Methodology and Instrumentation

## Methodology and Instrumentation Needs and Development

The new methods and technologies that now exist, or that are under development and being tested, will allow one to look at the physical and biological processes occurring on Georges Bank in ways not previously possible. These new developments will allow more complete spatial surveys, more detailed processes studies and longer time-series observations of the combined physical and biological parameters over Georges Bank. It is with this additional information, supplied by the combined observational data set and modeling efforts, that the complex questions outlined above can be addressed. This will aid our understanding of how physical processes influence the biological processes which control the distribution, abundance, production and population dynamics of planktonic organisms on Georges Bank.

Well established technologies such as CTDs, current meters, towed nets, and drifters should be used along with the new methods to extend our observational capabilities. Acoustical and optical instruments could be moored, towed and profiled along with established technologies to give a unique combination of biological and physical information. These new techniques are in a trial and growth period that requires additional field and laboratory testing and calibration. This development and testing program should be carried out within the context of an ongoing scientific program that can supply complementary and redundant information to evaluate and understand the performance of new instrumentation. This redundancy and overlapping data should strengthen the program considerably, as well as advance the evaluation of new technology. To be considered for funding, new instruments and methods should be in a fairly mature state of development that will allow field deployment in the early stages of the experiment. Some of the appropriate technologies and considerations are outlined and discussed below.

- Integrated Shipboard Profiled and Towed Survey Platforms: The broad-scale surveys and specific fine-scale process studies will require shipboard observations made with towed and profiled instruments to resolve the horizontal and vertical structure of the physical oceanographic variables and the biological environment, and to identify the number, size and type of biological organisms present. Existing shipboard platforms should be equipped with new combined sensing capability to provide the ability to sample from the surface to the bottom in the shallow waters of Georges Bank. The broad-scale survey should provide the gross spatial structure of the physical and biological parameters into which the more detailed fine-scale process studies fit.

  The vertical structure of the water column should be measured by profilers with CTD, bio-optical (Dickey, 1991), optical (Gardner et al., 1990), acoustical (GLOBEC, 1991e), and optical imaging (Davis, et al., in press) instrument suites. (Table 3 lists the various sensors included in each instrument suite.) These profiling systems should also have the ability to collect multiple water samples for shipboard or laboratory analysis of oxygen, salinity, nutrients, and chlorophyll. For biota identification, these profiling systems also should be integrated with pumped profilers. For the broad-scale shipboard surveys, 1-meter vertical spacing on most observations should be adequate. For turbulent mixing processes, centimeter or finer resolution may require free-fall microstructure (temperature, salinity and velocity) resolving instruments.

  To resolve the spatial distribution, size, and number of species present, towed multiple-net sampling systems (such as the MOCNESS and bongo nets) should be employed for the broad-scale surveys as well as some process studies. CTD sensors should be added to the towed nets systems to give a record of the depth and water properties where the nets were deployed and should be of a quality to supplement the profiled and towed platform (see below) data. Some bio-optical and acoustical sensors should be included on the towed nets as appropriate. Data on species, number, size and age structure from these net tows should be used for in situ calibration and intercalibration of acoustical imaging sensors (see below).

  Towed integrated biological/physical instrument platforms (such as SEASOAR) should sample the horizontal structure in more detail than shipboard profilers, as well as have the ability to sample the vertical, but with less resolution than shipboard or free-fall profiling systems. These towed platforms should have CTD, acoustical imaging, bio-optical, and optical imaging instrument suites (Table 3). They should be used in addition to vertical profilers for more detailed process studies of the spatial structure of the physical and biological environment over the Bank. For studies associated with fine-scale mixing processes, microstructure temperature, conductivity and velocity probes should be added to the towed platforms to measure the spatial structure of the turbulence and energy dissipation rates.

  To determine the abundance, taxonomy and size distribution of plankton on scales from a few micrometers to centimeters, optical imaging systems (such as video cameras with magnifying optics, holographic systems and range-gated laser systems) should be used as part of the broad-scale survey as well as in specific process studies. Development of image processing systems for pattern recognition should be a critical component of this program. In addition to automating the counting of plankton samples, moored optical imaging systems could collect optical time series of plankton abundance. Data compression and processing techniques should be developed to reduce the in situ storage requirements and to allow telemetry of video data from ships and moored platforms.

  For the measurement of fine-scale distributions and in situ rate processes such as swimming and feeding behaviors, and for microturbulence measurements, large Remotely Operated Vehicles (ROVs) and/or Autonomous Underwater Vehicles (AUVs) could also be used as stable remote platforms. ROVs and AUVs could be used to make process study measurements at specific locations within the water column and could be used for fine-scale three-dimensional mapping in frontal and high gradient regions as well as the bottom-boundary layer.

- Acoustic Doppler Current Profilers (ADCPs): ADCPs should be used to obtain the required vertical profiles of the velocity field as either a function of position (shipboard surveys) or as a function of time (moorings). The broad-scale shipboard surveys of the velocity structure should be used to complement the moored current observations by putting them in context of the flow field, while the moorings should put the shipboard surveys in temporal context. The new broadband ADCP (Brumley et al., 1991) will provide the finer resolution in both space (3 m vertically) and time (1 min) required to resolve smaller spatial scales associated with shear, internal wave mixing and cross-frontal exchange. To address the stability of the water column and relationship to velocity shear, moored ADCP data should be utilized with the density time series. For shipboard navigation, GPS positions integrated with bottom tracking should be used in depths to 200 meters. To assure that current meter observations are adequate for monitoring the small space and short time scales required for the process studies, tests and calibrations should be done in both shipboard and moored configurations.

  The broadband ADCP also monitors the amplitude of the backscattered signal and has the potential for producing profiles of scattering and biomass estimates by measuring profiles of volume scattering strength. This measurement could be used to estimate zooplankton abundance and biomass time series and profiles (see Flagg and Smith, 1989a, 1989b for results from older style, narrow-band ADCPs). When coupled with traditional biological sampling techniques on shipboard surveys, the spatial distribution of the zooplankton can be estimated. It is important that the moored time-series observations of backscatter profiles should provide qualitative time histories of biomass variations, although not as detailed as more advanced acoustical imaging instruments.

  As with all acoustical backscattering instruments, calibration and intercomparison with net tows, etc., should be made before any quantitative estimates of the spatial or temporal distributions of zooplankton and biomass are made. The calibration and testing of new ADCP systems for velocity profiling, spatial resolution, bottom tracking, and acoustical backscattering need to be completed before their results can be used in the Georges Bank program. This evaluation should be done in conjunction with the acoustical imaging systems discussed below. Research may be needed on algorithm development and on measurement of target strength of individual organisms.

- Acoustical Imaging: The recently held workshop on acoustical technology details the technological tools and advances required to support the U.S. GLOBEC research initiative (GLOBEC, 1991e). As indicated above, there are a variety of applications for acoustical imaging sensors where information is required about zooplankton biomass, numbers, and size information as a function of time and space. The development of acoustical imaging technology to estimate these parameters (i.e., as volume backscattering, numbers, and target strength) has increased markedly during the past several years, but there is a need for continued basic research and development to achieve a level of functionality in acoustical remote sensing of marine animals commensurate with the programmatic requirements. The Georges Bank Study will require a combination of moored, towed, and profiling acoustical instruments that use multi-frequency, dual-beam, or split-beam techniques.

  In addition to the development of new sensor packages and new methods for quantitative combination of multi-frequency acoustical data, there is a need for continued development of better models of acoustical scattering from all marine organisms (e.g., various taxa of zooplankton, with special emphasis on the target species) and testing of the models through acquisition of acoustical backscatter data from single individuals, as a function of orientation, and groups of individuals. Procedures to intercalibrate the various acoustical sensors likely to be used should be established at the beginning of the program. A bio-acoustics group should be established to provide technical assistance with instruments, data processing, and calibration services and should be coordinated with the bio-optical sensor calibrations.

- Integrated Moored Platforms with Adaptive Sampling and Telemetry: Time-series observations of atmospheric variables are required to measure the atmospheric forcing (momentum, heat and buoyancy fluxes) over Georges Bank. The more complete meteorological sensor suite (Table 3) on the U.S. GLOBEC mooring in the center of Georges Bank should provide the primary data for the experiment and should be used with observations made by the three NOAA NDBC buoys moored around the Bank (Figure 12) to resolve the spatial structure of the forcing. Technologies such as Doppler SODARs should be considered to improve momentum flux measurements by directly observing the vertical wind profiles rather than measuring the velocity at one height and applying bulk parameterization models to estimate stress.

  Moored buoys and bottom mounted platforms should be placed at specific locations around the Bank for the broad-scale, time-series observations as well as for fine-scale process studies (Figure 12). Various sensor systems, including CTD, bottom pressure gauges, bio-optical, current meter, ADCP (for velocity profiles), systems for acoustical biological profiling, and optical imaging systems (for taxonomic identification, size and number), should be placed at various depths below the surface to give combined physical/ biological time series at critical locations. Bottom-mounted tripods should measure variables in the the bottom-boundary layer, as well as resolve the surface wave field with bottom pressure sensors.

  Powering, controlling and recording data from these various sensors will require more complex data systems with higher capacity recorders and compression schemes for optimal use of the recording space and telemetry links. Power, supplied by solar panels and batteries, will have to be controlled and conditional sampling programs devised to provide long-term unaliased averages yet still sample any high frequency internal wave, turbulent mixing and biological events. Optical systems will require additional in situ image processing and compression developments (with digital signal processors) before they can be routinely used in moored applications.

- Real-Time Data Management and Distribution: To take advantage of the potential for increased understanding and better utilization of ship time, a data management component should develop and utilize two-way data links to routinely relay information from instrument systems in the field to a "real-time" data base in the Georges Bank data center. This data base should be accessable by project investigators in the field as well as in their laboratories. Developments in networking ships and remote platforms together (as is now done on land by computer networks such as INTERNET) should be used to relay data (i.e., satellite imagery, moored data, and preliminary model results) from the Georges Bank data management center to the ships in the field for real-time use in planning operations. Thus, the data management office should develop and/or adapt the methodology of receiving, cataloging, and processing and distributing the real-time data (with lower data quality but faster access time) to project investigators, and at the same time deal with the slower process of editing, checking data quality, archiving and distributing the high-quality, final data sets to the project investigators and the oceanographic community. As an integrated study, this will involve time series, spatial series, optical images, physical and biological samples, satellite data, etc., in the data base with directory.

  Subsurface instruments not connected directly to the surface should communicate to the data center through additional acoustical telemetry links to surface platforms. These devices have been demonstrated in test applications (Catapovic et al., 1991), and now need to be proven in general oceanographic applications. The protocol and interfacing with bottom instrumentation needs to be completed and tested. The telemetry schemes need to be reviewed, and integrated into the moored and shipboard instrumentation and into the data management plan.

- Drifters: Lagrangian drifters should play an important role in illustrating water movements such as the entrainment of source water from the Gulf of Maine, the circulation around the crest of Georges Bank, the leakage of water into the North Atlantic via cross-frontal exchange, and the movement of water down the New England shelf. They should be used to "tag" water masses or regions containing a biological population to aid in following and identifying changes in the water or biology with time. Drifters should be able to be drogued at varying depths, and have GPS tracking for the accurate, closely spaced positions required for detailed process studies. Drifters need to be developed that are not influenced by surface phenomena, otherwise they may "slip" away from a subsurface patch of animals that is being studied. The drifters should be deployable from both aircraft and ships to allow the optimum deployment distributions. The drifters should be low enough in cost that using large numbers should not be prohibitively expensive. Drifter life should exceed 3 to 6 months.

  In addition to position, the drifters should also measure sea surface temperature and conductivity to aid in the interpretation of the satellite surface temperature maps and shipboard surveys, and for characterization of water masses. Larger integrated drifting platforms could be constructed with the capability of carrying most of the sensors listed in Table 3. Consideration should be given to the development and use of a neutrally buoyant, subsurface drifter that can follow an isotherm or isolume to mimic the diel migration of animals, and can follow a patch of water or organisms to aid in studies of retention and exchange, and relationships between vertical distribution and current structure. These drifters should have the capacity to be tracked acoustically from ship.

- Molecular Technology: Recent advances in the fields of biochemistry, molecular biology, molecular genetics, and immunology offer considerable potential to provide unprecedented insight into the feeding success, growth rate, physiological status, and genetic makeup of individual organisms collected at sea (Powers et al., 1990; Powers, 1991). Advances in molecular technology should greatly increase our understanding of the relations among variability in the physical environment and biological responses at the molecular, organismal, and population levels.

  Much basic work must be done with the organisms of interest in order to put molecular biological approaches to work to answer ecological questions. Nucleic acid base sequence data are fundamental to a variety of technical approaches, and should be considereda foundation of molecular ecological work on Georges Bank. Base sequence data may allow the design of species- and population-specific molecular probes, and the development of rapid means of identification and discrimination of taxa. Nucleic acid hybridization probe technology can provide information on the identity, growth rate, physiological status, and reproductive condition of individual organisms, as well as information on the genetic relationships among individuals, cohorts, and populations. The concentration of RNA and the RNA-DNA ratio have been used to estimate feeding success and recent growth in a wide variety of marine organisms including larval fish (Buckley and Lough, 1987) and crustaceans (Wang and Stickle, 1986). Detection and measurement of specific RNAs using nucleic acid hybridization probes should provide more detailed information on growth rate and physiological status.

  The analysis of recruitment, production, and dispersal processes of the target species on Georges Bank may be addressed through population genetic approaches. Many fundamental population genetic processes for planktonic marine species are unknown, including effective population size, migration and gene flow patterns, population structure, and temporal stability of genetic characteristics. These questions could be addressed explicitly for the target species using current molecular and biochemical techniques. In addition, there should be a focussed effort to develop rapid and sensitive molecular techniques to enable screening of the many individuals necessary for ecological studies. While existing techniques can be used to assay molecular characteristics of formalin-preserved animals, these techniques need to be improved and enhanced so that existing collections can be utilized to develop the historical context for the population genetic information that will result from the Georges Bank Study.

  The field of biotechnology is developing at a rapid pace, and the successful application of new techniques to oceanographic research will require interdisciplinary planning, research and development. A posteriori assimilation of published techniques will not provide tools and answers at the desired pace; an accelerated plan to address specific oceanographic goals is needed. To this end, U.S. GLOBEC convened a workshop in Fall 1990 on the application of biotechnology to field studies of the plankton (GLOBEC, 1991d), and in 1992 initiated a program of funding for biotechnology research and development. Two studies have been funded:

  - Estimation of recent growth and physiological condition of marine fish larvae and zooplankton using nucleic acid hybridization probes: T. Durbin, A. Durbin and L. Buckley -- This project is an effort to use different roles, half-lives and properties of messenger RNA and ribosomal RNA to develop new indices of recent growth and physiological status of larval Atlantic cod (Gadus morhua) and the copepod Calanus finmarchicus. The concentration of rRNA and selected mRNAs will be determined using probes developed for these species. RNA concentrations will be determined under a variety of conditions imposed in culture.

  - Physiological status of zooplankton: Utilizing PCR to rapidly assess specific enzyme expression: D. Crawford and L.Incze -- Molecular techniques will be developed to rapidly identify and quantify the level of expression of physiologically significant genes in the copepod Calanus finmarchicus. The techniques take advantage of the specificity and sensitivity of the polymerase chain reaction (PCR) to quantitatively amplify species-specific mRNAs that encode enzymes regulating metabolic fluxes. Species-specific primers will be designed using phylogenetically conserved primary sequences of several enzymes. The effect of environmental parameters on levels of gene expression will be investigated using animals of various stages held in culture and captured at sea. The concentration of product from a physiologically invariant gene will be measured as a potential datum for normalizing results in order to perform analyses on taxonomically and developmentally complex assemblages of zooplankton.

- Biochemical Techniques: Feeding success, important for growth and survival of larval fish, can be studied by stomach content observations and by analysis of the condition and chemical composition of individuals. Stable isotope analyses could be used in feeding studies to detect the origins of carbon and nitrogen in the diet (Thayer et al., 1983). Isotopic studies usually encompass several tropic levels, and suggest patterns of chemical connectedness between organisms such as phytoplankton, zooplankton and fish. This connectedness is one of the only direct ways to estimate energy flow in planktonic food webs, and is a powerful way to check trophic relations inferred from stomach content studies (Fry, 1988).

  The distributional histories of larval cod on Georges Bank will be investigated during the spring and early summer period of developing thermal stratification, a time during which the intricacies of their vertical and areal distributions may hold important implications for larval survival and ultimately recruitment. The recently developed technique of otolith analysis (Sr/Ca ratios) (Radtke, 1988; Townsend, et al., 1989; Radtke et al., 1990) could be used to hindcast the temperature histories, and hence the distributional histories (from time of hatching to capture) of individual cod larvae collected in various hydrographic regimes in the Bank area. This technique could be advanced by employing electron energy loss spectroscopy, which will increase the resolution of the Sr/Ca technique to a level capable of resolving sub-daily temperature histories. These analyses could reveal diel migrations into or through the thermocline.

  With any new technique, intercalibration and comparison among the new measurement systems and techniques will be critical to assure highest data quality and enhanced interpretation. Pre- and post-cruise calibrations of all sensors will be necessary, especially extensive calibrations and validations of the acoustical sensing systems. In situ calibrations and comparisons between moored and profiling instruments, and net tow samples should be used to identity and correct for sensor malfunction and drift, and will increase the data quality required for U.S. GLOBEC integrated studies. The calibrations and acceptance of new biochemical techniques may require dedicated laboratory experiments.