Rare earth elements in drill cutting samples from off-shore oil and gas exploration activities in ultradeep waters.

Rare earth elements (REEs) are essential in high technology industries and have great economic value. The monitoring of REEs concentrations in rocks from oil well drill cuttings is critical to avoid environmental contamination and evaluate new sources of these elements. However, information is scarce about the REEs concentrations in drill cuttings. In this work, the concentration of REEs in drill cuttings from oil and gas exploration wells in ultradeep coastal water of Brazilian were investigated at different depths. The drill cutting samples were submitted to microwave-assisted acid digestion prior to the determination of concentration by ICP-MS, using Rh as internal standard for calibration. The limits of quantification (LoQ) ranged from 3.3 µg kg-1 for Ho to 198 µg kg-1 for Sm. The accuracy was evaluated by analyzing certified reference materials for rocks. The obtained REEs concentrations agreed with the certified values, reaching 83%-105% agreement. The drill cutting depth profile analysis indicates Ce, La, Nd, Sm, and Eu concentrations up to mg kg-1. The REEs concentrations obtained in drill cutting depth profile was analyzed by principal component analysis (PCA), and hierarchical cluster analysis (HCA) identified tendency and similarity between drill cutting samples. Three groups were formed according to the composition of the REEs. In addition, the concentration of these chemicals elements varied at different depths. The analysis of drill cuttings revealed REEs concentrations up to the mg per kg-range (ppm), potentially making this disposable material an alternative source for REEs extraction, and adding value to this material.

Recovery of lanthanides from hydrocarbon cracking spent catalyst through chemical and biotechnological strategies.

The aim of this work is to evaluate the rare earth elements (REEs) recovery from fluid catalytic cracking spent catalyst (FCC-SC) by chemical and biochemical strategies while also examining a route for the valorization of biodiesel-derived glycerin (RG), which is presently unprofitable to refine. Recovery tests for REEs were performed with no pretreatment of the FCC-SC. A chemical leaching investigation was carried out using HCl, HNO3, NaOH, CaCl2 and citric acid aqueous solutions (1 mol L-1, at 30, 50, 60 or 70 ± 1 °C). The leaching tests carried out with 1 mol L-1 citric acid at 50 °C provided the best recovery of La (27%). Subsequent bioleaching tests were carried out with four strains of Yarrowia lipolytica to evaluate their potential to produce organic acids using RG as the main carbon source. The FCC-SC contains some REEs, predominantly La. Remarkable biorecovery rates for REEs (namely, La (53%), Ce and Nd (both 99%)) were achieved using the Y. lipolytica IM-UFRJ 50678 fermented medium at 50 °C. Thus, here, a sustainable approach to recovering metals from spent cracking catalyst using RG under low-cost and non-energy-intensive processing conditions is reported.

Correction: Peripheral Organs of Dengue Fatal Cases Present Strong Pro-Inflammatory Response with Participation of IFN-Gamma-, TNF-Alpha- and RANTES-Producing Cells.

[This corrects the article DOI: 10.1371/journal.pone.0168973.].

Pseudomonas spp: contamination sources in bulk tanks of dairy farms / Pseudomonas spp: fontes de contaminação em tanques de expansão em fazendas leiteiras

This study focused on isolating Pseudomonas spp. during milking process in ten dairy farms with manual and mechanical milking systems during dry and rainy seasons, and evaluating DNA homology and patterns of distribution between isolates, in order to identify main sources of milk contamination by Pseudomonas spp. A total of 167 isolates of Pseudomonas spp. were obtained from water, milkers' hands, cows' teats, teat cups, cooling tanks and raw milk. Bacteria of Pseudomonas spp. genus were isolated from 85 and 82 sampling points in dairy farms with manual and mechanical milking system, respectively. A significant difference (p=0.02) on Pseudomonas spp. isolation was observed among samples of surface of cows' teats before and after pre-dipping, but no significant difference (p>0.05) was observed among milking systems or seasons. The possibility of the same Pseudomonas spp. patterns are distributed in different farms and seasons using Amplified Fragment Length Polymorphism (AFLP) technique was demonstrated. Milkers' hands, surface of cows' teats, teat cups and cooling tanks were associated with raw milk contamination with Pseudomonas spp. on farms with manual and mechanical milking system, showing that regardless of the type of milking system and season, proper hygiene procedures of equipment, utensils and workers' hands are essential to avoid contamination of the milk and, therefore, improve milk quality.(AU)

Este estudo se propôs a isolar Pseudomonas spp. durante o processo de ordenha em dez fazendas com sistemas manuais e mecanizados, durante as estações seca e chuvosa, além de avaliar a homologia do DNA e seus padrões de distribuição entre os isolados, a fim de se determinar as principais fontes de contaminação do leite. Cento e sessenta e sete isolados de Pseudomonas spp. foram obtidos a partir de amostras de água, mãos de ordenhadores, tetos, teteiras, tanques de resfriamento e leite cru armazenado, sendo 85 e 82 pontos de amostragem em fazendas com sistemas de ordenha manual e mecânico, respectivamente. Diferença estatisticamente significativa foi encontrada entre os isolados observados entre a superfície dos tetos antes e após o pré-dipping (p=0,02), mas nenhuma diferença foi encontrada entre sistemas de ordenha ou estações (p>0,05). A possibilidade do mesmo padrão de Pseudomonas spp. estar distribuído em diferentes fazendas ou estações foi avaliada pela técnica de Polimorfismo do Tamanho de Fragmento Amplificado (AFLP). As mãos de ordenhadores, superfície dos tetos das vacas, teteiras e tanques de resfriamento foram associados com a contaminação do leite cru, demonstrando que independente do tipo de ordenha e estação, a higiene adequada de equipamentos, utensílios e mãos dos ordenhadores é essencial para evitar contaminação do leite, e consequentemente aumentar sua qualidade.(AU)

Inhibition of pRB Pathway Differentially Modulates Apoptosis in Esophageal Cancer Cells.

Esophageal cancer is the sixth most common cause of cancer-related death worldwide. Current chemotherapy regimens include a combination of 5-fluorouracil (5-FU) and cisplatin, but more efficient therapy strategies are needed to increase 5-year survival. Alterations in the signaling pathway of the tumor suppressor gene Rb-1, which encodes a phosphoprotein (pRB) that negatively regulates the G1/S transition of the cell cycle, are present in 70% of all tumors, but its role in esophageal cancer is still unclear. Most of these are alterations leading to up-regulation of the activity of cyclin-dependent kinases (CDKs) to phosphorylate pRB, which suggests that keeping the wild type pRB phosphorylated might be advantageous. Besides proliferation, pRB also regulates apoptosis induced by tumor necrosis factor-alpha (TNF-α) and DNA-damage. We investigated the status of phosphorylation of pRB along esophageal tumorigenesis stages, as well as whether hyperphosphorylation of pRB could suppress apoptosis induced by cisplatin, 5-FU, or TNF-α in esophageal cancer cells. pRB phosphorylation increased progressively from normal esophageal tissue to metaplasia and adenocarcinoma, suggesting that pRB phosphorylation increases along esophageal tumor stages. When RB-1 was knocked down or CDK inhibitors reduced the levels of phosphorylated pRB, opposite apoptotic effects were observed, depending on the combination of drugs tested: whereas TNF-α- and cisplatin-induced apoptosis increased, 5-FU-induced apoptosis decreased. Taken together, these data suggest that pRB plays a role in esophageal adenocarcinoma and that, depending on the type of anti-cancer treatment, combining CDK inhibitors and chemotherapy has the potential to increase the sensitivity of esophageal cancer cells to cell death.

Peripheral Organs of Dengue Fatal Cases Present Strong Pro-Inflammatory Response with Participation of IFN-Gamma-, TNF-Alpha- and RANTES-Producing Cells.

Dengue disease is an acute viral illness caused by dengue virus (DENV) that can progress to hemorrhagic stages leading to about 20000 deaths every year worldwide. Despite many clinical investigations regarding dengue, the immunopathogenic process by which infected patients evolve to the severe forms is not fully understood. Apart from differences in virulence and the antibody cross reactivity that can potentially augment virus replication, imbalanced cellular immunity is also seen as a major concern in the establishment of severe dengue. In this context, the investigation of cellular immunity and its products in dengue fatal cases may provide valuable data to help revealing dengue immunopathogenesis. Here, based in four dengue fatal cases infected by the serotype 3 in Brazil, different peripheral organs (livers, lungs and kidneys) were studied to evaluate the presence of cell infiltrates and the patterns of local cytokine response. The overall scenario of the studied cases revealed a considerable systemic involvement of infection with mononuclear cells targeted to all of the evaluated organs, as measured by immunohistochemistry (IHC). Quantification of cytokine-expressing cells in peripheral tissues was also performed to characterize the ongoing inflammatory process by the severe stage of the disease. Increased levels of IFN-γ- and TNF-α-expressing cells in liver, lung and kidney samples of post-mortem subjects evidenced a strong pro-inflammatory induction in these tissues. The presence of increased RANTES-producing cell numbers in all analyzed organs suggested a possible link between the clinical status and altered vascular permeability. Co-staining of DENV RNA and IFN-γ or TNF-α using in situ hibridization and IHC confirmed the virus-specific trigger of the pro-inflammatory response. Taken together, this work provided additional evidences that corroborated with the traditional theories regarding the "cytokine storm" and the occurrence of uneven cellular immunity in response to DENV as major reasons for progress to severe disease.

Case-control study of pathogens involved in piglet diarrhea.

BACKGROUND: Diarrhea in piglets directly affects commercial swine production. The disease results from the interaction of pathogens with the host immune system and is also affected by management procedures. Several pathogenic agents such as Campylobacter spp., Clostridium perfringens, Escherichia coli, Salmonella spp., group A rotavirus (RV-A), coronaviruses (transmissible gastroenteritis virus; porcine epidemic diarrhea virus), as well as nematode and protozoan parasites, can be associated with disease cases. RESULTS: All bacterial, viral, protozoan, and parasitic agents here investigated, with the exception of Salmonella spp. as well as both coronaviruses, were detected in varying proportions in piglet fecal samples, and positive animals were equally distributed between case and control groups. A statistically significant difference between case and control groups was found only for Cystoisospora suis (p = 0.034) and Eimeria spp. (p = 0.047). When co-infections were evaluated, a statistically significant difference was found only for C. perfringens ß2 and C. suis (p = 0.014). CONCLUSIONS: The presence of pathogens in piglets alone does not determine the occurrence of diarrhea episodes. Thus, the indiscriminate use of antibiotic and anthelminthic medication should be re-evaluated. This study also reinforces the importance of laboratory diagnosis and correct interpretation of results as well as the relevance of control and prophylactic measures.

Determination of metals in Brazilian soils by inductively coupled plasma mass spectrometry.

The concentration of metals in Brazilian soil under no-tillage (NT) and an area under native vegetation (NV) was determined by inductively coupled plasma mass spectrometry. The applied method was based on microwave-assisted acid digestion using HNO3, HCl, H2O2, and HF. The accuracy of the method was evaluated by analyzing two certified reference materials (BCR-142 and RS-3). The relative standard deviation for all target elements was below 8% indicating an adequate precision and the limit of detection ranged from 0.03 µg g(-1) (Cd) to 24.0 µg g(-1) (Fe). The concentrations of Al, As, Ba, Cd, Cu, Fe, Mg, Mn, Ni, Pb, Sr, and Zn in the different layers (0-10, 10-20, 20-40, and 40-60 cm) were determined in two types of soils, located in Paraná State in Brazil. The soil layers analysis revealed a different behavior of metals concentrations in soil samples under NT and NV. The obtained results showed a clear impact of anthropogenic action with respect to specific metals due to many years of uncontrolled application rates of limestone and phosphate fertilizers.

The pathology of severe dengue in multiple organs of human fatal cases: histopathology, ultrastructure and virus replication.

Dengue is a public health problem, with several gaps in understanding its pathogenesis. Studies based on human fatal cases are extremely important and may clarify some of these gaps. In this work, we analyzed lesions in different organs of four dengue fatal cases, occurred in Brazil. Tissues were prepared for visualization in optical and electron microscopy, with damages quantification. As expected, we observed in all studied organ lesions characteristic of severe dengue, such as hemorrhage and edema, although other injuries were also detected. Cases presented necrotic areas in the liver and diffuse macro and microsteatosis, which were more accentuated in case 1, who also had obesity. The lung was the most affected organ, with hyaline membrane formation associated with mononuclear infiltrates in patients with pre-existing diseases such as diabetes and obesity (cases 1 and 2, respectively). These cases had also extensive acute tubular necrosis in the kidney. Infection induced destruction of cardiac fibers in most cases, with absence of nucleus and loss of striations, suggesting myocarditis. Spleens revealed significant destruction of the germinal centers and atrophy of lymphoid follicles, which may be associated to decrease of T cell number. Circulatory disturbs were reinforced by the presence of megakaryocytes in alveolar spaces, thrombus formation in glomerular capillaries and loss of endothelium in several tissues. Besides histopathological and ultrastructural observations, virus replication were investigated by detection of dengue antigens, especially the non-structural 3 protein (NS3), and confirmed by the presence of virus RNA negative strand (in situ hybridization), with second staining for identification of some cells. Results showed that dengue had broader tropism comparing to what was described before in literature, replicating in hepatocytes, type II pneumocytes and cardiac fibers, as well as in resident and circulating monocytes/macrophages and endothelial cells.

Phylogenetic analyses of the VP4 and VP7 genes of porcine group A rotaviruses in Sao Paulo State, Brazil: first identification of G5P[23] in piglets.

This study determined the group A rotavirus occurrence in pig farms from 7 different cities in São Paulo State, Brazil. Out of 143 samples, 70 tested positive. Sequence analyses of 37 strains indicated that the strains had the G3, G5, G9, and P[6], P[13]/P[22]-like, and P[23] genotypes.

The use of electrothermal vaporizer coupled to the inductively coupled plasma mass spectrometry for the determination of arsenic, selenium and transition metals in biological samples treated with formic acid.

A fast method for the determination of As, Co, Cu, Fe, Mn, Ni, Se and V in biological samples by ETV-ICP-MS, after a simple sample treatment with formic acid, is proposed. Approximately 75 mg of each sample is mixed with 5 mL of formic acid, kept at 90°C for 1 h and then diluted with nitric acid aqueous solution to a 5% (v/v) formic acid and 1% (v/v) nitric acid final concentrations. A palladium solution was used as a chemical modifier. The instrumental conditions, such as carrier gas flow rate, RF power, pyrolysis and vaporization temperatures and argon internal flow rate during vaporization were optimized. The formic acid causes a slight decrease of the analytes signal intensities, but does not increase the signal of the mainly polyatomic ions ((14)N(35)Cl(+), (14)N(12)C(+), (40)Ar(12)C(+), (13)C(37)Cl(+), (40)Ar(36)Ar(+), (40)Ar(35)Cl(+), (35)Cl(16)O(+), (40)Ar(18)O(+)) that affect the analytes signals. The effect of charge transfer reactions, that could increase the ionization efficiency of some elements with high ionization potentials was not observed due to the elimination of most of the organic compounds during the pyrolysis step. External calibration with aqueous standard solutions containing 5% (v/v) formic acid allows the simultaneous determination of all analytes with high accuracy. The detection limits in the samples were between 0.01 (Co) and 850 µg kg(-1) (Fe and Se) and the precision expressed by the relative standard deviations (RSD) were between 0.1% (Mn) and 10% (Ni). Accuracy was validated by the analysis of four certified reference biological materials of animal tissues (lobster hepatopancreas, dogfish muscle, oyster tissue and bovine liver). The recommended procedure avoids plasma instability, carbon deposit on the cones and does not require sample digestion.

Universal scaling rules predict evolutionary patterns of myogenesis in species with indeterminate growth.

Intraspecific phenotypic variation is ubiquitous and often associated with resource exploitation in emerging habitats. For example, reduced body size has evolved repeatedly in Arctic charr (Salvelinus alpinus L.) and threespine stickleback (Gasterosteus aculeatus L.) across post-glacial habitats of the Northern Hemisphere. Exploiting these models, we examined how body size and myogenesis evolve with respect to the 'optimum fibre size hypothesis', which predicts that selection acts to minimize energetic costs associated with ionic homeostasis by optimizing muscle fibre production during development. In eight dwarf Icelandic Arctic charr populations, the ultimate production of fast-twitch muscle fibres (FN(max)) was only 39.5 and 15.5 per cent of that in large-bodied natural and aquaculture populations, respectively. Consequently, average fibre diameter (FD) scaled with a mass exponent of 0.19, paralleling the relaxation of diffusional constraints associated with mass-specific metabolic rate scaling. Similar reductions in FN(max) were observed for stickleback, including a small-bodied Alaskan population derived from a larger-bodied oceanic stock over a decadal timescale. The results suggest that in species showing indeterminate growth, body size evolution is accompanied by strong selection for fibre size optimization, theoretically allowing resources saved from ionic homeostasis to be allocated to other traits affecting fitness, including reproduction. Gene flow between small- and large-bodied populations residing in sympatry may counteract the evolution of this trait.

Development temperature has persistent effects on muscle growth responses in gilthead sea bream.

Initially we characterised growth responses to altered nutritional input at the transcriptional and tissue levels in the fast skeletal muscle of juvenile gilthead sea bream. Fish reared at 21-22°C (range) were fed a commercial diet at 3% body mass d(-1) (non-satiation feeding, NSF) for 4 weeks, fasted for 4d (F) and then fed to satiation (SF) for 21d. 13 out of 34 genes investigated showed consistent patterns of regulation between nutritional states. Fasting was associated with a 20-fold increase in MAFbx, and a 5-fold increase in Six1 and WASp expression, which returned to NSF levels within 16h of SF. Refeeding to satiation was associated with a rapid (<24 h) 12 to 17-fold increase in UNC45, Hsp70 and Hsp90α transcripts coding for molecular chaperones associated with unfolded protein response pathways. The growth factors FGF6 and IGF1 increased 6.0 and 4.5-fold within 16 h and 24 h of refeeding respectively. The average growth in diameter of fast muscle fibres was checked with fasting and significant fibre hypertrophy was only observed after 13d and 21d SF. To investigate developmental plasticity in growth responses we used the same experimental protocol with fish reared at either 17.5-18.5°C (range) (LT) or 21-22°C (range) (HT) to metamorphosis and then transferred to 21-22°C. There were persistent effects of development temperature on muscle growth patterns with 20% more fibres of lower average diameter in LT than HT group of similar body size. Altering the nutritional input to the muscle to stimulate growth revealed cryptic changes in the expression of UNC45 and Hsp90α with higher transcript abundance in the LT than HT groups, whereas there were no differences in the expression of MAFbx and Six1. It was concluded that myogenesis and gene expression patterns during growth are not fixed, but can be modified by temperature during the early stages of the life cycle.

The parallel evolution of dwarfism in Arctic charr is accompanied by adaptive divergence in mTOR-pathway gene expression.

Ecological factors have a major role in shaping natural variation in body size, although the underlying mechanisms are poorly understood. Icelandic Arctic charr (Salvelinus alpinus L.) populations represent an ideal model to understand body-size evolution, because adult dwarfism has arisen independently on multiple occasions in response to parallel environmental pressures. The mechanistic target of rapamycin (mTOR) pathway transmits signals from the environment to control cellular growth and is a primary candidate to be under selection for the dwarf phenotype. To test this hypothesis, we modified 'inputs' to this pathway in five dwarf and two generalist populations (with ancestral life history and body-size traits), using a standardized manipulation of food intake in a common environment. The skeletal muscle transcript levels of 21 mTOR-pathway genes were quantified in 274 individuals (∼6000 datapoints), and statistical modelling was used to elucidate sources of variation. Constitutive expression differences between populations were the main component of variation for around three-quarters of the studied genes, irrespective of nutritional-state and body-size phenotype. There was evidence for stabilizing selection acting among populations, conserving the nutritionally dependent regulation of pathway genes controlling muscle atrophy. There were three genes (mTOR, 4E-BP-1 and IGFBP4), where the expression variation between dwarf and generalist populations exceeded the between-population variation. Divergence in the expression of these candidate adaptive genes was most evident during a period of rapid growth following sustained fasting and was directionally consistent with their functions regulating growth and protein synthesis. We concluded that selection has operated efficiently to shape gene expression evolution in Icelandic charr populations and that the regulation of certain mTOR-pathway genes evolved adaptively in locations favouring dwarfism, resulting in reduced muscle protein accretion under growth-favouring conditions.

The synergistic interaction between morphine and maprotiline after intrathecal injection in rats.

BACKGROUND: Antidepressant drugs act as potent inhibitors of norepinephrine and/or serotonin reuptake and are widely used with opioids for the treatment of chronic pain. The mechanism of this increased analgesic action is unclear. We compared the antinociceptive effects of the intrathecal administration of morphine with that of a nonselective (amitriptyline) or selective (maprotiline or citalopram) antidepressant drug using the thermal withdrawal test in rats. We also investigated the possible mechanisms involved in the interactions of these drugs. METHODS: Male Wistar rats were anesthetized with sevoflurane and administered morphine and antidepressant drugs, or saline, through intrathecal injection. The antinociceptive effect was evaluated using the thermal withdrawal test before and after drug administration. The time for the withdrawal reaction was expressed as percentage of maximum possible effect (MPE). Animals were also pretreated with yohimbine (a nonselective alpha2-adrenergic antagonist) and naloxone (a nonselective opioid antagonist) for mechanism of action studies. Pharmacologic interaction was evaluated using isobolographic analysis of simultaneous administration of fixed proportions of maprotiline and morphine. RESULTS: Single intrathecal administration of morphine (2 microg), amitriptiline (125 microg), citalopram (144 microg), and maprotiline (1.25 microg) produced 51.6% +/- 8.9%, 10.3% +/- 3.2%, 33.8% +/- 5.2%, and 48.5% +/- 9.2% MPE, respectively. The antinociceptive effect of morphine was increased when combined with amitriptyline (91.3% +/- 4.6% MPE) and maprotiline (86.9% +/- 9.2% MPE) but not with citalopram (40.6% +/- 4.6% MPE). Coadministration of maprotiline increased the antinociceptive duration of morphine by 4-fold (from 120 to 480 min), which was reversed by pretreatment with the alpha-2 adrenoceptor inhibitor, yohimbine, and the mu-type opioid receptor antagonist, naloxone. Isobolographic analysis demonstrated a synergistic interaction between morphine and maprotiline. CONCLUSIONS: Selective norepinephrine reuptake inhibitors can significantly increase the intensity and duration of morphine antinociceptive activity via both alpha(2)-adrenergic and opioid receptors. This interaction was not observed with the selective serotonin inhibitor, citalopram.

Análise da prontidão para o tratamento em alcoolistas em um centro de tratamento / Analysis of the readiness for the treatment in alcoholics in a treatment center

O objetivo desta pesquisa foi verificar a prontidão para o tratamento em alcoolistas participantes de uma intervenção terapêutica, seguindo o Modelo Minnesota. Participaram desta pesquisa 25 alcoolistas, de grau moderado e grave, com idade entre 23 a 60 anos, de ambos os sexos. Foram aplicadas as escalas SADD e SOCRATES na primeira entrevista, no início do tratamento. Três entrevistas adicionais realizadas no meio, no final e um mês após o tratamento, avaliaram a prontidão para o tratamento por meio da escala SOCRATES. Os resultados mostraram que, embora tenha havido 64 por cento de adesão, não houve diminuição da ambivalência dos alcoolistas. Houve correlação significativa entre os fatores da escala SOCRATES. Esta pesquisa aponta para a necessidade de se utilizar técnicas motivacionais para a diminuição da ambivalência, visando o aumento na adesão e prevenção de recaída.

Rapid evolution of muscle fibre number in post-glacial populations of Arctic charr Salvelinus alpinus.

Thingvallavatn, the largest and one of the oldest lakes in Iceland, contains four morphs of Arctic charr Salvelinus alpinus. Dwarf benthic (DB), large benthic (LB), planktivorous (PL) and piscivorous (PI) morphs can be distinguished and differ markedly in head morphology, colouration and maximum fork length (FL(max)), reflecting their different resource specialisations within the lake. The four morphs in Thingvallavatn are thought to have been isolated for approximately 10 000 years, since shortly after the end of the last Ice Age. We tested the null hypothesis that the pattern of muscle fibre recruitment was the same in all morphs, reflecting their recent diversification. The cross-sectional areas of fast and slow muscle fibres were measured at 0.7 FL in 46 DB morphs, 23 LB morphs, 24 PL morphs and 22 PI morphs, and the ages of the charr were estimated using sacculus otoliths. In fish larger than 10 g, the maximum fibre diameter scaled with body mass (M(b))(0.18) for both fibre types in all morphs. The number of myonuclei per cm fibre length increased with fibre diameter, but was similar between morphs. On average, at 60 mum diameter, there were 2264 nuclei cm(-1) in slow fibres and 1126 nuclei cm(-1) in fast fibres. The absence of fibres of diameter 4-10 mum was used to determine the FL at which muscle fibre recruitment stopped. Slow fibre number increased with body length in all morphs, scaling with M(b)(0.45). In contrast, the recruitment of fast muscle fibres continued until a clearly identifiable FL, corresponding to 18-19 cm in the dwarf morph, 24-26 cm in the pelagic morph, 32-33 cm in the large benthic morph and 34-35 cm in the piscivorous morph. The maximum fast fibre number (FN(max)) in the dwarf morph (6.97x10(4)) was 56.5% of that found in the LB and PI morphs combined (1.23x10(5)) (P<0.001). Muscle fibre recruitment continued until a threshold body size and occurred at a range of ages, starting at 4+ years in the DB morph and 7+ years in the LB and PI morphs. Our null hypothesis was therefore rejected for fast muscle and it was concluded that the dwarf condition was associated with a reduction in fibre number. We then investigated whether variations in development temperature associated with different spawning sites and periods were responsible for the observed differences in muscle cellularity between morphs. Embryos from the DB, LB and PL morphs were incubated at temperature regimes simulating cold subterranean spring-fed sites (2.2-3.2 degrees C) and the general lakebed (4-7 degrees C). Myogenic progenitor cells (MPCs) were identified using specific antibodies to Paired box protein 7 (Pax 7), Forkhead box protein K1-alpha (FoxK1-alpha), MyoD and Myf-5. The progeny showed no evidence of developmental plasticity in the numbers of either MPCs or muscle fibres. Juveniles and adult stages of the DB and LB morphs coexist and have a similar diet. We therefore conclude that the reduction in FN(max) in the dwarf morph probably has a genetic basis and that gene networks regulating myotube production are under high selection pressure. To explain these findings we propose that there is an optimal fibre size, and hence number, which varies with maximum body size and reflects a trade-off between diffusional constraints on fibre diameter and the energy costs of maintaining ionic gradients. The predictions of the optimal fibre size hypothesis and its consequences for the adaptive evolution of muscle architecture in fishes are briefly discussed.

Reduction in muscle fibre number during the adaptive radiation of notothenioid fishes: a phylogenetic perspective.

The fish fauna of the continental shelf of the Southern Ocean is dominated by a single sub-order of Perciformes, the Notothenioidei, which have unusually large diameter skeletal muscle fibres. We tested the hypothesis that in fast myotomal muscle a high maximum fibre diameter (FD(max)) was related to a reduction in the number of muscle fibres present at the end of the recruitment phase of growth. We also hypothesized that the maximum fibre number (FN(max)) would be negatively related to body size, and that both body size and size-corrected FN(max) would show phylogenetic signal (tendency for related species to resemble each other). Finally, we estimated ancestral values for body size and FN(max). A molecular phylogeny was constructed using 12S mitochondrial rRNA sequences. A total of 16 species were studied from the Beagle Channel, Tierra del Fuego (5-11 degrees C), Shag Rocks, South Georgia (0.5-4 degrees C), and Adelaide Island, Antarctic Peninsula (-1.5 to 0.5 degrees C). The absence of muscle fibres of less than 10 micro m diameter was used as the criterion for the cessation of fibre recruitment. FD(max) increased linearly with standard length (SL), reaching 500-650 micro m in most species. Maximum body size was a highly significant predictor of species variation in FN(max), and both body size and size-corrected FN(max) showed highly significant phylogenetic signal (P<0.001). Estimates of trait values at nodes of the maximum likelihood phylogenetic tree were consistent with a progressive reduction in fibre number during part of the notothenioid radiation, perhaps serving to reduce basal energy requirements to compensate for the additional energetic costs of antifreeze production. For example, FN(max) in Chaenocephalus aceratus (12 700+/-300, mean +/- S.E.M., N=18) was only 7.7% of the value found in Eleginops maclovinus (164 000+/-4100, N=17), which reaches a similar maximum length (85 cm). Postembryonic muscle fibre recruitment in teleost fish normally involves stratified followed by mosaic hyperplasia. No evidence for this final phase of growth was found in two of the most derived families (Channichthyidae and Harpagiferidae). The divergence of the notothenioids in Antarctica after the formation of the Antarctic Polar Front and more recent dispersal north would explain the high maximum diameter and low fibre number in the derived sub-Antarctic notothenioids. These characteristics of notothenioids may well restrict their upper thermal tolerance, particularly for Champsocephalus esox and similar Channichthyids that lack respiratory pigments.