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The effect of food composition on ingestion, development, and survival of a harpacticoid copepod, Tisbe cucumariae Humes
101
J. Exp. Mar. Biol. Ecol., 1984, Vol. 84, pp. 101-110
Elsevier
JEM 381
THE EFFECT OF FOOD COMPOSITION
ON INGESTION, DEVELOPMENT,
AND SURVIVAL
COPEPOD,
OF A HARPACTICOID
TZSBE CUCUMARZAE
Humes
L.D. GUIDI Laboratoire Arago. 66650 Banyuls-sur-Mer, France
Feeding experiments were carried out on the benthic harpacticoid copepod Tisbe cucumariue Humes, using seven different diets of various dried and ground macroalgae and marsh grass, algal Aufwuchs, diatoms, polychaete meat, and cereal. In short-term experiments (1 h), i4C-labelled foods were used to measure ingestion rate of non-ovigerous adult females (individual dry wt = 5.57 f 2.49 fig). No significant difference was found among the rates at which all foods, except polychaete meat, were ingested (12.7to 17.3x 10-2~gdrywt~ind-i~h~’ ). Pol yc h ae t e meat was consumed faster (23.9 x 10e2 ng dry wt ind - ’ . h - ‘). The nutritional value of the foods was estimated in long-term experiments (22 days) by measuring development time and survival of T. cucumariae. Both these variables were significantly correlated with the nitrogen, protein content, and C :N ratio of the foods. No relation was found, however, with the amount of carbon, calories and available calories in the diets. Thus, nitrogen (protein) content of the food was the factor limiting secondary production of the copepods.
Abstract:
Key words:
food composition; ingestion; development; survival; Harpacticoida
In the laboratory, harpacticoid copepods feed on just about anything - from algae (e.g., Lee et al., 1976; Sellner, 1976), bacteria (e.g., Rieper, 1978, 1982), detritus (e.g., Ustach, 1982), and meat of various origins (e.g., It& 1970), to more artificial foods such as Tetramin (Gaudy & GuCrin, 1977), rice bran (Gopalan, 1977), and garden vegetables (Kahan, 1979). While there is much evidence of ingestion of these foods (see review by Hicks & Coull, 1983), only a few studies have been designed to determine their actual nutritional value for copepods, or their impact on functional responses such as ingestion rate, growth, reproduction etc., or to relate these functional responses to some characteristic of the food. For example, Ustach (1982) showed that Heteropsyllus pseudonunk produced more eggs and nauplii when fed detritus or bacteria than when fed algae, and Sellner (1976) found that survival of adult females and offspring of Thompsonula hyuenae correlated with caloritic values measured for each food offered. The purpose of the present study was: (1) to test the effect of different foods (macroalgae, marsh grass, diatoms, algal Aufwuchs, polychaete meat, cereal) on ingestion, development, and survival of a harpacticoid copepod, Tisbe cucumariue Humes; 0022-0981/84/$03.00 0 1984 Elsevier Science Publishers B.V.
L. D. GUIDI
102
and (2) to try and relate these functional responses to food composition (in terms of carbon, nitrogen, protein, caloric, and available caloric content).
MATERIALS
AND
METHODS
T. cucumariae is a ubiquitous animal that is often found associated with macroinvertebrates, e.g. holothurians (Humes, 1957), tunicates (Lopez, 1982), polychaetes (the present study). For these experiments the copepods were collected by placing small dishes containing clean sand and pablum (Gerber’s mixed cereal) at the bottom of tanks used to culture the polychaete Capitella capitata (Fabricius) at the Skidaway Institute of Oceanography (Georgia, U.S.A.) where this study was made. FOOD
SOURCES
Seven foods, homogeneously labelled with 14C when needed, were tested. (1,2) Two stocks of the red macroalga Gracilaria tikvahiae McLachlan, differing in nitrogen content (G-20: high nitrogen; G-10: low nitrogen). (3) One stock of the marsh grass Spartina altemiflora Loisel (S-13). (4) One stock of periphyton associated with Gracilaria tikvahiae, including a high proportion of blue-green algae (G-13A). The preparation and labelling of these foods is given in Tenore et al. (1979). All stocks were ground to < 250 pm particle size and kept freeze dried. Prior to the experiments, the foods were leached in autoclaved filtered sea water for 4 to 5 h on a shaker table, then the water was decanted. Although drying and grinding is somewhat “artificial”, it made these foods similar in particle size and thus avoided confounding experiments with a particle-size effect. (5) The benthic diatom Navicula sp. (DIA) cultured on F/2 medium (Guillard & Ryther, 1962). The diatoms were labelled with [ 14C]bicarbonate after 2 to 3 days of culture, and used 5 days later. Experiments were carried out with freshly rinsed live cultures. (6) Freshly killed (by temperature shock) and ground polychaetes (Capitella capitata) that had previously been starved for 24 h (CAP). Worms were labelled by growing juveniles for 10 days with 14C-labelled G-20. (7) Pablum (PAB) was labelled with [ “C]acetic anhydride following the procedure described in Banks & Woltinbarger (1981). This technique results in labelling of primary amino-groups present on amino-acids, proteins or amino-sugars, and does not produce major changes in chemical composition of the food. Although the food is not homogeneously labelled by this method, it can be used to measure ingestion rate. Food composition was determined on weighed freeze dried samples. Carbon and nitrogen contents were measured using a Perkin-Elmer Elemental Analyzer (Model 240). Protein content was measured by the fluorescamine assay method (Bohlen et al., 1973), with modifications after Castell et al. (1979). With this method protein content also includes other primary amino-bearing groups, such as peptides and aminoacids.
FEEDING OF TISBE
103
Caloric content of foods was measured by microbomb calorimetry using benzoic acid as a standard. Available caloric content, i.e. that portion of total calories in the food that is readily utilizable by the consumer, was estimated by the caloric change in the sample after hydrolysis in 1 N HCl for 4 h at 20 “C, following the procedure described in Tenore (1981). FEEDING
EXPERIMENTS
All experiments were replicated three to five times at 20 “C under natural light conditions, in 3.5cm diameter covered Petri dishes containing 3.5 ml of autoclaved 0.45 pm-filtered sea water (S = 26%,), 1.5 ml of sieved (~300 pm) and ashed (400 “C overnight) clean beach sand, and food in excess (l-2 mg dry wt). Ingestion rate was measured for batches of 20 to 100 non-ovigerous adult females, after l-h incubations (gut passage time estimated with diatoms as food, was z 1 h) in the presence of the labelled foods. Prior to the experiments, the animals had been starved for only 2 to 3 h, because longer starving may lead to over-estimations of ingestion rate (see Grant et al., 1983). After each incubation, the labelled copepods were quickly rinsed in 10% HCL and in distilled water, and placed in a pre-weighed Oxidizer paper cone. The cones were dried ove~i~t at 80 “C, and dry weight (DW) of the copepods measured to the nearest ,ugper cone using a Mettler analytical microbalance. The effect of the different foods on development time (i.e., time from hatching to reproductive maturity) and survival, was tested in long-term (22 days) experiments. Ten ovigerous females were placed in each dish, and were removed as soon as they had deposited their egg-sac. Experiments started when the first nauplii hatched and ended when the first ovigerous females appeared. Non-labeled foods and water were changed every other day. Individuals were checked daily. In order to measure survival, for all foods, copepods were enumerated on the ninth day of culture. For this purpose, each dish was emptied into a glass tube containing 5 ml of sterile sea water (dilution factor: 2). Two sub-samples of 2 ml each, drawn off after the tubes had been gently vortexed for 20 s, were then mixed with 70% ethanol. Copepods were enumerated and, to estimate growth, measured under a dissecting microscope equipped with an ocular micrometer, and separated into 5 arbitrary size classes (N < 0.4 mm; 0.4 mm < S < 0.6 mm; 0.6 < MS < 0.8 mm; 0.8 mm g M < 1.0 mm; 1.0 mm d L; this last class included females only). If no gravid females were present, the remaining copepods were put back into Petri dishes and left to grow on the corresponding diet for up to an additional 13 days. TREATMENT
OF LABELLED
SAMPLES
Weighed dried copepods and food samples were burned in a Packard Tri-Carb Oxidizer, counted in a Packard Liquid Scint~ation Counter, and after correction for quenching, background, and machine efficiency, specific activity (DPM *pg DW - ‘) was calculated. Ingestion rate of dry food was calculated from the specific activities of the copepods and the foods.
L. D. GUIDI
104
RESULTS CHEMICAL
OF THE
COMPOSITION
FOODS
(TableI)
Carbon content (% DW), which ranged from 27.6 (DIA) to 44.8 (G-13A), did not differ greatly from one food to another. Nitrogen content (% DW), however, varied from 1.1 (G-10) to 8.4 (CAP), and seemed partly responsible for the important variations of the C : N ratio (from 4.4 in CAP, to 40.4 in G-10). Protein content ( y0 DW) TABLEI Chemical composition of the different foods offered to Tisbe cucumariae in the experiments: G-10, low nitrogen Gracilaria tikvahiae; S-13, Spartina alterniflora; PAB, Gerber’s mixed cereal; G-20, high nitrogen Gracilaria tikvahiae; DIA, Navicula sp.; CAP, Capitella capitata meat; G-13A, algal Aufwuchs; DW, dry wt; all determinations were performed in triplicate. G-10
s-13
PAB
G-20
DIA
CAP
G-13A
44.4
39.2
43.1
39.1
21.6
37.1
44.8
1.1 40.4
1.2 32.1
2.5 17.2
5.4 1.2
4.8 5.1
8.4 4.4
7.8 5.1
6.9
7.0
15.4
33.7
30.0
52.0
49.0
3.8
3.5
4.0
3.5
2.8
4.6
4.1
1.7
1.5
2.1
1.1
2.0
1.8
1.2
Carbon content (% DW) Nitrogen content (% DW) C : N ratio Protein content (% DW) Caloric content (cal.mg DW-‘) Available caloric content (cal.mg DW-‘)
varied from 6.9 (G-10) to 52.0 (CAP), and was proportional to nitrogen content. The different foods contained from 2.8 (DIA) to 4.6 (CAP) calories . mg DW ‘, and from 1.1 (G- 10) to 2.1 (PAB) available calories * mg DW - ‘. Regarding their over-all chemical composition, all diets, except G-10 and S-13, appeared quite different from one another. As for G-10 and S-13, it was merely the C : N ratio that showed a marked difference. INGESTION OF THE FOODS
(Table II)
Depending on the diet offered, mean ingestion rate of dry matter by non-ovigerous adult females of Tisbe cucumariue varied from 12.7 (S-13) to 23.9 (CAP) x lo-* pg. ind-‘.h-‘,or2.3 to4.3 x IO-*mg.mgDW Tisbe- ’ . h - ‘. Ingestion rate data from the different diets were compared using the Wilcoxon-Mann-Whitney W-test (Bradley, 1968). The rates at which G-10, S-13, PAB, G-20, DIA, and G-13A were ingested did not differ at the 5 y0 level of significance. CAP, which had the highest nitrogen, protein, and caloric content, was, however, ingested at a significantly faster rate.
FEEDING
OF TIME
105
TABLE II adult females of Tisbe cucumariae Ingestion (mean individual dry wt = 5.57 pg, SE = 2.49, n = 29) offered different “‘C-labelled diets: G-10, low nitrogen Gracilaria tikvahiae; S-13, Spartina altemiflora; PAB, Gerber’s mixed cereal; G-20, high nitrogen Gruciluria tikvahiae; DIA, Navicula sp.; CAP, Capitella capitata meat; G-13A, algal Aufwuchs; m, mean of n determlnations; SE, standard error rate of dry matter (lo-* pg. ind - ’ . h- ‘) by non-ovigerous
m SF
n
EFFECT
G-10
s-13
PAB
G-20
DIA
CAP
G-13A
14.6 3.6 5
12.7 3.9 3
16.3 3.4 5
16.’ 37 4
17.0 2.5 4
23.9 4.0 4
17.?
OF THE
FOODS
ON DEVELOPMENT
AND
II 4
(Fig. 1)
SURVIVAL
On all diets except G-10, the life cycle (from hatching to reproductive maturity) was completed in 8 (DIA, CAP, G-13A) to 15.3 (S-13) days. When the animals were cultured on G-10, no ovigerous females were found even after 22 days (when the experiments were stopped). Development time was correlated (Spearman’s rank correlation test: Siegel, 1956) with the nitrogen and protein content (I, = - 0.89, CI= 0.05) and C : N ratio (rs = 0.97, a = 0.0 1) of the foods. No relation was found, however, with the carbon, caloric, and available caloric content. After 9 days of culture (started with newly hatched nauplii), the mean total number of individuals that had survived on the different diets, ranged from 60 k 12 (G-10) to
Development
OL-
0
1
1
G-10
T~rne
s-13
PAB
G-20
OIA
CAP
G-13A
Fig. 1. Effect of the different diets on development time, i.e. time from hatching to reproductive maturity, and survival of Tisbe cucumarfue: survival was measured as the concentration of individuals 9 days alter the beginning of the experiments; values are means (with SE) of three different experiments started with newly hatched nauplii; G-10, low nitrogen Gracilariu tikvahiae; S-13, Spa&a altemifora; PAB, Gerber’s mixed cereal; G-20, high nitrogen Gracilatia tikvahiae; DIA, Navicula sp.; CAP, Capitella cupitata meat; G-13A, algal Aufwuchs.
L. D. GUIDI
106
237 f 36 (G-13A) *ml- I. A highly significant (a = 0.01) correlation was found between survival and the nitrogen, protein content (I, = 0.93), and C : N ratio (rs = - 0.94) of the foods. Survival was not related, however, to the amount of carbon, caloric, and available caloric content in the foods. On the ninth day, the populations offered G-10 and S-13 were largely dominated by medium-small (0.6 mm d MS < 0.8 mm) indi-
SIZE
CLASSES
17 04mmQ
S <0.6mm F&I 0.6 mm S MS < 0.8 mm 0 0.8 mm < M < 1.0 mm 0 l.OmmQ L
25
10 0
~
~ c-10
_ s-13
PA6
~ G-20
~ DIA
CAP
G-13A
Fig. 2. Proportion of individuals of TiEbe cucumariae found in each of the five size classes aher nine days ofculture on the different diets: proportion of ovigerous females is represented by a black area in the largest size class (L) that includes females only; values are means (with SE) of three different experiments started with newly hatched nauplii; G-10, low nitrogen Graciiuria tikvahiae; S-13, Spar&a a~terniflra; PAB, Gerber’s mixed cereaI; G-20, high nitrogen GraciZa~a ~~a~~ae; DIA, Navicda sp.; CAP, Capitella capitata meat; G-13A, algal Aufwuchs.
viduals, which made up to 78 and 70% of the total number, respectively (Fig. 2). On all the other diets, the three size classes MS, M, and L were more or less equally represented. Populations cultured on CAP and G-13A had the highest proportion of ovigerous females (13.6 and 8.9% of total number, respectively). Nauplii (N < 0.4 mm) were present in the dishes receiving G-10, CAP, and G-13A.
DISCUSSION
Although some harpacticoid copepods are known to be highly selective for certain foods (e.g. bacteria: Vanden Berghe 8~ Bergmans, 1981), in general if substances
FEEDING OF TISBE
107
remotely edible are presented to these animals, they invariably make attempts at ingestion (see Hicks & Coull, 1983). It is, therefore, not surprising that the non-ovigerous adult females of T. cucumariae ingested ail the foods tested in the present study. The rates at which the foods were consumed are equivalent to those reported by Rieper (1978) for T. holothuriae and Paramphiascella vararesis, i.e. 8.6 to 29.5 x lo-’ pg bacteria . ind - l . h - l. All foods, except CAP, were ingested at approximately the same rate. The faster consumption of CAP could have resulted from the higher digestibility of a diet of entire heterotrophic origin (flesh and presumably bacterial associates). Although some of the other diets, and particularly G-13A, included heterotrophic components, they all contained or originated from autotrophic organisms. As noted by Lampert (1977) and Tietjen & Lee (1977), the rate at which a food source is consumed does not necessarily reflect its nutritional value. For example, Gyllenberg & Lundqvist (1978) found that although the copepod Cyclops oithonoides ingested fresh bacterial cells (evidence from radioisotope experiments), it was not able to survive on such a diet. The nutritional quality of the seven foods ingested by Tisbe cucumariae was assessed according to the development time and survival of the copepods. Both these variables were affected by the type of food offered, and more precisely by the nitrogen (and protein) level in the foods. The importance of the regulatory r61e of nitrogen has been seen especially in detrital food chains. For example, both Tenore (1977,198 1) and Findlay (1982) found that the best index of nutritional value (measured as growth) was the amount of nitrogen in the detrital source supplied to a polychaete and a nematode. While other food characteristics, such as total caloric content (Sellner, 1976; Tenore, 1977) or available caloric content (Tenore, 198 1) have been related to nutritional quality, such a relationship could not be established in the present study. Available caloric content is, however, usually high in algal materials, and low in detritus derived from marine vascular plants (Tenore et al., 1982). Thus, available energy is typically potentially limiting in diets not greatly represented in the present investigation. The relationship between development time or survival of T. cucumariae and food quality, was not linear (particularly visible on the development time curve, Fig. 1). There seemed to be some threshold level of nitrogen in the food beyond which development time remained constant. Although a large number of ovigerous females were present after 9 days of culture on PAB (Fig. 2), according to development time values the best foods were DIA, CAP, and G-13A. These three foods were apparently of equivalent nutritional value, although the populations receiving CAP and G-13A did produce more ovigerous females (nauplii, probably of the second generation, were also present) than did that receiving DIA. The presence of heterotrophic components in both CAP and G-13A could be responsible for these minute differences. Experiments carried out over longer periods might have brought out greater discrepancies between the three diets since certain foods may become inadequate only after several generations (Shiraishi & Provasoli, 1959). G-13A, which was composed of algal Aufwuchs, contained blue-green algae, bacteria, diatoms, fungi, and mucilage, making it the only ‘mixed’ food offered to T. cucumariae in the experiments. It is likely that such a diet would give the best results
108
L. D. GUIDI
in multi-generation cultures, since it is now well established that the reproductive performance and health of copepod populations under culture are favoured by mixed foods (see Hicks & Coull, 1983), reflecting the need for a balanced diet. In the Cupitelh capitutu tanks, the copepods probably feed on a mixture of both PAB (used to maintain the cultures) and dead worms (over the course of seven years, dead polychaetes have very seldom been found in the cultures: Tenore, pers. comm.). Other populations of Tisbe cucumariae have been found to feed on the dead bodies of the organisms with which they are associated, e.g. the ttmicate Ciona intestinalis (Lopez, 1982). At the other end of the nitrogen scale, G-10 and S-13, which had a very similar chemical composition, did not produce the same effects on Tisbe cucumariae. G-10 may have been lacking some essential elements (minerals, vitamins) that limited growth (nauplii were still present in the cultures after 9 days) and development of the copepods. Lopez (1982) noted a slowing in nauplius development of T. cucumariue cultures receiving inadequate food. A number of authors have stressed the importance of trace nutrients in the food as a factor affecting nutritional value, e.g. Shiraishi & Provasoli (1959) showed that a supplement of vitamins to Tigriopus cultures corrected the nutritional inadequacy of certain algal foods. Animals react to changes in food value and supply at the ingestion and/or assimilation level. Indeed, some animals will increase ingestion rate of foods of low nutritional quality and thus grow at the same rate as when fed highly nutritional material, e.g. Nereis virens cultured on clam tissue and faeces (Tenore & Gopalan, 1974). Unlike this polychaete, Tisbe cucumariae, however, did not vary ingestion rate in response to food quality as a behaviour in trophic dynamics. In conclusion, the present study has shown that food composition, which had little, if any influence on ingestion rate, significantly affected development and survival of T. cucumariue. Nitrogen (protein) level in the food seems to play a dominant regulating role in the secondary production of the copepods.
ACKNOWLEDGEMENTS
I gratefully acknowledge Dr. K. R. Tenore for welcoming me in his laboratory, and supervising my research. His advice and comments on the manuscript were very helpful. I thank Dr. N. W. Phillips for assistance with chemical analyses. Financial support was from the French “Minis&e des Relations Exttrieures”, and from the Oceanography Section of the National Science Foundation, Grant No. OCE 82 - 00385 to Dr. K. R. Tenore. This work was presented at the Fifth International Meiofauna Symposium, 16-20 August 1983, Belgium, but no proceedings of the meeting are to be published.
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REFERENCES BANKS, C. W. & L. WOLFINBARGER,
JR., 1981. A rapid and convenient method for radiolabelling detritus with “C-acetic anhydride. J. Exp. Mar. Biol. Ecol., Vol. 53, pp. 115-123. BOHLEN,P., S. STEIN, W. DAIRMAN,& S. UDENFRIEND, 1973. Fluorometric assay of proteins in the nanogram range. Arch. Biochem. Biophys., Vol. 155, pp. 213-220. BRADLEY,J. V., 1968. Distribution-fee scacisticaltests. Prentice-Hall, London, 388 pp. CASTELL,J. V., M. CERVERA& R. MARCO,1979. A convenient micromethod for the assay ofprimary amines and proteins with fluorescamine. A reexamination of the conditions of reaction. Anal. Biochem., Vol. 99. pp. 379-391. FINDLAY, S.E.G., 1982. Effect of detrital nutritional quality on the population dynamics of a marine nematode (Diplolaimella chitwoodi). Mar. Biol., Vol. 68, pp. 223-227. GAUDY,R. & J.-P. GUBRIN, 1977. Dynamique des populations de Tisbe holothuriae (Crustacea: Copepoda) en tlevage sur trois regimes artiticiels differents. Mar. Biol., Vol. 39, pp. 137-146. GOPALAN,U.K., 1977. Experimental mass culture of a harpacticoid copepod Nicocra spinipes Boeck. In, Proc. Symp. Warm Water Zooplankton Spec. Public., UNESCO/NIO, National Institute of Oceanography, Goa, India, pp. 558-562. GRANT, I. F., E.A. EGAN & M. ALEXANDER,1983. Measurement of the rates of grazing of the ostracod Cyprinotus carolinensis on blue-green algae. Hydrobiologia, Vol. 106, pp. 199-208. GUILLARD,R. R. L. & J. H. RYTHER,1962. Studies on marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea (Cleve) Gran. Can. J. Microbial., Vol. 8, pp. 229-231. GYLLENBERG,G. & G. LUNDQVIST,1978. Utilization of dissolved glucose by two copepod species. Ann. Zool. Fenn., Vol. 15, pp. 323-327. HICKS, G. R. F. & B.C. COULL, 1983.The ecology of marine meiobenthic harpacticoid copepods. Oceanogr. Mar. Biol. Annu. Rev., Vol. 21, pp. 67-175. HUMES, A. G., 1957. Deux coptpodes harpacticoi’des nouveaux du genre Tisbe, parasites des holothuries de la Mediterranee. Vie Milieu, Vol. 8, pp. 9-22. ITB, T., 1970. The biology of a harpacticoid copepod, Tigriopusjaponicus Mori. J. Fat. Sci. Hokkaido Univ., Ser. 6, Zool., Vol. 17, pp. 474-500. KAHAN,D., 1979. Vegetables as food for marine harpacticoid copepods. Aquaculture, Vol. 16, pp. 345-350. LAMPERT,W., 1977. Studies on the carbon balance ofDaphniapulex as related to environmental conditions. 1 - Methodological problems of the use of ‘%Zfor measurement of carbon assimilation. Arch. Hydrobiol., Vol. 48 (Suppl.), pp. 287-309. LEE, J.J., J.H. TIETJEN & J.R. GARRISON, 1976. Seasonal switching in the nutritional requirements of Nirocru typica, a harpacticoid copepod from salt marsh Aufwuchs communities. Trans. Am. Microsc. Sot., Vol. 95, pp. 628-637. LOPEZ,G. W., 1982. Short-term population dynamics of Tisbe cucumariae (Copepoda : Harpacticoida). Mar. Biol., Vol. 68, pp. 333-341. RIEPER, M., 1978. Bacteria as food for marine harpacticoid copepods. Mar. Biol., Vol. 45, pp. 337-345. RIEPER,M., 1982. Feeding preferences of marine harpacticoid copepods for various species ofbacteria. Mar. Ecol. Prog. Ser., Vol. 7, pp. 303-307. SELLNER, B.W., 1976. Survival and metabolism of the harpacticoid copepod, Thompsonula hyaenae (Thompson) fed on different diatoms. Hydrobiologia, Vol. 50, pp. 233-238. SHIRAISHI,K. & L. PROVASOLI,1959. Growth factors as supplements to inadequate algal foods to Tigriopus japonicus. Tohoku J. Agric Res., Vol. 10, pp. 89-96. SIEGEL, S., 1956. Non parametric statisticsfor behavioral sciences. McGraw-Hill Kogakusha Ltd., Tokyo, 312 pp. TENORE, K.R., 1977. Growth of Capicella capitata cultured on various levels of detritus derived from different sources. Limnol. Oceanogr., Vol. 22, pp. 936-941. TENORE,K. R., 1981. Organic nitrogen and caloric content of detritus. I. Utilization by the deposit-feeding polychaete, Capitella capitata. Estuarine Coastal Sherf Sci., Vol. 12, pp. 39-47. TENORE,K. R., L. CAMMEN,S. E. G. FINDLAY& N. W. PHILLIPS,1982. Perspectives of research on detritus: do factors controlling the availability of detritus to macroconsumers depend on its source? J. Mar. Res., Vol. 40, pp. 473-490. TENORE,K. R. & U.K. GOPALAN, 1974. Feeding efficiencies of the polychaete Nereis virens cultured on hard-clam tissue and oyster detritus. J. Fish. Res. Board Can., Vol. 31. pp. 1675-1678.
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TENORE,K. R., R. B. HANSON, B. E. DORNSEIF& C. N. WIEDERHOLD,1979. The effect of organic nitrogen supplement on the utilization of different sources of detritus. Limnol. Oceanogr., Vol. 34, pp. 350-355. TIETJEN,J. H. & J. J. LEE, 1977. Feeding behavior of marine nematodes. In, Ecology ofmarine benrhos, edited by B.C. Coull, University of South Carolina Press, Columbia, U.S.A., pp. 21-35. USTACH, J.F., 1982. Algae, bacteria and detritus as food for the harpacticoid copepod, Heteropsyllus pseudonunni Coull and Palmer. J. Exp. Mar. Biol. Ecol., Vol. 64, pp. 203-214. VANDENBERGHE,W. & M. BERGMANS,1981. Differential food preference in three co-occuring species of Tisbe (Copepoda: Harpacticoida). Mar. Ecol. Prog. Ser., Vol. 4, pp. 213-219.
J. Exp. Mar. Biol. Ecol., 1984, Vol. 84, pp. 101-110
Elsevier
JEM 381
THE EFFECT OF FOOD COMPOSITION
ON INGESTION, DEVELOPMENT,
AND SURVIVAL
COPEPOD,
OF A HARPACTICOID
TZSBE CUCUMARZAE
Humes
L.D. GUIDI Laboratoire Arago. 66650 Banyuls-sur-Mer, France
Feeding experiments were carried out on the benthic harpacticoid copepod Tisbe cucumariue Humes, using seven different diets of various dried and ground macroalgae and marsh grass, algal Aufwuchs, diatoms, polychaete meat, and cereal. In short-term experiments (1 h), i4C-labelled foods were used to measure ingestion rate of non-ovigerous adult females (individual dry wt = 5.57 f 2.49 fig). No significant difference was found among the rates at which all foods, except polychaete meat, were ingested (12.7to 17.3x 10-2~gdrywt~ind-i~h~’ ). Pol yc h ae t e meat was consumed faster (23.9 x 10e2 ng dry wt ind - ’ . h - ‘). The nutritional value of the foods was estimated in long-term experiments (22 days) by measuring development time and survival of T. cucumariae. Both these variables were significantly correlated with the nitrogen, protein content, and C :N ratio of the foods. No relation was found, however, with the amount of carbon, calories and available calories in the diets. Thus, nitrogen (protein) content of the food was the factor limiting secondary production of the copepods.
Abstract:
Key words:
food composition; ingestion; development; survival; Harpacticoida
In the laboratory, harpacticoid copepods feed on just about anything - from algae (e.g., Lee et al., 1976; Sellner, 1976), bacteria (e.g., Rieper, 1978, 1982), detritus (e.g., Ustach, 1982), and meat of various origins (e.g., It& 1970), to more artificial foods such as Tetramin (Gaudy & GuCrin, 1977), rice bran (Gopalan, 1977), and garden vegetables (Kahan, 1979). While there is much evidence of ingestion of these foods (see review by Hicks & Coull, 1983), only a few studies have been designed to determine their actual nutritional value for copepods, or their impact on functional responses such as ingestion rate, growth, reproduction etc., or to relate these functional responses to some characteristic of the food. For example, Ustach (1982) showed that Heteropsyllus pseudonunk produced more eggs and nauplii when fed detritus or bacteria than when fed algae, and Sellner (1976) found that survival of adult females and offspring of Thompsonula hyuenae correlated with caloritic values measured for each food offered. The purpose of the present study was: (1) to test the effect of different foods (macroalgae, marsh grass, diatoms, algal Aufwuchs, polychaete meat, cereal) on ingestion, development, and survival of a harpacticoid copepod, Tisbe cucumariue Humes; 0022-0981/84/$03.00 0 1984 Elsevier Science Publishers B.V.
L. D. GUIDI
102
and (2) to try and relate these functional responses to food composition (in terms of carbon, nitrogen, protein, caloric, and available caloric content).
MATERIALS
AND
METHODS
T. cucumariae is a ubiquitous animal that is often found associated with macroinvertebrates, e.g. holothurians (Humes, 1957), tunicates (Lopez, 1982), polychaetes (the present study). For these experiments the copepods were collected by placing small dishes containing clean sand and pablum (Gerber’s mixed cereal) at the bottom of tanks used to culture the polychaete Capitella capitata (Fabricius) at the Skidaway Institute of Oceanography (Georgia, U.S.A.) where this study was made. FOOD
SOURCES
Seven foods, homogeneously labelled with 14C when needed, were tested. (1,2) Two stocks of the red macroalga Gracilaria tikvahiae McLachlan, differing in nitrogen content (G-20: high nitrogen; G-10: low nitrogen). (3) One stock of the marsh grass Spartina altemiflora Loisel (S-13). (4) One stock of periphyton associated with Gracilaria tikvahiae, including a high proportion of blue-green algae (G-13A). The preparation and labelling of these foods is given in Tenore et al. (1979). All stocks were ground to < 250 pm particle size and kept freeze dried. Prior to the experiments, the foods were leached in autoclaved filtered sea water for 4 to 5 h on a shaker table, then the water was decanted. Although drying and grinding is somewhat “artificial”, it made these foods similar in particle size and thus avoided confounding experiments with a particle-size effect. (5) The benthic diatom Navicula sp. (DIA) cultured on F/2 medium (Guillard & Ryther, 1962). The diatoms were labelled with [ 14C]bicarbonate after 2 to 3 days of culture, and used 5 days later. Experiments were carried out with freshly rinsed live cultures. (6) Freshly killed (by temperature shock) and ground polychaetes (Capitella capitata) that had previously been starved for 24 h (CAP). Worms were labelled by growing juveniles for 10 days with 14C-labelled G-20. (7) Pablum (PAB) was labelled with [ “C]acetic anhydride following the procedure described in Banks & Woltinbarger (1981). This technique results in labelling of primary amino-groups present on amino-acids, proteins or amino-sugars, and does not produce major changes in chemical composition of the food. Although the food is not homogeneously labelled by this method, it can be used to measure ingestion rate. Food composition was determined on weighed freeze dried samples. Carbon and nitrogen contents were measured using a Perkin-Elmer Elemental Analyzer (Model 240). Protein content was measured by the fluorescamine assay method (Bohlen et al., 1973), with modifications after Castell et al. (1979). With this method protein content also includes other primary amino-bearing groups, such as peptides and aminoacids.
FEEDING OF TISBE
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Caloric content of foods was measured by microbomb calorimetry using benzoic acid as a standard. Available caloric content, i.e. that portion of total calories in the food that is readily utilizable by the consumer, was estimated by the caloric change in the sample after hydrolysis in 1 N HCl for 4 h at 20 “C, following the procedure described in Tenore (1981). FEEDING
EXPERIMENTS
All experiments were replicated three to five times at 20 “C under natural light conditions, in 3.5cm diameter covered Petri dishes containing 3.5 ml of autoclaved 0.45 pm-filtered sea water (S = 26%,), 1.5 ml of sieved (~300 pm) and ashed (400 “C overnight) clean beach sand, and food in excess (l-2 mg dry wt). Ingestion rate was measured for batches of 20 to 100 non-ovigerous adult females, after l-h incubations (gut passage time estimated with diatoms as food, was z 1 h) in the presence of the labelled foods. Prior to the experiments, the animals had been starved for only 2 to 3 h, because longer starving may lead to over-estimations of ingestion rate (see Grant et al., 1983). After each incubation, the labelled copepods were quickly rinsed in 10% HCL and in distilled water, and placed in a pre-weighed Oxidizer paper cone. The cones were dried ove~i~t at 80 “C, and dry weight (DW) of the copepods measured to the nearest ,ugper cone using a Mettler analytical microbalance. The effect of the different foods on development time (i.e., time from hatching to reproductive maturity) and survival, was tested in long-term (22 days) experiments. Ten ovigerous females were placed in each dish, and were removed as soon as they had deposited their egg-sac. Experiments started when the first nauplii hatched and ended when the first ovigerous females appeared. Non-labeled foods and water were changed every other day. Individuals were checked daily. In order to measure survival, for all foods, copepods were enumerated on the ninth day of culture. For this purpose, each dish was emptied into a glass tube containing 5 ml of sterile sea water (dilution factor: 2). Two sub-samples of 2 ml each, drawn off after the tubes had been gently vortexed for 20 s, were then mixed with 70% ethanol. Copepods were enumerated and, to estimate growth, measured under a dissecting microscope equipped with an ocular micrometer, and separated into 5 arbitrary size classes (N < 0.4 mm; 0.4 mm < S < 0.6 mm; 0.6 < MS < 0.8 mm; 0.8 mm g M < 1.0 mm; 1.0 mm d L; this last class included females only). If no gravid females were present, the remaining copepods were put back into Petri dishes and left to grow on the corresponding diet for up to an additional 13 days. TREATMENT
OF LABELLED
SAMPLES
Weighed dried copepods and food samples were burned in a Packard Tri-Carb Oxidizer, counted in a Packard Liquid Scint~ation Counter, and after correction for quenching, background, and machine efficiency, specific activity (DPM *pg DW - ‘) was calculated. Ingestion rate of dry food was calculated from the specific activities of the copepods and the foods.
L. D. GUIDI
104
RESULTS CHEMICAL
OF THE
COMPOSITION
FOODS
(TableI)
Carbon content (% DW), which ranged from 27.6 (DIA) to 44.8 (G-13A), did not differ greatly from one food to another. Nitrogen content (% DW), however, varied from 1.1 (G-10) to 8.4 (CAP), and seemed partly responsible for the important variations of the C : N ratio (from 4.4 in CAP, to 40.4 in G-10). Protein content ( y0 DW) TABLEI Chemical composition of the different foods offered to Tisbe cucumariae in the experiments: G-10, low nitrogen Gracilaria tikvahiae; S-13, Spartina alterniflora; PAB, Gerber’s mixed cereal; G-20, high nitrogen Gracilaria tikvahiae; DIA, Navicula sp.; CAP, Capitella capitata meat; G-13A, algal Aufwuchs; DW, dry wt; all determinations were performed in triplicate. G-10
s-13
PAB
G-20
DIA
CAP
G-13A
44.4
39.2
43.1
39.1
21.6
37.1
44.8
1.1 40.4
1.2 32.1
2.5 17.2
5.4 1.2
4.8 5.1
8.4 4.4
7.8 5.1
6.9
7.0
15.4
33.7
30.0
52.0
49.0
3.8
3.5
4.0
3.5
2.8
4.6
4.1
1.7
1.5
2.1
1.1
2.0
1.8
1.2
Carbon content (% DW) Nitrogen content (% DW) C : N ratio Protein content (% DW) Caloric content (cal.mg DW-‘) Available caloric content (cal.mg DW-‘)
varied from 6.9 (G-10) to 52.0 (CAP), and was proportional to nitrogen content. The different foods contained from 2.8 (DIA) to 4.6 (CAP) calories . mg DW ‘, and from 1.1 (G- 10) to 2.1 (PAB) available calories * mg DW - ‘. Regarding their over-all chemical composition, all diets, except G-10 and S-13, appeared quite different from one another. As for G-10 and S-13, it was merely the C : N ratio that showed a marked difference. INGESTION OF THE FOODS
(Table II)
Depending on the diet offered, mean ingestion rate of dry matter by non-ovigerous adult females of Tisbe cucumariue varied from 12.7 (S-13) to 23.9 (CAP) x lo-* pg. ind-‘.h-‘,or2.3 to4.3 x IO-*mg.mgDW Tisbe- ’ . h - ‘. Ingestion rate data from the different diets were compared using the Wilcoxon-Mann-Whitney W-test (Bradley, 1968). The rates at which G-10, S-13, PAB, G-20, DIA, and G-13A were ingested did not differ at the 5 y0 level of significance. CAP, which had the highest nitrogen, protein, and caloric content, was, however, ingested at a significantly faster rate.
FEEDING
OF TIME
105
TABLE II adult females of Tisbe cucumariae Ingestion (mean individual dry wt = 5.57 pg, SE = 2.49, n = 29) offered different “‘C-labelled diets: G-10, low nitrogen Gracilaria tikvahiae; S-13, Spartina altemiflora; PAB, Gerber’s mixed cereal; G-20, high nitrogen Gruciluria tikvahiae; DIA, Navicula sp.; CAP, Capitella capitata meat; G-13A, algal Aufwuchs; m, mean of n determlnations; SE, standard error rate of dry matter (lo-* pg. ind - ’ . h- ‘) by non-ovigerous
m SF
n
EFFECT
G-10
s-13
PAB
G-20
DIA
CAP
G-13A
14.6 3.6 5
12.7 3.9 3
16.3 3.4 5
16.’ 37 4
17.0 2.5 4
23.9 4.0 4
17.?
OF THE
FOODS
ON DEVELOPMENT
AND
II 4
(Fig. 1)
SURVIVAL
On all diets except G-10, the life cycle (from hatching to reproductive maturity) was completed in 8 (DIA, CAP, G-13A) to 15.3 (S-13) days. When the animals were cultured on G-10, no ovigerous females were found even after 22 days (when the experiments were stopped). Development time was correlated (Spearman’s rank correlation test: Siegel, 1956) with the nitrogen and protein content (I, = - 0.89, CI= 0.05) and C : N ratio (rs = 0.97, a = 0.0 1) of the foods. No relation was found, however, with the carbon, caloric, and available caloric content. After 9 days of culture (started with newly hatched nauplii), the mean total number of individuals that had survived on the different diets, ranged from 60 k 12 (G-10) to
Development
OL-
0
1
1
G-10
T~rne
s-13
PAB
G-20
OIA
CAP
G-13A
Fig. 1. Effect of the different diets on development time, i.e. time from hatching to reproductive maturity, and survival of Tisbe cucumarfue: survival was measured as the concentration of individuals 9 days alter the beginning of the experiments; values are means (with SE) of three different experiments started with newly hatched nauplii; G-10, low nitrogen Gracilariu tikvahiae; S-13, Spa&a altemifora; PAB, Gerber’s mixed cereal; G-20, high nitrogen Gracilatia tikvahiae; DIA, Navicula sp.; CAP, Capitella cupitata meat; G-13A, algal Aufwuchs.
L. D. GUIDI
106
237 f 36 (G-13A) *ml- I. A highly significant (a = 0.01) correlation was found between survival and the nitrogen, protein content (I, = 0.93), and C : N ratio (rs = - 0.94) of the foods. Survival was not related, however, to the amount of carbon, caloric, and available caloric content in the foods. On the ninth day, the populations offered G-10 and S-13 were largely dominated by medium-small (0.6 mm d MS < 0.8 mm) indi-
SIZE
CLASSES
17 04mmQ
S <0.6mm F&I 0.6 mm S MS < 0.8 mm 0 0.8 mm < M < 1.0 mm 0 l.OmmQ L
25
10 0
~
~ c-10
_ s-13
PA6
~ G-20
~ DIA
CAP
G-13A
Fig. 2. Proportion of individuals of TiEbe cucumariae found in each of the five size classes aher nine days ofculture on the different diets: proportion of ovigerous females is represented by a black area in the largest size class (L) that includes females only; values are means (with SE) of three different experiments started with newly hatched nauplii; G-10, low nitrogen Graciiuria tikvahiae; S-13, Spar&a a~terniflra; PAB, Gerber’s mixed cereaI; G-20, high nitrogen GraciZa~a ~~a~~ae; DIA, Navicda sp.; CAP, Capitella capitata meat; G-13A, algal Aufwuchs.
viduals, which made up to 78 and 70% of the total number, respectively (Fig. 2). On all the other diets, the three size classes MS, M, and L were more or less equally represented. Populations cultured on CAP and G-13A had the highest proportion of ovigerous females (13.6 and 8.9% of total number, respectively). Nauplii (N < 0.4 mm) were present in the dishes receiving G-10, CAP, and G-13A.
DISCUSSION
Although some harpacticoid copepods are known to be highly selective for certain foods (e.g. bacteria: Vanden Berghe 8~ Bergmans, 1981), in general if substances
FEEDING OF TISBE
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remotely edible are presented to these animals, they invariably make attempts at ingestion (see Hicks & Coull, 1983). It is, therefore, not surprising that the non-ovigerous adult females of T. cucumariae ingested ail the foods tested in the present study. The rates at which the foods were consumed are equivalent to those reported by Rieper (1978) for T. holothuriae and Paramphiascella vararesis, i.e. 8.6 to 29.5 x lo-’ pg bacteria . ind - l . h - l. All foods, except CAP, were ingested at approximately the same rate. The faster consumption of CAP could have resulted from the higher digestibility of a diet of entire heterotrophic origin (flesh and presumably bacterial associates). Although some of the other diets, and particularly G-13A, included heterotrophic components, they all contained or originated from autotrophic organisms. As noted by Lampert (1977) and Tietjen & Lee (1977), the rate at which a food source is consumed does not necessarily reflect its nutritional value. For example, Gyllenberg & Lundqvist (1978) found that although the copepod Cyclops oithonoides ingested fresh bacterial cells (evidence from radioisotope experiments), it was not able to survive on such a diet. The nutritional quality of the seven foods ingested by Tisbe cucumariae was assessed according to the development time and survival of the copepods. Both these variables were affected by the type of food offered, and more precisely by the nitrogen (and protein) level in the foods. The importance of the regulatory r61e of nitrogen has been seen especially in detrital food chains. For example, both Tenore (1977,198 1) and Findlay (1982) found that the best index of nutritional value (measured as growth) was the amount of nitrogen in the detrital source supplied to a polychaete and a nematode. While other food characteristics, such as total caloric content (Sellner, 1976; Tenore, 1977) or available caloric content (Tenore, 198 1) have been related to nutritional quality, such a relationship could not be established in the present study. Available caloric content is, however, usually high in algal materials, and low in detritus derived from marine vascular plants (Tenore et al., 1982). Thus, available energy is typically potentially limiting in diets not greatly represented in the present investigation. The relationship between development time or survival of T. cucumariae and food quality, was not linear (particularly visible on the development time curve, Fig. 1). There seemed to be some threshold level of nitrogen in the food beyond which development time remained constant. Although a large number of ovigerous females were present after 9 days of culture on PAB (Fig. 2), according to development time values the best foods were DIA, CAP, and G-13A. These three foods were apparently of equivalent nutritional value, although the populations receiving CAP and G-13A did produce more ovigerous females (nauplii, probably of the second generation, were also present) than did that receiving DIA. The presence of heterotrophic components in both CAP and G-13A could be responsible for these minute differences. Experiments carried out over longer periods might have brought out greater discrepancies between the three diets since certain foods may become inadequate only after several generations (Shiraishi & Provasoli, 1959). G-13A, which was composed of algal Aufwuchs, contained blue-green algae, bacteria, diatoms, fungi, and mucilage, making it the only ‘mixed’ food offered to T. cucumariae in the experiments. It is likely that such a diet would give the best results
108
L. D. GUIDI
in multi-generation cultures, since it is now well established that the reproductive performance and health of copepod populations under culture are favoured by mixed foods (see Hicks & Coull, 1983), reflecting the need for a balanced diet. In the Cupitelh capitutu tanks, the copepods probably feed on a mixture of both PAB (used to maintain the cultures) and dead worms (over the course of seven years, dead polychaetes have very seldom been found in the cultures: Tenore, pers. comm.). Other populations of Tisbe cucumariae have been found to feed on the dead bodies of the organisms with which they are associated, e.g. the ttmicate Ciona intestinalis (Lopez, 1982). At the other end of the nitrogen scale, G-10 and S-13, which had a very similar chemical composition, did not produce the same effects on Tisbe cucumariae. G-10 may have been lacking some essential elements (minerals, vitamins) that limited growth (nauplii were still present in the cultures after 9 days) and development of the copepods. Lopez (1982) noted a slowing in nauplius development of T. cucumariue cultures receiving inadequate food. A number of authors have stressed the importance of trace nutrients in the food as a factor affecting nutritional value, e.g. Shiraishi & Provasoli (1959) showed that a supplement of vitamins to Tigriopus cultures corrected the nutritional inadequacy of certain algal foods. Animals react to changes in food value and supply at the ingestion and/or assimilation level. Indeed, some animals will increase ingestion rate of foods of low nutritional quality and thus grow at the same rate as when fed highly nutritional material, e.g. Nereis virens cultured on clam tissue and faeces (Tenore & Gopalan, 1974). Unlike this polychaete, Tisbe cucumariae, however, did not vary ingestion rate in response to food quality as a behaviour in trophic dynamics. In conclusion, the present study has shown that food composition, which had little, if any influence on ingestion rate, significantly affected development and survival of T. cucumariue. Nitrogen (protein) level in the food seems to play a dominant regulating role in the secondary production of the copepods.
ACKNOWLEDGEMENTS
I gratefully acknowledge Dr. K. R. Tenore for welcoming me in his laboratory, and supervising my research. His advice and comments on the manuscript were very helpful. I thank Dr. N. W. Phillips for assistance with chemical analyses. Financial support was from the French “Minis&e des Relations Exttrieures”, and from the Oceanography Section of the National Science Foundation, Grant No. OCE 82 - 00385 to Dr. K. R. Tenore. This work was presented at the Fifth International Meiofauna Symposium, 16-20 August 1983, Belgium, but no proceedings of the meeting are to be published.
FEEDING OF TISBE
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TENORE,K. R., R. B. HANSON, B. E. DORNSEIF& C. N. WIEDERHOLD,1979. The effect of organic nitrogen supplement on the utilization of different sources of detritus. Limnol. Oceanogr., Vol. 34, pp. 350-355. TIETJEN,J. H. & J. J. LEE, 1977. Feeding behavior of marine nematodes. In, Ecology ofmarine benrhos, edited by B.C. Coull, University of South Carolina Press, Columbia, U.S.A., pp. 21-35. USTACH, J.F., 1982. Algae, bacteria and detritus as food for the harpacticoid copepod, Heteropsyllus pseudonunni Coull and Palmer. J. Exp. Mar. Biol. Ecol., Vol. 64, pp. 203-214. VANDENBERGHE,W. & M. BERGMANS,1981. Differential food preference in three co-occuring species of Tisbe (Copepoda: Harpacticoida). Mar. Ecol. Prog. Ser., Vol. 4, pp. 213-219.