Marine Biology 97, 191-197 (1988) Marine . . . . . . . . . . . . . . . . BiOlOgy

�9 Springer-Verlag 1988

Ontogenetic feeding shifts in the meiobenthic harpacticoid copepod Nitocra iacustris

A. W. D e c h o * and J. W. Fleeger

Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana 70803, USA

Abstract

~4C-radiolabelling experiments indicate that adult stages of the salt-marsh harpacticoid copepod Nitocra lacustris (Schmankevitsch) receive a large part of their nutrition through the ingestion and assimilation of certain diatoms. An abundance of empty diatom frustules occurs in the gut- pellet contents of field-collected individuals. Naupliar stages do not ingest diatoms in the laboratory, and nauplii from the field do not contain frustules in their gut pellets. Ingestion of diatoms in the laboratory first occurs during the second or third copepodite stage. 3H-radiolabel exper- iments and grazing experiments using bacterium-sized beads adhering to the diatoms indicated that both adults and nauplii ingest bacteria adhering to the outer mucus coating of the diatoms (and probably ingest the diatom mucus itself). Adults ingest bacteria (and probably mucus exopolymer) coincidently while ingesting diatoms. The nauplii ingest these components by scraping the outer sur- face of the diatoms. SEM observations indicate that dia- toms are not punctured by the nauplii during feeding. While diatom mucus and associated bacteria play an (as yet unquantified) role in the nutrition of the adults, these components may comprise the bulk of food resources for naupliar stages.

Introduction

Investigations of food resource utilization by the meiobenthos have indicated both ingestion selectivity (Tietjen and Lee 1973, Brown and Sibert 1977, Lee et al. 1977, Rieper 1978, Rieper and Flotow 1981, Vanden

* Present address: CSIRO Marine Laboratory, P.O. Box 120, Cleveland, Queensland 4163, Australia

Berghe and Bergmans 1981, Rieper 1982, Montagna 1984, Trotter and Webster 1984, Carman and Thistle 1985, Decho 1986, Decho and Castenholz 1986) and digestion selectivity (Chua and Brinkhurst 1973, Tietjen and Lee 1973, Deutsch 1978, Decho and Castenholz 1986). Such studies are beginning to reveal the mechanisms of energy flow through the meiobenthos.

Most feeding studies have concerned adult stages be- cause of taxonomic problems in identifying immature stages and the logistical problems connected with exper- imentation using small organisms. Food sources may change during the course of development in planktonic copepods (Elster 1936, Marshall and Orr 1956, Lewis 1967, Smyly 1970, Paffenh/Sfer 197t, Maiy and Maty 1974, Allan et al. 1977, Fernandez 1979, Chow-Fraser and Wong 1986). Such dietary shifts are often a result of changes in (1) size, morphology and structure, and/or (2) developmental needs.

Because adult survival, distribution and abundance may ultimately depend on early life-history stages (Sellner 1976, Weinberg etal. 1986), it is of interest to know whether feeding patterns in the meiobenthos change throughout the life history of a given species. The purpose of this study was to investigate the feeding and resource utilization of the estuarine meiobenthic harpacticoid cope- pod Nitocra lacustris to determine (1) what constitutes the food resources of the species, and (2) if adults ingest and assimilate the same food resources as nauplii.

Materials and methods

Individuals of Nitocra lacustris (Schmankevitsch) were col- lected from salt-marsh sediments at Cocodrie, Louisiana, USA, and maintained for several generations in laboratory culture (Chandler 1986) until use. Additional individuals (both adult and naupliar stages) were collected from field sediments and immediately frozen in situ in liquid nitrogen for gut and fecal pellet analyses.

192 A.W. Decho and J. W. Fleeger: Food of adult and nauplii Nitocra lacustris

Feeding experiments

For diatom-feeding experiments, sediment diatoms were isolated from the same local habitat as Nitocra lacustris, and maintained in laboratory culture on Erdschreiber medium in 15%0 S Instant Ocean Seawater using a 16 h light:8 h dark cycle at 2 0 ~ C ~ The diatom used was Amphora tenerrima (Aleem and Hustedt) because (1) it is commonly found in the local habitat of N. lacustris, and is often found in gut contents of individuals collected from field sediments; (2) it is a very small diatom (16Hm• relative to the size of the harpacticoid nauplii and would reduce any size-selectivity effects; and (3) unialgal cultures of this diatom on agar plates can sub- stain vigorous growth rates of N. lacustris for many gener- ations (Decho unpublished data). Diatoms were labelled in their log phase of growth with NaH14CO3 (New England Nuclear, Boston, Massachusetts, 10 Hg/HCi; 50 HCi 1-1 fi- nal conc) for 24 h under constant light, washed and pre- pared according to the methods of Decho (1986), and placed in sediment microcosms.

The bacterial flora associated with the diatoms were labelled with all-methyl thymidine (New England Nuclear, 50 to 80 Ci mmol-1; 0.8 #Ci m1-1 final conc) for 4 h. Up- take of label by bacteria and adsorption by non-bacterial components was monitored over the 4 h period and com- pared with formalin-killed controls (Lessard and Swift 1985). Estimations of bacterial numbers were made using the direct-count method (Hobbie et al. 1977), as modified for sediments by Montagna (1982).

For feeding experiments, 75 adult and 375 naupliar in- dividuals of Nitocra lacustris were added to sediment microcosms containing labelled microbial flora and al- lowed to feed for 2 h. At the conclusion of the 2 h feeding period, the copepods were quickly removed by sieving and washed. Some of these individuals were immediately fixed, placed in scintillation vials containing 1 ml Protosol (New England Nuclear), and digested in darkness (65 ~ C) for 4 h. Samples were later counted on a Beckman LS8000 liquid scintillation counter using 10 ml Econofluor (New England Nuclear) as the scintillation fluor. Each vial contained either 7 adults or 20 nauplii. Six replicates were used for each group (adult and nauplii). Quenching was corrected for by the external-standards ratio method.

Another group of copepods (used in assimilation stud- ies) were immediately transferred to "cold-feed" sediment microcosms containing unlabelled microbial flora of the same type as mentioned above. These individuals were al- lowed to feed for 2 h, then removed and prepared for liquid scilitillation counting.

Respiration of 14CO2 during the labelled feeding incu- bations and "cold-feed" incubations was monitored ac- cording to the methods of Hobbie and Crawford (1969), as modified for use with meiobenthos by Decho (1986).

To control for uptake of label not due to ingestion of either diatoms or bacteria, adult and nauplii were placed in sediment containing filtered (0.22 Hm) exudates from 14C- labelled diatoms or sterile seawater containing 3H-methyl

thymidine (at the same concentration as used in the feeding experiments). Results were analysed by analysis-of-vari- ance (SAS Institute Inc. 1982).

Bacterial-sized particle ingestion

Bacteria-sized (0.75 Hm) fluorescent beads (Fluoresbite, Polysciences Inc.) were used as an inert tracer. The beads were mixed with diatom (Amphora tenerrima) cultures. The cultures were washed by centrifugation (3 000 rpm for 5 rain) to remove unattached beads, and then mixed with sediment. Adult and naupliar harpacticoids were added and allowed to feed for 1 h. Individuals were removed, washed, and examined for the presence of fluorescent particles in gut pellets by means of epifluorescence micros- copy (excitation 4= 458 nm; emission 4= 540 nm).

Gut-content analyses

Adults and nauplii were collected from field sediments and the gut-pellet contents observed by light microscopy. Gut pellets were removed from various portions of the adult digestive tracts. Naupliar gut pellets, too small to be re- moved, were observed intact using squash preparations. Specifically noted were the presence or absence of diatom frustules in the pellets. Because the silica frustules are not totally destroyed (and sometimes remain completely in- tact) during digestion, their presence in the gut- and fecal- pellets was easily noted. Identification of nauplii of Nitocra Iacustris nauplii from field samples was verified by com- parison with laboratory-hatched individuals.

Growth stage at which feeding shift occurs

To determine at which developmental stage ingestion of di- atoms began, single individuals of newly hatched Nitocra lacustris (Naupliar Stage I) were placed in agar in petri dishes (35 x 10 ram) containing unialgal cultures of the dia- tom Amphora tenerrima. The dishes were examined for exuvia every 4 to 8 h and the number of molts recorded. The gut pellets from several individuals of all naupliar and ear- ly copepodite stages were examined for the presence of di- atom frustules.

Effect on diatom frustules ofnaupliar grazing

Scanning electron microscopy was used to examine diatom frustules which had been grazed by nauplii. Thirty nauplii were added to an approx 5 x 5 mm patch of Amphora tenerrima and allowed to feed for 10 h. The diatoms were prepared for SEM by fixation in 2% glutaraldehyde buf- fered with sodium cacodylate buffer (adjusting for osmo- lality), for 2 h, rinsed in buffer, and then placed in 1% os- mium tetroxide for 1 h. The specimens were rinsed twice in

A. W. Decho and J. W. Fleeger: Food of adult and nauplii Nitocra lacustris 193

buffer (15 min each), then in dH20, and dehydrated in 3 ml 2,2-dimethoxypropane (Sigma Chemical Co.) with 400 y1 of concentrated HC1. The mixture was equilibrated to room temperature, and then replaced with three changes of 100% acetone (high-performance liquid chromatogra- phy-grade) for 15 min. Finally, the specimens were critical- point dried, mounted and sputter-coated with Au/Pd, and examined on a Hitachi S-500 scanning electron micro- scope. A control consisted of diatoms from the same cul- ture which were not exposed to naupliar grazing.

were 873 670 4- 8 100 cells cm -2 sediment. Specific activities of bacteria were 0.6 4-0.018 dpm cell-t

Results of grazing experiments showed significant up- take of tritium-labeled (3H) bacteria by adults (P< 0.05). After 2 h cold-feeding, most (78%) of this label was lost. These reduced amounts of label, however, still differed from controls (P< 0.05). Nauplii also showed significant uptake (P<0.05) of labelled bacteria; however, they re- tained a small, but significant (P<0.05) portion (18%) of this label after 2 h cold-feeding (Fig. 1).

Results

14C-bicarbonate (diatom) grazing experiments

Concentrations of Amphora tenerrima used in feeding ex- periments were 1 058 000___ 34 500 cells cm -2 sediment, with specific activities of 4.3 4-0.25 dpm cell -1.

Significant amounts of 14C-label (used to label diatoms) were consumed by adult Nitocra lacustris. After 2 h "cold- feeding" on unlabelled diatoms, the copepods still retained a significant amount (70%) of label, 20% having been re- moved by respiration of 14CO2 (Fig. 1). During naupliar grazing, a significant (P < 0.05), but small amount of label was taken up above control levels (Fig. 1). After 2 h cold- feeding, most (58%) of this label remained, indicating as- similation. Respiration accounted for 27% of the lost label.

Examination of adult gut-pellets after feeding exper- iments revealed the presence of many diatom frustules. No frustules were observed in nauplii gut-pellets.

3H-methylthymidine (bacterial-uptake) experiments

Uptake of 3H-methyl thymidine was linear over the 4 h incubation period compared with formalin-poisoned con- trols. Most of this uptake, correcting for adsorption, could be assumed to be due to bacterial incorporation (Lessard and Swift 1985). Direct counts of bacteria revealed 5.43 + 0.13 x 10 -7 cells cm -2 sediment; diatom densities

(J

E Q. ~3

700

500

3 0 0

100

I L 14C-diat~

~_,_~L f I

0 2

l : ime [h] Cold Feeding

500 [- 3H-bacter ia

.oof oot

0 2

T i m e (hi Cold - Feeding

Fig. 1. Nitocra lacustris. 14C-bicarbonate (diatoms) and ~H-thymi- dine (bacteria) grazing by adults and nauplii, dpm: disintegrations per minute; R: 14C respired; U: ~4C unaccounted for. Cold-feed- mg represents time spent feeding on unlabeled food, immediately after conclusion of labelled feeding experiments

Observational results

Adult and naupliar stages consumed diatoms with bacteria- sized fluorescent beads (adhering to diatom matrices) and had bacteria-sized beads in their gut (Fig. 2) and fecal pellets.

Harpacticoid gut-pellets collected from field sediments contained an abundance of diatom frustules (and sediment particles) in adult stages (Fig. 3 A). None of the naupliar- stage individuals examined had frustules in their gut con- tents (Fig. 3B) indicating that ingestion of Amphora tener- rima began at the second to third copepodite stage. Dia- toms were regularly found in gut contents during sub- sequent life stages.

Scanning electron microscopy of clusters of diatoms re- vealed that no breakage of diatoms frustules was apparent after they had been grazed upon by nauplii (Fig. 4).

Discussion

Ontogenetic feeding shifts occur in some calanoid cope- pods, which switch from herbivorous to omnivorous feed- ing as they develop from nauplii to adult (Lewis 1967, Smyly 1970, Chow-Fraser and Wong 1986). Feeding shifts in the meiobenthos have received little attention, despite the important role these shifts would have in maturation and reproduction (Phillips 1984), and in food-web in- teractions.

The results of the present study show important shifts in the ingestion and utilization of microbial food resources between the adult and naupliar stages of the meiobenthic harpacticoid copepod Nitocra lacustris. Adults ingested and assimilated diatoms (Amphora wnerrima) in the laboratory and field-collected adults had an abundance of empty diatom frustules in the gut-pellets. This indicates that these diatoms may comprise a large part of the food of the adult.

Naupliar stages did not ingest 14C-labeled diatoms in the laboratory and field-collected individuals did not have diatom frustules in the gut pellets. Direct determination of naupliar food resources (i.e., bacteria, mucus-exopolymer, dissolved organic matter, etc.) is difficult for several rea- sons. Nauplii consume a small amount of food, and gut-re- tention times are fast (5 to 20? min) relative to adults. Also, metabolic losses (i.e., excretion and respiration of as-

194 A.W. Decho and J. W. Fleeger: Food of adult and nauplii Nitocra lacustris

Fig. 2. Nitocra lacustris. Gut pellets of adults (A) and nauplii (B), showing ingest- ed bacterium-sized fluorescent beads (Fb). (Epifluorescent microscopy; scale bars-- 30 #m)

similated label) over a given time-frame will be much greater relative to adults. A small amount of 14C-label is taken up by nauplii, but this uptake is not due to ingestion of diatoms or even breakage of the diatoms (and sub- sequent ingestion of intracellular contents) by the nauplii. Some of the 14C in the diatoms is quickly metabolized and excreted as dissolved labelled-exudate products that are quickly incorporated by bacteria (Bell and Sakshaug 1980) growing in close association with these diatoms. Some of this metabolised 14C-label is incorporated into extracellu- lar mucus (exopolymer) of the diatom. Ingestion of the la- belled extracellular mucus-exopolymer (sensu Hobble and

Lee 1980) and secondarily-labelled bacteria, or even the direct uptake of dissolved exudates can result in uptake of 14C label (originally derived from diatoms) by nauplii (and adults). In addition, although the mouthparts are well-de- veloped, the nauplii can only manipulate and scrape the outsides of diatoms and cannot actually ingest the diatom cell (Decho, unpublished data). The absence of diatom frustules from the gut pellets, but the presence (in the gut pellets) of bacteria-sized fluorescent particles that had been affixed to the outer diatom mucus coat (from separate experiments) strongly support this interpretation. SEM ex- amination of diatoms previously grazed by nauplii did not

A. W. Decho and J. W. Fleeger: Food of adult and nauplii Nitoo'a Iacustris 195

Fig. 3. Nitocra lacustris. Gut contents of adults (A) and nauplii (B) collected from field sediments, gp: gut pellet; d: diatom frustule. (Scale bars = 30 ~m)

show any breakage of diatom frustules. The nauplii do not break open the diatoms during feeding to remove their in- tracellular contents, as do some nematodes (Jensen 1982).

The naupliar (through adult) stages of Nitocra lacustris can be cultured for many generations on monoxenic strains of the diatom Amphora tenerrima. Lee et al. (1976) were able to culture a congener (N. typica) on agar plates con- taining different microalgal strains. Since the nauplii do not ingest the diatoms themselves, some associated com- ponent of the diatoms (i.e., mucus-exopolymer coats, as- sociated bacteria, dissolved exudates, etc.), which is neces- sary for sustained growth, must be utilized. Although

thymidine-grazing experiments do not suggest assimilation of bacteria (which would be indicated by retention of label over time), utilization may still occur. The pathways by which the 3H-methyl group on the thymidine is transferred during metabolism vary unpredictably (Azam and Holm- Hansen 1973, Hollibaugh et al. 1980) and are difficult to quantify. Bacteria (or at least a portion of the bacteria) may be assimilated. Food resources of harpacticoids are not fully understood. Although diatoms are a commonly ingested (and assimilated) food item in the diet of adult N. lacustris, other components (i.e., associated mucus-se- cretions, bacterial flora, etc.) may be important. The in-

196 A.W. Decho and J. W. Fleeger: Food of adult and nauplii Nitocra lacustris

Fig. 4. Amphora tenerrima. Scanning electron micrograph of diatom clusters. (A) Ungrazed (control) patch; (B) patch previously grazed by Nitocra lacustris nauplii. Note that no breakage of diatom frustules is evident in "grazed" patches, b: bacterium; m: mucus-exopolymer. (Scale bars = 5 #m)

gestion of whole diatoms by adults will include the mucus- exopolymer slime and numerous bacteria associated with these diatoms (Fig. 4). However, the relative importance of these food components may change ontogenetically. For example, bacteria may act as an important supplemental food component (Phillips 1984) in adults, serving as a source of specific nutrients, e.g. B-complex vitamins (Kut- sky 1981). Some harpacticoids require the presence of bac- teria for continued reproduction (Provasoli et al. 1959). The role of bacteria and mucus secretions (of diatoms and bacteria) is potentially important, but as yet unquantified.

Food-resource utilization appears to be complex and variable, depending on the life stage. As stated by Hicks and Coull (1983): "It appears that the use of the terms dia- tom-feeder or bacteria-feeder will be far too general in scope to ultimately describe the utilization of food re- sources in the meiofauna.". Although some harpacticoids

may be labelled as diatom-feeders or diatom-specialists (i.e., preferentially ingest only certain species of diatoms), diatoms should not be considered their sole food resource. tt is more likely that a wide variety of other organic com- ponents (diatoms, mucus-exopolymer, bacteria, etc.) are of supplemental importance in their nutritional requirements and reproductive biology (Phillips 1984). Understanding how the relative importance of various utilized food com- ponents changes through ontogeny will require studies fo- cusing on the nutritional requirements and assimilation ef- ficiencies of these harpacticoid copepods.

Acknowledgements. The authors would like to thank Dr. M. J. Sul- livan, Mississippi State University, for identification of diatoms used in this study; Dr. R. C. Gayda, Louisiana State University, for valuable discussion on microbial aspects; Dr. M. Blackwell, Louisiana State University, for use of her epi-fluorescent microscope: R~ A. Roller, for technical assistance in S.E.M., and Professor J.

A. W. Decho and J. W. Fleeger: Food of adult and nauplii Nitocra lacustris 197

M. Lawrence and anonymous reviewers for constructive help on various portions of the manuscript. We also would like to thank the staff of the Louisiana Universities Marine Center for their helpful assistance and use of facilities. This research was supported by NSF (Dissertation Improvement) Grant No. OCE-8313109 to A. W. Decho.

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Date of final manuscript acceptance: October 2, 1987. Communicated by J. M. Lawrence, Tampa