The effect of biotic variables on culturing conditions of Calanoid copepod Acartia grani

The effect of abiotic and biotic variables on culturing conditions of Calanoid copepod Acartia grani Luis Bernardo dos Santos Sumares Dissertation for the Master in Marine Sciences – Marine Resources 2012 1 Luís Bernardo dos Santos Sumares The effect of abiotic and biotic variables on culturing conditions of Calanoid copepod Acartia grani Dissertation application to the master degree in Marine Sciences – Marine Resources submitted to the Institute of Biomedical Sciences Abel Salazar, University of Porto. Supervisor: Natacha Nogueira Researcher Mariculture Center of Calheta (CMC) Co-Supervisor: António Afonso Associate Professor Institute of Biomedical Sciences Abel Salazar, University of Porto Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares i Preface The work described in this document was made between the months of November 2011 and September 2012, initially on IPIMAR - Algarve, and later at the Mariculture Center of Calheta (CMC), in Madeira Island. The work was organized in two phases: one was to acquire knowledge of microalgae and copepods in IPIMAR; and the second phase, performed at CMC facilities, was the performance of all the experiments that gave rise to this thesis. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares ii Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares iii Acknowledgements To my super parents Paula e Angelino that give me all the support and love for successfully completes another important stage of my life. The wise words of my father who helped me a lot: “Depois da tempestade vem a bonança”. My sister Carolina and Rik for always being available to help me even in the hours of hard work always put my work first. To Catarina for all your love and affection that was always present when I needed. To my supervisor Natacha Nogueira who was the basis of this work, for the advices, for the difficult decisions, for their patience to answer the obvious questions, for being always present when I needed, for arrange the material needed for the experiments, doubtless the best supervisor that I could ever have. And to my co-supervisor Professor António Afonso for the excellent advices and availability that always manifested. I would like to thank the entire staff of Centro de Maricultura da Calheta (CMC) for helping me whenever I needed. To Dr. Carlos Andrade who provide a laboratory where I cloud make all my experiences and had my microalgae stock to feed the copepods. To all staff of IPIMAR-Algarve, in particularly Drª Maria Emília Cunha that spent all his knowledge of the Acartia grani, showing how fascinating is the world of zooplankton, and how this world has a lot to show us. To Paulo Jorge, without him I could not be able to produce any type of microalgae, and now I´m proud to have been able to produce the demanding microalgae Rhodomonas. To Drª Nereida Cordeiro from Centro de Competências de Ciências Exatas e de Engenharia – Universidade da Madeira that had available she laboratory and together with Miguel e Marisa who give me a precious help. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares iv Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares v Abstract Calanoid copepods, incluinding species of the genus Acartia are commonly used for larval diets of marine finfish. The interest in copepods for marine fish aquaculture is growing especially in large-scale culturing. However, studies quantifying the optimal conditions for intensive copepod production are generally lacking for most species. The present thesis was conducted to evaluate the effect of abiotic and biotic variables on culturing condition of Calanoid copepod Acartia grani. The egg hatching rate (EHR) at different temperatures showed a clear trend of increasing EHR in warmer temperatures; 36 hours after incubation, 74.7% of eggs had hatched in the 28°C treatment (Chapter I). In Chapters II to Chapter V we analyzed the egg hatching rate (EHR), egg production (EP) and population growth of the different variables imposed. The highest egg hatching observed within each variable analyzed was: (1) 60%, 72h after incubation at 24°C within the temperature experiment (Chapter II) ; (2) the photoperiod experiment (Chapter III) 21%, 48h after incubation with light regimen of 6L:18D within the photoperiod experiment. (3) 44.2% at 72h in the Rho+T-Iso diet treatment (Chapter IV) and finally (4) 30.4%, 48 hours after incubation in the diet experiments using heterotroph Oxyrris marina (Chapter V). Egg production results varied among variables and within each experiment best results were found for: 18ºC, which had an EP of 10.5±1.5 eggs female-1day-1; photoperiod of 12Light:12Dark achieved an EP of 9.5±1.9 eggs female-1day-1; diet with Rhodomonas marina obtained 4.7±0.9 eggs female-1day-1; and finally in Chapter V, the diet Rhodomonas marina + Oxyrrhis marina had 3.7±0.6 eggs female-1day-1. Regarding results of population growth when all developmental stages were included , we found that the temperature of 18°C produced a population increase from 12 to 663.0±52.2 individuals in 12 days (Chapter II); photoperiod 12Light:12Dark increased to a final population of 964.3±410.7 copepods. Last two experiments that evaluated the use of different diets revealed a population increase from 12 to 1783.0±560.6 individuals with the use of a binary diet composed by Rhodomonas marina + Tahitian strain of Isochrysis sp.; and in the last experiments (Chapter V) the best diet was once more, a binary diet composed by Rhodomonas marina and Oxyrrhis marina with a mean final population of 1795.0±451.8 copepods. Cannibalism occurred in all tested densities (125; 250; 500; 1000; and 2000 ind./L) (Chapter VII) and conclusion was that 125ind./L induced significantly lower cannibalistic rates. In Chapter VI we analysed the lipid and fatty acids composition of Acarti grani with different diets. The results show a moderate level of total lipids in the two samples. In the first diet (Rho) a total lipid content of 9.4±2.2% was found and for binary diet Rho+Oxy, Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares vi lipid content was of 8.6±0.1%. In the fatty acid composition no significance differences were observed (p>0.05). Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were present in both samples in high amounts, witn a mean ratio of DHA/EPA of 2.29 for copepods fed on Rhodomonas and 2.18 for copepods fed Rhodomonas + Oxyrrhis. In general, we concluded that the following parameters are a good choice for maximization of the culture of A.grani; Temperature: 24°C; Photoperiod: 12Light:12Dark; Diet: Rhodomonas marina + Oxyrrhis marina; Density: 150ind./L. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares vii Resumo Os copépodes Calanóides, em particular as espécies do género Acartia são geralmente usadas como dieta para larvas de peixes ósseos. O interesse nos copépodes em aquacultura de peixes marinhos tem aumentado, em especial na produção de culturas em grande escala. No entanto, para a maioria das espécies existem poucos estudos que refiram as condições óptimas de cultura. A presente tese foi realizada para avaliar o efeito de variáveis abióticas e bióticas nas condições de cultura do copépode Calanóide Acartia grani. As taxas de eclosão (EHR) em relação à temperatura mostraram que aumentavam com a subida da mesma; Após 36 horas de incubação, 74.7% dos ovos tinham eclodido no tratamento de 28°C. Do Capítulo II até ao Capítulo V analisamos as taxas de eclosão (EHR), a produção de ovos (EP) e o crescimento da população com as diferentes variáveis impostas. As maiores taxas de eclosão observadas entre as variáveis analisadas foram: (1) experiência da temperatura (Capítulo II) apresentou uma EHR de 60%, 72horas após incubação dos ovos a 24°C; (2) a experiência do fotoperíodo obteve uma EHR de 21%, 48h após incubação com um regime de 6Luz:18Escuro (Capítulo III); (3) na experiência das dietas (Capítulo IV) foi encontrada uma EHR de 44.2% às 72h na dieta Rho+T-Iso; (4) na experiência das dietas utilizando o organismo heterotrófico Oxyrrhis marina a maior taxa de eclosão foi verificada na dieta T-Iso (30.4%) 48h após a incubação (Capítulo V). Os resultados da produção de ovos variaram com as diferentes variáveis e entre as experiências. Os melhores resultados foram: 18°C, com uma produção de 10.5±1.5 ovos fêmea-1dia-1; para o fotoperíodo de 12Luz:12Escuro a EP foi de 9.5±1.9 ovos fêmea-1dia-1; finalmente na dieta de Rhodomonas marina + Oxyrrhis marina, a EP máxima foi de 3.7±0.6 ovos fêmea-1dia-1. Em relação aos resultados do crescimento da população, quando todas as fases foram consideradas (incluindo ovos), a temperatura de 18°C apresentou um crescimento dos 12 para 663.0±52.2 indivíduos em 12 dias (Capítulo II) e o fotoperíodo 12Luz:12Escuro teve uma população final de 964.3±410.7 copépodes. As últimas duas experiências em que foi analisado o papel das diferentes dietas, revelaram um aumento da população de 12 para 1783.0±560.6 indivíduos com o uso de uma dieta binária composta por Rhodomonas marina + Tahitian strain of Isochrysis sp. e na última experiência (Capítulo V) os melhores resultados foram uma vez mais com uma dieta binária composta por Rhodomonas marina e Oxyrrhis marina, onde foi verificado uma população média de 1795.0±451.8 copépodes, após 12 dias de cultura. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares xiv Chapter I: Egg hatching success at different temperatures of calanoid Copepod Acartia grani Figure 1 - The 36 hours cumulative egg hatching rate (CUM%) of Acartia grani eggs at four different temperatures (18°C, 22°C, 24°C and 28°C). Eggs were incubated under identical conditions of 38±1 psu and photoperiod 12L:12D. Data are presented as mean±SD. (One way ANOVA p=0.576) ................................... 25 Chapter II: The effects of temperature on population growth, egg production and egg hatching success of the calanoid copepod Acartia grani (Calanoida: Acartiidae) Figure 1 - The cumulative egg hatching rate (CUM%) of Acartia grani eggs produced by adults with four different temperatures, over 72 hours. Eggs were incubated under identical condition of 38±1psu and photoperiod 12L:12D ........................................................................................................................................ 35 Figure 2 - Mean egg production (eggs female-1day-1) of Acartia grani with 4 different temperatures on the 4 experimental days. Data are presented as mean±SD. (Day 1 p=0.86; Day 2 p=0.08; Day 3 p=0.32; Day 4 p=0.99) .............................................................................................................................................................. 36 Figure 3 - Mean final total population of Acartia grani cultured at four different temperatures for a 12 day period. Initial population was 8 females: 4 males Data are represented as mean±SD. All Stages bar: eggs, nauplii, copepodites, adults; All post-egg-stages bar: nauplii, copepodites, adults. Different letters on the tops of bars indicate significant differences (p<0.05). One way ANOVA: All stages included p=0.86; All Post-EggsStages p=0.82 ................................................................................................................................................... 37 Figure 4 - Sex ratio of A.grani in the population increase experiment after 12 days ......................................... 38 Chapter III: The effects of photoperiod on population growth, egg production and egg hatching success of the calanoid copepod Acartia grani (Calanoida: Acartiidae) Figure 1 - The cumulative egg hatching rate (CUM%) of Acartia grani eggs produced by adults at five different photoperiod over 48 hours. Eggs were incubated under identical conditions of 24±0.5°C and 38±1 psu ......... 49 Figure 2 - Mean egg production (eggs female-1day-1) of Acartia grani, with 5 different photoperiods on the 3 experimental days. Data are presented as mean±SD. (Day 1 p=0.54; Day 2 p=0.63; Day 3 p=0.68) ............. 50 Figure 3 - Mean final total population of Acartia grani cultured at five different photoperiods for a 12 day period. Initial population was 8 females: 4 males. Data are represented as mean±SD. All Stages bar (all life stages: eggs, nauplii, copepodites, adults); All post-egg-stages bar (nauplii, copepodites, adults). Different letters on the tops of bars indicate significant differences (p<0.05). One way ANOVA: All stages included p=0.94; All Post-Eggs-Stages p=0.07; (24Light: 0Dark; 18Light: 6Dark; 12Light: 12Dark; 6Light: 18Dark; 0Light: 24Dark................................................................................................................................................... 51 Figure 4 - Sex ratio of A.grani in the population increase experiment after 12 days ......................................... 52 Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares xv Chapter IV: Egg production, egg hatching success and population increase of the calanoid copepod, Acartia grani (Calanoida: Acartiidae), fed different microalgal diets Figure 1 - The cumulative egg hatching rate (CUM%) of Acartia grani eggs produced by adults fed four different microalgae diets, over 72 hours. Eggs were incubated under identical condition of 24±0.5°C, 38±1 psu and photoperiod 12L:12D ........................................................................................................................... 62 Figure 2 - Mean egg production (eggs female-1day-1) of Acartia grani, eggs produced by adults fed four different microalgae diets (Rho, Rho+T-Iso, Tet, Tet+T-Iso), on the 3 experimental days. Data are presented as mean±SD. (Day 1 p=0.01; Day 2 p=0.82; Day 3 p=0.62 .............................................................................. 63 Figure 3 - Mean final total population of Acartia grani cultured at four different microalgae diets for a 12 day period. Initial population was 8 females: 4 males Data are represented as mean ±SD. All Stages bar (all life stages: eggs, nauplii, copepodites, adults); All post-egg-stages bar (nauplii, copepodites, adults). Different letters on the tops of bars indicate significant differences (p<0.05). One way ANOVA: All stages included p=0.023; All Post-Eggs-Stages p=0.001; (Diet 1: Rhodomonas marina; Diet 2: Rhodomonas marina + Isochrysis sp. (T-Iso); Diet 3: Tetraselmis suecica; Diet 4: Tetraselmis suecica + Isochrysis sp. (T-Iso) ......... 64 Chapter V: Egg production, egg hatching success and population increase of the calanoid copepod, Acartia grani (Calanoida: Acartiidae), using various combinations of autotrophic and heterotrophic protists Figure 1 - The cumulative egg hatching rate (CUM%) of Acartia grani eggs produced by adults fed four different microalgae diets, over 72 hours. Eggs were incubated under identical condition of 24±0.5°C, 38±1 psu and photoperiod 12L:12D ........................................................................................................................... 76 Figure 2 - Mean egg production (eggs female-1day-1) of Acartia grani, eggs produced by adults fed four different microalgae diets (Diet 1: Rhodomonas marina + Oxyrrhis marina; Diet 2: Oxyrrhis marina; Diet 3: Isochrysis sp. (T-Iso); Diet 4: Isochrysis sp. (T-Iso) + Oxyrrhis marina), on the 3 experimental days. Data are presented as mean±SD. (Day 1 p=0.04; Day 2 p=0.06; Day 3 p=0.00) ........................................................... 77 Figure 3 - Mean final total population of Acartia grani cultured at four different microalgae diets for a 12 day period. Initial population was 8 females: 4 males. Data are represented as mean ±SD. All Stages bar (all life stages: eggs, nauplii, copepodites, adults); All post-egg-stages bar (nauplii, copepodites, adults). Different letters on the tops of bars indicate significant differences (p<0.05). One way ANOVA: All stages included p=0.062; All Post-eggs-etages p=0.071. (Diet 1: Rhodomonas marina + Oxyrrhis marina; Diet 2: Oxyrrhis marina; Diet 3: Isochrysis sp. (T-Iso); Diet 4: Isochrysis sp. (T-Iso) + Oxyrrhis marina) .................................. 78 Figure 4 - Sex ratio of A.grani in the population increase experiment after 12 days ......................................... 79 Chapter VII: Effect of Acartia grani culture density on cannibalism occurrence Figure 1 - Naupliar mortality rates at different Acartia grani stocking densities. Data are presented as mean±SD. Different letters on the tops of bars indicate significant differences (p<0.05) ................................. 99 1 1| Introduction Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 2 1.1 Importance of Production of Live Feed in Aquaculture Aquaculture is one of the fast growing food producing sectors in the world that plays an important role in satisfying the needs for seafood worldwide. In fact, in the period 19702008, the production of food fish from aquaculture increased at an average annual rate of 8.3 percent, while the world population grew at an average of 1.6 percent per year. The combined result of development in aquaculture worldwide and the expansion in global population is that the average annual per capita supply of food from aquaculture products for human consumption has increased by ten times, from 0.7 kg in 1970 to 7.8 kg in 2008, at an average rate of 6.6 percent per year (Grainger, 2010). Despite the increased production over the years, aquaculture production, particularly fish farming, will always depend on “seed” supplying of known quantity and quality at any time (Andrade et al., 2012). Once spawning of a fish species is under control larviculture protocols have to be established in order to meet the biological and nutritional demands of the larvae. The main bottleneck for fry production of many marine fish species is related to the high mortality rates associated with larval first feeding. Many finfish larvae rely on live prey for several reasons. Prey size may affect the prey ingestion by early fish larval (Planas and Cunha, 1999). The larvae of numerous marine fish require small prey (about 50– 100µm wide) at first feeding (Detwyler and Houde, 1970; Yúfera and Pascual, 1984) due to the small size of their mouth. In many cases, even the commonly used rotifer Brachionus rotundiformis, type ‘‘S’’, may be too large (Houde, 1973; May et al., 1974; Doi and Singhagraiwan, 1993). Moreover, moving prey seems to be more attractive to fish larvae than inert particles, which have a tendency for sinking or agglomerating, making it unavailable for a quick consumption. Moreover, the gut of many fish larvae (particularly altricial larvae) is not fully developed at the time of first feeding, and it is an advantage to get live prey which brings some exo-enzymes to the fish larvae (Conceição et al., 2010). High larval mortalities are also often associated to the lack of a nutritionally adequate diet (Nanton and Castell 1998). Major gap in knowledge of fish larval nutritional requirements still remains (Conceição et al., 2010). Nevertheless, it has become evident that fish larvae have specific nutritional requirements that can be linked to the biochemical characteristics of their natural prey. In most aquaculture enterprises, fish and crustacean larvae are reared using rotifers and Artemia nauplii as live prey. The rotifer, Brachionus plicatilis constitute an essential part of the feeding during the larval stages of marine fish and crustaceans. Its body size; high growth rate; good tolerance to culture conditions or handling and feeding by filtration of particles in suspension make this organism an appropriate prey to start feeding after reabsortion of vitelline reserves. The ready Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 3 availability of Artemia nauplii, through the purchase of cysts and subsequent hatching of their nauplii (Lavens and Sorgeloos, 2000) makes them the most convenient and least labour intensive live food available for aquaculture (Lavens and Sorgeloos, 2000). Metanauplius I (instar II) of Artemia are continuous non-selective filter feeding organisms, just as rotifers, which is an important characteristic and the basis of the bioencapsulation processes (Van Stappen, 1996). Despite significant research and development has been achieved on enhancing culture techniques or enrichment methods to improve the availability and nutritional value of rotifers (Rainuzzo et al., 1997; Sargent et al., 1997) and Artemia nauplii, the use of these live prey does not always lead to optimal larval growth, as they usually have an inadequate fatty acid profile (Olivotto et al., 2008a,b). Dietary lipids are recognized as one of the most important nutritional factors that affect larval growth and survival (Watanabe et al. 1983). In particular, the importance of n-3 HUFA for marine fish larvae has been widely studied and their requirements have been reported for some species (Planas and Cunha, 1999). Alternatives to rotifer culture have been used such as chicken egg yolk, frozen abalone larvae, copepods, processed krill and oyster ovaries (Koga and Naghama, 1981; Nagahama and Hidaka, 1982), but results have been unsuccessful on a large scale due to improper nutrition or difficulty in supplying the quantity of food required and when needed (Koga and Naghama, 1981). Despite recent progress in the development of inert diets for fish larvae (e.g., Lazo et al., 2000; Cahu and Infante, 2001; Koven et al., 2001), feeding of most species of interest for aquaculture still relies on live feeds during the early life stages. Even the ‘Artemia replacement’ products increasingly used in commercial operations are normally used in co-feeding with live feeds. For all the above, the selection of an adequate live food for larvae is considered as a key factor for the future expansion of marine aquaculture, unless breakthroughs in the development of microdiets for marine fish larvae are introduced as a viable alternative to live feed (Liu and Xu, 2009). Thus, the availability of live prey of high nutritional value for maximal growth and survival of fish and crustacean larvae is of fundamental importance (Drillet et al., 2008; Fleeger, 2005; Olivotto et al., 2008a,b; Sargent et al., 1997). Among these potential alternatives, copepods are the best prospective candidate and the development of many species is increasing their potential as live prey. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 4 1.2 Copepods 1.2.1 Copepods and their use in aquaculture The name copepod is derived from the Greek kope meaning ‘oar’ and podos meaning ‘foot’, and refers to the flat, paddle-like swimming legs. Around 200 families with some 1650 genera and 11.500 species were classified by 1993 (Humes, 1994). Copepods are aquatic animals, mostly marine, although many species occupy freshwater or estuarine habitats. In nature, they constitute a first vital link in the marine food chain leading from primary producers to fish. They can represent up to 80% of the zooplankton biomass in the water column (Mauchline, 1998). In the open water marine environment, calanoids dominate the herbivorous zooplankton and provide the food-chain base for practically all marine fish larvae and planktivorous fish (Pauly and Christensen 1995). The diets of pelagic copepods are characteristically broad (Kleppel, 1993) and strict herbivory copepods rarely exists in nature. Most copepods prefer feeding on microzooplankton due to their large size, easy perception, as well as the relatively high food quality (Batten et al., 2001; Gifford et al., 2007; Campbell et al., 2009). Moreover, copepods are capable of switching their feeding behavior depending on the prey composition, and particularly on the relative abundance of phytoplankton and microzooplankton (Landry, 1981). It is well accepted that many copepods are a valuable source of food for fish larval rearing although they are not often used in aquaculture industry. Interest in the use of copepods in aquaculture has grown since the 1980s (Schipp, 2006). Over the past few years there have been several review articles published and numerous conferences, conference sessions and workshops dedicated to discussions of copepod culture and the important role that copepods can play as feeds for marine fish larviculture (Bell et al., 1997; Støttrup, 2000; Kleppel and Hazzard, 2002; Lee et al., 2005). The three main freeliving copepod orders, Cyclopoida, Harpacticoida and Calanoida have each been investigated for their suitability as feeds for larval and juvenile fish (Marcus, 2005; Gaudy and Guerin, 1982; Ogle et al., 2005; Payne and Rippingale, 2001a; Phelps et al., 2005; Schipp et al., 1999; Støttrup and Norsker, 1997; Sun and Fleeger, 1995). Cyclopoids include pelagic, epibenthic, benthic and parasitic species and inhabit both freshwater and marine environments, but most abundant in freshwater. Cyclopoid copepods with obligatory pelagic nauplii are pelagic and are used occasionally in aquaculture and densities of ~5000 ind./L are possible to achieve in cultures (Phelps et al., 2005; Su et al., 2005). Cyclopoids definitely offer a great potential for aquaculture and aquarium trades but they have not been intensively studied maybe because of the Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 5 difficulties in harvesting nauplii from culture (Støttrup, 2006) and lack of storage possibilities for eggs. Harpacticoids include over 50% of copepod species and are primarily marine free living, benthic organisms. Harpacticoid copepods can be produced in high density cultures, but their benthic habitat may limit their availability to pelagic fish larvae (Drillet et al., 2008; Lee et al., 2006; Olivotto et al., 2008b; Puello-Cruz et al., 2009; Støttrup, 2006). Fleeger (2005) cites the advantages of using harpacticoid copepods in marine fish cultures. Amongst them, according to Lavens and Sorgeloos (1996), benthic harpacticoid copepods of the genera Tisbe and Tigriopus have characteristics that facilitate cultivation, such as high fecundity, short generation time, tolerance to extreme environmental changes (salinities ranging from 15 to 70psu and temperatures from 17 to 30°C), acceptance of a variety of food sources (microalgae, yeast, dry feeds) and high population densities can be achieved. Calanoids are predominantly pelagic, occurring at all depths, with some living near bottom benthic species. They are selective feeders, feeding on small phytoplankton cells by filtration or predators feeding on animal prey including copepod eggs. Several species belonging to the calanoida order have been proven as ideal food for many cultured marine larvae (Molejón and Alvarez-Lajonchère, 2003; Marcus, 2005; Schipp, 2006; Milione and Zeng, 2008; Camus and Zeng, 2009) (Table I), showing excellent nutritional value when compared to rotifers and Artemia. Moreover, calanoid copepods are also interesting due to their pelagic, produce resting eggs. However, many calanoids cannot be kept at high densities without negative Table I - Species of Calanoids used in aquaculture as live prey for marine fish species (Støttrup, 2003). Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 6 side effects, like a decrease in hatching success or high mortalities (Jepsen et al., 2007; Peck and Holste, 2006). Independently of the copepod order (Calanoida, Harpacticoida or Cyclopoida), it is well known that raised copepods as well as harvested zooplankton contain biochemical characteristics which makes them a good alternative or supplement of live prey for larval rearing (Naess et al., 1995; Shansudin et al., 1997; Støttrup and Norsker, 1997; McEvoy et al., 1998; Rønnestad et al., 1998; Payne and Rippingale, 2000; Støttrup, 2000; Payne et al., 2001; Evjemo et al., 2003). They are considered to be “nutritionally superior live feeds” for commercially important cultivable species, as they are a valuable source of protein, carbohydrates, enzymes (amylase, protease, exonuclease and esterase) and lipid (with particular regard to fatty acids) (Watanabe, 1979; Watanabe et al., 1983; Witt et al., 1984; Shansudin et al., 1997; Toledo et al., 1999), which are essential for larval survival, growth, digestion and metamorphosis (Støttrup, 2000; Molejón and Alvarez-Lajonchère, 2003; Kleppel et al., 2005). The fact that these organisms contain highly unsaturated fatty acids, especially n-3 fatty acids, in comparison to other live prey used in aquaculture is an advantage, not only because fatty acids are important components of biomembranes in fish, as well as providing energy (Cowey and Sargent, 1972; Rodriguez et al., 1993; Izquierdo, 1996; Barclay and Zeller, 1996; Sargent et al., 1997), but also because there is likely no need for further enrichment, as is required with rotifers and Artemia nauplii. Sargent et al. (1997) listed the major advantages in using copepods for marine finfish larval feeds as follows: (1) A preponderance of phospholipids rather than triacylglycerols; (2) Levels and ratios of fatty acids that more closely approximate the natural diet of marine finfish larvae; (3) The probability of optimal protection of polyunsaturated fatty acids (PUFA) by natural antioxidants against peroxidation and the delivery of optimal levels of natural antioxidants to the larvae. In addition, copepods are also known to have greater digestibility (Schipp et al., 1999) because of a slower passage through the gut of fish larval than Artemia spp., which leads to a more complete digestion and efficient nutrient uptake (Pedersen, 1984). This may be due to the fact that copepods have higher digestive enzyme contents than Artemia which can be used by the fish larvae as exo-enzymes (Munilla-Moran et al., 1990). Finally, the wide range of body sizes of copepods makes them more suitable to predation by larvae and juvenile fish. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 7 Støttrup (2000) and Payne et al. (2001) strongly suggest that the inclusion of copepods in the aquaculture industry may increase the number of successfully reared fish species. The future expansion of marine aquaculture may further encourage work on copepods towards the development of reliable production systems or, alternatively, the production of resting or diapause eggs for sale on a commercial scale (Støttrup, 2003). 1.2.2 Considerations of copepod culture Despite significant progress in copepod cultivation methods (Payne and Rippingale, 2001b; Støttrup, 2003; Lee et al., 2005), establishing cost-effective protocols for mass production are still a challenge. The progress towards mass-culture techniques of copepods has been slow and not thoroughly researched in larviculture. In addition, development of this field has been fragmentary (Støttrup, 2000). Intensive research is therefore needed in copepod culture engineering to enhance the appropriate culture techniques that will promote the feasibility and cost-effectiveness that allow their in the aquaculture industry (Ajiboye et al., 2011). 1.2.3 Factors affecting production The most important factors controlling stage duration of copepods are temperature, light, food quantity and quality (Cook et al., 2007), amongst others. The primary need for information with regards to large-scale and dense copepod cultures resides in the effects of these parameters during intensive cultivation. Temperature has to be adapted to each population of copepods and is often analogous to the conditions the population is facing in wild because both populations are adapted to these particular environments. Species inhabiting coastal environments are usually more tolerant to variations in salinity and have wider thermal tolerance. Temperature has been positively related to growth rate (MacLaren, 1965-1966; Landry, 1975) and egg production rate (Uye, 1981), but inversely related to body size (Deevey, 1960). Light rhythm is another important factor controlling the physiological performances of copepods, activating sometimes the production of diapause stages (Marcus, 1982; Alekseev et al., 2007). This factor affects egg production and hatching success of Acartia sp. cultures; longer light exposure increased the 48 hour hatching success of the eggs Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 8 (Camus and Zeng, 2008; Peck et al., 2008). However, cultivation procedures kept constant over time under particular conditions are likely to select for specific traits. For example, cultures of A. tonsa from the Danish Technical University (DTU-Aqua) have lost their diel feeding and egg production rhythm due to excess food availability and absence of predators through 150 generations (Tiselius et al., 1995). The amount and quality of food are also essential parameters that enhance the production of copepod cultures. Effects of food quantity and quality have been assessed in natural populations because of their importance at the ecological level. Most of the information present in the literature can be extrapolated and used for culture purposes. Quantity-wise, all copepods do not have the same need to reach their maximum growth and production potentials even when closely related (Jonasdottir, 1989). Too little food tends to slow down stage development, increase the competition for food between the individuals, and ultimately increases mortality (Berggreen et al., 1988). Food quality should be adjusted for the target number of copepods, and the size of the prey should be consistently adapted not only to the species but also to the development stages that need to be fed (Berggreen et al., 1988; Hansen et al., 1994). Previous research has shown that a mixture of different diets generally enhances the somatic growth and egg production of copepods in the laboratory (Harris, 1977; Jonasdottir, 1994; Klein Breteler, 1980), but also the delivery of right food item at the right development stage increases the overall success (Koski et al., 2006; Murray and Marcus, 2002). The food items should be adapted to the copepod feeding habits for example whether the copepods raised are raptorial feeders or suspension feeders. 1.2.4 Culture of Calanoids – Family Acartiidae The most frequently cultured calanoid species belong to the genera found in coastal waters, such as those of the genera Acartia, Centropages, Eurytemora and Temora. These copepods are small, with relatively short generation times and a wide thermal and salinity tolerance (Støttrup, 2003). On this work, we focused in one copepod of the family Acartiidae because they are common in coastal and estuarine habitats in all oceans of the world. They are thought to be mainly adapted to high food concentrations which are found in estuaries and upwelled waters. Their wide distribution in space and time may be due to the fact that a number of Acartia species are known (1) to produce resting eggs which lie dormant in the sediment and allow them to appear suddenly in the plankton when conditions are favorable (e.g. Uye, 1983; Lindley, 1990; Naess, 1991; Belmonte, 1992) and (2) to be transported in Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 15 1.3.2 Starter Culture of Rhodomonas marina Microalgae sterile starter cultures were supplied by the Institute Investigation of fisheries and sea (IPIMAR - Algarve). The culture began in test tubes with seawater filtered and UVtreated 25 psu (Fig.6A). From November 2011 to January 2012, several culture tests were performed in order to successfully cultivate Rhodomonas marina CMC facilities. In our first attempts, the culture “crashed” after 2-3 days or was contaminated with different kind of ciliates, which made difficult to proceed with the batch method. This algae is technically more demanding to culture than other algae traditionally used in aquaculture facilities (Knuckey et al., 2005). After some attempts, we successfully cultivated Rhodomonas in our facilities (Fig. 5), and used it as live feed to Acartia grani. 1.3.3 Algae Production Microalgal species can vary significantly in their nutritional value, and this may also change under different culture conditions (Enright et al., 1986; Brown et al., 1997). Nevertheless, a carefully selected mixture of microalgae can offer an excellent nutritional package for larval animals, either directly or indirectly (through enrichment of zooplankton) (Brown, 2002). The most important parameters regulating algal growth are nutrient quantity and quality, light, pH, turbulence, salinity and temperature (Table IV). Figure 6 - Microalgae sterile starter cultures. (A) The three main algae used in this thesis; Isochrysis sp. (T-Iso), Rhodomonas marina and Tetraselmis suecica respectively. (B) The stock culture. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 16 Figure 7 - Different microalgae and Oxyrrhis marina, in our laboratory, with controlled temperature (20±1°C). Table IV - Culture condition of microalgae. The most optimal parameters as well as the tolerated ranges are species specific. All microalgae used in this work (Fig.6B and Fig.7) were produced according to the batch methodology. A batch culture is a discontinuous culture, sterilized and inoculated by the microorganism wanted. It enables its growth until nutrients become limited. The medium has to contain at least one energy, carbon, nitrogen and mineral ions source. A single inoculation of cells into a container (10-6000ml) of fertilized seawater (filtered and UVtreated) followed by a growing period of several days 5-7 and finally harvesting when the algal population reaches its maximum or near-maximum density 20 - 40 x 105 cel.ml-1, depending the microalgae used. In practice, algae are transferred to larger culture volumes (Fig.8) prior to reaching the stationary phase and the larger culture volumes are then brought to a maximum density and harvested. Species Rhodomonas marina Tetraselmis suecica Isochrysis sp. (T-Iso) Temperature 20±1°C Salinity 25 psu Photoperiod 24 hours light Aeration Moderately Nutrient Nutribloom 1:1000ml Figure 8 - Production of microalgae in our laboratory. (A) Erlenmeyer (250ml) with Rhodomonas marina and Tetraselmis suecica. (B) Rhodomonas marina in 1L balloons. 17 2| Objectives Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 18 2.1 Aim of the study Numerous studies have demonstrated that copepods may have a higher nutritional value than Artemia, as the nutritional profile of copepods appear to match better the nutritional requirements of marine fish larvae. Furthermore, they can be administered under different forms, either as nauplii or copepodites at start feeding and as on grown copepods until weaning (Delbare, et al., 1996). Over the years several studies have focused in copepods culture, to understand the optimal biotic and abiotic factores that would satisfy the need of regular culture production. Most Acartia grani studies published to date do not cover important biological aspects of culture, including optimal temperature and photoperiod conditions or best diet. Some of these parameters have however been determined for other species of copepods. The aim of this thesis was to improve knowledge of the copepod Acartia grani, in order to determine the culture requirements and the most appropriate biotic and abiotic factores. This project was made in lab scale, but some of the culture conditions and requirements can be applied to large scale production. The thesis presents seven chapters, excluding the introduction, discussion-final remarks and each chapter is briefly described below: - To begin a copepod culture, is crucial to know the hatching rates (present in Chapter I), in order to prepare the correct food concentration, for supplying at the beginning of feeding stage. - Variability in temperature characterizes seasonal succession and directly affects copepod reproduction and development (Ianora et al., 1992). In Chapter II different temperatures were tested to evaluate the optimal culture temperature. - Photoperiod is one of the most significant cues for seasonality in nature (Hairston and Kearns, 1995) and could therefore be a key factor controlling female copepod reproductive status and population dynamics. This abiotic factor was investigated in Chapter III. - Food quality and quantity are probably the most important factors regulating the productivity of copepod culture: Chapter IV and V tested different diets to determine the most appropriate diet for A. grani based on the most common cultured microalgae species. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 19 - Copepods are a rich source of phospholipids, essential highly unsaturated fatty acids (HUFA), natural antioxidants and other essential compounds (Kraul et al., 1992; Sargent et al., 1997) Chapter VI describes the methodology used to identify the nutritional value of Acartia grani, based on lipid and fatty acids of adult copepod fed with Rhodomonas marina and Oxyrrhis marina. - High densities are considered difficult due to density-related stress factores (Jepsen et al., 2007). One of the stress factores of high densities is cannibalism. In Chapter VII we tested this factor at different culture densities. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 20 21 3| Chapters Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 22 Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 23 Chapter I Egg hatching success at different temperatures of calanoid Copepod Acartia grani 1. Introduction Copepods are primary consumers in the oceans and are perhaps the most numerous metazoans on earth (Ohman and Hirche, 2001). Calanoid copepods play a key role in the cycling of nutrients and energy in marine ecosystems by forming a trophodynamic link between primary (phytoplankton) and tertiary (e.g. planktivorous fish) production (DeYoung, 2004). The widespread distribution and abundance of members of this group partially result from adaptation of life history traits to match specific environmental (physical or chemical) conditions or constraints. Amongst these environmental conditions, temperature is often considered as the key external factor that affects life history traits and the population dynamics of copepods. Variability in temperature characterizes seasonal succession and directly affects copepod reproduction and development (Ianora et al., 1992). Changes in species abundance in the Baltic sea, throughout the year, have been related to wide range variations in salinity and temperature that exceed those of the preferred niche of the calanoid species found there (Holste and Peck, 2006). Egg hatching rate and success are also reported to be temperature dependent (McLaren, 1966; Uye and Fleminger, 1976; Ban, 1994; Holste and Peck, 2005). Furthermore, temperature dependent hatching patterns are related to spawning temperature (Landry, 1975; Uye and Fleminger, 1976). Unfortunately, the functional response of reproductive success (i.e., egg production and hatching) to temperature variations in many calanoid species is not well known, having been studied in only a handful of species such as Eurytemora affinis (Gonzalez and Bradley, 1994) and a number of Acartia congeners (Tester and Turner 1991; Chinnery and Williams, 2004). Koski and Kuosa (1999) found that unfavorable temperature might be the probable reason for low hatching in Acartia bifilosa and Milione and Zeng (2008) found that temperature significantly affected hatching times of Acartia sinjiensis. Acartia grani is typical of coastal, semi-confined ecosystems, conditioned by a high degree of physical conditions (temperature and salinity) (Calbet and Alcaraz, 1996). Hatching success is responsible for the growth of the culture, being crucial in the reposition of new copepods in the culture systems. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 24 2. Material and methods 2.1 Copepod stock culture Stock culture was kept according to the conditions described in chapter 1.2.7. Eggs used in this experiment came from our “egg bank”. The “egg bank” contains harvested and “cleaned” eggs, which were stored in 1µm filtered and UV-treated seawater in closed falcon tubes at 4ºC. 2.2 Experimental design and setup For this experiment, eggs from a falcon tube were rinsed in a mesh (55µm) with distilled water to wash out some of the accumulated feces that are stored with the eggs. This procedure is known not to affect viability of unhatched eggs (Knuckey et al., 2005). Eggs were then transferred into a 500ml beaker containing seawater. The beaker was agitated for 15 seconds waiting for a few seconds to allow air bubbles to surface. Then a sample of 1ml was taken from the beaker using an automatic pipette (Pipetman – Gilson 1000uL). This was repeated for five times and the number of eggs in each of these subsamples was counted using a Sedgewick-Rafter counting cell and a microscope (Zeiss – Axioskop 2 Plus). The variability of egg numbers between sub-samples was less than 10%. The average number of eggs in the sub-samples was calculated and used to estimate the concentration of eggs in the stock solution. Twelve 600ml beakers were incubated with 150 A. grani eggs at four different temperatures: 18.0, 22.0, 24.0 and 28.0 ±0.5°C, using water baths; each temperature treatment had 3 replicates. Throughout the experiment, great care was taken to ensure that the correct temperatures were maintained by continuously monitoring the temperature in all water baths and all other conditions were kept very similar in all replicates. Each beaker contained 300ml of seawater (1µm filtered - UV treated), with salinity 38±1 psu and 24 hours light (approximately 1200-1500lux). 2.2.1 Experimental procedure With the use of a plastic pipette, all hatched nauplii were carefully removed from the beakers and discarded. This procedure was repeated every two hours, until each treatment reached 50% individual hatching time. At the end of the experiment data were analyzed: 1. The cumulative egg hatch (CUM%) was calculated: EHR (CUM%) = [(sum of all replicates)*100] / nº total of eggs Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 31 Chapter II The effects of temperature on population growth, egg production and egg hatching success of the calanoid copepod Acartia grani (Calanoida: Acartiidae) 1. Introduction Identifying factors that control the growth of copepods is essential to understanding nutrient and carbon fluxes in the marine environment. There has been considerable debate in the literature about the relative importance of the two main factors that control copepod growth: food and temperature (Ban, 1994). The influence of temperature on growth rates in the wild has been well documented, especially in temperate seas (Middlebrook and Roff, 1986; Davis, 1987; McLaren et al., 1989). Variability in temperature characterizes seasonal succession and directly affects copepod reproduction and development (Ianora et al., 1992). In their review of field measurements of growth rate, Huntley and Lopez (1992) concluded that copepods grow at maximum rates in the field, with an exponential increase in growth rate with temperature over a wide range of habitats. The same authors have also shown that 90% of the growth variability in 33 copepod species can be explained by temperature alone. Research investigating the effects of temperature and salinity on the productivity of calanoid copepods has shown their clear effects on egg production (Devreker et al., 2009) and egg hatching rates (Hall and Burns, 2002; Peck and Holste, 2006; Holste and Peck, 2006). Most calanoids are broadcasters, shedding eggs singly into the water. The number of eggs spawned in a single event may vary from a few eggs to 50 or more eggs and each spawning event may occur about once every 24 h for extended periods. Free-spawning species such as various Acartia species may produce between 11 and 50 eggs female1day-1, producing a total of up >1200 from one single spawning (Støttrup, 2003). Rodriguez et al. (1995) on a seasonal scale showed that temperature did not serve to predict completely the egg production rate of Acartia grani. Instead, the coincidence of a temperature increase from the winter minima of 13.5 to 18°C appeared to act as synergetic triggers for an explosive reproduction (mean = 79 eggs female-1day-1) of the adult copepods present in the water column. Moreover, nauplii growth and development of various calanoid copepod species (Geiling and Campbell, 1978; Nagaraj, 1992; Takahashi and Ohno, 1996; Payne and Rippingale, 2001; Chinnery and Williams, 2004) have also been described to be temperature related. Despite, the effects of water temperature on hatching, growth, and fecundity have been well investigated in some Acartia species such as A. clausi (Landry, 1978), A. tonsa Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 32 (Heinle, 1969) and A. calforniensis (Johnson, 1980), optimum conditions for the development of A. grani are not well known. Evaluating the effects of temperature on overall copepod population dynamics after a certain period of culture rather than focusing on a single stage of the copepod life cycle is likely to provide more complete information for the purposes of aquaculture, as analysis of population growth provides a summary of the effects of environmental factors on the interrelated parameters mentioned above that affect ultimate productivity (Milione and Zeng, 2008). Furthermore, this abiotic parameter is relatively easy to be manipulated and has a strong impact in copepods culture. The major purpose of this study was to investigate the effects of water temperature on the development of A. grani based on rearing experiments in the laboratory, with special attention to the relationship between water temperature and mortality, fecundity and developmental rates of the species. 2. Material and methods 2.1 Microalgae culture Microalgal culture used for the experiment was Rhodomonas marina. For further details concerning algae culture conditions and maintenance protocols, please refer to chapter 1.3.2. 2.2 Copepod stock culture Stock culture was kept according to the conditions described in chapter 1.2.7. Adults used in this experiment were collected from a parental culture tank with 12 days, since eggs were incubated. They were collected with a 200µm sieve and transferred to the respective treatment. 2.3 Experimental design and setup Three separate experiments were carried out to assess the effect of temperature in A.grani culture productivity, i.e. (1) egg hatching rate, (2) egg production rate and (3) population increase over a 12 day culture period. Four treatments (4 replicates) were considered on each experiment: Treatment 1: A.grani culture at 18°C Treatment 2: A.grani culture at 22°C Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 33 Treatment 3: A.grani culture at 24°C Treatment 4: A.grani culture at 28°C Throughout all the experiments, great care was taken to ensure that the correct temperatures were maintained by continuously monitoring the temperature in all water baths. Experiments and acclimatization were carried out under similar conditions: seawater 1µm filtered - UV treated; salinity 38±1psu; photoperiod 12L:12D and fed with Rhodomonas marina at 1500μgCL−1, a carbon concentration known to saturate copepod feeding (Kiørboe et al., 1985). Carbon concentrations were calculated according to Strathmann (1967). 2.3.1 Acclimatization to different temperatures Approximately 200 A. grani adults were acclimatized to each temperature for 3 days in 5L aquariums filled with seawater (1µm filtered - UV treated), with salinity 38±1psu and gentle aeration. Each aquarium received 1500μgCL−1 of microalgae every day. Rhodomonas marina concentrations in each aquarium were determined daily using a haemocytometer under a microscope (Zeiss – Axioskop 2 Plus). Temperatures and oxygen were monitored daily with the use of a thermometer and oximeter (OxyguardHandy Polaris). 2.3.2 Egg Hatching Rate experiment (EHR) As in population increase experiment, previously acclimatized copepods were collected into 600ml beakers with a sieve of 200µm. Adult’s collection to new beakers was to warranty that the eggs used in the experiment had a maximum of 24 hours. The freshly produced eggs were carefully collected with a 55µm sieve of each treatment and total eggs were counted with Sedgwick-Rafter chamber. The eggs were rinsed and put in distilled water for 2 min to kill any possible nauplii. Then 40 to 60 eggs of each treatment were randomly distributed into 100ml beakers, a total of 5 replicate beakers per temperature were used. Egg hatching success was estimated for each treatment by calculating the difference between the number of eggs unhatched and the hatched nauplii, at 10, 20, 24, 30 and 48 hours after incubation. Counting was done with a Sedgwick-Rafter chamber, as above. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 34 2.3.3 Egg Production experiment (EP) Like in the two previous experiments, copepods were subjected to a three day period of acclimatization. With the sieve 200µm, 5 active mature females were collected and carefully transferred to 100ml beakers. There, were 3 replicates per temperature and hence a total of 12 beakers. 24 hours later, all the eggs produced by the females were collected with 55µm sieve and count using Sedgwick-Rafter cell counter and a microscope (Zeiss – Axioskop 2 Plus). Following the procedure described above, new females were randomly selected daily from the 5L aquarium culture and transferred into a new set of twelve 100ml beakers containing fresh filtered seawater and microalgae to obtain individual 24h egg output for each of 3 consecutive days. The daily replacement of females ensures that the new females are fertilized and healthy, being ready to the egg production experiment. 2.3.4 Population Growth experiment After 3 days of acclimatization, 12 healthy (actively swimming and intact appendages) adults (4 Males and 8 Females) were transferred into 600ml beakers filled with 300ml seawater and without aeration. A total of 4 replicate beakers were established for each treatment. Every morning 30% of the culture water was exchanged with a siphon of 55µm mesh to prevent the loose of eggs or nauplii. After water exchanged, a sample (2ml) was collected to determine microalgal concentration on the water column, and readjust if necessary the 1500μgC.L−1. A.grani is a suspension feeder, therefore it is important to supply phytoplankton in two meals (one in the morning and the second afternoon), to ensure that microalgae stay more time in the water column. From previous observations, we knew that a complete life cycle would be possible in 9-12 days. After 12 days, content from each beaker was drained through a 55μm sieve and all eggs, nauplii, copepodites and adults retained were fixed with 4% formaldehyde and stored at 4°C. The counting of A.grani samples was made using a Sedgwick-Rafter cell counter and a microscope (Zeiss – Axioskop 2 Plus). Sex ratio was determined based on a sample of all the collected adults (50% of population count). The specific population growth rate (K) of Acartia grani was calculated using the following formula (Omori and Ikeda, 1984; Hada and Uye, 1991): K = (lnNt - lnN0)/t Here, t is the culture days (12) and N0 and Nt are the initial and final density of copepods, respectively. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 35 0 10 20 30 40 50 60 70 80 90 100 12 24 30 48 72 Cumulative Egg Hatching Rate (CUM%) Experiment Running Time (h) T 28 T 24 T 22 T 18 In addition, doubling time (Dt) was calculated by dividing loge2 by the populations growth rate (K) according to the following formula (James and Al-Khars, 1986): Dt = (loge2)/K 2.4 Statistical analysis Data from all experiments were analyzed using one-way ANOVA. When significant differences (p<0.05) were found, Tukey's multiple comparisons test was used to determine specific differences among treatments (p<0.05). All statistical analyses were conducted using SPSS, version 20.0. Data are presented as mean±standard deviation (SD). 3. Results 3.1 Egg Hatching Rate (EHR) Figure 1 presents the results of the egg hatching rate experiment. Twelve hours after incubation hatching rates were very low in all temperature treatments. Figure 1 – The cumulative egg hatching rate (CUM%) of Acartia grani eggs produced by adults with four different temperatures, over 72 hours. Eggs were incubated under identical condition of 38±1psu and photoperiod 12L:12D. First eggs incubated at 18ºC hatched only 30 hours later (2%). At the same observation hour, highest mean hatching rate was of 12.2% at 24ºC. Until 48h, EHR at 18ºC was significantly lower than eggs produced at the remaining temperature treatments. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 36 0 2 4 6 8 10 12 0 1 2 3 4 5 Egg Production Averaged from 5 females over 4 days (egg female day) Days 28°C 24°C 22°C 18°C Compared to 48h EHR, 72h hatching rates improved across temperature treatments, except for eggs produced at 28ºC, which increased only to 15% final mean EHR. Egg hatching rate was highest at 24ºC, reaching a mean value of 60.0%, followed by eggs incubated at 22ºC (44%). 3.2 Egg Production (EP) Results of 24h egg production (EP; eggs female-1day-1) of Acartia grani during 4 consecutive days are presented in Figure 2. As different females were used for each experimental day, it is possible to observe that regardless of the temperature treatment there is variation in the mean egg number per females per day. Still, within days no significant differences were observed between treatments (p>0.05), and both maximum (10.5±1.5) and minimum (1.4±0.6) EP were found on eggs laid at 18ºC on day 4 and day 2, respectively. Since no significant differences were detected within each temperature treatment, data were then pooled to calculate the overall mean 24 EP of the four days (Table I). Though egg production rate was highest in A. grani cultured at 18ºC (7.0±2.7), again no significant differences were found on the EP of Acartia grani subjected to the four considered temperatures (p=0.76). Figure 2 – Mean egg production (eggs female-1day-1) of Acartia grani with 4 different temperatures on the 4 experimental days. Data are presented as mean±SD. (Day 1 p=0.86; Day 2 p=0.08; Day 3 p=0.32; Day 4 p=0.99). Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 37 0 100 200 300 400 500 600 700 800 28°C 24°C 22°C 18°C Population increase over 12 days Temperature All stages All Post-Egg-Stages Table I – Effects of temperature on averaged 24h egg production of Acartia grani. For each treatment, 24h egg production was averaged from 5 females over 4 days. Data are presented as mean±SD. (One way ANOVA p=0.76). Temperature Eggs female-1day-1 28°C 5.7 ± 4.0a 24°C 6.3 ±4.0a 22°C 6.1 ±3.6a 18°C 7.0 ±2.7a 3.3 Population Growth The results of population growth experiment showed that temperature did not have significant effect (p>0.05) on the population growth of A. grani (Fig.3) if All Stages were included (All Included-eggs, nauplii, copepodite and adults). Still highest mean final population was observed for A. grani cultured at 18ºC (663.0±52.2) followed by 28 ºC treatment with 640.3±74.7 individuals. On the other hand, if egg stage was not included (All post-egg-stages) highest mean final population number was observed at A. grani cultivated at 22ºC (216.3±25.0), while lowest population was observed for 24ºC temperature treatment (136.5±15.8), again without significance differences. Figure 3Mean final total population of Acartia grani cultured at four different temperatures for a 12 day period. Initial population was 8 females: 4 males Data are represented as mean±SD. All Stages bar: eggs, nauplii, copepodites, adults; All post-egg-stages bar: nauplii, copepodites, adults. Different letters on the tops of bars indicate significant differences (p<0.05). One way ANOVA: All stages included p=0.86; All Post-eggsstages p=0.82. Within the population, except for the adult stage, there were no notable differences in the distribution of the various life-stages, i.e. eggs, nauplii, copepodites of A.grani cultured Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 38 at different temperatures. Adult population was significantly higher (p=0.025) at 28ºC than mean adult population cultured at 18 ºC (Table II). Table II - Mean number of four life stages (eggs, nauplii, copepodites and adults) within the population of Acartia grani cultured for 12 days at four different temperatures (18ºC, 22ºC, 24ºC, 28ºC) from an initial number of 12 adults. (K) is the specific population growth rate, and (Dt) is the doubling time. Different letters indicate significant differences (p<0.05). Data are represented as mean±SD. Temperature Eggs Nauplii Copepodites Adults K Dt 28°C 455.0 ±299.9a 10.3 ± 4.7a 118.3 ±56.9a 56.7 ± 28.9a 0.32 ±0.04ª 2.16 ±0.30ª 24°C 310.3 ±271.8a 37.5 ±30.2a 82.5 ±102.1a 16.5 ±19.0ab 0.28 ±0.07ª 2.84 ±0.39ª 22°C 318.3 ±229.4a 97.7 ±66.0a 98.3 ±96.0a 20.3 ± 17.6ab 0.30 ±0.06ª 2.35 ±0.47ª 18°C 459.3 ±196.0a 155.3 ±99.3a 40.8 ± 27.4a 7.8 ± 1.0b 0.33 ±0.02ª 2.07 ±0.14ª p= 0.764 p= 0.051 p= 0.595 p= 0.031 p=0.489 p= 0.374 The specific population growth rate (K) of A. grani was calculated for all treatments (Table II). It ranged from 0.28 to 0.33, the lowest at 24°C and highest both at 18°C and 28°C. Population doubling time (Dt) showed that 24°C was the treatment that needed more time to grow (2.84) and that the 18°C treatment presented the lowest value (2.07). For all treatments no significance differences where found in K and Dt parameters. Sex ratio of A. grani after a 12 day culture period was similar (p>0.05) between different temperatures, except for adults cultured at 18ºC that presented a skewed tendency sex ratio toward males (80%F:20%M) (Fig. 4). Figure 4 – Sex ratio of A.grani in the population increase experiment after 12 days. 0 10 20 30 40 50 60 70 80 90 100 28°C 24°C 22°C 18°C Sex Ratio in Population Increase (Female:Male) Temperature Male Female Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 39 Mortality of the initial adult population was registered and results revealed that on the first three experimental days, between 10% (18ºC) and 21% (22ºC) of the initial adults had died. By the 8th experimental day, mortality reached 55.6% at 18ºC, followed by 22ºC with a mean mortality rate of 54.0%. From that day onwards mortality was not counted, due to possible misleading of initial incubated adults with born grown adults during the experiment. 4. Discussion Temperature can be relatively easily manipulated in aquaculture hatcheries, where mass culture of copepods is desirable as a means of providing live prey for culture animals (Milione and Zeng, 2007). The aim of this study was to evaluate the importance of temperature on the egg hatching rate, egg production and population increase. Aside from egg production, copepod productivity may also be linked to other factors, such as the hatching rate of eggs and subsequent survival and development of the nauplii and copepodites, as well as life expectancy and sex ratio of the adults (Knuckey et al., 2005). Egg production and hatching rate are normally lower at low temperatures (Ambler, 1985; Uriarte et al., 1998), and generally increase with increasing temperature up to a thermal threshold, after which decline begins. This was reported by Takahashi and Ohno (1996) in A. tsuensis; White and Roman (1992), Holste and Peck (2006) reported it for A. tonsa and Milione and Zeng (2008) for A. sinjiensis. Commonly A. grani was cultured at 18°C (Calbet and Alcaraz 1996, 1997; Guerrero and Rodriguez, 1998; Costa and Fernández, 2002). However, new data (Cunha, 2010 unpublished data) suggest that better production could be achieved with warmer temperatures. Leandro et al. (2006) found that Acartia clausi, in special the northern population, had a faster growth rate when reared at high temperatures. Also, Chinnery and Williams (2003) concluded that highest temperatures influenced survival of nauplii of Acartia species (A. bifilosa, A. clausi, A. discaudata and A. tonsa). Takahashi and Ohno, (1996) studying A. tsuensis showed that high hatching success, normal isochronal development, minimum mortality and high fecundity was obtained consistently at around 25.0°C, when compared with temperatures that ranged between 17.5°C and 30 °C. In our study, some of the data do not concur with the above mentioned studies. EHR increased with increasing temperature, though EHR in the 28°C treatment had the lowest rate after 72 hours of incubation. Contrary to our findings, Rodriguez et al. (1995) observed in August (22°C to 26°C) with A.grani that 89% of eggs produced hatched in 48- Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 40 72 hours. In the previous study that we realized with Acartia grani incubated at different temperatures (Chapter I), this same temperature showed the highest egg hatching rate, being included in the optimal range of temperatures for induction of better EHR of A.grani. We believe that low egg hatching rate at 28°C was due to either the appearance of bacteria in the culture medium or bad quality of the eggs. According to Peck and Holste (2006) it is possible that using eggs produced from different cohorts (or even from different days from the same cohort) could have contributed to variability in egg hatch success. Moreover, on our first experiment hatching rates were found as soon as eleven hours after incubation. On the current experiment, first hatch occurred further later (thrity hours) and within the temperature treatment of 18ºC. Egg production data revealed that averaged 24h EP was similar in A. grani cultured at different evalueted temperatures. Koski and Kuosa (1999) found that for 24ºC affected negatively survival and egg production of A. bifilosa. Similarly, Acartia tonsa egg production appears to be inhibited when temperature exceeds 27 ºC (White and Roman, 1992). However, in our experiment, on the first two days, the highest temperature (28°C) presented the maximum egg production, although no significances were found. Holste and Peck (2006) found for Acartia tonsa an increase in egg production rate with increasing temperature that was far stronger than that estimated from studies of other calanoid copepod species. Temperature also has a direct influence on the development rate and the survival of copepod nauplii (Mauchline, 1998; Dussart and Defaye, 2001; Peterson, 2001). In our study, population growth of Acartia grani was also not significantly affected by temperature. The highest mean final population was observed at 18ºC if all stages were considered, but the 28 ºC treatment had only less 23 individuals. Taking into account that the same cohort of copepods was used and analyzing egg hatching rate and egg production at 18 ºC, results of final population growth if all stages are included could be easily explained at this temperature. However, due to possible lower metabolic rates, adult population was only 7.8±1.0 individuals with significant differences to 56.7±28.9 adults of the 28 ºC treatment. Development time decreased as the temperature increased. According to Chinnery and Williams (2004), temperature is considered the most important fact affecting juvenile development and growth rate in Acartia congeners. The development time of P. elongata copepodites (Ozaki and Ikeda 1997) and S. tenellus nauplii (Kimoto et al. 1986) also decreased with increasing temperature. Landry (1975) explained this effect by assuming that development is controlled by a series of biochemical reactions, the rates of which are regulated by temperature. Moreover, if similar results to our previous egg hatching rate experience had been found (EHR at 28ºC was of 74.3±1.2%), the number of eggs and nauplii would have certainly increased. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 47 of Rhodomonas marina. Microalgal concentrations were determined daily using a haemocytometer (Malassez counting chamber) under a microscope (Zeiss – Axioskop 2 - plus). 2.3.2 Egg Hatching Rate experiment (EHR) Acclimatization of A. grani adults for the hatching rate experiment followed the same procedure as per population growth experiment. Adults were then collected with a 200µm sieve to new beakers to warrant that the eggs used in the experiment were fresh. After a 24h period, the freshly produced eggs of each photoperiod treatment were carefully collected with a 55µm sieve and total number of eggs was counted with a SedgwickRafter chamber. Samples of 40 to 60 eggs (counted under a microscope) were randomly distributed in each of the five replicates per treatment. Egg hatching success was estimated for each photoperiod by calculating the difference between the number of eggs unhatched and the hatched nauplii at 10, 20, 24, 30 and 48 hours after incubation. 2.3.3 Egg Production experiment (EP) With the 200µm sieve adults were collected from the acclimatization 5L aquarium and placed in a Petri dish with filtered seawater. Seventy five mature females were carefully transferred to fifteen 100ml beakers (five females per replicate; three replicates per treatment). Each replicate contained fresh filtered seawater and Rhodomonas marina. After 24 hours freshly produced eggs were collected with 55µm sieve and counted under a microscope (Zeiss – Axioskop 2 - plus) with a cell counter Sedgwick-Rafter. For three consecutive days, the same procedure was followed and new females were randomly selected from the acclimatization aquarium and transferred into a new set of twelve 100ml beakers. The daily replacement of females ensures that the new females are fertilized and healthy, being ready to the egg production experiment. 2.3.4 Population Growth experiment Adult A. grani were siphoned from the acclimatization aquarium onto a 200µm sieve and were placed in a Petri dish with a small amount of seawater. Individuals were randomly captured using a fine-tipped pipette. Twelve healthy (actively swimming and Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 48 intact appendages) adults (4 Males and 8 Females), were transferred into 600ml beakers filled with 300ml seawater and without aeration. A total of 20 replicate beakers were established with 4 replicates for each treatment. Every morning 30% of the culture water was exchanged with siphon 55µm mesh to prevent the loose of eggs or nauplii. After water exchanged, a sample was collected to determined microalgal concentrations on the water column, and readjust if necessary the 1500μgC.L−1, a carbon concentration known to saturate copepod feeding (Kiørboe et al. 1985). Carbon concentrations were calculated according to Strathmann (1967). A.grani is a suspension feeder, therefore it is important to supply phytoplankton in two meals (one in the morning and the second at afternoon), to ensure that microalgae stay more time in water column. The population growth experiment lasted 12 days, after which all the contents of each replicate beaker was drained onto a 55µm sieve mesh and all eggs, nauplii, copepodites and adults retained were fixed with 4% formaldehyde and stored 4°C until further counting. The counting of A.grani samples were made using a Sedgwick-Rafter cell counter and a microscope (Zeiss – Axioskop 2 - plus). The specific population growth rate (K) of Acartia grani was calculated using the following formula (Omori and Ikeda, 1984; Hada and Uye, 1991): K = (lnNt - lnN0)/t Here, t is the culture days (12) and N0 and Nt are the initial and final density of copepods, respectively. In addition, doubling time (Dt) was calculated by dividing loge2 by the populations growth rate (K) according to the following formula (James and Al-Khars, 1986): Dt = (loge2)/K 2.4 Statistical analysis Data from all experiments were analyzed using one way ANOVA. When significant differences (p<0.05) were found, Tukey's multiple comparisons test was used to determine specific differences among treatments (p<0.05). All statistical analyses were conducted using SPSS, version 20.0. Data are presented as mean±standard deviation (SD). Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 49 3. Results 3.1. Egg Hatching Rate (EHR) Figure I shows the results of the egg hatching rate experiment. No hatching occurred in the first two observation hours (12 and 24 hours after incubation) in all photoperiod conditions. For that reason, these results were excluded from statistical analysis. At 30h, A. grani eggs subjected to photoperiods of 24L:0D and 6L:18D, presented an EHR of 4.3% and 3.5% respectively. Two days after incubation, hatching had occurred in all photoperiods considered. Still, EHR was very low, not reaching 50%.The highest rate was observed for 6L:18D treatment (21.0%), being the lowest within the photoperiod of 12L:12D (2.8%). Figure 1 – The cumulative egg hatching rate (CUM%) of Acartia grani eggs produced by adults at five different photoperiod over 48 hours. Eggs were incubated under identical conditions of 24±0.5°C and 38±1psu. 3.2 Egg Production (EP) Under different photoperiod regimes, the daily egg production rates per female of A.grani over 3 consecutive days are showed in Figure 2. No clear trend was observed between different photoperiod treatments (p>0.05) per day. Oscillation of egg production between treatments and days was evident. Adults subjected to a photoperiod 12L:12D, presented the highest and lowest value of egg production: 9.5±1.9 (day 1) and 1.3±0.8 0 5 10 15 20 25 30 35 40 45 50 30 48 Cumulative Egg Hatching Rate (CUM%) Experiment Running Time (h) 24L:0D 18L:6D 12L:12D 6L:18D 0L:24D Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 50 (day 3), respectively. Second highest EP was registered with the photoperiod of 0L:24D (8.7±4,6 eggs.female-1day-1) in day one. Figure 2 – Mean egg production (eggs female-1day-1) of Acartia grani, with 5 different photoperiods on the 3 experimental days. Data are presented as mean±SD. (Day 1 p=0.54; Day 2 p=0.63; Day 3 p=0.68). Since no significant differences were detected within each photoperiod treatments, data were then pooled to calculate the overall mean 24 EP of the three days (Table I). Though egg production was highest in A. grani cultured at 12L:12D (6.5±4.6 eggs female1day-1), again no significant differences (p=0.62) were found on the EP of Acartia grani subjected to the five considered photoperiods. Table I – Effects of photoperiod on averaged 24h egg production of Acartia grani. For each treatment, 24h egg production was averaged from 5 females over 3 days. Data are presented as mean±SD (One way ANOVA p=0.62). Photoperiod Eggs female-1day-1 24L:0D 5.0 ±2.7a 18L:6D 5.1 ±2.3a 12L:12D 6.5 ±4.6a 6L:18D 3.9 ±1.8a 0L:24D 5.5 ±3.8a 0 2 4 6 8 10 12 0 1 2 3 4 Egg Production Averaged from 5 females over 3 days( egg female-1day-1) Days 24L:0D 18L:6D 12L:12D 6L:18D 0L:24D Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 51 3.3 Population Growth Figure 3 presents the results of population growth experiment after 12 days of culture at different photoperiods. The results showed that photoperiod did not have a significant effect on the population growth of Acartia grani (p>0.05). The average final population numbers are presented in two categories: All stages included (i.e. eggs, nauplii, copepodites, adults); and All post-eggs-stages (i.e. excluding eggs). The highest final population All stages included was found in treatment 12L:12D, reaching a mean population of 964.0±410.7 individuals, from 12 initial adults. Similar results were found under no light conditions (0L:24D) (951.0±310.4). At constant light, population was reduced to 658.0±187.1 individuals, though not statistically different. The results of population growth when all post-eggs-stages were considered significantly reduced the number of individuals in all photoperiod treatments. Moreover, highest number was now found under the photoperiod of 0L:24D, with a population of 253.3±58.4 individuals, while lowest population was observed at 18L:6D (98.5±15.2). Still, no significant differences were observed between photoperiod treatments. Figure 3Mean final total population of Acartia grani cultured at five different photoperiods for a 12 day period. Initial population was 8 females: 4 males. Data are represented as mean±SD. All Stages bar (all life stages: eggs, nauplii, copepodites, adults); All post-egg-stages bar (nauplii, copepodites, adults). Different letters on the tops of bars indicate significant differences (p<0.05). One way ANOVA: All Stages included p=0.94; All Post-Eggs-Stages p=0.07; (24Light: 0Dark; 18Light: 6Dark; 12Light: 12Dark; 6Light: 18Dark; 0Light: 24Dark). Within the population, there were also no significant differences (p>0.05) in the distribution of the various life-stages, i.e. eggs, nauplii, copepodites and adults of A.grani 0 200 400 600 800 1000 1200 1400 1600 24L:0D 18L:6D 12L:12D 6L:18D 0L:24D Population increase over 12 days Photoperiod (Ligth:Dark) All Stages All Post-Egg-Stages Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 52 0 10 20 30 40 50 60 70 80 90 100 24L:0D 18L:6D 12L:12D 6L:18D 0L:24D Sex Ratio in Population Increase (Female:Male) Photoperiod Male Female cultured at different photoperiods (Table II). The specific population growth rate (K) of A. grani was calculated for all treatments (Table II). It ranged from 0.32 to 0.35, the lowest was in 24L:0D and 18L:6D treatments and highest was observed in 12L:12D and 0L:24D. Population doubling time (Dt) (Table II) showed that 24L:0D was the treatment that needed more time to grow (2.18±0.36). For all treatments no significance differences where found in K and Dt parameters. Table II - Mean number of four life stages (eggs, nauplii, copepodites and adults) within the population of Acartia grani cultured for 12 days at five different photoperiods from an initial number of 12 adults. (K) is the specific population growth rate, and (Dt) is the doubling time. Different letters indicate significant differences (p<0.05). Data are represented as mean ±SD. Photoperiod Eggs Nauplii Copepodites Adults K Dt 24L:0D 440.3 ±197.4a 107.0 ± 93.3a 85.5 ±58.9a 25.0 ±13.5a 0.32 ±0.05a 2.18 ±0.36a 18L:6D 607.3 ±248.0a 49.5 ±45.2a 29.2 ±43.7a 19.8 ±12.6a 0.32 ±0.04a 2.09 ±0.26a 12L:12D 857.0 ±534.4a 39.5 ±15.4a 44.5 ±31.4a 23.3 ±13.6a 0.35 ±0.06a 2.03 ±0.44a 6L:18D 565.0 ±450.2a 86.5 ±76.6a 124.0 ±96.8a 31.8 ±17.3a 0.33 ±0.05a 2.07 ±0.26a 0L:24D 697.8 ±344.4a 115.8 ±71.2a 120.5 ±50.0a 17.0 ±6.3a 0.35 ±0.04a 1.97 ±0.26a p= 0.614 p= 0.413 p= 0.140 p= 0.582 p= 0.886 p= 0.913 Sex ratio of A. grani after a 12 day culture period was similar (p>0.05) between different photoperiods (Fig.4). Highest incidence of females was registered in 24L:0D treatment (57.9%) followed by 0L:24D (57.4%) and 18L:6D (55.7%). Photoperiods of 12L:12D and 6L:18D presented the highest percentage of males. Figure 4 – Sex ratio of A.grani in the population increase experiment after 12 days. Mortality of the initial adult population was registered and results revealed that on the first three experimental days, 4.2% of initial adults died in all treatments with the exception Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 53 of 24 light regime that had highest mortality percentage of 6.3%. By the 8th experimental day, mortality reached 14.6% at 12L:12D, followed by 6L:18D with a mean mortality rate of 12.5%. From that day onwards mortality was not counted, due to possible misleading of initial incubated adults with born grown adults during the experiment. 4. Discussion Over the years copepods are more promising in the aquaculture because of the potential to use as live feed. Interest in large-scale culturing copepods is therefore growing and recent reviews (Støttrup, 2003; Lee et al., 2005; Drillet et al., 2011) discuss culturing techniques and the application of copepods as live prey in marine fish aquaculture. Although photoperiod is a major environmental parameter that can be easily manipulated with minimum costs in aquaculture hatcheries (Chinnery and Williams, 2003), effects of photoperiod on copepod productivity have not been well examined (Peck and Holste, 2006). In the present study, we analyzed the photoperiod impact on the egg hatching, egg production and population increase of Acartia grani. In the egg hatching rate our data showed that, despite no significant differences were observed, Acartia grani eggs prefer to hatch in conditions with less light, since better results were obtained within the photoperiod of only 6 hours of light (21.0%). However the results are not satisfactory, when compared with other studies, thus egg hatching rate seemed to be very low in all photoperiod conditions. Moreover, and contrary to our findings, Camus and Zeng (2008), using A. sinjiensis obtained the lowest egg hatching rate (72.9±2.6%) at constant darkness (0L:24D) and the highest EHR (87.2±1.4%) at constant light (24L:0D), over 48hours. In 2006, Peck and Holste showed that egg hatching rates for Acartia tonsa was highest (85%) in the treatment exposed to more light hours. Results in egg production demonstrated that photoperiod did not affect egg production per female per day neither production per female over 3 days. Peck and Holste (2006) also reported to A. tonsa that total number of egg output was unaffected by photoperiod. Overall the highest averaged 24 hours egg production was obtained in the 12 hour light treatment, taking into account that in day 3, this same light period had the lowest EP. In the treatment without light, the averaged 24 hours egg production was the second highest. In agreement, Stearns et al. (1989) and Peck and Holste (2006) reported that for Acartia tonsa, hourly egg production in darkness tended to be more than twice than the hourly rate during light periods. Calbet and Alcaraz (1996) concluded that Acartia grani feeding on high food during the night produce more eggs than copepods fed on high Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 54 food during the day. Contrasting to our results, Camus and Zeng (2008) working with Acartia sinjiensis found with a similar protocol highest egg production in treatments with ≥18h illumination. Different results probably reflect species-specific responses to photoperiod. Some species of Acartia and several Calanus are known to spawn at night (Støttrup, 2003) but some species have no rhythm. According to Rodriguez et al. (1995), Acartia grani would produce eggs continuously, a few at time and not in distinct clutches (Rodriguez et al., 1995). Population increase over 12 days showed that photoperiod did not have a significant effect on the population growth of Acartia grani (p>0.05). Again, the 12L:12D photoperiod had the highest results when all stages are included (964±410.7). Within the population, there were also no significant differences in the distribution of the various life-stages. The 24L:0D had the lowest production of eggs in the population growth. Camus and Zeng (2008) suggested that under constant illumination, copepods were probably active 24 h around, which required higher metabolic rate to sustain. Such a high metabolic rate plus high daily egg production probably had depleted their energy reserves, leading to the decrease in egg production. This suggests that high metabolic rate probably influence the mortality percentage in the first 3 days (6.3%) and had influence too in the population growth, shortening the life expectancy. Sex ratio of A. grani after a 12 day culture period was similar (p>0.05) between different photoperiods. In 3 treatments (24L:0D, 18L:6D; 0L:24D) higher percentage of females was registered, and the results are in accordance with previous reports for other Acartia species (Fleminger, 1985; Medina and Barata, 2004; Camus and Zeng, 2008). 5. Conclusion In conclusion, based upon the results of this study, photoperiod does not exert a significant effect on the culture of Acartia grani. Still, this species seems to present good culture conditions with a photoperiod of 12 hours of light or less. With exception of the egg hatching rate (which was severely low in all treatments), the 12L:12D treatment presented mean better results of culture in order to maximize its productivity. This same photoperiod was suggested for Calanoid species (Støttrup, 2003) as being the most favorable regimen. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 55 6. References Ambler, J.A.,1986. Effects of food quantity and quality on egg production of Acartia tonsa Dana from East Lagoon, Galveston, Texas. Estuar. Coast. Shelf Sci. 23, 183–196. Calbet, A., Alcaraz, M., 1996. Effects of constant and fluctuating food supply on egg production rates of Acartia grani (Copepoda: Calanoida).Mar. Ecol. Prog. Ser. 140, 33–39. Camus, T., Zeng, C., 2008. Effects of photoperiod on egg production and hatching success, naupliar and copepodite development, adult sex ratio and life expectancy of the tropical calanoid copepod Acartia sinjiensis. Aquaculture 280, 220–226. Castro-Longoria, E., 2003. Egg production and Hatching success of four Acartia species under different temperature and salinity regimes. J. Crustac. Biol. 23, 89–299. Cervetto, G., Gaudy, R., Pagano, M., Saint Jean, L., Verriopoulos, G., Arfi, R., Leveau,M., 1993. Diel variations in Acartia tonsa feeding, respiration and egg production in aMediterranean coastal lagoon. J. Plankton Res. 15, 1207–1228. Chinnery, F.E., Williams, J.A., 2003. Photoperiod and temperature regulation of diapause egg production in Acartia bifilosa fromSouthamptonWater.Mar. Ecol., Prog. Ser. 263,149– 157. Drillet, G., Frouël, S., Sichlau, M.H., Jepsen P.M., Højgaard, J.K., Joarder, A.K., Hansen B.W., 2011. Status and recommendations on marine copepod cultivation for use as live feed. Aquaculture, 315, 155-166. Dussart, B.H., Defaye, D., 2001. Introduction to the Copepoda, 2nd ed. Backhuys Publishers, Leiden. Fleminger, A., 1985. Dimorphism and possible sex change in copepods of the family Calanidae. Mar. Biol. 88, 273–294. Hada, A., Uye, S., 1991. Cannibalistic behavior of the brackish water copepod Sinocalanus tenellus. J. Plankton Res. 13, 155–166. Hairston, N.G., Kearns, C.M., 1995. The interaction of photoperiod and temperature in diapause timing: a copepod example. Biol. Bull. 189, 42–48. James, C.M., Al-Khars, A.M., 1986. Studies on the production of planktonic copepods for aquaculture. Syllogeus 58, 333-340. Jepsen, P.M., Andersen, N., Holm, T., Jorgensen, A.T., Hojgaard, J.K., Hansen, B., 2007. Effects of adult stocking density on egg production and viability in cultures of the calanoid copepod Acartia tonsa (Dana). Aquacult. Res. 38, 764–772. Kiørboe, T., Møhlenberg, F., Hamburger, K., 1985. Bioenergetics of the planktonic copepod Acartia tonsa: relation between feeding, egg production and respiration, and composition of specific dynamic action. Mar. Ecol. Prog. Ser. 26, 85–97. Klepper, G.S., Burkart, C.A., Houchin, L., 1998. Nutrition and the regulation of egg production in the calanoid copepod Acartia tonsa. Limnol. Oceanogr. 43,1000–1007. Koski,M.,Kuosa,H.,1999. The effect of temperature, food concentration and female size on the egg production of the planktonic copepod Acartia bifilosa. J. Plankton Res. 21,1779– 1789. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 56 Leandro, S.M., Tiselius, P., Queiroga, H., 2006. Growth and development of nauplii and copepodites of the estuarine copepod Acartia tonsa from southern Europe (Ria de Aveiro, Portugal) under saturating food conditions. Mar. Biol. 150, 121–129. Lee, C.S., O´Brien, P.J., Marcus, N.H., 2005. Copepods in Aquaculture. Blackwell Publishing, Iowa Usa. pp. 269. Mauchline, J., 1998. The Biology of Calanoid Copepods. Elsevier Academic Press, Oxford. pp 710. McKinnon, D., Duggan, S., Nichol, P.D., Rimmer, M.A., Semmens, G., Robin, B., 2003. The potential of tropical paracalanoid copepods as live feeds in aquaculture. Aquaculture 223, 89–106. Medina, M., Barata, C., 2004. Static-renewal culture of Acartia tonsa (Copepoda: Calanoida) for ecotoxicological testing. Aquaculture 229, 203–213. Milione, M., Zeng, C., 2007. The effects of algal diets on population growth and egg hatching success of the tropical calanoid copepod, Acartia sinjiensis. Aquaculture 271, 656–664. Omori, M., Ikeda, T., 1984. Methods in zooplankton ecology. John Wiley and Sons Inc., New York. Peck, M.A., Holste, L., 2006. Effects of salinity, photoperiod and adult stocking density on egg production and egg hatching success in Acartia tonsa (Copepoda: Calanoida): optimizing intensive cultures. Aquaculture 255, 341–350. Rodriguez, V., Guerrero, F., Bautista, B.,1995. Egg production of individual copepods of Acartia grani Sars from coastal waters: seasonal and diel variability. J. Plankton Res.17, 2233– 2250. Stearns, D.E., Tester, P.A., Walker, R.L., 1989. Diel changes in the egg production rate of Acartia tonsa (Copepoda, Calanoida) and related environmental factors in two estuaries. Mar. Ecol., Prog. Ser. 52, 7–16. Støttrup, J., 2003. Production and Nutricional Value of Copepods. In: Støttrup, J., and McEvoy, L., (Eds.) Live Feeds in Marine Aquaculture. Blackwell Science Ltd. pp. 145205. Strathmann, R.R., 1967. Estimating the organic carbon content of phytoplankton from cell volume or plasma volume. Limnol. Oceanogr. 12, 411–418. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 63 0 1 2 3 4 5 6 0 1 2 3 4 Egg Production Averaged from 5 females over 3 days (egg female-1day-1 Days Rho Rho+T-Iso Tet Tet+T-Iso Figure 2 – Mean egg production (eggs female-1day-1) of Acartia grani, eggs produced by adults fed four different microalgae diets (Rho, Rho+T-Iso, Tet, Tet+T-Iso), on the 3 experimental days. Data are presented as mean±SD. (Day 1 p=0.01; Day 2 p=0.82; Day 3 p=0.62). On the remaining days no significant differences were found between treatments. Still, dietary treatment of Tetraselmis presented in all three days the lowest egg production per female per day. Clearly the presence of Rhodomonas algae affected the EP. When calculated the overall mean 24h EP (Table I), the egg production was significantly higher (p<0.05) in the Rho dietary treatment (3.66±1.40) followed by binary diet (Rho+T-Iso) with an EP of 3.30±1.34. The lowest EP was found in Tetraselmis treatment, which is in accordance with the Figure 2, being significantly lower than Rho+T-Iso and Rho treatments, except that of Tet+T-Iso (2.62±1.33). Table I – Effects of diets on averaged 24h egg production of Acartia grani. For each treatment, 24h egg production was averaged from 5 females over 3 days. Data are presented as mean±SD. (One way ANOVA p=0.023). Diet Eggs female-1day-1 Rho 3.66 ±1.40a Rho +T-Iso 3.30 ±1.34a Tet 1.40 ±0.84b Tet +T-Iso 2.62 ±1.33ab Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 64 0 500 1000 1500 2000 2500 Rho Rho + T-Iso Tet Tet + T-Iso Population increase over 12 days Diets All stages All Post-Egg-stages 3.3 Population Growth The results showed that diet significantly affected the population growth of A. grani after 12 days of culture on different microalgae diets. The averaged final population numbers of A.grani are presented in two categories, i.e. “All Stages included (i.e. including eggs); “All Post-Egg-Stages” (i.e. excluding eggs) (Fig. 3). Highest mean final population was observed for A. grani fed on diet Rho+T-Iso (1783.0±560.6) if all stages were considered. In fact, Tukey´s test reveled significance differences between this dietary treatment and Tetraselmis, which presented the lowest mean population increase (325.0±135.7) When analyzed without eggs (all post-egg-stages), the highest mean final population number was observed on adults fed microalgae diet Tet+T-Iso (511.0±94.3), being the second most productive diet followed closely by the Rho+Iso dietary treatment (508.0±114.1). The Tetraselmis diet produced the lowest registered results (p<0.05), with a mean final population of only 41.0±16.4 individuals. Figure 3Mean final total population of Acartia grani cultured at four different microalgae diets for a 12 day period. Initial population was 8 females: 4 males Data are represented as mean±SD. All Stages bar (all life stages: eggs, nauplii, copepodites, adults); All Post-Egg-Stages bar (nauplii, copepodites, adults). Different letters on the tops of bars indicate significant differences (p<0.05). One way ANOVA: All stages included p=0.023; All Post-Eggs-Stages p=0.001; (Diet 1: Rhodomonas marina; Diet 2: Rhodomonas marina + Isochrysis sp. (T-Iso); Diet 3: Tetraselmis suecica; Diet 4: Tetraselmis suecica + Isochrysis sp. (T-Iso). Analysis of population different stages revealed major differences in the number of eggs between dietary treatments, again with clear positive effects of Rhodomonas in egg production. Tetraselmis diet produced lower numbers (p<0.05) from the remaining treatments for both copepodite and adult stages. ab ac a x x a x x Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 65 Table II - Mean number of four life stages (eggs, nauplii, copepodites and adults) within the population of Acartia grani cultured for 12 days at four different diets (Rho; Rho+T-Iso; Tet; Tet+T-Iso) from an initial number of 12 adults. (K) is the specific population growth rate, and (Dt) is the doubling time. Different letters indicate significant differences (p<0.05). Data are represented as mean ±SD. Treatment Eggs Nauplii Copepodites Adults K Dt Rho 1029.0 ±339.4a 107.0 ± 42.1a 249.8 ±78.8a 83.0 ±47.3ª 0.39 ±0.02ª 1.73 ±0.07ª Rho +T-Iso 1275.0 ±385.6a 285.8 ±136.7ab 164.5 ±23.7a 57.8 ±19.0ab 0.41 ±0.01ª 1.66 ±0.05ª Tet 283.8 ±57.6b 32.5 ±37.,1ac 6.0 ±5.7b 2.5 ±1.,7b 0.27 ±0.01b 2.52 ±0.08b Tet +T-Iso 286.0 ±181.0b 260.0 ±138.2ab 179.3 ±74.0a 72.0 ±36.2ª 0.35 ±0.02c 1.99 ±0.11c p= 0.000 p= 0.011 p= 0.000 p= 0.015 p= 0.00 p= 0.00 The specific population growth rate (K) of A. grani was calculated for all treatments (Table II). It ranged from 0.27 on Tetraselmis diet to 0.41 in Rho+T-Iso. Population doubling time (Dt) demonstrates that Tetraselmis was the dietary treatment that needed more time to grow (2.52±0.08) and that the binary diet (Rho+T-Iso) had the lowest doubling time (1.66±0.05), which is in agreement with previous results. For both K and Dt significance differences were observed between these dietary treatments (p<0.05). Mortality of the initial adult population was registered and results revealed that on the first three experimental days, between 10% (Tet+T-Iso) and 15% (Tet) of the initial adults had died. By the 10th experimental day, mortality reached 68.8% at Tetraselmis, followed by Rhodomonas diet with a mean mortality rate of 37.5%. From that day onwards mortality was not counted, due to possible misleading of initial incubated adults with born grown adults during the experiment. 4. Discussion Calanoids copepods have a non-visual, active raptorial mode of feeding, capturing and ingesting a variety of animal prey (Tiselius and Jonsson, 1990). Successful growth and development of cultured species depends largely on the nature and content of biochemical constituents in the food provided. These constituents are used in the anabolic process for tissue production, in catalyzing metabolic process and in the creation of energy to power those processes (Matias-Peralta et al., 2012). This study evaluated the best diet to optimize the culture of Acartia grani. In all current experiments, A. grani were acclimatized to experimental diets for 3 days, period judged to be sufficient. Based on our results, the use of Rhodomonas algae affected positively all of the analyzed parameters, performing better than Tetraselmis. Moreover, our results show that the use of the binary diets seems to more appropriate, positively Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 66 affecting population dynamics of Acartia grani. Rho+T-Iso dietary treatment clearly presented a superior egg hatching rate over the 72 hours experiment, followed by diet Tet+T-Iso. Several authors (Koski et al., 1998; Broglio et al., 2003, Tang and Taal, 2005), refered the high nutritional value of these microalgae for copepods. Knuckey et al. (2005) demonstrated the superiority of Rhodomonas sp. as a mono-algal diet for Acartia sinjiensis. However, bear in mind that the use of Rhodomonas in a commercial aquaculture operation is limited by the instability of this species in mass culture. Cultures can fail to initiate exponential growth or prematurely enter stationary phase. Cultures also have a short stationary phase compared to other microalgae commonly used in aquaculture and can quickly die (Knuckey et al., 2005). On the other hand, Tetraselmis monoalgal diet did not seem suitable for Acartia grani. When compared to other diets, results were lower in most experiments, which is in accordance with several other authors that reported the same issue, i.e., Camus et al. (2009) for Bestiolina similis; Milione and Zeng (2007) in Acartia sinjiensis; and Koski et al. (1998) for Pseudocalanus elongatus. Egg production is one of the principal factors determining copepod culture productivity and has been linked to the maternal nutrition (Castro-Longoria, 2003). Averaged 24h egg production was favorable again for the treatment that had Rhodomonas included. In monoalgal treatment (Rho) the averaged egg production of 4 eggs female-1day-1 for Acartia grani was similar to values observed by Koski et al. (1998) for Pseudocalanus elongates (5 eggs female-1day-1), although Broglio et al. (2003) found for the same concentration of Rhodomonas (1500μgCL−1) a value of 25 eggs female-1day1 for Acartia tonsa and McKinnon et al. (2003) found that Rhodomonas supported high egg production rates of A. sinjiensis (up to 33 eggs female-1day-1). Analysis of population growth is probably more pertinent to the ultimate goal of improving productivity of copepod culture for hatcheries because it provides a summary of the dietary effects on a range of inter-related parameters, including egg production, egg hatching rates, nauplii and copepodite development time and survival (Milione and Zeng, 2007). Diet significantly affected the population growth of A. grani in our study. The binary diet Rho+T-Iso presented great growth when all stages were included with 1783 individuals, followed by the monoalgal treatment Rho (1469 individuals), exclusion made when analysis was performed on all post-egg stages where the binary diet Tet+T-Iso presented the highest population increase. This high growth can be related to the Isochrysis sp. microalgae, which is considered among the best food sources for filterfeeders, because of their high content of essential fatty acids that promote high survival and growth (Watanabe et al., 1983; Brown et al., 1989). On other hand, Tetraselmis microalgae has been reported to induce a high degree of deformities and complete lack of Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 67 development beyond the copepodite stage in A. sinjiensis (Knuckey et al., 2005). Another point of view for poor performance of Tetraselmis was the bad digestibility of this microalgae (Puello-Cruz et al., 2009). Moreover during the experiments, we observed that Tetraselmis deteriorates the quality of water relatively fast, and it is possible to see a superficial layer in water surface, that sometimes adheres to swimming appendages of copepods leading to death. When different stages were analyzed separately and in concurrence to our previous results of egg production and egg hatching rate, the population growth in the Rho and Rho+T-Iso had the highest number of eggs over the 12 days of experiment, (1029 and 1275, respectively). We also found that diets with T-Iso included had a high number of nauplii in concordance to what Knuckey et al., (2005) reported, that T-Iso supported better nauplii development. The poor performance of Tetraselmis was evident in adult’s stages over the 12 days, since final population was only of 3 individuals. This microalgae was responsible for high mortality, after 3 days of culture 15% of the adults died, and by the 10th experimental day, mortality reached 68.8%, which was crucial for the bad development of population. 5. Conclusion Of the many algal species that have been used in aquaculture, Rhodomonas and Isochrysis have been conspicuously successful as a food for rearing copepod species (Støttrup et al., 1986; Lacoste et al., 2001; Rippingale and Payne, 2001; Lee et al., 2006). In summary the presence of Rhodomonas microalgae clearly affect the development of Acartia grani, although the Isochrysis seems to be important complement algae in early stages, because of is nutritional value and maybe the small size of the cells. It is recommended that for the culture of Acartia grani the binary diet of Rhodomonas + Isochrysis is used to achieve a good culture production. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 68 6. References Azaiteiro, U.M., Marques, S.C., Vieira, L.M.R., Pasto-Rinho, M.R.D., Pereira, P.A.B., Ré, M.J., Moragado, F.M.R., 2005. 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Aquaculture 249, 339–351. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 69 Koski, M., Klein Breteler,W.C.M., Schogt, N., 1998. Effect of food quality on rate of growth and development of the pelagic copepod Pseudocalanus elongatus (Copepoda: Calanoida). Mar. Ecol. Prog. Ser. 170, 169– 187. Koski, M., Kuosa, H., 1999. The effect of temperature, food concentration and female size on the egg production of the planktonic copepod Acartia bifilosa. J. Plank. Res. 21, 1779– 1789. Lacoste, A., Poulet, S.A., Cueff, A., Kattner, G., Ianora, A., Laabir, M., 2001. New evidence of the copepod maternal food effects on reproduction. J. Exp. Mar. Biol. Ecol. 259, 85– 107. Leandro, S.M., Morgado, F., Pereira, F., Queiroga, H., 2007. Temporal changes of abundance, biomass and production of copepod community in a shallow temperate estuary (Ria de Aveiro, Portugal). Est Coast Shelf Sci 74: 215-222. Leandro, S.M., Tiselius, P., Queiroga, H., 2006. 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The Biology of Calanoid Copepods. Elsevier Academic Press, Oxford. pp 710. McKinnon, D., Duggan, S., Nichol, P.D., Rimmer, M.A., Semmens, G., Robin, B., 2003. The potential of tropical paracalanoid copepods as live feeds in aquaculture. Aquaculture 223, 89–106. Milione, M., Zeng, C., 2007. The effects of algal diets on population growth and egg hatching success of the tropical calanoid copepod, Acartia sinjiensis. Aquaculture 271, 656–664. Morehead, D.T., Battaglene, S.C., Metillo, E.B., Bransden, M.P., Dunstan, G.A., 2005. Copepods as a live feed for striped trumpeter Latris lineata larvae. In: Lee, C.S., O'Bryen, P.J., Marcus, N.H. (Eds.), Copepods in Aquaculture. Blackwell Scientific Publications Ltd, Melbourne, pp. 195–208. Omori, M., Ikeda, T., 1984. Methods in zooplankton ecology. John Wiley and Sons Inc., New York. Payne, M.F., Rippingale, R.J., 2000. Evaluation of diets for culture of the calanoid copepod Gladioferens imparipes. Aquaculture 187, 85–96. Peck, M.A., Holste, L., 2006. Effects of salinity, photoperiod and adult stocking density on egg production and egg hatching success in Acartia tonsa (Copepoda: Calanoida): optimizing intensive cultures. Aquaculture 255, 341–350. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 70 Puello-Cruz, A.C., Mezo-Villalobos, S., Gonález-Rodríguez, B., Voltolina, D.,2009. Culture of calanoide copepod Pseudodiaptomus euryhalinus (Johnson 1939) with diferente microalgal diets. Aquaculture 290, 317-319. Rippingale, R.J., Payne, M.F., 2001. Intensive cultivation of a calanoid copepod for live food in fish culture. Department of Environmental Biology, Curtin University of Technology, Perth. Saiz, E., Calbet, A., Atienza, D., Alcaraz, M., 2007. Feeding and production of zooplankton in the Catalan Sea (NW Mediterranean). Progr Oceanog 74, 313-328. Shin, K., Jang, M., Jang, P., Ju, S., Lee, T., Chang, M., 2003. Influence of food quality on egg production and viability of themarine planktonic copepod Acartia omorii. Oceanography 57, 265–277. Smith, L.L., Biedenbach, J.M., Lawrence, A.L., 1992. Penaeid larviculture: Galveston method. In: Fast, A.E., Lester, L.J. (Eds.), Marine Shrimp Culture, Principles and Practices. Elsevier Science, Amsterdam, pp. 171–191. Støttrup, J.G., Jensen, J., 1990. Influence of algal diet on feeding and egg production of the calanoid copepod Acartia tonsa Dana. J. Exp. Mar. Biol. Ecol. 141, 87–105. Støttrup, J.G., Norsker, N.H., 1997. Production and use of copepods in marine fish larviculture. Aquaculture, 155, 231–247. Støttrup, J.G., Richardson, K., Kirkegaard, E., Pihl, N.J., 1986. The cultivation of Acartia tonsa for use as a live food source for marine fish larvae. Aquaculture 52, 87–96. Strathmann, R.R., 1967. Estimating the organic carbon content of phytoplankton from cell volume or plasma volume. Limnol. Oceanogr. 12, 411–418. Tang, K.W., Taal, M., 2005. Trophic modification of food quality by heterotrophic protists: species-specific effects on copepod egg production and egg hatching. J. Exp. Mar. Biol. Ecol. 318, 85-98. Tiselius, P., Jonsson, P.R., 1990. Foraging behaviour of six calanoid copepods: observations and hydrodynamic analysis. Mar. Ecol. Prog. Ser., 66, 23–33. Turner, J.T., Ianora, A., Miralto, A., Laabir, M., Esposito, F., 2001. Decoupling of copepod grazing rates, fecundity and egg-hatching success on mixed and alternating diatom and dinolate diets. Mar. Ecol. Prog. Ser. 220, 187–199. Watanabe, T., Kitajima, C., Fujita, S., 1983. Nutritional values of live feed organisms in Japan for mass propagation of fish: a review. Aquaculture 34, 115–143. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 71 Chapter V Egg production, egg hatching success and population increase of the calanoid copepod, Acartia grani (Calanoida: Acartiidae), using various combinations of autotrophic and heterotrophic protists 1. Introduction Marine pelagic systems are characterized by the presence of a wide variety of autotrophic and heterotrophic species, which represent potential food items for omnivorous copepods. In the last two decades, appreciation for the importance of large heterotrophic protists (ciliates and dinoflagellates) in the copepod diet has grown, because they have been proposed as an intermediate link between the microbial loop and higher trophic levels (Verity and Paffenhöfer 1996; Klein Breteler et al., 1999). Several studies showed that copepods can ingest protozoa (Gifford and Dagg, 1991; Atkinson, 1994; Levinsen et al., 2000) at higher rates than phytoplankton (Dolan, 1991; Fessenden and Cowles, 1994; Zeldis et al., 2002), and may also preferentially select the former (Stoecker and Sanders, 1985; Stoecker and Egloff, 1987; Sanders and Wickham, 1993; Verity and Paffenhofer, 1996). Thus, the nutritional content of heterotrophic protists is equally, if not more, important as that of algae in regulating zooplankton growth and production (Tang and Taal, 2005), as some heterotrophic protozoa appear to provide essential copepod growth compounds that are not always found in phytoplankton (Klein Breteler et al., 1999). Tang et al. (2001) showed that heterotrophic protists as a trophic link between poor nutritional quality algae and copepods resulted in higher egg production and egg hatching success. Nowadays there is evidence that protozoa hold a key position and are an essential link in pelagic food webs (Calbet, 2008). As predators, heterotrophic protists consume, assimilate and repackage not only the biomass and nutrients of their prey, but, most importantly, upgrade their biochemical constituents, such as the longchain n-3 essential fatty acids (LCn-3 EFAs), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and sterols (Ackman et al., 1980; Sargent et al., 1987, 2002). Oxyrrhis marina is an extensively studied heterotrophic flagellate (Montagnes et al., 2011), that exhibits a wide geographic distribution (Watts et al., 2011), easily recognized (e.g. Dodge, 1982) and easy to isolate from the natural environment (Lowe et al., 2011). Klein Breteler et al. (1999) reported that this heterotrophic dinoflagellate grown on the EFA deficient alga D. tertiolecta supported rapid growth of the copepods Temora longicornis and Pseudocalanus elongatus from naupliar stages to adulthood. Calanoid Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 72 copepods (e.g. Acartia grani) are considered opportunistic omnivores (Turner, 1984; Kleppel, 1993) and have been proven to ingest the dinoflagellate Alexandrium minutum (Calbet, 2003). Also, the calanoid copepod Pseudocalanus has been successfully reared on the heterotrophic dinoflagellate Oxyrrhis (Klein Breteler et al., 1999). We have also learned that copepods can grow better on mixed-food diets than on single-food diets (e.g., Stoecker and Egloff, 1987; Kleppel and Burkart, 1995; Bonnet and Carlotti, 2001, Camus et al., 2009). Although comparative studies of the suite of effects of autotrophic, heterotrophic and mixed diets on the ingestion, growth and egg survival exist (e.g., Adrian and Frost, 1993; Kleppel and Burkart, 1995; Sanders et al., 1996; Koski et al., 1998; Bonnet and Carlotti, 2001), they remain relatively few. Yet, this comparative approach is useful in linking the functional and numerical responses, and hence, in determining the suitability of different diets for copepods. This approach is also helpful in understanding variability in coupling between primary and secondary production Based on previous results (Chapter IV) we decided to evaluate the effects of using Rhodomonas marina and Tahitian strain of Isochrysis sp. (the two algae that stimulated best growth performance of Acartia grani) combined with Oxyrrhis marina. The purpose of this experiment was to identify an optimal diet for culturing A.grani, with microalgae and dinoflagellates, to achieve maximum productivity of this copepod. 2. Material and methods 2.1 Microalgae culture All of the microalgae utilized in present experiments are common used algal species in aquaculture, therefore relatively easy to culture with the exception of Rhodomonas marina. Two algal species were used in this study: Rhodomonas marina (Rho); Tahitian strain of Isochrysis sp. (T-Iso). Rhodomonas was cultured according to the conditions and maintenance protocols described in chapter 1.3.2. Isochrysis sp. (T-Iso) was inoculated by starter cultures supplied by IPIMAR (Olhão) and was by batch method with Nutribloom medium (see Annex, Fig.1), at 20±1ºC salinity 25psu, 24h light in 1L carboys, with continuous aeration. Seawater was 1μm filtered and UV irradiated. The algal cultures were in their exponential growth phase when were used for feeding copepods. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 79 0 10 20 30 40 50 60 70 80 90 100 Rho + Oxy Oxy T-Iso T-Iso + Oxy Sex ratio in Population Increase (Female:Male) Diets Male Female Overall the binary treatment Rho+Oxy presented higher mean final population when all stages were considered, only due to significant differences (p=0.032) in the amount of eggs produced (Table II), which was three times higher than amount of eggs counted in TIso treatment. The remaining stages did not present differences between dietary treatments, though the highest number was always observed within Rho+Oxy treatment. Table II - Mean number of four life stages (eggs, nauplii, copepodites and adults) within the population of Acartia grani cultured for 12 days at four different diets (Rho+Oxy; Oxy; T-Iso; T-Iso+Oxy) from an initial number of 12 adults. (K) is the specific population growth rate, and (Dt) is the doubling time. Different letters indicate significant differences (p<0.05). Data are represented as mean±SD. Treatment Eggs Nauplii Copepodites Adults K Dt Rho + Oxy 1050.3 ±514.4a 541.3 ±514.8a 127.8 ±184.4ª 76.0 ± 64.8a 0.41 ±0.03ª 1.68 ±0.14ª Oxy 641.3 ±209.8ab 221.3 ±183.7a 35.5 ±32.5ª 48.3 ±17.2a 0.36 ±0.01b 1.91 ±0.08ª T-Iso 348.8 ±94.3b 17.0 ±10.0a 44.3 ±32.9ª 28.3 ±8.2a 0.29 ±0.01c 2.32 ±0.11b T-Iso + Oxy 795.5 ±132.0ab 198.8 ±118.9a 75.0 ±110.3ª 33.3 ±27.9a 0.38 ±0.01ab 1.84 ±0.07ª p= 0.032 p= 0.116 p= 0.642 p= 0.296 p= 0.00 p= 0.00 The specific population growth rate (K) of A. grani was calculated for all treatments (Table II). It ranged from 0.29 to 0.41, being lowest in T-Iso diet and highest on Rho+Oxy diet. Accordingly, Dt values (Table II) showed that T-Iso was the treatment that needed more time to double population (2.32±0.11) and the binary diet Rho+Oxy had the lowest value 1.68±0.14. For both K and Dt significance differences were observed (p<0.05). Sex ratio of A. grani after a 12 day culture period was similar between different microalgae diets (Fig. 4). Highest incidence of females was registered in Rho+Oxy treatment (53.3%) followed by T-Iso+Oxy (51.1%). The highest ratio of males was in T-Iso (54.9%) and Oxy (52.8%). Figure 4 – Sex ratio of A.grani in the population increase experiment after 12 days. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 80 Mortality of the initial adult population was registered and results revealed that on the first three experimental days, between 16.7% (T-Iso+Oxy) and 22.9% (Oxy) of the initial adults had died. By the 6th experimental day, mortality reached 41.7% with T-Iso+Oxy treatment, followed by Oxy diet with a mean mortality rate of 39.6%. From that day onwards mortality was not counted, due to possible misleading of initial incubated adults with born grown adults during the experiment. 4. Discussion Several authors attributed trophic upgrading to modification of algal fatty acid by heterotrophic protists (Klein Breteler et al., 1999; Broglio et al., 2003). The diets of pelagic copepods are characteristically broad (Kleppel, 1993) and strict herbivory of copepods rarely exists in nature. Most copepods prefer feeding on microzooplankton due to their large size, easy perception, as well as the relatively high food quality (Batten et al., 2001; Gifford et al., 2007; Campbell et al., 2009). As copepods are typically omnivorous, any deficiency resulting from a nutritionally poor diet could be compensated in the field by feeding on a wider spectrum of prey (Broglio et al., 2003). Many field studies also showed that heterotrophic protists contribute as much as 100% to a copepod’s diet. Thus, the nutritional content of heterotrophic protists is equally, if not more, important as that of algae in regulating zooplankton growth and production (Tang and Taal, 2005). Based on previous results of experiments performed with different algae diets on Acartia grani (Chapter IV) we decided to evaluate the effects of using Rhodomonas marina and Isochrysis sp. (T-Iso) (the two algae that stimulated best growth performance of Acartia grani previously) combined with the heterotrophic dinoflagellate Oxyrrhis marina. In the current study egg hatching rate was highest in treatments with Isochrysis (similar to what we found in our first experiment with Rho+T-Iso and Tet +T-Iso). Egg production was highest over the 3 days on Rho+Oxy diet, as expected. Rhodomonas supports high egg production efficiency and naupliar growth rate (Tang et al., 2001). Contrary to our findings, Tang and Taal (2005) refered for A. tonsa that R. salina + O. marina treatment resulted in trophic downgrading, meaning lower egg production efficiency. In our case the heterotrophic O.marina does not downgrade the quality of Rhodomonas. Higher food value is obtained from mixed diets, which are more likely to contain the diversity of biochemical’s to satisfy most nutritional requirements for growth (Whyte et al., 1989). In population growth the binary diet Rho+Oxy had the highest value of 1795 Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 81 individuals (all stages included), having the T-Iso treatment the lower population, confirming that Isochrysis cannot be provided as monoalgal diet. However, when T-Iso was complemented with O. marina, the diet was the second highest, with 1103 individuals over the 12 days. It is generally assumed that by preying on heterotrophic protists a copepod can diversify its diet and obtain a more balanced nutrition (Kleppel, 1993). In the mono diet with Oxyrrhis marina, the results were overall good, with 641 eggs, 221 nauplii and 48 adults. The population had a good development with this diet, although total number of individual (946 individuals) was lower than the binary diets. Analyzing all stages separately between treatments in population growth, the binary diets (Rho+Oxy; T-Iso+Oxy) had the best egg production (population growth) unlike our previous experiment on egg production with values for T-Iso + Oxy diet of only 0.29 eggs female-1day-1. Still, Rho+Oxy treatment, maintained the highest egg production revealing again as an optimal diet to obtain eggs. Copepod reproduction and subsequent development involve multiple life stages and biological processes for which the best suited diet may vary at different life stages or for different biological functions. On this basis, a single species of microalgae may become nutritionally limiting whereas appropriate combinations of algae are likely to offer better balance of required nutrients (Camus et al., 2009). Overall the binary diet Rho+Oxy was the most complete in all stages (i.e. eggs, nauplii, copepodites, adults) despite of the egg hatching rate that was the lowest of all treatments. This diet proves to be the most complete over the four diet treatments, being a good choice for the production of Acartia grani. 5. Conclusion In conclusion, the present study showed that all the microalgal food were able to support the production of A.grani, but clearly the copepod have preferences and a better production with a Rho+Oxy diet was observed. To achieve improved productivity of A.grani we suggest that using the binary diet Rho+Oxy was the best option. However and for further studies, a tri-algal diet (Rho + Oxy + T-Iso) probably will improve the production of Acartia grani, being more complete for all stages, due to their different requirements. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 82 6. References Ackman, R.G., Sebedio, J.L., Kovacs, M.I.P., 1980. Role of eicosenoic and docosenoic fatty acids in freshwater and marine lipids. Mar. Chem. 9, 157– 164. Adrian, R., Frost, T.M., 1993. Omnivory in cyclopoid copepods: comparisons of algae and invertebrates as food for three, differently sized species. J. Plankton Res. 15, 643– 658. Atkinson, A., 1994. Diets and feeding selectivity among the epipelagic copepod community near South Georgia in summer. Polar Biol. 14, 551– 560. Batten, S.D., Fileman, E.S., Halvorsen, E., 2001. The contribution of microzooplankton to the diet of mesozooplankton in an upwelling filament off the northwest coast of Spain. Prog. Oceanogr. 51, 385–398. Bonnet, D., Carlotti, F., 2001. Development and egg production in Centropages typicus (Copepoda: Calanoida) fed different food types: a laboratory study. Mar. Ecol. Prog. Ser. 224, 133–148. Broglio, E., Jónasdóttir, S.H., Calbet, A., Jakobsen, H.H., Saiz, E., 2003. Effect of heterotrophic versus autotrophic food on feeding and reproduction of the calanoid copepod Acartia tonsa: relationship with prey fatty acid composition. Aquat. Microb. Ecol. 31, 267–278. Calbet, A., 2008. The trophic roles of microzooplankton in marine systems. ICES J. Mar. Sci., 65, 325–331. Calbet, A., Vaqué, D., Felipe, J., Vila, M., Sala, M. M., Alcaraz, M., Estrada, M. 2003. Relative grazing impact of microzooplankton and mesozooplankton on a bloom of the toxic dinoflagellate Alexandrium minutum. Mar. Ecol. Prog. Ser. 259, 303–309. Campbell, R.G., Sherr, E.B., Ashjian, C.J., Plourde, S., Sherr, B.F., Hill, V., Stockwell, A., 2009. Mesozooplankton prey preference and grazing impact in the western Arctic Ocean. Deep Sea Res. II 56, 1274–1289. Camus, T., Zeng, C., McKinnon, A.D., 2009. Egg production, egg hatching success and population increase of the tropical paracalanid copepod, Bestiolina similis (Calanoida: Paracalanidae) fed diferent microalgal diets. Aquaculture 297, 169-175. Dodge, J. D., 1982. Marine Dinoflagellates of the British Isles. Her Majesty’s Stationary Office, London. Dolan, J.R., 1991. Microphagous ciliates in mesohaline Chesapeake Bay waters: estimates of growth rates and consumption by copepods. Mar. Biol. 111, 303–309. Fessenden, L., Cowles, T.J., 1994. Copepod predation on phagotrophic ciliates in Oregon coastal waters. Mar. Ecol. Prog. Ser. 107, 103–111. Gifford, D.J., Dagg, M.J., 1991. The microzooplanktonmesozooplankton link: consumption of planktonic protozoa by the calanoid copepods Acartia tonsa Dana and Neocalanus plumchrus Murkukawa. Mar. Microb. Food Webs 5, 161– 177. Gifford, S.M., Rollwagen-Bollens, G., Bollens, S.M., 2007. Mesozooplankton omnivory in the upper San Francisco Estuary Mar. Ecol. Prog. Ser. 348, 33-46. Hada, A., Uye, S., 1991. Cannibalistic feeding behavior of the brackish water copepod Sinocalanus tenellus. J. Plankton Res. 13, 155–166. James, C.M., Al-Khars, A.M., 1986. Studies on the production of planktonic copepods for aquaculture. Syllogeus 58, 333-340. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 83 Klein Breteler, W.C.M., Schogt, N., Baas, M., Schouten, S., Kraay, G.W., 1999. Trophic upgrading of food quality by protozoans enhancing copepod growth: role of essential lipids. Mar. Biol.135, 191–198. Kleppel, G.S., 1993. On the diets of calanoid copepods. Mar. Ecol. Prog. Ser 99, 183-195. Kleppel, G.S., Burkart, C.A., 1995. Egg production and the nutritional environment of Acartia tonsa: the role of food quality in copepod nutrition. ICES J. Mar. Sci. 52, 297– 304. Koski, M., Klein Breteler,W.C.M., Schogt, N., 1998. Effect of food quality on rate of growth and development of the pelagic copepod Pseudocalanus elongatus (Copepoda: Calanoida). Mar. Ecol. Prog. Ser. 170, 169– 187. Levinsen H., Turner, J. T., Nielsen, T. G., Hansen, B. W., 2000. On the trophic coupling between protists and copepods in arctic marine ecosystems. Mar. Ecol. Prog. Ser., 204, 66–77. Lowe, C.D., Martin, L.E., Watts, P.C., 2011. Collection, isolation and culturing strategies for Oxyrrhis marina. J. Plankton Res., 33, 569–578. Montagnes, D.J.S., Lowe, C.D., Roberts, E.C., 2011. An introduction to the special issue: Oxyrrhis marina, a model organism? J. Plankton Res., 33, 549–554. Omori, M., Ikeda, T., 1984. Methods in zooplankton ecology. John Wiley and Sons Inc., New York. Sanders, R.W., Wickham, S.A., 1993. Planktonic protozoa and metazoan: predation food quality and population control. Marine Microbial Food Webs. 7, 197-223. Sanders, R.W., Williamson, C.E., Stutzman, P.L., Moeller, R.E., Goulden, C.E., Aoki-Goldsmith, R., 1996. Reproductive success of ‘‘herbivorous’’ zooplankton fed algal and nonalgal food resources. Limnol. Oceanogr. 41, 1295–1303. Sargent, J.R., Parkes, R.J., Mueller-Harvey, I., Henderson, J., 1987. Lipid biomarkers in marine ecology. In: Sleigh, M.A., (Ed.), Microbes in the Sea. Ellis Horwood, Chichester, pp. 119– 138. Sargent, J.R., Tocher, D.R., Bell, J.G., 2002. The lipids. In: Halver, J.E., Hardy, R.W., Hardy, D.M. (Eds.), Fish Nutrition. Academic Press, San Diego, pp. 182– 257. Stoecker, D.K., Egloff, D.A., 1987. Predation by Acartia tonsa on planktonic ciliates and rotifers. J. Exp. Mar. Biol. Ecol. 110, 53–68. Stoecker, D.K., Sanders, N.K., 1985. Differential grazing by Acartia tonsa on a dinoflagellate and a tintinnid. J. Plankton Res. 7, 85–100. Strathmann, R.R., 1967. Estimating the organic carbon content of phytoplankton from cell volume or plasma volume. Limnol. Oceanogr. 12, 411–418. Tang, K.W., Jakobsen, H.H., Visser, A.W., 2001. Phaeocystis globosa (Prymnesiophyceae) and the planktonic food web: feeding, growth and trophic interactions among grazers. Limnol. Oceanogr. 46, 1860– 1870. Tang, K.W., Taal, M., 2005. Trophic modification of food quality by heterotrophic protists: species-specific effects on copepod egg production and egg hatching. J. Exp. Mar. Biol. Ecol. 318, 85-98. Turner, J.T., 1984. The feeding ecology of some zooplankters that are important prey items of larval fish. NOAA Tech Rep. NMFS, 7, 1-28. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 84 Verity, P.G., Paffenhöfer, G.A., 1996. On assessment of prey ingestion by copepods. J. Plankton Res. 18, 1767–1779. Watts, P.C., Martin, L.E., Montagnes, D.J.S., 2011. The distribution of Oxyrrhis marina: a global wanderer or poorly characterized endemic? J. Plankton Res., 33, 579–589. Whyte, J.N.C., Bourne, N., Hodgson, C.A., 1989. Influence of algal diets on biochemical composition and energy reserves in Patinopecten yessoensis (Jay) larvae. Aquaculture 78, 333–347. Zeldis, J., James, M.R., Grieve, J., Richards, L., 2002. Omnivory by copepods in the New Zealand Subtropical Frontal Zone. J. Plankton Res. 24, 9–23. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 85 Chapter VI Lipid and fatty acid composition of Acartia grani adults fed Rhodomonas marina and Oxyrrhis marina 1. Introduction Besides being the natural live prey for marine larval fish, copepods are a rich source of phospholipids, essential highly unsaturated fatty acids (HUFA), natural antioxidants and other essential compounds (Kraul et al., 1992; Sargent et al., 1997). It is generally accepted that copepods can meet the nutritional requirements of fish larvae and that the nutritional superiority of copepods for marine fish larvae to traditional live food such as rotifers Brachionus plicatilis and Artemia nauplii is well-established (Sargent et al., 1997; Støttrup and Norsker, 1997; Næss and Lie, 1998). Marine larvae of carnivore fish species are generally believed to exhibit high requirements for n-3 HUFA (Yone and Fuji, 1975; Watanabe, 1982; Koven et al., 1990; Rainuzzo et al., 1992; Sargent et al., 1999), especially docosahexaenoic acid (22:6n-3 - DHA) and eicosapentaenoic acid (20:5 n-3 - EPA) which are found to be of particular importance (Watanabe, 1988, 1993; Izquierdo et al., 1989; Estevez and Kanazawa, 1996). Moreover, these essential lipids, EPA, DHA and sterols, have a wide range of critical functions including being important structural components and precursors to bioactive molecules such as eicosanoids and steroids (Ackman et al., 1980; Sargent et al., 1987, 2002). Content of n-3 HUFA in traditional live food is low, unless they are fed diets rich in n-3 HUFA (Watanabe et al., 1983; Léger et al., 1986, 1987; Léger and Sorgeloos, 1991; Olsen et al., 1993; Dhert et al., 1993; Rainuzzo et al., 1994; Evjemo et al., 1997). On the other hand, several coastal copepods have a high content of both DHA and EPA (60% of total fatty acids) (Sargent and Henderson, 1986; Fraser et al., 1989; Evjemo and Olsen, 1997), and their nutritional feasibility judged by larval growth rate, survival, pigmentation and successful metamorphosis has been documented (Holmefjord et al., 1989; Næss et al., 1995; Næss and Lie, 1998; Shields et al., 1999). Though Watanabe et al. (1983) reported that culture media did not influence the copepod chemical composition, several authors have reported that levels and ratios of fatty acids in copepods reflected the culture diet (Sargent and Falk-Petersen, 1988; Græve et al., 1994; Delbare et al. 1996) and often reflect the lipid composition of algae which varies between taxonomic groups (Chuecas and Riley, 1969; Sargent and Falk-Petersen, 1988), the stage of development of the algal culture (Fernández-Reiriz et al., 1989) and on the stage of development of the copepods (Sargent and Falk-Petersen, 1988). Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 86 Calanoid copepods are unable to elongate and desaturate 18:3n-3 to produce significant amounts of longer chain HUFA. Norsker and Støttrup (1994) discovered that the harpacticoid copepod Tisbe holothuriae does have the ability to elongate and desaturate the 18:3n-3 fatty acid supplied by Dunaliella tertiolecta to produce significant amounts of the long-chain EFA’s, EPA and DHA. Similarly, Watanabe et al. 1978 had found that another copepod, Tigriopus sp., contained high levels of n-3 HUFA (12% DHA and 7% EPA) in its lipids even when fed exclusively with baker’s yeast. As important prey items for larval fish, information on the effects of diet on copepod biochemical composition is necessary to better understand fish recruitment and yield. The aim of the present study was to obtain information on the nutritional value of coastal copepod Acartia grani which may be used to rear marine fish larvae, and in particular it’s content of n-3 HUFA, more specifically DHA and EPA. Rhodomonas marina was the microalgae utilized, not only because of its nutritional value, rich in proteins and lipids but also because it has been proven to be consumed by Acartia grani. Oxyrrhis marina is an important nutritional feed for A. grani, as predators, heterotrophic protists consume, assimilate and repackage not only the biomass and nutrients of their prey, but, most importantly, upgrade their biochemical constituents, such as the long-chain n-3 essential fatty acids (LCn-3 EFAs), DHA and EPA and sterols (Chu et al., 2009). 2. Material and methods 2.1 Sample collection Starter culture tanks (500L) were initiated with ±35x104 A.grani eggs in order to obtain adults for determination of total lipid and fatty acid composition. Two distinct treatments were prepared. One tank was fed only with Rhodomonas marina, and the other with Oxyrrhis marina. The concentrations were the same provided in the previous experiments, 1500μgCL−1 of microalgae, and 110μgCL−1 of dinoflagellate. After 15 days, adults were collected with a 200µm sieve and washed with distilled water and ammonium formate 2%. Copepods were then gently transferred to cryopreservation tubes and kept at -80ºC (Fryka – Kaltetechnik – Esslingen) until further analysis. The aim was to collect at least 100mg of dry weight of adult’s copepods of each treatment. Samples were then lyophilized (Labconco – Freeze dry system – freezone 4.5) prior to lipid extraction. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 87 2.2 Lipid extraction Lipids were extracted with a chloroform-methanol mixture (1:2 v/v), containing 0.01% BHT, according to Bligh and Dyer (1959). Briefly, 100-300mg of freeze-dried sample was homogenized with 3ml of chloroform-methanol (1:2 v/v) solution, followed by the addition of saturated NaCl solution. After chloroform and desionized water the sample was ultrasonically extracted during 30min. After filtration the chloroform layer was separated from the methanol-water layer, and dried with anhydrous sodium sulphate. The lipid solution was transferred to a flask and the solvent was evaporated in a rotator evaporator kept at approximately 40ºC. Total lipid (TL) content was determined gravimetrically. 2.3 Transesterification and fatty acid methyl ester analysis For the determination of fatty acid profiles, methyl esters (FAMEs) will be prepared according to the Lepage and Roy method (1986) modifed by Cohen et al. (1988). The preparation of fatty acid methyl esters is carried out using 5ml of the acetylchloride/methanol reagent (1:19v/v). The reaction is done at 80ºC for 1 hour. After cooling, 1ml of water and 2ml of n-heptane is added to the mixture, stirred and centrifuged. The organic phase is collected, filtered and dried with anhydrous sodium sulphate. Solvent is removed under nitrogen and the methyl esters solubilized in 0.1ml of n-heptane. Each sample was made in duplicate. The quantitative analysis is performed in a gas chromatograph (Agilent HP 6890) equipped with a flame ionisation detector and a 5973 Agilent mass selective detector. The separation is performed in a polyethylene glycol capillary column Supercolwax with 30m of length, 0.25mm i.d. and 0.25µm film thicknesses from Supelco. The column is subjected to a temperature program starting at 140ºC for 5min, heating 4ºC min-1 at 240ºC. The injector (split ratio 100:1) and detector temperatures are kept constant at 250ºC during the analysis. Each aliquot was injected in duplicate. The presented results are the average of the values obtained for each sample. Identification of FAMEs was based on the comparison of their retention times with those of authentic standards and/or by the mass spectra. FAME was expressed as mg per g of ground tissue wet weight and as weight percent of the total fatty acids of each sample. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 88 2.4 Statistical analysis Statistics were performed on positively identified FAs that contain non-zero values in all groups. All groups containing greater than one sample are included. Significant differences in lipid data were confirmed by randomization testing on all data. Where appropriate, samples showing a significant difference were subjected to a Tukey´s multiple comparison test with a significance level set at 95% (p<0.05). 3. Results The diets used in this study were chosen based on previous result of experiments performed with different diets on Acartia grani (Chapter IV – V). There were no significant differences (p>0.05) in the lipids (%) content of Acartia grani adults fed the two diets. Analysis of the major fatty acids groups - saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) - revealed no significant differences (p>0.05) between the two diets. Nevertheless, levels of the fatty acids were superior when Acartia grani was fed with the mono diet Rhodomonas marina. SFA were dominated by palmitic acid (16:0), followed by stearic acid (18:0). PUFA dominated the fatty acid composition, accounting for 60% of total fatty acid detected in the Rho diet and 41% in the Rho+Oxy diet. Differences between the two dietary treatments, though no statistically different (mg.g-1), were on the expense of SFA of the Rho+Oxy diet. PUFA composition was dominated in both diets by docohexaenoic acid (DHA), followed by eicosapentaenoic acid (EPA). Again, though not statistically different, levels of DHA (22:6n3) were superior on the treatment where A.grani were fed with Rhodomonas marina (0.629mg.g-1). Levels of DHA/EPA were similar for the two diets: 2.327±0.094mg.g-1 for Rho diet, and 2.176±0.060mg.g-1 for the binary diet. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 95 Veloza, A.J., Chu, F.L.E., Tang, K.W., 2006. Trophic modification of essential fatty acids by heterotrophic protists and its effects on the fatty acid composition of the copepod Acartia tonsa. Mar. Biol., 148, 779–788. Venizelos, A., Benetti, D.D., 1999. Pigmentation abnormalities in flatfish. Aquaculture 176, 181– 188. Watanabe, T., Arakawa, T., Kitajima, C., Fukusho, K. and Fujita, S. 1978. Nutritional quality of living feed from the viewpoint of essential fatty acids for fish. Bull Jpn Soc Sci Fish 44, 1223-1227. Watanabe, T., 1982. Lipid nutrition in fish. Comp. Biochem. Physiol. 73B, 3-15. Watanabe, T., Kitajima, C., Fujita, S., 1983. Nutritional values of live feed organisms in Japan for mass propagation of fish: a review. Aquaculture 34, 115–143. Watanabe, T., 1988. Nutrition and growth. In: Shepard, C.J., Bromage, N.R. (Eds.), Intensive Fish Farming. BSP Prof. Books. Billing & Sons, Worcester, UK, pp. 154-197. Watanabe, T., 1993. Importance of docosahexaenoic acid in marine larval fish. Aquac. Soc. 24, 152-161. Yone, Y., Fuji, M., 1975. Studies on the nutrition of red sea bream: XI. Effect of n_3 fatty acid supplement in a corn oil diet on growth rate and feed efficiency. Bull. Jpn. Soc. Sci. Fish. 41, 73– 77. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 96 Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 97 Chapter VII Effect of Acartia grani culture density on cannibalism occurrence 1. Introduction Several abiotic and biotic factors are known to affect copepod culture to be used as live prey for the aquaculture industry. Amongst them, density of individuals is crucially important to the copepods culture. Despite all efforts in the development of a method for the semi-intensive culture of calanoid copepods, little emphasis is still placed on their mass production to commercial levels. Calanoids are mostly used in larviculture and their maximum density in culture is an important limitation. Calanoid copepods are generally believed to have low tolerance to poor water quality (Payne and Rippingale, 2001) therefore their cultivation at high densities is considered difficult due to density-related stress factors (Jepsen et al., 2007). Past studies investigating effects of copepod stocking density on their culture productivity have mainly assessed egg production and egg hatching success and used them as major indicators (Medina and Barata, 2004; Peck and Holste, 2006; Jepsen et al., 2007). However, other biological parameters, such as cannibalism represent other important criteria. Cannibalism rate is generally reported to increase with increased stocking density (Gallucci and Ólafsson, 2007) due to increased encounter rates of individuals in the rearing tanks. Similarly to the wild, in culture tanks, copepod populations consist of various developmental stages, often coexisting in the same water mass. In this case, it is highly likely that adults and later copepodites often encounter their own offspring. Smaller individuals (younger stages) are then more likely to be victims of the more developed stages (Uye and Liang, 1998, Ohman and Hirche, 2001). For example, Hada and Uye (1991) demonstrated that cannibalism rate increased asymptotically with naupliar and copepodite density (as prey) for Sinocalanus tenellus and Lazzaretto and Salvato (1992) reported the existence of cannibalistic behavior in Tigriopus fulvus females towards first-stage on-ralted nauplii. Although cannibalism is known to be common in Acartia spp. (Mauchline et al., 1998; McKinnon et al., 2003) no work has been done with Acartia grani. The main goal of this experiment was to evaluate the effect of adult stocking density on cannibalism rate toward newly hatched nauplii. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 98 2. Material and Methods 2.1 Experimental design and setup Newly hatched nauplii (hatched within 24h) of Acartia grani were isolated from unhatched eggs by attracting them to a light source utilizing their positive phototoxic behavior. The nauplii collected were subsequently counted and an identical number of 300 nauplii per liter were randomly introduced into each of twenty 50ml replicate vessels (5 replicates per treatment). Using a 250μm mesh sieve, the predators consisting of late copepodites (C-5) and adults, were pre-isolated from stock culture and suspended in Petri dishes with fresh algae. After nauplii had been distributed into each replicate vessel, the pre-isolated adults and copepodites were then counted and added to each replicate vessel to form 5 densities of 125, 250, 500, 1000 and 2000ind./L, respectively. Fresh Rhodomonas was added to all replicates at 1500μgCL-1. To avoid possible confounding effects of nauplii hatched out from eggs produced by adult females introduced as predators, cannibalism experiment lasted 8 hours. This experimental duration was selected based on previous hatching results (Chapter I) for Acartia grani eggs. At the end of the 8h experiment, content of each replicate was collected on 55μm mesh sieve and fixed with 10% formalin. The remaining nauplii were then counted using a dissecting microscope (Zeiss – Axioskop 2 Plus). The difference between initial and final number of the nauplii in each replicate was assumed due to cannibalism, as it is expected that within an 8 hour period, with this culture conditions, nauplii survival would be high(>95%). 2.2 Statistical analysis Data from all experiments were analyzed using one way ANOVA. When significant differences (p<0.05) were found, Tukey's multiple comparisons test was used to determine specific differences among treatments (p<0.05). All statistical analyses were conducted using SPSS, version 20.0. Data are presented as mean±standard deviation (SD). Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 99 3. Results Predation of A. grani adults and late copepodites on nauplii increased with increasing stocking density (p<0.05) (Fig.1). Statistical analysis showed that cannibalism rate on nauplii was significantly lower at the lowest stocking density (125 ind./L) when compared to the remaining treatments. Moreover, significant differences were also found between 250 ind./L density and the highest stocking density of 2000 ind./L (p<0.05). Figure 1 – Naupliar mortality rates at different Acartia grani stocking densities. Data are presented as mean±SD. Different letters on the tops of bars indicate significant differences (p<0.05). 4. Discussion Cultures techniques of copepods are to be improved to render the production of copepod reliable and useful to aquaculture businesses. We evaluated the effect of cannibalism of the copepod Acartia grani on nauplii, which could significantly impact culture productivity. Cannibalism rate is generally reported to increase with increased stocking density (Gallucci and Ólafsson, 2007). Results from current experiment confirmed a trend of A. grani for cannibalism regardless of the existence of other food items as Rhodomonas. Significantly higher predation was observed within densities as low as 250 ind./L. Towards the densest predator treatment of 2000 ind./L significant differences were again found when compared to that of the lower density treatments. Findings of our experiment are in accordance to what Camus and Zeng (2009) found for Acartia sinjiensis at the same densities. 0 10 20 30 40 50 60 125 250 500 1000 2000 Naupliar Mortality (%) Stocking Density (number of adults and copepodites / L) a b bc b bd Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 100 5. Conclusion Further investigation should be done in order to investigate the influence of density on population growth to obtain a more complete picture on the various factors contributing to cannibalistic rates of Acartia grani. Moreover, it would be interesting to determine cannibalistic rates over eggs, since species belonging to the same genera (Acartia) are known to prey upon eggs, eating twice as more eggs than they can produce themselves (Drillet, 2012). Finally it would also be useful to evaluate the degree to which other food items influence cannibalism. Tough in our case the amount of microalgae supplied to each treatment was enough to meet copepodite and adult demands, due to our methodology it is hard to ascertain whether there was an active selection of prey particle or direct effect of random grazing. 6. References Camus, T., Zeng C., 2009. The effects of stocking density on egg production and hatching success, cannibalism rate, sex ratio and population growth of the tropical calanoid copepod Acartia sinjiensis. Aquaculture 287, 145-151. Drillet, G., 2012.Copepod cultures: a key to the development of a diverse marine fish farming industry. Australasian Aquaculture Conference, Melbourne. Gallucci, F., Ólafsson, E., 2007. Cannibalistic behavior of rock-pool copepods: an experimental approach for space, food and kinship. J. Exp.Mar. Biol. Ecol. 342, 325-331. Hada, A., Uye, S., 1991. Cannibalistic feeding behavior of the brackish water copepod Sinocalanus tenellus. J. Plankton Res. 13, 155–166. Jepsen, P.M., Andersen, N., Holm, T., Jorgensen, A.T., Hojgaard, J.K., Hansen, B., 2007. Effects of adult stocking density on egg production and viability in cultures of the calanoid copepod Acartia tonsa (Dana). Aquacult. Res. 38, 764–772. Lazzaretto, I., Salvato, B., 1992. Cannibalistic behavior in the Harpacticoid copepod Tigriopus fulvus. Mar. Biol. 113, 579–582. Mauchline, J., 1998. The Biology of Calanoid Copepods. Elsevier Academic Press, Oxford. pp 710. McKinnon, D., Duggan, S., Nichol, P.D., Rimmer, M.A., Semmens, G., Robin, B., 2003. The potential of tropical paracalanoid copepods as live feeds in aquaculture. Aquaculture 223, 89–106. Medina, M., Barata, C., 2004. Static-renewal culture of Acartia tonsa (Copepoda: Calanoida) for ecotoxicological testing. Aquaculture 229, 203–213. Ohman, M.D., Hirche, H.J., 2001. Density-dependent mortality in an oceanic copepod population. Nature 412, 638–641. Payne, M.F., Rippingale, R.J., 2001. Intensive cultivation of the calanoid copepod Gladioferens imparipes. Aquaculture 201, 329–342. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 101 Peck, M.A., Holste, L., 2006. Effects of salinity, photoperiod and adult stocking density on egg production and egg hatching success in Acartia tonsa (Calanoida: Copepoda): optimizing intensive cultures. Aquaculture 255, 341–350. Uye, S.I., Liang, D., 1998. Copepods attain high abundance, biomass and production in the absence of large predators but suffer cannibalistic loss. J. Mar. Syst. 15, 495–501. Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 102 103 4| Discussion and Final Remarks Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 104 4.1 Final Discussion Copepods, the most numerous multicellular organisms on earth, are the major food source of numerous species of fish larvae. In 1978, Watanabe et al. analyzed fatty acid content of two marine copepods, Trigriopus and Acartia, and concluded they presented a high nutritional value for rearing juvenile fish. Years passed and several studies have been performed on copepods, so the amount of knowledge on these organisms has grown. Yet, a lot is still to be “discovered” in this small world of zooplankton. In aquaculture, they are known to improve fish larval quality and recently proved to be a key for the success of new species, such as Tuna and Grouper. However, due to difficulties in rearing sufficient quantities of copepods and the high associated costs, copepods are today not widely used in the aquaculture industry. On this work, we focused on the copepod Acartia grani because of the known value of the family Acartiidae as live feed, and the wide distribution of this specie. The results presented in this work increase the available knowledge about the copepod Acartia grani, being accomplished the aim that we proposed to. Results showed that Acartia grani could be produced in all of the experimented biotic and abiotic variables. Still, A. grani has its own set of requirements to gauge success and therefore it performed better within optimal sets of parameters. Observation of parental culture tanks and results found between different experiments also showed that differences between cohorts are evident, thus affecting development of the population. Peck and Holste (2006) referred that using eggs produced from different cohorts (or even from different days from the same cohort) could contribute to variability. Egg hatching is one of the factors that are related with copepods productivity. In Chapter I we tried to understand the optimal temperature to initiate culture. Highest temperatures seemed to encourage embryogenesis of the egg, resulting in satisfying egg hatching success. If lower temperatures were used then more time was needed until the first nauplii appeared, since the eggs seem to be in quiescence stage (Hansen et al., 2010). Yet, Guerrero and Rodriguez (1998) observed in their study that 18°C temperature seems to be favorable for egg leaving the dormancy phase in Acartia grani. As a result of this experiment, 24ºC was the chosen temperature for culturing A. grani in all studies performed during this work (Chapter III to Chapter VII). Comparison between the results of the best egg hatching rates at 48h in all the different tested parameters revealed that EHR was relatively low, but with similar values across experiments. Best EHR (38.5%) at 48hours was observed at 24ºC on the dietary experiment (Chapter IV) using binary diet Rho+T-Iso. Nevertheless, in Chapter I Master´s degree in Marine Sciences – Marine Resources | Bernardo Sumares 111 Campbell, R.G., Wagner, M.M., Teegarden, G.J., Boudreau, C.A., Durbin, E.G., 2001. Growth and development rates of the copepod Calanus finmarchicus reared in the laboratory. Mar. Ecol. Prog. Ser. 221,161–183. 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