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  Settlement cues
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Research study 1

photograph of larva of nudibranch Rostanga pulchra showing ciliar rejection tract for unwanted food
photograph of dorid nudibranch Rostanga pulchra crawling on it food sponge Ophlitaspongia pennata Dave Cowles, Washington Eggs of Rostanga pulchra hatch in the laboratory about 2wk after being laid and the veligers feed on phytoplankton for another 5-6wk (at 10-15oC) before settling and metamorphosing. Metamorphosis is readily induced in the presence of the sponge Ophlitaspongia pennata, which is its principal habitat and food in later life. Note the prominent groove on the ventral surface of the foot of the veliger larva, which is a ciliated rejection tract for unwanted food particles.  Chia & Koss 1978 Mar Biol 46: 109.



Dorid nudibranch Rostanga pulchra with its
food sponge Ophlitaspongia pennata 2.5X.
Photo courtesy Dave Cowles, Washington
and Seaslug Forum at seaslugforum

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Research study 2

histogram of % settlement by larval Hermissenda crassicornis with and without settlement inducersphotograph of aeolid nudibranch Hermissenda crassicornis
Laboratory studies at Woods Hole, Massachusetts using adult specimens obtained from California show that Hermissenda crassicornis veligers are competent to settle during a -wk period (at 12-17°C), with the best time for inducing metamorphosis being from 4-7wk following hatching.  As the larvae are able to metamorphose with several natural inducers, including hydroids (Pennaria sp.) and sea anemones (e.g., Metridium senile), and often spontaneously, metamorphosis appears to be non-specific.  Combinations of the presence of natural inducers with certain cations (e.g., K+, Mg2+) also produce high metamorphic yields, up to 25%.  Best metamorphic success, however, is attained with aqueous extracts of hydroids Tubularia crocea at high concentrations (yielding 50% metamorphosis), suggesting that the metamorphosis-inducing compound is water soluble.  Note in the histogram that additon of Tubularia extracts will induce early metamorphosis although, as noted above, spontaneous metamorphosis does occur in the absence of any inducer substance. Avila 1998 J Exp Mar Biol Ecol 231: 81; see also Avila et al. 1994 Biol Bull 187: 252 for a preliminary study on settlement induction in H. crassicornis. 

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Research study 3

photo series showing metamorphossis of the dorid nudibranch Onchidoris bilamellatatable showing % settlement of larval Onchidoris bilamellata onto different types of barnacle-type substrata
A study on settlement and metamorphosis of larvae of the barnacle-eating nudibranch Onchidoris bilamellata at Friday Harbor Laboratories, Washington and Bamfield Marine Sciences Centre, British Columbia indicates that the 2 behaviours are separate events.  This is unusual because in most or all other opisthobranchs they are continuous – once one starts, the other follows.  Metamorphic competency in Onchidoris is reached after about 30d at 11°C. 

table showing % metamorphosis of larval Onchidoris bilamellata onto different types of barnacle-type substrataResults of tests with different substrata or conditioned seawaters suggest that settlement is induced by chemical(s) diffusing from living barnacles (see Table upper Right).  Note that aging or boiling of barnacle-conditioned seawater considerably reduces the effectiveness of the inducing substance.  Barnacle plates treated with NaOH have no effect on inducing settlement, but if the plates are kept among living barnacles for 5d they become partially effective.  Finally, exposure of the competent veligers to seawater conditioned by the presence of other invertebrates induces some larvae to settle (14%).  Another interesting result from this part of the study is that settlement, involving descent to the bottom, foot contortions, and crawling, is reversible.  In comparison, metamorphosis, involving absorption of the velum and loss of shell, is irreversible. 

Whereas settlement is induced by some diffusible substance, metamorphosis is triggered by contact with living or dead barnacles – but only in seawater that has previously contained living barnacles (see Table on Left). The authors note that the reversibility in settlement in Onchidoris is unique among nudibranchs.  Chia & Koss 1988 Int J Invert Repr Dev 14: 53.

NOTE  invertebrates used include goose barnacles Pollicipes polymerus, mussels Mytilus trossulus, and crabs Hemigrapsus sp.  These invertebrates are kept for 24h in seawater, separately, then the seawater is used in tests of settlement

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Research study 4

photographs showing location of settlement receptors in the veliger larvae of the nudibranch Onchidoris bilamellatadrawing of dorid nudibranch Onchidoris bilamellata
There has been much research interest in identifying the chemical composition of metamorphosis-inducers in opisthobranch molluscs.  Many natural inducers are known, including presence of conspecifics and species associated with a suitable habitat (e.g., bacterial films, algae, invertebrates). Additionally, one cue that often induces settlement and metamorphosis in molluscan veligers is the prey food of the adult.  After identifying and characterising a metamorphosis-inducing chemical in a larva, the next task is to identify the receptor cells that respond to the chemical.  This has been done for veliger larvae of the nudibranch Onchidoris bilamellata, a mid-intertidal consumer of barnacles, at Bamfield Marine Sciences Centre, British Columbia.  A unique set of nerve structures, termed the anterolateral ganglia, are found to contain sensory receptor cells that depolarise on contact with a known settlement cue for the nudibranch, namely, barnacle-conditioned seawater.  The anteriolateral ganglia are located in 2 areas known as the settlement receptor fields - visible under a dissecting microscope as oblong, cilia-free cellular masses on either side near the anterior and middle regions of the larval foot.  The authors note that this is the first demonstration of electrophysiological properties of larval settlement-receptor cells. Arkett et al. 1989 Biol Bull 176: 155; for more information on the structure of these anterolateral ganglia in O. bilamellata see Chia & Koss 1989 Cell Tissue Res 256: 17.

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Research study 5
  photograph of sensory cell in the larva of Onchidoris bilamellata
In another paper by the same research group the authors are able to excise the anterolateral ganglia from the foot of competent veligers of the nudibranch Onchidoris bilamellata, dissociate the component cells including sensory cells, and maintain them alive in culture for up to 4d.  The sensory cells are more numerous than other cell types and are located on the outer, lateral perimeter of each ganglion.  Each sensory cell is about 2µm in diameter and characteristically has 2 neurites radiating from the cell body.  The aim of the work is to use the isolated cells in later electrophysiological studies of settlement/metamorphic cues, thereby avoiding the difficulty of working with whole-animal preparations.  The authors reiterate that the anterolateral ganglia in the foot of larval Onchidoris represents the only system where morphologically identified chemosensory receptor cells have been shown to respond electrophysiologically to a known settlement cue.  Chia et al. 1992 Biol Bull 182: 66.
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Research study 6
  histograms comparing settlement-inducing quality of several species of algae to larvae of sea hares Aplysia californicaExperiments done at the Scripps Institution of Oceanography, La Jolla, California reveal that larvae of sea hares Aplysia californica will settle and metamorphose not just on one or a few red-algal species, but on a number of red, brown, and green species. Best inducers to settle of the species offered are the red algae Rhodymenia californica, Corallina officinalis, Plocamium cartilagineum, and  Laurencia pacifica.  However, only the last 2 species promote feeding and growth, and juveniles that metamorphose on the other species soon crawl off them, presumably to seek out a better food source.  Pawlik 1989 Mar Ecol Progr Ser 51: 195.
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Research study 7

histogram showing % metamorphosis of larvae of the cephalaspidean opisthobranch Haminoea callidegenita exposed to different fractions of egg-mass jelly photograph of partly bleached green alga Chaetomorpha sp.
A natural metamorphosis inducer for the cephalaspidean Haminoea callidegenita is the green alga Chaetomorpha linum, which is a food for both juveniles and adults.  Additionally, earlier research1 shows that a water-soluble chemical in the egg-mass jelly not only induces competent larvae of Haminaea to metamorphose, but causes an unusual developmental pattern where both swimming veligers and crawling juveniles are produced from the same egg mass.  In the research considered here the authors have partially purified the substance2 and tested its metamorphic efficacy on Haminaea veligers and on other molluscan veligers (4 opisthobranchs and an oyster).  The substance is smaller than 1000 daltons3 in molecular mass, and is polar, non-protein, and stable to boiling water temperature and acid. It is effective only with Haminaea veligers and not with veligers of the other molluscs. Note in the graph that elution-fraction "E" is more effective at inducing metamorphosis than extract of egg-mass jelly.  The authors note that this is the first demonstration of larvae metamorphosing in response to a substance present in a maternal egg mass.  Gibson & Chia 1994 Biol Bull 187: 133.

NOTE1  see Research Study1 under Haminoea in HATCHING  & LARVAL DEVELOPMENT

NOTE2  the method involves first extracting the egg-mass jelly sequentially with organic solvents of increasing polarity, then evaporating each solvent, resuspending each residue in seawater, then testing the metamorphic-inducing ability of each with competent veligers.   Identification in this way of the inducer in the methanol extraction then allows isolation using high-performance liquid chromatography.  Each “fraction” shown in the graph is a 2-min capture of elution fluid (methanol extract plus buffer) as it flows through the chromatography column.  Each 2-min portion is dried, resuspended in seawater, and tested for its efficacy on competent veligers

NOTE3  named after an English chemist/physicist who formulated atomic theory and the law of partial pressures; a unit of molecular mass approximately equal to the mass of an hydrogen atom

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Research study 8

graph showing % metamorphosid of larvae of the cephalaspidean Haminoea callidegenita when exposed to different natura substrataThe attainment of metamorphic competency in sibling larvae of Haminoea callidegenita varies over a 2-wk period, but almost all larvae eventually metamorphose successfully.  In an experiment at Friday Harbor Laboratories, Washington, gastrulae within their individual capsules are teased from their egg masses and placed in culture wells containing filtered seawater and a single potential metamorphic inducer.  Treatments are continued for 2wk after hatching (17°C).  Each day the number of hatched veligers and new juveniles are tallied.  At first, the veligers are super-responsive, and the ones treated with egg-mass jelly metamorphose rapidly within their capsules in response to the presence of a naturally occurring inducer substance (see yellow line on graph). Some larvae in all treatments also metamorphose within the egg capsule, but at a slower rate.  Within a few days the larvae become less choosy, and begin to respond to other cues such as ones present in seagrass Zostera marina (dark green on graph) and filamentous alga Chaetomorpha linum (light green). In time, even larvae in seawater in the absence of a known metamorphic cue will spontaneously metamorphose. Ultimately, almost all the veligers metamorphose.  Rather than reflecting the slow development of a few “inferior” larvae, this intra-clutch variability in time of metamorphic competence may be adaptive in providing flexibility within the larval period.  Thus, the quick-metamorphosing veligers will settle close to their parents within or near favourable estuarine systems inhabited by them, while some slower-mtamorphosing siblings will undertake longer-range dispersal with its obvious advantages, and risks.  The author reminds us that feeding is not an issue here because the veligers of Haminoea are lecithotrophic.  Gibson 1995 J Exp Mar Biol Ecol 194: 9.

NOTE  the author also includes a treatment of 19 mMol K+ which is known to induce metamorphosis to a similar degree as natural egg-mass jelly; these results, and the results of a sediment treatment are not shown here

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Research study 9

histogram comparing the potential of several preparations of the green alga Vaucheria longicaulis to induce settlement in larvae of the sacoglossan Alderia modestaThe sacoglossan Alderia modesta1 is a specialist feeder on the alga Vaucheria longicaulis, and this is its only known food source.  Research studies at San Diego, California show that settlement of larval Alderia is initiated by chemical cues released from and present on the cell surfaces of the alga. Note from these data that the alga does not have to be alive to induce settlement. The soluble components are mostly extracted with single, but no more than double, treatments with boiling water, and the boiling has no effect on the potency of the extract.  Analysis reveals that the soluble cues are high and low molecular-mass carbohydrates2, while the insoluble algal cell-wall cues are high molecular-mass carbohydrates.  Larval Alderia exist in 2 forms3 in a single spawn mass.  Some sibling larvae metamorphose spontaneously withing 2d of hatching, while the remaining veligers delay metamorphosis until encountering chemical cues derived from the adult food-alga Vaucheria.  Only the latter type of larvae is used in the present experiments.  Although the results strongly suggest that the settlement cue for A. modesta is a carbohydrate, the authors note that definitive proof will require the isolation of a pure oligosaccharide that induces metamorphosis.  The study is the first to show that the same substratum produces both secreted and surface-associated forms of a larval-settlement cue, each sufficient to induce metamorphosis.  From the larval side of things, selection for receptors for both soluble and insoluble chemical cues from their single algal food source must favour a greater proportion of individuals recruiting successfully. Krug & Manzi 1999 Biol Bull 197: 94.

photograph of sacoglossan Alderia modestaNOTE1  recently the author has determined that the subject of this and other studies by he and his research group is actually a new species Alderia willowi.  It is this new species A. willowi, indigenous to California south of Bodega Harbor, that is poecilogonous (it produces both planktotrophic and lecithotrophic larvae), while Alderia modesta, a cosmopolitan species, produces strictly planktotrophic larvae.  Krug 2007 Amer Malac Bull 23: 99.

NOTE2  the soluble carbohydrates are <2,000Da, while the insoluble ones are >100,000Da

NOTE3  these are described elsewhere in the ODYSSEY:  HATCHING & LARVAL DEVELOPMENT/ALDERIA

Note the algal-packed digestive diverticula in this Alderia
. It has recently been feeding on Vaucheria longicaulis

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Research study 10

histogram showing that water from Vaucheria patches significantly enhances settlement in the sacoglossan Alderia modestaIn a follow-up paper to the foregoing Research Study the researchers focus on the simple carbohydrates, mannitol and glucose, as possible waterborne chemical cues involved in settlement and metamorphosis of the specialist herbivore Alderia modesta.  Both sugars are highly concentrated in the tissues of Alderia’s food alga Vacheria longicaulis and both are released from the absorbent mats of the alga into the overlying water when the tide comes in.  Tests show that this overlying water (diluted 1:5 with filtered seawater) induces changes in swimming behaviour and significantly increases metamorphosis in larvae of Alderia (see histogram comparing concentrations of mannito and glucose in overlying "patch" water in green-algal Vaucheria beds in Mission Bay, Californiahistogram upper Left).

Water samples taken from in and around Vaucheria patches at Mission Bay near San Diego, California show high concentrations of mannitol and glucose concentrations above the centre of a histogram comparing percentage metamorphosis in sacoglossan Alderia modesta larva in wataer from mats of different green-algal speciesVaucheria patch, diminishing towards the edges and outside of the patch (see histogram on Right). In comparison, water collected from pores in mats of co-occurring green alga Enteromorpha clathrata, a non-food alga for Alderia), does not affect swimming behaviour or rate of metamorphosis in the larvae (see histogram lower Left). The authors point to the strong correlation between responses of larval Alderia to the sugars mannitol and glucose (and also possibly to other complex carbohydrates) released from Vaucheria and natural concentrations of these chemicals around the algal patches, and suggest that their study may be the first to document the structure of a natural, water-soluble inducer of metamorphosis in a marine larva. They also note that their study is the first to suggest that larval settlement in an intidal habitat may vary both temporally and spatially owing to dilution of waterborne cues during an incoming tide.  Krug & Zimmer 2000 Mar Ecol Progr Ser 207: 283.

NOTE  other work by the senior author shows that mannitol comprises 1.5% of the dry mass of V. longicaulis and glucose represents 60% if the pool of low-molecular mass carbohydrates in the alga

NOTE  as to how the sugars get out of the alga, the authors suggest that leakage from woundings or other damage may be responsible.  Vaucheria is a siphonaceaous alga with a contiguous cytoplasm within each blade, so damage could lead to considerable leakage of intracellular products into the surrounding seawater

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Research study 11

histogram comparing settlement-inducing effectiveness of different dilutions of Vaucheria-conditioned seawater on larval activity in the sacoglossan Alderia willowiphotograph showing the two species of Alderia: modesta and willowi courtesy Patrick Krug, California State University, Los AngelesStudies at the Scripps Institution of Oceanography, California show that on contact with waterborne cues from the green alga Vaucheria longicaulis, the lavae of Alderia willowi increase their turning rate, change their swimming speed, and move in rapid hops or spirals along the sea bottom. Note in the graph the significant diminution in rate of direction change, expressed in number of degrees . sec-1, with increasing dilution of the settlement cue.  These behaviours tend to keep the larvae in the immediate region of the dissolved cue; hence, close to their adult host food Vaucheria.  While both lecithotrophic and planktotrophic types of larvae in this poecilogonous species behave similarly, the response of the long-lived planktotrophic type is stronger.  Metamorphosis in the presence of the dissolved cue from Vaucheria is also dose-dependent, as suggested by data from experiments using Vaucheria-conditioned seawater (data not shown here). These experiments show non-significant differences between the lecithotrophic and planktotrophic larvae.  Krug & Zimmer 2000 J Exp Biol 203: 1741. Photograph courtesy Patrick Krug, California State University, Los Angeles krugLab.

NOTE  the conditioned seawater is aspirated from the surface of a Vaucheria algal mat immediately after the tide has receded

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Research study 12

graph comparing histogram showing genetic variability among clutches of larvae from different adults of Alderia sp. with respect to % metamorphosis when dosed with Vaucheria waterThe close trophic relationship between Alderia sp. and its host alga Vaucheria longicaulis allows tests to be made of whether lecithotrophic larvae become less discriminating over time to settlement cues owing to depletion of energy reserves.  Because the larvae of Alderia can develop planktotrophically, response of larvae to settlement stimuli1 can be tested at different nutritional2 levels.  Results of experiments at the Scripps Institution of Oceanography, California show, as predicted, that sensitivity of fed (planktotrophic) larvae to dissolved chemical cues from Vaucheria does not change over time, while that of unfed larvae (lecithotrophic) becomes increasingly more responsive to settlement cues over time. This is shown in the graph on the Left.  Note that 3d-old larvae are significantly more responsive than 1d-old larvae.  Metamorphosis also becomes faster, with 4d-old larvae completing their metamorphosis up to 24h faster than 1d-old larvae.  Thus, energy-depleted larvae react to weaker cues and respond more quickly during habitat selection.  If, however, unfed larvae are not exposed to host extract they mostly die within 2wk without metamorphosing.  The authors explain that though a common behaviour in other species, spontaneous metamorphosis would be fatal for larvae of Alderia in the absence of their specific food host. An interesting side-experiment in the study reveals significant variation in response to the settlement cue in larvae from different adults, suggesting a heritable3 basis for cue-sensitivity in Alderia. Botello & Krug 2006 Mar Ecol Progr Ser 312: 149.

NOTE1 a stock “stimulus solution” is prepared by boiling 1.34g live tissues of Vaucheria in 50ml distilled water, then centrifuging to clarity.  This particular concentration is known from earlier studies to induce settlement and metamorphosis in Alderia larvae.  This stock solution is used to test dose responses of the larvae

NOTE2 in the laboratory the larvae are fed on a 1:1:1 suspension of Rhodomonas sp., Isochrysis galbana, and Pavlova lutheri at a concentration of 104 cells . ml-1.  Although the larvae of of the lecithotrophic type, they will feed on this suspension. The authors note that this is the first demonstration of feeding in these ‘lecithotrophic’ larvae

NOTE3  the authors note, however, that owing to sperm-storage ability in Alderia, the clutch-mates may not be full siblings, and further research will be needed to resolve this

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