Relationship between respiration and Electron Transfer System (ETS) in zooplankton

Relationship between Respiration and Electron Transfer System (ETS) in zooplankton Relationship between Respiration and Electron Transport System (ETS) in zooplankton Alumna Amelia Iguanira López López Curso 2014/2015 Tutor Santiago Hernández León Trabajo de Fin de Grado para obtener el título de Graduada en Ciencias del Mar Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 1 Facultad de Ciencias del Mar Trabajo de Fin de Título Grado en Ciencias del Mar Relationship between Respiration and Electron Transfer System (ETS) in zooplankton Este trabajo se presenta por la alumna Amelia Iguanira López López para la obtención del título de Graduada en Ciencias del Mar por la Universidad de Las Palmas de Gran Canaria, y es dirigido por el Dr D. Santiago Hernández León del Instituto de Oceanografía y Cambio Global (IOCAG). Universidad de Las Palmas de Gran Canaria En Las Palmas a 30 de Noviembre de 2015 La alumna Fdo.: Amelia Iguanira López López El Director Fdo.: Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 2 Index Abstract 3 Introduction 4 Material and Methods 5 Results 7 Discussion 8 Conclusions 10 Acknowledgments 10 References 11 Personal assessments 22 Tables Index I: Average of values of respiration, ETS, R/ETS, and GF. 14 II: Experimental conditions, including date, density of food, temperature of acclimation and incubation. 15 III: The respiration rates obtained by other authors and our range of respiration rates estimated in this study for comparison. 16 IV: Average values of respiration rates and GF of experiments performed with copepods. 17 Figures Index 1: Time-course of gut fluorescence (ng pigments·ind-1) and standard error for the different experiments. 18 2: Time-course of respiration rates (µlO2·ind-1·h-1) and ETS activity (µlO2·ind-1·h-1) for each experiment. 19 3: Time-course of the respiration/ETS ratio and standard error for the different experiments. 20 4: Oxygen consumption and gut fluorescence of copepods in the 100-200 µm (A and C) and 500-1000 µm (B and D) size fractions 21 Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 3 Abstract Respiration of zooplanktonic organisms is an significant fraction of the global carbon cycle. However, it estimation in order to obtain the data required in oceanography is still a problem. In this work, we studied respiration rates in laboratory and field experiments. Laboratory experiments using Daphnia spp. showed a significant decrease of respiration rates during starvation. In addition, we measured the gut fluorescence and enzymatic activity (electron transfer system, ETS). The former did not show the expected decrease probably due to the volume of the incubators. The relationship between respiration and ETS presented the classical variability ranging between 0.5 and 1 as observed in previous works. Copepod respiration rates were measured during RAPROCAN 1504 cruise around the Canary Islands. These organisms showed a high variability in oxygen consumption during incubation. Our study suggests that duration experiments to calibrate enzyme activities to obtain physiological rates should be considered. We suggest the use of experiments covering the metabolic scope of organisms under study. Taking this into consideration, our values of the relationship between respiration and ETS activity is close to a value of 1. Lower ratios could underestimate respiration in the ocean. Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 4 Introduction The ocean plays an important role in the global carbon cycle. Atmospheric carbon dioxide enters into the natural cycle of oceans and remains there for a variable period of time. The photosynthetic organisms start the biological pump capturing the carbon dioxide and producing organic matter and oxygen. This mechanism transport carbon dioxide from the ocean surface to the deep layers. Heterotrophic organisms, or consumers, take the organic matter from other organisms and use it as fuel for their metabolism. These organisms respire the metabolized carbon and return carbon dioxide to the environment. Understanding the mechanisms linked to respiration in the ocean are a fundamental step to study the carbon flux through the different communities. Therefore, the knowledge of oxygen consumption is essential for the study of the involved processes in production and energy transfer in the ocean due to the different components of marine ecosystems. The methods used to estimate respiration in heterotrophic organisms, specially in zooplanktonic organisms, are based on the measurement of oxygen consumption. The balance method (Omori and Ikeda, 1984) is normally used and consist in the incubation of zooplanktonic organisms in glass bottles filled with filtered seawater for several hours. During incubation, dissolved oxygen in experimental bottles (with organisms) decreases respect to control bottles (without organisms). The difference between both is the organisms respiration. This technique needs an intensive work on board oceanographic vessels and consume time, limiting the number of estimations. Because of this, Curl and Sandberg (1961) and Packard (1969) proposed the measurement of the enzymatic activities responsible of cellular respiration as an index to be used in oceanography. The measurement of the electron transfer system activity (ETS) was developed by Packard (1975) and it is also routinely used in oceanography. The ETS is the last enzymatic complex which takes part in the production of ATP and uses the oxygen as the last electron acceptor. However, the relationship between respiration and ETS presents an important variability. Hernández-León and Gómez (1996) observed that the Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 5 relationship between respiration and ETS activity presented the classic variability between 0.5 and 1 related to the previous feeding history. These authors also found that during the winter bloom (relatively high primary production) the R/ETS ratio was around 1, while out of the bloom, in a more oligotrophic situation, the values were near 0.5. Besides, this variability could also be explained because of the methology used to assess respiration rates using the classical balance method and the enzymatic response at different time intervals. Long or short incubations could modify this ratio as ETS activity is more conservative than respiration. In the present work, we studied respiration in the cladoceran Daphnia spp. and copepods, its relationship with the gut fluorescence (GF) and the enzymatic activity of the ETS. The main goal was to study the variability in respiration rate and the enzyme activity in experiments of short duration (8 hours). Specifically, we focused in the relationship between high respiration rates during the first hours of incubation and its enzymatic response. Material and methods Laboratory experiments The cladocerans used for experiments were incubated for 12 hours in a beaker of 2 liters at a temperature of 17.8ºC ± 0.1 in saturation of food with the algae Ankistrodesmus. Its density was counted with a Thoma device in an inverted microscope, taking an aliquot of 10 µl of the same culture (Table I). After the feeding period, 15 organisms were frozen in liquid nitrogen in order to obtain the time zero in each experiment for enzyme activity. To measure respiration, the organisms previously incubated in food saturation were incubated in filtered water (0.2 µm) in vials of 5 ml equipped with optodes. Incubations were carried out in triplicate for each incubation time, in the dark, and at 18ºC. The incubation lasted for approximately 8 hours. At different intervals, samples were frozen in liquid nitrogen for later analysis of ETS and gut fluorescence (GF). To obtain the values of respiration we designed an application using the statistical software R. The values of the first half an hour were discarded as it is the time required for stabilization of oxygen inside the incubators. Then, the slopes of experiment and control bottles were subtracted in order to obtain respiration every 0.5 h. Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 6 ETS analyses were carried out following the method proposed by Packard (1971). The homogenate was centrifuged at 4000 rpm and 0ºC during 10 minutes. An aliquot of the homogenate was used for the incubation at 18ºC, in darkness, in the presence of NADH, NADPH, succinate (SS) and INT as an artificial electron acceptor. After 20 minutes, the incubation was stopped by a quench solution. The ETS activity was estimated spectrophotometrically at 490 nm with a turbidity base-line of 750 nm. Biomass of each size fraction was determined as protein content using the method of Lowry et al. (1951) or the method of Peterson (1983) for samples with very low protein content and using bovine serum albumine (BSA) as standards. For the measurement of fluorescence, we used the method proposed by Nemoto (1968) and Mack and Bohrer (1976). An aliquot of the homogenate used for ETS measurement was introduced in a test tube with 10 ml of 90% acetone and kept at -20ºC during 24 hours. Pigments were measured using a Turner Design fluorometer, previously calibrated with pure chlorophyll (Yentsch and Menzel, 1963), before and after acidification with 10% HCl. Pigments were calculated using the equations given by Strickland and Parson (1972) slightly modified to: Chlorophyll a = k· (F0-Fa) · Individual-1 Pheopigments = k· (R· Fa-F0) · Individual-1 where k is the instrument calibration constant, F0 and Fa are the fluorescence readings before and after acidification and R is the acidification coefficient. The overall concentration of pigments refers to chlorophyll a and pheopigments expressed as ng pigments·ind-1. Field experiments Copepods were collected in six oceanographic stations on board the research vessel “Ángeles Alvariño” around the Canary Islands from 6th to 16th April, 2015. Capture of organisms was carried out using a double WP-2 net equipped with a 100 µm mesh net, from 200 m depth to the surface at about 40 m·minute-1. Three experiments were carried out with organisms of sizes between 100 and 200 µm and three other with copepods between 500 and 1000 µm. The organisms were picked up with a Pasteur pipette and Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 7 were incubated for triplicate in the dark at 18ºC for a total of 4 hours on seawater filtered through 0.2 µm. To measure respiration, was used a precision polarographic oxygen electrode (VWR DO210). Once experiments were completed the difference between the oxygen content in the control and experimental bottles was measured in order to obtain the respiration rate. These data were standardized for the protein weight of incubated organisms. Similar to the experiments carried out in the laboratory we graphed the results using the statistical program STATISTICA 7. Results Laboratory experiments Pigment content in the different experiments decreased during incubation (Fig. 1). However, this decrease was weak and variable (see Fig. 1b and d), and, in general, we did not observe a clear decrease as expected (Fig. 1f). By opposite, respiration rates showed a significant decrease during the first four hours of the experiment. The initial oxygen consumption ranged between 0.9 and 1.4 µlO2·ind-1·h-1 decreasing in all experiments reaching values of 0.27±0.01, µlO2·ind-1·h-1 after 3-4 h (Table II, Fig. 2). Therefore, the initial respiration was between 3 and 5 times the standard rates obtained after four hours. The electron transfer system activity (ETS) did not show high values at the beginning of the experiments as observed for respiration (Fig. 2). This activity showed values in the range 0.2-0.5 µl·O2·ind-1·h-1, being the average in the order of 0.36±0.04 (see Table II) As expected from respiration values, the ratio R/ETS showed a clear decline from the beginning of incubation stabilizing after the first four hours (Fig. 3). The values of the ratio decreased to an average value of 0.29±0.05 (see table II) after the first four hours of incubation. Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 8 Field experiments Small organisms (100-200 µm) showed relatively high values of gut pigment content (mean value of 197.2±106.2 ng pigments/mg protein, table III, Fig. 4a), while larger copepods (500-1000 µm) showed lower average values (93.9±38.1 ng pigments/mg of protein, table III, Fig. 4b). Small organisms showed a high variability in the gut content during incubation. However, the 500-1000 µm organisms showed an unexpected increase. (Fig. 4). Respiration in both size classes showed a decreasing respiration during incubation as expected. Average values for the 100-200 µm fraction was 0.58±0.28 µlO2·mg protein·h-1 (table III, Fig. 4C), while for the larger animals (500-1000 µm) was lower as also expected (0.31±0.06 µlO2·mg protein·h-1, table III, Fig. 4d). Discussion Respiration rates in our experiments with cladocerans decreased during incubation similar to other studies (Nival et al., 1974; Mayzaud, 1976; Skjoldal et al., 1984; Blažka et al., 1982; Abou, 1984; Ikeda, 1977; Tsuda, 1994; Thor, 2003). In the field experiments, copepods did not show this pronounced decrease probably due to the fact that they were not previously acclimated in food saturation. The results in cladocerans is in agreement with authors who argue that the decrease in metabolic rates after the start of incubation is due to food shortage rather than stress produced by the handling of organisms (Ikeda, 1977; Ikeda and Skjoldal 1980). Our average respiration for all experiments with Daphnia over 8 hours (0.32 ± 0.39 µlO2·ind-1·h-1) matched the values found by Valenciano (2010). In her experiments, standard metabolic rate was reached after 3.5 hours (mean value 0-3.5 h: 0.41 ± 0.07 µlO2·ind-1·h-1). In our study, this average value was quite similar (0.40 ± 0.10 µlO2·ind1·h-1). After four hours until the end of the experiments, respiration rate was 0.27 ± 0.01 µlO2·ind-1·h-1 (table I), while Valenciano (2010) obtained a rate of 0.29 ± 0.11 µlO2·ind-1·h-1. For copepods, we observed a high variability in the respiration rates for the six experiments (average total value of 0.44 ± 0.23 µlO2·mg protein-1·h-1). Valenciano (2010) also found a decrease in respiration rates during one hour, although the number of experiments performed by this author was quite short. Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 15 Table II. Experimental conditions, including date, density of food (cell/ml), temperature of acclimation and incubation. Total Resp Total ETS Total R/ETS Total GF Exp Date tª cells/ml tª inc. 0-8 h 0-8 h 0-8 h 0-8 h 1 19/03/15 17.9 1.5·106 18 0.26 ± 0.35 0.31 ± 0.09 0.71 ± 0.71 120.61 ± 45.99 2 20/03/15 17.9 1.5·106 18 0.38 ± 0.46 0.33 ± 0.12 0.95 ± 1.22 131.54 ± 47.08 3 23/03/15 17.8 1.5·106 18 0.31 ± 0.36 0.34 ± 0.13 1.26 ± 2.17 5.97 ± 6.58 4 24/03/15 17.8 5·105 18 0.35 ± 0.44 0.37 ± 0.09 0.87 ± 1.17 254.54 ± 98.43 5 25/03/15 17.9 5·105 18 0.28 ± 0.34 0.41 ± 0.10 0.58 ± 0.72 104.41 ± 32.36 TOTAL EXPERIMENTS 0.32 ± 0.39 0.35 ± 0.04 0.87 ± 0.26 123.41 ± 88.63 Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 16 Table III. Comparison of respiration rates (µlO2·ind-1·h-1) obtained by different authors in relation to results obtained in this study. References Organisms Respiration rates Units Temperature Acclimation time Ikeda (1976) Subtropical copepods 0.46-2.12 µlO2 ind-1 h-1 22ºC 36 hours Ikeda (1977b) Acartia tonsa 0.089-0.181 µlO2 ind-1 h-1 22ºC None Schmoker and Hernández-León (2003) Cladoceran (D. Magna) 0.5-4 µlO2 ind-1 h-1 19ºC 24 hours Li et al. (2004) Zooplankton 0.64-0.84 µlO2 ind-1 h-1 18-27ºC None Present study D. magna 0.2-1.3 µlO2 ind-1 h-1 17-18ºC 12 hours Copepods 0.27-0.78 µlO2 mg protein h-1 17-18ºC None Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 17 Table IV. Average values of respiration rates (µlO2·ind-1·h-1) and gut fluorescence (ng pigments·ind-1) of experiments performed with copepods COPEPODS EXPERIMENTS Respiration GF 0-3 h 0-3 h 100 µm 1 0.69 ± 0.94 303.22 ± 386.74 2 0.26 ± 0.29 197.45 ± 105.29 3 0.78 ± 0.39 90.87 ± 109.99 TOTAL 100 µm 0.58 ± 0.28 197.18 ± 106.18 500 µm 4 0.27 ± 0.32 115.16 ± 150.94 5 0.38 ± 0.32 49.85 ± 61.26 6 0.27 ± 0.25 116.59 ± 108.91 TOTAL 500 µm 0.31 ± 0.06 93.87 ± 38.13 TOTAL EXPERIMENTS 0.44 ± 0.23 145.5 ± 91.1 Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 18 Figure 1. Time-course of gut fluorescence (ng pigments·ind-1) and standard error for the different experiments. Figure 1A corresponds to experiment 1, B for experiment 4, C for the number 2, D for 5, E for 3, and F for 6. Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 19 Figure 2. . Time-course of respiration rates (µlO2·ind-1·h-1) and ETS activity (µlO2·ind-1·h-1) for each experiment. Figure 2A corresponds to experiment 1, B for experiment 4, C for the number 2, D for 5, E for 3, and F for 6. Vertical lines are standard error Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 20 Figure 3. Time-course of the respiration/ETS ratio and standard error for the different experiments. Figure 3A corresponds to experiment 1, B for experiment 4, C for the number 2, D for 5, E for 3, and F for 6. Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 21 Figure 4. Oxygen consumption and gut fluorescence of copepods in the 100-200 µm (A and C) and 500-1000 µm (B and D) size fractions Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 22 VALORACIÓN PERSONAL (PERSONAL ASSESSMENTS)  Descripción detallada de las actividades desarrolladas durante la realización del TFT Para poder abarcar sobre este punto, es necesario destacar que el TFT se realizó con todos los datos que se obtuvieron de las distintas prácticas realizadas tanto en el laboratorio como a bordo del buque oceanográfico, BIO Ángeles Alvariño. Estos datos fueron tratados de tal forma que son los resultados de los que en este estudio hablamos. Por tanto, se trata de un estudio experimental en el que hemos unido la realización de las prácticas con el Trabajo de fin de título. Durante la realización del TFT, se realizaron búsquedas de artículos científicos, los cuales fueron leídos para poder encontrar correlación con nuestro estudio y para saber qué diferencia aportaban nuestros experimentos a los ya realizados anteriormente. Además, como ya se ha mencionado anteriormente, se estudiaron casos de respiración tanto en el laboratorio como en el barco. Comenzando con los experimentos que se realizaron en el laboratorio, es importante señalar que era necesario dejar a los organismos en saturación de alimento, durante un periodo de 12 horas, hasta el día siguiente que era cuando se realizaban las medidas del consumo de oxígeno. Además, mientras los individuos permanecían incubados, se realizaba el contaje del número de células por mililitro que se le suministraba. Una vez acabados los experimentos, se limpiaba todo el material y se ordenaba el lugar de trabajo. Para finalizar, hablamos de los ensayos realizados a bordo del barco. En estos ensayos medíamos el consumo de oxígeno con un oxímetro diferente, puesto que éste nos daba medidas puntuales. El número de botellas donde se incubaban era menor a las de los estudios en laboratorio, pero de un volumen más grande (250 ml). Además, los organismos eran recién tomados de su medio natural y pasados a las botellas de incubación, por tanto, no conocemos con seguridad si venían de estar en saturaciones de alimento o no. Aunque este dato podríamos conocerlo debido a las condiciones oceanográficas, ya que las pescas para estos estudios fueron lanzadas en áreas más oligotróficas, lejanas al afloramiento. Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 23  Formación recibida (cursos, programas informáticos, etc.) En este apartado podemos destacar que si bien previamente se tenía conocimiento de cómo trabajar en un laboratorio en cuanto a orden, pulcritud y vestimenta, tuve fallos que poco a poco fui corrigiendo. Así mismo, en cuanto a cómo tratar algunos datos, se proporcionó también bastante información nueva. En cuanto al trabajo en el laboratorio, antes de realizar cualquier tipo de análisis, se me explicaba con detalle cómo funcionaban los instrumentos que se iban a utilizar en cada momento. Además, en el caso de la utilización del oxímetro con el que obtuve los valores de respiración para las Daphnia spp., se me facilitó un disco duro desde el que pude descargarme el software del programa que necesitaba dicho instrumento. Por otra parte, los datos que proporcionaba el oxímetro señalado anteriormente, fueron tratados con el programa Excel, con el que ya había trabajado. Sin embargo, antes de obtener ningún dato de mis experimentos, mi tutor me proporcionó otros datos con los que tuve que manejar en el programa estadístico R. Esto me ayudó a familiarizarme con esta herramienta que más adelante utilizaría con mis propios datos. Cabe destacar la formación que recibí con otros programas estadísticos, como el STATISTICA, con el cual obtuve los gráficos representados en el trabajo.  Nivel de integración e implicación dentro del departamento y relaciones con el personal. La integración en el departamento se puede considerar totalmente satisfactoria, puesto que desde el principio los técnicos del laboratorio se mostraron atentos ante cualquier duda que se me pudiera ocurrir en cuanto a la utilización de material, al lavado o simplemente a la colocación del mismo en el laboratorio. Además de ayudarme al traslado de materiales desde los laboratorios de la Facultad hasta los laboratorios de Taliarte, concretamente al IOCAG (Instituto de Oceanografía y Cambio Global). Además, obtuve el apoyo del equipo de investigación que trabaja dentro de esta institución como profesores, estudiantes de doctorado, personal de trabajo del instituto en general, desde el servicio de limpieza por suministrarme las llaves para acceder a los laboratorios hasta el servicio de seguridad por abrirme las puertas del parking y dejarme acceder los días que no se trabajaba en el instituto pero que yo tenía que ir para poder realizar los experimentos. Relationship between Respiration and Electron Transfer System (ETS) in zooplankton 24 Considero que he estado totalmente implicada en todas las tareas que he hecho a lo largo del estudio, ya que se me ha explicado cómo hacerlas y si bien en muchas ocasiones he fallado, a las siguientes lo he corregido y eso me ha hecho ganar conocimiento. En cuanto a las relaciones con el personal puedo decir que he aprendido de todos y cada uno de los que me han ayudado de una forma u otra en cada momento.  Aspectos positivos y negativos más significativos relacionados con el desarrollo del TFT En este apartado se debe ser más objetivo si cabe, ya que, es una forma de mejorar y así ayudar a los futuros estudiantes de TFT. En primer lugar, sobre los aspectos negativos durante el desarrollo del Trabajo de Fin de Título, al igual que se expuso en la memoria de prácticas externas, podemos decir que lo más destacado de este punto es el hecho de acudir durante días no lectivos hasta los laboratorios de la facultad o de Taliarte, para dar comienzo al experimento. Sin embargo, de este rasgo negativo he concluido en muchas cosas positivas. En cuanto al aprendizaje positivo durante el desarrollo de este trabajo, cabe destacar sin ninguna duda que el hecho de realizar un trabajo experimental contiene muchos aspectos positivos, puesto que además de ganar en conocimiento, he aprendido a ser paciente, ya que para llegar a obtener unos buenos resultados, realicé muchos ensayos anteriores.  Valoración personal del aprendizaje conseguido a lo largo del TFT. Como valoración personal puedo decir con certeza que la realización del Trabajo de Fin de Título, junto con las demás asignaturas de la carrera, me ha hecho crecer como persona. No sólo me he enriquecido académicamente después de tanta búsqueda de bibliografía, lecturas, prácticas, trabajos, etc, sino que además en lo personal me ha hecho crecer mucho, implantando un poco en mí una gran virtud de la que carecía, la paciencia. Para mí el TFT no sólo ha sido una asignatura más de la carrera, ha sido una etapa de mi vida, en donde he vivido momentos buenos y momentos malos. Puesto que lo he pasado mal cuando las cosas que he hecho no me han salido bien, en la que he