Research Study 1

Fig. 1.  Sea cucumber Psolus chitonoides amongst yellow cup corals Balanophyllia elegans

A single female Psolus chitonoides (Fig. 1) may release 35,000 eggs (each 400 x 600µm in size) in a single spawning.  Fertilisation is external.  Development is to a gastrula (Fig. 2) and then to a non-feeding swimming larva called a doliolaria (Figs.  2 - 6). Tube feet and tentacles begin to develop in the doliolaria.  Within 2wk a pentacula (Fig. 6) develops that has five primary tentacles and two early-stage tube feet. The pentacula preferentially settles on a conspecific adult or, lacking that, in a shady spot.  Metamorphosis is completed after about 4wk and involves secretion of ossicles to form the body plates, branching of the primary tentacles, flattening of the body, and proliferation of the tube feet.  The juvenile then seeks out a shady habitat and shortly begins to feed.  As an adult, Psolus chitonoides rarely moves, and is termed “functionally sessile” by the authors. 

NOTE non-feeding invertebrate larvae are rich in yolk and are termed lecithotrophic (lit. "yolk + food" G.).  Another larval type known as an auricularia is perhaps more common in holothuroids. It is a feeding larva and is termed planktotrophic 

NOTE lit. “five” L., referring to the presence of five primary tentacles

Fig. 2.  First in a series showing developmental stages of Psolus chitonoides: here, 48h gastrula

Fig. 3.  Early doliolaria: tentacles will emerge from the vestibule, and tube feet from the podial pit

Fig. 4. The aboral surface of the doliolaria larva bears the hydropore, or madreporite

Fig. 5.  On the oral surface the five primary tentacles and primary tube feet are emerging

Fig. 6. The now settled pentacula stage crawls about for a day or two, then begins to metamorphose

Research Study 2

Fig. 1.  The trend for smaller sized echinoderm larvae is to have transverse ciliated bands, and for larger sized ones to be uniformly ciliated

Fig. 2.  Relationship of buoyancy and drag forces in theoretical larvae of different sizes and with differing numbers of ciliary bands.  The pink area represents a cross-over size where drag and buoyancy forces exceed the propulsive forces theoretically generated by three or more bands of cilia

The early early doliolaria larva of Psolus chitonoides is 1.1mm in length and has whole-body ciliation for locomotion.  The later doliolaria larva is 900µm in length and has three transverse bands of cilia for locomotion. This is unremarkable in itself, but the author points out an interesting tendency in echinoderm larvae, including those of holothuroids, that small non-feeding larvae tend to have transverse cilitated bands, while larger non-feeding larvae tend to be ciliated all over, or uniformly ciliated.   Note in Fig. 1 that this pattern holds for crinoid and ophiuroid larvae, which tend to be small in size. Echinoid and holothuroid larvae are mixed sizes, but note that the larger larvae in these groups tend to have whole-body ciliation. The largest echinoderm larvae are possessed by asteroids, and tend to have whole-body ciliation.  But what is the significance of transverse-banded or whole-body ciliation? The author suggests that bands of cilia, while providing enough propulsion for small larvae, are insufficient for larger larvae that have greater buoyancy forces to overcome. The author calculates these theoretical forces and shows in a model for a theoretical larva a cross-over point at about 650 - 800µm in length, where drag and buoyancy forces exceed the propulsive forces theoretically generated by three or more bands of cilia (Fig. 2).  Note that this cross-over size is close to that of the transition size of 900 - 1100µm noted above for Psolus larvae.  If whole-body ciliation is so good for propulsion, why are three- and four-band patterns retained in smaller-sized larvae?  This, of course, can’t be answered without knowing a lot more about the kinematics of cilia and the water movements caused by them.  The author suggests that for the opposite situation, that is, for ciliary bands to generate sufficient propulsion in larger larvae, they would have to be disproportionately wide, and close-packing of the cilia in this way could create inter-ciliary interference and related water-movement problems.  The author also notes that the theory assumes that the cilia are for propulsion. If they were to be involved in some yet unknown energy-obtaining process, then these ideas would have to be reconsidered.  The theory is an interesting one and is deserving of further research. 

NOTE the author also includes frictional drag as a force that a larva has to overcome when swimming.  This is true.  However, as drag is directly related to surface area, it could be argued that small-sized larvae, having relatively greater surface area than large-sized larvae, would correspondingly have relatively greater frictional resistance to overcome; hence, perhaps should be the ones that possess whole-body ciliation...

Research Study 3

Fig. 1.  Two well-camouflaged Cucumaria piperata have burrowed into the sand so that just their mouths and tentacles are visible 

Several west-coast holothuroids are lecithotrophic, that is, their larvae, while free-living, subsist on yolk and do not feed from the plankton. Some examples are Cucumaria miniata, C. piperata (Fig. 1), Eupentacta quinquesemita, and Psolus chitonoides.  At least two other west-coast species, Cucumaria pseudocurata and C. curata, brood their eggs under their bodies until the juveniles can crawl out and take up life on their own

NOTE  now known as Pseudocnus curatus

Research Study 4

Fig. 1.  Early juvenile "pentacula" stage of development of Pseudocnus curatus

An early description of reproduction in a brooding species, Pseudocnus curatus collected near Dillon Beach, California includes simultaneous production of sperm and eggs in the same individual, suggesting hermaphroditism. The eggs are sticky on release and this, combined with secretion of mucus over the entire body by the adult, aids in attachment of the eggs to the ventral side of the body among the tube feet.  A gastrula stage is reached about 3d after fertilisation and, after 5d, this transforms to a stage that the author calls a “pentacula”, but is more likely to have been a post-metamorphosis stage; thus, an early juvenile (Fig. 1). This tiny juvenile has five primary tentacles and may use these to crawl onto the upper surface of the adult.  By 2wk of age the juvenile (no size given by the author) leaves the parent and takes up independent life. 

NOTE a pentacula stage is usually considered part of a pelagic developmental pattern in holothuroids, not a brooding one, but the stage shown here does, perhaps not surprisingly, bear close resemblance to the planktonic variety

Research Study 5

Fig. 1.  Pseudocnus lubricus

Courtesy Keoki & Yuko Stender, Hawai'i

Field and laboratory observations of sea cucumbers Pseudocnus lubricus (Fig. 1) at Friday Harbor Laboratories, Washington reveal that females spawn eggs from anterior gonopores, but capture them immediately in their tube feet before they drift away. The eggs are retained as a mass between the female’s body and the substratum, hemmed in on either side by tube feet. Although no sticky mucus appears to be involved, a female is able to move around without losing her eggs. Males spawn from a genital papilla extended several millimeters from the body and the sperm strands break up as they drift downwards, catching up on females on their way.  Spawning by females occurs during Nov - Jan in late afternoon, while that by males takes place in the same seasonal period at mid-day.  Females appear not to feed during the brooding period of autumn-late winter.  The eggs are large (almost 1mm diameter) and are full of yolk.  Egg numbers range between 120 - 150 depending upon habitat.  Eggs hatch after 6wk, but the young are non-feeding for another 4 - 8wk until they crawl free of the brood area.  Brooding commences when an adult reaches 1.5  -2.4cm in length, at 3 - 4yr of age. The author notes that brooding provides protection for the developing embryo and ensures that large numbers of young are recruited to a favourable habitat. 

NOTE formerly known as Cucumaria lubrica

NOTE the author describes an incident in which a brooding adult is reflected back to show its brood, at which time amphipods Parapleustes pugettensis immediately swarm in for the kill

Research Study 6

Fig. 1.  Auricularia larva of Apostichopus californicus showing ciliated bands and relevant body parts

Fig. 2.  Auricularia larva

Most west-coast holothuroids, including Apostichopus californicus, have an auricularia larva that feeds in the plankton for about 3 - 5wk (Figs. 1 - 2). During this time the larvae propel themselves about and feed using a single, sinuate, ciliary band.  A second, smaller ciliary band is located around the mouth. The bands are thickened regions of the epidermis densely packed with a row of ciliated cells. The bands are arranged so that a large proportion of the ciliary beat is directed posteriorly and the larva moves with its anterior end foremost.  As food and other particles pass over the ciliated bands, the beating direction reverses in those areas and the particles are deflected towards the oral region.  Inedible particles are sensed and ignored by the bands, or rejected wholesale by complete reversal of beating.  This causes the larva to move backwards and clears the particles from the oral area.

Research Study 7

Fig. 1. Auricularia stage of development in Apostichopus californicus

Fig. 2. Doliolaria stage of development in Apostichopus californicus

These scanning e-microscopical views show the considerable morphogenetic transformation required in the change from auricularia to doliolaria stages in Apostichopus californicus (Figs. 1 -2).  Note that the mouth shifts from mid-way along the body to the front of the animal.

Research Study 8

Fig. 1.  In this diagrammatic representation of an auricularia, water currents generated by the ciliary band are shown in blue, while movements of food particles are shown by thin black arrows (barely visible here)

Feeding in the auricularia larva stage is the same as in other echinoderm larvae, as shown in the Fig. 1 representation.  Phytoplankton cells are swept up in micro-currents generated by beating of cilia in the main ciliary band, which also provides for propulsion of the larva.

Research Study 9

Fig. 1.  Seasonal gonadal indices in Leptosynapta clarki in Barkely Sound, B.C.  Males spawn in late autumn/early winter and females release the juveniles in springtime

Fig. 2.  Development of pentaculae (early juveniles) in an ovary of Leptosynapta clarki takes about 30wk.  Note that the sequence goes from Right to Left.  The youngsters begin feeding immediately on their release from the parent

The viviparous sea cucumber Leptosynapta clarki inhabits burrows in intertidal mudflats along the west coast of North America from Haida Gwaii, British Columbia to central California.  In Barkely Sound, B.C. gonadal growth of L. clarki occurs during the summer, with spawning of males in Nov - Dec (Fig. 1). The female ovary consists of two bilaterally symmetrical sacs within which the eggs are packed. Fertilisation of the oocytes occurs in the ovary, although it is not known how the sperm gain entry, and development to a pentacula stage takes about 2wk (Fig. 2). The pentaculae have functional guts and move about in the ovarian fluid feeding on fluid droplets and organic matter.  Later, organic material such as unfertilised eggs and dead embryos is resorbed by the parent. After about 30wk in the ovary, the juveniles are released via rupture of the body wall and crawl out into the immediate habitat of the adult.  Leptosynapta clarki is protandrous, and most or all of these juveniles will function as males for the next reproductive season, after which many will transform to females. Some males remain so through their lifetime. In this way, at a size of about 500g, the sex ratio reaches 1:1. 

Research Study 10

Fig. 1.  Relationship of eggs and pentaculae (early juveniles) to female mass in Leptosynapta clarki

Further studies at the Bamfield Marine Sciences Centre, British Columbia provide more information on the reproductive cycle of Leptosynapta clarki. Juveniles of 1- 2mm length are released from the ovary in early spring and are reproductively active as males by November of the same year.  By the following year, some individuals continue to reproduce as males, while others transform to females. Transformation to female occurs at about 200mg live mass or 20mm body length.  Not all individuals change sex. In fact, the population appears to be divided into ones that change sex at a specific time, ones that change under the influence of some genetic or environmental factor, and ones that remain as males for their entire lifetimes. There is no allometric constraint on brood size in L. clarki because the brooding is done within the distensible ovary.  Thus, number of eggs or pentaculae scale linearly with mass of female (Fig. 1).  The author suggests that sequential hermaphroditism or protandry, which is not common in holothurians, may function to reduce inbreeding in a species with limited dispersal by reducing the chances of sibling crosses. 

NOTE the so-called allometric hypothesis, proposed initially for an external brooder, the sea star Leptasterias hexactis, predicts that brooding will not occur in large species because of spatial limitations on brood size with increasing individual size.  While applicable to external brooders where brooding space scales allometrically with animal mass, it does not apply to internal brooders where internal brooding volume scales isometrically with animal mass

NOTE for some reason the author uses drained mass of female, that is, with the coelomic fluid removed, as the abscissa axis of the graph. This would not change the slope of the relationship, but would affect the intercept.  Coelomic fluid is part of the living mass of a sea cucumber, and should not be confused with mantle-cavity fluid of a mollusc that is external to the body mass.  The authors would have known this

Research Study 11

Fig. 1.  Brood mortality in Leptosynapta clarki estimated from presence of calcareous rings in the ovary.  The coloured line is added for visual effect only. The graph should be read as anywhere from zero to 100% mortality in broods from two populations in Barkley Sound, B.C. during late winter/early spring

A follow-up study at the Bamfield Marine Sciences Centre, British Columbia by the same authors of the previous Research Studies assesses mortality of early-stage pentaculae over the 30wk brooding period in the viviparous sea cucumber Leptosynapta clarki.  Apparently, such a study has not been done before owing to the difficulty in determining the number of embryos at the start of brooding and not having a way to estimate how many embryos are resorbed. The project is made possible by the discovery that a calcareous ring is present in the pentacula at the base of the tentacles that remains after death and is not resorbed by the parental ovary.  Counts of rings and live embryos indicate that mortality, even up to 100% or total brood loss, is not uncommon in Leptosynapta (Fig. 1).  The dead larvae are not wasted, however, as their resorption in the ovary may provide nutrients and energy for general metabolic maintenance of the female, for the later production of eggs and, as the pentaculae feed on organic matter in the ovarian fluid, for nutrition of the brood.

NOTE this is a general problem encountered when estimating brood mortality in other marine invertebrates

NOTE the ring appears in the embryo about 14d after fertilisation; hence, pre-ring estimates of mortality are not possible. The author notes, however, that the ring is present for about 93% of the long brooding period, and this minimises underestimation of mortality 

Research Study 12

Fig. 1. Parental addition of organic nutrients to developing young in Leptosynapta clarki

Viviparity, where eggs are fertilized internally and develop for a time within the female reproductive system, is quite rare in marine invertebrates.  In the sea cucumber Leptosynapta clarki, recent findings at the Bamfield Marine Sciences Centre shows that the parent provides significant additional nutrients for development of the embryo during viviparous development. The authors describe histological changes in the structure of the ovarian wall, primarily an increase in size of the connective tissue/genital hemal sinus, suggestive of transfer of nutrients.  Embryos during development end up with significantly greater carbon content than they had in the egg stage, up to a factor of 10-fold, providing clear evidence for matrotrophy (Fig. 1). 

NOTE according to the authors, only 14 species are known, all echinoderms

NOTE this is termed matrotrophy, where the young first depend upon yolk and then on additional nutrients provided by the female, and is distinct from lecithotrophy, where the young depend solely upon the yolk for nutritional requirements during development

Research Study 13

Should you need to culture larvae of Apostichopus californicus, or perhaps another species, through to metamorphosis, you may find useful the results of a study at the Pacific Biological Station, Nanaimo, British Columbia.  The researchers determine growth, survival, and metamorphic competency of larvae on 8 single microalgal diets, then again on all possible binary combinations of the five best diets. Rearing is done at 13.5°C over 16d, at which time metamorphosis is well underway in most cultures.  Best overall growth, survival, and metamorphic rates are attained on a diet of diatoms Chaetoceros calcitrans, and this species also features prominently in the best binary-combination diets. In fact no bi-species diet performs better than C. calcitrans alone. 

NOTE microalgae species used are ones previously used in rearing experiments with other sea-cucumber species in other studies

Research Study 14

Fig. 1.  Larval development of Apostichopus californicus from fertilised egg through to doliolaria stage.  The series begins upper Left and across to Right, then down

Another aquaculture-oriented study on dietary effects on reproductive fitness in sea cucumbers Apostichopus californicus focuses on adults rather than larvae. The aim of the study, done by scientists at the University of Fairbanks, Alaska is to assess reproductive success of adult females fed on different microalgal diets.  Here, “success” is measured as gonadal growth, egg size, fertilisation efficiency, and larval growth. The diets, represented by the green alga Tetraselmis sp. and the diatom Thalassiosira sp., differ in types and amounts of fatty acids, and these differences represent the thrust of the study.  A few significant effects are noted (e.g., higher fecundity in for females eating Tetraselmis) but, in view of the fact that all larvae died within about 12d post-fertilisation, it is hard to give credence to any data on maternal-diet effects on larval development. The authors provide photographs of the larval stages (Fig. 1). 

NOTE this likely owes to the larvae being starved during development. The authors justify this decision in order “to test the carryover effects of maternal diet on larval starvation resistance, and to eliminate confounding effects of larval diet on results”.  A naive reader might counter with, “what could be more confounding than starving them?”.  Also, what if maternal-diet effects do not manifest until metamorphosis, or even post-metamorphosis? While the authors’ intentions are understandable, could a third treatment group not have been employed, one in which the larvae are fed, perhaps using a different microalga species than the ones used as food for the adult broodstock? Better still, is there no artificial diet devised for echinoderm larvae that could be formulated to contain no fatty acids, yet still provide sufficient nutrients for metamorphic transition and beyond?

Research Study 15

Fig. 1.  Warty sea cucumber Apostichopus parvimensis

Courtesy Peter Bryant/Nat Hist Orange County, CA

Researchers in California provide details of larval development of sea cucumbers Apostichopus parvimensis (Fig. 1) raised on a diet of Rhodomonas sp. at a temperature of 20^oC.  Selected developmental stages are shown in Fig. 2.  For whatever reason survival is poor, suggesting possible deficiency in the culture conditions. 

NOTE  the researchers' data show that an individual has only a 25% chance of surviving the 20d culture period

Figs. 2 - 5.  Selected developmental stages of sea cucumber Apostichopus parvimensis