Evechinus chloroticus (Val.), an Endemic New Zealand Echinoid*
[Received by the Editor, April 23, 1958.]
The anatomy of Evechinus chloroticus (Valenciennes, 1846) is compared with that of Heliocidaris erythrogramma and with published descriptions of Echinus esculentus. The differences between the three species are slight, but uphold the view that Evechinus and Heliocidaris should be placed together and apart from Echinus and its allies H. L. Clark (1912) first referred Evechinus to the Family Echinidae, and later (1925) referred both Heliocidaris, and Evechinus to the Family Strongylocentrotidae. Helicidaris, which has polyporous ambulacral plates and a circular ambitus, obviously conformed to Clark's Family Strongylocentrotidae. Evechinus, although oligoporous, was included because of the specialization shown by the larval form and the pedicellariae. Mortensen (1943) contends that both genera should be placed in the Family Echinometridae on account of the strongly developed single lateral tooth of the gemmiform pedicellariae, the paired nature of the poison glands, and the structure of the larval form.
Evechinus shares with Heliocidaris:
The large collateral canal, which is conspicuous in both forms; it has, however, also been described from Echinus, although not from all members of the Echinidae (Bonnet, 1925).
The gonads elongate from apex to lantern, although those of Evechinus are strongly coalesced while those of H. erythrogramma remain quite separate.
The genital papillae, which are of very similar form in the two genera; the genital pores are also of the same order of size.
A well defined ridge on the internal surface of the apical plates; in Evechinus the apical connection between the axial organ and the stone canal does not appear to be developed, although it has been described for Echinus (Chadwick, 1900,et al.)
The alimentary canal, which is voluminous and greatly convolutes in both species, unlike that of Echinus; both forms bear well developed processes on the epiphyses of the lantern, which in those members of the Echinidae which possess them are only slightly developed (Mortensen, 1943); there are also small differences between Echinus and Evechinus in the structure of the pharynx and in the histology of the alimentary canal.
Therefore it is considered that Evechinus and Heliocidaris are closely related genera, and although both Clark's and Mortensen's classifications have been admitted by their authors to be artificial, it is thought that Mortensen's criteria provide a more convenient basis for separation at the familial level than do those of Clark.
Evechinus chloroticus, the common littoral sea-urchin inhabiting the New Zealand coasts, is a regular echmoid belonging to the Family Echinometridae, which is included in the Order Camarodonta (Mortensen, 1943). Mature specimens are figured on Plates 12 and 13. This species is the largest known echinometrid. The largest on record is one described by Farquhar (1897) of 145 mm diameter, although an average sized specimen is smaller, rather less than 100 mm in diameter.
Despite the work of numerous authors from Valenciennes (1846) to Mortensen (1943), the systematic position of Evechinus chloroticus has not been fully estab-
[Footnote] * This study was undertaken in the Zoology Department, Victoria University of Wellington.
lished. No work, except for a brief mention by Mortensen (1943), has been done on the anatomy of the animal. As it is such a well-known member of our littoral fauna, and is a standard dissection during the Zoology course at the Victoria University of Wellington and probably also at other universities, it seemed time that its anatomy should be investigated. Accordingly, a detailed study has been made of the gross anatomy and morphology of the animal, together with histological study of the alimentary canal and the tissues of other organs where necessary. No parasites had previously been recorded from Evechinus, but during this study two endocommensals and one ectocommensal were found. At the same time five specimens of Heliocidaris erythrogramma (Valenciennes) were obtained from Sydney, so that a brief comparison between the anatomy of the two echinometrids could be made, although unfortunately the specimens of H. erythrogramma were not received in a good state of preservation. An endocommensal rhabdocoel, similar to that found in Evechinus chloroticus, was found present in large numbers in the gut of all these sea-urchins. They appear to represent two different and undescribed species of the genus Syndesmis, of which the species S. echinorum is commonly found in the gut of European echinoids. As text-books usually take species of Echinus, especially E. esculentus, as the example of a “typical” echinoid, I have made my main comparisons with this genus.
The families of the Sub-order Echinina all consist of camarodont echinoids with compound ambulacra of the echinoid type. The test is unsculptured, while the tubercles are imperforate and smooth. The gill slits are not sharp or deep, and the spines are solid. The families within the Sub-order are mainly distinguished by the structure of their gemmiform pedicellariae, which in all echinometrids possess a single, unpaired lateral tooth. Evechinus chloroticus is recognized by its almost horizontal pore-arcs, the pore-pairs of which are arranged in three vertical series, by the green colour of the test, and the fact that the spicules of the tube-feet are simply bihamate (Mortensen, 1943). However, as the result of closer investigation, it has been found that these spicules bear small distal projections, in much the same way as the closely related Selenechinus armatus, so that they can not now be regarded as good specific characters.
Fell (1953) has made certain suggestions as to the possible history of the genus. He states that Evechinus is known as a fossil from the Nukumaruan (mid-Pliocene), appearing, together with Arachnoides, Pseudechinus and Echinocardium, after the extinction of the early-Tertiary warm-water echinoid fauna. It is restricted to the New Zealand region, throughout which, however, it is widely distributed, reaching as far as the Kermadec Islands in the north and to Stewart Island in the south. It also occurs at the Chatham Islands. The fact that this endemic genus is eurytopic points to a relatively early differentiation within the New Zealand region, which is thus probably the original home of the genus. With its pelagic larvae, which have obviously been successful in distributing it widely within the New Zealand region, Evechinus would seem admirably equipped to take advantage of any dispersal mechanism operating between the Australian and New Zealand regions. Although it has been present in New Zealand since the Pliocene, it has not so far been able to traverse the Tasman, so that it has been deduced, on this and other evidence, that no such east to west dispersal mechanism can operate between the two regions.
Evechinus is usually referred to coloquially as the “sea-egg”, although the Maoris, who relish the gonad for food, refer to it as “kina”. It is eaten raw, preferably while the animal is still alive. Many Europeans have also acquired the taste for “kina”, and it is occasionally on sale in fish shops in Wellington, retailing usually at the price of sixpence each.
I would like to take this opportunity of thanking Professor H. B. Fell for his invaluable help and advice; Miss Isabel Bennett, of the Department of Zoology. Sydney University, for sending me preserved specimens of Heliocidaris erythrogramma
from Sydney; and Mr. M. D. King, for photography. I am also grateful to those members of the Zoology Department, Victoria University of Wellington, Mr. B. E. Maxwell, and Mr. J. C. Yaldwyn in particular, who have kindly collected material, and all whose advice and encouragement have helped me during the course of this study.
Materials and Methods
Specimens of Evechinus chloroticus were obtained from Oriental and Balena Bays, where they are present in such numbers that collection has never been a problem. They occur in about knee depth of water at low tide. It was found best to collect them in calm weather when they could easily be seen, in among the stones and clinging on to rocks. Their habit of holding stones and old shells on the aboral surface, and of nestling in between the stones, makes them difficult to detect if the surface of the water is broken.
Animals required for preservation were transported back to the laboratory in a plastic bag, where they were anaesthetized in tepid fresh water. A small hole was made in the test wall and the animal fixed and preserved in 8% formalin. Any which were required to be kept alive in an aquarium tank were transported to it in a billy of sea water. In general sea-eggs seem to be able to remain alive for quite a considerable time out of water. They may sometimes be seen in fish shops moving their spines quite actively, hours after they have been taken from the water.
Occasionally, when I was interested only in knowing the degree of maturity of the animals, specimens were opened at the collecting ground and gonad samples taken and preserved in alcohol. At the same time a measurement of the diameter of the animal was taken. All measurements of height, diameter, etc., of the test were taken as if the spines had been removed.
Dissections were always carried out under water. To obtain the correct orientation of the internal organs the animal was fixed to the dissecting dish with a small quantity of melted wax, in such a way that radius III was always the anterior ambulacrum. Once the test wall had been perforated by a sharp instrument such as a scalpel, coarse forceps were used for breaking away the remaining parts. A magnifying plate was found useful for examining dissections, drawings of which were made to scale by hand.
Whole tests were cleaned by boiling specimens for two or three minutes in 10% potassium hydroxide solution. It was found best to make a small hole in the peristome first, so that the solution could penetrate easily to the internal organs. Any adhering epithelium and spines were then washed away under a strong jet of water, and if necessary maceration continued for a few days in water. The apical ossicles are very delicate and likely to drop out if violent maceration is carried on for too long.
Whole specimens were also prepared following a method described by Bonnet (1925). A hole was pierced at the apex of the test of an animal previously preserved in formalin. It was made in a radial position to avoid penetrating the gut, and the liquid in the body cavity drawn off with a syringe. The interior of the animal was then washed out several times with warm water, until the wash water was no longer discoloured. A gelatine solution (3 parts gelatine: 1 part water) was then injected to completely fill the body cavity, and the hole at the apex plugged with cotton wool. The preparation was placed in a refrigerator overnight, or until the gelatine was set hard. It was found that a little of the hot liquid had leaked out before setting, and so more was added at this stage to completely fill the whole interior. After the second gelatine addition had set, the preparation was placed in a 10% formalin solution for one or two days to harden the gelatine. This was followed by a bath of 5% hydrochloric acid to dissolve the test, a process taking three or four days. The acid solution needed to be renewed at least once. When
all the calcareous plates had been dissolved away, the outer epithelium and membranes between plates were carefully picked away, so that the underlying organs, the gonads, coils of the gut and radial ambulacral canals and their associated ampullae, were revealed, visible through the gelatine. The whole preparation was then kept in 8% formalin. Evechinus chloroticus is, however, not a satisfactory subject for preparation in this way, as the gonads are large and coalesced and so obscure most of the other organs. Small specimens, in which the gonads are still quite small, should, however, make good preparations.
Material was prepared for all histological examination by fixation in Zenker's mixture, followed, after the appropriate washing, dehydration and clearing, by imbedding in rubberized paraffin, made according to the directions given by Sass (1940). This was found to give much better ribboning than ordinary parafin. Sectioning at from 6μ to 12μ was done with a Cambridge rocker microtome. To remove the mercuric salts left by the Zenker's fixative, the sections were treated before staining with tincture of iodine in 70% alcohol; the brown iodine coloration was removed by 50% alcohol. Ehrlich's acid haematoxylin and eosin in 95% alcohol for counterstaining was found the most useful general stain. Canada balsam was the usual mounting medium. For parts of the gut, especially the oesophagus and also the gonad, Mallory's triple stain was used with success. The sections, however, faded when mounted in an acid medium such as Canada balsam and so euparal had to be used instead. In general it was found that extremely rapid dehydration was necessary for sections stained with Mallory's. They were briefly washed in tap water, dipped in and out of 50% alcohol and taken straight to two successive washes of 95% alcohol, from which they could be mounted straight into euparal.
Decalcification of fixed material for histological purposes was accomplished by placing the material in Schurig's solution (95 parts of 80% alcohol, 2 parts of conc. hydrochloric acid, 3 parts of picric acid). The process took approximately two weeks, during which time the solution was changed two or three times. A 30% solution of sodium hexametaphosphate was also tried for decalcification, but was found too slow-acting for the thick test wall of Evechinus.
All histological drawings were made using a Watson-Victor monocular microscope and a camera lucida. A Watson-Victor low power binocular microscope was used for drawing of small anatomical structures.
Ambulacral and interambulacral areas. Divisions between ambulacral plates were often difficult to see, especially in large specimens, but one or two specimens from the College collection, which had been kept in formalin for some time, were found particularly useful in this respect. The formalin had evidently become acidic and attacked the plates, so that the membranes separating them were partially revealed. Correlation of the patterns of pore-pairs on the inner and outer surfaces of the test was achieved by passing a hair through every pore on the exterior of a plate and noting its position of emergence in the interior. Counts of the ambulacral plates were made using Mortensen's method (1943) of recognizing each member of the inner series of pore-pairs as a separate plate. Sections of interambulacral plates were obtained by mounting in Canada balsam and grinding to the required thinness. This was first done horizontally, and when no results were obtained, further sections were ground parallel to the median suture, and then parallel to the suture between plates of the same column.
Peristome. This was observed under a magnifying plate and drawn to scale by hand. The peristomial plates were obtained by boiling small pieces of the peristome in 10% potassium hydroxide for a few minutes.
Spines. Spines were removed from the test and cleaned in boiling potassium with a tubercle was obtained in the course of sectioning a decalcified ambulacrum hydroxide. A longitudinal section through the base of a spine at its articulation
Transverse and longitudinal sections of cleaned spines were obtained by mounting in Canada balsam and grinding to the required thinness.
Pedicellariae. Whole pedicellariae were studied, both in water under a binocular microscope and mounted in glycerine jelly under higher magnification. Separate valves were macerated by boiling for a few minutes in 10% potassium hydroxide. They were subsequently mounted in glycerine jelly. Fell's (1941) method of studying calcareous structures through polarized light was found useful in determining the nature of the valves, when they appear as glowing golden structures standing out against a deep blue background. Some gemmiform pedicellariae were decalcified, sectioned at 6μ and stained in Ehrlich's haematoxylin and eosin.
Sphaeridia. Sphaeridia were detected by scraping carefully with a scalpel along the centre of each ambulacrum near the peristome, all the time examining the area through a hand lens under strong light. Any sphaeridia detected were then cleaned by leaving in 10% potassium hydroxide solution for a few hours. The histological structure of the sphaeridia and their relationship to the test were discovered by decalcifying and sectioning part of an ambulacrum from near the peristome.
Alimentary System. Ossicles of the lantern were obtained by cutting away as much of the adhering muscle as possible, and macerating the whole structure in 10% potassium hydroxide. For details of the dissection of the gut see the section on the alimentary system. The histology of the alimentary canal was determined by sectioning and staining small portions from representative regions in both Mallory's triple stain and Ehrlich's acid haematoxylin and eosin.
Ambulacral System. To show the relationships between the ambulacral canals, their appendages and other radial structures, small sections of the ambulacra were placed in Schurig's solution (Hiraiwa, 1932) to decalcify the test. The decalcified material was then imbedded and sectioned. The relationships of the madreporite, stone canal and other apical structures were also demonstrated by this technique.
Glycerine jelly was found to be the most satisfactory mountant for the tube-feet, as they could then be taken straight from water, and thus prevented from shrivelling. The buccal tube-feet, however, were too dense to show the xtalline plate clearly when mounted in this way; so maceration by Fell's method (1940) was tried. The tube-foot was removed from the peristome and severed just below the sucker, so that the plate could lie face down in the bottom of an evaporating dish, and would not break up when the supporting tissues were removed. A few drops of 10% potassium hydroxide were then added, and the evaporating dish placed in the oven. Further drops of 10% potassium hydroxide were added as the original solution evaporated, until maceration had proceeded sufficiently to reveal the plate, usually 2–3 hours.
The ampullae were removed by carefully scraping them away from the interior of the test, and examining in water under a low power microscope. One from each region was then selected and mounted in glycerine jelly.
Coelomic Cavities. The larger coelomic cavities are visible to the naked eye. The smaller ones, such as the terminal sinus, aboral ring, etc., were revealed by sectioning a previously decalcified apical system.
Coelomic Fluid. The test of a living animal was perforated in the apical region of an ambulacrum to avoid the gonad and the coils of the gut, and several cubic centimetres of coelomic fluid drawn off with a syringe. This was then observed fresh under a high power microscope.
Axial Organ. Sections were cut and stained in both Ehrlich's haematoxylin followed by eosin, and Mallory's triple stain. To determine the apical relationships of the gland, an apical system, with its internal tissues and adhering stone canal and axial organ, was removed and decalcified. This material was then sectioned and stained with Ehrlich's haematoxylin and eosin.
Lacunal System. The description of the lacunal system of Echinus esculentus is apparently based to a large degree on injected specimens which were found quite easy to prepare by Cuénot (1948). “Le réseau absorbante de l'intestin, l'anneau oral et le réseau de la glande brune se remplissent facilement en poussant l'injection dans la lacune marginale interne, très visible au bord du mésentère, ou encore en perforant avec la canule la périphérie de la glande brune …” Echinus esculentus, however, attains the greatest size of all echinoids and so would possess haemal canals of dimensions suitable for injection. In Evechinus chloroticus the internal marginal canal, although quite readily visible, is only of the order of 0.1 mm in diameter and so quite impossible to inject by ordinary methods. Attention was then directed to the lacunal network of the brown gland, which Cuénot also found possible to inject. The first injection fluid tried consisted of a gelatine vehicle (1 part gelatine: 10 parts water) carrying a colouring mass of indian ink. The animal was anaesthetized and the aboral test removed, leaving the apical system and its connection to the brown gland intact. A small hand syringe with a very fine needle was used to make the injection. However, the sudden strong pressure was not suitable for penetration of the fluid. An added difficulty was the necessity to keep the whole apparatus warm to prevent the gelatine from congealing. A second animal was selected and prepared in the same way. The fluid used, however, was pure indian ink, and a slow pressure was supplied by an aspirator. It was still impossible, however, to obtain a good penetration of the gland, let alone the whole lacunal system, so it was concluded that the periphery of the axial organ is too dense and its lacunae too small for such a technique. Luckily, however, the internal and external marginal canals, and the collateral canal, were easily visible to the naked eye, while sectioning of the gonad, the gut and the axial organ had previously revealed the lacunal network within their walls. The haemal ring about the oesophagus at the top of the lantern still remained problematical. To observe this a lantern was decalcified and its upper portion imbedded and sectioned.
Nervous System. To observe the histology of the radial nerves and their exact relationship to other radial structures, small pieces of ambulacra were decalcified and sectioned. The nerve ring about the mouth, and parts of the deeper oral nervous system were similarly observed by decalcifying a lantern and sectioning its adoral half. It was hoped that it would be possible to demonstrate the peripheral nerves passing under the epithelium over the exterior of the test. For this both fresh and fixed material was used.
Fresh Tissue, Methylene Blue Method (Smith, 1949). A small section of the test was placed in a petrie dish containing 50 ml of sea water to which had been previously added 0.5 ml of 1% methylene blue. The material was left in this solution for about five hours.
Fresh Tissue, Silver Nitrate Method (Bolles Lee, 1950). Small sections of the test were washed for half an hour in a 5% solution of potassium nitrate to remove the unwelcome chlorides present in sea water. They were then washed in distilled water and placed in a 1% solution of ammoniacal silver nitrate, which was agitated under bright light until the material began to turn grey. It was then washed in distilled water.
Neither of these methods was successful, however. Evidently the nerves were neither sufficiently stained nor impregnated to show up through the darkly pigmented epidermis.
Fixed Tissue, Silver Nitrate Method. Ramón y Cajal's method, as described in “The Microtomist's Vade-Mecum”, was followed. Small pieces of test were fixed for 48 hours in ammoniacal alcohol (50 ccs of 96% alcohol to which 4 to 5 drops of ammonia have been added), the material having been previously dehydrated in 70% alochol for 6 hours, and 85% alcohol for one hour. It was then silvered by placing in a 1 5% solution of silver nitrate, which was kept at 32° to 35° C. in an
oven for five days. At the end of this time the material had become light grey in colour. It was washed for a few minutes in distilled water and placed in the following reducing solution for 24 hours:
Hydroquinone, 3 gms.
Distilled water, 100ccs.
Formalin, 5 to 10ccs.
This was followed by quick washing and hardening in successive concentrations of alcohol. After this procedure there was still no evidence of nervous structures; so bleaching of the epidermis was tried. Two or three drops of hydrochloric acid were added to a few crystals of potassium chlorate in a test-tube, and as soon as the green colour of evolving chlorine could be seen a few cubic centimetres of 70% alcohol were added. The material was left in this solution for one to two days but, although bleaching was successful, the process evidently removed any of the silver which may have been precipitated on the nerves.
After the failure of all these methods I decided that demonstration of the peripheral nerves was outside the scope of this thesis.
Reproductive System. For details of dissection see section on reproductive system. To determine the sex and also the degree of ripeness of animals, small pieces of the gonad, taken from near the aboral pole, were teased out on a slide and examined under a microscope. The histology was investigated by sectioning and staining in both Mallory's triple stain and Ehrlich's acid haematoxylin and cosin.