Comparing the response of the brown shrimp Crangon crangon (Linnaeus, 1758) to prolonged deprivation of food in two seasons

Comparing the response of the brown shrimp Crangon crangon (Linnaeus, 1758) to prolonged deprivation of food in two seasons Cláudia Filipa Devesa André Moreira Dissertação Mestrado em Recursos Biológicos Aquáticos Porto 2012 Faculdade de Ciências da Universidade do Porto Mestrado em Recursos Biológicos Aquáticos Comparing the response of the brown shrimp Crangon crangon (Linnaeus, 1758) to prolonged deprivation of food in two seasons Cláudia Filipa Devesa André Moreira Orientador: Prof. António Paulo Carvalho Co-orientador: Dra. Joana Campos Dissertação submetida à Faculdade de Ciências UP como requisito parcial para obtenção do grau de Mestre em Recursos Biológicos Aquáticos. Porto 2012 FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons II To my family and friends who were deprived of me for so long. To all my friends that help me achieve. Thank you all, you are amazing! FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons III Agradecimentos Desejo agradecer a todas as pessoas que contribuíram para a realização deste trabalho. Em primeiro lugar gostaria de agradecer á Doutora Joana Campos, pela forma como orientou o meu trabalho, pelo tempo que me dedicou e pelos conhecimentos e boa disposição que me transmitiu. Ainda ao professor António Paulo Carvalho pela sua disponibilidade e paciência na orientação deste trabalho. Gostaria ainda de agradecer a toda a equipa do Biotério de Organismo Aquáticos (BOGA) do CIIMAR, dirigido pelo Doutor Hugo Santos e pelos seus colaboradores Olga Martinez e Ricardo Lacerda, por disponibilizarem espaço e material, bem como pelo tempo despendido para me orientarem e aconselharem no processo experimental e pela sua boa disposição e pensamento positivo. Obrigado ainda a Maria João Almeida pela sua ajuda e apoio. Um bem-haja à Patrícia e ao José pelo apoio, força e pelos momentos divertidos passados na sua companhia. Quero agradecer ainda ao pessoal do Aquamuseu do Rio Minho pela simpatia com que sempre me receberam, em especial ao técnico Eduardo Martins pela sua ajuda no trabalho de campo. Gostaria ainda de agradecer a todo o departamento de Nutrição da Faculdade de Ciências da Universidade do Porto por me receberem e disponibilizarem todos os meios necessários à realização do trabalho laboratorial. Por fim, um grande obrigado á minha família, a minha mãe Maria Rosa e o meu irmão Flávio pelo apoio incondicional. E ao David Cruz por todo o apoio, paciência e boa disposição em tempos que nem sempre foram fáceis! FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons IV Abstract Crustaceans often undergo periods of starvation, due to natural food shortage or physiological aspects. During these periods several metabolic and behavioral changes can occur. This study evaluates how the brown shrimp Crangon crangon (L.) responds to prolonged deprivation of food in two seasons of the year, and how this species mobilizes its energetic reserves. Shrimps caught in June (summer) and October (autumn) 2010 in Minho estuary (North of Portugal) were placed in individual cages in experimental aquaria and kept in starvation until the last shrimp died or was sacrificed (six shrimps per aquarium every week). The caloric content, total lipids and total proteins, and the oxygen consumption rate were compared between seasons, sacrificed and naturally dead shrimps, and weeks of starvation. Summer shrimps were proven to be better prepared to endure stressful situations than those caught in autumn: they survived 2.5 times longer, had a higher Fulton’s condition factor and higher caloric, lipid and protein content at the beginning of the experiments. During the first week of starvation the percentage of total proteins decreased significantly and stabilized in the next four weeks to decrease again abruptly in the fifth week. The percentage of total lipids only started to decrease after four weeks. This suggests that, on one hand, C. crangon probably uses stored proteins as a first energetic recourse and after that carbohydrates and eventually lipids, but at much lesser extent; and on the other hand, that after four weeks under starvation a critical point is reached when structural components might be mobilized to pay for maintenance costs. Keywords: Energy reserves, Crangon crangon, starvation, biochemical analysis, oxygen consumption rate. FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons V Resumo Os crustáceos frequentemente enfrentam períodos de jejum devido à escassez natural de alimento ou a processos fisiológicos. Durante estes períodos podem ocorrer diferentes alterações metabólicas e comportamentais. Este estudo avalia como o camarão mouro Crangon crangon (L.) reage a períodos prolongados de jejum em duas épocas do ano e como esta espécie mobiliza as suas reservas energéticas. Os camarões foram capturados em Junho (verão) e Outubro (outono) de 2010 no estuário do Rio Minho (Norte de Portugal), mantidos individualmente em jejum nos aquários experimentais até que o último camarão morreu ou foi sacrificado (seis camarões por aquário todas as semanas). Os resultados referentes ao conteúdo calórico, lípidos totais, proteínas totais assim como o consumo de oxigénio foram comparados entre estações do ano, entre animais sacrificados e mortos naturalmente e entre semanas de jejum. Verificou-se que os camarões de verão se encontravam em melhores condições para enfrentar situações de stress do que os camarões capturados no outono: sobreviveram mais tempo, tinham um índice de condição de Fulton mais elevado e conteúdo calórico, lípidos e proteínas totais mais elevados no início das experiências. Durante a primeira semana de jejum a percentagem de proteínas totais diminui significativamente estabilizando nas seguintes até voltar a descer abruptamente na quinta semana. A percentagem de lípidos totais apenas decresceu na quarta semana de jejum. Estes dados sugerem que, por um lado as proteínas são a principal reserva energética desta espécie, seguidas de hidratos de carbono e só muito eventualmente lípidos; por outro lado depois de quatro semanas de jejum é atingido um ponto crítico a partir do qual os componentes estruturais provavelmente começam a ser mobilizados para suportar os custos de manutenção corporal. Palavras-chave: Reservas energéticas, Crangon crangon, jejum, análises bioquímicas, consumo de oxigénio. FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 1 Contents List of tables ................................................................................................................................ 2 List of figures ............................................................................................................................... 3 List of abbreviations ................................................................................................................... 5 Introduction .................................................................................................................................. 6 Chapter 1: Species description ............................................................................................ 6 Chapter 2: Availability of food ............................................................................................... 8 Chapter 3: Energetic reserves .............................................................................................. 9 Determination of an animal’s reserves ............................................................................ 9 Objectives .................................................................................................................................. 10 Material and Methods .............................................................................................................. 11 Starvation experiment .......................................................................................................... 11 Oxygen consumption rate ................................................................................................... 14 Biochemical analysis ............................................................................................................ 15 Data treatment ...................................................................................................................... 16 Results ....................................................................................................................................... 17 Natural mortality .................................................................................................................... 17 Oxygen consumption ........................................................................................................... 18 Dry weight, percentage of water and Fulton’s Condition factor ..................................... 20 Fulton’s Condition factor .................................................................................................. 25 Total lipids, total proteins and caloric content .................................................................. 27 Discussion ................................................................................................................................. 36 References ................................................................................................................................ 42 Appendix .................................................................................................................................... 48 FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 2 List of tables Table 1: Number of shrimps and their mean (±se) total length (TL) and wet weight (WW); mean (±se) temperature and salinity; and duration of both experiments; ANOVA results with significant differences in bold Table 2: Time till half of the shrimps were dead and maximum time in starvation in each aquarium Table 3: Tukey’s pairwise comparisons results on the oxygen consumption rate in the experiment I Table 4: Mean (±se) dry weight (DW, g) of the naturally dead and sacrificed shrimps in both experiments Table 5: Tukey’s pairwise comparisons results on the dry weight (DW)Table 6: Tukey’s pairwise comparisons results on the dry weight (DW) and percentage of water in the experiment I Table 7: Mean (±se) Fulton’s condition factor of the naturally dead and sacrificed shrimps at the beginning of the experiments Table 8: Mean (±se) Fulton’s condition factor of the naturally dead and sacrificed shrimps in the end of the experiments Table 9: Turkey’s pairwise comparisons results on the Fulton’s condition factor in the experiment I Table 10: Mean (±se) percentage of total lipids (%) of the naturally dead and sacrificed shrimps in both experiments Table 11: Mean (±se) percentage of total proteins (%) of the naturally dead and sacrificed shrimps in both experiments Table 12: Tukey’s pairwise comparisons results on the total proteins Table 13: Mean (±se) caloric content (cal.g-1) of the naturally dead and sacrificed shrimps in both experiments Table 14: Tukey’s pairwise comparisons results on the total proteins of the sacrificed shrimps in the experiment I Table 15: Tukey’s pairwise comparisons results on the total proteins of the naturally dead shrimps in the experiment I Table 16: Mean (±se) caloric content (cal.g-1) per week of the sacrificed shrimps in both experiments Table 17: Summary of the results in both experiments FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 3 List of figures Figure 1: Distribution of the brown shrimp Crangon crangon (after J. Campos 2009) Figure 2: Upper – Minho River estuary in the North of Portugal; Lower – detail of Coura saltmarsh (Google maps) Figure 3: Beam-trawl used to collect the shrimps for the experiments Figure 4: Individual cages for the shrimps Figure 5: Oxygen consumption rate (mg O2.g-1.h-1) in both experiments (* and ° are outliers) Figure 6: Oxygen consumption rate (mg O2.g-1.h-1) per week in the experiment I (* are outliers) Figure 7: Oxygen consumption rate (mg O2.g-1.h-1) per week in the experiment II; only one shrimp was available in the fourth week (* and ° are outliers) Figure 8: Oxygen consumption rate (mg O2.g-1.h-1) in relation to the wet weight (g) of the shrimps in both experiments Figure 9: Oxygen consumption rate (mg O2.g-1.h-1) in relation to the total length (mm) of the shrimps in both experiments Figure 10: Mean dry weight (DW, g) of the naturally dead and sacrificed shrimps in both experiments (* and ° are outliers) Figure 11: Percentage of water of the naturally dead and sacrificed shrimps in both experiments (* and ° are outliers) Figure 12: Mean dry weight (DW, g) of the naturally dead and sacrificed shrimps per aquarium (* and ° are outliers) Figure 13: Mean percentage of water of the naturally dead and sacrificed shrimps per aquarium (* and ° are outliers) Figure 14: Dry weight (DW, upper) and water content (%, lower) along the weeks of starvation in the experiment I (* and ° are outliers) Figure 15: Percentage of water per week in experiment II (* and ° are outliers) Figure 16: Initial Fulton’s condition factor of the shrimps in both experiments (* are outliers) Figure 17: Initial Fulton’s condition factor of the shrimps in each aquarium (* are outliers) Figure 18: Final Fulton’s Condition factor of the shrimps in both experiments (a) and in each aquarium (b) (* and ° are outliers) FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 10 Artificially induced fasting and starvation experiments might be the only way to determine the energy reserves and to enlighten the metabolic routes (in hierarchical order) used during fasting. These experiments have been used to describe the biochemical and physiological adaptation as well as to determine the energetic requirements of several crustaceans (Guderley et al. 2003, Comoglio et al. 2005, Comoglio et al. 2008, Zhang et al. 2009). In these experiments it is assumed that a well fed animal will have plenty of reserves, while an animal in low nutritional condition (low weight, low energy content) will have its reserves depleted. The amount of reserves is then estimated by comparing the decrease in weight or in physiological condition during a period of fasting or starvation. Animals have different strategies to cope with the lack of food. The hepatopancreas mainly accumulates lipids (Yepiz-Plascencia et al. 2000; Luvizotto-Santos et al. 2003) and to a lesser degree glycogen (Verri et al. 2001) which, in some crustaceans, are used during short-term food shortage such as moult (Sánchez-Paz et al. 2007). Besides the class of reserves mobilized, also the sequence of substrates used varies considerably (Sánchez-Paz et al. 2007). Although proteins are considered as the main reserve compound in most crustaceans (New 1976, Comoglio et al. 2005, Comoglio et al. 2008), it has been suggested that C. crangon uses glycogen as a first resource and protein as a last (Cuzon & Ceccaldi 1973). Yet previous studies on this last species had serious flaws because the authors did not accounted for cannibalism and other possible origins of food like bacterial and microalgae productivity within the aquaria system. Objectives The aim of this work was to study the response of the brown shrimp Crangon crangon to prolonged starvation in two distinct seasons: summer, when energetic reserves were assumed to be higher, and autumn. For that, starvation experiments were performed with shrimps from Minho estuary, north of Portugal. The energy reserves of the species were estimated directly by calorimetric analysis and the sequence of mobilized compounds were estimated directly by biochemical analysis of total lipids and total proteins, and indirectly through the oxygen consumption rate determination. FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 11 Material and Methods Starvation experiment Crangon crangon were collected at Coura saltmarsh (fig. 2) within Minho estuary in the beginning of summer (June 2010; 18.4ºC and 17.4 ups) for experiment I and in the beginning of fall (October 2010; 17.2ºC and 31.5 ups) for experiment II, with a 1m beam trawl (5mm mesh size) (fig. 3). Shrimps were transferred to a maintenance aquarium and gradually adapted to a closed circulation system of salt water at 29 ±1ups, with temperature controlled at 20 ±1ºC, and under artificial photoperiod (12/12h) for acclimation to laboratory conditions. Animals were maintained with ad libitum food to acclimate for a week. Figure 2: Upper – Minho River estuary in the North of Portugal; Lower – detail of Coura saltmarsh (Google maps) CAMINHA LANHELHAS Sampling site SPAIN FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 12 Figure 3: Beam-trawl used to collect the shrimps for the experiments After the acclimation period, 374 female shrimps, 180 for experiment I and 194 for experiment II, were randomly selected, measured (total length, from the tip of the scaphocerite to the end of the telson, to the nearest 0.5mm), weighed (wet weight, to the nearest 0.0001g), placed in individual cages (10 x 10 x 17cm) inside the experimental aquaria (see table 1 for total length and wet weight of the animals) and kept without food for 2 days to purge faeces and pseudofaeces. In each experiment, 4 aquaria (8 in total) were used (A to D in experiment I, and E to H in experiment II) and each aquarium was completed with 50 shrimps; exception were the aquaria D which had only 30 shrimps, and aquarium E which had 44 shrimps. These two aquaria were kept in the same conditions but were used exclusively to determine the oxygen consumption rate. Placing the animals in individual cages enabled to monitor each individual separately and eliminated the possibility of cannibalism (fig. 4). Figure 4: Individual cages for the shrimps FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 13 The experimental aquaria were kept in a closed circulation system, isolated and in total darkness so that no algae or microorganisms could grow and be used as food. The aquaria had a 2cm layer of sediment, which was previously burned (580ºC, 8h) to remove all organic matter. Water parameters such as temperature, salinity, ammonia and nitrites were determined daily. Temperature and salinity were maintained similar in the four aquaria (table 1) at 20.2 ±0.11ºC and 30.2 ±0.08 ups (mean ±se) respectively, with no differences between aquaria. The concentration of ammonia presented a mean (±se) of 0.32 ±0.03mg.L-1 with no significant differences between aquaria (p>0.05). Water was changed twice a week with UV treated water; partial changes were made whenever it was necessary to adjust parameters. Total length (TL) and wet weight (WW) of the animals at the beginning of the trials are shown in table 1. The animals in aquaria D and E were intentionally larger, so that the oxygen consumption rate could be measured. For these shrimps the total length was significantly larger in experiment II (ANOVA: F=55.35, p<0.0001), with values ranging from 31.1 and 41.5mm, while in the experiment I the values ranged between 31.2 and 36.5mm. Concerning the wet weight, shrimps from experiment II were also heavier than the ones in experiment I (ANOVA: F=46.62, p<0.0001), with the weight varying between 0.3124 and 0.4516g in experiment II and 0.2488 and 0.3995g in experiment I. Along the starvation time, TL and WW of the individuals used in experiment I did not change significantly, this is, shrimps used for the oxygen measurements in the first week had similar mean TL and WW as the ones used in the sixth week (p>0.05). In the experiment II, the shrimps’ size did not differ between the weeks (p>0.05). However, the shrimps used in the third week measurements were lighter than the ones used in the first and second week (ANOVA: F=5.83, p=0.0022). All observed animals in the other 3 aquaria in the experiment I had similar mean total length and wet weight (table 1). In one aquarium of experiment II (H) the mean total length and wet weight were significantly smaller (F=676.26, p<0.0001 and F=7.55, p=0.0008 respectively for TL and WW). Also animals used in experiment II were significantly larger and heavier than the ones of experiment I (table 1). Every day, besides registering the temperature and salinity, shrimps were checked for dead animals or exuvia, while every week six animals were sacrificed. In all aquaria, animals were kept in starvation until the last animal was sacrificed or died (maximum 49 days). Sex of the individuals was confirmed in the exuvia based on the endopodite of the first and second pairs of pleopods; exuvia was then returned to the animal to prevent mineral disequilibria. Dead and sacrificed individuals were measured, weighed FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 14 and frozen for later biochemical analyses. In the end, all shrimps were dried at 60ºC for two days, weighed (dry weight, DW) and then macerated for the biochemical analysis. Table 1: Number of shrimps and their mean (±se) total length (TL) and wet weight (WW); mean (±se) temperature and salinity; and duration of both experiments; ANOVA results with significant differences in bold Experiment Aquarium A/E Aquarium B/F Aquarium C/G Aquarium D/H ANOVA Between aquaria ANOVA between experiments TL (mm) I 27.1 ±0.16 26.9 ±0.16 26.9 ±0.15 33.9 ±0.21 0.54, p≥0.05* 622.85, p<0.0001* II 38.8 ±0.23 30.4 ±0.13 30.3 ±0.10 29.5 ±0.17 12.45, p<0.0001* WW (g) I 0.1654 ±0.004 0.1620 ±0.004 0.1674 ±0.005 0.3093 ±0.007 0.39, p≥0.05* 31.71, p<0.0001* II 0.4008 ±0.006 0.1961 ±0.004 0.1874 ±0.004 0.1726 ±0.004 7.55, p<0.05* TºC I 20.4 ±0.18 20.0 ±0.18 20.6 ±0.18 19.8 ±0.26 3.42, p<0.05 0.68, p≥0.05 II 20.3 ±0.31 20.2 ±0.33 19.7 ±0.04 19.5 ±0.32 1.57, p≥0.05 Salinity (ups) I 29.9 ±0.17 30.1 ±0.15 30.5 ±0.13 30.3 ±0.14 2.33, p≥0.05 4.03, p≥0.05 II 30.1 ±0.14 30.0 ±0.14 29.7 ±0.16 29.8 ±0.12 1.91, p≥0.05 Number of animals I 50 50 50 30** II 44** 50 50 50 Time of starvation (days) I 36 36 36 49 II 21 14 14 8 *ANOVA of only 3 aquaria, i.e. excluding the one used in the oxygen consumption rate. ** Aquaria used for the measurements of the oxygen consumption rate Oxygen consumption rate Animals from aquaria D and E were used for the determination of the oxygen consumption rate. In the first week O2 consumption was determined in three consecutive days (every 24h, at the same time), and afterwards measurements were made once a week. A number of shrimps (four and five, respectively in experiment I and II) were randomly selected, weighed and transferred to air tight containers, with substrate. An extra container without any shrimp was used as control. After 15 minutes of acclimation, the oxygen concentration was measured continuously for 45 minutes. FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 15 The containers consisted of glass chambers into which an HQd meter with a LDO101 probe was inserted. This probe monitored the environmental data every 30s transmitting that information to a pen drive where it was stored for later treatment. Consumed oxygen was taken as being the net difference between the measurements at the start and the end. Water in the containers came from the experimental aquarium. Measurements were made at the same temperature as in the experimental aquaria. The shrimps’ survival to starvation enabled to follow the oxygen consumption along six and four weeks respectively in the experiments I and II. Biochemical analysis Six individuals from each aquarium except D and E (the oxygen consumption study aquaria) were sacrificed, measured and weighed every week; at day zero also 6 individuals from the 3 aquaria were sacrificed to be used as initial estimates. About 3 sacrificed shrimps were used for caloric content determination in cal.DWg-1, in a bomb calorimeter (PARR 1261); other 3 sacrificed shrimps were used to determine total proteins and total lipids, respectively in an Isotope-Ratio Mass Spectrometer (IRMS), which determines the total nitrogen (N), and with a Spinreact® commercial kit. The same procedure was followed for the naturally dead animals. In the end, in total for both experiments, 145 individuals were analyzed for caloric content, 30 of them were naturally dead shrimps; 143 animals for total proteins and total lipids, from which 87 were naturally dead. The determination of total proteins requires only a small amount of sample (approximately 0.5 mg), so the remaining was used for the total lipids’ quantification. This means that data on total proteins and total lipids corresponds to the same individual. The samples were first homogenized in a chloroform-methanol-water (2:2:1) mixture and extraction of lipids was performed according to Sanchés-Paz et al. (2007) protocol, using a ratio of 20 v/w of buffer A and 20 v/w of the chloroform-methanol- water mix. For the calculus of total protein from total N, the standard factor of 6.25 was used (Mariotti et al. 2008). Both, total proteins and total lipids were presented as percentage of DW. FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 16 Data treatment The Fulton’s condition factor (K) was determined dividing the body mass (WW) by the cube of the body length (TL), assuming that the individual has an isometric growth. The Fulton’s Condition factor was calculated in the beginning and in the end of both experiments. This morphological index allows quantifying the health of an individual and comparing the morphometric data with the biochemical information. ANOVA tests were applied using Systat 13.0 software. Comparisons were made between experiments, between aquaria, between observation weeks (pooling data from the same week of starvation) and considering each aquarium as a replicate. Whenever differences were detected a Tukey’s comparison test was applied. Assuming that naturally dead animals were in lower condition than sacrificed ones, their results were analyzed separately and compared between each other. When comparing across time, at the start or beginning means at day “zero”, first week means “at the end of the first week” and the same for the following weeks. FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 17 Results Natural mortality Shrimps were sacrificed every 7 days for biochemical analysis in both experiments. Natural mortality in the experiment I started to occur after 6 days in starvation, except in the aquarium A, where dead animals were found on the second day. In the experiment II, shrimps started to die after 2 or 3 days in all aquaria. Half of the shrimps were dead after 18 to 31 days and after 3 to 12 days, respectively in the experiments I and II (table 2). Maximum duration of the starvation trials was observed in the experiment I (49 days), allowing observing temporal trends across 6 weeks. In contrast, in the experiment II, the maximum duration was 21 days in the oxygen consumption study aquarium enabling comparisons across 4 weeks. In the other aquaria, shrimps survived up to 14 days, and hence only the results from the first two weeks can be compared. Table 2: Time till half of the shrimps were dead and maximum time in starvation in each aquarium Experiment Aquaria Time in starvation (days) Time till 50% mortality (days) I A 36 22 B 36 20 C 36 18 D* 49 31 II E* 21 12 F 14 6 G 14 7 H 8 3 * Shrimps from these aquaria were used in the oxygen consumption study and were not sacrificed FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 18 Oxygen consumption The oxygen consumption rate differed significantly between the two experiments (ANOVA: F=9.06, p=0.0035). Higher rates were found in the experiment II, 0.79 ±0.14 versus 0.29 ±0.05mg O2.g-1.h-1 in experiment I (fig. 5). Figure 5: Oxygen consumption rate (mg O2.g-1.h-1) in both experiments (* and ° are outliers) The oxygen consumption rate was significantly different between the weeks of starvation in the experiment I (ANOVA: F=4.46, p=0.0037). These differences were found between the second, third and fourth weeks and the fifth week (table 3, fig. 6). The highest mean of oxygen consumption was found in the third week and the lowest in the fifth week, respectively 0.45 ±0.06 and 0.12 ±0.03mg O2.g-1.h-1. In the experiment II, the rate of consumption was similar along the weeks (p>0.05), with a maximum mean of 1.08 ±0.31mg O2.g-1.h-1 in the second week and a minimum of 0.48mg O2.g- 1.h-1 in the fourth week (fig. 7). FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 19 Figure 6: Oxygen consumption rate (mg O2.g-1.h-1) per week in the experiment I (* are outliers) Figure 7: Oxygen consumption rate (mg O2.g-1.h-1) per week in the experiment II; only one shrimp was available in the fourth week (* and ° are outliers) No clear trend was found relating the oxygen consumption rate with the animals’ weight and total length besides a tendency of the larger and heavier experiment II shrimps to present higher rates (fig. 8 and 9). week week p 2 5 0.0053 3 5 0.0292 4 5 0.0444 Table 3: Tukey’s pairwise comparisons results on the oxygen consumption rate in the experiment I FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 26 K ranged between 0.51 and 0.78 and between 0.37 and 0.87, respectively for the sacrificed shrimps and for the naturally dead (table 8). However, while in the experiment I mean final K was higher for the sacrificed shrimps, in the experiment II these shrimps had a lower final K than naturally dead shrimps (fig. 18a). Final K also significantly differed between aquaria (ANOVA: F=16.32, p<0.0001), with the exception of aquaria B and H (fig. 18b). Despite a great individual variability, shrimps that naturally died in all aquaria were in a similar final condition (p>0.05). Figure 18: Final Fulton’s Condition factor of the shrimps in both experiments (a) and in each aquarium (b) (* and ° are outliers) Table 8: Mean (±se) Fulton’s condition factor of the naturally dead and sacrificed shrimps in the end of the experiments Exp. I Exp. II Naturally dead 0.62 ±0.032 0.65 ±0.013 Sacrificed 0.74 ±0.008 0.62 ±0.011 a b FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 27 Over the time differences started to appear. Shrimps that were sacrificed had higher values of final K than those that naturally died (ANOVA: F=18.72, p<0.0001). For the sacrificed shrimps, differences were clear between the first and the fourth, fifth and sixth weeks (p<0.05, table 9) (fig. 19). The highest condition was found in the sacrificed shrimps’ first week in experiment I (0.91). In the experiment II, no statistical difference was found across time for both sacrificed and naturally dead shrimps (p>0.05). Table 9: Tukey’s pairwise comparisons results on the Fulton’s condition factor in the experiment I week week p 1 4 0.0001 5 0.0255 6 0.0461 Figure 19: Mean Fulton’s condition factor of the shrimps along the weeks of starvation in the experiment I (* are outliers) Total lipids, total proteins and caloric content For the quantification of the total lipids and total proteins, 143 shrimps were analyzed, but 10 lipids and 5 proteins’ values were discharged because the results were not reliable. Of the 145 shrimps analyzed for the caloric content only 72 produced reliable results, because some of the shrimps were lighter than the minimum required by the FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 28 calorimeter (0.150g DW), weighing less than 0.100g. For the lighter individuals an excipient of fishmeal (with a known and stable energy content of 5777cal.g-1) was used to make up for the weight, but nevertheless in several cases the equipment was not capable of detecting the low levels of energy produced in the combustion. Therefore, several errors and some outliers were obtained and only a small set of data provided reliable information. The percentage of total lipids and the caloric content of the sacrificed and the naturally dead shrimps did not differ considering the data from both experiments (p>0.05), while differences were found between the percentage of total proteins for the naturally dead shrimps in the experiment I and of the naturally dead and sacrificed shrimps in the experiment II (ANOVA: F=37.81, p<0.0001). However, in both experiments, the sacrificed shrimps presented mean values of lipids near half of the naturally dead’s levels (table 10), ranging from 0.004 to 1.593% in the experiment I and from 0.060 to 1.470% in the experiment II, while for the naturally dead shrimps the percentage ranged between 0.024 and 2.886% in the experiment I, and 0.013 and 4.265% in the experiment II (fig. 20). Regarding the percentage of total proteins, naturally dead shrimps from the experiment II showed the highest values (table 11), ranging from 4.2 to 66.2% (fig. 21). In the experiment I the percentage varied between 13.6 and 47.4%. For the sacrificed shrimps, differences were not significant between experiments, and total proteins ranged between 33.4 and 66.4% in the experiment II and 34.3 and 58.4% in the experiment I. The mean caloric content of the sacrificed shrimps varied between 774 and 6273cal.g-1 in the experiment I and 1143 and 4394cal.g-1 in the experiment II. The naturally dead shrimps presented mean values between 1634 and 2334cal.g-1 in the experiment I and 196 and 4060cal.g-1 in the experiment II (fig. 22). Table 10: Mean (±se) percentage of total lipids (%) of the naturally dead and sacrificed shrimps in both experiments Exp. I Exp. II Naturally dead 1.060 ±0.155 0.869 ±0.114 Sacrificed 0.602 ±0.059 0.567 ±0.109 FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 29 Figure 20: Percentage of total lipids of the naturally dead and sacrificed shrimps in both experiments (* and ° are outliers) Table 11: Mean (±se) percentage of total proteins (%) of the naturally dead and sacrificed shrimps in both experiments Exp. I Exp. II Naturally dead 31.2 ±1.55 46.8 ±1.66 Sacrificed 48.8 ±0.89 51.3 ±1.91 Figure 21: Percentage of total proteins of the naturally dead and sacrificed shrimps in both experiments (* and ° are outliers) FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 30 Figure 22: Mean caloric content (cal.g-1) of the naturally dead and sacrificed shrimps in both experiments (* are outliers) In both experiments, results of total lipids (fig. 23a) and total proteins (fig. 23b) of both sacrificed and naturally dead shrimps were not significantly different between aquaria (p>0.05). Regarding the caloric content, differences were only detected in the sacrificed shrimps from aquaria A and C (experiment I) (p<0.05) (fig. 24). a FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 31 Figure 23: Mean percentage of total lipids (a) and total proteins (b) of the naturally dead and sacrificed shrimps per aquarium (* and ° are outliers) Figure 24: Mean caloric content (cal.g-1) of the naturally dead and sacrificed shrimps per aquarium (* are outliers) Differences between aquaria were found in the total proteins of the naturally dead individuals (ANOVA: F=6.06, p<0.0001) (fig. 23b, table 12). Despite not significant (p>0.05), the mean caloric content was higher in shrimps from experiment II than in those from experiment I, for both naturally dead and sacrificed shrimps (table 13). b FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 32 Table 12: Tukey’s pairwise comparisons results on the total proteins Aquarium Aquarium p E B 0.0397 C 0.0031 D 0.0003 H C 0.0146 D 0.0049 G D 0.0427 Table 13: Mean (±se) caloric content (cal.g-1) of the naturally dead and sacrificed shrimps in both experiments Along the starvation time, data for the percentage of total lipids presented statistical differences between naturally dead and sacrificed shrimps in the experiment I (ANOVA: F=10.03, p=0.0024), the first with higher values (fig. 25). The percentage of total lipids of the sacrificed and naturally dead shrimps varied along the time of starvation though with no significant difference (p>0.05): mean percentage of total lipids rose till the fourth week and then decreased abruptly, especially in the naturally dead shrimps. The highest value was found in the third week in the experiment I (2.900%) and in the first week in the experiment II (4.270%), both for the naturally dead shrimps. Exp. I Exp. II Naturally dead 1419 ±269.7 2041 ±255.6 Sacrificed 2311 ±309.0 2734 ±235.5 FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 33 Figure 25: Percentage of total lipids of the shrimps along the weeks of starvation in the experiment I (* and ° are outliers) The percentage of total proteins varied along the time of starvation, being higher for the shrimps that were sacrificed than for those that naturally died (ANOVA: F=109.93, p<0.0001). Regarding the experiment I, significant differences were found between weeks for both the sacrificed shrimps (ANOVA: F=12.81, p<0.0001) and the naturally dead (ANOVA: F=4.09, p=0.0084). The main differences were obtained between all weeks and the final 6th week (p<0.05) (fig. 26). There were also differences between the first week and the second, fourth and fifth weeks (table 14). For the naturally dead individuals the differences were observed between the second week and the third and fifth weeks (table 15). Regarding the experiment II, no statistical differences were found between weeks, though sacrificed shrimps had always higher mean percentage of total proteins. FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 34 Figure 26: Percentage of total proteins of the shrimps along the weeks of starvation in the experiment I Table 14: Tukey’s pairwise comparisons results on the total proteins of the sacrificed shrimps in the experiment I week week p 1 2 0.0028 4 0.0077 5 0.0089 6 0.0000 2 6 0.0004 3 6 0.0000 4 6 0.0002 5 6 0.0002 Table 15: Tukey’s pairwise comparisons results on the total proteins of the naturally dead shrimps in the experiment I week week p 2 3 0.0456 5 0.0221 FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 35 Despite a decrease across time in the mean caloric content of the sacrificed shrimps in the experiment I, the difference was not significant (p>0.05) (fig. 27). Also the energy in the first week was higher in the experiment I than in the experiment II (table 16). Yet data was insufficient for a statistical sound analysis. Figure 27: Caloric content (cal.g-1) of the shrimps along the weeks of starvation in the experiment I (° are outliers) Table 16: Mean (±se) caloric content (cal.g-1) per week of the sacrificed shrimps in both experiments week Exp. I Exp. II 1 3343 ±747.6 2842 ±329.3 2 2722 ±143.0 2626 ±352.7 3 2354 ±673.2 4 1146 ±204.6 5 1949 ±1069.8 6 719 FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 42 References Abbott, OJ & Perkins, EJ (1977). The biology of the brown shrimp Crangon crangon in the Solway Firth. Scientific Report Cumbria Sea Fisheries Committee 77: 1-34. Al-Adhub, AHY & Naylor, E (1975). Emergence rythms and tidal migrations in the brown shrimp Crangon crangon (L.). Journal of the Marine Biological Association of the United Kingdom 55: 801-810. Amara, R & Paul, C (2003). 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FCUP Comparing the response of the brown shrimp, Crangon crangon, to prolonged deprivation of food in two seasons 48 Appendix Table 17: Summary of the results in both experiments Variables Experiment I Experiment II Inicial K Lower for sacrificed shrimps Similar between sacrificed and naturally dead shrimps Higher in exp I Final K Lower for naturally dead shrimps Similar between sacrificed and naturally dead shrimps Higher in exp I DW Lower for sacrificed shrimps Similar between sacrificed and naturally dead shrimps Higher in exp II Energy Lower for the naturally dead shrimps Lower for the naturally dead shrimps Higher in exp II % of Total Lipids Lower for sacrificed shrimps Lower for sacrificed shrimps Higher in exp I % of Total Protein Lower for naturally dead shrimps Lower for naturally dead shrimps Higher in exp II % of Water Lower for naturally dead shrimps Similar between sacrificed and naturally dead shrimps Higher in exp I Oxygen consumption rate Higher in exp II