Feeding adult of Artemia salina (Crustacea-Branchiopoda) on the dinoflagellate Gyrodinium corsicum (Gymnodiniales) and the Chryptophyta Rhodomonas baltica

BIOLOGICAL AND APPLIED SCIENCES

Feeding adult of Artemia salina (Crustacea-Branchiopoda) on the dinoflagellate Gyrodinium corsicum (Gymnodiniales) and the Chryptophyta Rhodomonas baltica

Rauquírio André Albuquerque Marinho da CostaI, ^*; Maria Luise Koening^II; Luci Cajueiro Carneiro Pereira^III

^ILaboratório de Plâncton e Cultivo de Microalgas (LPCM); Núcleo de Estudos Costeiros; Campus Universitário de Bragança; Universidade Federal do Pará; Alameda Leandro Ribeiro, s/n; Aldeia; 68600-000; Bragança - PA - Brasil

^IIDepartamento de Oceanografia; Universidade Federal de Pernambuco; Av. Arquitetura, s/n; Campus Universitário; Cidade Universitária; 50739-540; Recife - PE - Brasil

^IIILaboratório de Oceanografia Costeira e Estuarina; Campus Universitário de Bragança; Universidade Federal do Pará; Alameda Leandro Ribeiro, s/n; Aldeia; 68600-000; Bragança - PA - Brasil

ABSTRACT

Experiments were carried out on feeding performance and survival rates of adult Artemia salina exposed to no axenic strains of the dinoflagellate Gyrodinium corsicum and of the Chryptophyta Rhodomonas baltica. Filtration rates on R. baltica and G. corsicum varied from 3.35 to 7.14 ml.artemia^-1.h^-1 and from 2.97 to 15.86 ml.artemia^-1.h^-1, respectively. The ingestion rates observed for A. salina did not indicate any digestive dysfunction or physiological impairment for organisms fed on G. corsicum and their functional response were similar to those observed for other organisms like copepod fed on different food concentrations. Mortality rates oscillated from 2.5% to 100% when A. salina was fed on R. baltica or G. corsicum, respectively. Highest mortality rates observed for organisms fed on G. corsicum indicated that this dinoflagellate presented a hazard effect on A. salina that was not possible to confirm if it was related to toxin production or to nutritive inadequacy of this dinoflagellate as food for organisms of this species.

Key words: Feeding, Artemia salina, Rhodomonas baltica, Gyrodinium corsicum

RESUMO

Experimentos foram desenvolvidos para estudar as taxas de alimentação e de sobrevivência de Artemia salina alimentada com cepas não tóxicas do dinoflagelado Gyrodinium corsicum e da Chryptophyta Rhodomonas baltica. As taxas de filtração sobre R. baltica e G. corsicum variaram entre 3,35 e 7,14 ml.artemia^-1.h^-1 e 2,97 e 15,86 ml.artemia^-1.h^-, respectivamente. As taxas de ingestão observadas para A. salina não indicaram disfunção digestiva ou prejuízo fisiológico nos organismos alimentados com G. corsicum, sendo a resposta funcional destes organismos similar a observada em copépodos alimentados com diferentes concentrações de alimento. As taxas de mortalidade de A. salina oscilaram entre 2,5 e 100% quando alimentada com R. baltica e G. corsicum, respectivamente. As maiores taxas de mortalidade observadas para os organismos alimentados com G. corsicum indicam que este dinoflagelado apresenta algum efeito nocivo sobre A. salina, embora não tenha sido possível corfirmar se sua origem está relacionada com a produção de toxinas ou com a inadequação nutritiva deste dinoflagelado para alimentação de organismos desta espécie.

INTRODUCTION

Blooms of harmful algae increased in coastal waters around the world during the last decades (Anderson, 1989; Hallegraeff et al., 1988; Sournia et al., 1991; Smayda, 1992; 1997) which evidently would increase more in the future, given the increasing eutrophization (Lam and Ho, 1989; Okaichi, 1989; Hallegraeff, 1993) and global warming perspectives (Hallegraeff, 1993). Data available in the literature indicate that zooplankton grazing could generate a substantial impact on wild populations of toxic dinoflagellates (Turner and Anderson, 1983; Watras et al., 1985). However, other authors consider that production and liberation of toxic compounds would interfere with the feeding processes of some zooplanktonic organisms like copepods (Dutz, 1998; Teegarden, 1999; Maneiro et al., 2000). The potential reduction of toxic blooms and the transference of dinoflagellate toxins through the marine food webs by some zooplanktonic organisms must be considered of great importance when dealing with feeding experiments that involve PSP toxin producers. Hence, it would be important to know whether dinoflagellates are only toxic to fish or also toxic to organisms at lower trophic levels in the pelagic food webs.

Gyrodinium corsicum Paulmier is an unarmoured dinoflagellate that was first reported for the salt-water lake of Diana in Corsica from which originates its denomination. This new species belongs to the order Gymnodiniales (family Gymnodiniaceae) and it was responsible for the development of outbreaks related with the mortality of important fishes such as gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax), caged in intensive aquaculture systems during massive blooms in Diana Lake (Paulmier et al., 1995) and other fish species in Alfacs Bay (Garcés, 1998). These authors did not discard the possibility of ichthyotoxin (haemolysins or allotoxins) production by organisms of this species although negative results were observed for samples collected in Corsica.

Rhodomonas baltica was used in our work as a non-toxic control because it presented similar dimensions of those observed for G. corsicum and has been commonly offered as food supply in aquaculture systems. The present study aimed to determine the feeding patterns of adult Artemia salina (Branchiopoda - Crustacea) exposed to variable cell concentrations of Gyrodinium corsicum (toxic dinoflagellate) and to compare the results with those obtained for Rhodomonas baltica (non-toxic algae control).

MATERIAL AND METHODS

Food supply

G. corsicum and R. baltica were selected due to similar cell size and their toxic and non-toxic nature, respectively. The used stocks were obtained from the Institute of Marine Sciences of Barcelona (I.C.M. Barcelona, Catalonia- Spain). The algae selected were strains of the dinoflagellate G. corsicum Paulmier, GCORS1 (I.C.M. - Barcelona) and the non-toxic Cryptophyta R. baltica Karsten (CCAP 979/9 - UK from I.C.M. - Barcelona). The equivalente espherical diameter (ESD) of the used species were 12.61 µm to G. corsicum and 7.53 µm to R. baltica. The algae were batch-cultured at 15 ± 1 °C in an incubator under controlled temperature, light regime of 12:12 h light/dark cycle and a irradiance of 200 µmol. m^-2.s^-1 (day-light fluorescence tubes). The cultures were maintained in 200 mL flasks containing f/2 medium (Guillard, 1975) in seawater at 35 ‰ salinity.

Animal preparations for feeding

Dried eggs of Artemia salina (L.) were obtained from New Technology Laboratories LTD, United Kingdom. A stock culture of Artemia was reared on R. baltica at 27 ± 1 °C. The stocks were then transferred to the same controlled environmental chamber cited for the culture of phytoplankton and maintained with this cell food during one-generation time. All the Artemia used in the experiments were reared from adult females cultured under laboratory conditions. Before the experiments, adult organisms were sorted and placed in 1 liter beackers containing 800 ml of 0.45 µm filtered seawater, rinsed with filtered seawater and then transferred to the experimental bottles.

Resistance to starvation

To study the tolerance of adult individuals to the lack of food, 15 adult A. salina were transferred to 250 ml bottles filled with 0.45 µm Millipore filtered seawater and fixed to a rotate grazing wheel adjusted to 1 rpm. The bottles were examined every day and the dead organisms were recorded and discarded using a Pasteur pipette. All determinations were carried out with 4 replicates. A. salina adults were defined as dead when no movements were registered within 60 s. Mortality rates were calculated as:

M24H = (NMC/TC) x 100 (1),

where: M24H is the mortality rate after the 24 h trials, NMC is the number of non-motile Artemia in each flask after the 24 h trials and TC is the total number of organisms in each bottle. The M"X"H index was calculated in the same way for 48 h, 72 h and so on.

Feeding experiments

Fixed 6h grazing trials were carried out at 15 ± 1 °C and low light levels to limit algae growth.

Initial cell concentrations were performed using a Coulter electronic particle counter model TA II equipped with a 140 µm aperture. Dilutions were carried out in 0.45 µm filtered seawater to provide food suspensions that varied between 100 and 2000 cell.ml^-1 (Table 1). Experiments consisted of three bottles with grazers for each of the species (R. baltica or G. corsicum) and cell concentrations used, three growth control bottles for R. baltica (non-toxic algae) and three growth control bottles for G. corsicum. At the beginning of the experimental period the bottles were capped and affixed to a rotate grazing wheel, and rotated at 1 rpm until the end of the experiment. At the end of the experiment, containers were removed from the wheel and artemias were separated from the algae suspensions with mesh nets of 200 µm and transferred to Millipore 0.45 µm filtered seawater.

Filtration and ingestion rates were calculated according to Fernández (1979):

F = (Ln C_c-LnC_e)/t.(V/N) (2),

I = (C_c-C_e).(V/Nt) (3),

where:

F= filtration rate (ml of medium swept clear.artemia^-1.h^-1);

I= ingestion rate (number of cells ingested.artemia^-1.h^-1);

C_c= final algal concentration in control flasks;

C_e= final algal concentration in the experimental flasks;

t = duration of the experiment in hours;

V and N = the respective volume (ml) and number of Artemia in the experimental flasks.

Statistical analysis

One-way ANOVA was employed to verify the existence of significant differences between ingestion rates of A. salina on the different algae concentrations. Fisher's PLSD test was used to analyse differences between treatments using a significance level of 0.05.

RESULTS

Starved adults of A. salina showed total mortality rates after 288 h of incubation in 0.45 µm Millipore filtered seawater. Incubations with adult organisms fed on R. baltica demonstrated mortality rates of 2.5 % after the same incubation period (Fig. 1) showing that this alga was appropriated to maintain Artemia cultures under laboratory conditions. On the other hand, it was not possible to complete the life cycle of A. salina with G. corsicum as food. A. salina fed on this dinoflagellate showed 100 % mortality before attain adult stage.

Filtration rates obtained for A. salina fed on R. baltica and G. corsicum are given in Figures 2 A and 2 B. Filtration rates on R. baltica varied from 3.35 to 7.14 ml.artemia^-1.h^-1. For organisms fed on G. corsicum, filtration rates oscillated from 2.97 to 15. 86 ml.artemia^-1.h^-1.

Maximal ingestion rates on R. baltica (5.45 x 10³ cell.ml^-1) and G. corsicum (5.70 x 10³ cell.ml^-1) were observed at the maximal food concentrations employed during the experiments (@ 2000 cells.ml^-1). Significant differences (p<0.05) were found between ingestion rates on R. baltica and G. corsicum at low concentrations although it was not observed at high concentrations, except for 1500 cell.ml^-1. Saturating concentrations were not observed neither for R. baltica or G. corsicum (Figs. 3 A and 3 B).

DISCUSSION

Highest mortality rates observed for organisms fed on G. corsicum indicated that this dinoflagellate presented a hazardous effect on A. salina that was not possible to confirm if it was related to toxin production or to nutritive inadequacy of this dinoflagellate as food for organisms of this species. Results reported by Costa and Fernández (2002) using G. corsicum as food for Acartia grani and Euterpina acutifrons (Copepoda) showed no haemolytic activity for extracts of this dinoflagellate as it was suspected by other authors (Paulmier et al., 1995) indicating that the toxic effects of G. corsicum could be due to the presence of other toxins. The functional response of A. salina was similar to that observed for other organisms like copepod (Uye and Takamatsu, 1990; Liu and Wang, 2002) with higher filtration rates when fed on lower food concentrations (@ 8 x 10² cell.ml^-1) and low filtration rates when exposed to high cell concentrations (@ 4 x 10³ cell.ml^-1). Comparisons between Artemia filtration rates on both algae species were not carried out due to differences between alga dimensions. Some authors reported that differences in algae dimensions and shape (Frost, 1972; Nival and Nival, 1976) could interfere in the filtration efficiency of some organisms (copepod) capturing large cells efficiently than those of low dimensions.

For A. salina fed on planktonic algae, Reeve (1963) did not observe saturating ingestion rates until concentrations higher than 1 x 10⁴ cell.ml^-1. Saturating ingestion rates were also not observed in the present work where maximal cell concentrations were 2 x 10³ cell.ml^-1. This functional response was similar to those registered for copepods were maximal ingestion rates were obtained when organisms were fed on the highest food concentrations employed in the experiments (Dutz, 1998; Frangópulos et al., 2000).

The ingestion rates observed for A. salina did not indicate any digestive dysfunction or physiological impairment for organisms fed on G. corsicum. Reduced ingestion rates of adult A. salina on G. corsicum could be an artefact related to differences of cell dimensions that implies in a higher carbon concentration in this species. So, to satisfy carbon requirements A. salina needs to ingest a low number of G. corsicum cells than it could be expected to R. baltica cells.

In summary it was possible to confirm that A. salina fed on R. baltica and G.corsicum. However, consequences of the ingestion of the later could be the responsable for the death of organisms in few days. On the other hand, R. baltica showed to be very efficient as food suply for adult A. salina giving survival rates of 97.5 % after twelve days of incubation.

ACKNOWLEDGMENTS

This work was supported by CAPES ("Coordenação de Aperfeiçoamento de Pessoal de Nível Superior" - MEC, Brazil) through the concession of a Postgraduate fellowship. We are grateful to Dr. Maximino Delgado for providing the algae clones used in the experiments.

Received: September 22, 2003

Revised: June 17, 2004

Accepted: March 08, 2005

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*

Author for correspondence

Publication Dates

• Publication in this collection
  14 Sept 2005
• Date of issue
  July 2005

History

• Received
  22 Sept 2003
• Reviewed
  07 Jan 2003
• Accepted
  08 Mar 2003