### SUMMARY OF DISCUSSION, WORKING GROUP III: FEEDING RATES AND DIETARY COMPONENTS

Discussion Leaders: Gary Kleppel and Steven Hand

The discussion in this group was intended to contribute to the sections on physiological condition and rates. In the context of Global Change, the intent of GLOBEC is to understand fundamental mechanistic processes that are responsible for: (1) abundance of marine animals (2) fluctuation in abundance, and (3) secondary production in ocean ecosystems. In order to approach these broad issues, much more basic information is needed on the following topics:

1. Physiological condition and rates; population genetics and genetic markers

2. Viable biotechnology that can be applied at sea (i.e., on shipboard) to speed data acquisition in these research areas

One underpinning of physiological condition is the process of feeding and the nature of the associated dietary components. Definitive data and new experimental approaches to three simple questions are needed, particularly as related to zooplankton:

1. What methods are available to determine whether or not a population of organisms is feeding?

2. What are the most effective approaches for identifying dietary components?

3. What is feeding rate of populations in situ?

The first question is intended to stimulate technological development in high frequency data acquisition, so that feeding data can be interfaced more rapidly with oceanic physical/chemical data that are acquired on real time and near time bases. Because variations in both concentration and composition of food in the environment may influence zooplankton production, data on the composition of the diet are needed (question #2). Diet tends to be among the most poorly quantified aspects of feeding, yet it may be one of the most important.

Identification of gut contents by using taxon specific DNA probes (particularly above species level) is one promising approach. Probes should at least differentiate between carnivory and herbivory; additional levels of taxonomic detail are desirable. The ability to analyze small samples within a time scale of hours on board ship is an important goal. Various potential problems need to be addressed and resolved. For example, we need to evaluate the effect of transit time of food through the gut, the acidic gut pH, and the actions of nucleases and proteases on the degradation of the DNA and protein used for food identification. It may not be feasible to prepare large numbers of probes for use on predator species with a broad dietary intake. Application of this approach to adult stages which feed on the development stages of the same species (e.g., copepods eating their own nauplius larvae) may not be feasible. Are these approaches quantifiable? For example, can the number of gene copies be related to the number of cells ingested, providing the appropriate controls are performed (i.e., laboratory verification and calibration)?

Immunological identification of food also may be applicable. For example, antibodies raised against the yolk protein of fish eggs or yolksac-stage larvae could be used to follow ingestion by euphausiids. Antibodies raised against parvalbumin (lower vertebrate specific contractile protein in white muscle), against whole phytoplankton cells, or against ciliates may be useful. Will gut dissections be required in all cases to acquire a clean immunological signal? If so, the number of analyses performed per day would be compromised. The relative effectiveness of polyclonal vs. monoclonal antibodies should be considered.

Application of flow cytometry to zooplankton feeding studies may be helpful in speeding up the processing of gut content analysis, as well as for monitoring the rate of food removal by predators in "closed container" experiments. Other optical screening techniques may be useful.

Analytical techniques for identification of pigments, including measurement of carotenoids for determining diet of predators, already exist and are relatively rapid. Pigment content can be related to total carbon ingested. Further refinement of these techniques is necessary. The feasibility of developing immunofluorescent markers for carotenoprotein complexes would increase the sensitivity of pigment analyses enormously.

Finally, reliable indicators for feeding rates of populations in the field need to be developed. Attempts to use the level of digestive enzymes measured in predators as an indicator of recent feeding activity has not been overly successful. Often the induction of these enzymes in response to food intake is not detectable. The successful application of this approach will likely require laboratory verification for each predator species in question. New and more reliable enzyme/macromolecular markers that are induced rapidly and that can be used as indices of feeding and/or assimilation need to be identified. For example, can the level of enzymes associated with synthesis of the peritrophic membrane be used as an index of feeding activity? Unquestionably, more basic research on the physiology of zooplankton feeding and on the biochemistry of the zooplankton gut is absolutely essential to accomplish this objective.