Factors affecting the health and fitness of Juvenile winter flounder ( Pseudopleuronectes americanus ): Associations among data from the cellular to population level Anne McElroy and colleagues… 10th International Flatfish Symposium – St. Malo France, November 11-16 2017
Today – A tale of two projects both funded by NOAA Fisheries through the Saltonstall-Kennedy Program 2010-2013 & 2016-2018 This work lead by Mike Frisk and I has been done collaboratively with a large team over time Robert Cerrato (SBU), Demian Chapman (Florida Atlantic U.) Mark Fast (UPEI,), Kevin Feldheim (Mus. Nat. Hist. Chicago), Karin Limberg (SUNY ESF), Nolwenn Dheilly (SBU) Lyndie Hice (Ecol &Environ. Inc.) Shannon O’Leary (TX A&M), Brian Gallagher (UMD – CBL) and as you’ve heard Tara Dolan and Matt Siskey (SBU)
Winter flounder are in trouble! Recruitment into fishery appears to be limited by young of the year (YOY) survival
Objectives of the first project: Investigate factors influencing the survival of YOY winter flounder in Long Island bays Ø Biological- predation, individual condition, population structure Ø Environmental - temperature, oxygen stress, urban gradient “Cleaner” Degraded Hempstead Bays Napeague Harbor Shinnecock Bay Cold Spring Pond Moriches Bay Jamaica Bay
Overall project components • Environmental measures – Dissolved oxygen, salinity, temperature • Population surveys – Abundance, size • Mortality rates • Growth rates Comprehensive – Cage study- predator free survival and growth model & • Measures on individuals collected multivariate – Condition- Fulton’s K, HSI – Muscle RNA:DNA-proxy for recent growth statistics – Otolith analysis- settlement date, daily growth increment – Fin clip- population genetics (microsatellites) – Liver RNA- gene expression analysis
Population surveys • Beam trawl surveys every other week from May-October – 10 random stations per day, 1-2m depth – 2010 and 2011 YOY mortality relatively high, 3-6% per day – higher than in previous studies in nearby areas
Cage study to assess predator-free mortality – Four 1m x 1m x 0.5m cages at each site – 6-9 YOY per cage Continuous environmental monitoring – Temperature, salinity, dissolved oxygen – sonde attached to cage at each site taking measurements every 30 min
Hypoxia seemed to be most closely linked to mortality in free and caged fish – and in some bays predation also a factor Jamaica Bay Jamaica 5.0 4.5 4.0 Cage 3.5 3.0 Field LN(N) 2.5 2.0 1.5 1.0 0.5 0.0 5/17/2011 6/6/2011 6/26/2011 7/16/2011 8/5/2011 8/25/2011 Date
Individual growth and condition did not show site specific patterns, but did show strong temporal trends Trend in RNA:DNA best matched growth 10 Growth 9 Incremental growth ( µ m) 8 7 6 2010 5 2011 4 3 2 1 0 0 2 4 6 8 10 12 Month
Genetic population structure showed bay specific differences - pairwise F ST Jamaica Hempstead Moriches Napeague Shinnecock Cold Spring 0.0104 0.0036* 0.0075 0.0142** 0.0046 Jamaica 0.0116 0.0046 0.0134 0.0133* Hempstead 0.0079 0.0153* 0.0087* Moriches 0.0108** 0.0022** Napeague 0.0128** * P < 0.05 ** p<0.001
and more importantly a high level of internal relatedness Data indicates effective population size is between 60-260 per bay !!! O’Leary et al. 2013: PloS ONE 8(6):e66126.
Relative gene expression 1. Livers collected from YOY flash frozen in field 2. RNA extracted from individuals 3. Relative expression evaluated in pooled samples from adjacent sites with divergent condition (Shinnecock vs. Moriches) 4. Illumina paired-end reads of pools (RNA-seq) Napeague Hempstead Bays Harbor Shinnecock Bay Cold Spring Pond Moriches Bay Jamaica Bay
What happened? • Needed to create flounder gene database as no reference genome available – de novo assembly using SOAPdenovo-trans program • 187,354 contigs/scaffolds taken for further analysis – >100 bp, Mean = 579bp • DESeq used for differential analysis – reads mapped back to assembly using Tophat program • 253 Significant transcripts – Moriches > Shinnecock : 180 – Shinnecock > Moriches : 73 – Glucose and glycogen metabolism major responder
We then targeted transcripts coding for genes associated with contaminant exposure, immune response, and glucose and glycogen metabolism by qPCR in individuals Contaminant Exposure: Almost all showed Cytochrome P4501A (CYP1A) significant Vitellogenin (VTG) differences among Immune Response: sites, but most not Hepcidin II (HEP) clearly associated Complement C3 (C3) with stressed Pleurocidin (PLEUR) Phospholipase A2 (PLA2) degradation Glycolosis and Glucose Metabolism: Glycerol 3 phosphate dehydrogenase (GPDH) Glucokinase (GCK) Glutamate decarboxylase (GAD)
PCA analysis indicated relationships among variables, and some site specific patterns in expression McElroy et al.2015: Comp. Biochem.Physiol.Genomics
Hierarchical linear models used to see which variables were statistically associated with growth Incremental growth was significantly affected by: settlement date, age at capture, condition index (Fulton’s K and HSI), genes associated with immune response (PLEUR) contaminant exposure (CYP1A), and energy metabolism (GPDH). Both Shinnecock and Jamaica Bay were significant as well. Gallagher et al. 2016: Mar. & Coast. Fish.
To summarize, our first project demonstrated: Ø High YOY mortality rates in wild Ø Extremely low spawning stock Ø Survival negatively associated with both hypoxia and predation in some cases Ø A complex mix of individual responses at many levels Ø HLM as useful tool to identify associations among which of the wide diversity of parameters were associated with growth
The second project is focusing more on restoring Long Island’s winter flounder stocks, examining: Ø Stock structure using both otolith microchemistry (Siskey) and genetics (Dolan – in progress) Ø Predator exclusion as a restoration tool (Dolan) Ø Cellular markers of growth and condition (Dolan – in progress)
Otolith microchemistry in YOY and adults already has identified resident inshore, migratory, and offshore fish, may identify bay fingerprints and with genetic analysis will indicate what contingents are contributing to recruitment Adult YOY
Ø Multiple cohorts indicating wide diversity of spawning times Ø Caging studies have demonstrated influence of predation and we are examining influence of environmental factors Ø Genetic analysis will further evaluate and identify stocks and help further differentiate contingent structure
Gene analysis work underway on 2016 samples: ü We conducted more extensive sampling to obtain more similar size/age groups to compare among sites ü More extensive gene analysis underway: • Resampling Cyp1a, Vtg, Pleur, Hep, GPDH, GCK • Evaluating new transcripts to look hypoxic effects, more at contaminant exposure, and general stress response: pi-GST, Vegf, HSP70, and Cortisol in 2016 individuals as well as some reanalysis of earlier samples for new transcripts
In Summary Value of holistic multidisciplinary approach - Habitat contaminant exposure hypoxia, temperature - Responses at multiple levels of biological organization community – predation population – stock structure, interrelatedness, individual – survival and growth cellular – response patterns Hierarchical linear models as a promising tool to find statistical associations among diverse parameters and space and time
Thank you from team winter flounder!!!
Overall distribution of differentially expressed Relative Contribution of Expression genes Changes Fold change of each transcript used to weight relative contribution to expression change 57% have functional annotations from teleost genomes (50% flatfish)
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