Larval settlement of stalked barnacles (Pollicipes ...

Larval settlement of stalked barnacles (Pollicipes pollicipes) PhD in Marine Science and Technology

Sofia Franco

Prof. Anthony Clare Dr. Nick Aldred Prof. Teresa Cruz

Introduction Bio-economy of P. pollicipes Balancing exploitation and conservation • P. pollicipes is considered a delicacy in Portugal and Spain ▫ Collection is a risky activity and unreliable due to sea/weather conditions ▫ Supply is often insufficient to satisfy the demand (prices may reach 150 €/kg)

▫ Overexploitation of stocks has led to strict conservation measures ▫ Current search for new exploitation solutions

Could P. pollicipes be a candidate species for aquaculture?

Introduction Aquaculture of P. pollicipes (1)

(2)

(3)

Collection of juveniles

Potential and limitations

(4)

Grow-out of juveniles

[1] P. pollicipes production cycle based on the natural production of larvae by wild adults (1), development of larvae in the wild (2), settlement on artificial structures (3) and transfer to grow-out systems (4). (1)

Collection Collection of larvae larvae of

(2)

(3)

Larval culture to juvenile in hatchery

(4)


[2] P. pollicipes production cycle based on the collection of larvae from will adults (1), larval development in captivity, (2) settlement on artificial structures in captivity (3), followed by transfer to grow-out systems (4). (1)

Production of larvae by broodstock

(2)

(3)

Larval culture to juvenile in hatchery

(4)


[3] P. pollicipes production c cycle based on the production of larvae by broodstock in captivity (1), larval development in captivity, (2) settlement on artificial structures in captivity(3), and transfer to grow-out systems (4).

• Adults reproduce and spawn in captivity (fertilized eggs can be easily extracted)… … optimal conditions unknown • Larvae can be easily cultured … … optimal conditions unknown and no records of survival or growth • Settlement occurs massively on adults… … settlement on other substrata is residual

• Juveniles have high growth in the field… … further data required

Introduction Aquaculture of P. pollicipes Larval settlement of P. pollicipes • High interest on cyprid settlement in spite of disappointing results • P. pollicipes cyprids show high larval selectivity, mostly rejecting all surfaces other than the adult or in contact with the adults – settlement rates almost residual Larval growth

Larval Factors that affect settlement: settlement • Surface texture, chemistry, etc. • Biofilm • Conspecifics/Allospecifics • Chemical inductors Juvenile • Cyprid age and condition Broodstock growth • culture Environmental conditions

• How are new clusters formed? • Is this selection post-settlement? • What are the selection criteria?

• The conditions, to which the cyprids of P. Which conditions promote pollicipes respond to, remain vastly larval settlement? to unknown, in spite of theHow high knowledge on thethe settlement of other barnacles solve settlement problem?

Experimental work General material and methods Larval rearing conditions • Nauplii distributed by experimental rearing units: • Artificial seawater (34psu) /low aeration • Antibiotics full strength (Pen. and Strep.) • Photoperiod of 16/8 L/D

• Reared at the assigned temperatures and feeding regimes

a)

b)

[4] (a) Larval rearing unit on 10L buckets with weak bottom aeration, (b) Multiple larval rearing units on 500ml conical bottles with weak bottom aeration

Experimental work Experiments and research questions Cyprid morphology and settlement behaviour

• What are morphological characteristics of the cyprid? • What is the cyprid settlement behaviour?

Settlement on conspecifics, natural substrata and allospecifcs

• What are the settlement rates and post-settlement survival on adults and other natural substrata in captivity? And in the wild? • And in other species? Or colonized/uncolonized substrata?

Settlement on artifical substrata

• What is the settlement on different artificial substrata? How is it affected by surface properties?

Effects of environmental factors on settlement

• What are the optimal conditions to promote cyprid settlement?

Chemical cues inductors of settlement: SIPC and footprints

• Do adults of P. pollicipes produce SIPC? What is the effect of SIPC in inducing settlement? And the SIPC of other species? • Do the cyprids produce footprints? What is the effect of conspecific footprints in inducing settlement?

Exp.1 Cyprid morphology and settlement behaviour • What are the morphological characteristics of the cyprid and post-cyprid?

a)

b)

Cyprids Post-cyprids

30 replicates

Morphological description - Obs. structures - Photographed

c)

[5] Moulting process from naupli VI to cyprid larva, where (a) nauplii VI before moulting (b) cyprid moulting from nauplii VI, (c) fully moulted cyprid

Exp.1 Cyprid morphology and settlement behaviour

b)

a)

c)

d)

[6] Cyprid larva (a) full body, (b) posterior detail of swimming appendages, (c) anterior detail of frontal oil dropplets and conpound eye, (d) detail on antenulles and suckers morphology and movement

Exp.2 Settlement on conspecifcs, natural substrata and allospecifics • What are the settlement rates and post-settlement survival on adults and other natural substrata in captivity? Adults of P. pollicipes

Stalk skin of P. pollicipes

Natural rocks Natural rocks pre-colonized uncolonized Natural rocks No substrata w/ other spp. .

6 systems (5 replicates of each surface per system; 1 larva per 2ml; MCA)

Larval survival rate Temporary settlement Permanent settlement

a)

Post-settlement survival

- Obs. settlement/dislodgment -Est. larval quality - Est. survival b) [7] Cyprid larva (a) healty and (b) unhealthy

Exp.2 Settlement on conspecifcs, natural substrata and allospecifics • Settlement varied with substrata and reflected high larval selectivity • Temporary settlement was considerably higher than perm. settlement • The larva explore but do not attach permanently?

Graphs not shown, as research is pending publication. For further details about present or ongoing research, please email [email protected]

• Difference of 25 to 10 larva per adult!

• Permanent settlement only on the adults, while larva attached temporarily to all other natural substrata. No settlement on control or stalk skin.

[8] Number of temporary and permanently settled larva of P. pollicipes at 48h, according to substrata ( in MCA in captivity), in a total of app.500 larva per assay. Substrata included the adults of P. pollicipes (Adults Pp), natural rocks per-colonized (Rocks pre-col.), natural rocks un-colonised (Rocks un-col.), stalk skin of P. pollicipes (Skin Pp), natural rocks with other species (Rocks other spp), and cotrol (Control).

Exp.2 Settlement on conspecifcs, natural substrata and allospecifics • From the permanently settled larva, 82.4% did so in the first 48h


• After 8 days no permanent settlement was recorded, in spite of the continuation of temp. settling cyprids and high larval survival • With time cyprid activity decreases – in spite of stimulation by light/heat

[9] Percentage of permanetlly settled larva on the adults of P. pollicipes (in relation to the total number of settled larva), according to time at 0h, 48h, 96h, 144h and 192h.

• Depletion of lipidic reserves

Exp.2 Settlement on conspecifcs, natural substrata and allospecifics

• Permanent settlement only on the adults – high larval preference • From app. 500 larvae, 25% settled if given conspecifics • From the 60% of temporarily settled larvae, about 35% fail to attach permanently


• Full rejection of other natural surfaces and control • Is substrate preference more important than settling? What if it was a single choice situation?

• NB: Possible cannibalism (?)

[10] Total larval permanently settled (in relation to total number of larva per system) according to substrata (MCA in captivity), Substrata included the adults of P. pollicipes (Adults Pp), natural rocks per-colonized (Rocks precol.), natural rocks un-colonised (Rocks un-col.), stalk skin of P. pollicipes (Skin Pp), natural rocks with other species (Rocks other spp), and cotrol (Control).

Exp.3 Settlement on artificial substrata • What are the settlement rates and postsettlement survival on artificial substrata? Adults

Adults

Adults

Control

Control

Nat. rocks

Nat. rocks

Nat. rocks

Tufnol

Ceramic

Nat. Rocks + Ex

Glass epoxy

Cork

Control + Ex.

Carbon epoxy

Net

Stalk mim. I

PMMA

Plankton mesh

S. mim. I + Ex.

Nylon

Slate

Stalk mim. II

Calcium silicate

PVC

S. mim. II + Ex.

Glass Vynilest.

9 treatments per test (5 replicates; 1 larva /2ml; SCA)

Larval survival rate Temporary settlement Permanent settlement Post-settlement survival

- Obs. settlement/dislodgment -Est. larval quality - Est. survival

[11] Substrata tested (a) adults, (b) natural rocks, (c) artificial substrata, (d) stalk mimics

Exp.3 Settlement on artificial substrata

[12] Details of P. pollicipes (a) cirri and capitulum, (b) capitular plates, (c) base of capitulum and stalk , (d) stalk, (e) stalk base

[13] Examples of various types of stalk scales of P. pollicipes adults

[14] (a) Extrude medium and equipment to cast the moulds (b) mould of stalk

[15] Mimics of stalk of P. pollicipes adults on epoxy resin cast from moulds

Exp.3 Settlement on artificial substrata • Settlement rates validated previous results, showing only settlement on the stalk of conspecific adults or by the adults, contrary to all other surfaces and extract • Even when in single choice situations, larva do not settle on unsuitable substrata • No response to barnacle extract – high mortality – too high concentration or impurity? • No response to stalk mimics

• Larval survival varied with substrata – toxicity response


[16] Larval survival at 48h according to substrata (SCA in captivity), where adults of P. pollicipes (Adults Pp), control, control with extract (Control Ext.), natural rocks un-colonised (Rocks un-col.), natural rocks uncolnised with extract (Rocks Ext.), ceramic, cork, net, plankton mesh, slate, PVC,, stalk mimics in Epoxy type I (Mim.I), stalk mimics in Epoxy type I with extract (Mim.I Ext.), stalk mimics in Epoxy type II (Mim.II), and stalk mimics in Epoxy type II with extract (Mim.II Ext.). Composites not shown, as there was no negative effect on survival.

Exp.4 Effects of environmental factors on cyprid settlement • What are the optimal conditions to promote cyprid settlement? Movement:

Volume:

Static

3ml

Larval survival rate

Moving

50ml

Temporary settlement

500ml

Aeration:

Air-water interf:

Aerated

Present

Non aerated

Absent

6 treatments (10 replicates; 1 larva/2ml)

Permanent settlement

Further validation required

Post-settlement survival

- Obs. settlement/dislodgment -Est. larval quality - Est. survival

[17] Examples of containers used to test settement (a) 24 well plates IWAKI, (b) 500ml inverted conical bottles, (c) 200ml plates on moving platform, (d) 200ml glass bottles

Exp.4 Effects of environmental factors on cyprid settlement • Larval survival was considerably higher in higher volumes, moving conditions and with aeration - water volume was the determining factor

• Water movement led to better survival than aerating: • Aerated larvae tend to be propelled outside of water by the bubbling, becoming irreversibly stuck to the container walls • Larvae in moving water tend to get temporarily attached to the substrata on the water-air interface • Temporary settlement occurs often in the interface contact – Involuntary or strategy?

• The presence of air-water interface seems to lead to higher settlement than no interface, in spite of no effect on surivival – more results needed for validation!

[18] Settlement plates on shaking platform

Exp.5 Chemical cues inductors of settlement: SIPC and footprints • Do adults of P. pollicipes produce SIPC? Extraction and purification of SIPC

- Crude extraction - Ion exchange cromatography - Total protein essay - Chloroform-methanol precipitation

Purfication of SIPC

- SDS-Page electrophoresis - Western blotting - Immunostaining - Ultra filtration - Gel filtration cromatography

[20] Adults of P. pollicipes

Exp.5 Chemical cues inductors of settlement: SIPC and footprints • With B. amphitrite (c) it was visible the three characteristic bands of the SIPC at 98, 88 and 76kDa. • With P. pollicipes (d) there was a high concentration of protein of similar molecular weight to the larger units of the SIPC (>[Pp]?) and a less concentrated second band in the lower weight SIPC region

• The confirmation of the P. pollicipes SIPC has not been achieved due to immunoblotting problems – is there a P. pollicipes SIPC?

a) b) c) d) [21] Results of the purification of the settlement-inducing protein complex (SIPC) after SDS PAGE (a) molecular weight marker (from Matsumura et al., 1998b), (b) purified SIPC from Balanus amphitrite (from Matsumura et al., 1998b), (c) purified SIPC from Balanus amphitrite, (d) possible SIPC from Pollicipes pollicipes

Main conclusions • Cyprid morphology: • Highly specialized for settlement and surface recognition (results not shown) • High lipidic reserves to sustain long non-feeding period – allowing for surface selection

• Cyprid behaviour • Extensive exploratory behaviour and capacity to reject surfaces after exploration, not settling at all – adaptative strategy to survival? • Confirmed high permanent settlement rate in conspecifics • Response to artificial substrata: • All tested surfaces were rejected - need for chemical atractant? • Natural surfaces were rejected • Stalk mimics were rejected -not only texture • Barnacle extract incured in some toxicity need for better water quality

• Existence of SIPC: possible but unconfirmed

Main conclusions • Which options are now being pursued? • Considering the importance of the adult – Surface? Biofilm? Water retention properties?

• Field work testing – do results repeat themselves? • Further development of the SIPC option – need for high chemical stimulation? • Considering allowing for settlement on the adult and investigating removal options

Rearing of juvenile stalked barnacles (Pollicipes pollicipes)

Acknowledgements • Thanks to Prof. A. S. Clare, Dr. Nick Aldred and Prof. Teresa Cruz for their constant support and advice, which greatly contributed to the work developed. • Thanks to the colleagues and staff of the School of Marine Science and Technology.

• This work has been supported by Fundação para a Ciência e Tecnologia, doctoral grant SFRH/BD/63998/2009.

Thank you!

Any questions?