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Technological development of a high resolution, rapid survey capability to identify spawning habitat of large pelagic fishes

PI: Robert Cowen, University of Miami

PROJECT DESCRIPTION:
We proposed to improve the speed, accuracy and precision of ichthyoplankton surveys via the development and application of two technological advancements. Each is operable (and advances our capabilities) individually, but they are synergistically linked to maximize our ability to rapidly and quantitatively survey and identify spawning habitat of large pelagics based on early larval stages. Specifically – we propose to: 1) make operational a rapid, very high resolution imaging system capable of sampling sufficiently large volumes of water to accurately count larval fish in situ ; and 2) establish a high-throughput DNA sequencing protocol to rapidly identify larval fish samples taken for validation and biological sample purposes.

PROGRESS thru 10/31/2006:
1) Complete development of, and test ISIIS (In situ Ichthyoplankton Imaging for rapid survey of egg and larval scombroids).
We have completed the design of our prototype system, manufactured all components and assembled ISIIS. We have successfully deployed the system for one short trial (~ 1.5 hours in the water) as a proof of concept and to detect design short-comings. We were able to do this initial sea trial by piggy-backing on another colleague's cruise, where we were able to have several hours of wire time. We are pleased to report very positive results, though also recognize the need for several critical refinements prior to attempting a full field test (planned for year two). The critical refinement has to do with the best means of increasing the volume imaged. We originally proposed a second (duplicate) system wedded to the first thereby imaging twice the volume. We are now also examining an alternative approach that might yield an even greater increase while at the same time increasing resolution.

2) Develop high-throughput molecular sequencing of cytochrome B portion of mtDNA for all Atlantic scombroids.
During the first six months, the molecular identification technique was expanded to include Atlantic Thunnus, Auxis and Coryphaena species, in addition to its previous application with istiophorids. This choice of taxa includes those species with larvae that cannot be identified morphologically. We have continued to apply this technique to our large collection of pelagic larvae from the Straits of Florida . To date ~2000 larvae have been identified using this methodology. Ultimately this dataset will be used in a comparative study of the temporal and spatial distribution of spawning of pelagic species in the Straits of Florida.

PRELIMINARY DATA:
Obj. 1 – The best illustration of our success with ISIIS thus far is a set of images obtained in the Gulf Stream while towing the system at 5 knots! We estimated that our camera system was quantitatively imaging approximately 70 l/s which is equivalent to about 10% of that of a typical 1 m2 plankton net. Our estimated densities measured from the camera were comparable to larval fish density estimates obtained for the same water mass, same time of year (but different years) collected with a 1 m2 MOCNESS net (1200 larvae per 1000 m3 vs. 950 larvae per 1000 m3 , respectively; SD= 180 and 200, respectively) .

Obj 2 – The relevant sequence data for known adults of all Thunnus, Auxis and Coryphaena species ( Atlantic ) were established (and are to be published in Richardson et al. (in press)). Using this method, we have positively identified the following taxa in our SOF:

PLANS FOR THE NEXT SIX MONTHS TO YEAR:
Obj. 1 - We will be completing the transfer to full fiber-optic cable capability (including preparation of winch and slip-ring system). We will work on the internal optics to increase the depth of field imaged by ISIIS. We will also be refining our light source to increase light level, while decreasing the heat generated by the light, as well as provided a simple means of adjusting the light level for fine tuning. We are also incorporating the CTD sensors into our data stream, as well as developing an operator's computer interface for system set-up, operation, and data management. At the same time, we are working on the details of the best approach to take for further increasing the image volume (per discussion above in ‘Progress to date' section).

Obj. 2 - We will continue to apply the high thru-put sequencing to our samples from the SOF as a check on the success of the method – to see if we encounter any unknowns or other sample processing problems.

Obj. 3 – Field test the instrument by conducting a short survey of the SOF with simultaneous net samples to calibrate the image system. It is critical that both sampling approaches be spatially and temporally matched for critical comparison. This work is planned for the proposed two day cruise – probably during the summer of 2007

PUBLICATIONS:
Richardson , D.E., J.D. VanWye, A.M. Miyake, R.K. Cowen, D.L. Crawford. In Press. High throughput species identification: from DNA isolation tobioinformatics. Molecular Ecology Notes.

CONFERENCES:
30th Annual Larval Fish Conference , Lake Placid , NY . Sept 2006.
a) Robert K. Cowen and C. Guigand. In situ Ichthyoplankton Imaging System (ISIIS). [Plenary Talk]
b) Richardson , D.E., J.D. VanWye, A.M. Miyake, D.L. Crawford, R.K. Cowen. High throughput species identification: from DNA isolation to Binformatics. Poster Presentation. [Received BEST STUDENT POSTER Award]

Gulf and Caribbean Fisheries Institute (GCFI), Belize City, Nov 2006
Robert K. Cowen. Advances in the study of billfish early life history

MANUALS, PROTOCOLS:
Species Identifier: A character-based sequence analysis script developed as a part of the molecular species identification portion of the project. The script is freely available from the author (D. Richardson) and has the following characteristics: