Reproduction
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  Settlement, metamorphosis, & recruitment
  Considered in this section are topics relating to settlement, metamorphosis, & recruitment, while the topic of  SPAWNING, FERTILISATION, & LARVAL DEVELOPMENT is located in its own section.
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photograph of abalone Haliotis kamtschatkana

An abalone veliger is free-swimming for a short time, perhaps as little as 5-6d. Consequently, larval dispersal is limited to possibly just a few tens of meters from the parents.  Settlement involves a descent from the water column to the sea bottom, followed by bumping and gliding at the substrate surface as the larva tests for physical and chemical suitability.

 

Pinto or northern abalone Haliotis kamtschatkana perched on a
rock covered in encrusting coralline algae, possibly Pseudolithophyllum sp.
Coralline algae represent a favoured settling substratum for abalone larvae 0.5X

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Research study 1
 

A study in La Jolla, California on Haliotis rufescens shows that settlement and metamorphosis can be divided into a number of recognisable stages.  Hatching occurs 18-24h after fertilisation (at 14-16oC) to a trochophore larva.  Next comes an early veliger, followed by a succession of later veliger stages, including the post-torsional veliger (3days of age) shown here.Then comes the settling/crawling (6d) stage, which marks the end of the larval phase of development.  Metamorphosis is gradual, taking about 7d to complete, and leads to several post-larval stages, including the post-larva (10d) and asymmetric post-larva stages shown here. The circular-shell juvenile marks the transition from post-larva to juvenile.  Leighton 1974 Fish Bull 72: 1137.

 

NOTE the author lists 11 stages but, for brevity, only 7 are shown here

drawings of developmental stages of abalone Haliotis rufescens
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Research study 2
 
drawing of 14-d juvenile abalone photograph of 40-d juvenile abalone Cessation of larval swimming and initiation of metamorphosis in Haliotis species is induced by contact with any of several species of encrusting coralline algae (e.g., Pseudothophyllum spp.) but not, apparently, by contact with large kelps that later serve as food for the early adult.  Areas in which coralline algae grow are characterised by clean, strong water flows; hence, are healthy areas.  Additionally, the algae host films of diatoms and bacteria on which the post-metamorphic juveniles feed. Drawing from Crofts 1937 Phil Trans Roy Soc Lond B 228: 219.
 

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Research study 3

 

The inducer in coralline algae is a peptide molecule, of unknown structure, that is a mimetic molecular formula for GABA (gamma-aminobutryric acid)of gamma-aminobutyric acid (GABA); hence, GABA can be used to induce settlement in abalone larvae.  Typical test results in the laboratory are shown on the Right for larvae of the California red abalone Haliotis rufescens.  Note that GABA promotes 100% settlement, and this takes place in less than 10min under general laboratory conditions.  GABA appears to work with all species of abalone, and is used extensively by mariculturists the world over.  Successful settlement of thousands of larvae en masse in just a few minutes has obvious economic implications in abalone culture. Data from Morse et al. 1979 Science 204: 407.

NOTE  a small-molecular-weight molecule that acts as a neurotransmitter in many animals including humans
table showing settlement of larval abalone Haliotis rufescens on various substrata including GABA
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Research study 4
 

graph showing settlement-inducing effect of GABA on larvae of abalone Haliotis rufescens

Further studies on the influence of GABA on the development of Haliotis rufescens veligers show that in the presence of 10-3 M concentrations of the molecule, table showing metamorphic-inducing effect of various amino acids including GABA on larvae of abalone Haliotis rufescensmetamorphic competency is reached within 160h from fertilisation (graph on Left, 15oC). Control larvae not treated with GABA are still not competent to settle even as long as 240h after fertilisation.

Tests of other amino-acid neurotransmitters, some homologues of GABA and others differing by a little as one carbon atom in chain length from GABA, show variable results on 8d-old larvae (table on Right: asterisked substances are homologues of GABA). The authors note that the closely related GABA homologues and other amino acids are less effective, requiring higher concentrations and longer exposures to induce metamorphosis (data not shown here).  GABA is essentially 100% effective after 1h at concentrations of 10-3-10-4 M.  Morse et al. 1980 Fed Proc 39: 3237.

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Research study 5
 

graph showing effect of potassium ion addition to seawater on settlement of abalone Haliotis rufescens larvaeAnother chemical inducer for settlement and metamorphosis of larval abalone Haliotis rufescens is increased concentration of K+ in the surrounding seawater.  Studies at the Marine Sciences Institute, Santa Barbara, California show that addition of K+ in concentrations in excess of those already present in seawater of 6-12mM KCl or K2SO4 significantly increases the percentage attachment of larvae (see graph). Note the dose-dependency of the settlement response in the larvae.  The researchers also show that a decrease in external potassium ion concentration will inhibit the larval settling response to GABA.  The authors propose that increased K+ may act in a photograph of abalone Haliotis rufescens courtesy Kevin Lee, Fullerton, Californiamanner similar to GABA, by directly depolarising excitable cells involved in perception of inductive stimuli by the larva.  Baloun & Morse 1984 Biol Bull 167: 124. Photograph courtesy Kevin Lee, Fullerton, CA diverKevin.

NOTE  the authors choose not to have a control treatment in the experiment, perhaps because they know that without addition of potassium the larvae will not settle

 

Haliotis rufescens 1X

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Research study 6
 

map showing release and recovery locations for drift tubes in abalone-recruitment studyphotograph of green abalone Haliotis fulgens courtesy Kevin Lee, Fullerton, CALike most species of Haliotis, larvae of green abalone H. fulgens are free-living for just a few days.  Consequently, their potential for dissemination over any but short distances is severely limited.  This is tested by researchers from the Scripps Institution of Oceanography, La Jolla by releasing drift tubes from several abalone-populated sites in the Channel Islands and recording time taken to arrive at the mainland.  At the time of publication of this work, mainland stocks had been largely depleted, and it was thought that recruitment might potentially come from Channel-Islands stocks.  Of 1225 tubes released in June, only 4% are recovered on the mainland (see map).  More importantly, only a handful (about 5 in total) is recovered within the normal competency period of the larvae (average crossing time is about 16d, while larvae of H. fulgens would be expected to settle within about 10d at 21oC, the seawater temperature at the time).  The authors remark that while some transport occurs between isolated mainland populations within a realistic time period, the potential for this to have any significant impact is likely slight.  In comparison, a high proportion of tubes released from both island and mainland sites travel just a few km to suitable sites within biologically realistic times.  These observations suggest that the present fishery closure (dating from 1977) is unlikely to promote recovery of mainland populations any time soon.  The study is an interesting one, and is sure to have been pursued in later papers.  Tegner & Butler 1985 Mar Ecol Progr Ser 26: 73. Photograph courtesy Kevin Lee, Fullerton, California.

NOTE  the “larval mimics” are clear plastic test-tubes of 15 x 1.6cm size, sealed and weighted to float upright just at the sea surface.  A stamped and addressed card inside instructs the finder to provide date, time, and precise location of recovery.  Tubes are released from both island and mainland sites in June and October, but only data for the island releases are reported here.

NOTE  a partial assumption made by the authors with regard to these data is that recovery is simultaneous with landing on the shore, which is unlikely.  Also, of the 5 tubes crossing to the mainland in a realistic time, only “a portion” is found near suitable rocky habitat.  The authors do not speculate on the actual number of larvae that might be represented by this “portion”, based on numbers of individuals spawning on the islands, nor on what size population of upstream spawners would be required under any circumstances to provide “adequate” numbers of larvae for an “x-sized” estimated recruitment. Perhaps 5 out of 1225 drift tubes would actually represent multi-billions of larvae being transported

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Research study 6.1
 

beating cilia of a veliger larva of the trochid snail Calliostoma ligatumWhen swimming a veliger larva of an abalone or related trochid snail bumps into things, such as other swimming organisms, floating objects or, in shallow water, even the sair-water interface  itself.  In all cases, swimming is likely to stop  immediately through cessation of beating of the velar cilia.  Similarly, when a competent larva is swimming/crawling along the sea bottom in search of a settling spot, a ciliary-photograph of Calliostoma ligatum in an aquarium tank feedingbeat interruption precedes each crawling phase.  Researchers at Friday Harbor Laboratories, Washington identify in competent larvae of the trochid snail Calliostoma ligatum 2 types of neuronal input from the central nervous system that control ciliary beating.  The first is a high-frequency input that generates long-duration slow depolarisations of the ciliated cells that slows ciliary beating and diminishes swimming or feeding; the second is a rapid velum-wide ciliary arrest that stops these activities entirely.  The ciliated cells are electrically coupled to one another, a feature that ensures the synchrony of these slowings or stoppages.  Arkett et al. 1987 Biol Bull 173 (3): 513.

Calliostoma ligatum seen from below 1.5X

Side view of a single ciliated cell from C. ligatum
showing an effective beating stroke from left to
right.  The bumps on the cell are lipid vacuoles

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sResearch study 6.1
 

Settlement behaviour in abalone larvae involves cessation of swimming and initiation of exploration of the substratum by foot.  As we have seen in earlier research on abalone larvae, these responses are identical whether the settlement inducer is natural coralline algae or GABA (gamma-aminobutyric acid).  An aim of a researcher at the University of Washington, Seattle is to determine the  location of the chemoreceptive sites that mediate settlement, focusing initially on the ciliated cells of the larval swimming organ, the velum.  Through intracellular recordings from these cells and simultaneous videotapes made of larval behaviour, the researcher determines that larvae, whether pre-competent or competent, respond to GABA in similar ways with respect to swimming behaviour and, more importantly, that the receptors for GABA are not present on the ciliated cells of the velum as first hypothesised. Tests of larvae of increasing age show that they become increasingly sensitive to GABA, something known for larvae of other species tested with their own specific settlement-inducing stimuli.  The author thinks that this increased sensitivity may delineate the beginning stage of metamorphic competency, an event that occurs some time after the time the larva is competent to settle.  Barlow 1990 Bull Mar Sci 46 (2): 537.

NOTE  determined through a series of experiments showing that GABA does no alter the net membrane conductance of the velar cells

 

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Research study 7

 

photograph of model used in study of rugosity/current effects on settlement in abalone Haliotis rufescensAs the larvae move along in currents at the time of settlement, are they active or passive on a small scale?  This is tested in laboratory flow tanks in Santa Barbara, California where settlement of Haliotis rufescens larvae is compared with settlement of larval-mimicking particles under different current intensities flowing over plastic casts modeled after natural rock substrata.  The current carries the larvae and mimic-particles over high-velocity regions of the model (on the tops of projections, indicated in yellow on the model at Right) and past low-velocity regions (in and around depressions, indicated in pink).  In each location chips of coralline algae are placed as cues to induce settlement. histogram showing settlement percentages of larvae of Haliotis rufescens under different conditions of substratum rugosity and current speeds In low-velocity regions of the model where deposition of mimic-particles is great (left pink bar in histogram), the author favours a prediction that larval settlement will be low because of poor contact with the settlement-inducing algal chips.  An alternative prediction is that it would be high because particles would settle out. In fact, larval settlement is actually high, indicating that the larvae behave more like passively deposited particles.  In comparison, in high-velocity areas of the model where deposition of mimic particles is small and where we predict that larval settlement will be great because of good contact with the settlement-inducing algal substrata (right yellow bar in histogram), larval settlement is much lower than prediction but significantly more than predicted for a passive particle.  The author sums up this part of a much larger study by noting that the pattern of larval settlement in the flow tanks closely resembles that of a passively deposited particle, but one that still responds to a cue when able.  Boxshall 2000 J Exper Mar Biol Ecol 254: 143.

NOTE the mimicking-"particles" are actually dead or inactive larvae, and empty shells, most of a size of about 320µm

NOTE  veliger larvae are able swimmers, but would be swept along in even the slowest current velocities used in the flow tanks (3.5 . sec-1) were it not for boundary-layer flow effects.  Shear forces may be high away from the substrate surface, but they diminish as the substrate surface is approached until, just at the substrate-water interface, they ultimately decrease to zero.  This means that even on the tops of projections on the model where the current is fast, near-zero velocities are accessible close to the substratum, just a short distance away.  For the tiny veliger larvae, the algal surfaces represent an oasis of calm within the storm

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Research study 8
 

Settlement of a veliger larva requires that swimming be stopped, and this is accomplished by cessation of beating of the cilia located on the larval swimming organ, the velum, by nerve impulses generated in associated ganglia.  Researchers at Friday Harbor Laboratories, Washington discover that all ciliated cells on the velum of a slipper limpet Crepidula fornicata are electrically coupled.  While a single action potential causes temporary disruption of ciliary beating, a burst of impulses causes complete cililary arrest, sometimes accompanied by retraction of the velar lobes. Recordings via a fine wire electrode tethered to the shells of swimming and crawling larvae show that these cessation-type impulses are photograph of veliger larva of a slipper limpet tethered to a platinum recording wiremore frequent during bouts of exploratory crawling on the substratum than during bouts of pre-settlement swimming.  Tests with larvae of different ages show that it is contact with the substratum, rather than age of larva, that is the main trigger for ciliary-arrest spikes.  Having said this, younger pre-competent larvae are significantly less responsive to substratum contact than  older competent larvae.  Contrary to expectation, pulse rates do not differ significantly in larvae crawling on clean plastic than on surfaces coated with natural settlement cues (e.g., mucus from conspecific adults).  The authors include in their study  considerable immunohistochemical detail on possible neuromodulators in the velum and their responses to different neurotransmitter chemicals, and discuss generally the neurochemical control of velar functions involved in settlement behaviour.  Penniman et al. 2013 Invert Biol 132 (1): 14.

Veliger larva of Crepidula fornicata
tethered byits shell to an amplifier/
oscilloscope. The purple arrows indicate
metachronal waves of cilliary beating

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