J. Mar. Biol. Ass. U.K. (2000), 80, 85^93 Printed in the United Kingdom

Reproductive aspects of females of the pugilator (Crustacea: : ) from southern Spain

M.E. Manjo¨ n-Cabeza and J.E. Garc|¨a Raso Departamento Biolog|¨a , Universidad de Ma¨ laga, Campus de Teatinos s/n, 29071 Ma¨ laga, Spain. E-mail: [email protected], [email protected].

The ovary structure of the hermit crab (Crustacea: Decapoda), is described in the di¡erent phases using electron microscopy. Sexual maturity was reached at a very small size; females from 0.74 mm shield length (SL) could be found in vitellogenesis stage and even 0.6 mm SL ovigerous females exist. The biometric study of the ovigerous females by independent size-class (non-accumulative) shows that 1.3 mm SL is the size at which 50% of females are ovigerous; this value is increased a little (1.7^1.8 mm) if the population of females, or ovigerous females, is analysed from an accumulative point of view. In addition, an optimal reproductive size (between 2.1 and 2.3 mm SL) and a reproductive decrease in the maximum size-classes have been detected. The relation between brood size (measured through egg number) and the hermit crab size follows a potential function. The number of eggs by brood varies in a wide range (9^2838), with a mean diameter of 0.35 0.03 mm. No relationship between egg size and shield length has been found. Æ

INTRODUCTION MATERIALS AND METHODS Electron microscopic studies have recently drawn Sampling attention to the morphology of decapod reproductive Samples were taken using a small, heavy dredge, structures, a subject studied since the 18th and 19th similar to that used for rock-dredging, with a rectan- centuries in some decapods including hermit crabs gular frame of 42 22 cm and double net, the size of the (Carayon, 1941). inside mesh beingÂ4.5 mm knot to knot, to get a good At the beginning of the 20th century, there were some representation of small specimens. The samples were interesting studies on anomuran (Jackson, 1913; taken o¡ Barbate (Ca¨ diz, southern Spain) between 15 Bloch, 1935; Krainska, 1938; Carayon, 1941; Kamalaveni, and 24 m depth. 1947), including works related to reproductive aspects of In the laboratory, the sediment was washed over a Diogenes pugilator (Roux, 1829) (Bloch, 1935). More recent graded sieve column with mesh sizes between 1cm and literature illustrates speci¢c aspects of the composition 1mm. The fauna was separated, and the hermit crabs and structure of the reproductive system, using electron were identi¢ed using the keys provided by Zariquiey microscopy (Aiken & Waddy, 1982; Meusy et al., 1983; (1968) and Ingle (1993). Hinsch & Cone, 1969; Komm & Hinsch, 1985, 1987). Reviews of decapod reproduction written in the past decade include studies of general aspects of reproduction Anatomy and morphology making comparisons among (Charniaux- The specimens used for scanning electron microscopy Cotton, 1980; Johnson, 1980; Adiyodi & Subramonian, (SEM) or transmission electron microscopy (TEM) 1983; Adiyodi & Anikumar, 1988; Meusy & Payen, 1988; were ¢xed for 2^3 h in freshly prepared 1% glutaralde- Kroll et al., 1992). hyde and 1% paraformaldehyde diluted in PBS (phos- In addition, studies of sexual maturation in decapods phate bu¡ered saline). They were then rinsed in the same examine three other aspects: (1) egg counting in bu¡er, post¢xed in 1% OsO4 in the PBS for 2 h at 48C ovigerous females, related to the size of the animal, i.e. and washed in distilled water. Specimens were then dehy- biometrical studies about fecundity patterns (Diaz et al., drated in an ethanol series ¢nishing in 100%. 1983; Grant & Tayler, 1983; Haynes et al., 1976, among The specimens used for conventional histology were others); (2) changes in the pattern of relative growth of ¢xed in methanol-acetone-distilled water (2:2:1) for 8^ di¡erent structures, i.e. chelipeds, abdomen (Teissier, 12 h or in 4% formaldehyde. 1960; Hartnoll, 1974, 1978, 1982); and (3) dynamics of Scanning electron microscopy. External structures structures related to mating in males (sexual tubes, gopo- The traditional protocol has been used. podes and appendix masculina) or with egg incubation in females (setation of the pleopods, etc.). Para¤n-cut sections The aim of this work is to review, update and give new The samples were ¢xed and post¢xed. After washing, information about reproduction in female D. pugilator. the specimens were dehydrated in an ethanol series

Journal of the Marine Biological Association of the United Kingdom (2000) 86 M.E. Manjo¨ n-Cabeza and J.E. Garc|¨a Raso Reproduction in female Diogenes pugilator

Figure 1. (A) Scheme of the internal anatomy of a female Diogenes pugilator in previtellogenesis phase. (B) Previtellogenetic ovary. (B1) Light microscopy photography of a semi-thin section, toluidine stained; *, proliferation zone. (B2) Detail of B1. (B3) Scanning electron microscopy. (B4) Detail of B3, follicular cell. (B5) Detail of B3, oocyte; arrowhead, follicular cells. Scale bars: B2, 10 mm; B1, B4, 50 mm; B3, B5, 100 mm.

¢nishing in butanol; para¤n-embedded and cut in a ¢nishing in acetone, and embedded in araldite 502. Semi- microtome exposing sagittal or frontal sections of the thin sections (1 mm) were cut with a Ultracut E Reichert^ individual. They were dewaxed in xylene and washed Jung ultramicrotome and stained with toluidine blue. several times in 100% ethanol. Ultra-thin sections were obtained by the same method. Samples (external structures and para¤n-cut sections) These sections were contrasted with lead citrate and were critical point dried using ethanol as the transitional uranyl acetate, observed and photographed with a JEOL £uid and CO2 as the exchange £uid. The dried specimens JEM-100 CX transmission electron microscope. were sputter coated with gold to a layer of about 450 Ð. Observations were made using a JEOL JSM-840 scanning electron microscope. Conventional histology After ¢xation, the individuals were dehydrated in an Transmission electron microscopy ethanol series ¢nishing in butanol, and para¤n- The samples were ¢xed and post¢xed. After washing, embedded. Serial sections were cut with a microtome and the specimens were dehydrated in an ethanol series collected on poly-l-lysine coated slides.

Journal of the Marine Biological Association of the United Kingdom (2000) Reproduction in female Diogenes pugilator M.E. Manjo¨ n-Cabeza and J.E. Garc|¨a Raso 87

Figure 2. (A) Scheme of the internal anatomy of a female abdomen in vitellogenesis phase of Diogenes pugilator. (B) Vitello- genetic ovary. (B1) Light microscopy photography of a para¤n section, Mallory's stained. (B2) Detail of B1, oocyte. (B3) Detail of B1, proliferation line. (B4) Scanning electron photography of early vitellogenesis phase. (B5) Detail of B4, oocyte. (B6) Scanning electron microscopy of later vitellogenesis phase. (B7) Detail of B6, vesicles. Scale bars: B1, 10 mm; B7, 25 mm; B4, B5, B6, 100 mm; B3, 200 mm; B2, 600 mm.

Journal of the Marine Biological Association of the United Kingdom (2000) 88 M.E. Manjo¨ n-Cabeza and J.E. Garc|¨a Raso Reproduction in female Diogenes pugilator

Figure 3. Female gonopore area. (A1) Scanning electron microscopy of female of 0.55 mm SL. (A2) General aspect of the ventral zone (female of 2 mm SL). (A3) Detail of A2. (A4) Detail of A3, pores on the antero^inner margin. (A5) Detail of A4, pore. (A6) Light microscopy photography of a semi-thin transverse section of the third periopodal coxa, toluidine stained. (A7) The operculum retractor muscle (m), toluidine stained. (A8) Detail of A6, the cuticle (c) breakage (b) in connection with the acini coxa gland (a), toluidine stained. (A9) Detail of A6, empty rosette-like. (A10) Detail of A6, full rosette-like. o, operculum; r, real ori¢ce; v, atrium; g, glandular tissue. Scale bars: A5, 1 mm; A1, 10 mm; A4, A8, 25 mm; A9, A10, 50 mm; A7, 150 mm; A3, 250 mm; A6, 600 mm; A2, 1 mm.

Journal of the Marine Biological Association of the United Kingdom (2000) Reproduction in female Diogenes pugilator M.E. Manjo¨ n-Cabeza and J.E. Garc|¨a Raso 89

Figure 4. Percentage of ovigerous females (OF(%)) during the periods studied.

Figure 5. Percentage of ovigerous females (over total Figure 6. Fecundity curves: SL(mm), shield length by number of females) by independent or non-accumulative size-classes; OF(%), percentage of ovigerous females. size-classes. SL(mm), shield length by size-classes; OF(%), Ovigerous females accumulated percentage by size-classes: ovigerous females percentage. (A) over total number of females; (B) over total ovigerous females. The sections were dewaxed in xylene, hydrated in an ethanol series, washed in distilled water, and Mallory, Feulgen and/or haematoxylin-eosin stained.

Biometric studies

The anatomical structures were analysed with a VID V eggs computer programme that processes stereoscopic micro- of scope images taken by a video camera, with a measure- No. ment error of 0.001mm. Four dimensions were measured: cephalothoracic shield length (SL, maximum length, from rostrum to posterior part of shield). The cephalo- thoracic shield length was considered as a reference measurement of the total length, because it is the hard Figure 7. Fertility curve: SL(mm), shield length by structure most frequently used to de¢ne size in hermit size-classes; no. of eggs, number of eggs produced by each crabs (McLaughlin, 1974; Blackstone, 1986a,b; Lancaster, ovigerous female. 1990; Ingle, 1993; Fotheringham, 1976; Bertness, 1981; 2A). When the females are inactive, the ovaries are etc.) cavities surrounded by simple, thin and almost trans- In order to determine the breeding period, the parent epithelium which is very di¤cult to identify ovigerous females were counted every month as well as among the hepatopancreatic acini. It is very di¤cult to the eggs they carried. To estimate the quantity of eggs observe di¡erences between an inactive ovary (mature) and their mean diameter, an Image Analysis method and juvenile ovary (immature) because the arrangement based on a SUN computer system was used. of both phases is very similar, displaying the same histo- logic structure of the somatic and germinate cells. RESULTS In later periods, when the ovaries are active, two The ovaries of adult females of Diogenes pugilator are di¡erent phases can be distinguished, previtellogenesis located in the dorsal side of the abdomen (Figures 1A & and vitellogenesis. During these phases the ovary shows

Journal of the Marine Biological Association of the United Kingdom (2000) 90 M.E. Manjo¨ n-Cabeza and J.E. Garc|¨a Raso Reproduction in female Diogenes pugilator

Figure 8. (A & B) Pleopod, light microscopy photography of a semi-thin sagittal section: (A) glandular structures; (B) detail of the glandular duct; (C) scanning electron microscopy of the female ventral zone around gonopore ( , female gonopore; s, setae; ., spermatophore peduncle; *, spermatophore ampulla). Scale bars: 100 mm. two lobes which are joined and arranged so as to occupy of both lobes (proliferation line). In this line small most of the abdominal volume at the expense of the hepa- oogonias appear with heterochromatic nuclei (Figure 1B1). topancreas (Figure 2A). In previtellogenesis, the ovary When the cell matures, the nucleus becomes large and presents a gradient of maturation from the medium line the nucleolus is visible. The cytoplasm is very small and

Journal of the Marine Biological Association of the United Kingdom (2000) Reproduction in female Diogenes pugilator M.E. Manjo¨ n-Cabeza and J.E. Garc|¨a Raso 91 some vesicles begin to appear. It is also possible to observe which corresponds to a range of SL between 2.1 and the follicular cells placed around the previtellogenesis 2.3 mm and a decrease in the maximum size-classes oocytes (Figure 1B2^5). In vitellogenesis phase, (Figure 2), (Figure 5). However, the size at which 50% of females the follicular cells cannot be seen (Figure 2B2). The are ovigerous is a little increased (1.7^1.8 mm of SL) if the vesicles, which are stained strong orange by Mallory's population (total females, Figure 6A; or ovigerous trichrome, become larger and the nuclei have a more females only, Figure 6B) is analysed from an accumula- euchromatic aspect. There are two kinds of vesicles: small tive point of view. and large. Brood size (egg number) was measured in females Dissection results have shown that the oviducts are which apparently did not lose any eggs. Its relation with simple ducts enclosed by a thin transparent epithelium. the body size follows a potential function (Figure 7). The Their route through the abdomen (from the ovary to the number of eggs by brood varies in a wide range, from 9 to gonopores, located in the coxa of the third periopods) 2838, with a mean diameter of 0.35 0.03 mm. seems to be asymmetric, the left oviduct folds over the The setae of females are very larÆge and feathery just right one in their route through the `waist' (thorax^ before mating (precoupling period) when the ovary can abdomen transition); however their morphology is be found in a late vitellogenesis phase. During mating, identical. the spermatophores are attached on the females sternum The cuboidal epithelium that wraps the oviducts is and coxae (near gonopores). The spermatophores open covered by a connective layer. These oviducts seem to along the broken line, allowing the spermatozoa to exit as have the same anatomy and morphology along all of their the ovules liberated from the female gonopores and tract. attached to the setae of pleopods (Figure 8C). On these When the oviduct reaches the coxal area, it widens to pleopods (Figure 8A,B) and on the coxae (Figure conform the atrium, then becomes narrow again in the 3A6,8,9,10) we have observed a glandular system, which proximity of the gonopore. The walls of this atrium have produces secretions that could be related to the attach- cuticular origin, because they show sclerotization ment of the eggs. (Figure 3A6). In this last portion of the oviduct it is possible to observe a complete glandular system, where we have found `full glands' (¢lled by a secretory substance) and `empty glands' (the stage after secretion); DISCUSSION also, it is possible to observe cuticle breakages which Previtellogenesis in Diogenes pugilator shows the same seems to correspond to the aperture of the glands characteristics as in other species of decapods. The route (Figure 3A6,8,9&10). of the proliferation line (or germinative zone) into the The muscle involved in the aperture of the gonopore ovary depends on the hermit crab species which used to has been found in a transverse position to the coxa be in a ventral position (Jackson, 1913; Bloch, 1935; (Figure 3A7). It is a large muscle which is divided in two Carayon, 1941; Kamalaveni, 1947). However, our results near the cuticle. From outside, the gonopore appears as a in vitellogenesis phase (Figure 2B1) have showed us that little depression on the surface of the cuticle, which has a this zone is more dorsal. circular or ellipsoidal contour. There is also a small bulge The previtellogenic oocytes show a of nuclear nearby completely covered by setae (Figure 3A1,2,3). The condensation, which could correspond to the synapto- bottom of this depression is occupied by the chitin walls demic complex described by Komm & Hinsch (1985) in of the obturator device, which is movable. The disk is less Coenobita clypeatus (Herbst, 1791). The follicular cells do not chitinous than the coxa wall, which contains more chitin seem to be elements of the germinative tissue, because layers. The operculum seems to open as a hinge, as it is they appear to have a somatic function, constituting part joined to the cuticle of the coxa only by one side. A study of the epithelium of the wall of the follicle as Meusy et al. of the presence of this obturator device has been made in (1983) described for other species. These cells are a females ranging from 0.55 mm (specimen of Figure 3A1) arranged at the periphery of the oocytes and possibly to 2.90 mm of SL. The obturator device is present in all exchange di¡erent substances that form part of the females studied, in both sides, and it is independent of the nutrient stock of the egg (proteins, vitellogenin and reproductive stage. lipids). Some authors suggest that the oocytes are autono- mous regarding the synthesis of these substances (Telfer, 1954, in Komm & Hinch, 1987, studies developed in Brooding time and fecundity insects), while others talk about a extra-oocytic origin Females of this species brood during the whole year, (Hinsch & Cone, 1969). although with oscillations. There is a maximum of abun- In this D. pugilator, vitellogenesis is macroscopically dance of ovigerous females during the summer (Figure 4). characterized by a change in the colour of the ovary to The fecundity studies show that sexual maturity is orange. This change has been mentioned in other species, reached at very small sizes. The smaller ovigerous females and it seems to be caused by a carotene accumulation found had a 0.6 mm SL and the histologic results showed (Charniaux-Cotton, 1980). On the other hand the micro- females in the vitellogenesis phase with a size of 0.74 mm scopic aspects of this stage in this species has the same SL. characteristics described for other species (Adiyodi & The biometrical analysis of the ovigerous females by Subramonian, 1983; Komm & Hinsch, 1985). size-class (non-accumulative) (Figure 5) shows that The results obtained by dissection have shown the 1.3 mm is the size at which 50% of females are ovigerous. asymmetric site of the oviducts. This asymmetry has been These results also show an optimal reproductive size discussed by some authors (Carayon, 1941) who suggest

Journal of the Marine Biological Association of the United Kingdom (2000) 92 M.E. Manjo¨ n-Cabeza and J.E. Garc|¨a Raso Reproduction in female Diogenes pugilator the idea that this asymmetry is related to the general able sizes such as bernhardus (Linnaeus, 1758) torsion of the body, as hermit crabs adapted their body (ovigerous females around 1.7 mm; Lancaster, 1990). shape to the internal structure of the gastropod shell There are many studies on fecundity and puberty size where they live. in females of di¡erent species (D|¨az et al., 1983; Grant & When the oviduct is near the gonopore it broadens, as Tayler, 1983; Haynes et al., 1976; Rodr|¨guez, 1977, some authors have described for the atrium of other Gonza¨ lez-Gurriara¨ n, 1985; Demestre & Fortuno, 1992) species (Carayon, 1941 in Clibanarius erythropus (as where the number of ovigerous females in relation to the C. misanthropus)). This is associated with a complex individual size is analysed. In these studies, the accumu- glandular system, which presents a structure composed of lated percentages of ovigerous females is used to assess acini, very similar to the rosette-shaped cement glands fecundity, assuming that the presence of 50% of described by Aiken & Waddy (1982) in the female pleo- ovigerous females in the ¢rst size-class means that the pods of the Homarus. Carayon (1941) described the population puberty size is possible at this size. We have same glandular system in Clibanarius erythropus. These studied size at puberty in relation to the total number of `rosette-shaped glands' are connected to small ducts that females or ovigerous females for each size-class, and have open to the external part of the cuticle as Carayon (1941) found slight di¡erences in relation to the method used. In described for Clibanarius erythropus. addition, the data showed, in the natural habitat The function of this glandular tissue appears to be (Manjo¨ n-Cabeza & Garc|¨a Raso, 1998a,b), that there related to mucilage secretions, that cover the egg. was an optimal reproductive size and a reproductive Whether this secretion represents an actual egg covering decrease in the maximum size-classes. or a means by which the eggs are attached to the pleo- The relation between egg number and the female size pods is uncertain. Some authors (Aiken & Waddy, 1982; (SL) shows a potential trend, which is in accordance with Lowe, 1961; Stephens, 1952; Andrews, 1906) have indi- the results found in some species of Brachyura (Haynes cated that the glandular tissue responsible for these secre- et al., 1976) and hermit crabs (i.e. tions is always found in the pleopods. However Carayon (Linnaeus, 1767) Manjo¨ n-Cabeza & Garc|¨a Raso, in (1941) found secretions of mucus throughout the entire press) and in disagreement with the data obtained from terminal portion of the oviduct of Clibanarius erythropus. other small hermit crab species such as Cestopagurus Mouchet (1931) developed several chemical experi- timidus (Roux, 1830) and Calcinus tubularis (Linnaeus, ments that re£ect the need of acid substances in the 1767) (Manjo¨ n-Cabeza & Garc|¨a Raso, 1994, 1996), medium in order to open the spermatophores by the which have shown linear trends. broken line. These arguments make us think that these Egg size seems to depend more on the size of the substances could also be in relation with this other func- species, than on the number of eggs or the species' life tion. We have found opened spermatophores on the strategy. Therefore, Diogenes pugilator and the other small female sternum, but we can not determine if it is conse- species (such as those belonging to the genus Anapagurus: quence of chemical ¢xation or post-¢xation process A. hyndmanni (Bell, 1845) and A. alboranensis Garc|¨a- (needed in SEM protocol). Go¨ mez, 1994; Manjo¨ n-Cabeza & Garc|¨a Raso, in press) Another structure described earlier (Carayon, 1941) for have a smaller egg size than other larger species such as other species is the gonopore operculum, which is moved Paguristes eremita or . by a system of muscles. The gonopore has been found open in all the size classes studied. However, this is not easy to examine using stereomicroscopy in some specimens. This research is included within the Project PB92-0415. In relation to the gonopore-opening muscle system, we `Comunidades bento¨ nicas del Litoral de Ca¨ diz. Estructura y have observed as Carayon (1941) did two bundles, which dina¨ mica de las taxocenosis de crusta¨ ceos deca¨ podos, moluscos y are inserted in the enlarged zone of the opercular cuticle. algas. Biolog|¨a de las especies dominantes', supported by Spanish These observations contradict Mouchet (1931) who M.E.C., DGICYT funds. We wish to thank J.L. Schib MSc, D. Macias, PhD and the SEM andTEM technicians G. Mart|¨ nez suggested that the aperture of the operculum was due to and J.J. Canca for assistance in sectioning and the photographic contractions of the smooth muscles of the oviduct. The material and M. Vega for her help in the image analysis data. function of this operculum would appear to serve as a safeguard to protect the oviduct. REFERENCES

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Journal of the Marine Biological Association of the United Kingdom (2000)