### APPENDIX II: DRAFT RFP FOR PHYSIOLOGICAL RATES AND CONDITION

GLOBEC CALL FOR RESEARCH PROPOSALS CONCERNING THE EXPLOITATION OF BIOCHEMICAL AND MOLECULAR TECHNIQUES FOR GAUGING PHYSIOLOGICAL STATE OF MARINE ZOOPLANKTON

General Goals

GLOBEC seeks to encourage the development of biochemical and molecular methods which can be used in laboratory and shipboard analysis of the physiological state of marine zooplankton. Methods are sought for obtaining accurate estimates of rates of metabolism, growth, reproduction, and physiological status--locomotory ability, dietary state, life stage, reproductive condition and morbidity--that can be applied to both invertebrates and fishes. The methods should be capable of assessing how changes in the environment, for example in seawater temperature, impact secondary production in different marine systems.

Specific Objectives

The rapid development of biochemical and molecular methods provides biological oceanographers with a rich potential for using new procedures to address long-standing questions about fundamental marine processes. Included among these are rates of secondary production and the characteristics of the organisms responsible for this production. Biochemical and molecular methods show potential for rapidly advancing our understanding of each of the following areas.

1. Indices of rate processes. The rates of metabolism, growth, and reproduction of zooplankton are amenable to analysis by simple biochemical and molecular methods, e.g., enzymatic indices. Enzymatic indicators appropriate to these rate processes need to be developed for major classes of zooplankters. These enzymatic indicators should be developed for shipboard analysis of large numbers of samples. Consideration should be given to the development of indices that can be applied to analysis of individual organisms, including very small species, and to unsorted collections of organisms.

2. Indices of physiological state. Characterizing the physiological state of marine zooplankton is critical for analyzing the factors governing secondary production. Biochemical and molecular approaches to indexing such key physiological characteristics as growth rate, reproductive state, developmental stage, and morbidity are necessary. The regulatory mechanisms and environmental factors governing diapause and quiescence in copepods need to be understood. Among the potential methods for addressing these diverse questions are: (i) procedures for quantifying nucleic acids, e.g., RNA:DNA ratios; (ii) enzymatic techniques for quantifying nucleic acid synthesis, e.g., DNA polymerase activity; (iii) antibody techniques for identifying and quantifying the egg populations in seawater samples; (iv) high resolution gel electrophoretic techniques,e.g., 2- dimensional gels, for characterizing the taxonomic compositions of populations and plankters, (v) enzymatic, immunochemical, molecular genetic, flow cytometric approaches and other highly sensitive chemical methods for assessing nutritional state and dietary composition; (vi) methods for quantifying the rate of chitin synthesis; and (vii) high sensitivity chemical techniques for estimating such growth- and reproduction-related chemicals as molting hormones and egg-releasing factors.

Target Species

There is a dearth of biochemical and molecular data on most marine invertebrate zooplankters. The development of biochemical and molecular indicator methodologies must entail substantial initial characterization of the biochemistries and molecular biology of key species of invertebrate zooplankters like copepods. Study species should include organisms for which one or more life stages is apt to be strongly impacted by global warming.

Training Components of Research Proposals and Cross-Calibration Among Laboratories

The successful design and implementation of biochemical and molecular indicator methodologies for use with marine zooplankton will require training regimens for biological oceanographers not familiar with these methods. Investigators submitting proposals should address the issue of necessary training in their submissions. Because it is desirable that different workers employ biochemical and molecular methods that will allow cross-calibration of data sets, investigators are encouraged to consult with colleagues who plan to use similar approaches, perhaps with different species or in different marine environments. Proper design (e.g., using physiologically realistic in vitro measurement conditions) of protocols which can be standardized among laboratories will greatly facilitate the exploitation of biochemical and molecular indicator methods for addressing major questions concerning secondary production and the impact of environmental change on this production.