ORIGINAL ARTICLE

Ultrastructural changes of the midgut epitheliumin Isohypsibius granulifer granulifer Thulin, 1928(Tardigrada: Eutardigrada) during oogenesis

Magdalena M. Rost-Roszkowska & Izabela Poprawa &

Maria Wójtowicz & Łukasz Kaczmarek

Received: 15 June 2010 /Accepted: 14 July 2010 /Published online: 27 July 2010# Springer-Verlag 2010

Abstract The midgut epithelium of Isohypsibius granu-lifer granulifer (Eutardigrada) is composed of columnardigestive cells. At its anterior end, a group of cells withcytoplasm which differs from the cytoplasm of digestivecells is present. Probably, those cells respond to crescent-like cells (midgut regenerative cells) described for sometardigrade species. Their mitotic divisions have not beenobserved. We analyzed the ultrastructure of midgutdigestive cells in relation to five different stages ofoogenesis (previtellogenesis, beginning of the vitellogen-esis, vitellogenesis—early choriogenesis, vitellogenesis—middle choriogenesis, late choriogenesis). In the midgutepithelium cells, the gradual accumulation of glycogengranules, lipid droplets and structures of varying electrondensity occurs. During vitellogenesis and choriogenesis,in the cytoplasm of midgut cells we observed theincreasing number of organelles which are responsiblefor the intensive synthesis of lipids, proteins and saccha-rides such as cisterns of endoplasmic reticulum and Golgi

complexes. At the end of oogenesis, autophagy alsointensifies in midgut epithelial cells, which is probablycaused by the great amount of reserve material. Midgutepithelium of analyzed species takes part in the yolkprecursor synthesis.

Keywords Epithelial cells . Midgut . Reserve material .

Autophagy . Isohypsibius . Tardigrada

Introduction

Tardigrada, also known as water bears, are small inverte-brates, closely related to arthropods and other members ofEcdysozoa, which are widespread in terrestrial, freshwaterand marine environments (Dewel et al. 1993; Kinchin1994; Dunn et al. 2008; Nelson et al. 2010). Their digestivesystem is composed of the foregut, midgut and hindgut(Kinchin 1994; Pirch and Greven 1994; Avdonina andBiserova 2003; Nelson et al. 2010). The foregut, whichconsists of a buccal apparatus (salivary glands, stylets,sucking pharynx) and esophagus, is responsible for drawingnourishments into the midgut (Dewel and Clark 1973a,1973b; Eibye-Jacobsen 1996, 1997, 2001; Dewel andEibye-Jacobsen 2006); the hindgut empties with the anusor cloaca (Dewel and Dewel 1979; Kinchin 1994; Pirch andGreven 1994). The midgut fulfills the role of a digestiveorgan, where processes of secretion, absorption andexcretion appear. The midgut epithelium of Tardigrada, asof the other invertebrates, is the first line of defense againstchanges of the environment, pathogens and excessive levelsof, for example, heavy metals and toxic substances. Inmany invertebrate groups, e.g. insects, arachnids, annelids,it is also important for the proper course of oogenesis,especially in the synthesis of vitellogenins (Kessel and

Handling Editor: Reimer Stick

M. M. Rost-Roszkowska (*) : I. Poprawa :M. WójtowiczDepartment of Animal Histology and Embryology,University of Silesia,Bankowa 9,40-007 Katowice, Polande-mail: magdalena.rost-roszkowska@us.edu.pl

I. Poprawae-mail: izabela.poprawa@us.edu.pl

Ł. KaczmarekDepartment of Animal Taxonomy and Ecology,A. Mickiewicz University,Umultowska 89,61-614 Poznań, Polande-mail: kaczmar@amu.edu.pl

Protoplasma (2011) 248:405–414DOI 10.1007/s00709-010-0186-9

Beams 1980; Eckelbarger 1983; Coons et al. 1989; Roselland Coons 1992).

Midgut ultrastructure has been analyzed in severalspecies of Tardigrada, Eutardigrada, e.g. Isohypsibiusprosostomus Thulin, 1928 (Parachela) (Greven 1976;Avdonina and Biserova 2003), Milnesium tardigradumDoyère, 1840 (Apochela) (Dewel and Clark 1973c; Dewelet al. 1993), Ramazzottius tribulosus Bertolani andRebecchi, 1988 (Parachela) and Paramacrobiotus richtersi(Murray, 1911) (Parachela) (Avdonina et al. 2007), Dactylo-biotus dispar (Murray, 1907) (Parachela) (Ząbczyk 2000;Rost-Roszkowska and Poprawa 2008), and Heterotardigrada,e.g. Echiniscus testudo (Doyère, 1840) (Pirch and Greven1994) and Echiniscus viridissimus Pèterfi, 1956 (Dewel et al.1988). Those studies are still performed in Tardigradaaccording to diet and the environment, but little is knownabout the correlation between midgut ultrastructure and thecourse of oogenesis. For this reason, our study is focused onsearching for morphological features of the midgut epitheli-um of Isohypsibius granulifer granulifer Thulin, 1928 whichis a hermaphroditic species (Węglarska 1987). We analyzedchanges of the midgut epithelium organization duringoogenesis at the ultrastructural level to decide if it takes partin oogenesis, especially in vitellogenesis and choriogenesis.The process of oogenesis of I. g. granulifer has beendescribed precisely by Węglarska (1987) and Poprawa(2010).

Materials and methods

Materials

Adult specimens I. g. granulifer were collected in ponds ofthe Botanical Garden of Jagiellonian University, Cracow,Poland.

Light and transmission electron microscopy

Specimens were fixed with 2.5% glutaraldehyde in 0.1 Mphosphate buffer at pH 7.4 (room temperature, 2 h),postfixed with 2% OsO4 in 0.1 M phosphate buffer (pH7.4, temp 4°C, 2 h), dehydrated in a graded series ofethanol (50%, 70%, 90%, 96%, 100% each for 15 min) andacetone (15 min) and then embedded in Epon 812. Semi-thin and ultra-thin sections were cut on a Leica ultracutUCT25 ultramicrotome. Semi-thin sections were stainedwith 1% methylene blue in 0.5% borax and examined withan OLYMPUS BX60 microscope. Ultra-thin sections afterstaining with uranyl acetate (10 min) and lead citrate(10 min) (Reynolds 1963) were examined with a HitachiH500 electron microscope at 75 kV.

A total of 47 specimens were analyzed, among which tenoccur in the previtellogenic stage, nine beginning ofvitellogenesis, ten vitellogenesis—early choriogenesis,eight vitellogenesis—middle choriogenesis and ten latechoriogenesis.

Histochemical methods

Detection of lipids

Semi-thin sections were stained with Sudan Black B atroom temperature (20 min) (Litwin 1985).

Detection of glycogen and polysaccharides (PAS method)

Semi-thin sections were treated with 2% solution ofperiodic acid to remove osmium (10 min at roomtemperature) and stained with Schiff’s reagent (24 h at37°C) (Litwin 1985).

Detection of proteins

Semi-thin sections were treated with 2% solution ofperiodic acid to remove osmium (10 min at roomtemperature) and stained with bromophenol blue (BPB)(24 h at 37°C) (Litwin 1985).

Results

The midgut of I. g. granulifer possesses a tube-like shapewithout distinct anterior and posterior parts. Its epithelium(Fig. 1a), which lies on the thin non-cellular basal lamina, iscomposed of columnar digestive cells (Fig. 1b). Theultrastructure of midgut digestive cells changes followingthe process of oogenesis.

In the anterior end of the midgut, a group of cells(Fig. 1a) with electron-lucent cytoplasm, which is poor inorganelles, is observed (Fig. 1d). The group lies on the thinbasal lamina of the midgut epithelium. Their cytoplasmpossesses only a single mitochondria, single autophago-somes, and cisterns of smooth and rough endoplasmicreticulum (RER) (Fig. 1d). The nucleus is oval with smallamounts of heterochromatin. Their ultrastructure does notchange according to oogenesis. Mitotic divisions of thosecells were not observed.

Stages of oogenesis in I. g. granulifer

Oogenesis has been divided into five stages according toWęglarska (1987) and Poprawa (2010) (Fig. 2a–e).

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Stage A: previtellogenesis

The germ cells localized in the vitellarium are connectedwith cytoplasmatic bridges. There is no reserved material inthe cytoplasm. At the end of this first stage, lipid dropletsare observed in the cytoplasm (Fig. 2a).

Stage B: beginning of the vitellogenesis

All cells localized in the vitellarium begin to synthesizereserve materials. The volume of each cell increases. It isdifficult to say which cell will be an oocyte (Fig. 2b).

Stage C: vitellogenesis—early choriogenesis

Some cells localized in the vitellarium start to grow intensely.These cells become oocytes. Oocytes and gonad wall cellssynthesize and secrete precursors of the chorion. In this stage,small parts of the chorion are produced (Fig. 2c).

Stage D: vitellogenesis—middle choriogenesis

The cytoplasm of the oocyte is rich in yolk. In this stage,the parts of the chorion are connected into a permanentlayer (Fig. 2d).

Fig. 1 Midgut epithelium of I.g. granulifer. a A fragment ofthe epithelium with digestive (e)cells. A group of cells (r) at theanterior end of the midgut.Midgut lumen (l). LM, bar=9 μm. b Midgut epithelium withits basal lamina (arrow), diges-tive cells (e). Microvilli (mv),nucleus of the digestive cell (n).TEM, bar=2.9 μm. c Midgutepithelium (e), midgut lumen (l),microvilli (mv), nucleus (n) withnucleolus (nu) in the digestivecell. TEM, bar =1.4 μm. d Theanterior part of the midgut. Agroup of crescent-like cells (r)with single mitochondria (m),autophagosomes (asterisk) andcisterns of SER (SER), basallamina (arrow), digestive cells(e), nucleus of the crescent-likecell (n). TEM, bar=0.8 μm

Ultrastructure of the midgut epithelium during oogenesis 407

Stage E: late choriogenesis

The completely developed three-layered chorion appears onthe surface of the oocyte. In this stage, oocytes secretprecursors of the vitelline envelope (Fig. 2e).

Ultrastructural alterations in the midgut epithelium of I. g.granulifer during oogenesis

Stage A: previtellogenesis (Fig. 3a–d)

The entire cytoplasm of digestive cells possesses mitochon-dria, cisterns of RER, free ribosomes and numerous Golgicomplexes (Fig. 3a). Regionalization in organelles distri-bution, which is characteristic for digestive epithelia, is notobserved. The lobular nucleus with a large homogenousnucleolus is shifted near the basal membrane (Fig. 3b). Thebasal membrane does not form folds (Fig. 3c). Near the

apical membrane, single lipid droplets and secretoryvesicles appear (Fig. 3d).

Stage B: beginning of the vitellogenesis (Fig. 3e, f)

The appearance of glycogen granules occurs and thenumber of small vesicles near the apical membraneincreases. In the posterior part (near the hindgut) of themidgut epithelium, droplets of varying electron densitybegin to accumulate. In some of the epithelial cells, severalmitochondria lose their crista. Simultaneously in such cellssporadic autophagosomes appear (Fig. 3e, f).

Stage C: vitellogenesis—early choriogenesis (Figs. 4a–eand 5a)

The apical cytoplasm of the midgut epithelial cells is rich inmitochondria, cisterns of rough endoplasmic reticulum,

Fig. 2 Stages of oogenesis in I.g. granulifer. a Previtellogene-sis. Germ cells connected withcytoplasmatic bridges (asterisk).Mitochondria (m), nucleus (n).TEM, bar=0.8 μm. b Beginningof the vitellogenesis. Germ cellsin the vitellarium with cisternsof RER (RER), mitochondria(m), nucleus (n) and yolk (y).TEM, bar=1 μm. c Vitellogen-esis—early choriogenesis. Afragment of oocyte (o) coveredwith small parts of chorion (ch).Yolk (y). TEM, bar = 1.2 μm.d Vitellogenesis—middle cho-riogenesis. A fragment of oocyte(o) covered with a permanentlayer of chorion (ch). Yolk (y).TEM, bar=0.54 μm. e Latechoriogenesis. A fragment ofoocyte (o) during secretion ofvitelline envelope (arrows).Chorion (ch), yolk (y). TEM,bar = 0.3 μm

408 M.M. Rost-Roszkowska et al.

multivesicular bodies and small vesicles with electron-lucent content. Those vesicles are usually observed near theapical membrane where they take part in endocytosis(Fig. 4a). In the perinuclear cytoplasm, numerous mito-

chondria without crista (Fig. 4b), typical mitochondria,Golgi complexes and lipid droplets appear. The basalcytoplasm possesses mainly numerous cisterns of roughendoplasmic reticulum and mitochondria. The entire cyto-

Fig. 3 Midgut epithelium of I.g. granulifer during A (a–d) andB (e–f) stages of oogenesis.a The cytoplasm with cisterns ofRER (RER), Golgi complexes(G) and mitochondria (m).Nucleus (n) with nucleolus (nu).TEM, bar=1.9 μm. b The lobu-lar nucleus (n) with a largehomogenous nucleolus (nu) nearthe basal membrane (arrow).Golgi complexes (G), mito-chondria (m).TEM, bar =1.3 μm. c The basal membrane(arrow) does not form folds.Cisterns of RER (RER), mito-chondria (m), nucleus (n),nucleolus (nu). TEM, bar =1.8 μm. d Single lipid droplets(arrowhead) and secretoryvesicles (arrow) near the apicalmembrane. Microvilli (mv), mi-tochondria (m), midgut lumen(l). TEM, bar=0.8 μm.e Glycogen granules (g), thenumber of small vesicles(arrow), several mitochondria(m) undergoing transformation(asterisk), sporadic autophago-somes (au). Midgut lumen (l),microvilli (mv), reserved mate-rial (rm). TEM, bar = 0.6 μm.f The posterior end of themidgut epithelium with dropletsof varying electron density (rm).Autophagosomes (au), mito-chondria (m) and transformingmitochondria (asterisks). TEM,bar = 0.7 μm

Ultrastructure of the midgut epithelium during oogenesis 409

plasm is rich in free ribosomes and glycogen granules(Fig. 4c). The basal membrane does not form folds andinvaginations. In the cytoplasm of the cells in the posteriorpart of the midgut, numerous, large electron-dense droplets

(Fig. 4d) occur (probably proteins or glycoproteins)(Fig. 5a). Together with the increasing number of suchdroplets, the number of autophagosomes and autolyso-somes is also increasing (Fig. 4e).

Fig. 4 Midgut epithelium of I.g. granulifer during C (a–e) andD (f) stages of oogenesis. a Theapical cytoplasm rich in mito-chondria (m), cisterns of RER(RER), multivesicular bodies(mvb) and small vesicles (arrow)with electron-lucent content.Microvilli (mv), midgut lumen(l), endocytosis (arrowhead).TEM, bar=0.4 μm. b Perinu-clear cytoplasm with numerousmitochondria without crista(asterisk), typical mitochondria(m), cisterns of RER (RER),Golgi complexes (G), reservedmaterial (arrow), autolysosome(al). TEM, bar = 0.3 μm. c Thebasal cytoplasm with numerouscisterns of RER (RER), mito-chondria (m), glycogen granules(g), reserved material (rm). Nu-cleus (n) with nucleolus (nu).TEM, bar = 0.6 μm. d Cisternsof RER (RER) and SER (SER),large electron-dense droplets ofreserved material (rm), lipiddroplets (ld), mitochondria (m).Basal membrane (arrow). TEM,bar = 0.8 μm. e Numerousautophagosomes (au), Golgicomplexes (G), cisterns of SER(SER), mitochondria (m), nucle-us (n). TEM, bar = 0.7 μm.f Intensive endocytosis (arrow)in the digestive cells. Cisterns ofRER (RER), Golgi complexes(G), midgut lumen (l), lipiddroplets (ld), microvilli (mv),mitochondria (m), multivesicularbodies (mvb). TEM,bar = 0.6 μm

410 M.M. Rost-Roszkowska et al.

Stage D: vitellogenesis—middle choriogenesis (Figs. 4fand 5b, c)

The number of free ribosome cisterns of rough and smoothendoplasmic reticulum and Golgi complexes increases and,

as a result, the cytoplasm becomes electron-dense.Electron-dense droplets and lipid droplets are very frequent(Fig. 5b, c). The process of endocytosis is intensified inrelation to stage C (Fig. 4f).

Stage E: late choriogenesis (Fig. 6a–g)

The midgut lumen, devoid of nourishments, is narrowing(the specimen does not feed). The entire cytoplasm is stillrich in free ribosomes, Golgi complexes, cisterns of roughand smooth endoplasmic reticulum, glycogen granules(Fig. 6a) and droplets of varying density (Fig. 6b, c). Atthe end of this stage, just before oviposition in severaldigestive cells, numerous mitochondria undergo transfor-mation. They lose crista and swell (Fig. 6d). In their matrix,the remains of crista and flocculent material are observed.The remains of mitochondria and of the reserve material aresurrounded with the phagophore (Fig. 6e) and largeautophagosomes (Fig. 6f, g) and autolysosomes are formed.The number of reserve material starts to decreases and,gradually, the ultrastructure of the remaining midgutepithelial cells resembles the cytoplasm of the stage A withsingle mitochondria, cisterns of rough endoplasmic reticu-lum and free ribosomes.

Discussion

The midgut (mesenteron) epithelium of Tardigrada isresponsible for digestion, secretion, absorption, excretionand probably osmoregulation in Heterotardigrada (Greven1976; Dewel et al. 1988, 1993). It is composed of digestivecells (Kristensen 1976; Pirch and Greven 1994; Ząbczyk2000; Avdonina et al. 2007; Rost-Roszkowska andPoprawa 2008), but in some specimens also regenerativecells have been described (Bertolani 1970; Dewel and Clark1973c; Greven 1976; Ząbczyk 2000). In D. dispar, threedistinct regions have been distinguished: pro-, meso- andmetamesenteron (Ząbczyk 2000), but in I. g. granulifer themidgut has a tube-like shape without any distinct regions. Itis composed of columnar digestive cells. Among epithelialcells, regenerative cells were not observed, but at theanterior end of the midgut a group of cells with cytoplasmdifferent from that of digestive ones was observed. Thosecells might respond to crescent-like cells described byBertolani (1970), Dewel and Clark (1973c), Greven (1976)and Ząbczyk (2000), but due to the fact that their mitoticdivisions and differentiation were not observed in I. g.granulifer we do not know if they fulfill the role of midgutstem cells (regenerative cells). It is also probable that thosecells would divide at a definite period of the life cycle, e.g.just after oviposition, which would be connected with themolting period. In the midgut epithelium of many insect

Fig. 5 Histochemical staining of the midgut epithelium of I. g.granulifer. Midgut epithelium (e), gonad (gn), midgut lumen (l). aStage C of oogenesis. PAS-positive granules of reserved material(arrow). Light microscope, PAS staining, bar=9.1 μm. b Stage D ofoogenesis. Sudan Black B-positive granules of reserved material(arrow). Light microscope, Sudan Black B staining, bar=8.8 μm. cStage D of oogenesis. BPB-positive granules of reserved material(arrow). Light microscope, BPB staining, bar = 8.6 μm

Ultrastructure of the midgut epithelium during oogenesis 411

species, regenerative cells divide intensively just beforeeach molting, which is combined with the cyclic midgutepithelium degeneration (Garcia et al. 2001).

During oogenesis, the process of yolk formation occursand it leads to the storage of lipids, proteins, saccharides orother minor components in the oocyte. In the process ofyolk accumulation, extra-ovarian tissues take part inproducing their yolk precursors (Eckelbarger 1983; Raikheland Dhadiala 1992).

Midgut epithelium has been described as one of theorgans which, together with fat body, storage cells,trophocytes and oocytes, are involved in the synthesis ofyolk and chorion components in invertebrates, e.g. insects,annelids, arachnids (Kessel and Beams 1980; Węglarska1987; Rosell and Coons 1992; Poprawa 2005, 2006;Świątek 2006). A comparison of the alterations in themidgut epithelial ultrastructure of I. g. granulifer with itsoogenesis suggests that both of these processes are

Fig. 6 Stage E of oogenesis onI. g. granulifer. a The cytoplasmstill rich in Golgi complexes(G), cisterns of RER (RER) andreserved material (rm). Midgutlumen (l), microvilli (mv). TEM,bar=0.8 μm. b Reserved mate-rial (arrows) in the midgut epi-thelium (e). Midgut lumen (l).Light microscope, bar=9.5 μm.c Droplets of the varying density(rm), cisterns of RER (RER),Golgi complexes (G), mito-chondria (m). TEM, bar=0.85 μm. d Digestive cells withnumerous mitochondria under-going transformation (asterisk).Typical mitochondria (m), cis-terns of SER (SER), microvilli(mv). TEM, bar = 0.6 μm. e Theremains of mitochondria andremains of the reserve material(rm) surrounded with phago-phore (arrow). Microvilli (mv),autophagosomes (au). TEM, bar= 0.45 μm. f Large autophago-somes (au) in the cytoplasm ofdigestive cells. Mitochondria(m). TEM, bar=1 μm. g Thecytoplasm of digestive cells richin large autophagosomes (au).Basal lamina (arrow), gonad(gn), midgut lumen (l), micro-villi (mv). TEM, bar = 1.3 μm

412 M.M. Rost-Roszkowska et al.

connected. Together with the accumulation of the reservematerial in the oocyte and the synthesis of chorioncomponents, the number of reserve material in the midgutcells also increases. During vitellogenesis and chorio-genesis (stages C–E), we observed in the epithelial cellsthe increasing number of organelles which are responsiblefor the intensive synthesis of lipids, proteins and saccha-rides. Simultaneously, the gradual accumulation of reservedmaterial appears. At the end of choriogenesis, just beforelaying eggs, specimens of I. g. granulifer do not feed andtheir midgut epithelium possesses numerous structures withreserve material, but their number is gradually decreasing.At the end of oogenesis, specimens of I. g. granuliferprobably do not feed because their midgut lumen is devoidof nourishments. It might be caused by the fact that thegrowing ovary occupies the most of the body cavity andcompresses the midgut (a cost of the reproduction). Anotherpossibility is preparing the organism for molting, when allprocesses connected with digestion are breaking. ManyTardigrada species lay eggs during molting. At thebeginning of the new oogenesis cycle, midgut epithelialcells are devoid of reserve material. That suggests that thematerial gathered in the cytoplasm of midgut epithelial cellsmight be exploited not only during vitellogenesis but alsoduring choriogenesis. Additionally, the reserve materialaccumulated in the midgut epithelial cells might also beused in the absence of food. The accumulation of thereserve material, e.g. lipid droplets and glycogen, in storagebodies has been described previously (Węglarska 1957,1975; Poprawa 2006). The authors state that this material isaccumulated in order to survive a period of hunger, whichis also probably for the participation of midgut epithelialcells in lipid storage (Greven 1976; Pirch and Greven 1994;Rost-Roszkowska and Poprawa 2008).

The reserve material gathered in the midgut epitheliumof I. g. granulifer during oogenesis might be eventuallyremoved from its cells by autophagy because this processplays a role in cell death (Levine and Yuan 2005; Giusti etal. 2007; Tettamanti et al. 2007; Rost-Roszkowska et al.2008, 2010a, 2010b) and enables exploiting the material ororganelles gathered in the cytoplasm. Autophagy is grad-ually intensified (from the beginning of vitellogenesis tillthe end of choriogenesis), which might be connected withthe increasing requisition for, for example, chorion layersformation or even for nutrition of the animal which doesnot feed. The more autophagosomes is gathered in the cell,the more material and organelles are removed from the cell(Rost-Roszkowska et al. 2008, 2010b).

Acknowledgements We would like to express our gratitude to Dr.Danuta Urbańska-Jasik (University of Silesia, Poland) for hertechnical assistance and to Professor Clark Beasley (USA), ProfessorPaul Bartels (USA) and the reviewers for valuable remarks on themanuscript.

Conflicts of interest The authors declare that they have no conflictsof interest.

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