genome sequencing of hyalella azteca · genome sequencing of hyalella azteca: a model for...
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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
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
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.
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
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!