Shark bioluminescence control possibly evolved/co-opted from vertebrate skin pigmentation control Duchatelet Laurent1,*, Pinte Nicolas1, Delroisse Jérôme2, and Mallefet Jérôme1 1 Laboratory
of Marine Biology, Université catholique de Louvain, Louvain-la-Neuve, Belgium 2 Biology of Marine Organisms and Biomimetics, University of Mons, Mons, Belgium *Corresponding author. E-mail:
[email protected]
INTRODUCTION Etmopterus spinax is a small deep-sea bioluminescent shark. This lanternshark emits a blue-green light thanks to thousands of tiny epidermal light organs called photophores. These luminous organs are composed of a cup-shaped pigmented cells, which form a pigmented sheath enclosing the emitting cells, the photocytes, surmounted by one or several lens cells. A multilayer cell zone called the iris-like structure (ILS) is present between the lens cells and the photocytes and is used as a shutter of the light organ(1, 2). Recent studies highlight that shark light emission is mainly under hormonal control: melatonin and prolactin triggering luminescence, while ⍺-MSH inhibits light emission(3). Interestingly, the hormones that regulate the shark light emission, are also been demonstrated as regulator of the skin pigmentation in shallow-sea sharks(4-7). As photophores display pigmented cells (ILS) to possibly mechanically regulate the light emission, we analyzed the possible co-option of the vertebrate pigmentation pathway to control the shark luminescence.
MATERIALS & METHODS Transcriptomic analysis
cAMP assay
hormones involved in the vertebrate pigmentation pathway
Comparaison
action on the cAMP concentration
Organism collection (Bergen, Norway)
Pharmacological studies implication in the luminescence control mechanism
between luminous shark bioluminescence control and swallow water shark pigmentation regulation
Immunodetection tissue localisation (IHF)
RESULTS & DISCUSSION PHOTOPHORE MORPHOLOGY
A
•
RNA-seq performed on ventral skin (i.e. containing photophores)
•
Sequence analysis allowed us to identify melatonin and ⍺-MSH / ACTH receptors (GPCR) involved in the bioluminescence control mechanism
•
G protein ⍺ subunits (i, o, t, s) were investigated. These proteins are linked to GPCR and have a key role in the intracellular transduction cascade.
•
MCH receptor was also investigated due to his implication in vertebrate pigmentation regulation
B l
e
IN SILICO TRANSCRIPTOME ANALYSIS
e
i ph
ps
IMMUNOHISTOFLUORESCENCE •
d
Etmopterus spinax emit a blue-green light thanks to thousands of photophores (A), which are composed of a cluster of photogenic cells (photocytes) enclosed in a pigmented sheath and topped by a shutter-like structure and a lens (B, [2]). d: dermal denticle ; e: epidermis ; i: iris-like structure cells ; l: lens ; ph:
•
The presence of the studied shark proteins was confirmed in the epidermal tissues using anti-mammal antibodies. Epidermis Lens Iris like Photocytes structure Cryosection immunodetections highlighted the Melatonin ✓ ✓ ✓ ✗ co-expression of some proteins involved in the receptor photoemission within the photophore. ⍺-MSH ✗ ✗ ✓ ✓ receptor
photocytes ; ps: pigmented sheath cells. Scale bar : 50µm
G⍺s
✗
✗
✓
✓
G⍺i
✓
✓
✓
✓
NEW HORMONAL CONTROL
CAMP CONCENTRATION • cAMP concentrations vary between treatments
•
ACTH ✓ new inhibitor of the light emission
•
MCH ✗
***
Induced intracellular cAMP level of 6 shark ventral skin patches (1cm2) after 20min for each treatment. melatonin and ⍺-MSH used concentration 10-6mol. L-1. Light represents blue light (around 486nm) exposition to mimic the E. spinax natural luminescence.
Relative decrease of light emission of 20min melatonin 10-6mol.L-1 (A) and prolactin 10-6mol.L-1 (B) stimulated lantern shark skin patches under various treatments (control, MSH, ACTH at different concentration). Results are expressed in pourcentage of the maximum of light emission after the first stimulation. ⍺-MSH 10-6mol.L-1 and ACTH 10-5mol.L-1 show a same inhibition curve.
CONCLUSION
PERSPECTIVES Melatonin
α-MSH ACTH
Adenylate cyclase
Adenylate cyclase αs
γ
αs
αi
β
γ β
G protein
Hormones Pigment dispersion
αi
melatonin
G protein
ATP
1
ILS cells mechanical control of the light emission will be investigated through the implication of PKA, kinesin and dynein activities. Additional data on the relationship between pigmentation regulation and bioluminescence control pathway need to be highlight to support the co-option hypothesis during evolution.
hv
⍺-MSH
cAMP
ACTH
Inactivate PKA 2
Activate PKA
Light emission
✗ ✓ ✓
✓ ✗ ✗
REFERENCES
➚ Light Mel G⍺i ➘ cAMP ➘ PKA activity
Melanin pigment dispersion kinesin
3
Intracellular effect
Gene expression
Melanin pigment aggregation dynein
Pigment aggregation MSH / ACTH G⍺s ➚ cAMP ➚ PKA activity
➘ Light Pigment dispersion
[1] Claes J.M., Mallefet J. (2009) JEB; [2] Renwart M. et al. (2014) Zoomorphology; [3] Claes J.M., Mallefet J. (2009) JEB; [4] Visconti M.A. et al. (1999) JEB ; [5] Mizusawa K. et al. (2012) General and comparative endocrinology ; Nery L.E.M, de Lauro Castrucci A.M (1997) Comparative Biochemictry and Physiology; Sugimoto M. (2002) Microscopy research and technique. DL, PN are FNRS/FRIA PhD student Fellow. DJ and MJ are Postdoctoral Fellow and Research Associate FNRS respectively.
