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Test of sequential feeding regimes for larvae of Elminius modestus Darwin (Cirripedia: Balanomorpha)
J. Exp. Mar. Biol. Ecol., 1988, Vol. 115, pp. 41-51
41
Elsevier
JEM 01003
Test of sequential feeding regimes for larvae of EZminius modestus Darwin (Cirripedia : Balanomorpha) Carolyn J. Stone Marine Research Group, School of Biological Sciences, University College of Swansea, Swansea, West Glamorgan, U.K.
(Received 12 May 1987; revision received 28 September 1987; accepted 12 October 1987) Abstract: The larvae of the intertidal barnacle Elminiuf modest Darwin were reared using six different feeding regimes comprising a large and/or a small flagellate fed to the larvae at different times during the course of their development. The relative value of the feeding regimes was ascertained by using the rate of development, survival, and size attained by the larvae as a measure of success or failure. The best feeding regime was found to be that providing a unialgal diet of the small flagellate initially followed by a unialgal diet of the large flagellate in later stages of development, while the reverse situation gave the worst results. The increase in mesh size of the antennal Nter during growth of the nauplii is suggested as a reason for the varying success of feeding regimes emplo~ng foods of different sizes during development. This is seen as confirmation of the idea that cirriped nauplii collect their food by means of filtering appendages rather than by localized currents alone. Key words: Antenna; Cirriped; Feeding; Filter; Nauplius
The variation in the mesh size of the antennal filter has been suggested as a reason for the differing food requirements of cirriped larvae (Moyse, 1963,1964; Stone, 1986). However, the increase in size of the larvae with each successive moult may result in a slight increase in the mesh size in the later stages of development (Stone, 1986) and it is possible that the size of particle filtered may vary not only between the species but also within one species at different stages of development. Lewis (1975) suggested that an optimum diet for the larvae of the lepadomorph Pollicipespolymeras Sowerby might be a small flagellate initially with the addition of a large dinoflagellate in the later stages of development. Lewis did not test this feeding schedule, but it is possible to apply the same principle to other cirripede species. Elminius modestus Darwin is a suitable species to use not only because of its wide availability, but also because it grows well on both smaller and larger algae (Moyse, 1963; Stone, 1986), thus enabling comparisons of different sequences of feeding to be made.
Correspondence address: C. J. Stone, Marine Research Group, School of Biological Sciences, University College of Swansea, Singleton Park, Swansea, West Glamorgan SA2 8PP, U.K. 0022-0981/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
42
C.J. STONE
MATERIALS AND METHODS Elminius modestus adults were collected from Mumbles Pier, Swansea, U.K., and the larvae were hatched in the laboratory. For each feeding regime replicate culture vessels (A and B) were set up, each containing a total of 200 ml of filtered sea water (0.45~pm Whatman glass libre filter paper) and algae, and 100 newly hatched nauplii. Two species of algae were employed - the small (5-6-pm diameter) flagellate Isochrysti gal&a Parke (z 10 x lo5 cells - ml- ’ when used alone or NN 5 x IO5cells. ml ~ ’ when used in a mixture) and the large (17 x 6-pm) flagellate ~~~d~rn~nu~ sp. (w 12 x lo4 cells * ml - ’ when used alone or z 6 x lo4 cells 1ml - ’ when used in a mixture). Both algal species were cultured using Conway medium (Walne, 1970a) to eliminate possible variation in the chemical composition of the algae due to using different culture media (Parsons et al., 196 1; Cowey & Corner, 1963). In order to make the total volume of food equivalent in all the diets employed, 10 ml of each algal culture were centrifuged at 3000 rpm for 15 min and the resulting packed cell volume was measured. Cell counts of the cultures were made using a haemocytometer and the concentrations needed to produce equivalent packed cell volumes were calculated. The amount of algal culture added to each larval culture could then be adjusted so that the total volume of algae would be equal in all the cultures. Antibiotics (penicillin-streptomycin solution from Gibco) were added to the rearing vessels at a concentration of 50 U penicillin and 0.05 mg streptomycin sulphate per ml. Black tape was attached to the outside of the rearing vessels at the water level to prevent the positively phototactic nauplii concentrating at the meniscus with the inherent risk of being stranded on the sides of the vessel (Tighe-Ford et al., 1970). The temperature was maintained at 18 + 0.2 “C and the salinity remained constant at 35%,. Light was supplied by 40 W fluorescent tubes on a 16-h on/&h off cycle. For each replicate culture vessel the sea water and algae were changed on the 5th day of the experiment. The feeding regimes were as follows:
Algal food
Abbreviation used in figures
-_ Days O-4
Days 5-9 -
.I R 14-R I/I + R w WI
fsoc~s~ Rhodomonas Isochrysis + Rhodomonas Isochrysis Isochtysis Rhodomonas
Isochvsk Rhodomonas Isochrysis + Rhodomonas Isochrysis + Rhodomonas Rhodomonas Isochrysis
After 9 days, the surviving larvae were filtered off using a nylon mesh and were counted, staged, and measured.
SEQUENTIAL FEEDING REGIMES FOR LARVAE OF ~~I~IN~US
~~~ES~US
43
RESULTS RATE OF DEVELOPMENT
with replicates showed that the stage of development reached after 9 days differed significantly with the different feeding regimes employed, with F = 2277.94 (df = 5,6). Development was fastest on an initial diet of unialgal Isochrysis followed after Day 4 by unialgal Rhodomonas (Tables I, II). The next fastest rates of development were obtained in feeding regimes where both Isochrysis and Rhodomonas were available in the latter half of the experiment, with either unialgaI Isochryis or Isoch~s~ mixed with Rhodom#~ in the fust half of the experiment. Unialgal Isoch~sis ~rou~out the experiment gave a slightly slower rate of development, but unialgal Rhodomonas throughout gave a much reduced rate. A feeding regime of unialgal Rhodomonas initially followed by unialgal Isochrysis later gave the slowest rate of development. A l-way
ANOVA
TABLE I Elminius modestus larvae reared for 9 days under six different feeding regimes.
_ Feeding regime
Stage of development I
II
III
IV
V
VI
-.-Zsochrysis
1
A B
3
4 4
20 16
43 49
Rhodomonas
A B
17 15
5 3
30 34
11 5
2 2
Isochrysis + Rhodomonas
A B
7 11
68 60
2 4
Zsochrysis/Rhodomonas
A B
3
57
12
5
53
16
Zsochrys~lisoch~s~ + Rhodomonas
A B
15 17
37
13
43
11
~odomonaslZs~~~s~
A B
11 13
14 18
13 19
14 10
SURVIVAL
There were also significant differences in survival under the six different feeding regimes, with F = 8.15 (df = 5~3).The highest survival was obtained on a mixed diet of Isochrysis with Rhodomonas throughout the experiment (Table II). Isochrysis followed by Rhodomonas gave better survival than unialgal Isochrysis throughout the experiment. Lowest survival was associated with a diet of unialgal Rhodomo~as followed by
44
C.J. STONE TABLEII
Values for survival and mean stage of development (It SD) of Elminius modes&s larvae reared for 9 days under six different feeding regimes. Feeding regime
Survival
Mean stage of development --
Isochrysb
A B
0.68 0.72
4.54 ( f 0.68) 4.54 ( f 0.79)
Rhodomonas
A B
0.65 0.59
3.63 (f 1.14) 3.59 (+ 1.07)
Isochrvsif + ~adomo~us
A B
0.71 0.75
4.94 (+ 0.34) 4.91 ( * 0.44)
~s~h~s~~~odarnonas
A B
0.72 0.74
5.13(iO.44) 5.15 (k 0.52)
Isochrysis/Isochrysis + Rhodomonas
A B
0.65 0.71
4.97 ( f 0.66) 4.92 ( f 0.63)
Rhodomonas/Isochrysis
A B
0.52 0.60
3.50 (f 1.15) 3.50 (k 1.23)
Isoch~~is in the latter half of the experiment. UnialgaI Rhodomonas throughout the experiment also gave lower survival compared with diets where Isochrysis was available initially. SIZE OF NAUPLII
Differences were apparent in the carapace length and width of stage IV and stage V nauplii fed on different diets (F = 38.9 for length, F = 11.47 for width of stage IV larvae; F = 87.46 for length, F = 7.74 for width of stage V larvae; df = 5,6 in all). The feeding regimes where only Isochrysis was available in the latter half of the experiment (R/I or I) gave noticeably shorter and narrower stage IV and V nauphi (Fig. l), although the ratio of carapace length : width did not differ significantly between the diets (F = 0.43 for stage IV, F = 0.20 for stage V, both with df = 56). The dimensions of the stage VI nauphi did not differ si~~c~tly (F = 2.61 for length, F = 2.79 for width, I: = 0.03 for the ratio of carapace length : width, df = 3,4 in all), but only those larvae fed on a regime containing Rhodomonas in the latter half of the experiment reached this stage of development.
DISCUSSION
Both Isochrysis galbana and Rhodomonas sp. are suitable foods for Elminius modestus larvae - development will proceed on a unialgal diet of either of these algae. Previous experiments (Moyse, 1963, 1964; Stone, 1986) have indicated that the celI size of the algal food is important, and the aim of this study was to provide conflation of this
SEQUENTIAL FEEDING REGIMES FOR LARVAE OF EL~~I~I~S
320
MODESTUS
4001 380-
460-
360.
450-1
340
440-
1
-
_ I:
270-
340
260-
330
250
320
240-
310
1.607
1.60-
1.50-
l-50-
I 1.40
1.40-
1.30
1.30-
l-l a
c
a
I
cl
rt
Stage IV Fig. 1. Mean size attained ( f
Stage V SD)
by El~i~i~
Stage VI
modestus larvae reared under different feeding regimes.
45
C.J. STONE
46
by looking for subtle differences obtained using different size classes of food in varying sequences. For this purpose, it is necessary to use foods that are known to support development of cirripede larvae but differ in cell size. ~~~c~~s~ g&ma (5-6 pm) and Rhodomonas sp. (17 x 6 pm) are two such foods. Both species were used while cultures were in the exponential phase of growth as the chemical composition of algae varies with the age of the culture (Antia et al., 1963 ; Pugh, 197 1; Myklestad, 1974). The differences observed when unialgal Isochrysis was available in the first half of the experiment and unialgal Rhodomo~~ in the latter half compared with unialgal ~hodomon~ followed by unialgal fsockrysi~ suggest that biochemical diierences between the algal species could not on their own account for the results of this experiment. On these two feeding regimes the nutritional components available to the larvae over the nine days were the same, but the differences lay in whether the larvae were fed the smaller cells first followed by the huger cells or whe~er the larger cells were used before the smaher cells. This wound seem to suggest that size is an important factor in this experiment. On a unialgal diet of the small flagellate Isochtysis, development proceeded steadily
I
I
It
60-
60-
40-
40.
20-
20-
L-
i
R/l
Ifl+R
60-
Stage
of
development
of
naupiii
modestw larvae after 9 days on six different feeding Fig. 2. Abundance of developmental stages of E~rninitcs regimes.
SEQUENTIAL FEEDING REGIMES FOR LARVAE OF ELI~~~~US
~~~E~TU~
47
as far as stage V during the 9 days of the experiment. However, on a unialgal diet of the large flagellate Rhodomonas the mean stage of development after 9 days was less than with lsochrysis, although a few stage VI nauplii were obtained with Rhodomonas. There were two peaks of abundance of developmental stages found on a diet of Rhodomonas, at stage II and stage IV, compared with one peak at stage V for Zsochrysis (Fig. 2). It seems likely that the earlier stages have difliculty in capturing the large cells of Rhodomonas, hence many larvae do not develop beyond stage II. The encounter rate with the larger cells is probably lower due to the lower concentration used, but the larger size of the cells should balance the lower encounter rate so that the overall gain is appro~ately equal with either alga. Those larvae that do progress beyond stage II on a diet of Rhodomo~~ are easily able to capture the large cells in the later stages and so development will proceed well on this food once the earlier stages of development are passed. The difficulty of capturing the larger cells in the earlier stages probably also accounts for the lower survival on Rhodomonas (62%) than on Zsochrysis (70%). A mixed diet of these two algae throughout the experiment provided the benefits of both size classes as well as the nutritional benefits of a mixed diet - several workers have found that there is a synergistic effect of mixed diets fed to the larvae of marine invertebrates (Davis & Guillard, 1958; Bayne, 1965; Nassogne, 1970; Walne, 1970b; Gruffydd & Beaumont, 1972; Lewis, 1975; Helm, 1977; Lang, 1977). On the mixed diet the larvae developed well, with all the larvae being at stage IV or later stages of development after 9 days. This was also the case where the mixed diet was available in the latter half of the experiment but only Zsochrysis was available initially - indeed on this feeding regime more stage VI nauplii were obtained than on a mixed diet throughout the experiment (an average of 12 as opposed to 3). It seems therefore that the addition of Rhodomonas in the earlier stages hindered development, possibly due to the larvae wasting energy in trying to capture the large cells, while at the same time the smaller ZsochrysD cells are available in lower concentrations, hence lowering the encounter rate. The fastest rate of development was obtained when Zsochrysis was available initially and Rhodomonas later (mean stage of development 5.14 after 9 days). This feeding regime not only gave faster development but also higher survival (73%) than Zsochrysis initially followed by the mixed diet later (68%) - the addition of Zs~h~sis in later stages may hinder development slightly due to the later stages being less easily able to filter the small cells. Moyse (1964) has emphasised the importance of the antennal endopodite in the feeding of cirripede larvae. In Elminius modestus setules spaced at < 5 pm apart cover 36.39% of the total area covered by the setae of the endopodite in the stage II nauplius, whereas in the stage VI nauplius the area covered by these closely spaced setules is only 13.92% (Fig, 3). Although there is a fringe of closely spaced hairs along the pre-axial edge of the exopodite in the stage VI nauplius, it appears that such fringes are unimportant in the feeding of cirripede nauplii (Stone, 1986). Several workers have suggested that in copepods the limbs act more like paddles than sieves due to their small size and the relative viscosity of the water (Koehl & Strickler,
C.J. STONE
48 a
/
endopodite
c
I
100pm
b
endopodi te
Fig. 3. Set&ion and setulation of the antenna of Elmin& modestus nauplii. a, stage II; b, stage VI. Scale bar = 100 cm, intersetole distances in pm.
SEQUENTIAL
FEEDING
REGIMES
FOR LARVAE
OF ELIMINIUS
MODESTUS
49
1981; Palfenhoffer et al., 1982). Schram (1986) believes that this may also apply to cirripede nauplii. However, Patfenhofer et al. (1982) believed that this only applied to larger particles and that a switch to a mode of feeding more closely resembling passive filtering occurred with small particles of 5 pm diameter and smaller. The secretion of labral or cephalic glands in cirripede larvae (Walker, 1973), rather than being sticky, may act as a “detergent” or decrease viscosity to improve filtration on the appendages. Filtration may also occur due to the compression of water confined between the two beating antennae or between the antennae and the body mass. The feeding regime providing the larger cells of Rhodomonus initially and then the smaller cells of Zsochrysislater proved to be the worst of the six feeding regimes tested, both in terms of rate of development and survival. Lewis (1975) postulated that an optimum feeding schedule for Pollicipes polyments larvae might be a small flagellate initially with the addition of a large dinoflagellate in the later stages. This experiment shows that smaller cells initially followed by larger cells later is a successful feeding schedule for Elminius modestus larvae and the reverse situation of Rhodomonas followed by Zsochrysis gives the poorer results that would be expected. The larger Rhodomonas may be difficult to capture in the earlier stages of development while the smaller Zsochrysis may be less easily filtered than a large cell would be in the later stages of development, hence this feeding regime combines the disadvantages of both size classes of food to give poorer results than obtained with either species used unialgally throughout the experiment. With regards to the size obtained by the nauplii under different feeding regimes, the apparent trend is that larger nauplii are obtained, both in terms of carapace length and carapace width, where the large celled Rhodomonas is available in the later stages, whether unialgally or in a mixture. It seems, therefore, that large cells in the later stages of development lead not only to a faster rate of development and better survival, but also to a large size being reached by the larvae. The size of the food cell appears here to be an important factor contributing to the success or failure of a particular feeding regime. Whether the same principle would be seen in the natural environment is a matter for speculation, as the densities used here were far higher than would be found in the sea. However, the apparent importance observed here of the cell size of the food relative to the size of the larvae sheds some light on the mechanism of food collection by cirripede nauplii. The fact that larger cells are less useful in the earlier stages of development is probably a reflection of the difficulty of capture. But the apparent advantage of larger cells over smaller ones in later stages is not so easily explained. If localized currents alone are responsible for the collection of food particles, as suggested by Lochhead (1936) and favoured by Rainbow & Walker (1976) then smaller cells would presumably be as beneficial as larger cells - however, it appears that the larger cells are better, which would seem to favour the idea of smaller cells escaping from a filter. Norris & Crisp (1953) were the first workers to suggest such a filter, while Moyse (1963, 1964) expanded this idea to show the importance of the endopodite setae of the antenna in food collection. The experiment presented here
50
C.J. STONE
shows that there is mechanistic selection of larger sized particles as the larvae become larger, which supports the idea of feeding using filtering appendages.
ACKNOWLEDGEMENTS
I would like to thank Dr. John Moyse for his interest and discussion of this work, and Professor J. S. Ryland for providing facilities in the School of Biological Sciences, University College of Swansea.
REFERENCES ANTIA, N. J., C. D. MCALLISTER,T.R. PARSONS,K. STEPHENS& J. D.H. STRICKLAND,1963. Further measurements of primary production using a large volume plastic sphere. Limnol. Oceanogr., Vol. 8, pp. 166-183. BAYNE,B. L., 1965. Growth and the delay of metamorphosis of the larvae of Mytil~ edulis (L.). Ophelia, Vol. 2, pp. l-47. COWEY,C. B. & E. D S. CORNER,1963. On the nutrition and metabolism ofzooplankton. II. The relationship between the marine copepod Calanus helgolandicur and particulate material in Plymouth sea water, in terms of amino acid composition. J. Mar. Eiol. Assoc. U.K., Vol. 43, pp. 495-511. DAVIS, H. C. & R. R. GUILLARD,1958. Relative values of ten genera of micro-organisms as foods for oyster and clam larvae. U.S. Fish Wildl. Serv. Fish. Bull., Vol. 58, pp. 293-304. GRUFFYDD,L. D. & A. R. BEAUMONT,1972. A method for rearing Pecten maximus larvae in the laboratory. Mar. Biol., Vol. 15, pp. 350-355. HELM, M.M., 1977. Mixed algal feeding of Osfrea edulis larvae with Isochrysk galbana and Tetraselmis suecica. J. Mar. Biol. Assoc. U.K., Vol. 57, pp. 1019-1029. KOEHL, M. A.R. & J. R. STRICKLER,1981. Copepod feeding currents: food capture at low Reynolds numbers. Limnol. Oceanogr., Vol. 26, pp. 1062-1073. LANG,JR., W. H., 1977. The barnacle larvae of North Inlet, South Carolina (Cirripedia : Thoracica). Ph.D. thesis, University of South Carolina, Columbia, South Carolina, 179 pp. LEWIS, C.A., 1975. Some observations on factors affecting embryonic and larval growth of Pollicipes polymerus (Cirripedia : Lepadomorpha) in vitro. Mar. Biol., Vol. 32, pp. 127-139. LOCHHEAD,J. H., 1936. On the feeding mechanism of the nauplius of Balanus perfratus Bruguihe. Proc. Linn. Sot. London, Vol. 39, pp. 429-442. MOYSE,J., 1963. A comparison of the value of various flagellates and diatoms as food for barnacle larvae. J. Cons. Cons. Int. Explor. Mer., Vol. 28, pp. 175-187. MOYSE,J., 1964. Feeding in barnacle nauplii. Rep. Challenger Sot., Vol. 3, No. 16. MYKLESTAD,S., 1974. Production of carbohydrates by marine planktonic diatoms. I. Comparison of nine different species in culture. J. Exp. Mar. Biol. Ecol., Vol. 15, pp. 261-274. NASSOGNE,A., 1970. Influence of food organisms on the development and culture of pelagic copepods. Helgol. Wiss. Meeresunters., Vol. 20, pp. 333-345. NORRIS, E. & D.J. CRISP, 1953. The distribution and planktonic stages of the cirripede Balanusperjivatus BruguiBre. Proc. 2001. Sot. London, Vol. 123, pp. 393-409. PAFFENH~FER,G.-A., J. R. STRICKLER& M. ALCARAZ,1982. Suspension-feeding by herbivorous calanoid copepods: a cinematographic study. Mar. Eiol., Vol. 67, pp. 193-199. PARSONS,T. R., K. STEPHENS& J. D. H. STRICKLAND,1961. On the chemical composition of eleven species of marine phytoplankters. J. F&h. Res. Board Can., Vol. 18, pp. 1001-1016. PUGH, P.R., 1971. Changes in the fatty acid composition of Coscinodiscus eccenbicus with culture age and salinity. Mar. Biol., Vol. 11, pp. 118-124. RAINBOW,P. S. & G. WALKER, 1976. The feeding apparatus of the barnacle nauplius larva: a scanning electron microscope study. J. Mar. Biol. Assoc. U.K., Vol. 56, pp. 321-326. SCHRAM,F.R., 1986. Crustacea. Oxford University Press, London, U.K., 606 pp.
SEQUENTIAL FEEDING REGIMES FOR LARVAE OF ELIMZNIUS MODESTUS
51
STONE, C. J., 1986. The effects ofvariations in diet, temperature and salinity on the development ofcirripede
nauplii. Ph.D. thesis, University of Wales, U.K., 340 pp. TIGHE-FORD, D. J., M. J. D. POWER & D.C. VAILE, 1970. Laboratory rearing of barnacle larvae for antifouling research. Helgol. Wiss. Meeresunfers., Vol. 20, pp. 393-405. WALKER,G., 1973. Frontal horns and associated gland cells of the nauplii of the barnacles, Balanus humeri, Balanus balanoides, and Elminius modestus (Crustacea : Cirripedia). J. Mar. Biol. Assoc. U.K., Vol. 53, pp. 455-463. WALNE,P.R., 1970a. Studies on the food value of nineteen genera of algae to juvenile bivalves of the genera Ostrea, Crassostrea, Mercenaria and Mytilus. Fish. Invest. Mintit. Agrik. Fish. Food (G.B.) Ser. II Salmon Freshwater Fish., Vol. 26, No. 5. WALNE, P.R., 1970b. Present problems in the culture of the larvae of Ostrea eduks. Helgol. Wiss. Meeresunfers., Vol. 20, pp. 514-525.
41
Elsevier
JEM 01003
Test of sequential feeding regimes for larvae of EZminius modestus Darwin (Cirripedia : Balanomorpha) Carolyn J. Stone Marine Research Group, School of Biological Sciences, University College of Swansea, Swansea, West Glamorgan, U.K.
(Received 12 May 1987; revision received 28 September 1987; accepted 12 October 1987) Abstract: The larvae of the intertidal barnacle Elminiuf modest Darwin were reared using six different feeding regimes comprising a large and/or a small flagellate fed to the larvae at different times during the course of their development. The relative value of the feeding regimes was ascertained by using the rate of development, survival, and size attained by the larvae as a measure of success or failure. The best feeding regime was found to be that providing a unialgal diet of the small flagellate initially followed by a unialgal diet of the large flagellate in later stages of development, while the reverse situation gave the worst results. The increase in mesh size of the antennal Nter during growth of the nauplii is suggested as a reason for the varying success of feeding regimes emplo~ng foods of different sizes during development. This is seen as confirmation of the idea that cirriped nauplii collect their food by means of filtering appendages rather than by localized currents alone. Key words: Antenna; Cirriped; Feeding; Filter; Nauplius
The variation in the mesh size of the antennal filter has been suggested as a reason for the differing food requirements of cirriped larvae (Moyse, 1963,1964; Stone, 1986). However, the increase in size of the larvae with each successive moult may result in a slight increase in the mesh size in the later stages of development (Stone, 1986) and it is possible that the size of particle filtered may vary not only between the species but also within one species at different stages of development. Lewis (1975) suggested that an optimum diet for the larvae of the lepadomorph Pollicipespolymeras Sowerby might be a small flagellate initially with the addition of a large dinoflagellate in the later stages of development. Lewis did not test this feeding schedule, but it is possible to apply the same principle to other cirripede species. Elminius modestus Darwin is a suitable species to use not only because of its wide availability, but also because it grows well on both smaller and larger algae (Moyse, 1963; Stone, 1986), thus enabling comparisons of different sequences of feeding to be made.
Correspondence address: C. J. Stone, Marine Research Group, School of Biological Sciences, University College of Swansea, Singleton Park, Swansea, West Glamorgan SA2 8PP, U.K. 0022-0981/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
42
C.J. STONE
MATERIALS AND METHODS Elminius modestus adults were collected from Mumbles Pier, Swansea, U.K., and the larvae were hatched in the laboratory. For each feeding regime replicate culture vessels (A and B) were set up, each containing a total of 200 ml of filtered sea water (0.45~pm Whatman glass libre filter paper) and algae, and 100 newly hatched nauplii. Two species of algae were employed - the small (5-6-pm diameter) flagellate Isochrysti gal&a Parke (z 10 x lo5 cells - ml- ’ when used alone or NN 5 x IO5cells. ml ~ ’ when used in a mixture) and the large (17 x 6-pm) flagellate ~~~d~rn~nu~ sp. (w 12 x lo4 cells * ml - ’ when used alone or z 6 x lo4 cells 1ml - ’ when used in a mixture). Both algal species were cultured using Conway medium (Walne, 1970a) to eliminate possible variation in the chemical composition of the algae due to using different culture media (Parsons et al., 196 1; Cowey & Corner, 1963). In order to make the total volume of food equivalent in all the diets employed, 10 ml of each algal culture were centrifuged at 3000 rpm for 15 min and the resulting packed cell volume was measured. Cell counts of the cultures were made using a haemocytometer and the concentrations needed to produce equivalent packed cell volumes were calculated. The amount of algal culture added to each larval culture could then be adjusted so that the total volume of algae would be equal in all the cultures. Antibiotics (penicillin-streptomycin solution from Gibco) were added to the rearing vessels at a concentration of 50 U penicillin and 0.05 mg streptomycin sulphate per ml. Black tape was attached to the outside of the rearing vessels at the water level to prevent the positively phototactic nauplii concentrating at the meniscus with the inherent risk of being stranded on the sides of the vessel (Tighe-Ford et al., 1970). The temperature was maintained at 18 + 0.2 “C and the salinity remained constant at 35%,. Light was supplied by 40 W fluorescent tubes on a 16-h on/&h off cycle. For each replicate culture vessel the sea water and algae were changed on the 5th day of the experiment. The feeding regimes were as follows:
Algal food
Abbreviation used in figures
-_ Days O-4
Days 5-9 -
.I R 14-R I/I + R w WI
fsoc~s~ Rhodomonas Isochrysis + Rhodomonas Isochrysis Isochtysis Rhodomonas
Isochvsk Rhodomonas Isochrysis + Rhodomonas Isochrysis + Rhodomonas Rhodomonas Isochrysis
After 9 days, the surviving larvae were filtered off using a nylon mesh and were counted, staged, and measured.
SEQUENTIAL FEEDING REGIMES FOR LARVAE OF ~~I~IN~US
~~~ES~US
43
RESULTS RATE OF DEVELOPMENT
with replicates showed that the stage of development reached after 9 days differed significantly with the different feeding regimes employed, with F = 2277.94 (df = 5,6). Development was fastest on an initial diet of unialgal Isochrysis followed after Day 4 by unialgal Rhodomonas (Tables I, II). The next fastest rates of development were obtained in feeding regimes where both Isochrysis and Rhodomonas were available in the latter half of the experiment, with either unialgaI Isochryis or Isoch~s~ mixed with Rhodom#~ in the fust half of the experiment. Unialgal Isoch~sis ~rou~out the experiment gave a slightly slower rate of development, but unialgal Rhodomonas throughout gave a much reduced rate. A feeding regime of unialgal Rhodomonas initially followed by unialgal Isochrysis later gave the slowest rate of development. A l-way
ANOVA
TABLE I Elminius modestus larvae reared for 9 days under six different feeding regimes.
_ Feeding regime
Stage of development I
II
III
IV
V
VI
-.-Zsochrysis
1
A B
3
4 4
20 16
43 49
Rhodomonas
A B
17 15
5 3
30 34
11 5
2 2
Isochrysis + Rhodomonas
A B
7 11
68 60
2 4
Zsochrysis/Rhodomonas
A B
3
57
12
5
53
16
Zsochrys~lisoch~s~ + Rhodomonas
A B
15 17
37
13
43
11
~odomonaslZs~~~s~
A B
11 13
14 18
13 19
14 10
SURVIVAL
There were also significant differences in survival under the six different feeding regimes, with F = 8.15 (df = 5~3).The highest survival was obtained on a mixed diet of Isochrysis with Rhodomonas throughout the experiment (Table II). Isochrysis followed by Rhodomonas gave better survival than unialgal Isochrysis throughout the experiment. Lowest survival was associated with a diet of unialgal Rhodomo~as followed by
44
C.J. STONE TABLEII
Values for survival and mean stage of development (It SD) of Elminius modes&s larvae reared for 9 days under six different feeding regimes. Feeding regime
Survival
Mean stage of development --
Isochrysb
A B
0.68 0.72
4.54 ( f 0.68) 4.54 ( f 0.79)
Rhodomonas
A B
0.65 0.59
3.63 (f 1.14) 3.59 (+ 1.07)
Isochrvsif + ~adomo~us
A B
0.71 0.75
4.94 (+ 0.34) 4.91 ( * 0.44)
~s~h~s~~~odarnonas
A B
0.72 0.74
5.13(iO.44) 5.15 (k 0.52)
Isochrysis/Isochrysis + Rhodomonas
A B
0.65 0.71
4.97 ( f 0.66) 4.92 ( f 0.63)
Rhodomonas/Isochrysis
A B
0.52 0.60
3.50 (f 1.15) 3.50 (k 1.23)
Isoch~~is in the latter half of the experiment. UnialgaI Rhodomonas throughout the experiment also gave lower survival compared with diets where Isochrysis was available initially. SIZE OF NAUPLII
Differences were apparent in the carapace length and width of stage IV and stage V nauplii fed on different diets (F = 38.9 for length, F = 11.47 for width of stage IV larvae; F = 87.46 for length, F = 7.74 for width of stage V larvae; df = 5,6 in all). The feeding regimes where only Isochrysis was available in the latter half of the experiment (R/I or I) gave noticeably shorter and narrower stage IV and V nauphi (Fig. l), although the ratio of carapace length : width did not differ significantly between the diets (F = 0.43 for stage IV, F = 0.20 for stage V, both with df = 56). The dimensions of the stage VI nauphi did not differ si~~c~tly (F = 2.61 for length, F = 2.79 for width, I: = 0.03 for the ratio of carapace length : width, df = 3,4 in all), but only those larvae fed on a regime containing Rhodomonas in the latter half of the experiment reached this stage of development.
DISCUSSION
Both Isochrysis galbana and Rhodomonas sp. are suitable foods for Elminius modestus larvae - development will proceed on a unialgal diet of either of these algae. Previous experiments (Moyse, 1963, 1964; Stone, 1986) have indicated that the celI size of the algal food is important, and the aim of this study was to provide conflation of this
SEQUENTIAL FEEDING REGIMES FOR LARVAE OF EL~~I~I~S
320
MODESTUS
4001 380-
460-
360.
450-1
340
440-
1
-
_ I:
270-
340
260-
330
250
320
240-
310
1.607
1.60-
1.50-
l-50-
I 1.40
1.40-
1.30
1.30-
l-l a
c
a
I
cl
rt
Stage IV Fig. 1. Mean size attained ( f
Stage V SD)
by El~i~i~
Stage VI
modestus larvae reared under different feeding regimes.
45
C.J. STONE
46
by looking for subtle differences obtained using different size classes of food in varying sequences. For this purpose, it is necessary to use foods that are known to support development of cirripede larvae but differ in cell size. ~~~c~~s~ g&ma (5-6 pm) and Rhodomonas sp. (17 x 6 pm) are two such foods. Both species were used while cultures were in the exponential phase of growth as the chemical composition of algae varies with the age of the culture (Antia et al., 1963 ; Pugh, 197 1; Myklestad, 1974). The differences observed when unialgal Isochrysis was available in the first half of the experiment and unialgal Rhodomo~~ in the latter half compared with unialgal ~hodomon~ followed by unialgal fsockrysi~ suggest that biochemical diierences between the algal species could not on their own account for the results of this experiment. On these two feeding regimes the nutritional components available to the larvae over the nine days were the same, but the differences lay in whether the larvae were fed the smaller cells first followed by the huger cells or whe~er the larger cells were used before the smaher cells. This wound seem to suggest that size is an important factor in this experiment. On a unialgal diet of the small flagellate Isochtysis, development proceeded steadily
I
I
It
60-
60-
40-
40.
20-
20-
L-
i
R/l
Ifl+R
60-
Stage
of
development
of
naupiii
modestw larvae after 9 days on six different feeding Fig. 2. Abundance of developmental stages of E~rninitcs regimes.
SEQUENTIAL FEEDING REGIMES FOR LARVAE OF ELI~~~~US
~~~E~TU~
47
as far as stage V during the 9 days of the experiment. However, on a unialgal diet of the large flagellate Rhodomonas the mean stage of development after 9 days was less than with lsochrysis, although a few stage VI nauplii were obtained with Rhodomonas. There were two peaks of abundance of developmental stages found on a diet of Rhodomonas, at stage II and stage IV, compared with one peak at stage V for Zsochrysis (Fig. 2). It seems likely that the earlier stages have difliculty in capturing the large cells of Rhodomonas, hence many larvae do not develop beyond stage II. The encounter rate with the larger cells is probably lower due to the lower concentration used, but the larger size of the cells should balance the lower encounter rate so that the overall gain is appro~ately equal with either alga. Those larvae that do progress beyond stage II on a diet of Rhodomo~~ are easily able to capture the large cells in the later stages and so development will proceed well on this food once the earlier stages of development are passed. The difficulty of capturing the larger cells in the earlier stages probably also accounts for the lower survival on Rhodomonas (62%) than on Zsochrysis (70%). A mixed diet of these two algae throughout the experiment provided the benefits of both size classes as well as the nutritional benefits of a mixed diet - several workers have found that there is a synergistic effect of mixed diets fed to the larvae of marine invertebrates (Davis & Guillard, 1958; Bayne, 1965; Nassogne, 1970; Walne, 1970b; Gruffydd & Beaumont, 1972; Lewis, 1975; Helm, 1977; Lang, 1977). On the mixed diet the larvae developed well, with all the larvae being at stage IV or later stages of development after 9 days. This was also the case where the mixed diet was available in the latter half of the experiment but only Zsochrysis was available initially - indeed on this feeding regime more stage VI nauplii were obtained than on a mixed diet throughout the experiment (an average of 12 as opposed to 3). It seems therefore that the addition of Rhodomonas in the earlier stages hindered development, possibly due to the larvae wasting energy in trying to capture the large cells, while at the same time the smaller ZsochrysD cells are available in lower concentrations, hence lowering the encounter rate. The fastest rate of development was obtained when Zsochrysis was available initially and Rhodomonas later (mean stage of development 5.14 after 9 days). This feeding regime not only gave faster development but also higher survival (73%) than Zsochrysis initially followed by the mixed diet later (68%) - the addition of Zs~h~sis in later stages may hinder development slightly due to the later stages being less easily able to filter the small cells. Moyse (1964) has emphasised the importance of the antennal endopodite in the feeding of cirripede larvae. In Elminius modestus setules spaced at < 5 pm apart cover 36.39% of the total area covered by the setae of the endopodite in the stage II nauplius, whereas in the stage VI nauplius the area covered by these closely spaced setules is only 13.92% (Fig, 3). Although there is a fringe of closely spaced hairs along the pre-axial edge of the exopodite in the stage VI nauplius, it appears that such fringes are unimportant in the feeding of cirripede nauplii (Stone, 1986). Several workers have suggested that in copepods the limbs act more like paddles than sieves due to their small size and the relative viscosity of the water (Koehl & Strickler,
C.J. STONE
48 a
/
endopodite
c
I
100pm
b
endopodi te
Fig. 3. Set&ion and setulation of the antenna of Elmin& modestus nauplii. a, stage II; b, stage VI. Scale bar = 100 cm, intersetole distances in pm.
SEQUENTIAL
FEEDING
REGIMES
FOR LARVAE
OF ELIMINIUS
MODESTUS
49
1981; Palfenhoffer et al., 1982). Schram (1986) believes that this may also apply to cirripede nauplii. However, Patfenhofer et al. (1982) believed that this only applied to larger particles and that a switch to a mode of feeding more closely resembling passive filtering occurred with small particles of 5 pm diameter and smaller. The secretion of labral or cephalic glands in cirripede larvae (Walker, 1973), rather than being sticky, may act as a “detergent” or decrease viscosity to improve filtration on the appendages. Filtration may also occur due to the compression of water confined between the two beating antennae or between the antennae and the body mass. The feeding regime providing the larger cells of Rhodomonus initially and then the smaller cells of Zsochrysislater proved to be the worst of the six feeding regimes tested, both in terms of rate of development and survival. Lewis (1975) postulated that an optimum feeding schedule for Pollicipes polyments larvae might be a small flagellate initially with the addition of a large dinoflagellate in the later stages. This experiment shows that smaller cells initially followed by larger cells later is a successful feeding schedule for Elminius modestus larvae and the reverse situation of Rhodomonas followed by Zsochrysis gives the poorer results that would be expected. The larger Rhodomonas may be difficult to capture in the earlier stages of development while the smaller Zsochrysis may be less easily filtered than a large cell would be in the later stages of development, hence this feeding regime combines the disadvantages of both size classes of food to give poorer results than obtained with either species used unialgally throughout the experiment. With regards to the size obtained by the nauplii under different feeding regimes, the apparent trend is that larger nauplii are obtained, both in terms of carapace length and carapace width, where the large celled Rhodomonas is available in the later stages, whether unialgally or in a mixture. It seems, therefore, that large cells in the later stages of development lead not only to a faster rate of development and better survival, but also to a large size being reached by the larvae. The size of the food cell appears here to be an important factor contributing to the success or failure of a particular feeding regime. Whether the same principle would be seen in the natural environment is a matter for speculation, as the densities used here were far higher than would be found in the sea. However, the apparent importance observed here of the cell size of the food relative to the size of the larvae sheds some light on the mechanism of food collection by cirripede nauplii. The fact that larger cells are less useful in the earlier stages of development is probably a reflection of the difficulty of capture. But the apparent advantage of larger cells over smaller ones in later stages is not so easily explained. If localized currents alone are responsible for the collection of food particles, as suggested by Lochhead (1936) and favoured by Rainbow & Walker (1976) then smaller cells would presumably be as beneficial as larger cells - however, it appears that the larger cells are better, which would seem to favour the idea of smaller cells escaping from a filter. Norris & Crisp (1953) were the first workers to suggest such a filter, while Moyse (1963, 1964) expanded this idea to show the importance of the endopodite setae of the antenna in food collection. The experiment presented here
50
C.J. STONE
shows that there is mechanistic selection of larger sized particles as the larvae become larger, which supports the idea of feeding using filtering appendages.
ACKNOWLEDGEMENTS
I would like to thank Dr. John Moyse for his interest and discussion of this work, and Professor J. S. Ryland for providing facilities in the School of Biological Sciences, University College of Swansea.
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SEQUENTIAL FEEDING REGIMES FOR LARVAE OF ELIMZNIUS MODESTUS
51
STONE, C. J., 1986. The effects ofvariations in diet, temperature and salinity on the development ofcirripede
nauplii. Ph.D. thesis, University of Wales, U.K., 340 pp. TIGHE-FORD, D. J., M. J. D. POWER & D.C. VAILE, 1970. Laboratory rearing of barnacle larvae for antifouling research. Helgol. Wiss. Meeresunfers., Vol. 20, pp. 393-405. WALKER,G., 1973. Frontal horns and associated gland cells of the nauplii of the barnacles, Balanus humeri, Balanus balanoides, and Elminius modestus (Crustacea : Cirripedia). J. Mar. Biol. Assoc. U.K., Vol. 53, pp. 455-463. WALNE,P.R., 1970a. Studies on the food value of nineteen genera of algae to juvenile bivalves of the genera Ostrea, Crassostrea, Mercenaria and Mytilus. Fish. Invest. Mintit. Agrik. Fish. Food (G.B.) Ser. II Salmon Freshwater Fish., Vol. 26, No. 5. WALNE, P.R., 1970b. Present problems in the culture of the larvae of Ostrea eduks. Helgol. Wiss. Meeresunfers., Vol. 20, pp. 514-525.