 1.15 M-0.68, where M is body mass (g). The concept of allometric scaling is an important consideration in studies investigating the function Of osmoregulatory organs. (C) 2003 the Fisheries Society of the British Isles.

Seasonal changes in the diel surfacing behaviour of the bimodally respiring turtle Rheodytes leukops

The purpose of this study was to determine whether a relationship existed between the diel surfacing trends of the bimodally respiring freshwater turtle Rheodytes leukops and daily fluctuations in specific biotic and abiotic factors: The, diel surfacing behaviour of adult R. leukops was recorded over four consecutive seasons (Austral autumn 2000 - summer 2001) within Marlborough Creek, central Queensland, Australia, using pressure-sensitive time-depth recorders. Additionally, diurnal variations in water temperature and aquatic Po-2 level, as well as the turtle's behavioural state (i.e., active versus resting), were monitored. In autumn and summer, surfacing frequency increased significantly during the daylight hours, with peak levels normally occurring around dawn (0500-0700) and. dusk (1700-1900). However, no consistent diel surfacing trend was recorded, for the turtles in winter or spring, owing to considerable variation among individual R. leukops. Diurnal surfacing trends recorded for R. leukops in, autumn and summer are attributed to periods of increased activity (possibly associated with foraging) during the daylight hours and not to daily variations in water temperature or aquatic Po-2 level. Turtles generally remained at a depth greater than 1 m throughout the day, where the effect of diel fluctuations in water temperature, (

Urea based osmoregulation and endocrine control in elasmobranch fish with special reference to euryhalinity

Since the landmark contributions of Homer Smith and co-workers in the 1930s there has been a considerable advance in our knowledge regarding the osmoregulatory strategy of elasmobranch fish. Smith recognised that urea was retained in the body fluids as part of the 'osmoregulatory ballast' of elasmobranch fish so that body fluid osmolality is raised to a level that is iso- or slightly hyper-osmotic to that of the surrounding medium. From studies at that time he also postulated that many marine dwelling elasmobranchs were not capable of adaptation to dilute environments. However, more recent investigations have demonstrated that, at least in some species, this may not be the case. Gradual acclimation of marine dwelling elasmobranchs to varying environmental salinities under laboratory conditions has demonstrated that these fish do have the capacity to acclimate to changes in salinity through independent regulation of Na+, Cl- and urea levels. This suggests that many of the presumed stenohaline marine elasmobranchs could in fact be described as partially euryhaline. The contributions of Thomas Thorson in the 1970s demonstrated the osmoregulatory strategy of a fully euryhaline elasmobranch, the bull shark, Carcharhinus leucas, and more recent investigations have examined the mechanisms behind this strategy in the euryhaline elasmobranch, Dasyatis sabina. Both partially euryhaline and fully euryhaline species utilise the same physiological processes to control urea, Na+ and Cl- levels within the body fluids. The role of the gills, kidney, liver, rectal gland and drinking process is discussed in relation to the endocrine control of urea, Na+ and Cl- levels as elasmobranchs acclimate to different environmental salinities. (C) 2003 Elsevier Inc. All rights reserved.

Molecular modelling of human CYP1B1 substrate interactions and investigation of allelic variant effects on metabolism

Molecular modelling of human CYP1B1 based on homology with the mammalian P450, CYP2C5, of known three-dimensional structure is reported. The enzyme model has been used to investigate the likely mode of binding for selected CYP1B1 substrates, particularly with regard to the possible effects of allelic variants of CYP1B1 on metabolism. In general, it appears that the CYP1B1 model is consistent with known substrate selectivity for the enzyme, and the sites of metabolism can be rationalized in terms of specific contacts with key amino acid residues within the CYP1B1 heme locus. Further-more, a mode of binding interaction for the inhibitor, a-naphthoflavone, is presented which accords with currently available information. The current paper shows that a combination of molecular modelling and experimental determinations on the substrate metabolism for CYP1B1 allelic variants can aid in the understanding of structure-function relationships within P450 enzymes. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

Role of glutamic acid 216 in cytochrome P450 2D6 substrate binding and catalysis

Human cytochrome P450 (P450) 2D6 is an important enzyme involved in the metabolism of drugs, many of which are amines or contain other basic nitrogen atoms. Asp301 has generally been considered to be involved in electrostatic docking with the basic substrates, on the basis of previous modeling studies and site-directed mutagenesis. Substitution of Glu216 with a residue other than Asp strongly attenuated the binding of quinidine, bufuralol, and several other P450 2D6 ligands. Catalytic activity with the substrates bufuralol and 4-methoxyphenethylamine was strongly inhibited by neutral or basic mutations at Glu216 (>95%), to the same extent as the substitution of Asn at Asp301. Unlike the Asp301 mutants, the Gln216 mutant (E216Q) retained 40% enzyme efficiency with the substrate spirosulfonamide, devoid of basic nitrogen, suggesting that the substitutions at Glu216 affect binding of amine substrates more than other catalytic steps. Attempts to induce catalytic specificity toward new substrates by substitutions at Asp301 and Glu216 were unsuccessful. Collectively, the results provide evidence for electrostatic interaction of amine substrates with Glu216, and we propose that both of these acidic residues plus at least another residue(s) is (are) involved in binding the repertoire of P450 2D6 ligands.

Ancient colour vision: Multiple opsin genes in the ancestral vertebrates

Molecular investigation of the origin of colour vision has discovered five visual pigment (opsin) genes, all of which are expressed in an agnathan (jawless) fish, the lamprey Geotria australis. Lampreys are extant representatives of an ancient group of vertebrates whose origins are thought to date back to at least the early Cambrian, approximately 540 million years ago [1.]. Phylogenetic analysis has identified the visual pigment opsin genes of G. australis as orthologues of the major classes of vertebrate opsin genes. Therefore, multiple opsin genes must have originated very early in vertebrate evolution, prior to the separation of the jawed and jawless vertebrate lineages, and thereby provided the genetic basis for colour vision in all vertebrate species. The southern hemisphere lamprey Geotria australis (Figure 1A,B) possesses a predominantly cone-based visual system designed for photopic (bright light) vision [2. S.P. Collin, I.C. Potter and C.R. Braekevelt, The ocular morphology of the southern hemisphere lamprey Geotria australis Gray, with special reference to optical specializations and the characterisation and phylogeny of photoreceptor types. Brain Behav. Evol. 54 (1999), pp. 96–111.2. and 3.]. Previous work identified multiple cone types suggesting that the potential for colour vision may have been present in the earliest members of this group. In order to trace the molecular evolution and origins of vertebrate colour vision, we have examined the genetic complement of visual pigment opsins in G. australis.

Limitations of the widely used GAM42a and BET42a probes targeting bacteria in the Gammaproteobacteria radiation

The 23S rRNA-targeted probes GAM42a and BET42a provided equivocal results with the uncultured gammaproteobacterium 'Candidatus Competibacter phosphatis' where some cells bound GAM42a and other cells bound BET42a in fluorescence in situ hybridization (FISH) experiments. Probes GAM42a and BET42a span positions 1027-1043 in the 23S rRNAand differ from each other by one nucleotide at position 1033. Clone libraries were prepared from PCR products spanning the 16S rRNA genes, intergenic spacer region and 23S rRNA genes from two mixed cultures enriched in 'Candidatus C. phosphatis'. With individual clone inserts, the 16S rDNA portion was used to confirm the source organism as 'Candidatus C. phosphatis' and the 23S rDNA portion was used to determine the sequence of the GAM42a/BET42a probe target region. Of the 19 clones sequenced, 8 had the GAM42a probe target (T at position 1033) and 11 had G at position 1033, the only mismatch with GAM42a. However, none of the clones had the BET42a probe target (A at 1033). Non-canonical base-pairing between the 23S rRNA of 'Candidatus C. phosphatis' with G at position 1033 and GAM42a (G-A) or BET42a (G-T) is likely to explain the probing anomalies. A probe (GAM42_C1033) was optimized for use in FISH, targeting cells with G at position 1033, and was found to highlight not only some 'Candidatus C. phosphatis' cells, but also other bacteria. This demonstrates that there are bacteria in addition to 'Candidatus C. phosphatis' with the GAM42_C1033 probe target and not the BET42a or GAM42a probe target.

Interactions between ecology, demography, capture stress, and profiles of corticosterone and glucose in a free-living population of Australian freshwater crocodiles

In this study we examined three aspects pertaining to adrenocortical responsiveness in free-ranging Australian freshwater crocodiles (Crocodylus johnstoni). First, we examined the ability of freshwater crocodiles to produce corticosterone in response to a typical capture-stress protocol. A second objective addressed the relationship between capture stress, plasma glucose and corticosterone. Next we examined if variation in basal and capture-stress-induced levels of plasma corticosterone was linked to ecological or demographic factors for individuals in this free-ranging population. Blood samples obtained on three field trips were taken from a cross-sectional sample of the population. Crocodiles were bled once during four time categories at 0, 0. 5, 6, and 10 h post-capture. Plasma corticosterone increased significantly with time post-capture. Plasma glucose also significantly increased with duration of capture-stress and exhibited a positive and significant relationship with plasma corticosterone. Significant variation in basal or stress induced levels of corticosterone in crocodiles was not associated with any ecological or demographic factors including sex, age class or the year of capture that the crocodiles were sampled from. However, three immature males had basal levels of plasma corticosterone greater than 2 standard deviations above the mean. While crocodiles exhibited a pronounced, adrenocortical and hyperglycaemic response to capture stress, limited variation in adrenocortical responsiveness due to ecological and demographic factors was not evident. This feature could arise in part because this population was sampled during a period of environmental benigness. (C) 2003 Elsevier Science (USA). All rights reserved.

Feeding ecology of the fish ectoparasite Gnathia sp. (Crustacea: Isopoda) from the Great Barrier Reef, and its implications for fish cleaning behaviour

The feeding rate of a parasitic gnathiid isopod on fish was examined. Individual fish, Hemigymnus melapterus, were exposed to gnathiid larvae and sampled after 5, 10, 30, 60, and 240 min. I recorded whether larvae had an engorged gut, an engorged gut containing red material, or had dropped off the fish after having completed engorgement; variation among sampling times and larval stages was analyzed using generalized linear mixed model analyses. The likelihood that larvae had an engorged gut increased with time and varied with larval stage. First stage (1.45 mm) larvae. After 30 min, however, most (>93%) larvae had an engorged gut regardless of their larval stage. The likelihood of red material in the gut of third stage larvae increased over time (46% after 30 min, 70% after 60 min, and 86% after 240 min) while that of first and second stage larvae remained relatively low (