Characterizing Winter Flounder (Pseudopleuronectes americanus) Nursery Areas Using Otolith Microstructure and Microchemical Techniques

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Elizabeth Fairchild UNH - Department of Biological Sciences Principal Investigator
Linda Kalnejais UNH - Department of Earth Sciences Collaborator
Scott Elzey Massachusetts Division of Marine Fisheries Collaborator
Vincent Manfredi Massachusetts Division of Marine Fisheries Collaborator
Jason MacNamee Rhode Island Dept. of Environmental Management Collaborator
Stephen Dwyer Millstone Power Station Collaborator
Dr. Chris Chambers National Oceanic and Atmospheric Administration Collaborator

Students Involved:

Erin Ducharme University of New Hampshire
David Bailey UNH - Department of Biological Sciences
Journal article
Bailey, D., E. Fairchild and L. Kalnejais. Microchemical signatures in juvenile winter flounder otoliths provide identification of natal nurseries. Transactions of the American Fisheries Society 144(1):173-183, January 2015.
2013 Accomplishments

Favorable winter flounder nursery habitats identified using fish growth and condition indices
Winter flounder populations on the eastern seaboard of the U.S. have reached historically low levels, despite increasing fishing regulations intended to preserve the species. Little is known about the quality of nursery habitats for winter flounder and how variations in quality affect recruitment into the adult populations. In 2013, N.H. Sea Grant-funded researchers calculated flounder growth and condition indices to indirectly evaluate the quality of twelve nursery areas ranging from New Jersey to New Hampshire. The data indicated that two sites — Boston Harbor, Mass., and Great Bay, N.H. — serve as the best nursery sites, while the Niantic River in Connecticut ranked as the worst nursery site. This research will help resource managers and scientists to better understand the effectiveness of indirect measurements like fish growth and condition to determine nursery quality for winter flounder recruitment.

Researchers identify two indices that most accurately assess winter flounder habitat quality
Following the recent collapse of the winter flounder fishery on the U.S. east coast, researchers are trying to determine how variations in the fish nursery habitat affect recruitment into the adult population. Because of the cost and time-intensive requirements of direct measurements such as otolith microchemistry analyses, it is likely that resource managers will only have access to indirect measurements, such as fish growth and condition. In 2013, N.H. Sea Grant-funded researchers evaluated four different indirect indices — length day-1, weight day-1, Fulton’s K and relativized weight — to assess the differences in winter flounder habitat quality in twelve locations ranging from New Jersey to New Hampshire. Based on these calculations, researchers determined that two indirect indices — length day-1 and Fulton’s K — most effectively and accurately determine the quality of winter flounder nursery habitats. These results provide resource managers with guidelines for the most effective tool choice to determine which sites that are most suitable for focusing their winter flounder population management efforts in the future.

Otolith microchemistry helps researchers trace adult winter flounder back to their natal nurseries
In light of historically low winter flounder populations on the U.S. east coast, scientists are seeking methods of assessing flounder nursery habitat quality to determine their contribution to the overall population. In 2013, N.H. Sea Grant-funded researchers conducted the first in a series of studies to determine if adult flounder found in offshore waters can be linked back to their natal estuarine nurseries solely based on unique estuarine water chemistry markers incorporated into the fish’s otoliths. The researchers developed a technique using solution-based inductively coupled mass spectrometry (ICP-MS) to more accurately analyze winter flounder otoliths. The results indicate that otolith elemental signatures are site-specific and vary on a small spatial scale (5-10 km). In addition, juvenile winter flounder can be classified with 73% accuracy to their natal nursery using this technique. This level of accuracy provides justification for further development of winter flounder otolith microchemistry as a tool to assess population connectivity and help resource managers identify and protect the most productive flounder nurseries.

Project Proposal

Winter flounder are an important commercial and recreational species; unfortunately populations have experienced drastic reductions throughout their range. Despite ever increasing fishing regulations, winter flounder populations are not rebounding. Therefore, the viability of winter flounder as a marine resource not only depends on the enforcement of regulated fishing practices but the protection of their habitat as well (Pereira et al, 1999). That is why the concept of Essential Fish Habitat (EFH) was incorporated into the 1996 Sustainable Fisheries Act. EFH is defined as “those waters and substrates necessary to fish for spawning, breeding, feeding or growth to maturity” (Pereira et al, 1999). Certain estuaries and embayments that serve as winter flounder spawning and nursery areas have been classified as EFHs and are extremely important to population sustainability. EFHs are critical for winter flounder because they can support the tenuous maturation beyond early life stages where the mortality rate can reach 99% (Pearcy 1962). The year class strength of winter flounder is determined primarily during these early life stages (Sogard, 1991). Therefore, it is critical that methods for determining the most productive nursery areas (sites that contribute the greatest number of recruits into the adult population) be identified, so these areas can be researched and protected. We will test a fairly modern technique (otolith microchemistry) that has not been used on winter flounder to determine if a unique chemical imprint due to natal nursery (estuary) ground is discernible in juvenile winter flounder otoliths. In addition, we will utilize otolith microstructure analyses to learn more about latitudinal variations in winter flounder stocks and populations.

The chemical composition of otoliths may provide a natural fish tag (Gillanders and Kingsford 1996; Campana 1999; Campana and Thorrold 2001). Numerous studies have shown that juvenile fish collected from different geographic areas can be distinguished based on the chemical composition of their otoliths (Thorrold et al. 1998; Gillanders and Kingsford 2000; Rooker et al. 2001). Establishing natural tags through microchemical analysis of trace elements allows for an effective method to assess stock structure, migration patterns, and connectivity between adult populations and nursery sources (Elsdon et al. 2008). Determining the scale at which the elemental signatures are site-specific allows for the creation of an elemental signature index of nursery areas based on otolith composition. This index then can be used for stock identification and to trace adults back to natal nursery areas. For winter flounder, this would allow for the increased protection of those nursery grounds that significantly contribute to the adult population, thereby potentially reducing early-stage mortality and increasing the resiliency of the adult population.
Although otolith microchemical techniques have the potential of serving as the most important proxy for the significance and quality of the nursery habitat, an immediate proxy can be determined through otolith microstructure analysis. Otolith microstructure analysis determines the timing of life history events and the growth rate of the juvenile fish. Since growth rates have been directly linked to the survival of the juvenile fish, identifying the growth rates allows the quality of each nursery area to be evaluated at a specific point in time (Houde, 1987; Gibson, 1994). Understanding the timing of life history events in specific nursery areas also can be used to promote successful recruitment by indicating when and where habitat protection is most critical.
For this preliminary study winter flounder will be collected from 10 estuaries from Machias, ME to Narragansett, RI (including both New Hampshire estuaries - Great Bay and Hampton-Seabrook) during the summer of 2012. Sample sites will be chosen based on the presence of routine state and private agency surveys, existing winter flounder data, and water quality monitoring (buoys, sondes, and routine surveys). Juvenile winter flounder will be collected using a beach seine (17 m x 2 m; swept area 550m2) at low tide. All winter flounder caught will be measured (TL) and weighed (g). Total length measurements will be used to separate fish into two different cohorts (young-of-the-year (YOY) and age 1; Correia, 2012). The range of the total length measurements of each cohort will be determined at each location using existing age and size frequency data for the specific area. Water quality parameters (salinity, temperature, and dissolved oxygen) will be recorded prior to and upon the completion of seining using a YSI 6920 sonde. The habitat type and benthic composition also will be recorded at each site.
Both sagittal otoliths will be extracted using plastic forceps, cleaned in distilled water, and stored in plastic vials. Otoliths then will be separated right from left for each sample. If there are any discrepancies as to which side they were removed from, they will be separated according to the position of the sulcus acusticus and the rostrum. The left sagittae otolith, previously found to provide the best correlation between somatic and otolith growth (Sogard, 1991) will be used for microstructure analysis, and the right sagittae otolith will be used for microchemical analysis. 
As conducted by Sogard (2001), 10 of the 35 left otoliths from the YOY flounder at each site will be randomly selected to undergo microstructure analysis. The otolith will be mounted on a slide with thermoplastic glue and the sagittal plane will be polished on both sides to the core. Images of each otolith will be taken using a digital compound microscope camera and stitched together using imaging software. The imaging software then will be used to count the increments deposited from the anterior most accessory primordium to the edge. These increment counts will provide an estimate of the data of mid-metamorphosis, because the accessory primordia appears at the midpoint of eye migration (Sogard, 1991). Since settlement occurs shortly after metamorphosis, the timing of settlement to the benthos can be determined (Chambers and Leggett, 1987). Daily growth rates in length also will be estimated from otolith increment widths, based on a formula relating somatic growth to otolith growth for oxytetracycline-marked individuals (Sogard, 1991). The micro-structure of the age 1 otoliths will not be examined during this part of the study, because daily growth rings are less visible as the age of the fish increases. Examinations of the age 1 will only occur if there is discrepancy in cohort identification. An analysis of variation (ANOVA) and a Tukey post hoc test will be used to explore the variations in growth rates and timing of life history events among the nursery areas.       
As conducted by Correia et al. (2012), 30 of the 35 right otoliths from each cohort at each site will be randomly selected for microchemical analysis. Solution-based Inductively Coupled Plasma Mass Spectrometry (ICP-MS) at UNH will be used to perform the microchemical analysis. Otolith preparation, elemental analysis, and instrument calibration will follow the procedures detailed in Zolick (2011). Otoliths will be prepared in haphazard order and analyzed in complete randomized block fashion with respect to location of capture to eliminate any bias resulting from preparation and instrument drift (Hamer et al. 2003).
To ensure that any size differences among samples did not influence differences in the otolith chemistry, linear regressions between element:Ca ratios and otolith mass will be performed (Campana et al., 2000). Otolith element:Ca ratios may then be adjusted depending on the results of the linear regression analysis. An ANOVA and Tukey post hoc tests will be used to explore variation in individual elements and multi-element otolith compositions temporally and geographically. Regression analysis will be used to evaluate the relationship between element:Ca ratios and water quality parameters.