Acontia and mesentery nematocysts of the sea anemone Metridium senile (Linnaeus, 1761) (Cnidaria: Anthozoa)

Acontia and mesentery nematocysts of Metridium senile (Linnaeus, 1761) are described from interferencecontrast light micrographs (LMs) and scanning electron micrographs (SEMs). The acontia have 2 nematocyst categories grouped into small, medium and large size-classes, including 5 types: of these, large b-mastigophores and large p-amastigophores are the largest and most abundant. Mesenterial tissues, characterised by small p-mastigophores and medium p-amastigophores, have 3 nematocyst categories grouped as small and medium, including 6 types. Attention is given to nematocyst maturation, especially to the differentiation of the shaft into proximal and main regions as helical folding of the shaft wall proceeds. Groups of differentiating nematoblasts occur along acontia, and near the junction between acontia and mesenterial filaments. Nematoblasts are sparsely found throughout mesenterial tissues.


INTRODUCTION
Metridium senile (Linnaeus, 1761) is a common sub-tidal sea anemone on the Swedish west coast, growing on hard substrates (Wahl, 1985).Three species of sea anemones referred to the genus Metridium occur along the coasts of North America; M. senile, M. exilis (Hand, 1956) and M. farcimen (Brandt, 1835).Biochemical genetic studies corroborate morphological evidence that the European Metridium is best referred to the subspecies M. s. senile (Linnaeus) (Bucklin and Hedgecock, 1982;Fautin et al., 1990).In the present paper Scandinavian Metridium will be referred to M. senile (L.).According to Carlgren (1945) the genus Metridium has, in addition to spirocysts, 4 categories of nematocysts: basitrichs, microbasic b-and p-mastigophores, and amastigophores, with size differences in different parts of the anemone.The basitrichs and b-mastigophores were difficult to distinguish (Carlgren, 1940, pp. 52-54).The nematocyst's size in different anemone structures is regarded as important taxonomically (Fautin, 1988).Hand (1956) thought that most basitrichs recorded in the literature were b-mastigophores.Westfall (1965) noted the similarity between basitrichs and microbasic b-mastigophores, though they differed in size.Each had a straight shaft with dense spines and a long, narrow tubule.
The present study illustrates nematocysts from acontia and mesenteries by light photomicrographs (LMs) and scanning electron micrographs (SEMs), including maturing stages of the nematoblast, capsule, tubule and shaft.Especially, we focus on the structure and differentiation of the inverted shaft.Our findings are compared with those of Cutress (1955), Skaer and Picken (1965), Picken and Skaer (1966), Westfall (1966), Schmidt (1969) and Kramer and Francis (2004).Hopefully, our illustrations will facilitate identification of Metridium nematocysts stored in nudibranchs, especially immature ones, if stored in the cnidosacs, as reported by Greenwood and Mariscal (1984).

MATERIALS AND METHODS
Metridium senile were collected from the Gullmarsfjord on the Swedish west coast (58°15´N, 11°28´E) in the period June to August 1996-2003.M. senile grew on hard substrates and appeared in two size-groups and several colour morphs (Anthony and Svane, 1994).Small red-brown individuals (pedal disc diameter (PDD)<20 mm) form mass aggregations in surface water down to 2 m.Small to large, reddish to white individuals (PDD up to 45 mm) grew solitary or in small presumed clones from the surface down to ca. 20 m.Nematocyst samples, including all colour morphs and sizes, were taken from aggregated and solitary individuals.Anemones were examined soon after collection or after keeping in running seawater for a few days without feeding.
The size of acontia and mesentery nematocysts of M. senile and of M. farcimen (as M. s. fimbriatum) (see Hand, 1956) is shown in Table 1.Regression analysis were done on the undischarged capsule length from small (PDD ca 5 mm) to large anemones (PDD ca 45 mm) to determine whether the nematocyst size increased with increasing sea anemone size.The extended tentacle crown of undisturbed anemones was measured with a ruler to an accuracy of 5 mm, and then related to the PDD.We consider it more accurate to measure the tentacle crown than the PDD, which could be bent around the substrate.
Squash preparations of living acontia and mesenteries for nematocyst studies in LM and SEM were made following the methods of Östman (1987) and Östman et al. (1991).To study detail structures in LM, some squash preparations were freeze-dried at 70 o C for 24 h.A Leica DMRBE light microscope equipped with interference-contrast optics, a 100x /1.30PL, fluotar objective and a camera connected to a video recorder was used, and also a Philips XL 30 scanning electron microscope.LM measurements of the nematocysts were accurate to ±0.5 µm.For each nematocyst type studied, at least 20 undischarged capsules were measured from each tissue of usually 5 or more individuals (Table 1).If possible, acontia and mesentery nematocysts were measured from the same specimens.The abundance was estimated by counting  (Hand, 1956).
Type of nematocysts from acontia and mesenterial filaments, N, number of samples, each taken from a different anemone; 1, abundance of nematocysts: + present, +++++ very abundant; absent to rare, nematocysts not found always in the structures examined; *, data on filaments includes adjacent mesenterial tissues (Fig. 3a) nematocysts in the field of view using the 100x objective or from the video screen.Some measurements were made on SEMs.
In fully developed nematocysts the inverted shaft reached or slightly exceeded 3/4 of the capsule length (Fig. 1d).Some shafts were slightly undulating (Fig. 5d).The inverted shaft of large b-mastigophores and p-amastigophores were differentiated into a proximal region with tight helices of the shaft wall and the remaining main shaft with spaced helices (Figs. 5c-e,g, 6e,h).
The broad p-amastigophore shaft enabled its end to invaginate and form the characteristic V-shaped notch (Figs.3c-e, 6e).A tiny tubule was emerging from the notch and made one or two irregular coils towards the posterior capsule (Fig. 6e).The tubule was not always visible in undischarged capsules.After discharge the broad shaft-tubule abruptly narrowed to the tiny tubule (Fig. 2a,c), reaching 30 mm, or the tubule broke, and the shaft ended as a pointed cone (Fig. 2b,d, h,j).A few coils of the broken tubule were often seen inside discharged, flattened capsules (Fig. 2i,j).In freeze-dried preparations the tiny tubule appeared more distinct (Fig. 2j) than in wet preparations (Fig. 2f,i).
The discharged shaft and capsule remained swollen (Fig. 2a,b,g,h) until the internal capsular fluid had left.This took longer in mesobasic p-amastigophores than in microbasic p-amastigophores and mesobasic  b-mastigophores.Swollen mesobasic p-amastigophore shaft were more conspicuous than flattened short shafts.Spines on the proximal shaft were sparse in spaced right-handed spine-rows (Fig. 2c,e).On the main shaft spines were dense in dense spine-rows (Fig. 2c,d).Regression analysis made on large b-mastigophores and p-amastigophores indicated a modest positive correlation between increasing capsule length and increasing anemone size (p<0.0001,R-Sq = 0.29 and p<0.001,R-Sq = 0.087, respectively).

Mesentery nematocysts
In the mesenterial filaments nematocysts were dense (Fig. 3a-c), and oriented with their capsule apex towards the filament margin (Fig. 3b).Small microbasic p-mastigophores with drop-shaped capsules dominated, followed by medium microbasic p-amastigophores, mesenterial (Fig. 3b,c, Table 1).The latter differed from the ordinary medium p-amastigophore (Fig. 3c,d) by its shorter inverted shaft, reaching 1/2 to 1/3 of the capsule length, by its broader, distal capsule and by lack of an internal structure of capsular matrix, characteristic for p-amastigophores (Fig. 3d).In addition, small and medium b-mastigophores and pamastigophores were sparsely scattered in the filament.In the adjacent tissue (Fig. 3a) nematocysts were sparse and small b-mastigophores were most abundant.Drop-shaped p-mastigophores and medium pamastigophores, mesenterial, were characteristic for mesenteries.The narrow distal tubule of drop-shaped p-amastigophores (Fig. 3g) was vaguely seen coiled around the inverted shaft, if capsular fluid had left (Fig. 3f).The spines (Fig. 7k) were often detached from discharged shafts (Fig. 3g) and the distal tubule reached 225 µm.The distal tubule of the medium p-amastigophores, mesenterial, was vaguely seen making a coil towards the apical capsule (Fig. 7f,g).The discharged tubule (Fig. 7j) reached 30 µm.

Acontia nematoblasts; formation of b-mastigophores and p-amastigophores
Nematoblasts and immature nematocysts (Fig. 4) were most abundant within the acontia near their attachment site to the mesenterial filaments, but were also scattered along the acontia (Fig. 2a).Round nematoblasts with U-shaped capsule and external tubule already formed (Fig. 4a), and irregularly shaped nematoblasts enclosing broad folded capsules (Fig. 4b), were found among differentiating large b-mastigophores   and p-amastigophores at later stages of development.
We interpret the nematoblasts in Figures 2a and 4b as b-mastigophores and those in Figure 4a,c as p-amastigophores.Figure 4c shows the external tubule.
During maturation the broad, folded capsules evidently became narrow and U-shaped and later straightened to the shape of mature capsules (Fig. 4a-f).By then the external tubule had inverted, the shaft had grown distinct, and spines had formed.Shafts, early in differentiation, were seen within folded capsules (Fig. 4b,e).They were longer than the capsules, flexed or coiled, and reached the distal capsule.
During the early stage of development the spines formed a V-shaped pattern pointing in the direction of eversion (Fig. 4e).As a nematocyst matured, the shaft apparently became gradually more pleated, its helices grew denser, and the shaft was shorter and straighter (Fig. 4f).The V-shaped spine pattern (Fig. 6b,c) was replaced by left-handed helices (Fig. 6e,h) and, in the p-amastigophores, the characteristic V-shaped notch was formed at the shaft end (Fig. 4f).In mature capsules the shaft was straight or slightly curved (Figs.1d, 6e).Mature, undischarged p-amastigophores were recognised by their internal asymmetrical U-shaped structure of capsular matrix (Fig. 6e).This structure was not seen in immature p-amastigophores (Fig. 6bd) and in discharged or partly discharged capsules (Fig. 2a,b,g,h).

Large mesobasic b-mastigophores; shaft and tubule
In broken capsules of b-mastigophores the tubule coils around the shaft are shown (Fig. 5a-c).The border between the proximal shaft with dense helices and the main shaft with more spaced helices was indistinct in LM (Fig. 5c,d) and SEM (Fig. 5e).Differences in pitch between the helices on the proximal and main shaft are shown with white lines (Fig. 5e).The left-handed helices on the inverted shaft and tubule, formed by ridges and pleats, corresponded to the three spines-rows (Fig. 5b,f, 1, 2, 3).Additional small pleats and ridges were visible on the main ridges (Fig. 5f).The helices were more extended and the pleats or folds deeper on the distal tubule than those on the shaft (Fig. 5f).Differences in pitch between the helices on the shaft and on the distal tubule were large (Fig. 5f, white lines).Figure 5c shows broken capsules with still-inverted, extended, long shafts and enlarged tubule coils.

Large p-amastigophores; differentiation of inverted shaft
The inverted shaft, when viewed in SEM (Fig. 5g), was clearly separated into the proximal shaft with tight helices and the main shaft with spaced helices.In LM the two shaft regions were more obvious earlier in development (Fig. 6a-d) than in mature p-amastigophores (Fig. 6e,h).
Figure 6a shows the inverted shaft of an immature p-amastigophore extended outside a capsule, interpreted as ruptured.The V-shaped pattern of dense spines is clear on the main shaft, which ends abruptly in a small spineless cone.On the proximal shaft (Fig. 6a, prox) the V-shaped pattern of sparse spines is seen in the inset.
Figure 6b-e is interpreted as a time series and illustrates the differentiation of the two shaft regions.Figure 6f,g,h shows enlargements of Figure 6c,d,e.Probably the inverted shafts in Figures 6a and 6b are close to the same state of development.In Figure 6b the inverted, not yet helically pleated shaft reaches the capsule end.The shaft is coiled, flexed and undulating, and the two shaft-regions are shown.The V-shaped pattern is not discernable on the proximal coiled shaft (Fig. 6b, prox), but is distinct on the main shaft, except at its basal part (marked inv).
Figure 6c-e shows the increasing distance between shaft and distal capsule end as the nematocysts mature and the helically more-pleated shaft becomes shorter.The somewhat helically pleated shaft (Fig. 6c,f) is shorter and later in development then the shaft in Fig- ure 6b.More capsular space is given to the undulating proximal shaft (Fig. 6c,f, prox), in which the V-shaped spine-pattern is distinct.The border between the proximal and main shaft is clear.On the nearly straight main shaft the V-pattern is partly replaced by helices.The Vshaped notch (Fig. 6c), formed by invagination of the shaft end (Fig. 6a,b, inv), is close to the distal capsule end.The tiny tubule is indistinct.
Figure 6d,g shows a p-amastigophore late in development with straight, shortened shaft.The V-shaped notch is on a clear distance from the distal capsule end.The border between the proximal and main shaft is distinct (Fig. 6g).Dense helices of the shaft wall are visible on the proximal straight shaft and around the V-notch (Fig. 6, inset).On the proximal shaft a narrow axial rod-shaped structure is seen, which broadens towards the main shaft.
Figure 6e,h shows a mature p-amastigophore with a shortened, straight shaft.The border between the proximal and main shaft is less obvious than earlier in development (Fig. 6c-d).The proximal shaft (Fig. 6h, prox) with tight helices has become shorter than the main shaft with wider-spaced helices.The narrow axial rod-shaped structure on the proximal shaft is indistinct.From the V-shaped notch the tiny tubule extends distally.The internal U-shaped matrix is partly discernable.

Differentiation of mesentery nematocysts
The mesentery nematocysts followed the same development (Fig. 7a-i) as large acontial p-amastigophores, except that in mature, small, drop-shaped p-mastigophores (Fig. 3b) and in p-amastigophores, mesenterial (Fig. 3b,c), no regular internal structure of a capsular matrix was visible, and their shaft was not differentiated into proximal and main regions.Nematoblasts and developing nematocysts were scattered in the tissue adjacent to the mesenterial filament (Fig. 7a-h).Figure 7a shows an extended external tubule in connection with a capsule (the nematoblast had probably ruptured).The same photograph shows an early developing small, drop-shaped p-mastigophore with V-shaped spine pattern on its inverted shaft.
The V-shaped spine pattern in small drop-shaped p-mastigophores (Fig. 7b) and in medium p-amastigophores (Fig. 7b-d) was replaced by helices during later stages of development.Their shaft wall became more pleated, and their shaft shorter (Fig. 7b,e-i).The border between proximal shaft and main shaft was only seen in the ordinary p-amastigophores (Fig. 7e), and so was the narrow axial rod along the proximal shaft.To left an immature p-mastigophore, small, drop-shaped, showing inverted shaft and nematoblast.Border between capsule and nematoblast faintly seen (tiny arrows).b) 3 p-mastigophores, small drop-shaped (most differentiate to right) and a pamastigophore, medium.Note V-shaped spine pattern.c,d) p-amastigophores, medium, showing V-shaped spine pattern.V-shaped notch at shaft end not developed.e) p-amastigophore, medium; proximal and main shaft distinct (see Fig. 6d).Proximal shaft with dense helices.Main shaft with spaced helices and V-notch.f,g,h.i)p-amastigophores, medium, mesenterial; late differentiation.The shaft shortens within capsule as its helices tighten.j) Discharged p-amastigophores, medium, mesenterial.Shaft end slightly damaged.k) Discharged p-mastigophore, small drop-shaped.p-medium, p-amastigophore medium; t, tubule; prox, proximal shaft; v, V-shaped notch.
As mentioned above (Skaer and Picken, 1965;Picken and Skaer, 1966;Carré, 1972Carré, , 1980;;Carré and Carré, 1973;Skaer, 1973), the tubule became more tightly folded after its arrival in the capsule.Our LM series of p-amastigophores (Fig. 6b-e) showed that the helical pleating process of the shaft continued late in differentiation.The shaft was noticeably shorter in mature nematocysts than early in shaft development, hence the gradually increasing distance between the Vshaped notch and distal capsule end during maturation (Fig. 6c-e).
Similar to Skaer and Picken (1965, pp. 139-141, plates 16-20), the pleats and ridges of the inverted shaft and tubule formed left-handed helices (our Fig. 5b,eg).Further, Skaer and Picken reported that the spines were attached to pockets in the folds of the shaft.The attachment sites of spines and spine-rows might correspond to the pleated ridges in our Figure 5 f,g marked 1, 2, 3.The pitch of the helices increased as the diameter of the tubule diminished, as illustrated by Skaer and Picken (1965, p. 138, Fig. 3).On the narrow distal tubule (our Fig. 5f) the pitch-angles of the elongated helices were hence greater than those on the broad shaft.The pleated, undischarged tubule could expand some three times in length by smoothing the pleats when discharged (Picken and Skaer, 1966).In ruptured capsules (our Fig. 5c), resulting in the absence of capsular pressure, the inverted shafts and tubule coils were extended.
The difference in density and size of the spines explains the difference in the helical pattern on the inverted proximal shaft (= "faltstuck", Schmidt, 1969) and the main shaft (="hauptstuck", Schmidt, 1969), especially in the p-amastigophores (our Figs. 5g,6e,h,7e).Shafts with small, spare spines (Fig. 2c,e) can be more tightly folded than shafts with long, densely-set spines (Fig. 2c,d).In mature nematocysts proximal shafts with dense helices (Fig. 6e,h) are thus more shortened (Fig. 6f-h) than the main shafts with wider-spaced helices (Fig. 6c-e).Schmidt (1969, p. 293, Fig. 4a-c) showed the tiny tubule at the inverted shaft end before the invagination of the V-notch had started.The tiny tubule was only seen by us after the V-notch was shaped (Figs. 6c,e,7f,g).We interpret that the cone-shaped shaft end (Fig. 6a) forms the V-notch as it invaginates into the short, flexed distal part of the main shaft (Fig. 6b, inv).After eversion the V-notch (Fig. 6c-e) is obviously equivalent to the cone at the end of the discharged shaft (Fig. 2b,d,h,j).
The helices and folds around the V-notch (our Fig. 6g, inset) are denser than those on the remaining main shaft (see Schmidt, 1969, p. 295, Fig. 5).SEM pictures of a discharged p-amastigophore shaft of the sea anemone Sagartiogen viduatus (Östman et al., in preparation) showed that spines close to and on the distal cone were shorter than spines on the remaining main shaft.
The capsular U-shaped matrix in our mature pamastigophores (Fig. 3d) was certainly identical to the cup-shaped contents around the shaft in the undischarged amastigophore drawn by Weill (1934, p. 34, Fig. 17) and also to the cup-shaped mass of uniform granular matrix of amastigophores reported by Westfall (1965, p. 390, Fig. 9).Although irregular, the shape of the capsular matrix, from round U-shaped to square U-shaped, and with different length and thickness of the U (Fig. 3d, e, 6e), is a valuable characteristic in distinguishing mature p-amastigophores from M. senile and from S. viduatus (Östman et al., in preparation).
We assume that the capsular matrix is only visible in fully mature p-amastigophores.It is certainly ejected during discharge, since the structure was not visible in discharged and partly discharged capsules (Fig. 2a,b,g,h).Numerous undischarged large p-amastigophores without this regular internal structure were found in proliferation zones of the acontia (Fig. 4a), their shafts being noticeably longer (Figs.4f, 6d) than the shafts in capsules with visible capsular matrix (Fig. 6e).We have regarded these p-amastigophores as being in late development.
We had some doubts that the mesenterial p-amastigophores were immature p-amastigophores, ordinary type, since no characteristic capsular matrix was visible and they seldom discharged.However, due to their short inverted shaft, which only reached half the length of the capsule (Figs.3b,c, 7i), we regarded them as mature and a variety of medium p-amastigophores.Moreover, the shaft was not differentiated into proximal and main regions (Fig. 7f-i), and when discharged the shaft armature seemed homotrichous and mostly the distal tubule was present (Fig. 7j).
The large amastigophore of Metridium senile pictured by Kramer and Francis (2004, p. 137, Fig. 4A) was certainly immature and close to the same age of development as our p-amastigophore (Fig. 6d).No capsular matrix was visible and the shaft was long.Its V-notch was closer to the distal capsule end than the V-notch in the amastigophore of M. farcimen (Kramer and Francis, 2004, p. 137, Fig. 4C), which we regard as mature, since part of the U-shaped capsular matrix was clearly visible.
The narrow axial rod-shaped structure in the inverted proximal shaft of p-amastigophores (Figs. 6d,g,7e) seemed to be some kind of condensed capsular matrix.During eversion it formed the pointed rod at the end of the everting shaft (Figs.2g, lower left, 4a, upper left) (see Östman et al., 2010).In the amastigophores of M. farcimen and M. senile (see Kramer and Francis, 2004, p. 137, Fig. 4A,C) this rod-shaped structure was also visible in the proximal shaft.As in our material (Fig. 6e,h) the rod-shaped structure was most conspicuous in immature nematocysts (Kramer and Francis, Fig. 4A,  C, G).
In early development the b-mastigophore shaft was typically coiled inside the folded, irregularly shaped capsules (Fig. 4b).Many mature b-mastigophores with a coiled shaft remained undischarged (Fig. 1d,e); coiled shafts might prevent discharge.We were not able to distinguish b-mastigophores late in their development from mature ones.

Nematocyst function and size
Large acontial nematocysts may be particularly advantageous when the predation level is high (Kramer and Francis, 2004).We assume that large p-amastigophores are the most potent penetrating nematocysts of M. senile.Certainly their function is purely penetrating, since their coarse shaft is armed with long, densely-set spines, and its tiny distal tubule often remained in the discharged capsule.A long distal tubule might be used to entangle prey (see Östman and Hyman, 1997).
As mentioned (p.486), the capsular fluid of the p-amastigophores does not leave the shaft at once after discharge, and stays longer inside the mesobasic p-amastigophore shafts (Fig. 2a,b) than in the shorter microbasic shafts (Fig. 2c,j).The internal hydrostatic pressure might be lower at the distal end of the mesobasic shaft, as it is spread over a longer distance than in the shorter microbasic shafts.The lower pressure may prolong the time for the fluid to open the cone at end of the discharge shaft (Fig. 2d).The hydrostatic pressure maintains the stiffness of the shaft until the fluid has left, and may increase the penetrating efficiency.
Small, drop-shaped microbasic p-mastigophores may be penetrators.The fluid stays for a long time in their discharged shaft, and the diameter differences between the broad shaft and the thin tubule may slow the internal fluid leaving the shaft.
The large b-mastigophores are the largest and most abundant acontia nematocysts in M. senile (Table 1).They certainly have a penetrating function since their shaft is armed with numerous large spines (Östman et al., 2010).The spines on their long distal tubules, however, are small and slender and not hook-shaped like those of penetrating isorhizas (Östman and Hydman, 1997;Östman, 2000).They are more similar to the spines on long distal tubules used to entangle prey (Östman and Hydman, 1999).Some nematocyst types of M. farcimen are larger than corresponding nematocysts in M. senile (see Carlgren, 1933;Hand, 1956;Kramer and Francis, 2004), possibly because M. farcimen are larger than M. senile.A larger sea anemone might enable larger cells to produce larger cnidae (Francis, 2004).Regression analyses indicated (p.490) that the large acontia nematocysts increased slightly in size with increasing sea anemone size (see also Kramer and Francis, 2004).
Interestingly, according to Carlgren (1933), Hand (1956) (our Table 1) and Francis (2004) the acontia amastigophores (= p-amastigophores), which solely has a penetrating function, are the largest nematocysts in M. farcimen.Large acontial nematocysts may be particularly advantageous if predation level is high (Kramer and Francis, 2004).In the anemone S. viduatus the acontia p-amastigophores are significantly larger than the acontia b-mastigophores and their abundance is higher than in M. senile (Östman et al., in preparation).The large size and abundance of the p-amastigophores in S. viduatus is perhaps related to the high predation by the nudibranchs Aeolidia glauca.
Hopefully, our detailed illustrations of mature and immature nematocysts will enable us to identify nematocysts stored in the cnidosacs of nudibranchs feeding upon M. senile.

Fig. 5 .
Fig. 5. -Intact and broken capsules of acontia nematocysts.a-f) Mesobasic b-mastigophores, large (as Fig. 1), and g) a p-amastigophore, large (as Fig. 2).a,b) Broken capsules showing shaft and tubule (SEM).b) Note helical folding of shaft and tubule.c) 4 broken capsules (1 intact).Their shafts and tubules have elongated without everting.Proximal shaft (p) straight.d) Intact capsule showing proximal and main shaft.e) Apical portion of a broken capsule (SEM).Helical folds of shaft wall appear to change pitch towards proximal shaft.f) Broken capsuleshowing helical folds on shaft and tubule walls (SEM).On the shaft these appear as pleated ridges.Lines show different pitches of shaft and tubule helices.g) Shaft of a p-amastigophore towards capsule apex (SEM).Proximal shaft with tight helices (has sparse spines), main shaft with spaced helices (has dense spines) (see Fig.2c-e).Long arrows show direction of eversion; p, prox, proximal shaft; thin white lines show pitch of helices on shaft and tubule; 1,2,3, correspond to the 3 spines-rows on everted shaft (b,f,g) and tubule (f).

Fig. 6 .
Fig. 6. -Acontia p-amastigophores, large.Inverted shaft differentiated into proximal and main regions.b-e) Illustrate differentiation of the shaft in capsules, interpreted as a time series.a) This empty capsule has ruptured (not seen) releasing the still inverted, elongated shaft.Spines on main shaft particularly clear.Inset) Enlargement of proximal shaft to shows spines (as Fig. 2e).b) Undischarged capsule, immature, with flexed shaft appearing longer than capsule.Proximal shaft strongly flexed, main shaft undulating.Spines on main shaft form clear V-pattern.Distal shaft flexed (marked inv) will later form the V-shaped notch.c,f) Immature capsule; shaft straightening, shorter, regions clear; V-pattern of spines still visible; distinct V-notch at shaft end.d,g) Immature capsule; shaft straight, slightly shorter.Note proximal shaft with dense helices or corrugations and thin rod along centre of proximal shaft (small arrows).Spines no longer visible.Dense helices around V-notch (g inset).e,h) A mature capsule; shaft fully shortened and rod-shaped.Boundary between regions of shaft indistinct; proximal shaft with tight helices or corrugations, narrow structure faintly visible.Main shaft with spaced helices.V-notch distinct.Arrows point at apical capsule end;small arrows point at thin rod; i, internal structure; prox, proximal shaft; t, tubule; v, V-shaped notch; * indicates area of inset.

Table 1 .
-Size in mm and abundance of undischarged nematocysts from acontia and mesenterial filaments of Metridium senile(L.)