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Volume 75, 1945-46
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Pollicipes spinosus Quoy and Gaimard. II: Embryonic and Larval Development

[Read before the Otago Branch, April 9, 1945; received by the Editor, August 20, 1945; issued separately, March, 1946.]

Introduction, Materials and Methods.

The life histories of barnacles, whose two types of larvae differ so markedly from one another and from the adult form, have attracted the attention of many in the past. Earlier accounts have dealt with scattered stages rather than presenting a continuous picture. In the present instance, material has been reared continuously in vitro from the one-cell egg to the post-cypris form, and the metamorphosis from nauplius to cypris has on a number of occasions been observed. This provides the basis for the present paper, which follows a previous description of the adult barnacle (Batham, 1945, pp. 359–374).

After many initial failures it has been found that the embryos and larvae of this species can be reared in vitro without undue difficulty and with no elaborate apparatus. Ovarian lamellae were removed from adults as soon as these were collected and immediately placed in jars of clean sea-water, one pair per jar. The embryology was chiefly studied from a pair of lamellae collected at the one-cell stage, another pair collected at the two-cell stage. These were kept in covered jam-jars half full of sea water. They received daily changes of freshly-collected, well-aerated sea water for the first fortnight, less frequent changes subsequently. Any visible debris on lamellae or jars was removed, as the successful rearing of embryos and larvae seems largely to depend on preventing the cultures from becoming unduly contaminated with protozoa. During their development, numerous samples of embryos were removed from the lamellae, examined, and fixed.

Nauplii on hatching were removed to a small dish until the first moult had occurred, then transferred to large covered petri dishes. About 5–10 nauplii per dish proved most satisfactory. The dishes were usually examined twice or thrice daily, when moult skins were counted and removed. As the nauplii of P. spinosus do not eat, the problem of supplying them with food did not arise.

For features visible externally, embryos, nauplii and cypris larvae were chiefly examined alive, this giving better results than whole mounts or dissections of carmine-stained specimens. For sectioning, alcoholic Bouin's fixative proved satisfactory for embryos, but hot 70% alcohol and Zenker-formalin caused less distortion of larvae. Mallory's triple and haematoxylin and eosin were the chief

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stains used. Drawings were made with the aid of a camera lucida, kindly loaned by the Otago University Zoology Department.

The times given in days for embryonic development refer to the two pairs of lamellae mentioned, collected on February 18, 1944, and reared contemporaneously. To what extent temperature variations and other factors affect embryo development has not been studied.

The writer wishes to express her appreciation of the facilities enjoyed at the Portobello Marine Biological Station during the latter part of this work.

Embryonic Development. (Plates 36–38.)

The embryos, as is general among thoracie cirripedes, undergo natural development in the mantle cavity of the adult, being cemented together into the pair of ovarian lamellae. Each lamella is irregularly oval, orange-coloured, and contains, on an average, about 1500 embryos. Scattered individuals show lamellae from December until July. The main breeding season, however, is from February to April, when two-thirds of the adults are carrying embryos. Typically, all the embryos in a lamella are at approximately the same stage of development.

The fertilised, one-cell egg, oval, telolecithal, and 550–600 microns long, nearly fills the transparent egg-shell (fig. 1). Sections show at the animal pole a hemispherical plug of eytoplasm embedded amongst the granular yolk of the rest of the egg (fig. 10). The nucleus is in the centre of this plug, while above, at the extreme animal pole, is an almost clear covering. These regions give the egg externally a zoned appearance.

By the next day the first cleavage has occurred, segmentation being complete, but very unequal (fig. 2). Further divisions oecur rapidly, so that by the third day some ten to fifteen epiblast cells were present in specimens examined, these apparently having been formed both by subdivisions of the first two epiblast cells and also from the large yolk-cell (figs. 3 and 4). The 4th day shows numerous tiny epiblast cells covering an antero-lateral quarter of the embryo, while the large yolk-cell has divided into approximately equal halves. This yolk-cell division was usually transverse or oblique, sometimes longitudinal. The division is at first not complete, a bridge holding the two large cells together (fig. 5). The next two days show further divisions of the large yolk-cells, occurring in almost any plane, and a gradual spreading-over of the epiblast cells. These are now so small and cover the yolk cells so closely that they are difficult to see; a casual glance strongly suggesting that the embryo consists only of from four to sixteen large cells of equal size (fig. 6–7). By the 6th day, the solid yolk is completely covered by an extremely thin layer of ectoderm cells (fig. 11); i.e., the blastopore has now closed. Mesoblast first appears a day later, sections showing limited areas of the embryo with tissue two cells thick outside the yolk. On the 8th day, slight thickenings are apparent externally, which 24 hours later can definitely be recognised as three pairs of limb rudiments (fig. 8). The 10th day shows these six simple lobes

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directed posteriorly (fig. 9). Two days later, slight notches develop in the tips of the second and third pair—the initial forking of the biramous nauplius limbs (fig. 12). The three pairs of limbs are now ranged in a semi-circle postero-ventrally on the embryo. Between the two mandibles (third pair) is a median protrusion—subsequently to become the caudal spine and furcae of the nauplius, overlying the site of the cypris limbs, and the point of origin of the “body” of the adult. Apart from these seven rudiments, the embryo appears as yet to be an undifferentiated mass of yolk-cells thinly covered with epiblast. Internally, however, transverse sections show a thickened solid rod of tissue mid-ventrally towards the posterior end. This, a day later, develops a cavity for a short distance, marking it out as the rudiment of the nauplius oesophagus (fig. 13).

Also, on the 13th day, there appear externally minute “blisters” at the tips of the limb-rudiments, which within the next 24 hours become recognisable as short stumpy setae (fig. 16). The antennules carry 4, the antennae 4 on each anterior ramus, 2 longer (and 1 or 2 shorter) on each posterior ramus; while the tips of each mandibular ramus have 4, of the posterior ramus 2. There also appears on the 14th day the nauplius labrum, placed between the bases of the antennules. At this juncture, all the organs so far visible are crowded together on the posterior half of the ventral surface. During the subsequent development of the embryo, a longitudinal expansion of this area occurs, bringing the antennules forward to the anterior quarter of the body, whilst leaving the caudal spine and furcae at the posterior extremity (compare figs. 16 and 18); whereas the frontal horns of the nauplius, which first appear on the embryo on the 15th day as short, backwardly-directed stumps about halfway down the ventral surface (fig. 16, dotted). soon migrate forward to the extreme anterior margin (fig. 18). The frontal horn glands promptly become active, densely-staining blocks of secretion soon accumulating (fig. 14).

The caudal rudiment divided into a blunt tail-spine and a pair of ventral furcae just as the limb setae first appeared (fig. 16). These almost from their start are armed with short, sturdy spines. Both furcae and spines slowly elongate, curving back dorsally where they come in contact with the egg-shell.

During the first 12 days the embryos have a dull orange pigment uniformly distributed through their yolk cells. On the 13th day slightly darker granules begin forming a shallow U around the postero-ventral region, at the bases of the limb rudiments (fig. 16, stippled). During the next few days, light yellow bands of pigment develop along the lateral margins. At the same time the uniform orange colour of the yolk cells becomes progressively paler; so that by the 24th day the central part of the embryo's body is almost colourless, with yellow and orange pigment bands forming the colour pattern that characterises the nauplius to be (fig. 18: yellow pigment coarsely stippled, orange finely).

Externally, by the 15th day, the embryo shows most of the nauplius features—setose limbs, caudal spine and furcae, frontal

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horns and a labrum. Internally, however, there is as yet but slight histological differentiation; marked changes occurring in this respect during the subsequent 8 or 9 days.

Anteriorly and inside the limbs, cells begin to elongate to form muscle fibres, their nuclei becoming oval and their cytoplasm granular. Closely associated with the differentiation of these fibres is the development of movement. Seventeen-day embryos show undifferentiated cell masses, and are motionless. By the 19th day (fig. 15), considerable elongation of cells has occurred, and some perhaps show traces of striations. On this day the first feeble twitching of nauplius limbs was observed. By the 24th day, vigorous movements occur within the egg-shell, both of the limbs and of the embryo body as a whole. The main fibres by now are sharply striated, while anatomically a well-developed muscular system is present (figs. 17, 19). Extensor muscles pass to the limbs from the dorsal surface. Flexors to the antennules and antennae take origin from endosternites just posterior to the stomach rudiment. A short, stout, transverse muscle joins these endosternites, while from their corners fibres run to the dorsal and ventral body wall. Flexors runs to the mandibles, and additional fibres to the antennae, from a more posterior point of origin above and behind the oesophagus. Also present are median and lateral dorso-ventral body-wall muscles. Most of these are shown in figs. 17 and 19, but an additional lateral pair occurs more posteriorly.

The oesophagus by the 17th day is stout and conspicuous, with a continuous lumen. Between it and the yolk lies a small, round, hollow dilatation—the rudiment of the “stomach” or mid-gut of the larva, which increases in size as the yolk becomes less. The intimate contact between its cells and yolk of altered staining properties suggests that at this stage they have a yolk-absorbing function. Series of transverse and longitudinal sections show no trace of intestine or anus, either in embryo or larva until almost the end of the nauplius phase.

The labral gland also is formed during the 15–24 days period (fig. 17, l.gl.). In P. spinosus, only the tip of the labrum is free, serial sections showing that the mouth lies but little distance from it.

On the 16th day a small colourless square appears between the bases of the frontal horns (fig. 16, dotted). The following day, light brown pigment marks it as the nauplius eye, which darkens to black on the 19th or 20th day. In sections, the 17-day embryo shows the eye rudiment immediately anterior to a dense mass of cells as yet not separated from the epiblast. This marks the “brain” or supraoesophageal ganglion. More posteriorly is a similar rudiment, the sub-oesophageal ganglion. By the 24th day both these are more sharply defined (fig. 17, br and s.o.g.), and are connected by a stout circum-oesophageal commissure.

From the 19th or 20th day onwards the transparent anterior margin of the embryo becomes crinkled, the triangular carapace being compressed by the oval egg-shell. By the 24th day the embryos

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appear as if almost ready to hatch. Each is thin, flat, and transparent, stretching the enclosing egg-shell to 770 × 565 microns, instead of the original 600 × 445. In actual volume, however, these embryos are probably no greater than the one-cell eggs, as a decrease in depth accompanies the greater breadth and length. Already the limb setae are shortly plumose and protrude well beyond the posterior end of the body (fig. 18). But a specimen dissected out of its shell at this stage swam only very feebly.

Further specimens were liberated from their shells on the 26th and 27th days. These swam more actively, but their first moults were belated, only a minority developing further. Finally, however, 3 specimens hatched naturally on the 30th day, a considerable number on the 31st, and a few more on the 32nd. The behaviour and subsequent straightforward development of these suggests that their hatching was at the natural time.

Nauplius. (Plates 39 and 40.)

The nauplius on hatching promptly commences to swim about actively, usually with its ventral surface uppermost. Within the next few minutes, a noticeable expansion of its carapace occurs, so that the crinkled appearance of the late embryo soon vanishes. The caudal spine and furcae, curved back dorsally by the egg-shell, now become posteriorly directed. They are still, however, short and stumpy, and the frontal horns are as yet pointed backwards. Frontal filaments are present from the late embryo onwards. The newly-hatched nauplii crowd to the side of the vessel nearest the light —a tropism displayed (though with progressively less intensity) throughout the nauplius phase, but not by the cypris.

The first moult occurs soon after hatching, usually from half an hour to two hours later. Firstly, the old “skin” splits in the antero-dorsal region, and within 6 minutes the larva is free of its shed outer integument. The frontal horns, now pointing outwards, are soon directed more anteriorly, while at the same time the caudal spine and furcae elongate slightly.

The second moult does not occur until 3 to 4 days later. It is characterised by no marked increase in size or change in structure. In its internal anatomy the nauplius as yet differs but slightly from the late embryo. Decrease in the yolk volume leaves more space between this and the ectoderm, the so-formed body cavity being traversed by loose connective tissue. At this juncture, however, a change arises that foreshadows the development of the cypris—namely, an active proliferation of cells beneath the caudal furcae, causing them to protrude ventrally. Transverse ridges soon indicate externally its segmental nature. By the third moult, a day or two later, a longitudinal furrow divides it into the rudiments of the 6 pairs of limbs and the abdomen of the cypris.

The fourth moult follows equally closely, usually about two days after the third. The antennules, hitherto tapered to fine points (fig. 21), now are thickened distally, so that they are of approximately the same diameter throughout their length. From the fifth moult

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emerge nauplii whose antennules are bulbous distally, this foreshadowing the development of the suckers on the cypris antennules (figs. 20, 35).

The period from the second to the fifth moult occupies little more than one-third of the nauplius phase. During the next period—over half of the nauplius life—no moults occur; but a number of new structures develop which prelude the change to the cypris.

A day or two after the fifth moult two spherical outgrowths develop laterally from the bilobed brain (fig. 20, l.e.). These during the next few days gradually pigment, passing from pale brown to black and becoming surrounded by a number of facets—the paired compound eyes of the cypris.

Before the paired eyes blacken, a new and thicker covering to the carapace is secreted within the old. This is characterised by numerous longitudinal ridges on its surface.

Meantime the cypris limb rudiments have been slowly elongating and developing setae (fig. 23). Just anterior to them form two further pairs of appendages—to become the maxillules and maxillae of the adult. The former carry palps (fig. 20, mxu, mxa). During this period the region carrying the cypris limbs is becoming separated from behind forwards from the carapace. Thus does the “body” of the adult-to-be become surrounded by a mantle cavity (fig. 24, m.c.). Internally, the tissues of this region is becoming differentiated into cypris limb muscles and a stout ventral nerve cord (fig. 22). Finally, a solid narrow rod of tissue is formed—the rudiment of the post-cypris intestine (fig. 24, int.).

A stout transverse muscle is formed before the sixth moult. It passes through the bend of the gut, between the oesophagus and the yolk mass, and is attached laterally to the margins of the carapace. This is the cypris adductor muscle (figs. 24, 28, c.ad.). At the same time the yolk-mass is undergoing a curious rearrangement in shape. Anteriorly it remains continuous in transection. But in passing dorsally over the cypris adductor it separates into three tongues (fig. 24), divided posteriorly by the developing mantle cavity. The narrow median one runs towards the intestine, while the stout lateral pair occupy most of the margins of the carapace, both posteriorly and, to some extent, anteriorly above and below the cypris adductor (fig. 28, y).

Towards the end of the nauplius phase, the body of the larva becomes rather opaque. In its general appearance, however, it is not markedly different in size or shape from the newly-hatched nauplius. For instance, the various moult skins, from the first to the fifth, differ but slightly. On the biramous limbs the lateral setae become progressively more strongly developed, but those on the anterior margin of the antennules become reduced (cf. figs. 20 and 21). Posteriorly, later moult skins show a greater bulging-out in the region of the cypris limbs than do the earlier ones. The sixth nauplius moult skins (from which the cypris larvae emerge) differ from earlier ones in the bulbous antennules, the striated carapace, and the short paired processes, just anterior to the caudal furcae, into which the cypris limb setae protruded.

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Factors Affecting Rate of Nauplius Development.

Various batches of larvae, collected as embryos about to hatch, were reared at different seasons. Whereas controlled constant temperature conditions were not available, it was apparent that temperature affected the length of nauplius life. In late summer, when noted temperatures were around 18° C., the nauplius phase was completed in as short a time as 12 days; whereas in a cold room in winter-time 20–21 days were required. Most specimens, reared in autumn, took 2–2 ½ weeks to pass from hatching nauplii to eypris larvae.

Another factor found to affect nauplius development was the length of embryonic life. As the batches of specimens about to be considered were mostly reared side by side at the same time (the average of noted temperatures being between 14° and 15° C. for each batch), the following results are probably practically independent of temperature.

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Table I

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Comments: Of the 5 artificially-liberated specimens in the first column, 4 soon became pathological and did not advance beyond the first moult. Moult-times of the fifth were not observed. It commenced to become a cypris on the 30th day, but died a few days later before this was completed. One 27-day specimen also died without moulting a second time. Among other groups, only two deaths occurred before the metamorphosis to the cypris stage was reached. The 30, 31, and 32-day specimens all underwent their first moult within half to two hours of hatching. In this as in other respects they agree with specimens freshly collected in a state ready to hatch.

Deductions: Under a given set of environmental conditions, prematurely-liberated specimens take longer to develop than naturally-hatching ones, if they develop at all. During a hatching-range of 6 days, the total maximum and minimum periods of embryonic and nauplius life are remarkably constant (foot Table 1, columns 2–5). A considerable extension of the period within the egg-shell does not further accelerate the nauplius phase. Whereas absolute lengths of different nauplius phases vary considerably, the relative periods between moults and appearance of organs show a marked regularity.

Metamorphosis From Nauplius to Cypris. (Plate 41.)

The dramatic change from nauplius to cypris form has been observed completely in seven specimens, in part in many others. The two main features are the closing-up of the broad nauplius carapace by the permanent contraction of the cypris adductor muscle, and the rapid resorption of the biramous nauplius limbs. The time taken to pass from the typical nauplius to the typical cypris varies from half an hour or less in the first and most active specimens of a batch to over an hour in later ones; whereas weaker specimens may remain in a half-closed state for many days before slowly dying.

The onset of metamorphosis is first indicated by the outward migration of the lateral eyes. These throughout the late nauplius phase are placed with some constancy at a distance of 160 microns apart (measuring from the centre of one pigmented mass to that of the other). Then suddenly, within a matter of minutes, they migrate out laterally till they are nearly twice as far apart as before (namely, 290–300 microns). They are now placed on the outer sides of the antennule bases, each protruding ventrally and being connected with the brain by a rather attenuated optic nerve.

Further external change in the larva is not apparent until the lateral eyes have almost completed their migration. At this juncture, however, the anterior margin of the carapace becomes pushed in medianly and bulges forwards at each side. This is perhaps brought about by the contraction of a small pair of muscles which, attached mid-ventrally between the median eye and the brain, run to the carapace anteriorly. Then, presumably due to the contraction of the cypris adductor muscle, the lateral margins of the carapace begin closing in from the nauplius skin, at first from the nauplius frontal horns (two slight knobs representing for some time the vestiges of these), and later from the more posterior margins of the carapace.

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This gives an oval larva with widely-placed eyes. Its antennules are held stiffly outwards, remaining almost immobile for some minutes. By contrast, however, the nauplius antennae and mandibles are very active, usually, sending the larva scudding round on its back. The cypris limb rudiments, although well developed and with long plumose setae, have as yet shown no sign of movement.

At this point, several changes begin to occur more or less together (figs. 25, 27, 29). The antennae and mandibles start shrinking in diameter, and soon also in length. The antennules, immobile for the past several minutes, now regain activity, pulling themselves forwards out of their nauplius coverings. The previous lack of movement was probably associated with the breakdown and rearrangement of tissue that permits their change in function. For whereas in the nauplius they were swimming appendages beating together like a pair of very rapid oars, in the cypris they become prehensile walking limbs, moving alternately and deliberately.

Before the antennae and mandibles completely lose motility, during their early stages of shrinking, they pull the larva anteriorly part-way out of the old nauplius “skin.” But they are now being rapidly resorbed, changing within sometimes as short a period as 6 minutes from active swimming limbs to minute, squirming, opaque, biramous stumps. Next, however, the cypris limbs begin kicking, soon to complete the work of pushing the larva out of the sixth nauplius moult-skin.

Shortly before this occurs, the trophi rudiments that developed just in front of the cypris limbs migrate forwards and medianly towards the shrunken mandibles, to become the maxillules and maxillae of the adult. Internally the oesophagus seems to disappear, serial sections during metamorphosis showing that at least it is no longer the continuous tube that it was in the nauplius. It becomes reformed in the cypris before long, opening a little more posteriorly than previously.

As the narrowing of the nauplius carapace has been progressing, its anterior and lateral margins have considerably thickened, the tissues humping themselves up into a stout pigmented band that almost overhangs the lateral eyes. Thus there occurs a three-quarters closed-up cypris larva, still largely contained within the old nauplius skin. The cypris limbs meantime have been kicking more and more actively, pushing themselves forward from the caudal furcae of the nauplius. A few more vigorous kicks from these send the whole cypris larva out of the old nauplius covering, just as its carapace margins are brought almost together. The tips of the minute vermiform stumps of the antennae and mandibles may remain visible externally between the margins of the carapace for a few minutes longer (fig. 26). But to all other appearances the larva that kicks itself away from the old nauplius skin is now a typical cypris.

Its increase in length as compared with the late nauplius is caused more by the re-shaping of the carapace than by actual growth at this juncture. In fact, in total volume it is doubtful whether the cypris is larger than the one-cell egg; for while longer, it is very much narrower.

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Cypris. (Plate 42.)

The young cypris larva, 85 mm. long, can maeroscopieally be picked at a glance from the late nauplius, either by its changed shape or by its different mode of progression.

To swim, the 6 pairs of cypris limbs kick rapidly, the anterior ones slightly ahead of the posterior. This sends the larva along on its side in a jerky motion. Usually it swerves round dorsally in an are, sometimes ventrally, but only rarely does it travel forwards in anything approaching a straight line. The limbs themselves are small and biramous, but each carries a number of long, plumose setae (fig. 31). Behind the last pair, a small segmented outgrowth, commonly known as the abdomen (fig. 32), also assists with the swimming.

To walk, the antennules grip the substratum by their suckers (fig. 35), and the cypris progresses like a person walking on his hands.

In colour, the young cypris is light yellow, partly due to the straw-coloured chitin of the thick carapace, partly to the bands of bright yellow pigment down the sides. At each end is an area of orange-red pigment, with scattered spots of this in other parts. Later the arrangement of these pigments alters somewhat, and a diffuse pink, becoming brown, tints the posterior half of the carapace. Both median and large lateral eyes are readily visible by transparency.

Transverse and longitudinal serial sections were examined of the cypris immediately after the metamorphosis from the nauplius, two days after, and eleven days after. In the first of these an oesophagus was almost indistinguishable, a slender rod of solid tissue in the region of the nerve commissure and adductor muscle probably representing it. Gradually it becomes better developed, until by the 11-day specimens it is clearly shown and has a lumen for most of its length (fig. 30). The previous division of the yolk, into median and lateral masses connected by a bridge, has already been described. The median mass, which in the 2-day cypris runs from the stomach to the intestine (fig. 34, y), subsequently becomes absorbed. Hence by the 11th day the stomach ends and the intestine begins blindly, an empty space lying between the two (fig. 30, sp). The beginning of the intestine has a hollow cavity, for a while filled with amorphous contents similar to those of the stomach. The rest of it is solid, and no anus is yet present.

The lateral yolk-masses have a different fate, becoming converted into the two cement-glands of the post-cypris. On each side, a broad duct from the posterior mass (fig. 36, c.d.) joins another from the anterior, the fused product running down the antennule to its end (fig. 30, c.d.).

In the early cypris the lateral margins of the carapace are folded in ventrally, but are still quite free from each other (fig. 26). But in the 11-day cypris the tissues have actually fused ventrally for nearly a quarter the length of the larva, a thick layer of chitin being secreted beneath the join (fig. 36). Anteriorly and posteriorly,

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however, openings remain, leaving the antennules and cypris limbs with access to the outside world (fig. 33). The nature of the attachment of the cypris eyes is puzzling at a glance. But on considering their migration and ventral protrusion at the onset of metamorphosis, the subsequent folding of the carapace (fig. 27), and finally its fusion ventrally, their relation to the antennules and brain can readily be visualised.

Strong muscles running to the antennules and cypris limbs are a conspicuous feature. Others pass dorsally from the body forward to the carapace. The cypris adductor, already referred to, radiates out from a median endosternite, lying in the bend of the oesophagus between the mouth and stomach. Its fibres are thick and unstriated. The nerve-cord is short but deep, the stout commissures connecting it with the brain passing on each side of the oesophagus just below the stomach (figs. 30, 34, 36, n.c. and br).

Dorsally, the mantle cavity extends more anteriorly than in the late nauplius. By the 11-day cypris stage, however, it does not bisect the larva, so that as yet no peduncle is recognisable.

The trophi now bear the relationship to one another that they show in the adult barnacle (fig. 33).

Post-cypris. (Plate 43.)

Of the various cypris larvae reared from earlier stages, most died in the course of days or weeks without undergoing further apparent development. Small stones were placed in their dishes, but larvae did not attach themselves either to these or the glass surface. Some factor necessary for their further straightforward development appeared lacking. Eventually, however, it was found that two specimens, which had hatched on 28.2.44 had secreted chitinous prevalves and developed cirri by 30.7.44. Then a single specimen, which was collected as a one-cell egg on 18.2.44 and showed no valves by mid-August, appeared opaque on August 24 and secreted prevalves a couple of days later. Finally, another, by 15 weeks after its change from nauplius to cypris, showed not only the chitinous terga, scuta and carina of the post-cypris, but beneath them the beginnings of the corresponding calcareous valves, together with a tiny rostrum. This was before any trace had appeared of latera or peduncular spines. The total length was by now as great as that of its shed cypris carapace, and a week later the first “adult” moult occurred; the delicate chitinous covering of the cirri and “body” being cast out through the mantle opening.

The length of the cypris phase in these artificially-reared specimens is doubtless abnormally extended. Structurally, however, they appear similar to a single post-cypris collected from a Pollicipes colony on 26.5.40 (fig. 39).

Unfortunately, the course of the second metamorphosis cannot be followed on living specimens in the way that the first one could be seen; and extra specimens for fixing at various stages were not available. Hence its course in this species cannot be described here.

In external appearance (fig. 37) the post-cypris or pupal forms are typical young scalpelliform cirripedes. The cirri, stomach, and

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mantle-cavity are faintly visible by transparency. The lateral cypris eyes are empty save for some black pigment. In two, the antennules were surrounded by a mass of amorphous substance—presumably cement.

The arrangement of the viscera is shown in fig. 38. As stages intermediate between this and the cypris were not examined, a comparison of the two is deferred to the discussion. But when, on the other hand, the post-cypris is compared with the adult (mirror-image of fig. 10, previous paper, Batham, 1945), the essential similarity is apparent. The relative positions of food-canal, brain and median-eye, nerve-cord and adductor muscle are identical. In other words, although the tissues are as yet but slightly differentiated, the main lay-out of the adult barnacle has been achieved.

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Table 2.
Pollicipes spinosus: Dimensions of Embryos and Larvae (measurements in microns, each based on average of at least five living specimens).
Stage Length. Length of Egg-shell Maximum Width
One-cell egg 600 660 445
Embryo just before hatching 690 (+caudal spine = 710) 770 565
Length Length excluding including Caudal Spine Caudal Spine
Nauplius before first moult 670 730 580
Nauplius after third moult 690 805
Nauplius directly after fifth moult 710 81
Nauplius four days after fifth moult 725 825
Depth
Cypris directly after metamorphosis 850 385
Cypris three weeks later 860

Discussion.

Parts of the early development of cirripedes have been described by a number of workers, and general accounts are given by Darwin (1854), Gruvel (1902) and others. The most detailed account that has been available is that of Groom (1892, pp. 119–232), whose description is based chiefly on species of Balanus, Cthamalus, Lepas and Conchoderma. From these, Pollicipes spinosus differs in the

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following respects: the early cleavage of the ovum is much more unequal; nauplius limbs are first marked out on the ventral surface, and no intestine develops until the end of the nauplius stage (whereas Groom describes this organ in early nauplii as being more conspicuous than the oesophagus). The nauplius differs from that of most species figured (except Scalpellum villosum, Darwin, 1854, pl. 29, fig. 8) in having a relatively large carapace and body, causing the limbs, tail-spine and caudal furcae to appear dwarfed and inconspicuous by comparison. The newly-hatched nauplius is unusually large, but growth between successive moults is slight; so that the cypris and post-cypris stages are smaller than those of many other cirripedes. In fact, the numerous larval moults in close succession would seem to represent a phylogenetic retention of the primitive number of cirripede nauplius moults rather than a mechanism for permitting increase in size in this species (Table 2).

In all these features, the development of P. spinosus appears specialised as compared with many cirripedes. Each, however, is probably a direct corollary of the large amount of yolk in the egg, enabling larval development to occur without the taking of food. For the yolk would affect early cleavage and also dispense with the need for a continuous food canal in the larva; while the dependence on yolk instead of external food would prevent the nauplius from increasing much in size. Finally, if one took a typical cirripede embryo whose limb rudiments extended round dorsally, and considerably increased the volume of yolk above them, they would then scarcely extend beyond the ventral surface, and would seem small in proportion to the nauplius body, as in Pollicipes.

The term “metanauplius” has been variously applied to a nauplius with cypris limb rudiments, a nauplius with pigmented lateral eyes, and one with maxillules and maxillae. As at any rate in this species, the cypris limbs are marked out before the third moult, whereas the other features develop only well after the fifth, it is seen that the term is somewhat indefinite. If retained, it is best applied to the nauplius between the fifth and sixth moults, distinguishable from earlier stages by having antennules of greater diameter distally than proximally.

As intermediate stages between cypris and post-cypris were not examined, a comparison of the two forms is left meanwhile to deduction. If the mantle cavity of the cypris is extended in the direction of the arrow in figure 30, the “body” to the right of it at the same time being rotated through 90°, the similarity of plan is apparent. On this basis, the median nauplius eye, brain and nerve cord, oesophagus, stomach and intestine retain their relative positions in the post-cypris; though the last two have become connected and form a continuous tube. But assuming this, its adductor muscle cannot be the homologue of that of the cypris. Further, the two are quite distinct structurally, the one consisting of a number of parallel strands (fig. 39), the other of fibres radiating from a median endo-sternite.

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In view of the emphasis that has been laid on the additional latera and scales of Pollicipes adults in discussions on the phylogeny of cirripedes, it is of interest to note that the only valves represented in the post-cypris are the terga, scuta and carina. Broch has called attention to the same point in Pollicipes polymerus (1922, p. 153).

Literature Cited.

Batham, E. J., 1945. Pollicipes spinosus, I—Biology and Anatomy of Adult Baruacle. Trans. N.Z. Roy. Soc., vol. 74, pp. 359–374.

Broch, H., 1921. Studies in Pacific Cirripedes. Mortensen's Pacific Expedition, 1914–16, Kristiana.

Darwin, C., 1854. A Monograph of the Cirripedia. II. Balanidae. Ray Society.

Groom, T. T., 1894B. On the early Development of Cirripedia. Philsophical Transactions, pp 119–232.

Gruvel, A., 1905. Monographie des Cirripedes ou Thecostraces. Paris, Masson et Cie.

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Early Embryos. × 72.
Fig. 1.—First day (one-cell egg)
Fig. 2.—Second day.
Fig. 3.—Luter second da.
Fig. 4.—Third day.
Fig. 5.—Fourth day.
Fig. 6.—Fifth day.
Fig. 7.—Sixth day.
Fig. 8.—Ninth day.
Fig. 9.—Eleventh day. a1 antennules; a2 antennae; mn. mandibles.

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Fig. 10—L.S., anterior end of one-cell egg. × 120. FIG. 11.—L.S., 6-day embryo, × 110.
Fig. 12.—Ventral view posterior end of 12-day embryo. × 90. a1. antennules, a2, antennae; mn. mandibles.
Fig. 13.—T.S., ventral section of 13-day embryo. × 340. Lettering as above.
Fig. 14.—Early frontal horn. in L. horizontal S. of 19-day embryo. × 225.
Fig. 15.—Anterior dorso-ventral muscle fibres, in 19-day embryo. × 225.
Fig. 16.—Ventral view of 14-day embryo, × 195. a1. antennules; a2, antennae; mn, mandibles, e.sp, caudal spine. (Dotted lines indicate positions of frontal horns and median eye, first apparent one and two days later respectively.)

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Twenty-four day Embryos, × 145.
Fig. 17.—Longitudinal section.
Fig. 18.—Ventral view. (Coarse stippling: yellow pigment, fine, orange)
Fig. 19.—Reconstructed thick transverse section throgh limb muscle region viewed from behind, a1 antennules; a2, antennae; br, brain; e.f, caudal furcae; c.o.e., circum-oesophageal commissure; e.sp. caudal spine, c, eye; end, endosternite; fr.h, frontal horn; l.d.v, lateral dorso-ventral muscles; l.gl, labral gland; mn. mandible; oes, oesophagus, s.o.g., suboesophageal ganglion; st, stomach; y, yolk.

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Fig. 20.—Nauplius between 5th and 6th moults, just prior to pigmentation of laterl eyes, ×120, a1 antennules; a2, antennle; e,f, caudal furca; c.sp, caudal spine; d, d,ducts of pairedglands; e, nauplius eve, l.f, frontal fliments; fr,h frontal horn; l, labrum; l,e, lateral eye rudiment; mn, mandible; mxa, maxilla; msu, maxillule, (Yellow pigment stippled; orange pigment, small loops,)
Fig. 21.—Right antennule of newly-hatched nauplius, × 120, Setae lettered to correspond with those in Fig, 20.

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Nauphus. Fig. 22.—L.S. nauplius just after 5th moult. × 150. abd. cypris abdomen: b.c., body cavity; b [ unclear: ] brain; c.l.r. cypris limb [ unclear: ] udiments: mxa, maxilla, oes, oesophagus; s.o.g., suboesophageal ganghon. st. Stomach: y. yolk.
Fig. 23—Lateral view, nauphus with lateral eyes lightly pigmented. Lettering as in Fig. 20
Fig. 24.—Nauphus just before metamo [ unclear: ] phosis—reconstruction based on longitudinal horizontal [ unclear: ] ion × 150 [ unclear: ] add. cypris adductor muscle; e. nauplius eys; int, intestine rudiment; l.e., lateral (cypris) eye: m c., mantle cavity; st, stomach; y, yolk.

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Nauplius-cypris Metamorphosis
Fig. 25.—Nauplius metamorphosing to cypris. ventral view of living specimen, × 112.
Fig. 26.—T.S. cypris 10 minutes after emergence from nauplius “skin”: reconstruction. × 180.
Fig. 27.—T.S. larva at same stage as Fig. 25. through lateral eye region. × 90.
Fig. 28.—T.S. nauplius due to metamorphose. × 90. (Oesophagus and mouth are more anterior, maxillules and maxillae more posterior.)
Fig. 29.—T.S. larva at approx. same stage as Fig. 25. × 180. (Figs. 26, 28, 29 are all through cypris adductor muscle.) a1, antennules; a2 antennae; br, brain: c.ad. cypris adductor muscle; c.l.m. cypris limb muscles; i.e. lateral eye; m.c. mantle cavity; m, mouth; mn, mandible; mxa, maxilla; mxu, maxillule; n.c, nerve cord; o, oesophagus; st, stomach; y, yolk.

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Cypris.
Fig. 30.—Eleven-day cypris—reconstruction of right [ unclear: ] alt. based on L.S. and T.S. series. × 140.
Fig. 31.—Fifth right cypris limb. × 280.
Fig. 32.—“Abdomen” of cypris. × 250.
Fig. 33.—Ventral view of late cypris
Fig. 34.—L.S. two-day cypris. × 90.
Fig. 35.—End of cypris antennul [ unclear: ] .
Fig. 36.—T.S. 11-day cypris, × 160. a1, antennule; ab. “abdomen”; a.m., muscles to antennules; br. brain. c.ad. cypris adductor: e.d. cement duct; cem. cement; c.l, cypris limbs; c.l.m. cypris limb muscles: e. nauplius eye; int. intestine; m. mouth; m.c. mantle cavity; mn mandible; mxa, maxilla; mxu. maxillule; n.c, nerve cord; st, stomach; y, yolk.

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Post-Cypris.
Fig. 37.—Late post-cypris, reared in ritro; side view. × 120.
Fig. 38.—L.S. post-cypris, reared in ritro. × 130.
Fig. 39—L. horizontal S., post-cyris collected from P spiao [ unclear: ] us colony. × 130. A1. antennules; a. anus; ad, adult adductor muscle: br. brain: C. carina pre-valve; cem, cement: e, nauplius eye (= median eye of adult): m.c., mantle cavity; n.c. nerve cord, oes. oesophagus; ov. ovary rudiment; S, scutum pre-valve; st, stomach; T. te [ unclear: ] ga pre-valve.