Genome Sequencing of Hyalella azteca: a model for evolutionary toxicology and

ecological exposure

Helen Poynton, Donald Weston, Gary Wellborn, Micheal Lydy, Bonnie Blalock, Kaley Major, Jim Lazorchak

An other genome? Why H. azteca?

Overview:

• Exposure model for ecotoxicology

• Ecological model

• Model of evolutionary toxicology

• Hyalella azteca genome project

Exposure model for ecotoxicology

Poynton et al.2013 ES&T : 47: 9453

Hyalella azteca: sediment toxicity “lab rat”

• H. azteca are epibenthic, aquatic crustaceans and live in close contact with sediments

• H. azteca – sensitive indicator of toxicity in freshwater contaminated sediment assessments

• Standard protocols for acute (10-day) and chronic (42-d) toxicity

Model in Ecotoxicology Sediment: Quality Triad

Chapman (1990) Science of the Total Environment.

Chemical Analysis

Toxicity Testing

Bio- assessments

Ecology Toxicology

Chemistry

Sediment Quality

Hyalella azteca as a model of Nanoparticle sediment exposure

• Standard Ecotox organism for sediment toxicity testing

• Epibenthic amphipod – scavenges at sediment surface

• Highly sensitive to metals

H. azteca acutely sensitive to ZnO NPs

Why is H. azteca so sensitive to ZnO NPs? - determine the effect of Zn+2 in toxicity - determine the role of settling in toxicity

Gene expression patterns indistinguishable between ZnO NPs and ZnSO4

Hierarchical clustering

• Total of 71 differentially expressed genes, only 23 were annotated. • Hierarchical clustering of replicate exposures shows that control samples are distinct, but ZnSO4 (blue) and ZnO NP (orange) exposures cluster together.

9.0-10.0

10.0-11.0

11.0-12.0

12.0-13.0

13.0-14.0

14.0-15.0

15.0-16.0

log2 expression

level

Sequence ID Predicted Function

1/10 LC50

ZnSO4

1/10 LC50

ZnONP

LC25

ZnSO4

LC25

ZnONP

Chitin metabolism

contig51444 cuticular protein -0.13 0.53 2.09 1.26

contig05159 cuticular protein 0.54 0.98 1.50 0.85

contig58143 cuticular protein 0.02 0.00 -0.88 -0.90

DNA Damage Repair/ Cell Cyc le Arrest

contig38148 suppressor of tumorigenicity -0.33 -0.17 -0.40 -0.42

contig37414 ribosomal protein -2.91 -1.18 -3.67 -3.66

contig56426 ATP-dependent RNA helicase 0.17 -0.06 0.08 -0.24

contig56149 DNA damage-inducible trascript 0.26 0.25 0.94 0.51

contig08441 TNF receptor-associated protein -0.07 -0.20 -0.16 -0.29

Response to Stress/ Response to Env ironment

contig65399 kairomone-inducible transcript -1.50 -0.11 -3.33 -2.98

contig18799 chorion peroxidase 0.10 0.39 1.11 0.64

contig06681 spermidine synthase -0.19 -0.33 -0.05 -0.43

RNA metabolic processes

contig20295 transcription elongation factor S-II -0.25 -0.20 -0.19 -0.56

contig23436 oculomotor apraxia protein 2 0.03 0.14 0.69 0.68

contig54523 DEAD-box protein, RNA processing -0.11 0.08 -0.23 -0.72

other metabolic processes

contig00905 pg1 protein -0.09 0.30 0.85 0.92

contig47457 Mitochondrial ornithine transporter 0.13 -0.02 0.72 0.15

contig60912 hydrolase -1.26 0.07 -2.03 -1.97

other funct ions

contig13382 projectin -0.15 -0.23 -0.14 -0.23

contig30627 niloticus neuralized-like protein 4 0.03 -0.33 0.01 -0.28contig01785 retrotransposon -0.26 -0.16 0.03 -0.19

contig02261 delta-type opioid receptor 0.68 0.38 0.90 0.91

unknown funct ion

contig61071 unknown function 0.75 1.79 1.07 1.10

contig63165 unknown function -0.59 -0.43 -1.03 -0.84

cont

rol-

1

cont

rol-

2

cont

rol-

4

cont

rol-

5

cont

rol-

3

cont

rol-

6

ZnSO

4-01

ZnSO

4-05

ZnSO

4-06

ZnSO

4-02

ZnSO

4-04

ZnO

NP-

01

ZnO

NP-

02

ZnO

NP-

03

ZnO

NP-

04

ZnSO

4-03

ZnO

NP-

05

ZnO

NP-

06

ZnSO

4-11

ZnSO

4-12

ZnSO

4-14

ZnO

NP-

11

ZnO

NP-

13

ZnO

NP-

14

ZnO

NP-

15

ZnSO

4-13

ZnSO

4-16

ZnSO

4-15

ZnO

NP-

12

9.0-10.0

10.0-11.0

11.0-12.0

12.0-13.0

13.0-14.0

14.0-15.0

15.0-16.0

log2 expression

level

Se

qu

en

ce

IDP

red

icte

d F

un

ctio

n

1/10 LC50

ZnSO4

1/10 LC50

ZnON

P

LC25

ZnSO4

LC25

ZnON

P

Ch

itin m

eta

bo

lism

contig51444 cuticular protein

-0.130.53

2.091.26

contig05159 cuticular protein

0.540.98

1.500.85

contig58143 cuticular protein

0.020.00

-0.88-0.90

DN

A D

am

ag

e R

ep

air/ C

ell C

yc

le A

rres

t

contig38148suppressor of tum

origenicity-0.33

-0.17-0.40

-0.42

contig37414ribosom

al protein-2.91

-1.18-3.67

-3.66

contig56426A

TP

-dependent RN

A helicase

0.17-0.06

0.08-0.24

contig56149D

NA

damage-inducible trascript

0.260.25

0.940.51

contig08441T

NF

receptor-associated protein-0.07

-0.20-0.16

-0.29

Re

sp

on

se

to S

tres

s/ R

es

po

ns

e to

En

viro

nm

en

t

contig65399 kairom

one-inducible transcript-1.50

-0.11-3.33

-2.98

contig18799chorion peroxidase

0.100.39

1.110.64

contig06681 sperm

idine synthase-0.19

-0.33-0.05

-0.43

RN

A m

eta

bo

lic p

roc

es

se

s

contig20295 transcription elongation factor S

-II -0.25

-0.20-0.19

-0.56

contig23436oculom

otor apraxia protein 20.03

0.140.69

0.68

contig54523D

EA

D-box protein, R

NA

processing-0.11

0.08-0.23

-0.72

oth

er m

eta

bo

lic p

roc

es

se

s

contig00905pg1 protein

-0.090.30

0.850.92

contig47457M

itochondrial ornithine transporter0.13

-0.020.72

0.15

contig60912hydrolase

-1.260.07

-2.03-1.97

oth

er fu

nc

tion

s

contig13382projectin

-0.15-0.23

-0.14-0.23

contig30627niloticus neuralized-like protein 4

0.03-0.33

0.01-0.28

contig01785retrotransposon

-0.26-0.16

0.03-0.19

contig02261delta-type opioid receptor

0.680.38

0.900.91

un

kn

ow

n fu

nc

tion

contig61071unknow

n function0.75

1.791.07

1.10

contig63165unknow

n function-0.59

-0.43-1.03

-0.84

control-1

control-2

control-4

control-5

control-3

control-6

ZnSO4-01

ZnSO4-05

ZnSO4-06

ZnSO4-02

ZnSO4-04

ZnO NP-01

ZnO NP-02

ZnO NP-03

ZnO NP-04

ZnSO4-03

ZnO NP-05

ZnO NP-06

ZnSO4-11

ZnSO4-12

ZnSO4-14

ZnO NP-11

ZnO NP-13

ZnO NP-14

ZnO NP-15

ZnSO4-13

ZnSO4-16

ZnSO4-15

ZnO NP-12

Maybe it’s their lifestyle. . .

Zn2+ Zn2+

Zn2+

Because of their feeding behavior as an epibenthic amphipod, H. azteca may take up more NPs than ions. Once in the gut, or within cellular compartments, the ZnO NPs dissolve, releasing Zn2+ which causes cellular toxicity (Trojan Horse mechanism; Park et al. 2010)

Because of their unique lifestyle and feeding behavior, H. azteca can provide important exposure information especially for emerging

contaminants such as Nanomaterials

Poynton et al.2013 ES&T : 47: 9453

How will a genome inform Ecological Exposure?

Annotated genome will: • Allow gene expression response to better inform mode of toxicity and adverse outcome pathways • Aid in the selection of molecular biomarkers for exposure of emerging contaminants

Ecological Model

Description of Hyalella azteca

Lake Catemaco

Gonzalez and Watling (2002) Journal of Crustacean Biology, 22:173-183.

The species H. azteca was first described from Mexico by Saussure in 1858 and was later redescribed from the original library collection by Gonzales and Watling in 2002.

Diversification of Hyalella sp.

Over the last 11 million years, the species complex has diversified over North America (Witt and Hebert, 2000).

Major et. al. 2013 ET&C. 32: 2637

Lab Clone

Wild populations

Diversification of Hyalella sp.

Photo credit: Gary Wellborn

“Similar phenotypic solutions comparable to ecological challenge” Wellborn et al. (2005) Biol J Linn Soc. 84:161.

In North Amercia, Hyalella aztec is a cryptic species complex. Two major ecomorphs exist that appear to have evolved multiple times in under similar ecological conditions. Within each Ecomorph, there are several species. Recent evidence shows that although all species within an Ecomorph are similar morphologically, they do differ in other traits such as surivial under starvation and ecological stress (Soucek et al., 2013)

•The whole genome sequence for H. azteca would allow researchers to understand the genotypes that lead to similar Ecomorphs.

•They have a similar phenotypic solution, is it also a similar genetic solution?

A Genome for ecological studies:

Model for Evolutionary Toxicology: Evolution of pyrethroid insecticide resistance

Weston et al. 2013. PNAS 110:16532

Pyrethroid Insecticides

Don Weston, UC Berkeley

Class of pesticide derived from permethrin, affect insects by inhibiting nerve transmission. High insect mortality, low mammalian toxicity, not persistent in environment. cyfluthrin

GC

PG

MS

CH

MO BR LL

Sacramento

San Jose

San Francisco

40 miles

San Joaquin River Watershed

Sacramento River Watershed

San Francisco Bay Watershed

Tulare Lake Watershed

Pyrethroid concentrations in sediments: Non-detect Low Medium High Don Weston &

Michael J. Lydy

Species diversity in wild populations

Clade C: all laboratory strains

Clade D: resistant populations

Clade B: sensitive populations

Clade A: Laguna Lake (sensitive) population

Gary A. Wellborn

Toxicity of the pyrethroid pesticide to wild populations of Hyalella azteca:

Don Weston

Species

Group Collection Site

Cyfluthrin 96-h

LC50 (ng/L)

Relative

Tolerance

Lab cultures

C UCB 4.8 (3.9-6.2) 1

Wild Populations

A Laguna Lake (LL) 4.8 (3.7-5.8) 1

B Blodgett Reservoir (BR) 1.3 (1.1-1.5) 1

Pleasant Grove Creek

(PG)

11.8 (8.8-14.7) 3

D Morrison Creek (MO) 132 (63.5-174) 30

Mosher Slough (MS) 211 (176-244) 50

Grayson Creek (GC) >691 >175

Chualar Creek (CH) 535 (403-650) 100

Davies, Field, Usherwood and Williamson. (2007) IUBMB Life. 59:151-162.

Mechanics of the sodium channel in nerve transmission

Pyrethroid Insecticides bind inside the channel, and inhibit the H-gate from closing. Channel is left open, causing repetitive firing, and eventual exhaustion

Interesting findings: •Two different alleles present • Two populations containing individuals with multiple alleles • Across the populations, individuals wt and mut individuals exist for each clade.

Evolution in Action at Pleasant Grove Creek

• Between 2010 and 2013, sensitivity of Pleasant Grove Population decreased by an order of magnitude. • The presence of the L925I mutation was found in all organisms with reduced sensitivity. • In the unexposed population, about 30% of the animals carried the L925I mutation, up from non-detectable in 2010.

Loss of genetic diversity in populations subjected to anthropogenic stress (pollution). Evolved resistance may even cause a “genetic bottleneck.” (van Straalen and Timmermans, 2002)

Adaptation event

Are their costs to their adaptation? Genetic Erosion Hypothesis

Unique system for unraveling questions in Evolutionary Toxicology

System attributes: Evidence for independent , convergent evolution resulting in two resistant alleles across two species groups

Basic questions in Evolutionary Toxicology:

• Are there “costs” to adaptation?

• Do these “costs” differ depending on the mutation that is acquired?

• Do these “costs” differ across different species?

• When resistance occurs through similar mutations, does selection also follow a similar pathway? Should we expect the same “costs” when the same mutations appear?

Unique system for unraveling questions in Evolutionary Toxicology

System attributes: Evidence for independent , convergent evolution resulting in two resistant alleles across two species groups

Basic questions in Evolutionary Toxicology:

• Are there “costs” to adaptation?

• Do these “costs” differ depending on the mutation that is acquired?

• Do these “costs” differ across different species?

• When resistance occurs through similar mutations, does selection also follow a similar pathway? Should we expect the same “costs” when the same mutations appear?

A genome for Hyalella azteca would allow us to study the cost of adaptation to genetic diversity.

A Genome for Hyalella azteca

Current genomic resources for H. azteca

•Results of first round of 454 sequencing produced approximately 65K contigs, with a total of 205 Mb •These sequences were assembled with additional 454 and Illumina sequencing, yeilding around 30,000 contigs. This data is now available through GenBank.

Genome sequencing

Hyalella azteca is part of the first round of genomes to be sequenced through the i5k project

Genome sequencing

http://www.arthropodgenomes.org/wiki/i5K

Genome sequencing

Hyalella azteca is part of the first round of genomes to be sequenced through the i5k project

•“Lab clone” underwent four generations of inbreeding, and gDNA was isolated. •Through the pilot project at Baylor College, four different libraries are constructed and will utilize short read (180 bp library) to long-read (8k library) NGS technologies. •Sequencing – as we speak! Raw data will be available soon!

Genome sequencing: Get involved!

http://www.arthropod genomes.org/wiki/ Hyalella_azteca

Genome sequencing: Get involved!

http://www.gmod.org/wiki/WebApollo

Genome sequencing: Get involved!

Join Hyalella-azteca-genome@google.groups.com for project updates and announcements.

Acknowledgements:

Don Weston, UC Berkeley

Gary A. Wellborn, University of Oklahoma

Michael J. Lydy, Southern Illinois University,

Jim Lazorchak, U.S. EPA

Bonnie J. Blalock, graduate student

Kaley Major, graduate student

Early Career Development Grant

Stephen Richards