2001 identification of respiratory and ion transporting epithelia in the phyllosoma larvae of the slipper lobster scyllarus arctus

A thick 5 µm mitochondria-rich epithelium covers the ventral side of the cephalic shield; its cells are characterized by the presence of well-devel-oped apical infoldings adjacent to the

Abstract Phyllosoma larvae of the Palinura lack a

bran-chial cavity and gills In the phyllosoma, gas and ion

ex-changes that occur at the level of the gill in the adult

must occur in other parts of the body or through the

en-tire body The objective of this study was to localize

epi-thelia bordering the body of the phyllosoma larvae that

had features comparable to those of the gill epithelia of

adult decapods The first phyllosoma instar of the small

Mediterranean slipper lobster Scyllarus arctus was

studied First, we used a silver nitrate staining method

to identify parts of the body with high ionic

perme-ability Confocal laser scanning microscopy with a

fluorescent vital stain for mitochondria,

dimethyl-aminostyrylmethylpyridiniumiodine (DASPMI), was

then used to localize cells with a high density of

mito-chondria Next, an ultrastructural study of selected

epi-thelia was carried out A thick (5 µm) mitochondria-rich

epithelium covers the ventral side of the cephalic shield;

its cells are characterized by the presence of

well-devel-oped apical infoldings adjacent to the cuticle This part

of the body has a high ionic permeability as indicated by

a positive silver nitrate staining The ventral

mitochon-dria-rich epithelium might be involved in active ion

transport The rest of the body, particularly the dorsal

side of the shield and the appendages, shows a lower

ionic permeability (no positive silver nitrate staining)

and is limited by a thin (1 µm) epithelium with low

num-bers of mitochondria This epithelium exhibits features

of a typical respiratory epithelium

Keywords · Phyllosoma · Ultrastructure · Confocal

laser scanning microscopy · Ion-transporting epithelium ·

Lobster, Scyllarus arctus (Crustacea)

Introduction Phyllosoma larvae are characteristic of the larval devel-opment of the Palinura, a group of decapods consisting mainly of palinurid and scyllarid lobsters The morphol-ogy of the phyllosoma differs greatly from that of the adult In particular, the larvae are devoid of a branchial cavity and of gills throughout their planktonic larval life, which generally is very long, from 3 months to more than 1 year as in some spiny lobsters (Phillips and Sastry 1980) Gill buds appear only in the last phyllosomal

in-star, as described for the scyllarid lobsters Scyllarus americanus (Robertson 1968), Ibacus peroni (Marinovic

et al 1994) and Thenus orientalis (Mikami and Green-wood 1997), and for the palinurid lobster Jasus verre-auxi (Kittaka et al 1997).

In adult decapod crustaceans, gills are organs special-ized for gas and ion exchange between hemolymph and the external medium, as well as for waste excretion (Lockwood et al 1982; Péqueux 1995) According to their functional differentiation, two main types of ex-change epithelia are found in gills, a thin one mainly in-volved in gas exchange, and a thick one, made up of ionocytes that are involved in osmoregulation (Mantel and Farmer 1983; Gilles and Péqueux 1985; Taylor and Taylor 1992; Péqueux 1995; Haond et al 1998)

The first postembryonic developmental stages of de-capod crustaceans generally lack gills or carry only bran-chial buds (Felder et al 1986, Hong 1988; Bouaricha et

al 1994) In the crab Portunus trituberculatus, the buds

of gills, present in the three zoeae, develop into

function-al gills after metamorphosis (Nakamura 1990; Nakamura and Sakagushi 1991) However, decapod larvae (except for the nauplii of penaeid shrimps) develop a branchial cavity, limited on each side of the body by the thoracic pleurites and by lamellar expansions of the carapace, the

This work was supported by the Research School of

Environmen-tal Chemistry and Toxicology, Wageningen University, The

Nether-lands

C Haond · G Flik · S.E.W Bonga (✉)

Department of Animal Physiology, University of Nijmegen,

Toernooiveld, 6525 ED Nijmegen, The Netherlands

e-mail: wendelaar@sci.kun.nl

G Charmantier

Laboratoire d'Ecophysiologie des Invertébrés,

Université Montpellier II, place Eugène Bataillon,

34095 Montpellier Cedex 5, France

Cell Tissue Res (2001) 445–455

DOI 10.1007/s004410100405

R E G U L A R A R T I C L E

C Haond · G Charmantier · G Flik

S E Wendelaar Bonga

Identification of respiratory and ion-transporting epithelia

in the phyllosoma larvae of the slipper lobster Scyllarus arctus

Received: 20 October 2000 / Accepted: 2 April 2001 / Published online: 1 June 2001

© Springer-Verlag 2001

branchiostegites There is consensus that in larvae the

whole body or specialized areas of the carapace such as

the inner side of the branchiostegite represent efficient

exchange surfaces, replacing gills (Bouvier 1890;

Wolvekamp and Watermann 1960; Vuillemin 1967)

Ex-trabranchial osmoregulatory epithelia cover the pleurites

and the inner side of the branchiostegite in developing

larvae of the penaeid shrimps Penaeus aztecus (Talbot et

al 1972) and P japonicus (Bouaricha et al 1994), and of

the thalassinid shrimp Callianassa jamaicense (Felder et

al 1986) In small crustaceans devoid of gills,

special-ized ion-transporting epithelia have been localspecial-ized on

different parts of the body (Conte et al 1972; Lake et

al 1974; Hosfeld and Schminke 1997; Kikuchi and

Matsumasa 1997) No data are available on the

localiza-tion of specialized respiratory epithelia in decapod larvae

or in small crustaceans devoid of gills and/or a branchial

cavity

Phyllosoma larvae, with their particular morphology,

absence of gills and of a branchial cavity, provide a

unique model in decapod crustaceans It seems

reason-able to assume that at least some of the boundary

epithe-lia of the phyllosoma larvae should present

ultrastructur-al characteristics of an exchange epithelium for water

and ions The aim of the present study was to structurally

characterize the epithelia bordering the body of the first

phyllosomal instar of the small Mediterranean slipper

lobster Scyllarus arctus First, investigations were

con-ducted using two fast exploratory techniques, a silver

ni-trate staining and confocal laser scanning microscopy of

tissue vitally stained for mitochondria The silver nitrate

staining was used to localize areas of the body of

the phyllosoma with a high ionic permeability (Kikuchi

and Matsumasa 1997; Kikuchi and Shiraishi 1997;

Tsubokura et al 1998) Vital fluorescent dye for

mito-chondria has been used to localize mitomito-chondria-rich

cells in fish (Wendelaar Bonga et al 1990; Li et al 1995;

Van Der Heijden et al 1997) and has recently been

ap-plied by us for the localization of osmoregulatory

epithe-lia in crustaceans (Haond et al 1998) Subsequently,

his-tological and ultrastructural studies were conducted on

the specific areas of the body identified by the silver

ni-trate staining method and by confocal laser scanning

mi-croscopy

Materials and methods

Animals

Adult lobsters Scyllarus arctus were collected in the

Medi-terranean sea near Sète, France, and held at the Station

Méditerranéenne de l'Environnement Littoral, Sète, France, in a

fi-berglass tank supplied with running seawater at a salinity close to

35–36‰ Adult females carrying eggs were then transferred to the

Nijmegen laboratory, The Netherlands, and kept in 100-l glass

aquaria filled with artificial seawater (Instant Ocean) aerated and

recirculated through Eheim pumps and filters Salinity was

main-tained at 35‰ Temperature varied according to season and was

around 20°C when first hatching occurred in spring Adult lobsters

were fed with cooked mussels The first phyllosomal instar larvae

were collected in the aquaria after hatching and transferred in 1-l glass beakers filled with the same artificial aerated seawater Wa-ter was changed twice daily Larvae were fed ad libitum with

new-ly hatched nauplii of Artemia sp and kept for a maximum of

3 days At 20°C the first molt does not occur before 1 week (C Haond, unpublished); observations were conducted in larvae esti-mated to be in intermolt stage C (Drach 1939; Drach and Tchernigovtzeff 1967).

Silver nitrate staining Silver nitrate staining was performed according to the slightly modified procedure of Kikuchi and Matsumasa (1993a) Live lar-vae were briefly washed in demineralized water and then placed in

a 0.5% silver nitrate/0.2 M nitric acid solution for less than 10 s or more than 30 s to 1 min After removal of the excess silver nitrate

by rinsing with demineralized water for 1 min, larvae were soaked

in 0.2 M nitric acid for 5 min, rinsed again with demineralized wa-ter, and placed in a photographic developer (Ilford) for 5 min Af-ter rinsing with demineralized waAf-ter, they were soaked in a photo-graphic fixative (Ilford) for 2 min, and finally kept in a 4% form-aldehyde solution until observation and photography using a bin-ocular microscope.

Confocal laser scanning microscopy Live larvae were incubated in seawater containing 0.5 mmol/l di-methylaminostyrylmethylpyridiniumiodine (DASPMI, Molecular Probes; excitation 472 nm, emission 609 nm), a vital fluorescent dye for mitochondria (Bereiter-Hahn 1976) Samples were then washed for 30 min in seawater and mounted in a silicone grease ring prepared on a microscope slide and filled with seawater Lar-vae were observed with a confocal laser scanning microscope (MRC-600 Bio-Rad) equipped with an argon ion laser and a Ni-kon Optiphot microscope We used the 488-nm line of the argon ion laser as the excitation wavelength and a 515-nm emission bar-rier filter set (BHS).

Light microscopy and transmission electron microscopy Larvae were fixed in a 2.5% glutaraldehyde solution in 0.1 M ca-codylate buffer, and postfixed in 1% (w/v) OsO4in the same

buff-er The osmolality of the fixatives was adjusted with NaCl to the osmolality of the artificial seawater (1030 mosmol/kg) measured

on a Roebling osmometer After washing in demineralized water and staining for 1 h in 2% (w/v) aqueous uranyl acetate, larvae were dehydrated in ethanol baths and embedded in Spurr's resin using propylene oxide Semithin sections for histological observa-tions were made with glass knives and stained with toluidine blue Ultrathin sections for transmission electron microscopic (TEM) observations were cut using a diamond knife (Diatom), contrasted with lead citrate, and examined with a JEOL 100 CXII transmis-sion electron microscope.

Results General morphology of the first phyllosoma

The general morphology of the first phyllosoma instar of

S arctus is illustrated in Fig 1A The body of the larvae,

which is strongly dorsoventrally flattened, is composed

of two main parts, the cephalic and thoracic parts The cephalic part bears the well-developed eyestalks and the mouth The different lobes of the digestive gland can be observed through the transparent cuticle, which forms a 446

cephalic shield (Fig 1A) The thorax of the larvae bears

the different appendages, the maxillipeds and the three

pairs of pereiopods developed at this stage The abdomen

is also present at the posterior part of the body, but,

com-paratively, it is very small (Fig 1A)

Silver nitrate staining

After a short exposure time (less than 10 s) of the larvae

in the silver nitrate solution, a black staining, designated

positive silver staining, was seen on the ventral side of

the cephalic shield (Fig 1B) The thorax and appendages

were negative In some cases, apparent positive areas

were observed at different locations of the appendages, but these could be attributed to damage caused by the forceps handling of the larvae Dissection of the stained larvae confirmed that only the ventral side of the

cephal-ic shield showed positive silver staining (Fig 1C) Some

nauplii of Artemia sp., used as food, were accidentally

stained simultaneously with the phyllosoma These na-uplii showed a blackening of the neck organ (i.e., the salt gland) as previously described by Conte et al (1972), which confirmed the validity of the staining procedure Longer exposure time of the phyllosoma in the silver ni-trate solution (30 s to 1 min) led to a blackening of the entire surface of the larvae (result not shown) Similar

exposure of the nauplii of Artemia sp did not change the

specific staining pattern of the neck organ

Confocal laser scanning microscopy Larvae stained with DASPMI and observed at low mag-nification exhibit a very intense labeling of the antennal glands and an intense labeling of the midgut gland (Fig 2A) A similarly intense labeling was not observed

in the body surfaces at low magnification, although a faint labeling was seen on the ventral side of the cephalic

447

Fig 1A–E Light microscopy micrographs from the first

phylloso-ma larvae of Scyllarus arctus A Morphology of the phyllosophylloso-ma.

B, C Silver nitrate staining Whole-mounted larvae in B and

dis-sected piece of the cephalic part in C Note the blackening of the

ventral side of the cephalic shield D Semithin transverse section

through the cephalic shield Note the thick epithelium of the

ven-tral side and the thin one of the dorsal side E Semithin transverse

section through an appendage Note the thin lining epithelium

(ar-rowhead) (ab abdomen, ap appendage, cs cephalic shield, ds

dor-sal side, e eyestalk, hp hepatopancreas, m muscle, th thorax, vs

ventral side) Scale bars 500 µm (A–C), 20 µm (D, E)

448

shield (Fig 2A) At high magnification, individual

mito-chondria were observed on this part of the surface of the

body (Fig 2B–D) Indeed, the cells of this epithelium

show a high density of mitochondria around the nuclei

(Fig 2C, D) These mitochondria are mostly elongated

and curved, without any specific orientation within the

cytoplasm (Fig 2C), and make bulges at some locations

(Fig 2D) In contrast, the epithelium of the dorsal side of

the cephalic shield, observed at similar magnifications,

displays a low density of mitochondria (Fig 2E–G)

They are generally very elongated and located around

the nuclei (Fig 2E, F) or organized as a network

(Fig 2G) Their orientation is mainly parallel to the

sur-face of the epithelium, which points to a low thickness of

the epithelial layer Confocal laser scanning microscopic

(CLSM) analyses of the rest of the body surfaces of the

larvae revealed that the other limiting epithelia resemble

the dorsal side of the cephalic shield with respect to

mi-tochondrial density and arrangement; this is illustrated in

Fig 2H with a view of the pereiopod epithelium.

Ultrastructure of the boundary epithelia

Following the preceding observations, we focused our

ultrastructural investigations on: (1) the

mitochondria-rich and silver-positive epithelium covering the ventral

side of the cephalic shield, and (2) the epithelia covering

the dorsal side of the cephalic shield and the appendages

Mitochondria-rich epithelium

The epithelium covering the ventral side of the cephalic

shield is generally approximately 5 µm thick (Figs 1D,

3A) It is thinner (2–3 µm) or thicker (up to 7 µm) at

some locations It is lined externally by a thin cuticle

ap-proximately 500 nm thick and internally by a very thin

basal lamina approximately 20 nm thick (Fig 3B) The

nuclei of the cells, taking up more than half the height of

the epithelial layer, are slightly flattened (Fig 3A–C)

TEM confirms the presence of numerous mitochondria in

the cytoplasm (as observed by DASPMI staining) with a

very high density in some parts of the cytoplasm (Fig 3C); the cytoplasm further contains an abundant rough endoplasmic reticulum mostly located at the basal side of the cells (Fig 3B, C), numerous free ribosomes and microtubules observed in transverse (Fig 3B) or lon-gitudinal sections (Fig 3D), multivesicular bodies (Fig 3C, D) and lamellar bodies (Fig 3E) The cells also display a Golgi apparatus usually located between the mi-tochondria and at sites where electron-dense coated vesi-cles are frequently observed (Fig 3F) The plasma mem-brane of the basal side (Fig 3B, C) and of the lateral side (Fig 3E) of the epithelial cells is not folded and adjacent cells exhibit scarce or no interdigitations (Fig 3E) The apical plasma membranes of the cells display well-devel-oped infoldings approximately 500 nm–1 µm high (Fig 3A–C, E, G) Some of the apical infoldings are lon-ger, up to 2 µm, oriented in parallel to the cuticle (Fig 3B) or forming circular structures when observed in transverse sections (Fig 3G, H, J) At some locations, the apical infoldings are closely associated with mitochondria (Fig 3C), which can be surrounded by several layers of membrane infoldings (Fig 3G, H) The cytoplasmic side

of the apical infoldings is coated by 10-nm particles (Fig 3K) The cytoplasm near the apical microvilli con-tains small vesicles (Fig 3C–E) Some of them display 10-nm particles at their cytosolic side (Fig 3I)

Mitochondria-poor epithelium

The epithelial layer covering the dorsal side of the ce-phalic shield is thin and varies in thickness from 0.5 to

2 µm (Figs 1D, 4A, B) The nuclei appear generally flat-tened against the cuticle (Fig 4A) TEM revealed that the epithelial cells contain few mitochondria (Fig 4B), confirming CLSM observations The cytoplasm contains

an abundance of rough endoplasmic reticulum, numer-ous free ribosomes and microtubules (Fig 4B) The

bas-al plasma membrane does not present infoldings and is bordered by a thin basal lamina (Fig 4B) The apical plasma membrane shows regular indentations, approxi-mately 20 nm high (Fig 4B)

The epithelium limiting the appendages is also thin, approximately 1 µm thick (Figs 1E, 4C, D) Its ultra-structure is very similar to that of the thin epithelium of the dorsal cephalic shield described above Nuclei of the epithelial cells are very flat and the cytoplasm is granu-lar Scarce mitochondria are observed within the cyto-plasm The basal plasma membrane is not infolded and is bordered by a thin basal lamina The apical plasma mem-brane forms regular small indentations The cuticle is also very thin, approximately 1 µm thick

The ultrastructure of these epithelia differs at some locations, notably where muscles are attached to the cuti-cle (Fig 4E) At these places the epithelium is generally about 4 times thicker than the thin epithelia described above and it can exhibit a well-developed rough endo-plasmic reticulum and some apical indentations but no apical infoldings or an abundance of mitochondria

449

Fig 2A–H Confocal laser scanning micrographs of the first

phyllosoma larvae of Scyllarus arctus after in vivo labeling of the

mitochondria with DASPMI A Cephalic part of the larvae at low

magnification Note the intense staining of the hepatopancreatic

tubules (hp) and of the antennal glands (ag) The ventral side

of the cephalic shield appears slightly stained (arrowhead).

B–H Tangential optical sections through the epithelia covering the

ventral side of the cephalic shield (in B–D), the dorsal side of the

cephalic shield (in E–G), and the appendages (in H) Individually

labeled mitochondria are visible on the micrographs; the round- or

oval-shaped unstained areas observed between the mitochondria

are the nuclei The epithelium of the ventral side of the cephalic

shield hosts numerous mitochondria (in B–D); the intensely

la-beled cells in B are digestive cells from the hepatopancreas The

epithelia of the dorsal side of the cephalic shield (in E–G) and of

the appendages (in H) are mitochondria-poor Scale bars 100 µm

(A), 20 µm (B, E), 10 µm (C, F–H), 5 µm (D)

450

Discussion Silver nitrate staining and CLSM with a fluorescent vital stain for mitochondria were combined with light micros-copy and TEM to study boundary epithelia in

phylloso-451

Fig 3A–K TEM micrographs of the thick mitochondria-rich

epi-thelium lining the ventral side of the cephalic shield from the first

phyllosoma of Scyllarus arctus A–C General views of the

epithe-lium Note the well-developed and electron-dense apical

infold-ings, the abundant rough endoplasmic reticulum at the basal side

of the epithelial cells and the numerous mitochondria D–K

Ultra-structural details of the mitochondria-rich cells Structures

com-monly observed are multivesicular bodies (in D), lamellar bodies

(in E), Golgi apparatus with electron-dense coated vesicles

(ar-rowheads in F), apical infoldings closely associated with

mito-chondria (in G, H), and coated vesicles near the apical infoldings

(arrowhead in I) J–K Details of the apical infoldings The

elec-tron-dense apical infoldings (in J) show 10-nm-sized particles

at-tached to the cytosolic side of the plasma membranes (arrowheads

in K) (ai apical infoldings, bl basal lamina, c cuticle, cb cell

bor-der, g Golgi apparatus, l lacunae, lb lamellar body, m

mitochon-dria, mb multivesicular body, mt microtubule, n nucleus, r

ribo-some, rer rough endoplasmic reticulum, v vesicle) Scale bars

2 µm (A), 500 nm (B–H, J), 100 nm (I, K), 500 nm (J)

Fig 4A–E TEM micrographs of the epithelia lining the dorsal side

of the cephalic shield and the appendages from the first phyllosoma

of Scyllarus arctus A, B Epithelium of the dorsal side of the ce-phalic shield The epithelium is very thin (arrowhead in A) and

mi-tochondria-poor Note the structure of the apical plasma membrane

with small indentations (arrowheads in B) C–E Epithelium of the

appendages The epithelium is also very thin and

mitochondria-poor The apical plasma membrane displays small indentations

(ar-rowheads in C, D) The epithelium becomes thicker near muscle

attachments (in E) (bl basal lamina, c cuticle, cb cell border, dc

di-gestive cell, l lacunae, m mitochondria, n nucleus) Scale bars 5 µm

(A), 500 nm (B), 1 µm (C), 500 nm (D), 2 µm (E)

ma larvae of the slipper lobster S arctus, which are

de-void of gills and a branchial cavity All analyses infer the

existence of two kinds of boundary epithelia The ventral

side of the cephalic shield, which was stained positively

with the silver nitrate method, was covered by a thick

mitochondria-rich epithelium The rest of the body,

dor-sal side of the cephalic shield, thorax and appendages,

which were not stained positively by silver nitrate, was

almost exclusively limited by a thin epithelium hosting

few mitochondria

Silver nitrate staining

Silver nitrate staining has been used in insects and

crus-taceans to visualize areas of the body surface interpreted

to be involved in active ion transport such as the anal

pa-pillae of Diptera larvae or the salt gland of Artemia

sali-na (Koch 1934, 1938; Croghan 1958b) Croghan (1958b)

clearly showed that the specific staining is due to passive

diffusion of Ag+ ions through the cuticle and not to the

direct ion-transporting activity of the underlying

epithe-lium Thus, this staining procedure reveals areas of the

body with high ionic permeability The correlation

be-tween a positive silver nitrate staining and the

occur-rence of an ion-transporting epithelium has been

demon-strated by TEM in insects (Copeland 1964; Sohal and

Copeland 1966; Meredith and Phillips 1973) and in

many species of crustaceans: in freshwater species

(Morse et al 1970; Kikuchi 1983; Dickson et al 1991;

Andrews and Dillaman 1993; Kikuchi and Shiraishi

1997), in euryhaline species (Copeland and Fitzjarrel

1968; Barra et al 1983; Felder et al 1986; Kikuchi and

Matsumasa 1993a), or in species living in brine

(Cope-land 1966a; Conte et al 1972; Hootman and Conte

1975) The ion-transporting epithelia revealed by

posi-tive silver nitrate staining in crustaceans are involved or

believed to be involved in osmoregulation

In the first phyllosoma of S arctus, the blackening of

the entire body surface obtained after 30 s in the silver

ni-trate solution indicates that the complete body of the

lar-vae is highly permeable In the nauplii of Artemia sp used

in our experiment, a species considered to be strongly

im-permeable (Croghan 1958a), the procedure exclusively

stained the salt gland independent of incubation time One

of the most important adaptations of an animal facing

os-motic challenge is the reduction of the permeability of the

body surface This adaptive mechanism is apparently

lack-ing in the phyllosoma of S arctus, which is consistent

with its adaptation to live in open ocean water (Phillips

and Sastry 1980) The high permeability of the body

sur-face indicates that diffusion of gases and ions could easily

occur throughout the body surface However, the specific

staining of the ventral side of the cephalic shield observed

after 10 s in the silver nitrate solution indicates that the

cu-ticle covering this part of the body shows a higher

perme-ability than the rest of the body surface We can thus

con-clude that the ventral side of the cephalic shield probably

is covered by an ion-transporting epithelium

Ultrastructure of the thick mitochondria-rich epithelium

The epithelium limiting the ventral side of the cephalic

shield from the phyllosoma of S arctus presents

ultra-structural characteristics of an ion-transporting

epitheli-um In animals, salt-transporting tissues commonly dis-play an abundance of mitochondria and infoldings of the plasma membrane closely associated with mitochondria (Copeland 1964, 1966b; Berridge and Oschman 1972; Cioffi 1984)

The ventral epithelium of the cephalic shield of the phyllosoma is characterized by the presence of numerous mitochondria and of well-developed apical infoldings that are frequently observed closely associated to the mi-tochondria located near the apical side of the cells This epithelium presents some of but not all the characteris-tics of the osmoregulatory epithelia described in the branchial cavity of decapod crustaceans (for reviews, see Mantel and Farmer 1983; Péqueux et al 1988; Taylor and Taylor 1992; Péqueux 1995) Osmoregulatory epi-thelia are generally thick (10–20 µm) and made up of ionocytes, highly differentiated cells with apical infold-ings, generally called apical microvilli, and extensively developed infoldings of the basolateral membranes closely associated with numerous mitochondria

In the phyllosoma of S arctus, the mitochondria-rich

epithelium is less thick (~5 µm) than a typical branchial ion-transporting epithelium but it is still thicker than the thin epithelium covering the rest of the body of the lar-vae The numerous mitochondria are not associated with infoldings of the basolateral plasma membranes Al-though basolateral membrane infoldings associated with mitochondria are almost ubiquitous in the

osmoregulato-ry epithelia of crustaceans, their absence has been

report-ed in epithelia assumreport-ed to be osmoregulatory in the

co-pepod Parasteocaris vicesima (Hosfeld and Schminke

1997) and to be of the ion-transporting type in the gill

lamina of the crayfish Astacus leptodactylus (Dunel-Erb

et al 1997) In both species, a coat of 10-nm particles, similar to that observed in the mitochondria-rich

epithe-lium of the phyllosoma of S arctus, is present on the

cy-toplasmic side of the apical plasma membranes The

freshwater amphipod Sternomoera yezoensis (Kikuchi et

al 1993) and the tanaid shrimp Sinelobus stanfordi (Kikuchi and Matsumasa 1993b) present heterogeneous

ion-transporting epithelia, in which one cell type dis-plays typical basolateral infoldings associated with

mito-chondria and another an apical infolding system Apical

infoldings increase the exchange surface of the cells (Towle 1984) and their number and/or height increase in the osmoregulatory epithelia of crustaceans acclimated

to dilute media (Gilles and Péqueux 1985; Haond et al 1998), thus reflecting a function in osmoregulatory ex-change processes

The apical infolding system observed in the

mito-chondria-rich epithelium of S arctus is similar to the

apical infolding system described in the ion-transporting

epithelia of the syncarid crustaceans Allanaspides hel-onomus and A hickmani, in which apical membranes are

452

also closely associated with mitochondria (Lake et al.

1974) Insect ion-transporting epithelia further exhibit

this ultrastructural characteristic, for instance the

ion-transporting cells of the anal papillae of mosquito larvae

(Copeland 1964; Sohal and Copeland 1966; Meredith

and Phillips 1973), of the rectal pads of Periplaneta

americana (Oschman and Wall 1969), and of the

rectal papillae of Calliphora erythrocephala (Gupta and

Berridge 1966) In these cells (Gupta and Berridge 1966;

Oschman and Wall 1969; Meredith and Phillips 1973),

but also in cells of the Malpighian tubules (Berridge and

Oschman 1969) and in the goblet cells of the midgut

(Cioffi 1979), in which the apical infoldings form

micro-villi-like structures, the apical infoldings exhibit a coat

of approximately 10-nm particles similar to those found

in S arctus According to immunohistochemistry and

immunogold studies, these particles, which are called

portasomes (Harvey et al 1981; Cioffi 1984), are

be-lieved to be the cytoplasmic domain of an H+-ATPase

(Klein and Zimmermann 1991; Klein et al 1991; Klein

1992; Russel et al 1992) It is thus indicated that an H+

-ATPase is located in the apical infoldings of the

mito-chondria-rich cells of the phyllosoma of S arctus.

Other features of the ion-transporting cells in

crusta-ceans are the presence of small vesicles near the apical

microvilli, and of multivesicular bodies present in the

same area or scattered in the cytoplasm, as in the

osmo-regulatory epithelia of the lobster Homarus gammarus

(Haond et al 1998) In the phyllosoma of S arctus, we

also observed similar small vesicles, and multivesicular

bodies In addition we frequently observed lamellar

bod-ies Externally coated vesicles were also present near the

Golgi area and the apical infoldings The coat is made

up of approximately 10-nm particles similar to the ones

of the apical infoldings They also could be the

cytoplas-mic domain of an H+-ATPase

Potential function of the thick mitochondria-rich

epithelium

In the phyllosoma larvae of S arctus, results from the

ul-trastructural study and the silver nitrate staining indicate

that active ion transport probably occurs at the ventral

side of the cephalic shield No data are available about

the physiology of the ionic and osmotic regulation in

the phyllosoma Adult S arctus is an osmoconformer

(Vilotte et al 1980), but because the pattern of

osmoreg-ulation can change throughout the development of

crus-taceans (Charmantier 1998), we do not know if the

phyllosoma osmoregulates or osmoconforms However,

we can expect that the ion transports taking place in the

mitochondria-rich epithelium are comparable to those of

the gills in adult decapods

The model of ion transport in the branchial

osmoregu-latory epithelia of crustaceans is based on the presence of

Na+/K+-ATPase, located on the basolateral membranes of

the ionocytes and involved in active Na+ uptake (Towle

1984, 1993; Towle and Kays 1986; Péqueux 1995; Lignot

et al 1999) The absence of basolateral infoldings in the

mitochondria-rich epithelium of the phyllosoma of S arc-tus indicates the absence of a similar mechanism for ion

uptake In insect Malpighian tubules, the model of active ion transport is based on the presence of an H+-ATPase located on the membranes of the apical microvilli, which provides the driving force for ion transport (Klein et al 1991; Wieczorek 1992; Beyenbach et al 2000) In verte-brates, apically located H+-ATPase is involved in acid-base balance in kidney epithelial cells (Brown et al 1992; Gluck and Nelson 1992), in sodium absorption by the frog skin (Harvey 1992), and in acid-base balance and

Na+uptake in freshwater fish (Lin et al 1994; Sullivan et

al 1995; Perry and Fryer 1997; Fenwick et al 1999; Wilson et al 2000) In freshwater crustaceans, an apical membrane H+-ATPase could be involved in active Cl–

up-take in the Chinese crab Eriocheir sinensis (Riestenpatt et

al 1994, 1995; Onken and Putzenlechner 1995, Onken and Riestenpatt 1998), and in active Na+ uptake in the

crayfish Cherax destructor (Zare and Greenaway 1998).

However, uptake of ions such as Na+ and Cl–, and ener-gized by an H+-ATPase, is a mechanism present in fresh-water rather than seafresh-water animals Therefore, the

epithe-lium limiting the ventral surface of the cephalic shield from the phyllosoma of S arctus is unlikely to be

in-volved in ion uptake It might be inin-volved in the acid-base balance by actively secreting protons Phyllosoma

larvae are planktonic but active swimmers (Macmillan et

al 1997) They adopt a specific swimming behavior with

a continuous looping motion when they are in contact

with food (nauplii of Artemia sp in our experiment, C.

Haond, unpublished) In such intense activity, an active mechanism for proton excretion might be necessary for

the pH regulation of the hemolymph.

Ultrastructure of the thin epithelium

The thin epithelium lining the body of the phyllosoma of

S arctus is similar to the thin epithelia described in the

gills of adult decapods (Copeland and Fitzjarrell 1968; Barra et al 1983; Compère et al 1989; Dickson et al 1991; Haond et al 1998) The function of these epithe-lia, characterized by their thinness (less than 1 µm to

5 µm), the absence of numerous mitochondria and the absence or poor development of membrane infoldings, is presumed to be mainly respiratory In the phyllosoma of

S arctus, the epithelium covering the dorsal side of the

cephalic shield and the appendages is very thin and it does not present membrane infoldings or apical

micro-villi The thin epithelium of the phyllosoma of S arctus

is thus probably involved in respiratory gas exchanges

Acknowledgements The authors wish to thank the Station Méditerranéenne de l'Environnement Littoral in Sète, France, and its technical team for taking care of the lobsters, Tom Spanings for

providing Artemia nauplii, Huub Geurts for his assistance with the

electron microscopy and the Department of General Instrumenta-tion of the University of Nijmegen for providing confocal micro-scope and technical assistance.

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