JOURNAL OF MORPHOLOGY 259:340–346 (2004)

Comparative Electron Microscopic Study of the Stomach of cavimana and sylvaticus (Crustacea: )

Jasna Sˇ trus1* and Volker Storch2

1Department of Biology, University of Ljubljana, 1001 Ljubljana, Slovenia 2Zoologisches Institut, D 69120 Heidelberg, Germany

ABSTRACT The functional morphology of stomachs of and also from that of other , Euphausia- the European semiterrestrial amphipod Orchestia cavi- cea, and . Terrestrial isopods are well mana and of the Australian terrestrial species Arcitalitrus known for their complex stomach morphology. Dif- sylvaticus was studied by electron microscopy. The stom- ferences in the structure and function of the stom- ach of the two amphipod species is divided longitudinally achs in amphibious and terrestrial isopods were de- into a spacious dorsal food channel and two ventral filtra- ˇ tion channels. Additionally, a prominent helically oriented scribed by Strus et al. (1995). Studies by Coleman circulation channel is situated on each lateral side of the (1992) show that the stomachs of amphipods in gen- stomach, forming a semicircular channel separated from eral have a basic pattern, which in certain groups the food channel by spines. The food channel conveys could be related to food strategies. He described the coarse food particles directly into the midgut through a setation of lateralia as an important character that funnel. The filtration channels receive fine material fil- might indicate phylogenetic proximity among the tered through primary and secondary filters. Material families he examined. Comparative anatomical forced through the secondary filters by the pressure of the study of the stomach of hyperid amphipods laterally located inferolateralia eventually reaches the openings of the midgut glands. Washing of filters and (Coleman, 1994) showed that its structure is soaking of ingested food items with enzymes probably is strongly aberrant and the filtering capacity is re- achieved by a forward stream of digestive juice from the duced. It was proposed that the foregut morphology midgut glands and conveyed through the circulatory chan- could be helpful in amphipod classification. nels. The specializations of the stomach of the two species Since the amphipod stomach has been studied of Amphipoda investigated are described and compared to mainly in aquatic species, a study of a semiterres- the pertinent structures of Mysidacea and . J. trial species, Orchestia cavimana, and a truly ter- Morphol. 259:340–346, 2004. © 2004 Wiley-Liss, Inc. restrial species, Arcitalitrus sylvaticus, was under- taken. O. cavimana has proliferated along European KEY WORDS: Orchestia cavimana; Arcitalitrus sylvati- cus; amphipods; stomach; electron microscopy river banks, starting from southeast Europe (Kin- zelbach, 1972; Fischenich, 1979). Our investigations showed that one reason for their success is that they feed indiscriminately on a range of food items. As Morphology of peracaridean alimentary systems interpreted from the ultrastructure of the R-cells of has attracted the attention of several the midgut glands, this species can metabolize cel- biologists (Schmitz and Scherrey, 1983; Schmitz, lulose (Storch and Burkhardt, 1984, 1986) better 1992; Wa¨gele et al., 1981; Wa¨gele, 1992; Coleman, ˇ than terrestrial isopods (Beck and Friebe, 1981), 1994; Strus et al., 1995; De Jong-Moreau, 1998). although the amphipods have been less successful However, our understanding of the most complex ecologically. Most semiterrestrial Amphipoda are re- part of the alimentary canal, the stomach, is still insufficient from the ultrastructural and functional as well as phylogenetic viewpoints. Only a few work- ers (Icely and Nott, 1984; Storch, 1987, 1989; Storch Contract grant sponsor: Deutsche Forschungsgemeinschaft; Con- ˇ tract grant number: DFG Sto 74/4 and 11; Contract grant sponsor: and Strus, 1989; Kobusch, 1998) have used electron Slovenian Ministry of Education, Science and Sport; Contract grant microscopy to analyze the peracaridean stomach. number: MSˇ ZSˇ PO-0525. The structure of the stomachs was studied in differ- ent species of Mysidacea and it was shown that the *Correspondence to: Jasna Sˇ trus, Department of Biology, Univer- morphology of the stomach is characteristic of the sity of Ljubljana, Vecˇna pot 111, 1001 Ljubljana, Slovenia. genus and not related to the ecology of the species E-mail: [email protected] (De Jong, 1996; De Jong-Moreau and Casanova, 2001). In Lophogastrida the fine structure of the stomach differs within genera of the same suborder DOI: 10.1002/jmor.10216

© 2004 WILEY-LISS, INC. EM STUDY OF AMPHIPOD STOMACH 341 stricted to fairly narrow niches. Landhoppers, truly terrestrial species of the family , inhabit the leafmold of tropical and cold-temperature forests (Hurley, 1968). Where they are present, talitrids form an important element of the leaf-litter fauna, e.g., in the wetter forests of Australia and other regions of the southern hemisphere. About 120 spe- cies live independently of water bodies (Friend and Richardson, 1986). Most of these are as terrestrial as woodlice; their diet is generally unspecialized and consists principally of decayed plant material. The purpose of the current research was to de- scribe the complexity of the stomachs of the two ecologically different amphipod species and compare it to the stomachs of other peracaridean crusta- ceans.

MATERIALS AND METHODS Orchestia cavimana Heller, 1865, was collected from the banks of river Rhine in Germany, and Arcitalitrus sylvaticus Haswell, 1978, was sampled from rotten logs at Mt. Tomah in New South Wales, Australia. Dissections of the stomachs were conducted under a binocular dissecting microscope. Complete unopened and opened stomachs were prepared for light and electron microscopy. For electron microscopy, whole stomachs and isolated parts were fixed in 3.5% glutaraldehyde in So¨rensen phosphate buffer (pH 7.5) for2hat4°C.Thefixed material was repeatedly rinsed in buffer, postfixed in buffered 1% osmium tetroxide, and dehy- drated through a graded series of ethanol. For scanning electron microscopy, opened stomachs were critical point-dried, sputter- coated with gold, and examined with a Cambridge SEM S4-10 microscope. The samples for transmission electron microscopy were embedded in Araldite. Semithin sections were stained with methylene blue-Azure II after Richardson et al. (1960). Ultrathin Fig. 1. a: Schematic presentation of amphipod stomach based sections were stained for 5 min with uranyl acetate and lead on micrographs of the foreguts in Orchestia cavimana and Arci- citrate (Reynolds, 1963). They were examined in a Zeiss EM 9-S2 sylvaticus. b: Gross morphology of the stomach in O. electron microscope. cavimana. The food channel (FC) is filled with food, the filtering channel (FiC) situated ventrally drives filtrate to the secondary filter (SF) region. Circulation channel (CC) is free of food parti- RESULTS cles. Scale bar ϭ 150 ␮m. Abbreviations for Figures 1–3: C, cardiac chamber; E, esophagus; F, funnel; IL, inferolateralia; IM, The following description of the stomach is inferomedianum; L, lateralia; PF, primary filter; P, pyloric cham- based on freshly dissected specimens, serial sec- ber; SF, secondary filter. tions, and scanning electron microscopic prepara- tions. Although we have selected to follow the descriptive scheme of Kanneworff and Nicolaisen the publications of Agrawal (1965) and Schmitz and (1969) and Icely and Nott (1984), phylogenetic Scherrey (1983). A longitudinal section of the stom- considerations require a terminology that may ach of Arcitalitrus sylvaticus shows that it is subdi- eventually make it possible to understand homol- vided into cardiac, pyloric, and funnel regions (Fig. ogies among the . The anatomical no- 2a). However, to better understand the functioning menclature is based primarily on the work of of the stomach the subdivision into food, filtering, Scho¨nichen (1899), Scheloske (1976), and Licˇar (1977), who performed detailed studies on the and circulation channels should be taken into ac- anatomy of the isopod stomach. count (Fig. 1b). Coarse food particles are transported The sac-like stomach of Orchestia cavimana and through the food channel running along the roof of Arcitalitrus sylvaticus comprises about one-third of the stomach and are eventually conveyed to the the alimentary canal and extends from the esopha- midgut. Fluid may pass through complicated ventral gus to the funnel region (Fig. 1a,b) which protrudes filtration channels (Fig. 3a) leading to the midgut into the midgut. The whole stomach is lined by a glands where it is absorbed. In the midgut glands, cuticle of variable thickness and structure. Several digestive enzymes are produced and transported by folds project into the lumen, thus making cross- the circulatory channels which begin on each ven- sections difficult to interpret, as can be seen from trolateral side, surround the stomach helically, and 342 J. Sˇ TRUS AND V. STORCH

Fig. 2. a: Longitudinal sec- tion of the stomach in Arcital- itrus sylvaticus consisting of the cardiac (C), pyloric (P), and fun- nel (F) regions with lateralia (L), inferolateralia (IL) and filtering compartments (PF, primary fil- ter; SF, secondary filters). SEM. Scale bar ϭ 150 ␮m. b: Parallel rows of filtering setae of paired secondary filter. Scale bar ϭ 10 ␮m. c: Filtering setae of the pri- mary filter (PF) along the an- teromedianum (A). Scale bar ϭ 20 ␮m. d: Basal parts of the pri- mary filter setae on the ventro- lateral wall of the stomach. Scale bar ϭ 5 ␮m. merge anteriorly into a single dorsomedial channel grooves on each side of the inferomedianum are cov- (Fig. 3b). ered by a secondary filter consisting of two sets of The cardiac region is characterized by two large filtering surfaces composed of parallel rows of comb- lateral outpocketings, the lateralia, with several like filter bars about 1 ␮m apart (Fig. 2a,b). The bars rows of setae and two rows of strong spines (Fig. are Y-shaped, with the setous arms of Ys inter- 4a–d). They have been described as “lateral ridges” twined at their tips (Fig. 5a–c,e). The width of the (Agrawal, 1965), “lateral ampullae” (Icely and Nott, slits between the setae is ϳ0.1 ␮m. On either side of 1984), or “pushers” (Kanneworff and Nicolaisen, the inferomedianum are ventrolateral ridges, the 1969). Another essential feature of the cardiac re- inferolateralia, which are covered medially by gion is a ventrally located primary filter consisting densely arranged setae (Fig. 5d,e). They occupy a of two parallel channels separated by a ventrome- major portion of the ventral side of the pyloric re- dial ridge, the anteromedianum, and covered by par- gion. The space between the inferomedianum and allel rows of filtration setae (Fig. 2c,d). Posteriorly, inferolateralia receives fluid from the primary filter these channels gradually change position and con- grooves. vey their contents either to the midgut glands ven- The border between the pyloric and the funnel trally or into the dorsal unpaired channel, the func- regions is made up of long bristles forming a coarse tion of which is explained below. sieve (Fig. 2a). The bristles separate the two com- The pyloric region is characterized by a large ven- partments of the stomach and delimit two circula- tromedial ridge, the inferomedianum. Paired filter tory channels running helically around the stomach. EM STUDY OF AMPHIPOD STOMACH 343 isopod Porcellio scaber, on the other hand, the mas- ticatory teeth lie on the ventral side of the lateralia opposite to corresponding teeth on the ventral face of the stomach. However, this was not observed among terrestrial isopods in general (Storch and Sˇ trus, 1989; Sˇ trus et al., 1995). Lateralia equipped with spines are present in stomachs of various peracarid- ean , but their armature differs among the species. The armature of lateralia seems to be related to the feeding strategies and can also offer a good trait for phylogenetic reconstructions (Coleman, 1992). Peracaridean crustaceans have a primary filter of different complexity in the ventral wall of the car- diac region. In Orchestia and Arcitalitrus, the pri- mary filter consists of simple spines that are ar- ranged at a distance of several ␮m, whereas the primary filter in isopods is a complex straining de- vice made of intertwined Y-shaped spines allowing only very small particles to pass through the meshes (Storch and Sˇ trus, 1989). The inferomedianum with lateral secondary fil- ters is another feature that pericaridean groups have in common. In Amphipoda, each side has a two-tiered filter, whereas in isopods there is only one on each side of the inferomedianum. The filter meshes of isopod filters are finer than those of amphipods. The straining and pressing mecha- Fig. 3. a: The ventrolateral part of the stomach in Orchestia nisms, by which inferolateralia exert pressure cavimana with laterale (L) positioned anteriorly and a ventral upon the secondary filter, seem to be identical in channel (arrow) covered with setae. SEM. Scale bar ϭ 20 ␮m. both groups. b: The roof of the stomach in O. cavimana with paired dorsal The main difference between the stomach of Am- channels (arrows point anteriorly) merging into single dorsome- dial channel. SEM. Scale bar ϭ 50 ␮m. phipoda and Isopoda can be found in the architec- ture of the dorsal wall with the circulatory channel and in the existence of the long funnel region in the stomach of amphipods. The circulatory channel is Following the interpretation of Icely and Nott absent in isopods which, on the other hand, have an (1984), they begin laterally, allowing digestive fluids unpaired, posteriorly oriented lamella dorsalis and to enter the stomach and run dorsally where they paired lateral lamellae annulares that protrude into enter the cardiac region. The funnel region of the the hindgut (Sˇ trus et al., 1995). stomach is a long tube surpassing the anterior re- Based on these findings and earlier observations gion in its length and projecting deeply into the on the stomachs of three species of the Mysidacea midgut. Midventrally it is open, allowing food to (Storch, 1989) and four species of the Isopoda pass directly into the midgut. (Storch, 1987; Storch and Sˇ trus, 1989; Sˇ trus et al., 1995), the following seems a plausible explanation of DISCUSSION how the stomachs of these Peracarida and especially of land-dwelling Amphipoda function in general. The stomachs of the semiterrestrial amphipod, Food is conveyed from the anterior to the posterior Orchestia cavimana and the terrestrial species Ar- stomach through the dorsal food channel. The lat- citalitrus sylvaticus have many features in common. eralia, which lie close to the junction of esophagus However, their external design is different. Both and stomach, seem to be very effective in masticat- have lateralia composed of a multilayered epithe- ing and pressing coarse food items. This is indicated lium covered with elaborate cuticular setae and by their very strong spines and the muscle fibers, strong spines. The midventrally oriented strong which allow effective strokes. In this respect, the spines of the lateralia in both species may serve as a lateralia of Orchestia cavimana and Arcitalitrus syl- crushing structure. Since they lie directly behind the vaticus seem to be more effective than those of My- junction of the esophagus and the stomach, they are sidacea and Isopoda. Ventrally, primary and second- in the best position to fulfill this putative function. ary filters retain first coarse, and second fine Extrinsic muscle fibers allow the lateralia to move particles. The paired secondary filter surfaces of am- apart or to approach each other. In the terrestrial phipods are probably involved in even better sepa- 344 J. Sˇ TRUS AND V. STORCH

Fig. 4. a: Lateralia in the stomach of Arcitalitrus sylvati- cus, ventral surface with two rows of spines and several rows of setae. SEM. Scale bar ϭ 50 ␮m. b: A lateral spine in Orches- tia cavimana. TEM. c: An epi- thelial cell of the inferomedia- num in O. cavimana with large amounts of glycogen (G) and con- centric membranous bodies of endoplasmic reticulum (ER). TEM. d: The region below the inferolaterale in O. cavimana. TEM. Interdigitation (arrows) of muscle fiber (MF) and epithelial cell (EC) with numerous setae (S) and microtubules (M). Scale bars in b,c,d ϭ 1 ␮m. ration of fluid food items. The filtered food eventu- been well established in the Decapoda (Vogt et al., ally is conveyed into the midgut glands. However, 1989). amphipods differ from mysids and isopods in their The study of the fine structure of stomachs in two well-elaborated circulatory channel system, which ecologically different talitrid species shows that gross allows digestive fluids to reach the anterior portion morphology as well as their ultrastructure are very of the food channel also, and which are recirculated much alike. Differences in the fine structure of the through the ventral channels. The spines covering amphipod stomach compared to other Peracarida were the medial faces of the inferolateralia possibly pre- observed predominantly in the armature of the latera- vent the fine secondary filters from clogging. The lia, position and ultrastructure of the filters, arrange- spines differ considerably from the brush-like struc- ment of lamellae, and the presence of circulatory chan- tures in the Mysidacea (Storch, 1989) and are simi- nels and a funnel region. From a phylogenetic lar to those found in the Isopoda (Storch, 1987). viewpoint, the description of gradual transformation of Whether the initial steps of chemical breakdown of the complex stomach within Peracarida may contrib- food items really takes place in the stomach of the ute to a better understanding of the phylogeny of this Peracarida has not been proven. However, this has group and of all other . EM STUDY OF AMPHIPOD STOMACH 345

Fig. 5. Secondary filters of Arcitalitrus sylvaticus and Or- chestia cavimana. a: Setae of the filtering surface (see Fig. 2b) in A. sylvaticus SEM. Scale bar ϭ 0.5 ␮m. b,c: Y structures of filter setae and inferomedianum (ar- row) in O. cavimana. TEM. Scale bars ϭ 1 ␮m. d: The surface of the inferolaterale covered with setae in A. sylvaticus. SEM. Scale bar ϭ 20 ␮m. e: Y struc- tures of secondary filter and se- tous inferolaterale (arrow) in A. sylvaticus. TEM. Scale bar ϭ 1 ␮m.

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