Abstract

Diel vertical migration in marine zooplankton may be a bi-phasic feeding strategy involving the alternate exploitation of particulate and dissolved organic material. Active uptake of dissolved material may be pressure sensitive and occur to a significant extent at greater depths. In diving crustaceans this function may be uniquely optimized as the descent of the rigid exoskeleton induces a hydrostatic pressure gradient across the integument. It is generally believed that crustaceans have no significant ability to absorb dissolved organic material directly, but studies have not included normalization of hydrostatic pressure. Anatomical and behavioral evidence support the hypothesis that ammonia excretion by zooplankton occurs largely in surface waters, thereby potentially providing a biological vector for the stimulation of new production.

Introduction

An accurate model of the feeding ecology of zooplankton is critical to understanding the marine nutrient cycle, but explanations for the extensive diel vertical migration of zooplankton have failed to completely account for the observed behavior (1, 2, 3, 4, 5). Descent through the water column as a strategy to optimize dissolved organic material (DOM) uptake based on pressure dynamics is an idea that has not been explored, and the hypothesis that crustaceans have a significant capacity for direct DOM uptake appears to have been rejected prematurely.

Zooplankton typically descend hundreds of meters at dawn and rise toward the surface at dusk. Such a prominent behavior feature, involving a significant energy expenditure (6), must confer an adaptive advantage to the organism. A simple model of "hunger" has been suggested as the trigger for upward migration (7), since feeding on particulate material, including phytoplankton, occurs near the surface. "Satiation," however, seems less compelling as the trigger for the active descent of animals to extreme depths. And the "hunger" hypothesis fails to explain some observations, such as a downward migration in response to a shortage of particulate food at the surface (7).

It is suggested here that "hunger" may also be the trigger for the downward migration or ‘diving’ behavior in zooplankton, and that this hunger may be satisfied by the direct uptake of DOM during descent and the time spent at depth. If a feeding advantage for zooplankton exists in deeper water, it is more likely related to dissolved, than to particulate organic material (POM), since the availability of POM decreases with depth while the DOM concentration remains relatively constant (8). DOM availability to organisms adapted to exploit a gradient in hydrostatic pressure, however, might increase significantly with increasing depth and pressure.

Effects of Hydrostatic Pressure on Crustaceans

The metabolic rate (oxygen uptake) of vertically migrating crustaceans has been measured under elevated hydrostatic pressure, and no great pressure effect has been detected (9). Therefore it has been considered that metabolic processes measured at 1 atmosphere can reasonably be extrapolated to the depths normally occupied by zooplankton (10). No evidence exists, however, that a similar extrapolation can be made from studies of DOM uptake conducted at atmospheric pressure.

Along with temperature, salinity and pH (11), hydrostatic pressure is an environmental variable with the potential to modify characteristics of living membranes such as permeability and the function of active transport mechanisms (11, 12). Behavioral responses to small changes in hydrostatic pressure have been demonstrated in a variety of marine invertebrate species, including crustaceans (12, 13), and it has been suggested that the physiological mechanism mediating the behavioral responses might be pressure-induced alterations in these functional properties of living membranes (12). Pressure-induced physiological changes might reasonably be expected to alter membrane functions involved in nutrient uptake as well. Since zooplankton may experience diel pressure changes of 20 atm. or more, DOM uptake capabilities at depth may vary significantly from those measured at atmospheric pressure.

Beyond the potential alteration in DOM uptake capacity that may occur with depth, diving crustaceans may have the potential to develop a pressure gradient across the integument as they descend. This dynamic may also affect the efficiency of DOM uptake.

Internal Pressure Equalization: Potential Relevance to DOM Uptake

Although the compressibility of seawater is very low, the rigidity of the exoskeleton is such that crustaceans are significantly less compressible than seawater (12). Rising external hydrostatic pressure on descent, if unaccompanied by compression of the exoskeleton, will induce a relatively lower internal pressure, which the animal will most easily be able to equalize by the ingestion of water. Although the volulme of water ingested for this purpose will be very small, crustaceans appear to have the ability to actively control the degree of internal pressure equalization by regulating the movement of water into the digestive tract. (Small crustaceans have been observed to take water in through both mouth and anus (14).) An optimal pressure gradient could therefore conceivably be maintained across the crustacean integument during diving. Such a pressure gradient, occurring on even a very small scale, may have a significant effect on membrane transport functions of the softer regions of the exterior (the dermal glands, and possibly also the membranes involved in gas exchange).

Chapman (1981) demonstrated direct DOM uptake at atmospheric pressure through the dermal glands in a marine copepod, and concluded that "the presence of an exoskeleton, although it may restrict uptake to specific sites, does not necessarily restrict DOM uptake" (15). A rigid exoskeleton may prove to be advantageous if its rigidity allows the enhancement of DOM uptake by the induction of a slight pressure gradient across the integument during descent. An ability to exploit the pressure gradient in the water column in this manner may be an unrecognized adaptive feature of the hard exoskeleton of crustaceans.

The compressibility of water increases as temperature is lowered, therefore exploitation of the compressibility differential between water and crustacean may be enhanced at the lower temperatures associated with deeper water. Zooplankton may also find an advantageous decline in viscosity of water at depth, if conditions are encountered where the viscosity increase induced by lowered temperature is outweighed by the viscosity decrease associated with increasing pressure. (This is an anomalous property of water at ocean temperatures (16).) Subtle physical properties of water may therefore be used to greater advantage in DOM uptake in deeper water. These factors may come into play and help explain the extreme depths to which these tiny organisms descend, or membrane transport capacity may simply continue to increase with ambient pressure.

Pressure Equalization during Feeding: Potential implications for N-cycling

During ascent, the compressibility differential will induce a reverse pressure gradient across the integument. Although it appears unlikely due to the very small volume involved, if the animal becomes stressed by rising internal pressure from this source, equalization will easily be accomplished by the expulsion of space-occupying organ contents. Besides water and solids ingested and retained in the gut, crustaceans can release controlled amounts of liquid metabolic waste to equalize internal pressure. The internal anatomy of crustaceans includes paired bladders into which nitrogenous metabolic waste is channelled and significant volumes can be retained (14). The existence of these bladders implies that excretion of the waste product to the environment is controlled and intermittent in nature, rather than occurring continuously as it is produced by metabolic processes, and experimental observations confirm this (14). It is hypothesized that the discharge of bladder contents is delayed during ascent, in part due to positive buoyancy offered by the major nitrogenous waste product, ammonia. Ammonium has been observed to be used for flotation by other marine organisms (17). Retaining ammonia in the bladders during ascent would therefore be energetically advantageous, as would be discharging this waste prior to descent.

In a study reported by Norfolk (1978), internal hydrostatic pressure in the crab, Carcinus maenas, increased in response to the osmotic effect of a transfer from 100% to 50% seawater. Several times, as the pressure increased, sharp decreases in internal hydrostatic pressure were noted to occur, which were associated with the release of urine from the bladders (14). The pattern of urine release in response to steadily rising internal pressure was intermittent, and a degree of internal pressure elevation was tolerated before the release was triggered. Release of bladder contents by ascending zooplankton is therefore likely to be triggered primarily by rising internal pressure associated with the oral ingestion of POM.

In contrast, the pressure dynamics during descent after cessation of oral feeding appear most likely to inhibit discharge of bladder contents. And if, as argued above, urine release is also delayed during the initial phase of ascent, the excretion of ammonia will be concentrated in areas relatively closer to the surface, where upward migrating zooplankton resume oral feeding. The suggested spatial pattern of waste elimination offers the ecological benefit of stimulating phytoplankton productivity by the net delivery of nitrogen to the surface water.

Research on DOM Uptake by Crustaceans

In recent decades very little scientific research has focused on the question of direct DOM uptake by marine crustaceans. Early in the twentieth century, however, this was a topic of great interest (19). Most notably, the hypothesis was described by August Pütter who, in 1909, concluded that DOM must be the major energy source for marine organisms, since the ocean did not appear to contain enough particulate food to meet their needs (19, 20). The quantity of organic material in the DOM pool has been consistently estimated to be approximately an order of magnitude greater than that contained in POM (8).

While the issue stimulated considerable debate initially, the technology did not exist to directly test Pütter’s hypothesis until later in the century. In 1961, Stephens and Schinske demonstrated the general ability of marine invertebrates to remove glycine from solution in seawater (21). The single notable exception in their study were the Arthropods; six species were tested and all failed to remove measurable amounts of glycine from solution. In 1966, however, McWhinnie and Johannek briefly reported apparently contradictory evidence, that Antarctic euphausiids were capable of direct uptake of acetate and glucose from seawater (22). Subsequently, Anderson and Stephens demonstrated in 1969 that an apparent ability to accumulate labelled glycine from seawater by four small crustacean species, was an artifact only of direct uptake of DOM by microorganisms colonizing their exoskeletons (22). Anderson and Stephens (1969) acknowledged that the limitations of their study included the testing of only a single dissolved organic compound, and also the lack of testing on any arthropod belonging to the deepwater zooplankton (22). Nevertheless, it appears that, based on these few studies, it subsequently became widely accepted and reported as "a general rule" that crustaceans lack the ability to uptake DOM directly from seawater (1, 3, 10, 11, 15, 19, 23, 24). It was assumed that the rigid and relatively impermeable exoskeleton was the primary reason that crustaceans lacked this capacity (3, 11, 22).

It is a further important limitation of the studies of Stephens and Schinske, and Anderson and Stephens, that their experiments were conducted in very small volumes of water (50-1000ml) (21, 22). The crustaceans tested were consequently unable to migrate along any significant pressure gradient, as most zooplankton do in their natural environment. This factor, which may potentially affect their ability to uptake DOM, was not considered in these early studies.

A very few studies conducted since the late 1960s have suggested that direct DOM uptake does play a role in the nutrition of marine crustaceans (25). However, although the topic has been considered a "controversial" one (25), research interest has apparently waned. In the last 2 decades there seems to have been no significant published additions to this limited body of knowledge. Recent comprehensive reviews on calanoid copepods and general zooplankton biology have discussed POM ingestion at length while dismissing the potential for DOM uptake by these marine crustaceans as trivial (1, 10). Citations indicate that justification for this position still appears to be largely based on the work of Anderson and Stephens (1969).

In the most recent experimental study that I am aware of (also conducted at atmospheric pressure, in 500 ml of water), Chapman (1981) used autoradiographic studies to demonstrate the direct uptake of low concentrations of dissolved glucose from seawater by the marine copepod, Neocalanus plumchrus (15). The anatomical regions of uptake were shown to be the midgut gland and the dermal glands. Transport of glucose through the circulatory system and incorporation into somatic tissue were also demonstrated. Dermal glands are common to all crustaceans, but their function remains poorly understood (1, 15). They are, however, small areas where soft tissue extends through pores which penetrate the rigid exoskeleton. Mauchline (1998) discounted the potential significance of Chapman’s finding based on the observation that the anatomical regions involved in DOM uptake were areas "with thin or no cuticle, forming a small region of the body surface, (therefore) little nutritional advantage would be expected" (1). This may be inadequate grounds to draw this conclusion, however, if those small areas are specially adapted for DOM uptake under the fluctuating hydrostatic pressure conditions encountered in nature.

Recent investigations into the direct DOM uptake capabilities of marine invertebrates have focused exclusively on the "soft-bodied" species, and highly efficient uptake abilities have been demonstrated (11, 23, 26, 27). In contrast to crustaceans, some soft bodied invertebrate species seem able to absorb DOM directly through virtually the entire integument (11, 23). In bivalves, the gill is an important site of DOM uptake as well as gas exchange (26).

Supportive Findings in Zooplankton Research

Difficulties commonly encountered in working with zooplankton may be rooted in a nutritional deprivation imposed when diving animals are held in the relatively shallow, low pressure tanks used in laboratory experiments. Even with the addition of concentrations of particulate food far greater than those occurring in nature, zooplankton have been unusually difficult animals to maintain in laboratories. Similar difficulties are encountered in rearing the young of benthic crustaceans (which also migrate vertically during the planktonic stages), in the laboratory (3). A common observation is that the metabolic rates of zooplankton are initially relatively high immediately after capture, but that rates decrease markedly in the following hours and days. This pattern was initially believed to be due to physical trauma, or "capture stress," but Ikeda and Skjoldal (1980) demonstrated that the pattern of declining metabolism post-capture is an effect of the onset of starvation (18). In incubations in natural seawater, it has been noted that one common "bottle effect" is a decline in the growth rate of phytoplankton, and the procedure used to counteract this problem is the addition of ammonia to the incubation (10). These observations suggest that a nutrient impoverishment exists in the usual experimental set-ups in which small vertically migrating crustaceans are fed exclusively on POM at low hydrostatic pressure. Also suggested is that the stimulation of phytoplankton growth in nature involves the frequent injection of a nitrogenous fertilizer (possibly ammonia) into the surface water.

Low natural availability of POM in the ocean, and the difficulty in providing adequate nutrition to zooplankton via POM alone in the laboratory, seem inconsistent with the common finding of empty guts in a high proportion of apparently healthy, freshly captured organisms (20, 28). Observations of deep living copepod species have at times included seemingly unlikely generalizations, such as "adult males do not feed" (28). Mature zooplankton at higher latitudes descend to deeper water for months during the winter season. While, as is generally believed, they may maintain themselves largely on energy stores accumulated during the months of extensive migration and active oral feeding, these organisms may also continue to absorb DOM directly during this time. They may continue to undergo lesser degrees of vertical migration, confining themselves to the deeper water layers during the months of low phytoplankton productivity, and rely more heavily on DOM-acquired nutrients during these times. In 1916, Esterly reported his observations of one abundant pelagic copepod, Eucalanus elongatus, in which "most specimens (were) devoid of any intestinal contents," and the small bit of fecal material occasionally seen "never (contained) the recognizable remains of any ingested organism" (20). Might some species be more heavily oriented toward the exploitation of DOM, and might the more important function of the digestive tract in crustaceans sometimes be its utility in regulating the hydrostatic pressure gradient across the integument as the organism migrates vertically? As noted by Chapman (1981), the fact that any capacity for direct DOM uptake has been demonstrated in a copepod that "does not appear to feed during the last 7 months of its life cycle" (15), strongly suggests that further research into this question is warranted.

Bi-phasic feeding strategies may be a common trait of many aquatic invertebrates. Research into the nutritional requirements of marine invertebrate larvae has led to the conclusion that the optimum balance of nutrients for these organisms is provided by a "bi-phasic diet (algae and DOM)" (3). A diel bi-phasic feeding strategy (benthic and pelagic) has recently been described by Wilhelm et al (1999) in a vertically migrating freshwater copepod (29). Interestingly, these authors concluded that the behavior pattern of this copepod causes a net regeneration of "new" phosphorus, the limiting nutrient in the lake system, into the pelagic zone from the sediment, thus enhancing primary production. This echoes the ecological role suggested here for vertically migrating marine zooplankton, that of augmenting marine primary production by the physical transport of "new" nitrogen from deeper water to the photic zone.

Current understanding of the marine nutrient cycle holds that DOM uptake plays an important role in zooplankton nutrition, but only via the microbial loop or other physical or chemical processes that convert DOM into POM (e.g. 24, 30). Studies of planktonic marine crustacean nutrition today are focused exclusively on the ingestion of POM (1, 10). The enhancement of phytoplankton growth by ammonia excretion by zooplankton has long been considered to be significant, although it has been viewed only as the "regeneration" of nutrients that were originally obtained by grazing in the euphotic zone (e.g. 8). The acquisition of organic material and energy from lower parts of the water column and the active upward transport of nitrogen by zooplankton has not been considered. If this dynamic occurs, then part of the ammonia excreted by zooplankton in surface waters results in the stimulation of "new" rather than "regenerated" production. Therefore, this question has major implications for our understanding of the global carbon cycle as well as the marine nutrient cycle, and should become a research priority.

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