Diet-tissue discrimination factors and turnover of carbon and nitrogen stable isotopes in tissues of an adult predatory coral reef fish, Plectropomus leopardus.

RATIONALE: Stable isotope ratios (δ(13)C and δ(15)N values) provide a unique perspective into the ecology of animals because the isotope ratio values of consumers reflect the values in food. Despite the value of stable isotopes in ecological studies, the lack of species-specific experimentally derived diet-tissue discrimination factors (DTDFs) and turnover rates limits their application at a broad scale. Furthermore, most aquatic feeding experiments use temperate, fast-growing fish species and few have considered medium- to large-sized adults with low growth rates from tropical ecosystems. METHODS: A controlled-diet stable isotope feeding trial was conducted over a 196-day period for the adult predatory reef fish leopard coralgrouper (Plectropomus leopardus). This study calculated δ(13)C and δ(15)N DTDFs and turnover rates in five tissues (liver, plasma, red blood cells (RBC), fin, and muscle) using a continuous flow isotope ratio mass spectrometer equipped with an elemental analyzer. In addition, the effect of chemical lipid extraction (LE) on stable isotope values was examined for each tissue. RESULTS: Turnover was mainly influenced by metabolism (as opposed to growth) with LE δ(15)N half-life values lowest in fin (37 days) and plasma (66 days), and highest in RBC (88 days) and muscle (126 days). The diet-tissue discrimination factors for δ(15)N values in all tissues (Δ(15)N: -0.15 to 1.84‰) were typically lower than commonly reported literature values. Lipid extraction altered both δ(15) N and δ(13)C values compared with untreated samples; however, for the δ(15)N values, the differences were small (mean δ(15)N(LE-Bulk) <0.46‰ in all tissues). CONCLUSIONS: This study informs future interpretation of stable isotope data for medium- to large-sized fish and demonstrates that DTDFs developed for temperate fish species, particularly for δ(15)N values, may not apply to tropical species. Sampling of muscle and/or RBC is recommended for a relatively long-term representation of feeding habits, while plasma and/or fin should be used for a more recent indication of diet.

Age and growth parameters of shark-like batoids.

Estimates of life-history parameters were made for shark-like batoids of conservation concern Rhynchobatus spp. (Rhynchobatus australiae, Rhynchobatus laevis and Rhynchobatus palpebratus) and Glaucostegus typus using vertebral ageing. The sigmoid growth functions, Gompertz and logistic, best described the growth of Rhynchobatus spp. and G. typus, providing the best statistical fit and most biologically appropriate parameters. The two-parameter logistic was the preferred model for Rhynchobatus spp. with growth parameter estimates (both sexes combined) L(∞) = 2045 mm stretch total length, LST and k = 0·41 year⁻¹. The same model was also preferred for G. typus with growth parameter estimates (both sexes combined) L∞ = 2770 mm LST and k = 0·30 year⁻¹. Annual growth-band deposition could not be excluded in Rhynchobatus spp. using mark-recaptured individuals. Although morphologically similar G. typus and Rhynchobatus spp. have differing life histories, with G. typus longer lived, slower growing and attaining a larger maximum size.

Spatial variation in the effects of size and age on reproductive dynamics of common coral trout Plectropomus leopardus.

The effects of size and age on reproductive dynamics of common coral trout Plectropomus leopardus populations were compared between coral reefs open or closed (no-take marine reserves) to fishing and among four geographic regions of the Great Barrier Reef (GBR), Australia. The specific reproductive metrics investigated were the sex ratio, the proportion of vitellogenic females and the spawning fraction of local populations. Sex ratios became increasingly male biased with length and age, as expected for a protogyne, but were more male biased in southern regions of the GBR (Mackay and Storm Cay) than in northern regions (Lizard Island and Townsville) across all lengths and ages. The proportion of vitellogenic females also increased with length and age. Female P. leopardus were capable of daily spawning during the spawning season, but on average spawned every 4·3 days. Mature females spawned most frequently on Townsville reserve reefs (every 2·3 days) and Lizard Island fished reefs (every 3·2 days). Females on Mackay reefs open to fishing showed no evidence of spawning over 4 years of sampling, while females on reserve reefs spawned only once every 2-3 months. No effect of length on spawning frequency was detected. Spawning frequency increased with age on Lizard Island fished reefs, declined with age on Storm Cay fished reefs, and declined with age on reserve reefs in all regions. It is hypothesized that the variation in P. leopardus sex ratios and spawning frequency among GBR regions is primarily driven by water temperature, while no-take management zones influence spawning frequency depending on the region in which the reserve is located. Male bias and lack of spawning activity on southern GBR, where densities of adult P. leopardus are highest, suggest that recruits may be supplied from central or northern GBR. Significant regional variation in reproductive traits suggests that a regional approach to management of P. leopardus is appropriate and highlights the need for considering spatial variation in reproduction where reserves are used as fishery or conservation management tools.

Population structure and residency patterns of the blacktip reef shark Carcharhinus melanopterus in turbid coastal environments.

This study examined the characteristics of a blacktip reef shark Carcharhinus melanopterus population in turbid coastal habitats through a multi-year fishery-independent sampling and tag-recapture programme. Results revealed a highly structured population comprised almost entirely of juveniles and adult females with individuals between 850 and 1050 mm total length effectively absent. Mature males were also rarely encountered with adult sex ratio highly biased towards females (female:male = 7:1). Mating scars were observed on adult females between December and April, and parturition was observed from December to March. Regression analysis showed that catch rates were significantly higher during the summer wet season between November and May. Recapture data suggested a highly resident population with a recapture rate of 21% and a mean recapture distance of 0·8 km. In addition, 33% of recaptured animals were captured multiple times, indicating long-term residency. Most recaptures were, however, of adults with few juveniles recaptured. Widespread sampling at the study site and in adjacent areas suggested that the population was highly localized to a specific bay. The bimodal and sex-segregated population structure observed here differs from previous reports for this species, and in combination with reproductive observations, suggests population structuring to facilitate reproductive and recruitment success. These data also highlight the potential ecosystem functions performed by coastal habitats in sustaining C. melanopterus populations.

Comparison of the reproductive ecology of two sympatric blacktip sharks (Carcharhinus limbatus and Carcharhinus tilstoni) off north-eastern Australia with species identification inferred from vertebral counts.

Precaudal vertebral counts were used to distinguish between 237 morphologically similar Carcharhinus limbatus and Carcharhinus tilstoni and were congruent with differences in reproductive ecology between the species. In addition to differing lengths at maturity and adult body size, the two species had asynchronous parturition, were born at different sizes and the relative frequencies of neonates differed in two coastal nursery areas. Despite evidence that hybridization can occur, these differences suggest the species are largely reproductively isolated.

Modulating autoimmune responses to GAD inhibits disease progression and prolongs islet graft survival in diabetes-prone mice.

In nonobese diabetic (NOD) mice, beta-cell reactive T-helper type 1 (Th1) responses develop spontaneously and gradually spread, creating a cascade of responses that ultimately destroys the beta-cells. The diversity of the autoreactive T-cell repertoire creates a major obstacle to the development of therapeutics. We show that even in the presence of established Th1 responses, it is possible to induce autoantigen-specific anti-inflammatory Th2 responses. Immune deviation of T-cell responses to the beta-cell autoantigen glutamate decarboxylase (GAD65), induced an active form of self-tolerance that was associated with an inhibition of disease progression in prediabetic mice and prolonged survival of syngeneic islet grafts in diabetic NOD mice. Thus, modulation of autoantigen-specific Th1/Th2 balances may provide a minimally invasive means of downregulating established pathogenic autoimmune responses.

Huntington's disease: the challenge for cell biologists.

Huntington's disease (HD) is one of eight inherited neurodegenerative diseases caused by expansions of (CAG)(n) tracts that encode polyglutamine segments in expressed proteins. Studies of pathogenic mechanisms for all these late-onset diseases suffer from a common drawback: experimental studies require massive acceleration of a process that, in affected humans, usually takes decades. But is the rapid-onset disease of transgenic mouse models and in cells the same as the slow-onset disease in humans? We review recent work on HD, noting several issues whose significance is likely to be crucial - but which are as yet unresolved. We discuss these in light of the distinction between disease-specific pathogenic mechanisms and artifacts of polyglutamine overexpression. We suggest that the initial stages of HD result from dysfunction rather than death, and we consider the potential discovery of compounds that might interfere with early pathogenic events.

Brain glutamate decarboxylase cloned in lambda gt-11: fusion protein produces gamma-aminobutyric acid.

Glutamate decarboxylase (GAD; E.C. 4.1.1.15) converts glutamate to gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the vertebrate central nervous system. This report describes the isolation of a GAD complementary DNA clone by immunological screening of a lambda gt-11 brain complementary DNA expression library. The fusion protein produced by this clone catalyzes the conversion of glutamate to GABA and carbon dioxide, confirming its identity as GAD. Antibodies to beta-galactosidase remove GAD enzymatic activity from solution, showing that this activity is associated with the fusion protein. In immunoblotting experiments all three available antisera to GAD reacted with the fusion polypeptide and with two major polypeptides (molecular size, 60,000 and 66,000 daltons) in brain extracts.

The abnormal cerebellar organization of Weaver and reeler mice does not affect the cellular distribution of three neuronal mRNAs.

We used in situ hybridization of 35S-labeled antisense RNAs to study the cellular distribution of three neuronal mRNAs. We compared the expression of these RNAs in cerebellar Purkinje neurons in wild-type (C57Bl-6J) mice and in two mutants (Weaver and reeler) known to have abnormal cerebellar morphologies. In normal mice, GAD mRNA is present in four sets of neurons in the cerebellar cortex while calbindin mRNA is present only in Purkinje neurons. Proenkephalin mRNA is present in Golgi II neurons as well as in a set of neurons in the deep part of the molecular layer. Despite the dramatic differences in structural organization and inputs of Purkinje neurons in the cerebella of adult Weaver and reeler mice, the expression of these RNAs appears unchanged. These results support the hypothesis that Purkinje cell cytodifferentiation proceeds autonomously after its inception in early embryonic life.

Long-term increase of glutamate decarboxylase mRNA in a rat model of temporal lobe epilepsy.

Behavioral changes following injury, neural degeneration, and aging partly reflect the synaptic plasticity of the nervous system. Such long-term plastic changes are likely to depend on alterations in the production of proteins involved in synaptic structures and neurotransmission. We have studied the regulation of the mRNA encoding one such protein, glutamate decarboxylase (GAD), the rate limiting enzyme of GABA synthesis, after a unilateral lesion in the hippocampus that leads to increased seizure susceptibility. Quantitative in situ hybridization reveals a long-term increase in GAD mRNA in several bilateral structures, as well as in specific neurons in the ipsilateral dentate gyrus. Our data do not support the often stated hypothesis that seizure susceptibility depends on the malfunction of GABA neurons.

Two genes encode distinct glutamate decarboxylases.

gamma-Aminobutyric acid (GABA) is the most widely distributed known inhibitory neurotransmitter in the vertebrate brain. GABA also serves regulatory and trophic roles in several other organs, including the pancreas. The brain contains two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD), which differ in molecular size, amino acid sequence, antigenicity, cellular and subcellular location, and interaction with the GAD cofactor pyridoxal phosphate. These forms, GAD65 and GAD67, derive from two genes. The distinctive properties of the two GADs provide a substrate for understanding not only the multiple roles of GABA in the nervous system, but also the autoimmune response to GAD in insulin-dependent diabetes mellitus.

A novel alpha subunit in rat brain GABAA receptors.

Two cDNAs (alpha 1 and alpha 4) from rat brain cDNA libraries encode isoforms of the alpha subunit of the GABA/benzodiazepine receptor, which differ at 30% of their amino acid residues. Northern blot analysis and in situ hybridization histochemistry show that alpha 1 and alpha 4 mRNAs have distinct sizes and distinct regional and cellular distributions in rat brain: both mRNAs are found in the cortex and hippocampus; however, only the alpha 1 mRNA is detected in the cerebellum. We injected RNA transcribed from alpha 1 and alpha 4 cDNAs into Xenopus oocytes, together with an RNA for a rat beta subunit. We obtained GABA-dependent inward currents that were reversibly blocked by picrotoxin. Picrotoxin alone, applied to oocytes producing the alpha and beta polypeptides, elicited an outward current. We suggest that these polypeptides together produce GABA-gated ion channels that can also open spontaneously.

Islet cell cytoplasmic autoantibody reactivity to glutamate decarboxylase in insulin-dependent diabetes.

Individuals with or at risk for insulin-dependent diabetes (IDD) frequently have autoantibodies against an islet cell cytoplasmic (ICA) antigen thought to be a sialoglycolipid. However, we now report that preabsorption of ICA-positive sera with recombinant glutamate decarboxylase (human GAD 65 and/or GAD 67) reduced or blocked the ICA reactivity of 5/18 (27%) new-onset IDD patients and 7/18 (39%) prediabetics. Interestingly, nondiabetic subjects with ICA of > or = 5 yr in duration had GAD-reactive ICA significantly more often (16/24, 67%, P < 0.04) than the diabetic groups. ICA reactivity to GAD was not related to serum ICA titer nor the age of the individual, and in all cases tested was blocked by GAD 65 or GAD 67 with equivalent efficiency. The ICA observed in 21/25 (84%) IDD patients with ICA long after clinical onset of disease (9-42 yr) was reactive to GAD. A natural history analysis of three individuals showed conversions from ICA which was reactive to GAD to a non-GAD-reactive ICA nearer to their clinical onsets of IDD. This study further defines the autoantigens reactive to ICA, and suggests that, whereas ICA that are not reactive to GAD may identify an advanced and more prognostic lesion, GAD-reactive ICA may typify the early or inductive lesion that may or may not progress to clinically significant beta cell injury.

Autoimmunity to two forms of glutamate decarboxylase in insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is thought to result from the autoimmune destruction of the insulin-producing beta cells of the pancreas. Years before IDDM symptoms appear, we can detect autoantibodies to one or both forms of glutamate decarboxylase (GAD65 and GAD67), synthesized from their respective cDNAs in a bacterial expression system. Individual IDDM sera show distinctive profiles of epitope recognition, suggesting different humoral immune responses. Although the level of GAD autoantibodies generally decline after IDDM onset, patients with IDDM-associated neuropathies have high levels of antibodies to GAD, years after the appearance of clinical IDDM. We note a striking sequence similarity between the two GADs and Coxsackievirus, a virus that has been associated with IDDM both in humans and in experimental animals. This similarity suggests that molecular mimicry may play a role in the pathogenesis of IDDM.

Reproductive benefits of no-take marine reserves vary with region for an exploited coral reef fish.

No-take marine reserves (NTMRs) are expected to benefit fisheries via the net export of eggs and larvae (recruitment subsidy) from reserves to adjacent fished areas. Quantifying egg production is the first step in evaluating recruitment subsidy potential. We calculated annual egg production per unit area (EPUA) from 2004 to 2013 for the commercially important common coral trout, Plectropomus leopardus, on fished and NTMR reefs throughout the Great Barrier Reef (GBR), Australia. Geographic region, NTMR status, fish size, and population density were all found to affect EPUA. The interactions among these factors were such that, EPUA on NTMR reefs compared to reefs open to fishing was 21% greater in the southern GBR, 152% greater in the central GBR, but 56% less in the northern GBR. The results show that while NTMRs can potentially provide a substantial recruitment subsidy (central GBR reefs), they may provide a far smaller subsidy (southern GBR), or serve as recruitment sinks (northern GBR) for the same species in nearby locations where demographic rates differ. This study highlights the importance of considering spatial variation in EPUA when assessing locations of NTMRs if recruitment subsidy is expected from them.

The heterogeneous nuclear RNA of chicken erythroblasts.

Heterogeneous nuclear RNA (hnRNA) from chicken erythroblasts has a modal molecular weight of 1.6 -10(6) in 99% dimethylsulfoxide. When erythroblasts are labeled continuously with [14C]uridine, nuclear RNA is labeled as a single kinetic component with a half-life of 18 min. After a 10--20 min lag, label appears in cytoplasmic RNA at about 1% of the initial rate of total RNA synthesis. Of the hnRNA sedimenting faster than 28 S ribosomal RNA in both an aqueous sucrose gradient and a subsequent fructose gradient in 99% dimethylsulfoxide, about one-third is polyadenylated, although only about one in 2000 (i.e. about four molecules per cell) contain a globin messenger sequence. The hnRNA of erythroblasts isolated from 5.7- and 11-day chick embryos have the same content of globin messenger sequences as erythroblaasts from anemic adults.

Large predatory coral trout species unlikely to meet increasing energetic demands in a warming ocean.

Increased ocean temperature due to climate change is raising metabolic demands and energy requirements of marine ectotherms. If productivity of marine systems and fisheries are to persist, individual species must compensate for this demand through increasing energy acquisition or decreasing energy expenditure. Here we reveal that the most important coral reef fishery species in the Indo-west Pacific, the large predatory coral trout Plectropomus leopardus (Serranidae), can behaviourally adjust food intake to maintain body-condition under elevated temperatures, and acclimate over time to consume larger meals. However, these increased energetic demands are unlikely to be met by adequate production at lower trophic levels, as smaller prey species are often the first to decline in response to climate-induced loss of live coral and structural complexity. Consequently, ubiquitous increases in energy consumption due to climate change will increase top-down competition for a dwindling biomass of prey, potentially distorting entire food webs and associated fisheries.

Comparative localization of mRNAs encoding two forms of glutamic acid decarboxylase with nonradioactive in situ hybridization methods.

Nonradioactive in situ hybridization methods with digoxigenin-labeled cRNA probes were used to localize two glutamic acid decarboxylase (GAD) mRNAs in rat brain. These mRNAs encode two forms of GAD that both synthesize GABA but differ in a number of characteristics including their molecular size (65 and 67 kDa). For each GAD mRNA, discrete neuronal labeling with high cellular resolution and low background staining was obtained in most populations of known GABA neurons. In addition, the current methods revealed differences in the intensity of labeling among neurons for each GAD mRNA, suggesting that the relative concentrations of each GAD mRNA may be higher in some groups of GABA neurons than in others. Most major classes of GABA neurons were labeled for each GAD mRNA. In some groups of GABA neurons, the labeling for the two mRNAs was virtually identical, as in the reticular nucleus of the thalamus. In other groups of neurons, although there was substantial labeling for each GAD mRNA, labeling for one of the mRNAs was noticeably stronger than for the other. In most brain regions, such as the cerebellar cortex, labeling for GAD67 mRNA was stronger than for GAD65 mRNA, but there were a few brain regions in which labeling for GAD65 mRNA was more pronounced, and these included some regions of the hypothalamus. Finally, some groups of GABA neurons were predominantly labeled for one of the GAD mRNAs and showed little or no detectable labeling for the other GAD mRNA, as, for example, in neurons of the tuberomammillary nucleus of the hypothalamus where labeling for GAD67 mRNA was very strong but no labeling for GAD65 mRNA was evident. The findings suggest that most classes of GABA neurons in the central nervous system (CNS) contain mRNAs for at least two forms of GAD, and thus, have dual enzyme systems for the synthesis of GABA. Higher levels of one or the other GAD mRNA in certain groups of GABA neurons may be related to differences in the functional properties of these neurons and their means of regulating GABA synthesis.

Comparative distribution of mRNAs for glutamic acid decarboxylase, tyrosine hydroxylase, and tachykinins in the basal ganglia: an in situ hybridization study in the rodent brain.

Neurotransmitter-related messenger RNAs were detected by in situ hybridization in sections of rat and mouse brains by using 35S-radiolabelled RNA probes transcribed from cDNAs cloned in SP6 promoter-containing vectors. The distribution of messenger RNAs for glutamic acid decarboxylase, tachykinins (substance P and K), and tyrosine hydroxylase was examined in the striatum, pallidum, and substantia nigra. Dense clusters of silver grains were observed with the RNA probe complementary of the cellular messenger RNA for glutamic acid decarboxylase (antisense RNA) over most large neurons in the substantia nigra pars reticulata and medium-sized to large neurons in all pallidal subdivisions. A few very densely and numerous lightly labelled medium-sized neurons were present in the striatum. Among the areas examined, only the striatum contained neurons labelled with the antisense tachykinin RNA. Most of these neurons were of medium size, and a few were large. With the antisense tyrosine hydroxylase RNA, silver grains were found over neurons of the substantia nigra pars compacta and adjacent A10 and A8 dopaminergic cell groups. No signal was observed with RNAs identical to the cellular messenger RNA for glutamic acid decarboxylase or tachykinin (sense RNA). These results show a good correlation with immunohistochemical studies, suggesting that documented differences in the distribution and the level of glutamic acid decarboxylase, tyrosine hydroxylase, and substance P immunoreactivities in neurons of the basal ganglia are related to differences in the level of expression of the corresponding genes rather than to translation accessibility, stability, or transport of the gene products.

Differential localization of two glutamic acid decarboxylases (GAD65 and GAD67) in adult monkey visual cortex.

Adult monkey primary visual cortex contains a diverse population of stellate neurons that utilize the neurotransmitter gamma aminobutyric acid (GABA). Two glutamic acid decarboxylase (GAD) enzymes that synthesize GABA, GAD65 and GAD67, were localized within these stellate neurons by in situ hybridization of 35S or digoxigenin (DIG) labeled riboprobes. Double labels were done by using 35S GAD67 riboprobe and GABA immunocytochemistry on the same section to verify that the neuronal population identified by immunocytochemistry was the same one studied in the in situ hybridization experiments. We find that GAD65 mRNA and GAD67 mRNA are widely distributed in the cortex, with four bands of heavily labeled neurons in upper layer 2, lower 3, 4C, and 6. GAD67 labeled neurons were more obvious in layer 4C beta, while GAD65 containing neurons were common in layer 1 and white matter. Northern blots and in situ hybridization on sections with both 35S and DIG riboprobes indicate that cortical neurons typically contain more GAD67 mRNA. Cell counts show that 18% of all cortical neurons contain GAD67 mRNA and 13% contain GAD65 mRNA, suggesting that a small population of GABA neurons might lack GAD65. Cell bodies that contain high amounts of GAD65 mRNA are prominent in layers deep 3, 4B, 4C alpha, and 6 and often are the largest cells in their respective layers. Double labels demonstrate that 96% of all GABA+ neurons contain GAD67 mRNA. Neurons heavily labeled for GABA tend to have smaller cell bodies and contain less GAD67 mRNA, while lightly labeled GABA neurons are larger and contain more GAD67 mRNA. These data indicate that most GABA neurons in monkey striate cortex contain both GAD enzymes. Although the differences in GABA content, cell size, laminar distribution, and GAD mRNA concentration suggest different requirements for GAD67 and GAD65 in cortical circuits, our experiments do not reveal what different roles these two enzymes subserve within GABAergic stellate neurons.