THE OCEAN IS NOT DEEP ENOUGH

Pressure tolerance of Mytilus edulis early life stages

Major Question

How were deep sea environments colonized?

• High pressure environment• Noxious environment• Temperature extremes

Le Chatelier’s Principle

Background

“If a chemical system at equilibrium experiences a change in concentration, temperature, volume or total pressure, the equilibrium will shift in order to counteract the imposed change” (Mestre et al. 2009)

Pressure

Effects biochemical reactions and membrane functionality

• Keq = [C][D]/[A][B], G = -RTlnKeq and v = k[s]

• P sensitivity of reactions: Kp = K1e(-PV/RT) and kp = k1e-PV‡/RT

• P therefore affects both Keq and k (Kinsey 2009)

• Increased pressure will move the equilibrium to the side with lowest volume

Limits depth range of marine organisms Pressure tolerances are different for different life stages

Some life stages are more suited for deep-sea colonization

Along with pressure also plays an important role in biochemical reactionsCan speed up or slow down metabolism

Response also varies with life stage Can counteract pressure effects

If membrane is more compressed because of pressure can be somewhat decompressed by higher temperature

Temperature

What would limit organisms to a depth and temperature range in early ontogeny?

Biochemical reactions during fertilization

Program for post-fertilization changes in egg of sea urchins

Epel 1975

Ca-Wave After Fertilization

Biochemical pathways are sensitive pathways Could organisms, like mussels, with

sensitive biochemical pathways colonize deep-sea habitat?

How were deep sea environments colonized by

mussels?Mestre et al. chose a shallow-

water relative of a deep-sea inhabitant

Phylogenetic Relationship

subfamilys Mytilinae and Bathymodiolinae

Shallow water species Mytilus edulis found here

The rest are associated with one of the following:

Hydrothermal ventCold-water seepWood/bone

(Kyuno et al. 2009)

Free-spawning marine invertebrates

Worldwide distribution

Found in intertidal zones and estuaries

Endure a wide range of temperatures and physical challenges

Mytilus edulis

Origin of deep-sea mussels 2 Hypotheses:

Deep sea species evolved from shallow-sea species in step-wise fashion via wood/bone habitat

Direct colonization via larval transport from shallow-sea to deep-sea habitats.

Origin of deep-sea mussels 2 Hypotheses:

Deep sea species evolved from shallow-sea species in step-wise fashion via wood/bone habitat

Direct colonization via larval migration from shallow-sea to deep-sea habitats○ Determine larval functional tolerance of

pressure and temperature

Methods

3 ExperimentsTemperature effect on embryonic larvae and

developmentPressure effect on embryonic larvae and

development with fertilization under pressure

Pressure effect on embryonic larvae and development with fertilization at atmospheric pressure

Staging criteria for larvae:

Embryonic stages

D-larvae

Fertilized eggSixteen-cell

stage

Multi-cell stage

Early blastula

Two-cell stage

Four-cell stage

Temperature effect on embryonic larvae and development

5 temperature treatments5, 10, 15, 20, and 25C, at atmospheric pressure

Incubated until all treatments had reached D-larvae stage.

Pressure Vesselsa) Plastic vial filled with the

egg suspension and the microcentrifuge tube hald-filled with sperm suspension

b) Pressure vessel showing the plastic vials inside

Figure 1 from Mestre et al. 2009

Pressure Experiments

Pressure effect on embryonic and larvae development with fertilization under pressure Placed sperm in separate vial which ruptured at

pressure Resulting embryos were incubated at different

temperature/pressure treatments Pressure/Temperature treatments

Temperature treatments○ 10, 15, and 20C

Pressure treatments○ 1, 100, 200, and 300 atm ○ + 400 and 500atm for 4 hour treatments

○Incubated for 4 and 24 hours

Pressure effect on embryonic and larval development with fertilization at atmospheric pressure

Fertilization at atmospheric pressure, at 15C

Resulting embryos were incubated at 4 different pressures and 5 different temperaturesTemperature treatments

○ 5, 10, 15, 20, and 25CPressure treatments

○ 1, 100, 200, and 300 atm

Incubated for 50 hours

Results Divided results from 3 methods into 2

categories:Temperature effects on embryonic and larval

developmentPressure effects on embryonic and larval

development

Temperature effects on embryonic and larval development

Mytilus edulis embryos develop faster at higher temperatures

Effect in the proportion of abnormally developing embryos

Pressure effects on embryonic and larval development with fertilization at atmospheric pressure after 50hrs

Pressure effects with fertilization under pressure at 4 hours

Pressure effects with fertilization under pressure at 24 hours

Krustal-Wallis analysis of variance

Conclusions Temperature tolerance window is from

approximately 10-20C Embryo development possible up to

500atm (~5000m)Hypothesized pressure presents no barrier to

fertilization Slower development with increasing

pressure Increase in abnormal cells with increasing

pressure due to membrane rupture

“reasonable to hypothesize that the invasion of the deep sea by M. edulis is possible in terms of pressure tolerances in embryos and larvae

Was hypothesis correct?

References Kyuno, Akiko; Shintaku, Mifue; Fujita, Yuko; Matsumoto,

Hiroto; Utsumi, Motoo; Watanabe, Hiromi; Fujiwara, Yoshihiro; Miyazaki, Jun-Ichi. 2009. Dispersal and Differentiation of Deep-Sea Mussels of the Genus Bathymodiolus (Mytilidae, Bathymodiolinae). Journal of Marine Biology. Vol. 2009. pp. 15.

Epel, David. 1975. The Program of and Mechanisms of Fertilization in the Echinoderm Egg. American Zoologist. 15, 3. pp. 507-522.

Mestre, Nelia C.; Thatje, Sven; Tyler, Paul A. 2009. The ocean is not deep enough: pressure tolerances during early ontogeny of the blue mussel Mytilus edulis. Proc. R. Soc. B 276, pp. 717-726.

Discussion Questions

What other effects of pressure could cause developmental problems?

Do their results support direct colonization?Last section of discussion implies it does