Appendix D

# Appendix D. Technology Working Group Report

Chair: Peter Ortner

Participants: Ann Bucklin, Tim Cowles, Gus Paffenhofer

## Introduction

From our perspective U.S. GLOBEC is an effort to understand the relationship between physical forcing processes and the dynamics of marine animal populations. The northern Arabian Sea would be an especially appropriate study site because it is dominated by regular but highly dynamic physical processes (monsoonal wind reversals and oceanic upwelling). These are closely correlated with a dramatic biological signal (enhanced plant pigment in CZCS images). The amplitude of this signal exceeds that in any other oceanic regime. One cannot but suppose that population variability at higher trophic levels must be, at least in part, regulated by this periodicity. Moreover the Arabian Sea/Indian Ocean is scheduled to be the site of extensive study under the aegis of the U.S. JGOFS, WOCE and ONR/ARI programs. This represents an enormous practical opportunity. At absolutely no cost to U.S. GLOBEC, the physical variability and primary production cycle in the Arabian Sea will be characterized. Moreover these same programs will support extensive modelling efforts aimed at elucidating the dynamics of the Arabian Sea/Indian Ocean. The level of detailed understanding anticipated greatly exceeds that available to date for any other major oceanographic system.

The zooplankton and fish working groups have posed a set of interrelated biological-physical questions that might be addressed by an U.S. GLOBEC program in the Arabian Sea. The primary charge of the technology working group was to consider how these questions can best be answered, what tradeoffs would be involved and what priorities we would assign to specific technical alternatives. There are a number of practical considerations. First, to be co-extensive with U.S. JGOFS and WOCE means U.S. GLOBEC will be in the Arabian Sea as soon as the winter of 1994/95. This implies the use of currently available technology, or that already being field tested, in preference to technologies requiring considerable further development followed by field testing. Second, during the SW monsoon, winds are routinely in excess of 35 kt and the sea state is correspondingly high. This implies ruggedness and sea-state independence are important considerations in selecting sampling gear and experimental methods. Third, at some seasons the Arabian Sea varies between near eutrophy and extreme oligotrophy over relatively short distances. This implies that a wide dynamic range in instrumentation sensitivity is an important consideration. Last, fieldwork already planned in U.S. JGOFS and WOCE seemingly offers U.S. GLOBEC practical cost-effective sampling opportunities. However, inherent constraints associated with these include predetermined tracklines and mooring locations as well as limited wiretime and bunkspace. These opportunities have to be closely scrutinized in light of specific U.S. GLOBEC scientific objectives.

## Mesopelagic Fish Sampling Issues and Recommendations

Given the lack of basic knowledge about both abundance and distribution and basic biology (reproduction, growth, feeding, and mortality) in the Arabian Sea, there is absolutely no question that purely indirect sampling methods, e.g., acoustics, will be insufficient for U.S. GLOBEC. Net sampling will be required since individual organisms will need to be collected. It is possible no single sampling device will suffice. Vertical distribution studies will certainly require some sort of multiple net sampler like an 8 square meter (or larger) RMT net. To be sure that avoidance is not a significant problem, a larger net, perhaps a 100 m2 line rope trawl with multiple cod ends, may be desirable. Another alternative might be a larger 25 m2 RMT. While the RMT nets can be deployed from a properly equipped oceanographic vessel, a trawl is almost certain to require a commercial dragger.

Whatever net sampler is selected we strongly recommend the use of a self-contained CTD with an oxygen probe on every tow. These data are commonly collected in conjunction with plankton sampling but rarely with mesopelagic fish sampling. In the U.S. GLOBEC context these data are critical. Moreover, the net sampling needs to be guided by state-of-the-art fish sonar (mapping) and echo sounder (integrator) records. All ships used as sampling platforms will have to be suitably equipped.

Use of U.S. JGOFS or WOCE platforms is likely to be limited in regard to mesopelagic fish studies. Assuming ADCP units are calibrated (see zooplankton section below) their backscatter intensity may yield some scattering layer information (large-scale patterns). At the likely frequencies (>= 150 kHz), a substantial part of the ADCP return will be associated with zooplankton rather than larval or adult fish. Moreover, at these relatively high frequencies, no data will be obtained at the lower part of the oxygen minimum, nor below it. Yet some of the most significant biological questions posed by the zooplankton and fish working groups relate to the dysoxic zone. We recommend that every effort be made to equip all research vessels in the study area, including those used by U.S. JGOFS and WOCE, with a calibrated acoustical sensor suite with the appropriate frequencies. These data will greatly extend the generality of inferences to be made from specifically U.S. GLOBEC sampling efforts.

If a dragger is leased it too will need similar acoustic capability. It could then be used for acoustic surveys in the interstices between regular trawl sampling cruises. This may be essential to ground-truth the acoustic data. Moreover, for the data to be quantitative renewed efforts will have to be made to measure, or more accurately estimate, target strength at the chosen frequency for the dominant mesopelagic species found in the trawls. Whatever acoustical system is selected must have the capacity for digital data storage rather than yielding a simple hardcopy record.

An option worth consideration is acoustical instrumentation on U.S. JGOFS surface moorings or using Lagrangian drifter packages. The former will be deployed for extended periods throughout the annual cycle, i.e., between whatever U.S. GLOBEC process cruises are taken, and will be regularly serviced. A formidable constraint would appear to be data transmission of moored and drifting instruments. Considerable thought and testing would be required given the distance from the coast of planned mooring sites. Current satellite data links are not adequate for most acoustic data given its relatively high density, but the situation could change over the next several years. Quite likely only expendable Lagrangian devices would be practical and it is not yet evident that these will be affordable.

Last, there has been discussion of direct visualization of feeding and/or in situ physiological measurements using ROVs or submersibles. While these alternatives are feasible they are very expensive. Given the remote study site, logistical costs would be considerable. Prior use of such devices has required either a devoted tender vessel or a considerable number of days of multipurpose research vessel shiptime. The latter is likely to be incompatible with other sampling requirements for U.S. GLOBEC cruises. Moreover, ROV or submersible deployment and retrieval may be hampered by severe weather; a likely condition in the Arabian Sea, at least during the SW monsoon.

## Plankton Sampling Issues and Recommendations

As with the mesopelagic fish, indirect sampling will, by itself, be insufficient to meet U.S. GLOBEC objectives. Conventional net-sampling will be required to obtain animals for determination of reproduction, growth, feeding, and physiology, as well as to provide definitive taxonomic identification. It is expected that microzooplankton as well as juvenile and adult stages of the dominant mesozooplankton will have to be collected, identified and enumerated. This implies the use of more than one net sampler and perhaps bottle samples as well. As with mesopelagic fish all such conventional sampling gear will have to be fully instrumented (temperature, salinity, depth, fluorescence, oxygen). Deployment should be guided where possible by real-time feedback of these environmental parameters as well as of zooplankton biomass distribution. The latter can be obtained with available high frequency acoustics.

ADCP biomass estimation must be made a routine aspect of all U.S. JGOFS, WOCE and U.S. GLOBEC cruises. This requires a complex system calibration and the choice of appropriate software options from the manufacturers applications package. It can be accomplished with minimal interference with other aspects of the U.S. JGOFS or WOCE cruises. Given our present lack of knowledge of overall zooplankton biomass distribution patterns and their relationship to Arabian Sea hydrography, a considerable effort should be devoted to quantifying general patterns along proposed transects. The ADCP approach and any other that can obtain data underway will be invaluable in mapping efforts as well as subsequent hypothesis testing.

We believe it will be necessary to employ the latest developments in acoustical and optical zooplankton distribution assessment. As discussed in U.S. GLOBEC Report 4 from the Workshop on Acoustical Technology and the Integration of Acoustic and Optical Sampling Methods, these are often most efficiently deployed in conjunction. Specifically, optical approaches can be used to identify acoustic targets. Since U.S. GLOBEC is interested in populations, not simply biomass distribution or particle size, this capability is essential. An alternate approach is to use optical particle counting and sizing in conjunction with image visualization. Both approaches have already been used in the field. With imaging video the major remaining technical difficulty is efficient data processing (automated image analysis). In its present state, however, the technology is thought to be closer to satisfying the target identification objective than functioning as a routine primary sampler. Prototypes have a suitable wide dynamic range and have, in some cases, already been integrated with the recommended suite of U.S. GLOBEC environmental sensors. Suitable tow vehicles would have to be adapted for U.S. GLOBEC that permit long-term virtually unmonitored deployment and tow-yo cycling in relatively high sea states, but such vehicles are now commercially available, e.g., Batfish, Seasoar, Aquashuttle, etc. The patterns indirectly observed and mapped will be ground-truthed by systematic use of conventional samplers for U.S. GLOBEC process studies. Indirect and direct (or traditional) sampling are not alternatives but rather mutually essential complements. With the former rigorous relationships to physical parameters and extensive data sets may be efficiently collected at high spatial and/or temporal resolution. With the latter species identification may be validated and specimens collected.

In addition to the survey sampling discussed above the questions raised imply the desirability of obtaining a more extended series of observations within the various hydrographic domains within the northern Arabian Sea, e.g., an open ocean upwelling site, an oligotrophic downwelling site, and a coastal upwelling site. Time-series data is essential to fulfilling some of the U.S. GLOBEC objectives. There are basically three non-exclusive alternatives. Each has advantages and disadvantages. Ship-based studies could be made at fixed location near one of the U.S. JGOFS moorings, acoustical or optical sensors could be incorporated into these or other moorings, or Lagrangian drifters could be constructed specifically for U.S. GLOBEC. As discussed above, the realistic constraints are likely to be data transmission and "recoverability", rather than equipment sensitivity, ruggedness, etc. Prototype acoustical and optical systems have already been tested in the field, but have not been used in sites so remote for long periods.

A range of techniques are available to evaluate physiology, growth, reproduction and feeding rates of micro- and mesozooplankton. While in situ methods have been discussed it is unlikely these will be ready for routine use by 1994/95. Indeed they may never be suitable for use in the demanding conditions anticipated. The measurements must be usable despite extreme sea states, high ambient animal densities, and high ambient temperatures (> 25 deg C). This may require considerable adaptation of existing techniques (e.g., consider the adaptability of large volume incubation methods). These problems require addressing before any proposed field study. These practical considerations also suggest that if they can be made sufficiently specific and rigorous, biochemical and molecular approaches may be a critical component of an Arabian Sea U.S. GLOBEC since they can be performed on appropriately preserved specimens. We encourage sample preservation and curation sufficient to permit subsequent molecular systematic analysis. The optimum platform for zooplankton work is likely to be a UNOLS research vessel. Unfortunately, the work requires extensive shiptime and would in its entirety be incompatible with U.S. JGOFS and WOCE field plans. Beneficial use might be made of ships-of-opportunity. For example, equipping the U.S. JGOFS CTD system with a simple (2-4 frequency) acoustic sensor would appear to be a cost-effective way to obtain better data coverage. The same would apply to WOCE efforts within the northern Arabian Sea, but not to WOCE cruises considerably further south in the Indian Ocean.

U.S. JGOFS apparently plans to fund limited zooplankton studies. Rather than pursuing a token effort, we recommend an integrative exhaustive study that can satisfy U.S. JGOFS basic requirements and as well the more extensive demands of U.S. GLOBEC. While this implies somewhat more of an imposition (bunks, wiretime) upon U.S. JGOFS cruises it likely represents a more efficient overall utilization of limited NSF Global Climate Change Program resources. Conversely, a U.S. GLOBEC vessel should certainly be equipped with continuous sensing capability (chlorophyll, irradiance, meteorological parameters, etc.) that will assist U.S. JGOFS (and possible WOCE) to address their scientific objectives.

## Molecular and Biochemical Approaches to Arabian Sea Studies

Molecular approaches may be appropriate for use in the study of a variety of biological phenomena, including physiological rates and conditions, feeding rates and dietary components, the genetic basis of phenotypic variation, genetic exchange across species distributions, recruitment processes, and taxonomic identification of species (see U.S. GLOBEC Report Number 3, U.S. GLOBEC Workshop on Biotechnology Applications to Field Studies of Zooplankton, 1991).

The questions posed by the zooplankton and fish working groups of the Arabian Sea workshop provide the opportunity to apply molecular-based techniques in a fruitful way. Only molecular methodologies and approaches that will be ready for implementation (i.e., already field-tested) by fall 1994 should be considered for inclusion in the program. However, we must prepare ahead so that zooplankton and fish sample collection, preservation, and curation are done appropriately so that molecular and biochemical methods can be used later.

Fundamental information is needed to continue the transfer of biotechnology to the ocean sciences. Examples are DNA base sequencing of targeted regions of the genome and careful determination of levels of intra- and inter-specific variation. This need is particularly critical for marine invertebrates, for which very little molecular information is available. Once DNA sequence data are available, appropriate strategies may be developed to address physiological, ecological, and evolutionary questions.

Although automated taxonomic identification of plankton may not be achieved in the near future, molecular characteristics may be helpful in identifying zooplankton or fish species that are difficult or impossible to distinguish using morphological characters and in resolving systematic questions regarding the limits of a species. The U.S. GLOBEC Arabian Sea program offers an opportunity to study an ecologically important, but little-known, fauna. The zooplankton samples in particular should be collected and curated carefully to allow analysis of molecular diversity within and between species.

Molecular approaches to discriminating conspecific populations of fish and zooplankton will be useful for the U.S. GLOBEC Arabian Sea program. Prior U.S. GLOBEC workshops have identified a need for basic information on the amount of molecular genetic variation within and between populations of marine organisms. This information will lay the groundwork for studies of the effects of global climate change on marine populations and for analysis of the genetic basis of phenotypic variation. In the Arabian Sea, it is essential to determine whether Arabian Sea populations of widespread species (such as the myctophid fishes) are isolated from populations outside the region. For endemic species, analysis of population genetic structure will address questions of recruitment patterns and dispersal.

We expect that molecular analysis of nutritional status and metabolism will be possible as part of the U.S. GLOBEC Arabian Sea program. Molecular studies are ongoing to quantify the level of expression of physiologically significant genes in zooplankton, and RNA/DNA ratios are now used in field studies of growth and condition in ichthyoplankton. These and related condition factors can address questions of how plankters and fish respond to physical forcing and cope with seasonal variation in their environment.

There are molecular approaches which offer promise for the analysis of dietary components. Taxon-specific probes for identification of gut contents are feasible, and will discriminate herbivores and carnivores. However, potential difficulties with this approach indicate that implementation in 1994/95 is unlikely. Biochemical approaches, such as immunological and pigment-based identification of prey species and isotopic analysis to determine trophic level, are better developed and may provide indications of trophic relationships in the Arabian Sea ecosystem. Additionally, priority should be given to the development of molecular and/or biochemical analysis of feeding rates in field populations.

Population genetic approaches can be used to address significant questions in the marine environment: recruitment variation, ontogenetic shifts in gene frequency, geographic structure of the ecosystem at different scales, etc. Protein and nucleic acid polymorphisms are powerful markers of population structure. U.S. GLOBEC should encourage molecular studies of oceanic life processes; a specific goal should be the sequencing of DNA for targeted regions of the genome of marine, especially invertebrate, species.

U.S. GLOBEC is also an opportunity to institute changes in the way that biological samples are collected and preserved on oceanographic cruises. Formalin preservation, the norm, is not appropriate for molecular analyses. Biochemical analyses require frozen tissue, which can also by used for molecular studies. Alcohol preservation is suitable for many studies as well. Splitting of collections and preservation by a variety of means is highly desirable. At the minimum, 1/8 to 1/4 of all samples should be preserved in alcohol to allow a variety of molecular and genetic techniques to be used for future study. U.S. GLOBEC should emphasize the importance of existing sample archives and plan for molecular and biochemical analyses of future collections. As new techniques are developed, appropriately archived samples may become useful and important in ways not envisioned now. Time-series collections of plankton from around the world may prove essential for unraveling questions about global climate change.