RESUMO
Research using stable isotopes analysis (SIA) of carbon (δ13 C) and nitrogen (δ15 N) in blood components is lacking, because of the challenge of sample collection, processing, and storage in remote areas. There also is a paucity of information regarding the effect of tissue biochemical composition on isotopic ratios with few comparisons among taxa. We collected blood samples from shortfin mako sharks (n = 70; 2016) and Guadalupe fur seals (n = 25; 2017). All samples were centrifuged to obtain plasma from sharks and serum from the Guadalupe fur seals, and all the samples were prepared for SIA and analyzed using a Costech 4010 elemental analyzer interfaced with a Delta V Plus isotope ratio mass spectrometer. We found significant differences between plasma δ13 C values of shortfin mako sharks (-17.6 ± 0.9) and serum of Guadalupe fur seals (-20.3 ± 1.2), but we did not find any differences for δ15 N values between the two species. The differences in δ13 C values between species are probably due to the specific blood composition and to the different biochemical characteristics and different adaptations within taxa. These findings highlight the importance of further research on the influence of biochemistry features on isotopic results, in this way a more accurate assessment will be possible for this factor, separating it from the dietary influences on stable isotopic values.
Assuntos
Isótopos de Carbono/sangue , Otárias/sangue , Isótopos de Nitrogênio/sangue , Tubarões/sangue , Animais , Feminino , Otárias/fisiologia , Masculino , Espectrometria de Massas , México , Tubarões/fisiologiaRESUMO
The knowledge of how temperature influences elasmobranchs reproductive physiology allows a better understanding of their reproductive patterns. This study describes the relationship between temperature fluctuations and the plasmatic changes of the sex steroids related to reproduction: testosterone (T), estradiol (E2) and progesterone (P4), throughout the female reproductive cycle of the shark Mustelus schmitti. A total of 123 adult females were bi-monthly sampled in Buenos Aires, Argentina, coastal waters. Bottom temperatures were recorded at each sampling point and blood samples were taken from each female for plasma sex steroids measurement. Sex steroid plasma levels were analyzed in relation with maximum follicular diameter (MFD), uterosomatic index (USI, as indicator of pregnancy) and temperature using Generalized Additive Models. Plasmatic E2 and T increased during follicular growth until MFD reached 1.34 and 1.46â¯cm, respectively. Peak of T occurred at the follicular stage associated with parturition (MFD, 1.4-1.6â¯cm), just prior to final maturation and ovulation (MFD, 1.6-2.0â¯cm). Progesterone significantly increased at this last ovarian phase, while T and E2 decreased. The increase of USI with pregnancy was associated to a decrease in T and mainly E2 levels, while P4 remained unaffected. Prior to ovulation, T plasma levels decreased with temperature below to 13⯰C and then increased progressively with a pronounced elevation above 17⯰C, while E2 presented an opposite pattern. Progesterone plasma levels changed with temperature showing a similar pattern to that observed for T. Using M. schmitti shark as model species, this study shows a clear picture of how seawater temperature variations can affect the reproductive physiology in elasmobranch females. A hypothetical mechanism (based on T elevation driven by temperature increase and its connection by feedback with a P4 rise and parturition/ovulation induction) is proposed as evidence to support that the increase in temperature can trigger reproductive events in elasmobranchs. In addition to its ecological scope, this work contributes to reinforce the relatively scarce general knowledge of elasmobranchs reproductive physiology.
Assuntos
Tubarões/fisiologia , Temperatura , Animais , Feminino , Geografia , Folículo Ovariano/crescimento & desenvolvimento , Ovulação/fisiologia , Reprodução/fisiologia , Tubarões/sangue , América do Sul , Esteroides/sangueRESUMO
Seasonal fluctuation in environmental parameters can influence immune responses of vertebrates and consequently influence their health and disease resistance. Although seasonality of immune function is well documented in a broad range of vertebrate taxa, this information remains virtually unexplored in cartilaginous fish. Here we examine seasonal variation in immune and general-health parameters of free-living adult broadnose sevengill sharks, Notorynchus cepedianus, along an annual cycle. We sampled sharks during autumn/winter (i.e., coolest temperatures and nonreproductive period) and spring/summer (i.e., warmest temperatures and active reproductive period) and assessed aspects of immunity, general condition, and reproductive hormone levels. A seasonal influence was observed in some, but not all, parameters evaluated. Lower lymphocyte counts and higher heterophil counts and granulocyte to lymphocyte (Gâ¶L) ratios were observed in sharks sampled during autumn/winter than in those sampled during spring/summer. On the other hand, total leukocyte counts, eosinophil counts, bacterial agglutination mediated by natural antibodies, and hematocrit did not vary seasonally. The observed seasonal patterns could be explained as (1) greater levels of stress based on the Gâ¶L ratio, (2) a sign of immunosuppression or depressed immune investment based on the low lymphocyte counts, and/or (3) a sign of ongoing infection based on the higher heterophil counts in the colder seasons with respect to the warmer ones. In addition, the pattern is in line with the notion that while acquired components are usually depressed by lower temperatures, some innate components might increase to offset that reduction. Immune and health-state parameters were mostly independent of reproductive hormone levels, providing little support for a trade-off with reproduction. Overall, the observed seasonal pattern in immunity of broadnose sevengill sharks could be related to changes in abiotic environmental condition, such as water temperature and photoperiod, although other factors such as availability of high-quality food may play a part.
Assuntos
Estações do Ano , Tubarões/imunologia , Testes de Aglutinação , Animais , Argentina , Escherichia coli/imunologia , Feminino , Hormônios Gonadais/sangue , Hematócrito , Contagem de Leucócitos , Masculino , Reprodução/fisiologia , Tubarões/sangueRESUMO
Sharks are very sensitive to stress and prone to a high mortality rate after capture. Since approximately 50 million of sharks are caught as bycatch every year, and current recommendations to reduce the impact of commercial fishing strongly support immediate release, it is imperative to better understand post-release mortality caused by the stress of capture and handling. Blood samples allow the assessment of stress levels which are valuable tools to reduce mortality in commercial, recreational and scientific fishing, being essential for the improvement in those conservation measures. Biochemical analyses are widely used for sharks as stress indicators, with secondary plasma parameters (lactate, glucose and ions) being the most often employed assays. However, it is virtually impossible to determine baseline plasma parameters in free-ranging sharks, since blood withdrawal involves animal capture and restrain, which are stressful procedures. This study aims at analyzing secondary parameters of five healthy tiger sharks captured with circular hooks and handlines in Fernando de Noronha (Northeastern Brazil) and comparing them with secondary parameters of three dead tiger sharks caught off Recife (also Northeastern Brazil). The results showed that the analysis of some plasma constituents in dead animals may be an efficient tool to assess stress and lethality. However, traditional parameters such as glucose and calcium, need to be used with caution. The results also demonstrated the extreme importance of urea and phosphorus for assessing stress response and mortality in tiger sharks, both parameters frequently neglected and of utmost importance for shark's homeostasis.
Assuntos
Tubarões/sangue , Estresse Fisiológico , Estresse Psicológico/sangue , Animais , Autopsia , Glicemia/análise , Proteínas Sanguíneas/análise , Cloretos/sangue , Feminino , Proteínas de Peixes/sangue , Concentração de Íons de Hidrogênio , Ácido Láctico/sangue , Masculino , Metais/sangue , Concentração Osmolar , Fósforo/sangue , Ureia/sangueRESUMO
In addition to conventional antibodies, cartilaginous fish have evolved a distinctive type of immunoglobulin, designated as IgNAR, which lacks the light polypeptide chains and is composed entirely by heavy chains. IgNAR molecules can be manipulated by molecular engineering to produce the variable domain of a single heavy chain polypeptide (vNARs). These, together with the VHH camel domains, constitute the smallest naturally occurring domains able to recognize an antigen. Their special features, such as small size, long extended finger-like CDR3, and thermal and chemical stability, make them suitable candidates for biotechnological purposes. Here we describe the generation of two mouse monoclonal antibodies (MAbs), MAb 370-12 and MAb 533-10, that both specifically react with vNAR domains of the horn shark Heterodontus francisci. While the former recognizes a broad spectrum of recombinant vNAR proteins, the latter is more restricted. MAb 370-12 precipitated a single band from whole shark serum, which was identified as IgNAR by mass spectrometry. Additionally, we used MAb 370-12 to follow the IgNAR-mediated immune response of sharks during immunization protocols with two different antigens (complete cells and a synthethic peptide), thus corroborating that MAb 370-12 recognizes both isolated vNAR domains and whole IgNAR molecules. Both MAbs represent an affordable molecular, biochemical, and biotechnological tool in the field of shark single-domain antibodies.
Assuntos
Anticorpos Monoclonais Murinos/genética , Proteínas de Peixes/genética , Região Variável de Imunoglobulina/genética , Imunoglobulinas/isolamento & purificação , Tubarões/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/metabolismo , Especificidade de Anticorpos , Células Cultivadas , Epitopos/química , Eritrócitos/imunologia , Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Humanos , Hibridomas/metabolismo , Imunidade Humoral , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/metabolismo , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Tubarões/sangueRESUMO
Starch gel electrophoresis pH 8.6, or PAGE pH 8.9, of the scalloped hammerhead shark hemolysates showed three hemoglobins (Hb). An additional Hb between the two most mobile electrophoretic components was seen in starch gel electrophoresis, pH 8.1, and also in highly loaded PAGE gels. The relative concentration of these Hbs was variable among individuals, when accessed at pH 8.1. Dilution of hemolysates led to a redistribution of the Hb tetramer subunits. Under denaturing conditions, the unfractionated hemolysate was resolved in 3 Hb subunits. Isolated Hbs, named SL I-SL IV, showed unusual subunit compositions: SL I, the least mobile, is "b3c"; SL II is "a2bc"; SL III and SL IV are composed only by "a" subunits. Hemoglobins in the whole hemolysate have an average of two reactive cysteines per tetramer, which were not easily S-thiolated by glutathione, as is the case for related species. After hemoglobin denaturation, six additional -SH groups were titrated by Ellman's reagent. Methemoglobin content was low in the erythrocytes of nine examined specimens, 1.13 +/- 1.90%. High values for total erythrocyte glutathione (GSH) were found: 4.5 +/- 0.7 mM; n = 7. The ratio of 1.4 +/- 0.4 GSH/Hb is higher than usually reported for mammalians.
Assuntos
Glutationa/sangue , Hemoglobinas/química , Tubarões/sangue , Compostos de Sulfidrila/análise , Animais , Eletroforese/métodos , Eritrócitos/química , Glutationa/análogos & derivados , Glutationa/química , Dissulfeto de Glutationa , Hematócrito , Concentração de Íons de Hidrogênio , Metemoglobina/análise , Metemoglobina/química , Amido , Compostos de Sulfidrila/químicaRESUMO
1. Resolution of the fraction of sandbar shark (Carcharhinus plumbeus) serum that was soluble in 50% saturated ammonium sulfate by gel-immobilized metal-affinity chromatography allowed the isolation of a novel disulfide-bonded heterodimer of intact mass 70 kDa. 2. Following reduction, the molecule could be resolved into two chains of apparent mass 36 and 24 kDa. 3. The molecules were glycoproteins as determined by an observed reduction in molecular weight following enzymatic glycosylation. 4. The two separate chains were related to one another on the basis of amino-acid composition analysis and by comparison of the N-terminal amino acids (seven out of 10 identities). 5. The exact relationship of this molecule to characterized heterodimers of higher vertebrates is unknown. 6. Cross-linked agarose-acetate was synthesized and proved to be an efficient concentrating agent and also a hydrophobic interaction adsorbant.