Development of Biomarkers for Stress in Octopus Rachel Thompson School of Aquatic and Fishery Sciences May 15, 2009

Background Important part of local marine ecosystem Popular attraction at zoos and aquariums Population declines due to environmental stressors Previous stress physiology work Giant Pacific Octopus ( E. dofleini ) Red Octopus ( O. rubescens )

Important part of local marine ecosystem

Popular attraction at zoos and aquariums

Population declines due to environmental stressors

Previous stress physiology work

Objectives Develop non-invasive sampling techniques for octopus 2. Identify biomarkers of stress a) Discovery-based protein analysis b) Targeted protein analysis c) Behavioral changes

Develop non-invasive sampling techniques for octopus

2. Identify biomarkers of stress

a) Discovery-based protein analysis

b) Targeted protein analysis

c) Behavioral changes

Non-Invasive Techniques Epidermal mucus collected using 3 methods: 1. Underwater 2. Skin 3. Sampling container

Epidermal mucus collected using 3 methods:

1. Underwater

2. Skin

3. Sampling container

Objectives Develop non-invasive sampling techniques for octopus 2. Identify biomarkers of stress a) Discovery-based protein analysis b) Targeted protein analysis c) Behavioral changes

Develop non-invasive sampling techniques for octopus

2. Identify biomarkers of stress

a) Discovery-based protein analysis

b) Targeted protein analysis

c) Behavioral changes

Protein Gel Electrophoresis Goals: - Verify protein content - Visualize protein Success varied with sampling technique Container Underwater Skin

Goals:

- Verify protein content

- Visualize protein

Success varied with sampling technique

Protein Identification Prominent protein bands excised Sequenced using mass spectroscopy

Prominent protein bands excised

Sequenced using mass spectroscopy

Mass Spectroscopy Bands digested into smaller peptides Mass of peptides determined, compared to known proteins Sequences of proteins likely found in sample obtained Description Sequence (P02662) Alpha-S1-casein precursor YLGYLEQ (P02662) Alpha-S1-casein precursor EPMIGVNQELAYFYPELFR (P02808) Statherin precursor RIGRFGYGYGPYQPVPEQPLYPQPYQPQYQQYTF (P81605) Dermicidin precursor [Contains: Survival-promoting peptide; DCD-1] DAVEDLESVGK

Bands digested into smaller peptides

Mass of peptides determined, compared to known proteins

Sequences of proteins likely found in sample obtained

Proteins Isolated from Mucus Casein : Antioxidant peptide -Radical scavenging activity -Inhibits growth of E.coli on skin Dermicidin : Antimicrobial, limits skin infection by potential pathogens

Casein : Antioxidant peptide

-Radical scavenging activity

-Inhibits growth of E.coli on skin

Dermicidin : Antimicrobial, limits skin infection by potential pathogens

Objectives Develop non-invasive sampling techniques for octopus 2. Identify biomarkers of stress a) Discovery-based protein analysis b) Targeted protein analysis c) Behavioral changes

Develop non-invasive sampling techniques for octopus

2. Identify biomarkers of stress

a) Discovery-based protein analysis

b) Targeted protein analysis

c) Behavioral changes

Heat Shock Proteins Class of proteins High production during stressful conditions: - Temperature - Pathogens - Hypoxia Functions: - Chaperone: Membrane transport, maintain conformations of proteins - Monitor: Transportation to proteasomes

Class of proteins

High production during stressful conditions:

- Temperature

- Pathogens

- Hypoxia

Functions:

- Chaperone: Membrane transport, maintain conformations of proteins

- Monitor: Transportation to proteasomes

Western Blot - HSP 70 Target HSP 70 using antibody: Not detected in mucus Small leg tissue samples taken weekly HSP 70 detectable

Target HSP 70 using antibody:

Not detected in mucus

Small leg tissue samples taken weekly

HSP 70 detectable

Objectives Develop non-invasive sampling techniques for octopus 2. Identify biomarkers of stress a) Discovery-based protein analysis b) Targeted protein analysis c) Behavioral changes

Develop non-invasive sampling techniques for octopus

2. Identify biomarkers of stress

a) Discovery-based protein analysis

b) Targeted protein analysis

c) Behavioral changes

Behavior Monitoring “ Cephcam” time lapse videos Online viewer surveys Activity categories Cephcam watch Check appropriate boxes Lights on Moving Out of frame Tenacles extended On side of aquaria On bottom

“ Cephcam” time lapse videos

Online viewer surveys

Activity categories

Check appropriate boxes

Lights on

Moving

Out of frame

Tenacles extended

On side of aquaria

On bottom

Behavior Observations Time lapse video s analyzed at random time intervals Baseline activity level established

Time lapse video s analyzed at random time intervals

Baseline activity level established

Objectives Develop non-invasive sampling techniques for octopus 2. Identify biomarkers of stress a) Discovery based protein analysis b) Targeted protein analysis c) Behavioral changes 3. Investigate influence of environmental stress on biomarkers

Develop non-invasive sampling techniques for octopus

2. Identify biomarkers of stress

a) Discovery based protein analysis

b) Targeted protein analysis

c) Behavioral changes

3. Investigate influence of environmental stress on biomarkers

Egg Development Laid January, 2009 Refrain from imposing stressful conditions

Observation of Developmental Stages Early: 8 weeks

Late: 12 weeks - Appearance of chromatophores - Movement within egg

Egg Development Opportunity to explore gene expression over time Egg samples collected weekly RNA extracted, cDNA made via reverse transcription Designed primers for 2 developmental genes

Opportunity to explore gene expression over time

Egg samples collected weekly

RNA extracted, cDNA made via reverse transcription

Designed primers for 2 developmental genes

Developmental Genes Orthodenticle-like protein (OTX) Involved in brain morphogenesis Expressed in early stages of development Hedgehog (Hh) Mediate development, growth, patterning and morphogenesis Muscle development in cuttlefish Expressed early in development

Orthodenticle-like protein (OTX)

Involved in brain morphogenesis

Expressed in early stages of development

Hedgehog (Hh)

Mediate development, growth, patterning and morphogenesis

Muscle development in cuttlefish

Expressed early in development

Real-Time PCR (qPCR) Used to amplify/quantify targeted gene Fluorescent dyes bind to double stranded DNA product Fluorescence can be measured to determine relative amounts of DNA sequences produced

Used to amplify/quantify targeted gene

Fluorescent dyes bind to double stranded DNA product

Fluorescence can be measured to determine relative amounts of DNA sequences produced

Orthodenticle-like protein (OTX) -Expressed early in development -100,000 fold increase over time

Hedgehog (Hh) -Expressed later in development <100 fold increase over time

Results-Gene Expression Orthodenticle-like protein expressed early on in development (after ~1 month) - Consistent with literature Hedgehog protein expressed later in development (~2 months) - Expressed throughout development - Low levels of fluorescence may not have been detectable

Orthodenticle-like protein expressed early on in development (after ~1 month)

- Consistent with literature

Hedgehog protein expressed later in development (~2 months)

- Expressed throughout development

- Low levels of fluorescence may not have been detectable

Behavior Observations Prior to Egg-Laying Post Egg-Laying

Summary Non-invasive methods successful to an extent Possible protein biomarkers identified Changes in behavior related to senescence and parental care, similar methods could be used to evaluate stress Opportunity to look at basic gene expression patterns in larval development

Non-invasive methods successful to an extent

Possible protein biomarkers identified

Changes in behavior related to senescence and parental care, similar methods could be used to evaluate stress

Opportunity to look at basic gene expression patterns in larval development

Applications and Future Work Assess physiological condition of octopuses in wild and in captivity Test effectiveness of biomarkers in indicating stress (experiment) Compare protein expression between individuals

Assess physiological condition of octopuses in wild and in captivity

Test effectiveness of biomarkers in indicating stress (experiment)

Compare protein expression between individuals

Acknowledgements Dr . Steven Roberts Sam White Dr. Greg Jensen Shawn Larson and Roland Anderson of the Seattle Aquarium

Dr . Steven Roberts

Sam White

Dr. Greg Jensen

Shawn Larson and Roland Anderson of the Seattle Aquarium