### SUMMARY OF DISCUSSION, WORKING GROUP IV: SAMPLING

Discussion Leaders: Bruce Sidell, Peter Ortner and Lewis Incze

#### General

Developing techniques in molecular biology now make it possible to assess certain physiological properties of individual marine planktonic organisms. New discoveries and methodological advancements almost certainly will expand the present list of capabilities to include smaller samples sizes, additional physiological parameters and more convenient procedures that can be taken to sea. There should be collateral development of techniques for sample collection and processing so that the methods are complementary and, to the extent possible, can be physically combined to enable frequent sampling in the field. There are three parts to the problem: sample acquisition, identification and sorting of taxa, and application of desired chemical or molecular assays. The latter two topics were discussed by the other three working groups at this workshop. It was the objective of this Sampling Working Group to consider how the molecular tagging and various assays might be combined and to consider how these jointly might be incorporated into a sampling scheme that could satisfy the needs of the oceanographic field program.

As background and stimulus for discussion, a schema of an "ideal" sampling system was presented. This "pipe-dream" schema had been developed earlier for general discussion by members of the GLOBEC Steering Committee. It was recognized that the final technology might not even closely resemble the depicted flow diagram, but the central goals were clear and should be maintained. To wit: small-volume samples should be obtainable from well-defined depths; biological sampling should be combined, as much as possible, with physical and optical instrumentation; samples delivered to the surface should be "tagged" in some manner to enable rapid and accurate sorting; and tagged (that is, identified and sorted) animals should be used for the numerous shipboard analyses that might be desired. The central questions which arise from this sampling and processing strategy are: 1) what sampling frequency is desirable and feasible for the physiological/ecological work; 2) can the experimental and physiological work be done in tandem with the taxonomic marking and sorting; and 3) are there genetic or physiological objectives that cannot be met by such a sampling scheme? These questions formed the basis for our discussions, which are summarized accordingly.

#### Specific Comments

Sampling Frequency, Scale and Methods. The ability to sample physical properties of the ocean with fine spatial (especially vertical) and temporal resolution is recognized. It also is recognized that biological sampling must routinely be conducted at finer spatial and temporal scales than presently possible in order to meet GLOBEC objectives. Developing applications such as multi- frequency acoustic devices, imaging systems and acoustic current profilers are approaching the scales of physical observations with respect to estimates of zooplankton biomass and approximations of community composition based on size and shape. A pumping system could deliver samples with fairly small spatial resolution and could be used to sample frequently. It was felt that animals probably could be delivered to the ship's laboratory relatively unharmed. The question is: is it reasonable or desirable to try to approach this frequency of observation with the genetics or physiological assessments?

Most discussants felt that initially they should examine physiological parameters at only a few depths per station and concentrate on sampling a larger number of individuals from each depth. This is because differences between individuals even under the same conditions can be great and may play a significant role in survival and recruitment. More must be learned about these differences before it will be useful to sample for physiology at more depths. Group members were more positive about the immediate benefits of fine-scale genetic (taxonomic) information. This could provide valuable insights by itself and would be useful in considering how to plan physiological and experimental work in the future.

The advantages of a pumping system were recognized as saving ship time and providing small volumes from well-defined depths with accompanying physical and other (e.g., bioacoustical, fluorescence) information. Such systems should prove useful in many respects for the types of field objectives being discussed here. However, such systems probably are inadequate for sampling larval fish and other comparatively sparsely distributed fauna, and they likely introduce unacceptable levels of trauma to most organisms for subsequent live experimentation on board. The latter should be investigated, but the consensus was that specialized sampling designed for specific organisms and questions will remain a necessary feature of field programs. It was felt that most enzyme assays are relatively robust with respect to pump sampling per se, but that macromolecules in zooplankton may be subject to rapid degradation due to release of lytic enzymes (e.g., proteases, lipases, RNAses, etc.) and the total handling time may be a problem. Consequently, protocols for processing (and/or storage) of samples will be dictated by the particular organism under study and/or the nature of measurement being performed. Much has yet to be explored. Consolidation of sampling design, instrumentation and techniques should be sought wherever possible, but flexibility also is needed. A generalized and unified sampling scheme probably cannot be devised a priori.

Joining of Taxonomic Tagging with Physiological Assays. Molecular labeling of zooplankton is needed for more detailed and accurate taxonomic identification. This can be the basis for increased sample sorting speeds (e.g., with optically labeled tags) and for asking more detailed questions about the physiology and behavior of zooplanktonic organisms and their response to environmental conditions and change. There would be obvious benefit to performing behavioral studies and molecular assessments of physiology on animals that have already been sorted and identified. Specifically, this would reduce the number of "superfluous" tests and would ensure that you obtained intended sample sizes for the targeted taxa. The question is to what extent and in what manner can taxonomic tagging and sorting be coupled to the other procedures. It was felt that there may be ways to execute molecular physiological assays on individuals that had been tagged previously for identification (a serial coupling of the two procedures). For example, it is possible that taxon-specific molecular surface tags (e.g., optically active immunochemical tags) may be compatible with some physiological assays (e.g., enzyme activity determinations) that could be executed after tagging and identification. Alternatively, because of the high sensitivity of many molecular techniques, separate sample aliquots from moderately small organisms may be able to support both types of measurement. For example, taxonomic identification by 2-dimensional electrophoresis could be done in parallel with some enzyme assays requiring little tissue. However, this cannot be predicted a priori even with extant assay techniques because it depends on the chemical nature of the tag(s) used, and these tags have not yet been determined for the taxa of interest. This suggests that development of genetic tags and physiological assays will have to proceed independently for awhile, recognizing that a marriage of the two ultimately is wanted.

Present tagging techniques require destruction of the organism. Consequently, it would not be available for studies which require live or at least intact animals. In the near future, such tagging would have to take place after the behavioral or other experimental work was conducted. This would impose the same difficulties cited above with respect to superfluous samples and/or inadequate sample sizes. Some assays may themselves be destructive of the animal tissue. For these reasons, it is important to develop techniques for tagging of live and intact zooplankters to permit identification before, and without interference to, other analyses.

Genetic or Physiological Objectives Not Met by the Above Sampling Scheme. Discussions held by the three other working groups at this meeting focused on measuring the response of individual organisms to physically forced conditions of the environment. The sampling scheme discussed above attempts to make such measurements feasible within the framework of an oceanographic cruise and the desired coupling of biological and physical data. The focus is clearly on the small- scale for the individual, the medium-scale (that is, the study "site") for the population-level response (e.g., recruitment) and the present. Not all of the study can be restricted to these scales, however. Significant variations in biomolecular characters probably will be encountered not only within the study area, but also beyond its boundaries. For the development of reliable genetic markers and for understanding possible shifts in genetic structure and physiology of local populations in response to climatically related changes in the environment, it will be necessary to conduct some sampling well beyond the boundaries of specific study sites. GLOBEC must recognize these needs in addition to the focused effort on processes within designated areas.

#### Conclusions

The following summarizes the main points of the above discussions.

1. We are not ready to try to couple completely physiological measurements to the meter-scale depth resolution achievable in the physics. The next generation of developing molecular techniques should concentrate on measuring the range of individual variations at a relatively few depths per station, but doing so rapidly and efficiently enough to enable much improved resolution in the horizontal and over short time intervals. The use of a pump attached to a CTD package is valuable (see discussion for details) but should not be construed to suggest that physical and biological data will always be obtained at the same spatial and temporal scales. Abundance and compositional data (see discussion) are better suited to such 1:1 comparisons in the near future.
2. Some specialized sampling probably will be necessary regardless of efforts to streamline operations. A view to such flexibility must be maintained. Even within the realm of molecular techniques, it may not be possible to identify a generalized and unified sampling scheme a priori. Considerable work must be done first on the tagging and assay methodologies.
3. Genetic labeling techniques capable of supporting small-scale vertical investigations will likely precede the development of comparably facile physiological assays. This is not harmful; genetic information on vertical distribution patterns should lay the groundwork for asking physiological questions that demand the smaller vertical scale resolution.
4. The coupling of molecular tagging with subsequent molecular assay techniques is a reasonable goal and will probably be achievable at least for some assays and organisms. Methods for assaying physiological parameters should be developed with such a coupling in mind, but should not be constrained by such a requirement.
5. Methods should be developed for molecular labeling of live and intact zooplankters.
6. Some sampling for biomolecular characters will have to be conducted beyond the boundaries of designated study areas in order to develop reliable genetic markers and understand possible shifts in genetic structure and physiology of local populations.