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 projectsboth 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)

Recruitment into fishery appears to be limited by young of the year (YOY) survival

Winter flounder are in trouble!

Objectives of the first project:Investigate factors influencing the survival of YOY winter flounder in Long Island bays

ØBiological- predation, individual condition, population structure

ØEnvironmental - temperature, oxygen stress, urban gradient

Jamaica Bay Moriches Bay

Shinnecock Bay

Cold Spring Pond

Napeague Harbor

Hempstead Bays

Degraded“Cleaner”

Overall project components

• Environmental measures – Dissolved oxygen, salinity, temperature

• Population surveys– Abundance, size

• Mortality rates• Growth rates

– Cage study- predator free survival and growth• Measures on individuals collected

– Condition- Fulton’s K, HSI– Muscle RNA:DNA-proxy for recent growth – Otolith analysis- settlement date, daily growth

increment– Fin clip- population genetics (microsatellites)– Liver RNA- gene expression analysis

Comprehensive model &

multivariate statistics

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 cageContinuous 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

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5/17/2011 6/6/2011 6/26/2011 7/16/2011 8/5/2011 8/25/2011

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Jamaica

Cage

Field

Individual growth and condition did not show site specific patterns, but did show strong temporal trends

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20102011

Growth

Trend in RNA:DNA best matched growth

Jamaica Hempstead Moriches Napeague ShinnecockColdSpring 0.0104 0.0036* 0.0075 0.0142** 0.0046Jamaica 0.0116 0.0046 0.0134 0.0133*Hempstead 0.0079 0.0153* 0.0087*Moriches 0.0108** 0.0022**Napeague 0.0128**

Genetic population structure showed bay specific differences - pairwise FST

* P < 0.05 ** p<0.001

and more importantly a high level of internal relatedness

O’Leary et al. 2013: PloS ONE 8(6):e66126.

Data indicates effective population size is between 60-260 per bay !!!

Jamaica Bay Moriches Bay

Shinnecock Bay

Cold Spring Pond

Napeague Harbor

Hempstead Bays

1. Livers collected from YOY flash frozen in field 2. RNA extracted from individuals3. Relative expression evaluated in pooled samples

from adjacent sites with divergent condition (Shinnecock vs. Moriches)

4. Illumina paired-end reads of pools (RNA-seq)

Relative gene expression

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:Cytochrome P4501A (CYP1A)Vitellogenin (VTG)

Immune Response: Hepcidin II (HEP)Complement C3 (C3)Pleurocidin (PLEUR)Phospholipase A2 (PLA2)

Glycolosis and Glucose Metabolism:Glycerol 3 phosphate dehydrogenase (GPDH) Glucokinase (GCK)Glutamate decarboxylase (GAD)

Almost all showed significant differences among sites, but most not clearly associated with stressed degradation

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:

ØHigh YOY mortality rates in wild ØExtremely low spawning stock ØSurvival negatively associated with both

hypoxia and predation in some cases ØA complex mix of individual responses at

many levelsØHLM 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:

ØStock structure using both otolith microchemistry (Siskey) and genetics (Dolan –in progress)

ØPredator exclusion as a restoration tool (Dolan) ØCellular 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 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

Ø Multiple cohorts indicating wide diversity of spawning times

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 exposurehypoxia, 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!!!

Relative Contribution of Expression ChangesOverall distribution of differentially expressed genes

Fold change of each transcript used to weight relative contribution to expression change

57% have functional annotations from teleost genomes (50% flatfish)