The morphology and vasculature of the lungs and gills of the soldier crab,Mictyris longicarpus

JOURNAL OF MORPHOLOGY 193:285-304 (1987)

The Morphology and Vasculature of the Lungs and Gills of the Soldier Crab, Mktyris longicarpus

CAROLINE FARRELLY AND PETER GREENAWAY School of ZooEog.y, Uniuersity of New South Wales, Kensington, New Soulh Wules, Australia 2033

ABSTRACT The five gill pairs of Mictyris longicarpus have the lowest weight specific area reported for any crab. The cuticle of the gill lamellae is lined with epithelial cells which have structural features characteristic of ion- transporting cells. Pillar cells are regularly distributed in the epithelium and serve to maintain separation of the two faces of the lamellae. The central hemolymph space is divided into two sheets by a fenestrated septum of connective tissue cells. The dorsal portion of the marginal canal of each lamella receives hemolymph from the afferent branchial vessel and distributes it to the lamella while the ventral portion of the canal collects hemolymph and returns it to the efferent branchial vessel.

The lung is formed from the inner lining of the branchiostegite and an outgrowth of this, the epibranchial membrane. Surface area is increased by invagination of the lining which forms branching, blind-ending pores, giving the lung a spongy appearance. The cuticle lining the lung is thin and the underlyng epithelial cells are extremely attenuated, giving a total hernolymph/ gas distance of 90-475 nm. Venous hemolymph is directed close to the gas exchange surface by specialised connective tissue cells and by thin strands of connective tissue which run parallel to the cuticle. Air sacs are anchored in position by paired pillar cells filled with microtubules. Afferent hemolymph is supplied from the eye sinus, dorsal sinus, and ventral sinus. Afferent vessels interdigitate closely with efferent vessels just beneath the respiratory membrane. The two systems are connected by a “perpendicular system” which ramifies between the airways and emerges to form a sinus beneath the carapace and then flows back between the air sacs to the efferent vessels. The afferent side of the perpendicular system is the major site of gas exchange. Efferent vessels return via large pulmonary veins to the pericardial cavity. P , 0 2 levels were high (95.5 Torr), indicating highly efficient gas exchange.

It has long been recognised that air-breathing in crabs is associated with a reduction in the number and surface area of the gills (Pearse, ’29). This has been accompanied by the development of alternative gas exchange surfaces (von Raben, ’34; Diaz and Rodri- guez, ’77; Taylor and Greenaway, ’79; Cam- eron, ’81). In most air-breathing crabs these surfaces take the form of lungs formed by modification of the inner lining of the branchial chamber. Generally the branchiostegal lining is the principle gas exchange site but the thoracic wall may also become modified (Greenaway and Farrelly, ’84).

Three types of branchiostegal lungs are recognised. In the simplest, the branchiostegite has a smooth lining, e.g., in the freshwater/ land crabs of the families Sundathelphusi- dae, Trichodactylidae and Pseudothelphusi- dae and terrestrial crabs of the families Gecarcinidae and Grapsidae (Dim and Rod- riguez, ’77; Taylor and Greenaway, ’79; C.A. Farrelly, unpublished). In these species the branchial chambers are characteristically in-

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286 C. FARRELLY AND P. GREENAWAY

flated to increase surface area and lung volume is consequently large (Diaz and Rodriguez, '77; Greenaway, '84). In the other two types surface area is amplified in various ways. Thus, in Ocypode cordimanus and Bir- gus latro, the respiratory surface is evagin- ated by means of tufts, folds, and corrugations of the lining (Harms, '32; Dim and Rod- riguez, '77; Greenaway and Farrelly, '84). The third type of lung modification, which occurs in various pseudothelphusids, is an invaginated lining of the branchiosteite (Dim and Rodriguez, '77, Innes and Taylor, '86a,b). Here, there is a small but distinctive perforated area of the lining located on the roof of the branchial chamber. The perforations open into air channels, of irregular cross-section, which run towards the outer cuticle of the carapace (Diaz and Rodriguez, '77). It has been suggested that anastamoses exist between channels, and in Pseudothelphusa g a r mani channels are reported to open into air sacs below the carapace and are ventilated actively in a flow-through manner (El Haj et al., '86). This results in very high PaOz and low PaCO2 levels and gas exchange appears not to be diffusion-limited (Innes and Taylor, '86a). Lung volume in these species has been reduced as surface area is derived largely from invaginations rather than branchiostegal area (Diaz and Rodriguez, '77; El Haj et al., '86).

The soldier crabs (Mictyridae) also have invaginated lungs but here invaginations extend over the whole lung. Mictyrids are small, round-bodied crabs, rarely exceeding 12 g in body weight. At high tide, the estua- rine tidal flats on which they live are covered by water and the crabs remain below the surface of the sediment in a chamber partially filled with air. At low tide, when the sand flats are exposed, the crabs emerge from their chambers and feed on the surface. Mic- tyrids are flotation feeders, and water for feeding is absorbed from the sand via setae fringing the abdomen, (Quinn, '80). The water then passes over the pericardial sacs and contacts the free margin of the epibranchial membranes (outgrowths from the roof of the branchiostegites which divide the branchial chambers into two halves). The ventral margin of these membranes forms a "capillary tube" through which water passes over the gills to the bases of the scaphognathites (Quinn, '80). Thus, the inner branchial chamber forms a capillary tube, houses the gills, and acts as a store of water for feeding.

The outer branchial chamber, however, is air- filled and functions as a lung.

Invaginated lungs were first described in the Pseudothelphusidae by Diaz and Rodri- guez, ('77), and their light microscopic observations and several brief reports on Pseu- dothelphusa garmani (El Haj et al., '86; Innes and Taylor, '86a,b) are the only information available on this lung type. In view of our poor understanding of this type of lung and the more complete invagination seen in the Mictyridae, the morphology, ultrastructure, and vasculature of the lungs and gills of Mio tyris longicarpus have been examined.

MATERIALS AND METHODS

Mictyris longicarpus Latreille 1806 were collected from intertidal sand flats in Botany Bay, Sydney, Australia. They were trans- ported to the laboratory and were generally used within a few h of collection. If not used immediately, crabs were maintained in damp sand without food, at 25°C until used.

The gross morphology of the branchial chamber and gills was elucidated by dissec- tion of fresh and preserved crabs under a Wild M5 binocular microscope. Gill and lung tissue was also fixed in glutaraldehyde (2.51%)~ critical-point dried, and, after metal coating, examined in a Cambridge Stereos- can S4-10 scanning electron microscope.

For transmission electron microscopy, tissue was fixed in 2.5% glutaraldehyde buff- ered with 0.1 M 1-l Na cacodylate. After washing in 0.1 M 1-l Na cacodylate buffer, tissue was post-osmicated in a solution of 2% OsO4 in the same vehicle and embedded in Spw's resin. In a few instances Sorensen's phosphate buffer was used in place of Na cacodylate. Ultrathin sections were double- stained using uranyl acetate and lead citrate and examined using a Philips E.M. 300 electron microscope. Sections for light microscopy (0.5 prn epoxy sections) were fixed as for electron microscopy and stained with 1% methylene blue.

Corrosion casts of the vascular system using Batson's No. 17 Anatomical Corrosion Compound (Polysciences Inc., Warrington, PA) using previously described techniques (Greenaway and Farrelly, '84). Final diges- tion of decalcified cuticle was carried out in sodium hypochlorite (household bleach). This dissolved cuticle surrounding gill lamellae and other delicate structures, avoiding the need for microdissection to remove cuticle, a process often damaging to fine casts. Casts

A COMPLEX LUNG IN AN AIR-BREATHING CRAB 287

were examined with a binocular microscope and their structure was documented by pho- tography and drawing using a camera lu- cida. The microvasculature of the casts was then examined using scanning electron microscope (SEM), after metal coating.

Casts of the invaginated airways in the lung were made as follows. The epipodites which seal the afferent openings of the lungs (Milne-Edwards apertures) were removed and Batsons No. 17 was injected into the main cavity of the lung. The crabs were then sub- merged in the liquid plastic in a glass tube and placed in a vacuum chamber. Evacua- tion of the chamber helped remove gas from the airways and facilitated penetration of the plastic. The resultant casts were metal coated and examined with the SEM.

The surface area of the gills was measured using the method of Greenaway (‘84). Crabs covering as wide a range of body size as possible (3-13 g) were used.

An estimation of the surface area of the lung (both branchiostegite and epibranchial membrane) was attempted. This was calculated in three parts, using data sets from three different, though similarly sized crabs (7 g). Firstly, the surface area of the fine terminat.icns of the air sacs was estimated from scanning electron micrographs of casts of the airways, b counting the number of

erage length and diameter of the branches. (Surface area was calculated by treating the air sacs as cylinders open at one end). Sec- ondly, the surface area of the main trunks was estimated from light micrographs of fresh tissue by counting the approximate number of openings from the lumen into the lung and measuring the length and average diameter of the openings. Finally, the flat surface area of the lung, less the area of the openings, was calculated using mm2 graphs paper cut to the shape of the branchiostegite and epibranchial membrane and then counting the squares on the graph paper according to the method of Greenaway (‘84). The sum of these three calculations was then doubled to give total area of the lungs.

POz and PCOZ measurements were made at 25°C using Radiometer blood gas equip- ment (BMS 3 mark 2 and PHM 73). Samples of arterialised hemolymph were taken from the pericardial cavity through a small hole in the carapace drilled down to but not through the epidermis. Drilling was done 24 h before sampling to allow crabs to recover

branches per mm H and by measuring the av-

from disturbance. Venous hemolymph was sampled from the infrabranchial sinus at the base of the chela. Only one sample was taken from each crab. Measurements were made on crabs removed from burrows they had con- structed in sand (resting), crabs naturally active on the sand surface, and disturbed crabs removed from burrows and made to walk.

RESULTS Gross morphology of the branchial chamber The branchial regions of mictyrid crabs are

delineated by the cervical and cardiobran- chial grooves (Fig. 1A). The lower margins of the branchiostegite of M. longicarpus do not overlap the bases of the walking legs but fit closely against cuticular scallopings of the epimera above the coxae (Fig. 1A). The afferent opening to the branchial chamber Nilne- Edwards aperture) is a semicircular notch in the margin of the carapace at the base of the cheliped. The opening can be sealed by the concave valvelike expansion of the epipodite of the third maxilliped (Fig. lA,B). The efferent channels, in which the scaphognathites lie, open anterodorsally and run from the inner gill compartments of the branchial chamber to the mouthparts.

The gills lie on the floor of the branchial chamber, partially covered by the semitrans- parent epibranchial membrane Fig. lB,C). The epibranchial membrane is a dorsal outgrowth from the lining of the branchial chamber. It arises anteriorly, just dorsal to the Milne-Edwards aperture and continues along the dorsal margin of the branchiostegite to the lower posterior margin. Its free ventral margin curves from the anterior to the posterior, partially covering the gills and dividing each branchial chamber into an inner gill compartment and an outer lung compartment (Fig. 1B,C).

Structure of the gills External morphology

M. longicarpus has five gills and two epipodites on each side (Lazarus, ’45). The gills are phyllobranchiate with rigid, well-spaced lamellae extending anteriorly and posteriorly from the central gill shaft. The periph- ery of each lamella is expanded to form a marginal canal. Spacing between lamellae is maintained by rows of nodules on the corners of the marginal canals (Fig. 2A). Surface area of gills

The gill area of M. longicarpus increased with increasing body weight following a


Branc hios tegal Epi branc hial

Fig. 1. A) Lateral view of M. longicurpus showing branchiostegite (B) (outlined dorsally by the cervical car- diobranchial groove), the dorsal notch in the branchiostegite CDN) and the Milne-Edwards aperture sealed by an expanded epipodite (E). x 2.5. B) Lateral view with branchiostegite removed to reveal the spongy epibranchial membrane and gills. x 3. C) Diagrammatic vertical section showing the branchial chamber divided into an inner gill compartment and an outer lung compartment by the epibranchial membrane.

C Gills

relationship of the type y = amb, where y is gill area (mm2), m is the body mass (g), b the slope of the regression of log y on log m, and a is the value of y when m = 1. The relationshi obtained for gill area was

Thus, weight-specific gill area (mm2 g-l) decreased with increasing body size. Gill area in the species is lower than values reported in the literature for other air-breathing crabs (Hughes, '83; Greenaway, '84) and lower than measured in Ocypode cordimanus, Geograp- sus grayi, Gecarcoidea natalis, and Cardt soma hirtipes (P. Greenaway, unpublished data). The exponent, b, however, is similar to those for other air-breathing crabs.

y = 328.1 mo.61 B .

Ultrastructure

The gills of M. Zongicarpus are covered by thin cuticle, 0.7-1.4 pm thick, and are lined by a single layer of epithelial cells (Fig. 2B). The cell layers on each side of the lamellae are connected periodically by pillar cells which separate the two faces of the lamellae and form a junction with the septum which runs through the middle of the lamella, parallel to the cuticle (Fig. 2C). This central septum divides the lamellar hemocoel into two compartments (Fig. 2C). The septum is not complete but is characterised by small apertures which may allow movement of hemolymph between the two compartments.


Fig, 2. A) SEM of gill no. 3. Lamellae are few and are widely spaced. Nodules ensure regular spacing while the thickened marginal canals provide rigidity. x 15. B) General ultrastructure of principal epithelial cells of gill lamellae showing apical microvilli, basal infoldings, and abundant mitochondria (M). BL, basal lamina; C, cuticle. x 11,600. C) Pillar cells (P) form a zigzag function with

the lamellar septum (S) in the midline of the lamella. Bundles of microtubules (M) extend through the septum and pillar cells, anchoring them to the cuticle (C). x 2,400. D) SEM of cast of the vascular system in the gill showing afferent vessel (A), efferent vessel (El, and strong vascular restriction occurring at point (R) in the marginal canal (MC). x 36.

The epithelial cells possess an extensive system of apical microvilli which are attached to the cuticle at their tips by hemidesmo- somes (Fig. 2B). The basal surface is characterised by deep infoldings (Fig. 2B). The intercellular channels are closed apically and basally by tight junctions. Closely associated with the apical microvilli and with the basal infoldings are numerous mitochondria (Fig.

28). The lateral membranes between adjacent cells interdigitate extensively and possess septate apical junctions. All epithelial cells in the gill examined (no. 3) are of a relatively uniform type, 2.5-5.5 p m thick, with flattened nuclei. The epithelial cells which line the marginal canal are slightly thinner than those in the central lamella. The cuticle of the marginal canal, however,


is thicker than elsewhere, and therefore he- molympWgas diffusion distances are much the same.

Among the epithelial cells are pillar cells (Fig. 2C). The pillar cells have an arrangement of apical microvilli and distribution of mitochondria similar to that of normal epithelial cells. Some also have rods of electron- dense extracellular material which extend along the villi anchoring the pillar cells to the cuticle. Bundles of microtubules are also present in the cytoplasm of the pillar cells and run in a direction perpendicular to the cuticle. Basally, the pillar cells interdigitate with the septum (Fig. 2C). The opposite side of the septal cells are similarly anchored by other pillar cells extending from the opposite surface of the lamella. The junctions between the pillar cells and the septal cells are similar to those described in H. transversa between paired pillar cells (Taylor and Green- away, ,791, in that they are acutely zig- zagged, and material containing collagenlike fibres is interposed between the membranes of these junctions (Fig. 2C). The septal cells (Fig. 2C) are characterised by extensive systems of membrane infoldings and tortuous interdigitations with neighboring cells and are rich in mitochondria and what appears to be glycogen. Bundles of microtubules also extend through the septal cells, anchoring the pillar cells at both ends (Fig. 2C). The lamellar septum, pillar cells, and epithelial cells are surrounded by a common basal lamina which lines the hemocoelic space.

Vmculature Hemolymph is supplied to the lamellae

from the infrabranchial sinus via the dorsal afferent vessel and is drained by the ventral efferent vessel (Fig. 2D). These two vessels lie very close together in M. longicarpus and, as a result, the median gill shaft, characteristic of species in which the afferent and efferent vessels are more widely separated, e.g., Holthuisana transversa (Taylor and Greena- yay, "79), Eriocheir sznensis (Barra et al., 83), and Carcinus maenas (Taylor and Tay- lor, '861, is absent. All of the hemolymph in the afferent vessel is routed directly into the marginal canal which is then responsible for distributing it to the whole lamella. Casts of the vasculature of the gill show the marginal canal to be composed of two parts, with major vascular restriction occurring between the dorsal (afferent) and ventrolateral (efferent) portions (Fig. 2D). The dorsal portion distrib-

utes venous hemolymph to the lamella and the lateral and ventral portions collect and convey it to the main efferent vessel. Hemo- lymph flows through each lamella in two sheets separated by the septum. In M, longt carpus the septum is not confined to a particular area as in E. sinensis (Barra et al., '83), C. maenas (Taylor and Taylor, '861, Cardz- soma hirtipes, and Geograpsus crinipes (C.A. Farrelly, unpublished) but extends through- out the whole lamella.

Structure of the lung External morphology

The lung of M. longicarpus comprises the inner lining of the branchiostegite and the outer surface of the epibranchial membrane (Fig. 10. In M. longicarpus, the surface area of both these structures has been increased by invagination of the lining to produce a series of branching, blind-ending pores, giving both lung surfaces a spongy appearance (Figs. lB, 3A,C). The branchiostegal lung bears a number of large, protruding surface blood vessels which are orientated vertically down the lung (Fig. 3A). Lateral branches from these form a multilayered, horizontal mesh of blood channels which surround the openings to the air sacs (Fig. 3A). The air sacs are observed to branch just below the lumen surface (Fig. 3B).

The epibranchial membrane is also spongy, although the depth of penetration of the air sacs is much more variable than in the branchiostegal lung (Fig. 1B). The anterior dorsal region is relatively thin with only shallow perforations present and the larger vessels protrude (Fig. 3C). In contrast, the median region is highly invaginated with very large openings at the lumen surface and branching evident below the surface (Fig. 3C). The most ventral portion of the epibranchial membrane, which covers the gills, is very thin, lacks perforations and is poorly vascularised. (Figs. 1A,3C). The inner surface of the epi-

Fig. 3. A) The left branchiostegite removed and viewed from the lumen side. Note spongy appearance and large surface vessels. The lower scalloped portion contacts the leg bases. x 8. B) SEM of the lumen surface of the branchiostegite showing openings of air channel. Branching of air sacs is indicated by arrows. x 34. C) SEM of the left epibranchial membrane showing surface vasculature and openings of air channels. Spongy tissue tapers ventrally into a thin membrane which covers gills. x 15. D) Left branchiostegite removed with the epibranchial membrane (EM) still attached. The back of the epibranchial membrane which faces the inner gill compartment is smooth and heavily pigmented. x 5.



branchial membrane, which faces the inner branchial compartment, is quite smooth and does not appear to be modified €or gas exchange (see below) (Fig. 3D).

In longitudinal section the walls of the air sacs are thrown into diagonal pleats, with inclusions or pits studded at various inter- vals over the surface (Fig. 4A). Casts of the air sacs better illustrate their three-dimensional nature and are reminiscent of gardens of staghorn coral (Fig. 4B). Thick trunks give rise to multiple secondary and tertiary branches, forming minute respiratory trees (Fig. 4Cf. The casts emphasise that the air sacs are not simple tubes but bear pyramidal projections and ridges which greatly increase

their surface area. The maximum length of the air sacs is around 1.0 mm. There is no indication that air sacs of different respiratory trees are connected.

Vasculature of the lung Brunchiostegite. The afferent vascular

system of the lung is supplied anteriorly from the eye sinuses, with lesser inputs from the dorsal and ventral sinuses. The branchiostegite receives three large vessels from the eye sinus which enter the lung just above the dorsal notch of the branchiostegite (Fig. 5A). Two of these afferent branchiostegal vessels (VAV 1 & 2) run vertically down towards the ventral margin of the lung. The third and

Fig. 4. A) SEM of cross-section through the branchiostegite showing air sacs (A) in longitudinal section. Car- apace has been removed from the dorsal surface (D). L, lumen side of lung. X 140. B) SEM of cast of the airways of the branchiostegal lung viewed laterally from the carapace side. X 36. C) Magnified view of cast of air sacs. Note branching to form respiratory trees and projections and folds which amplify surface area. X 145.

Fig. 5. A) Diagrammatic representation of the main vasculature of the branchiostegal lung. DN, dorsal notch; E, epipodite; PV, pulmonary vein; 1 & 2, vertical afferent vessels; 3, dorsal afferent vessel. B) SEM of vasculature at the lumen surface of the branchiostegal lung. A, afferent vessel; E, efferent vessel. x 36.


A

From eye sinus 0 Afferent

Figure 5


largest vessel, the dorsal afferent vessel (DAV), runs below the cervical cardiobran- chial groove of the carapace to the lower posterior margin of the branchiostegite Fig. 5A). This main afferent vessel branches ventrally along its length in a vertical plane to supply the epibranchial membrane and in a lateral plane to supply the branchiostegite.

The descending vessels of the afferent system WAV 1 & 2) give off many lateral branches (Fig. 5A). Some of these form surface sinuses which pass directly around the openings of the air channels and then into the corresponding vessels of the efferent system (Fig. 5B). However, most of the hemolymph is directed towards the carapace in vessels which run perpendicularly to the main vasculature, ramifying between the air sacs until they emerge just beneath the carapace (Fig. 6A,B). The vessels in the afferent perpendicular system branch and anastamose and have multiple restrictions along their length. These vessels also possess extremely thin “plates” of hemolymph which form the peripheral blood spaces in closest contact with the lumen of the air sacs (Fig. 6C). Clearly, the resistance produced by multiple restrictions and branching in the perpendicular system is high enough to force hemolymph through the peripheral path- ways where maximum gas exchange must take place. At the carapace the vessels di- verge, branching around the terminations of air sacs and contacting the arms of adjacent vessels Fig. 6E,F). Thousands of these join to form a large, flat sinus, the subcarapacial sinus (Fig. 6E,F).

The efferent system arises as vessels which drain the subcarapacial sinus (Fig. 6D). Here hemolymph travels back down between the air sacs in vessels which lack peripheral lacunae and enters lateral collecting vessels near the main lumen of the lung. These in turn form large, parallel, vertically orientated vessels which descend into one very large collecting vessel, the pulmonary vein (Fig. 5A). This runs along the ventral perim- eter of the branchiostegite and empties into the pericardial cavity.

The afferent perpendicular system pro- vides the most efficient sites for gas exchange. It is possible, however, that gas exchange occurs in both afferent and efferent perpendicular systems and in the subcarapacial sinus.

Epibranchial membrane. The afferent vascular supply to the epibranchial mem-

brane consist of two large branching vessels arising from the eye sinus (Fig. 7A). The DAV supplies both branchiostegite and epibranchial membrane and traverses the dorsal margin of the branchiostegite (cervical bran- chiocardial groove), branching along its length until it terminates near the posterior edge of the membrane (Fig. 7A). The second vessel, the medial afferent vessel (MAW, descends and curves along the anterior and mid-ventral margin of the membrane (Fig. 7A).

The efferent system of the epibranchial membrane is a palmately branched complex that interdigitates with vessels from the afferent system (Fig. 7A,B). It drains the entire membrane, starting from the anterior end and extending laterally across the membrane. Posteriorly, it feeds the pulmonary vein from the branchiostegite, just before it empties into the pericardial sinus.

The arrangement of the afferent and efferent vasculature of the epibranchial membrane differs from that of the branchiostegite. This is because the epibranchial membranes are less uniform in thickness and have a lower density of air channels than in the branchiostegal lung. The perpendicular vessel component, characteristic of the vasculature of the branchiostegite, is not as well developed in the epibranchial membrane which has a more two-dimensional arrangement of lacunar exchange beds.

The dorsal regions of the epibranchial membrane are relatively thin, though corm- gated, with many small, very shallow air sacs. Here the afferent vessels give off many

Fig. 6. A) SEM of cross-section of vascular cast showing relationship between major afferent (A) and efferent (E) vessels, the perpendicular system (PS) and the subcarapacial sinus (&). L, lumen side of lung. x 16. B) Light micrograph of a semithin epoxy section through branchiostegite. Large vessel near lung lumen (I,). Smaller vessels branch between airways (A) towards carapace which has been torn away (top left). x 150. C) SEM of cast of afferent channels in the perpendicular system. Note the presence of many thin peripheral lacunae (PL). L, lumen side of lung; Sc, subcarapacial sinus. x 75. D) SEM of cast of efferent channels in the perpendicular system. Note generally thicker vessels with fewer thin peripheral lacunae. L, lumen side of lung; Sc, subcarapacial sinus. X 75. E) SEM of vascular cast of the subcarapacial sinus viewed from the carapace side. The large holes represent the terminations of air sacs and where the lung matrix joins with the outer subepidermal connective tissue. The small holes mark the former position of pillar cells. x 90. F) Semithin epoxy section through the branchiostegite showing the subcarapacial sinus (Sc). A, air sacs; C, membranous layer of external cuticle; P, pillar cell. x 185.



lateral branches which open into flattened, fanlike lacunae (Fig. 7C). These flow around a number of air sacs before passing into the larger return vessels of the efferent system (Fig. 7 0 . In the spongy region of the membrane, the air sacs are deep and highly branched. Here the lateral branches of afferent vessels fork around the large outer openings and then send small vessels down between the smaller branches of the air sacs which combine into a central pool (Fig. 7D). From this central sinus emerges a similar set of efferent vessels which travel back up between the air sacs to form a corresponding fork around the outer opening of the air sac. This hemolymph then flows into secondary branches of the palmate efferent system.

Ultrastructure The branchiostegites are lateral extensions

of the carapace and thus have an epidermis and cuticle on both outer and inner surfaces, the inner surface forming the lung. The epibranchial membrane is an outgrowth from this inner surface and it too has cuticle on both sides. The external cuticle of the branchiostegite is 65-90 pm thick, calcified, and structurally resembles the cuticle of the carapace of other decapods (Skinner, '62; Green and Neff, '72). The epidermal cells which se- crete this cuticle are associated with a subepidermal connective tissue containing chro- matophores similar to other crustaceans (Fig. 8A). Below this layer lies the subcarapacial sinus (Fig. 8A).

The internal cuticle covering the gas exchange surface of the branchiostegite is very thin indeed, 100-265 nm in the branchiostegite (Fig. 8B) and 70-375 nm on the respiratory face of the epibranchial membrane (Fig. 9A). The nonrespiratory surface of the epibranchial membrane has a cuticle thickness of almost 1.3-2.8 pm Fig. 9A). The inner epidermal cells of the lung are extremely attenuated, and distal to the perikaryon the cell thickness is 20-100 nm (Fig. 8B). Atten- uation of the epidermal cells has been achieved by restricting the perinuclear part of the cells to a trunk which projects into the hemocoel, with a thin cytoplasmic sheet extending all around it and lining the cuticle (Figs. 8B, 9B). These flattened flanges have virtually no organelles. The, total airhemo- lymph distance is 120-365 nm in the branchiostegal lung and 90-475 nm in the

epibranchial membrane, thus ranging between 3 and 7% of the waterhemolymph distance in the gills. The perikaryon of the epidermis is an-

chored to the cuticle by bundles of microtubules and rods of electron dense material which extend along short, simple apical projections (Fig. 8B). It contains a heterochro- matic nucleus, a small number of mitochondria and rough cisternae of the endo- plasmic reticulum. Basally, the perikary-on is in continuity with connective tissue which forms the structural framework of the lungs. Both are lined by a common basal lamina. Similar epidermal cells occur in the lungs of Ocypode ceratophthalrnus and in Birgus latro (Storch and Welsch, '75, '84). Pillar cells (modified epidermal cells) occur between the outer projections of adjacent air sacs (Fig. 9C) and also between the ends of the air sacs and the thick outer cuticle of the branchiostegite (Fig. 6F). The pillar cells are filled with microtubules and filaments running at right angles to the cuticle (Fig. 9D). Apically, these cells have thick rods of electron dense material anchoring them to the cuticle. Basally, each pillar cell forms a strong interlacing junction with another pillar cell extending from the opposite cuticle of the adjacent air sac. Rods of electron dense material are also present in the basal junctions Fig. 9D).

The connective tissue consists of collagen fibers and cells of irregular shape containing glycogen particles, profiles of rough endo- plasmic reticulum, a few mitochondria, and light vacuoles (Fig. 10A,B). Many of the connective tissue cells near the respiratory cuticle possess little footlike processes and bear microvilli which direct hemolymph close to the respiratory surface (Fig. 10A). These are similar to the fimbriated connective tissue cells described in Holthuisana transversa (Taylor and Greenaway, '79). Specialized fimbriated cells are also present in Gecarcoidea natalis, Cardisoma hirtipes, Geograpsus grayi, and Geograpsus crinipes (C.A. Far- relly, unpublished). Thin peripheral blood spaces are also formed in M. longicarpus by thin strands of connective tissue, broken up periodically by rounded cell bodies which run parallel to and in close proximity to the respiratory cuticle (Fig. 10B).

The fine structure of tissues in the epibranchial membrane is basically similar to that in the branchiostegite.

From

A

A COMPLEX LUNG IN AN AIR-BREATHING CRAB

A\ To heart

==i 7r, &- Pulmonary vein

297

Fig. 7. A) Diagrammatic representation of the main vasculature of the epibranchial membrane. DAV, dorsal afferent vessel; MAV, medial afferent vessel. B) SEM of cast of the vasculature in the epibranchial membrane viewed from the lumen, showing primarily the palmately branched efferent system. Only the posterior (P) and middle portions of the afferent system have filled. A, afferent vessel beginning to fill. x 12. C) Enlarge- ment of thin gas exchange lacunae in the thin dorsal region of the epibranchial membrane. Round holes show the position of air channels. A, afferent vessel; E, efferent vessel. X 150. D) Enlargement of afferent vessels (A) and efferent vessels (E) in the thick spongy region of the epibranchial membrane. x 66.



Lung surface area The surface area of the lungs of a 7 g crab

was estimated to be 1640 mm2, with 16-20 perforations per square mm. This figure rep- resents a minimum value as the air sacs were treated as perfect cylinders and the con- tribution from projections which cover the air sacs was not taken into account. Further- more, the intermediate branching which occurs between the thick trunks and the fine terminations could not be easily visualised and measured, so the fine branches were ar- bitrarily extended into this zone. The epibranchial membrane formed 45% of the lung surface area. The area of the lungs repre- sented approximately 1.5 times the gill area in a similarly sized crab.

Blood gas measurements Pa02 levels were uniformly high (Table 1)

although values for resting crabs were signif- icantly lower than in active or disturbed crabs (E' < 0.05). P,Oz levels were also quite high for air-breathing crabs. PaC02 levels were low in all states but P,CO2 was signifi- cantly higher in disturbed than in resting crabs (P < 0.001).

DISCUSSION

Mictyris longicarpus has the smallest gill number (five) recorded for any crab, most aquatic species having nine pairs of gills. The lamellae are also few and widely spaced and consequently weight-specific gill area is the lowest recorded for any crab. This un- doubtedly reflects a structural economy made possible by the development of a complex lung and the consequent reduction in importance of the gills in gas exchange. However, the gills of M. longicarpus drain readily on emersion, without clumping, thus permitting convective air flow between the lamellae, and no doubt contribute to aerial gas exchange. There is certainly a rich vascular supply to the gill lamellae. During flotation feedings,

Fig. 8. A) Electron micrograph of the thick external cuticle (EC) and its epidermis (E), melanin cells (MI, and associated connective tissue (CT). Hc, hemocyte; Sc, subcarapacial sinus. x 3,000. B) TEM of thin internal cuticle (C) bordering the lumen of an air sac. Epidermal cell body (Epi) projects into the hemoeoel (H) and joins with connective tissue partitions (CT). Rods of electron dense material (DM) anchor epidermal cell body to cuticle. The thin double membrane (E) lining the cuticle is an exten- sion of the epidermal cells. Hc, hemocyte. x 6,600.

the gills are covered with water (Quinn, '80) and may function in aquatic gas exchange in these periods. Thus, while the surface area has been reduced, the gills are still probably capable of significant gas exchange and are also modified for ion-regulatory functions.

The pattern of hemolymph flow through the gill lamellae differs from that in other crabs as all the blood is routed initially into the marginal canal which then directs it into the central lamellar region. The constriction point in the marginal canal which seems to separate the gill vasculature into distinct afferent and efferent parts also raises the possibility of regulating the flow of hemolymph through different lamellar routes by altering the diameter, and thus resistance, in the marginal canal. However, this possibility has not been investigated.

The lamellar septum found in the gills of M. longicarpus has been observed in a number of other crustaceans (Kimus, 1900; Ber- necker, '09; Drach, '30; Chen, '33; Nakao, '74; Barra et al., '83) and in the land crabs 0. cordimanus, G. natalis, C. hirtipes, G. gruyi, and G. crinipes, (C.A. Farrelly, unpublished), yet its function remains unclear. By splitting the hemolymph flowing through the lamella into two thinner sheets, the maximum diffusion distance for gases is reduced, and this, in addition to increased turbulence of flow, may achieve more efficient oxygenation of the hemolymph. The septum is also found in aquatic species (Chen, '33; Taylor and Taylor, '86) and it may confer no advantage specifi- cally to air-breathing forms. However, the lamellae here may be thicker due to requirements for rigidity and the septum may thus be used as a convenient way of reducing thickness of the hemolymph layer. Alterna- tively, it may contribute directly to the rigidity of the lamellae, thus reducing the thickness of the cuticle and shortening hemolymph gas diffusion distances. The septum is also used as a storage site for glycogen and thus may be involved in the regulation of hemolymph sugar levels and metabolism (Barra et al., '83).

The surface area of the branchiostegal lung in M. longicarpus is supplemented by the epibranchial membrane and by invagination of both these surfaces to produce a spongy structure. Clearly, the surface area is very large compared to a similarly sized lung with a smooth lining. A rough calculation sug- gests invaginations may enhance surface


Fig. 9. A) Section through epibranchial membrane. A thick cuticle (Cl), associated with a thick epidermis and connective tissue layer, lines the nonrespiratory side of the membrane facing the gill compartment (0). A thin cuticle (CZ) and epidermis (E) line the lumen side of the membrane. H, hernolymph; Hc, hemocyte. X 2,500. B) Cell body of epidermis (E) forms junctions with connective tissue cells (CT) which form the structural framework of the lungs. H, hemocoel, x 3,700. C) Semithin

epoxy section through branchiostegite showing dark pillar cell junctions (P) between adjacent air sacs (A). (Car- apace removed from tissue at top). CT, connective tissue. x 195. D) Enlarged view of pillar cells (P) joining adjacent air sacs (A). Note bundles of microtubules and filaments running at right angles to the cuticle (C), and dense anchoring material (DM) at basal and apical junctions. H, hemolymph; Hc, hemocytes. x 2,900.

A COMPLEX LUNG IN AN AIR-BREATHING CRAB

TABLE 1. Oxygen and carbon dioxide tensions (Tor f SEM) in the hernolymph of active and resting crabs In = 7-10)

301

Conditions Pa02 P"O2 PaCO2 P"C02 Burrowed 79.6 f 4.3 20.5 f 2.97 5.5 0.5 5.6 f 0.43

Active on 95.5 f 6.1 5.1 + 0.65

Disturbed 95.2 & 5.34 27.7 f 7.0 6.74 f 6.3 14.1 + 1.58

in sand

surface

active on surface

area by 300%. In M. longicarpus, the invaginations show multiple branching but all the branches are blind-ending. Given this structure, ventilation of the invaginations must be tidal or by diffusion. The main lumen of the lung is ventilated in the usual way by means of scaphognathites. The walls of the invaginations do show accordionlike pleats (Figs. 4A, 9C) but it is unlikely that they can be actively ventilated, as no muscle tissue has been found in the lung and the pillar cells are probably not contractile. However, bundles of nonstriated fibres were observed to traverse the epibranchial membrane and could conceivably cause ventilatory contrac- tions. Some change in the volume of the air channels could perhaps be effected by changes in dilation of the vasculature but this possibility has not been investigated. It is probable that gas movement in the airways is by diffusion from the ventilated lumen of the lung. Given that the airways seldom exceed 1.0 mm in length this process should be quite efficient and this is borne out by blood gas measurements.

In contrast to this situation in M. longicarpus, the invaginated airways of the lung of Pseudothelphusa garmani are reported to be ventilated actively. The airways in this species run from the lumen of the lung and anastamose with bellowslike air sacs which apparently produce a flow of air through the air channels (El Haj et al., '86; Innes and Taylor, '86a) resulting in a very high arterial PO2 (Innes and Taylor, '86a,b). However, there are problems with their descriptions. The lung system is lined with cuticle and this must be shed at the molt. It is difficult to understand how this could be achieved in the structure described by El Haj et al. ('861, as a complete cuticle cannot be withdrawn from an anastamosing structure unless special break zones are present. Thus the cuticle of the alimentary canal of crustaceans lines

the foregut and hindgut but is absent in between. More detailed descriptions of the anatomy of P garmani are needed to clarify this matter. In contrast, the cuticle of blind- ending, nonanastamosing channels described in M. longicarpus could readily be shed at ecdysis.

During feeding periods, the bottom of the branchial chamber in M. Zongicarpus is filled with water. However, the narrow air channels in the lungs are not filled with water by capillarity. Since the air sacs are closed at one end, a water circuit is not complete and, as the air sacs are already filled with air, water cannot enter. However, like all respiratory surfaces, the cuticle covering the lung is moist. All animals have a high water con- tent and the lung of the soldier crab has an extremely thin epidermis and cuticle, so that water is extended through the membrane onto the outer respiratory surface. Gases are dissolved in this thin layer of moisture and thus pass easily, in solution, through the cuticle and epidermis and into the blood. He- molymph/gas diffusion distances in the lung of M. longicarpus are exceedingly short (125- 550 nm) and in the thinnest areas of the lung are generally less than those of other crabs, e.g., H. transuersa, 250-300 nm (Taylor and Greenaway, '79); 0. ceratophthalmus, 250- 500 nm (Storch and Welsch, '75); and F! garmani 400 nm (El Haj et al., '86) and compare to those values found in the lungs of higher vertebrates, 300-1,700 nm (Weibel, '64).

The respiratory organs of M. longicarpus appear to be extremely efficient in oxygen- ating the hernolymph. The resting P,Oz values are at the top of the range reported in other air-breathing species (McMahon and Wilkens, '83; Greenaway et al., '83) while active and disturbed animals have even higher levels. At the same time P a C 0 2 is maintained at low levels in all activity states and there is a large drop in PCOz during the



passage of hemolymph through the respiratory surface in disturbed animals. As the area of the gills of M. longicarpus is small, it is likely that the lungs are responsible for the bulk of gas exchange. PaOz values of 120- 140 Torr are reported in another crab with an invaginated lung, Pseudothelphusa garmani. The higher values here may be due to the reportedly active ventilation of the air channels (Innes and Taylor, '86a,b).

The structure of the lung of M. longicarpus also shows some similarities with that of Ocypode cordimanus. The vascularised outgrowth of the thoracic wall in 0. cordimanus (Greenaway and Farrelly, '84) is analogous to the epibranchial membrane, and the folds and corrugations on its surface enhance surface area much as do the invaginations in M. longicarpus. The arrangement of the major vessel systems in the lung, as they appear at the lumen surface, are also similar. Thus, both have a relatively simple system with a single afferent system interdigitating with an efferent system. In contrast, multiple por- tal systems have been found in H. transversa, G. natalis, C. hirtipes, G. grayi, and G. crt nipes (C.A. Farrelly, unpublished). However, the interdigitating system of M. longicarpus is more complex than that of 0. cordimanus, as the invaginated airways add another di- mension of surface area and, therefore, addi- tional vasculature to the lung. The main vessels of the afferent and efferent systems of the lungs of M. longicarpus lie at the luminal surface where they can be clearly seen (Fig. 3A,C). However, the perforated area of the lung of the Pseudothelphusidae is restricted to a small patch in the ceiling of the branchiostegite. In contrast to those of M. longG carpus, the perforations in the lungs of these species are fairly regular in size, shape, and density and are all at the same level on the surface of the lung, thus producing a smooth, uniform, but perforated surface. This sug- gests that the main vasculature of the invaginated lung of the pseudothelphusid crabs lies within the lung near the carapace.

Fig. 10. A) Connective tissue cell (CT) with fimbria- tions. The footlike processes or microvilli (Mic) extend towards but do not attach to the respiratory cuticle (C). Col, collagenlike fibres; Hc, hermocyte; M, mitochondria. X 8,000. B) The main body of connective tissue cells (CT) are often joined together by thin strands of cytoplasm which run parallel to the cuticle (C), forming thin peripheral blood spaces. Col, collagenlike fibres; Hc, hemacyte. X 10,400.

M. longicarpus has developed a relatively complex lung for air-breathing, compared to other crabs, and it may well be asked why this crab is so specialized when other species, considerably more terrestrial in habits, have relatively simple lungs. The answer to this may be related to body size and the particular lifestyle adopted. M. longicarpus is small and globular and selective pressure has ob- viously been for the lungs to be compact with a small volume. Smooth lungs necessarily have a large volume to achieve adequate surface area (Diaz and Rodriguez, '77). The other important factor is the limited period of time at low tide available for feeding. This means that their feeding and trekking activities are energetically intensive, and so the metabolic rate is probably high. Therefore, an increased surface area for gas exchange is needed. Clearly, one way to accommodate the combined requirements of a small body size and a large respiratory surface is to produce a spongy lung. In this way, M. longicarpus has achieved a more efficient use of the space of the gill chamber for respiratory purposes while at the same time remaining very small.

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Documents

The morphology and vasculature of the lungs and gills of the soldier crab,Mictyris longicarpus