Oleospheres of the cave-dwelling shrimp Troglocaris schmidtii: A unique mode of extracellular lipid...

JOURNAL OF MORPHOLOGY 211:31-39 (1992)

Oleospheres of the Cave-Dwelling Shrimp Troglocaris schmidtii: A Unique Mode of Extracellular Lipid Storage

GUNTER VOGT AND JASNA STRUS Department of Zoology 1 (MorphologylEcology), University of Heidelberg, W-6900 Heidelberg, Germany (G. V.); Department of Biology, Biotechnical Faculty, University of Ljubljana, 61 001 Ljubljana, Yugoslavia (J.S.)

ABSTRACT The cave-dwelling shrimp, Troglocaris schmidtii, has a unique mode of lipid storage. The lipid lies extracellularly in specialized compartments of the hepatopancreas, named oleospheres. The lipid is synthesized in the R-cells of the hepatopancreatic epithelium and accumulates in lipid droplets which fuse to form bigger globules. Mature lipid globules display moderately electron dense centers probably comprising triglycerides, and a broad electron dense boundary presumably consisting of lipoproteins. The globules are discharged into the lumen of the hepatopancreatic tubules by a kind of apocrine secretion. There, they coalesce to form larger masses. Finally, these lipid masses are transported into the oleospheres through a valve-like structure. The continual accumulation of lipid results in a drastic expansion of the oleospheres up to 500 pm in diameter. The absence of food in the digestive tract and the inactivity of the digestive enzyme producing F-cells indicate that digestion is suspended in the period of oleosphere formation. The curious mode of lipid storage in T. schmidtii may represent an adaptation to the extreme environmental conditions of a cave.

The hepatopancreas (midgut gland, digestive gland) of decapod crustaceans is a major storage organ (Gibson and Barker, '79; Dall and Moriarty, '83). The most frequent storage products are the nutrient reserves, namely glycogen and lipids (Loizzi, '71; Storch and Welsch, '77). The hepatopancreas is further able to deposit heavy metals (Gibson and Barker, '79). The nutrient reserves serve to compensate for short-term starvation (Storch and Anger, '83; Vogt et al., '851, and to supply energy and metabolites for moulting (Al-Mohanna and Nott, '89) and vitello- genesis (Adiyodi and Subramoniam, '83).

The reserves of the hepatopancreas are synthesized and stored in the absorptive R-cells (Vogt et al., '85; Al-Mohanna and Nott, '87). In the other hepatopancreato- cytes, the embryonic E-cells, the digestive enzyme producing F-cells, and the excretory B-cells, lipids and glycogen occur only rarely or under extreme nutritional conditions such as refeeding after prolonged starvation (Vogt et al., '85). The type of storage product seems to depend on both the species and the food. In most decapods, glycogen and lipids are found together in the R-cells (Loizzi, '71; Storch and Welsch, '77). However, Penaeus mon-

odon never contains considerable amounts of glycogen, even when the individual has been fed a diet rich in carbohydrates. Upon feeding on lipid-enriched diets the R-cells accumulate enormous lipid reserves, whereas nei- ther glycogen nor lipids are stored after a pure protein food (Vogt, '85; Vogt et al., '85; '86).

In all decapod species investigated thus far, the reserve lipids are stored intracellu- larly as oil globules (Gibson and Barker, '79; Dall and Moriarty, '83). Similar mechanisms of lipid deposition are well known from other invertebrates (e.g., insects: Odhiambo, '67; molluscs: Walker, '70) or vertebrates (e.g., fish: Segner and Witt, '90; mammals: Chao et al., '86). In this paper, we report on a completely different mode of lipid storage which seems unique in the animal kingdom. In the cave-dwelling shrimp, Troglocaris schmidtii, reserve lipids are deposited extracellularly in specialized compartments of the hepatopancreas.

MATERIALS AND METHODS

Specimens of the shrimp T. schmidtii Dor- mitzer 1853 (Crustacea: Decapoda: Atyidae) examined in their natural habitat (Planina-

Q 1992 WILEY-LISS. INC.

32 G. VOGT AND J. STRUS

Cave, Slovenia, Yugoslavia) exhibited an unusual “bubbled” appearance of the hepatopancreas as observed through the transparent cuticle. Due to the geographically limited distribution (Balss et al., ’61) and potential en- dangering of the species, we took out only one animal to investigate the reason for the curious appearance of the hepatopancreas. This specimen was a male with immature gonads.

The gross morphology of the hepatopancreas was examined under a stereomicro- scope and photographed with Leitz Aristo- phot and Wild M420 macroscopes. For this purpose fresh and glutaraldehyde fixed material was used. The histology of the organ was evaluated by paraffin and resin sections. For paraffin methods, the tissues were fixed either in glutaraldehyde or Bouin’s fluid for several days, dehydrated in ethanol, and transferred through methylbenzoate into paraplast. Sections of 5 pm were stained with haematoxylin-erythrosin (HE). Semithin resin sections (0.5-1 pm) were cut with glass knives from the blocks processed for electron microscopy and stained with methylene blue- azur I1 (Richardson solution). Documenta- tion was performed with a Leitz-Aristoplan light microscope.

For electron microscopy, tissues were fixed in 3.5% glutaraldehyde in 0.1 M Sorensen’s buffer at pH 7.4 overnight, rinsed in Soren- sen’s buffer, postfixed in 1% osmium ferrocy- anide for 2 h, successively washed in Soren- sen’s buffer and 0.05 M maleate buffer at pH 5.2, en bloc stained with 1% uranyl acetate in maleate buffer for 2 h, dehydrated in a series of ethanol, and embedded in Spurr’s resin. Ultrathin sections were prepared with a dia- mond knife on a Reichert-Jung OM-2 ultrami- crotome and contrasted for 5 min with lead citrate prior to examination under a Zeiss EM 9-S2 electron microscope.

RESULTS

The shape of the anterior digestive tract of T. schmidtii principally fits the anatomical scheme published for other shrimps. In the specimen investigated no food was found in the stomach. In contrast, the gross morphology of the hepatopancreas differs from other decapods in respect to large oil-containing compartments (Fig. l a ) which are referred to as “oleospheres” in the following. These compartments are located either at the blind ending distal tips of the hepatopancreatic tubules or laterally a t the distal tubular re-

Fig. 1. Gross-morphology of the hepatopancreas of Troglocaris schmidtii. Macrophotographs were taken from glutaraldehyde fixed material. a: Oleospheres (0) dominating the macroscopic appearance of the organ. ~ 1 5 . b: Mature (MO) and immature (10) oleospheres located near the blind ending tips of the hepatopancreas

tubules (HT). ~ 2 6 . c: Growing oleosphere. Each oleosphere consists of a central lipid mass (OL) surrounded by an epithelium (OE) with only few small lipid droplets. Arrow depicts lipid globules in the hepatopancreas epithelium. x72.

EXTRACELLULAR LIPID STORAGE IN SHRIMP 33

Fig. 2. Histology of the oleospheres and structural relation with the hepatopancreas tubules. Light microscopy. a: Terminal oleosphere. The oleosphere cavity (OC) which appears collapsed due to lipid extraction is separated from the tubule lumen (TL) by a valve-like structure (V) with an iris-shaped aperture (arrow). HS, hemolymph space; OE, oleosphere epithelium. Longitudinal paraffin section, ~380. b: Oleosphere epithelium with several nuclei (N). Paraffin section, X530. c: Hepatopan-

creas tubule with oleospheres (01, luminal lipid masses (LL), and intracellular lipid globules (arrowheads). The oleosphere epithelium appears broken due to lipid expansion by osmication. Resin section, x 125. d Continuity of lipid masses of oleosphere (OL) and tubule lumen. The electron dense boundary of the lipid masses extends from the oleosphere through the valve into the tubule lumen (arrow). BC, B-cell; LG, lipid globule. Resin section, x 480.


Figure 3


gions (Fig. lb). They can reach diameters up to 500 Fm. Both mature and maturing oleospheres can be observed along the same hepatopancreatic tubule (Fig. lb). They are composed of a central lipid mass surrounded by a narrow epithelium. This construction is particularly visible in growing oleospheres (Fig. lc). The oleospheres of fresh tissues release an oily fluid when punctured with a needle.

Both lipid extracted paraffin sections and lipid conserving resin sections were used in order to evaluate the structural relation of the hepatopancreas tubules and the oleospheres. The paraffin material is suitable for an investigation of the general organization of the organ and particularly of the oleosphere epithelium. Resin sections give better insight into intracellular synthesis of the lipids, and their discharge and extracellular storage.

On paraffin sections, each oleosphere appears as large lipid-extracted cavity that is surrounded by a narrow multinucleate epithelium (Fig. 2a,b). The oleosphere epithelium is continuous with the epithelium of the hepatopancreas tubules (Fig. 2c), and both are enveloped by a common basal lamina located at the hemolymph side. Resin sections indicate that the cavities of mature oleospheres are completely filled with enormous lipid masses (Fig. 2c) which principally display the same organization as the intracellular lipid globules. An extended electron light center is limited by an electron dense rim (Fig. 2c,d). The cavity is separated from the lumen of the hepatopancreas tubule by a valve-like struc-

Fig. 3. Intracellular synthesis and apocrine secretion of lipid globules. Light (LM) and electron microscopy (EM). a: Overview of the hepatopancreas epithelium containing B-cells (BC) with electron lucent vacuoles, R-cells (RC) with lipid, and F-cells (FC) with large nuclei (N). The lipid globules (LG) are limited by electron dense boundaries. HS, hemolymph space; LL, luminal lipid; LM, resin section. ~ 7 3 0 . b: Formation of large lipid globules by integration of smaller lipid droplets (arrowheads). LM, resin section. ~ 8 2 0 . c: Membrane profiles (arrowheads) terminating at the periphery of lipid droplets (LD). EM, x 15,710. d Small lipid droplet coalescing with a larger one (arrow). Although very small, the depicted droplets display already a differentiation in a light center and a dense periphery. Arrowheads: membrane profiles; G1, glycogen. EM, ~ 3 8 , 7 0 0 . e: Apocrine secretion of a lipid globule. The microvillous border and apical cytoplasm portions (arrows) are discharged into the tubule lumen (TL) together with the lipid globule. LM, resin section. x 700. f: Apocrine secretion of a lipid globule and fusion with luminal lipid masses. The basal cell body is separated from the extruded apical part by a plasma membrane (arrows). CD, cell debris. EM, x 7,130.

ture (Figs. 2a,d). Such structures are generally free of cell nuclei as investigated by serial sections of numerous tubules. On longitudinal sections, continuous lipid masses were found extending from the cavity through the opening of the valve into the lumen of the tubules (Fig. 2d).

Electron microscopy reveals that intracellular lipid is found only in the R-cells and, in much smaller amounts, in the oleosphere epithelium. The R-cells can clearly be identi- fied by cytological characteristics such as the amount of cell organelles, their distribution within the cell, and the basal tubule system (details: Vogt, ’85). In the following, the small intracellular lipid inclusions are referred to as “lipid droplets,” and those measuring several p,m as “lipid globules.” The lipid globules are located mostly in the apical and medium parts of the cells, and the nuclei at the cell bases (Fig. 3a,e). Mature lipid globules consist of a homogenous and relatively light central part of several p,m in diameter and a smaller, 0.5-2 km thick electron dense periphery (Fig. 3a,e). They can reach a total diameter of 50 Frn and more. Growing lipid globules are mostly surrounded by small and medium-sized lipid droplets (Fig. 3b) and/or numerous membrane profiles (Fig. 3c). The small lipid droplets seem to fuse with each other or with the bigger globules (Fig. 3b,d). The membrane-like structures most fre- quently occur in close association with growing lipid droplets that have not yet gained their electron dense boundary, and often in- terconnect neighbouring lipid compartments (Fig. 3c). Even those lipid droplets and globules which already have a broad electron dense periphery display connections with membrane profiles (Fig. 3d). These membranous structures are free bilayers and not part of membrane limited cell compartments such as the endoplasmic reticulum.

Mature lipid globules are released into the lumen of the hepatopancreatic tubules by a kind of apocrine secretion (Fig. 3e,fJ. Prior to lipid extrusion, the apical part of the R-cells is separated from the basal region by a plasma membrane (Fig. 3fJ. In the course of apocrine secretion of the lipid globules, the microvillous border ruptures and smaller portions of the apical cytoplasm are expelled together with the lipid (Fig. 3e,f). Nuclei are never included in this cell debris. As the discharged lipid globules fuse with other lipid masses of the tubule lumen, the outer electron dense material remains always at the periphery


(Fig. 30. The expelled cell remnants are reab- sorbed by the B-cells by endocytosis and are apparently degraded (Fig. 4b).

The digestive enzyme producing F-cells display numerous profiles of rough endoplasmic reticulum. Compared to synthesizing F-cells (Vogt et al., '89a), the dictyosomes are small and inconspicuous and enzyme containing vacuoles are lacking (Fig. 4a). These cytological signs indicate an inactive status of the F-cells.

DISCUSSION

The storage of lipids as energy reserve is a widespread mechanism in animals. Lipids are deposited either in the epithelia of the digestive tract or in specialized organs. Particu- larly, invertebrates show the former process (Walker, '70; Hammersen and Pokahr, '72). In vertebrates, lipids are mainly stored in the adipose tissue (Johnson and Greenwood, '88). In fish, considerable amounts of lipid can be deposited also in the liver (Segner and Witt, '90).

Arthropods show various modes of lipid storage. Insects deposit glycogen and lipids mainly in a specialized organ, the fat body (Odhiambo, '67). Arachnids have a similar organ, the intermediate tissue, but additionally accumulate lipids in the digestive cells of the midgut gland (Ludwig and Alberti, '90). Crustaceans display a wide variety of storage mechanisms due to their great diversity of body architecture. The cladocerans, for in-

stance, store lipid in the enterocytes (Elendt and Storch, 'go), isepods in the large cells of the midgut gland (Strus et al., '851, and am- phipods (Storch and Burkhardt, '84) and decapods (Vogt et al., '85) in the R-cells of the hepatopancreas.

In the cave-dwelling shrimp Troglocaris schmidtii the decapod scheme of lipid storage is extended. The intracellular amount of lipid observed in the hepatopancreas cells already exceeds that of other decapods. Additionally, huge lipid sources are deposited in specialized organs, the oleospheres. Oleosphere cavities have to be considered as extracellular since they are continuous with the lumen of the hepatopancreas tubules. They are, to our best knowledge, the first example of extracellular lipid storage in animals. The closest similarity to this unusual mode of lipid storage is found in calanoid copepods. There, comparably large amounts of lipids are stored in so-called lipid sacs, which are located either around the anterior midgut or in the posterior region of the metasome without direct contact to the midgut. This kind of lipid storage is considered intracellular. The lipid sacs are not structurally related to the digestive tract, and there is no apocrine lipid secretion (Blades-Eckelbarger, '91). Consid- ering the huge homogenous lipid mass, they resemble the oleospheres of T. schmidtii; considering all other aspects, they more resemble the fat body of insects or the intermediate tissue of arachnids.

Fig, 4. Role of F- and B-cells during lipid secretion. a: endocytosis of membranous material from the tubule Central area of an F-cell characterized by rough endoplas- lumen. MB, microvillous border; TL, tubule lumen. mic reticulum (rER) and a comparatively small Golgi X13,480. body (GI. ~ 2 3 , 5 3 0 . b: B-cell apex. The arrow denotes the


In fresh tissues, the oleosphere lipid of T. schmidtii has an oily consistency. According to Chang and O'Connor ('83) the principal storage products in crustaceans are triglycerides, with the exception of wax esters in planktonic copepods. The moderately electron dense lipid in the center of the lipid compartments of T. schmidtii closely resem- bles the intracellular lipid droplets of other decapods (Vogt, '85; Al-Mohanna and Nott, '87); therefore, it may be composed of triglycerides. The electron dense peripheral lipid, however, has a completely different ultra- structural appearance. Its high electron density and peripheral position near the aqueous cytoplasm suggest that it represents phospholipids.

The gross morphology, histology, and cytology of the hepatopancreas of T. schmidtii permit one to deduce the mechanisms of synthesis, discharge, and extracellular storage of lipid. The lipid droplets arise and grow in the R-cells of the hepatopancreatic epithelium. The initiation of the formation of lipid droplets remains obscure, as in other lipogenic tissues (Slavin, 72; Bell et al., 81; Nedergaard and Lindberg, '82). Their further growth is ensured by fusion with other lipid droplets.

A rather curious feature is the aposition of the electron dense boundary of the lipid droplets and globules which is regularly associated with the presence of membrane structures. Such membranes are often interpreted as artifacts. However, we assume that these membranes may represent aggregations of phospholipids which assemble in the cytoplasm and disintegrate at the surface of the lipid droplets to build up an electron dense boundary. Bishop and Bell ('88) and Blanchette-Mackie and Scow ('81) reported on the capability of phospholipids and fatty acids to form membrane-like bilayers if present in high concentrations. Such membrane structures are thought to provide the structural basis for lateral lipid transport occurring in the membrane leaflets. This mode of lipid transport is highly effective and much faster than other transport mechanisms (Blanchette-Mackie and Scow, '81; Sleight, '87). The electron dense peripheral layer of the lipid droplets may function as interface between the lipid and the aqueous cytoplasm, particularly since i t is found around almost all lipid deposits of animals, however, in much smaller dimensions (Odhi- ambo, '67; Johnson and Greenwood, '88; Seg- ner and Witt, '90). Furthermore, it may be

important for the coalescence and stability of the lipids in the extracellular areas.

Apocrine secretion discharges the mature lipid globules into the lumen of the hepatopancreatic tubules. The apocrine type of secretion generally includes loss of cytoplasmic portions (Saacke and Heald, '74; Kurosumi et al., '84). In T. schmidtii the secretion process starts with the protrusion of the lipid globule toward the lumen. At the same time, a horizontal membrane is formed which sep- arates the cell apex from the nucleus-containing cell base. Then, the microvillous border ruptures and the lipid globule and apical cytoplasmic constituents are released into the tubule lumen. There, the lipid fuses with further lipid masses. The discharged cell debris seems to be endocytosed by B-cells for degradation. The R-cell apices may be regen- erated by the cell bases, as the discharged material never contains cell nuclei. No holo- crine or merocrine secretion of lipid was observed.

The lipid masses are then transported into the oleospheres through an opening in a valve-like structure. Oleospheres consist of a multicellular epithelium, a central cavern, and a valve without nuclei. They can be considered as an integral part of the hepatopancreas as their epithelium is continuous with that of the hepatopancreatic tubules; both are enveloped by a common basal lamina. The present observations lead us to assume that the oleospheres derive from the hepatopancreatic epithelium. The cell bases may differentiate into the oleosphere epithelium, and the apices into the valve. However, more material has to be examined finally to answer this question.

The mode of lipid synthesis, secretion, and storage in T. schmidtii strictly excludes par- allel food ingestion since secretion of digestive enzymes and absorption of food takes place in the hepatopancreas tubules as well (Vogt et al., '85; Al-Mohanna and Nott, '87; Vogt et al., '89a). Consequently, in our specimen the stomach was free of food and the F-cells lacked the typical cytological signs of enzyme synthesis (Vogt, '85; Vogt et al., '89a). If no food is absorbed during lipid synthesis, the metabolites must derive from other sources such as transient glycogen or amino acid stores in the hepatopancreas, in the blood, and in other organs in which they may have been deposited during an earlier feeding event. According to Chang and O'Connor ('83) lipid synthesis in crustaceans is inti-


mately associated with the catabolism of carbohydrates. In T. schmidtii electron lucent areas typically surround growing lipid droplets. These areas may reflect remnants of glycogen stores emptied by lipogenesis, particularly because they occasionally display small glycogen deposits.

The last topic to be discussed is the puta- tive physiological and ecological relevance of the curious mode of lipid storage. As men- tioned in the opening paragraphs, lipid deposits in crustaceans are set up to provide energy and metabolites for starvation periods, moulting, and reproduction. As oleospheres were found only in T. schmidtii thus far, they seem to represent an adaptation to the cave environment. The total amount of pure lipid accumulated in the oleospheres of one individual approximates somewhat more than 10 mm3. This appears to be an enormous energy reserve for an animal with a maximal total length of only 3 cm (Dormitzer, 1853). The huge amount of stored lipid is even more impressive if it is considered that troglobites generally have a lower level of activity than related epigean species (Ahearn and Howarth, '82). Because the food in caves will only be available at irregular intervals, troglobites likely take up much greater amounts of food per feeding interval than do their epigean relatives (Trimmel, '68). The excess energy is mostly stored as lipid (Howarth, '83).

The parallelism of oleosphere formation and the early maturation stage of the testis in our specimen could also suggest that the lipid stores have a possible role in gonadal maturation. Results obtained from other decapods indicate a major role of the hepatopancreas in reproduction, particularly in vitello- genic females (Adiyodi and Subramoniam, '83; Vogt et al., '89b). The large lipid sacs in calanoids are interpreted both in terms of starvation and reproduction, depending on the species (Blades-Eckelbarger, '91).

In T. schmidtii the precise role of the oleospheres will require the investigation of more animals in their habitat. As the internal organs are visible through the transparent cuticle, the evaluation of the relationship between oleosphere abundance and develop- mental stage of the gonads should be feasible without killing the animals. Further fields of future research are the analysis of the lipid composition, the cytology and histochemistry of its synthesis, the breakdown of the oleosphere lipid, and the origin of the oleospheres. Such problems could be resolved by

investigating a limited number of specimens selected in the framework proposed here.

ACKNOWLEDGMENTS

The authors thank A. Brancelj for the guid- ance into Planina cave, and Marika Suhm and Gisela Adam for careful technical and photographic assistance.

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