Reproduction
  Fertilisation occurs in the open ocean and hatching is to a 4-arm pluteus larva, known as an echinopluteus.  Successive 4-, 6-, and 8-arm stages feed for 5-8 weeks on phytoplankton.  After this time, the 8-armed pluteus larva begins to show adult features internally and, a few days later, spines and tubefeet are visible externally.  Metamorphosis is complete within a week and shortly thereafter the little juvenile is crawling about on the sea bottom.  This section includes fertilisation. GONAD GROWTH & SPAWNING, LARVAL FEEDING GROWTH & LIFE SPAN, LARVAL SKELETON, and SETTLEMENT METAMORPHOSIS & RECRUITMENT can be found in other sections. 

NOTE
lit. “later form”  About 99% of all marine invertebrates produce a free-living larva.  Because the adaptations for a floating planktonic life are so different from those for a bottom-dwelling adult life, a complex metamorphosis must be passed through at the end of larval developmenti
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  Fertilisation
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Research study 1
 
graph showing fertilisation success in 3 sea-urchin species Strongylocentrotus droebachiensis, S. franciscanus, and S. purpuratus as a function of sperm concentration

Egg size in free-spawning marine invertebrates is usually thought to involve a trade-off between quantity and quality.  Many small eggs are metabolically cheaper to produce, but have slower growth, spend longer time in the plankton, and incur greater risk of mortality.  Production of fewer large eggs leads to greater proportional survival, but with smaller overall recruitment.  But what about another idea: that larger eggs present a larger target for sperm and thus are fertilised at a higher rate?  This is tested with 3 species of sea urchins Strongylocentrotus droebachiensis, S. franciscanus, and S. purpuratus at the Bamfield Marine Sciences Centre, British Columbia.  Egg sizes in these species are 148,130, and 78µm in diameter, respectively. 

Results show that fertilisation success as a function of sperm concentration falls off as predicted, from highest levels in the large-egged species S. droebachiensis to lowest levels in the small-egged species S. purpuratus, with S. franciscanus  being intermediate (see accompanying graph). When swimming speeds of the sperm are also considered, these data have further ecological implications.  Thus, the species possessing the smallest eggs and fastest sperm, S. purpuratus, lives in shallow water and tidepools – both situations leading to a concentrating of sperm and concomitant higher levels of fertilisation.  The species with the largest eggs and slowest sperm, S. droebachiensis, lives subtidally but often in high population density.  Strongylocentrotus franciscansus, with intermediate egg size and sperm swimming speed, also lives subtidally and often in high densities.  However, it produces an order of magnitude more sperm than the other two species.  The paper is interesting and provides a provocative insight into evolution of egg size in marine invertebrates.  Levitan 1993 The Amer Nat 141: 517.

NOTE  sperm swimming speeds are 145, 130, and 88µm . sec1 for S. purpuratus, S. franciscanus, and S. droebachiensis, respectively.  Sperm half-life is expected to relate to swimming speed.  However, data presented by the author appear to show only a non-significant trend of longer-lived sperm in S. droebachiensis as compared with the other species

 

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Research study 2

 
schematic showing factors influencing fertilisation success in red urchins Strongylocentrotus franciscanus

To get a sense of what it would be like to be a sea urchin trying to accomplish fertilisation, visualise an aggregation of sea urchins of mixed sexes, and think of factors relating to the group or to its immediate environment that would enhance fertilisation.  Some ideas are listed.  Ideas from Levitan et al. 1992 Ecology 73: 248.

Group size.  The more animals there are and the larger the area that they inhabit, the more liklihood there is that sperm and eggs will meet.

Density.  Closer packing reduces sperm dilution.

Current flow.  By allowing longer time for sperm and egg to meet, a slower rate of current flow leads to greater fertilisation success.

Downstream location.  A central and/or downstream location will favour fertilisation success.

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Research study 3
 

There are several studies in which the sperm-dilution process in sea urchins is simulated using syringes filled with sperm and downstream sperm-permeable containers filled with eggs, and the general conclusion is that sperm limitation may be severe unless spawnings are simultaneous, sexes in close proximity, water currents slow, and time-spans short. 

In one example of the use of this method, the effect of current velocity on fertilisation success in green sea urchins Strongylocentrotus droebachiensis is assessed in the San Juan Islands, Washington.  Here, ten 10ml syringes containing eggs from a spawning female are arrayed downstream from single gravid males.  The first five are positioned at 10, 20, 40, 60, and 80cm distance, and the second five at 1-5m distance.  A CONTROL syringe with eggs is located just upstream of the male.  The male is then induced to spawn by injecting it with KCl.  Once the spawning stream has reached the last downstream syringe at 5m the plungers of all syringes are opened to draw in a fixed volume of seawater.  The syringes are returned to the lab where % fertilisation of the contained eggs is assessed.   The procedure is done in fast and slow current flows.  In the fast-flow tests about 25% of eggs are fertilised at 10-cm distance, but only 5-10% at distances beyond this.  In the slow-flow tests, fertilisation within the first meter is about 20-60%, but at distances over 1m, is less than 20%.  In the sperm-rich zones just above the spawning males, fertilisation is about 90%.  No eggs are fertilised in the upstream CONTROL syringes.  In other tests with 3 spawning males, fertilisation success increases such that at 3-4m distance, % fertilisation exceeds 20%.  The author concludes that if free-spawning adults of a species do not aggregate prior to spawning then percentage fertilisation will often be low.   However, it is not known if sea urchins do aggregrate prior to spawning.  Pennington 1985 Biol Bull 169: 417.

NOTE  fast current: >0.2m per sec; slow current: <0.2m per sec (a 1m per sec current is equivalent to 2 knots)

graphs showing fertilisation success in green urchins Strongylocentrotus droebachiensis in different current speeds and at varying distances downstream from a spawning male
 

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Research study 4

 
photograph of red urchins Strongylocentrotus franciscanusdiagram of syringe array used in sea-urchin fertilisation experiment at Bamfield, British ColumbiaIn a similar type of experiment with red urchins Strongylocentrotus franciscanus conducted near the Bamfield Marine Sciences Centre, British Columbia, size of spawning group and degree of aggregation are mimicked in field arrays.  Sperm are contained in 20ml syringes and unfertilised eggs are contained in fine-mesh sperm-permeable containers. The syringes and containers are positioned in arrays 15cm above the substratum to mimic the height of an adult urchin (see diagram on right).  The arrays are 2x2 and 4x4 to test effects of spawning-group size (i.e., small or large), and are dispersed or aggregated to test effects of density.  All sperm syringes are depressed within 1min and the egg histogram showing fertilisation success in red urchins Strongylocentrotus franciscanus relative to group size degree of dispersion of indivuals containers are left for 30min before being returned to the laboratory where fertilisation success is assessed.

Results show that fertilisation success increases significantly with increased group size and degree of aggregation.  In another set of experiments using all 4 treatments, current velocity is shown to affect fertilisation success negatively. The study provides supportive evidence as to the importance of population parameters and flow conditions on fertilisation success.  It is especially valuable because it is done in the field.  Levitan et al. 1992 Ecology 73: 248.

NOTE  the “nearest-neighbour” distances between “males” and “females” for “bunched” is 0.5m and for “dispersed”, 2m.  These correspond to equivalent field densities of 4 and 0.25 individuals per sq meter, respectively.  The arrays are positioned at 9m depth

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Research study 5
 

Fertilisation in broadcast-spawning bottom-dwelling marine invertebrates is a haphazard process owing to sperm dilution, and sea urchins are no exception.  What appears to happen is that one sex or the other (possibly the female?) begins to spawn and then the other sex follows suit.  There may also be pheromones released that mutually stimulate many individuals in a group to spawn en masse, although if such chemicals exist they have yet to be identified.  Fertilisation can only occur if the eggs are downstream from a spawning male, and sperm dilution, especially in currents, may be enormous. The graph shows an apparently significant effect of increasing current velocity on fertilisation percentage in red urchins Strongylocentrotus franciscanus. Levitan et al. 1992 Ecology 73: 248.

NOTE  studies on tropical echinoids show that sperm concentrations may decrease by 5 orders of magnitude over a distance of just 2m in moderate current conditions (equivalent to a 100,000-fold dilution)

graph showing fertilisation success of red urchins in different current velocities
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Research study 6
 

photograph of red urchins Strongylocentrotus franciscanusOther laboratory studies at the Bamfield Marine Sciences Centre, British Columbia on fertilisation kinetics in Strongylocentrotus franciscanus show that sperm concentration is the most important factor related to fertilisation success, followed by contact time, and then sperm age.  A 4th parameter tested in the study, egg concentration, is found to be not as important.  This last may be explained by the fact that under usual circumstances of sperm and egg dilution, contact time, sperm age, and so on, there are still plenty of spermatozoa available for fertilisation.  Levitan et al. 1991 Biol Bull 181: 371.

 

Red urchins Strongylocentrotus franciscanus. It is not possible to tell the sex of a sea
urchin externally 0.25X

 
Research study 7
 

topographical view of a small surge channel in Pacific Grove, Californiaphotograph of surge channelThe turbulent mixing associated with wave-swept rocky shores is usually thought to be detrimental to fertilisation success of broadcast spawners owing to dilution effects on gametes.  Does the same constraint apply in surge channels?  Although these are well mixed, field experiments by researchers at Hopkins Marine Station, California show that water exchange between a surge channel and the outside ocean is remarkably low.  In fact, the “containment-vessel” effect of surge channels can be shown actually to enhance fertilisation success of sea urchins (e.g., Strongylocentrotus franciscanus) theoretically up to 80-100% levels.  The researchers conduct 4 experiments on different surge channels in Pacific Grove using release of fluorescein dye to monitor rates of exchange between the channels and outside ocean (see topography of one of the 4 sites: Seagull Channel in diagram on Right).  Wave heights and frequencies are measured, and after many trials, a mixing-model for blind-ended surge channels is formulated.  Data on fertilisation kinetics of red urchins Strongylocentrotus franciscanus and other species are then used to predict probabilities of fertilisation success in such “containment vessels”.  Results for a typical surge channel of 10m3 volume indicate that a single male could potentially fertilise 2-3 orders of magnitude more eggs than had been predicted in an earlier study for a male urchin residing outside of the surge channel.  In a larger channel of 50m3 with perhaps 200 males total, 90% of eggs are predicted to be fertilized. On a shore with abundant surge channels, then, the effectiveness of external fertilisation may be great. In comparison, in the infinite dilution of the uncontained offshore waters, fertilisation success falls off quickly. Denny et al. 1992 Biol Bull 183: 220.

NOTE  see Denny & Shibata 1989 The Amer Nat 134: 859


Surfy shore near Botanical Beach, British
Columbia with a surge channel

 
Research study 8
 

graph showing water-turbulence effects on fertilisation in purple urchins Strongylocentrotus purpuratuseffect of water turbulence on early development in purple urchins Strongylocentrotus purpuratusTurbulent surf with high levels of hydrodynamic-shear stress can have deleterious effects not just on fertilisation success in sea urchins, but also on early development.  This is shown for purple urchins Strongylocentrotus purpuratus by researchers at Hopkins Marine Station, Pacific Grove using special devices to recreate different levels of environmentally relevant shear stresses in the laboratory.  As eggs move in a velocity gradient they rotate, thus creating a secondary velocity gradient around the surface of the egg.  Sperm can swim, but not fast enough to keep up with an egg’s rotational speed in high shear stresses, and their elongated shape means that they “go with the flow”, that is, are carried tangentially to the egg’s surface.  In high flow rates, then, these contrary movements of sperm and egg would be predicted to hinder contact between them.  Results from the simulation tests show, indeed, that low shear stresses improve fertilisation, likely  through favourable mixing effects, while high shear stresses (as in the surf zone) not only significantly decrease fertilisation success (see graph on Left), but also lead to abnormalities in development through the blastula stage (see graph on Right).  The authors consider several possible explanations for the shear-induced decrease in fertilisation success, including gamete damage, decreased encounter rate between gametes, and stripping of sperm from the egg’s surface after attachment but before actual fertilisation, but conclude that more experiments will need to be done.  Mead & Denny 1995 Biol Bull 188: 46.

NOTE  as a wave breaks on the shore, it degenerates into turbulent eddies whose interaction causes the water to be sheared.  The friction between these shearing layers causes the wave’s energy to be lost as heat, a process known as energy dissipation rate, measured in Watts . m3.  Values in habitats favoured by S. purpuratus range up to about 2000 W . m3, depending upon water depth and wave height

 
Research study 9
 

graph showing revised estimates of water turbulence on fertilisation success in purple urchins Strongylocentrotus purpuratusIn a later paper, the authors of Research Study 7 above identify an error based on an incorrect prediction of the rate at which energy dissipates in the test apparatus.  So, instead, they directly measure the rate at which water in the apparatus is heated from energy dissipation, as turbulence-induced shear stress is experimentally increased.  The new results are shown with the old in the accompanying graph, and indicate that things may not be so grim in the purple-urchin world.  In fact, fertilisation is now seen to remain above 80% until dissipation rates exceed 2200 W . m3, a value higher than predicted for wavey conditions in purple-urchin habitat Botanical Beach, B.C.2m-high waves typical of surf conditions on rocky shores.  These new values suggest that in anything less than storm conditions, mixing associated with turbulence may be advantageous in bringing eggs and sperms into contact. Denny et al. 2002 Biol Bull 203: 275.

NOTE using a thermister device

Surfey conditions in urchin
habitat at Botanical Beach, B.C.

 
Research study 10
 

graph showing current velocities at different heights above the substratum in relation to fertilisation success of eggs of sea urchins Strongylocentrotus droebachiensisIn a study on green sea-urchins Strongylocentrotus droebachiensis in laboratory flume-systems in Maine, scientists note that both sexes may release gametes in a viscous fluid that clings to the spines and resists dilution for hours.  Moreover, measurements of current flow-rates around spawning sea urchins show marked reductions in velocity at the height of the urchins, and near-zero velocities in the boundary layer close to the substratum surface.  Note that the effect is even more marked at higher current velocities.  The eggs of this species are apparently negatively buoyant and could stay in the bottom boundary layer for some time.   Whether this viscous-fluid release is common behaviour in field animals is not known.  Yund & Meidel 2003 L&O 48: 795.

NOTE  the thickness of the boundary layer is not static.  In quiet-water conditions it may be quite thick, even up to 1m or so.   On rough rock surfaces typical of wave-swept shores, however, current velocity need be only a small fraction of a meter per second for the flow in the boundary layer to be turbulent.   Denny & Shibata 1989 The Amer Nat 134: 859

 
Research study 11
 

In areas around Bamfield, British Columbia 3 sea-urchin species Strongylocentrotus franciscanus, S. purpuratus, and S. droebachiensis may live in closely intermixed populations.  In this area, however, which is near the southern limit of its distribution, Strongylocentrotus droebachiensis is much lgraphs showing cross-fertilistion success between eggs and sperm of different species of sea urchins Strongylocentrotusess populous than the other 2 species.   Observations reveal that a single S. droebachiensis is much more likely to be surrounded by individuals of the other species than by conspecifics. In such aggregations, simultaneous spawning of all species is common.  What is the risk of heterospecific fertilisations occurring in these intermixed populations?  Laboratory data for these species generally show that females whose eggs are more easily fertilised by conspecific sperms are also more susceptible to heterospecific fertilisation.  For example, S. droebachiensis females require an order of magnitude less conspecific sperm for fertilisation, and heterospecific fertilisation in this species is high (see graphs above).  In contrast, note photograph of 3 species of sea urchins Strongylocentrotus: purpuratus, franciscanus, and droebachiensisthat S. purpuratus and S. franciscanus rarely cross-fertilise. Genetic analysis of S. droebachiensis larvae in the field show a preponderance of hybrids if male conspecifics are more than 1m from a spawning female.  Are these hybrids characterised by extraordinary "hybrid vigour"? Apparently not, as mortality is much higher in hybrid larvae than in conspecifically produced ones.  The author remarks that this susceptibility of S. droebachiensis for hybrid production, and its consequences, may be a factor limiting its southward distribution. However, under conditions of sperm limitation in mixed spawnings, the benefits of having easily fertilised eggs could possibly outweigh the costs of losing some offspring to hybrid fertilisations. Levitan 2002 Evolution 56: 1599.

NOTE  the author reports that it actually requires an order of magnitude less S. purpuratus sperm to fertilise 50% of S. droebachiensis eggs than of S. purpuratus sperm to fertilise the same proportion of eggs of its own species


Three common west-coast species of Strongylocentrotus (clockwise
from 7 o'clock: droebachiensis, purpuratus, and franciscanus

 
Research study 12
 

graph showing reproductive success (fertilisation success) of a female urchin in relation to density of malesgraph showing proportion of males in a mixed spawning event that produce offspring with every femaleFertilisation success in sea urchins obviously depends on the density of males in a population and, thus, on density of sperm, but can there be too many males?  The question of fertilisation success in a broadcast-spawning invertebrate is investigated at the Bamfield Marine Sciences Centre, British Columbia for the red sea-urchin Strongylocentrotus franciscanus.  During a spawning event, eggs from a single female may be simultaneously fertilised by sperm from numerous males (polyspermy).  If this happens, an egg may fail to develop.  The author uses microsatellite markers to track reproductive success in mating urchins, defined for a male as the extent of fertilisation of a female’s eggs. Thus, a value of 1 represents full fertilisation of all eggs of a female by a male. For a female, reproductive success is defined as the extent to which her eggs are fertilised.  While the value for a female can range only from 0 to 1, that for a male could greatly exceed 1, depending upon how many females are involved in a spawning event and the proportion of all eggs fertilised by that single male.  An individual sea urchin will release 1million eggs, or 100 billion sperm.  The graph on the Right shows that the mean reproductive success of females spawning with different numbers of males increases with density of males but then falls off.  Success is lower at low density because of sperm limitation and also at high density because of increased incidence of polyspermy. 

For both sexes fertilisation success depends strongly on proximity to mates, and on the extent of mixing and dispersal of gametes in differing water conditions.  In sea-urchin mating systems polygamy is the norm with, in the study, multiple paternity being detected in 98% of females and multiple maternity in 83% of males. This is perhaps to be expected in a species with broadcast spawning. However, more striking is the proportion of individuals that mate with each other.  For example, in a spawning event an average of 65% of the males will produce at least some offspring with every female (see graph on Right).  Note that when only a few males spawn, most males produce offspring with all females, but this proportion falls off as density of males increases.  The author remarks that the high degree of multiple paternity noted in S. franciscanus could indicate that fertilisation results from random mixing.  If so, then variation in reproductive success may not result from sexual selection, but will simply reflect random variation in positioning of males and females during spawning. The author discusses this and other aspects of the study in the context of reproductive success in other animal mating systems.  Levitan 2005 Integr Comp Biol 45: 848; for other comparisons see also Levitan 2004 Amer Nat 164: 298 and Levitan 2002 Ecology 83: 464. 

NOTE density of males is measured as number of individuals per square meter

 
Research study 13
 

histogram showing % fertilisation of eggs in sea urchins Strongylocentrotus franciscanus in relation to time of release of upstream spermhistogram showing fertilisation success of male urchins Strongylocentrotus franciscanus in relation to timing of sperm releaseStudies to date on sea urchins Strongylocentrotus spp., as well as on other externally fertilising marine invertebrates, indicate that males mostly spawn first, followed by females.  But what are the consequences, if any, of males spawning after females?  This is tested in field experiments with red sea-urchins S. franciscanus at locations around the Bamfield Marine Sciences Centre, British Columbia using syringes1 filled with either sperm or eggs, as well as inducing males to spawn at different intervals within a population of spawning females by injecting them with KCl.  Afterwards, eggs are collected with a subtidal plankton pump and the fraction of eggs fertilised (fertilisation success = reproductive success) and the paternity2 share of each male assessed. 

Results show that a female’s fertilisation success is the same whether sperm is released before or after the eggs are released, but that significantly more eggs are fertilised if sperm is released both before and after (see histogram on Left).   For a male, fertilisation success does not significantly differ if its sperm is released before or after the eggs are released but, when sperm from 2 males is released, the sperm released before the eggs gains an advantage (see histogram on Right). Thus, spawning after the females imposes a cost on the males, but only when there is sperm competition. Because spawning late is only costly to males, selection should favour males that spawn before females, perhaps explaining the “male-first” spawning observed in sea urchins and other invertebrates.  Alternatively, but not exclusively, the sperm may stimulate the females to release eggs. The author comments that the use of broadcast-spawning sea urchins in such studies allows for investigation of how mating behaviour, including timing of gamete release, forming of aggregations, and so on, can influence mating success without the complications imposed by morphological differences between the sexes3 or post-mating investments by either sex.  Levitan 2005 Amer Nat 165: 682.

NOTE1  the syringes also contain fluorescein dye to enable SCUBA-divers to position the syringes in the correct orientation to currents and females

NOTE2  microsatellite markers are used to identify the males

NOTE3  a common feature of broadcast-spawning invertebrates is this lack of sexual dimorphism

 
Research study 14
 

graph showing fertilisation success of female sea urchins Strongylocentrotus purpuratus in relation to density of malesgraph showing fertilisation success of female sea urchins Strongylocentrotus purpuratus in relation to distance from malesIn follow-up studies to the ones above the same researcher compares fertilisation success1 of 2 sympatric sea urchins Strongylocentrotus franciscanus and S. purpuratus in field2 experiments near the Bamfield Marine Sciences Centre, British Columbia.  Techniques involve collecting eggs from spawning females using an underwater plankton pump3 and inspecting them later for fertilisation success, sampling tissues of both sexes for microsatellite genotyping, and measuring densities of both species.  Tagged individuals of both sexes are induced to spawn by injection of 0.5M KCl.  Results show that S. purpuratus requires higher sperm concentrations for fertilisation, but are more resistant to polyspermy than S. franciscanus (see graph on Left and compare with comparable data for S. franciscanus in Research Study 9 above). Note in the graph that female fertilisation success in S. purpuratus increases with increasing density of males up to and exceeding 150 individuals . m-2 without the polyspermy exhibited by S. franciscanus.  The author reminds us of the trade-offs relating to polyspermy, in that females producing eggs more resistant to polyspermy are at a disadvantage when sperm is limited; conversely, if eggs are easy to fertilise, then they risk polyspermy if sperm concentrations are high. Both species display high variance in male fertilisation success at all densities and high variance in female success at low densities.  They differ, however, in the level of female variance at high densities (only S. franciscanus displays high levels of variance).  Fertilisation success in both females and males, as expected, falls off quickly with distance (see graph on Left; only data for females shown here, males are similar).  Other features of the study relate to sexual conflict, female control over fertilisation, and a discussion of sexual selection in sea urchins, not included here.  Levitan 2008 Evolution 62: 1305; see also Levitan et al. 2007 Evolution 61: 2009.

NOTE1  fertilisation success, reproductive success, and sometimes mating success, may seem to be used interchangeably in these several Research Studies on Strongylocentrotus spp., but the author is usually careful to separate the proximal process of fusion of gametes (= fertilisation success) from other processes, such as larval survival, settlement, recruitment, and maturation, that ultimately lead to continuence of a genetic line over time (= reproductive photograph showing aggregation of mixed species of sea urchins Strongylocentrotus spp.success).  The author uses the term “mating success” only to compare broadcast-spawning species like sea urchins with truly mate-selecting species like vertebrates (as in assessing the validity of Bateman’s principle as applied to sea urchins, a topic not included here) 

NOTE2  the study is conducted entirely in the field using SCUBA.  The author discusses potential artifacts of field sampling, such as effects of water turbulence, current vagaries, sperm and egg dilution once out of the gamete plumes, natural vs. induced spawning events, and so on, but none detracts from the impact of the data. In fact, their "naturalness" of these potential field artifacts serves to make the author's studies all the more remarkable and valuable

NOTE3  this ingenious device enables particles to be collected in situ and partitioned into separate chambers, thus permitting eggs of up to 12 females, in this case, to be collected and isolated for inspection for fertilisation success.  This is done 3h after their collection and embryos are again monitored 48h later to determine if polyspermy at high sperm concentrations has affected survival


Mixed aggregation of sea urchins Strongylocentrotus
purpuratus
and S. franciscanus, but with 4 S.
droebachiensis
mixed in as well (can you spot them?)

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