Structure of Cyst Wall Precursors and Kinetics of Their Appearance During the Encystment of Laurentiella acuminata (Hypotrichida,Oxytrichidae)1

The encystment of Laurenliella acuminata was divided into five stages: stage A (precystic semitransparent cell with dark-globules), stage B (precystic transparent cell), stage C (precystic pigmented cell), stage D (spherical shape without cyst wall) and stage E (young resting cyst), on the basis of observations of changes in morphology and pigmentation during encystment. The duration of these stages was also established. Observations by electron microscopy confirmed that the cyst wall, composed of four layers, is derived from different kinds of precursors which are synthesized “de novo.” The ectocyst precursors are composed of stacks of between 5 and 12 small thin plates or discs; these stacks are about 0.9 μm in length and 0.06 μm in height. The mesocyst precursors are fibrillar bodies of variable shapes, about 2.4 μm in maximum length and 0.12–0.16 μm in diameter. These precursors appear in the cytoplasm of the precystic cell during the first precystic stage (stage A). The endocyst precursors are rounded bodies surrounded by a fine membrane, and their contents appeared similar to the endocyst. The granular layer precursors are spherical bodies about 0.1–0.2 μm in diameter, surrounded by a double membrane presenting ribosomes adhering to its outer membrane. Both endocyst and granular layer precursors are observed in the precystic cytoplasm from stage B. On the basis of ultrastructural studies, a formation and growth model of the cyst wall of the hypotrichous ciliate Laurentiella acuminata is proposed.     

Ultrastructural Changes During Encystment of Schizopyrenus russelli*

Ultrastructural changes associated with the encystment of Schizopyrenus russelli have been studied by electron microscopy. Before encystment small “black bodies” appear in the cytoplasm and later migrate toward the periphery. The outer cyst wall is secreted at this stage as a thin discontinuous layer which thickens and subsequently becomes continuous. Concomitant with this, the endoplasmic reticulum surrounds the mitochondria. The inner cyst wall later appears as a multilayered structure which presumably is cast off from the plasma membrane. Between the inner and outer layers of the cyst wall, there is a middle, less electron-dense layer wherein extruded cytoplasmic material is found embedded at certain places.     

Evaluation of membrane-bound black bodies in trophozoites and cysts of Naegleria spp

Various Naegleria strains were examined to determine the possible origin and significance of membrane-bound black bodies that were found in all exponentially growing cell populations. The bodies, 40–80 nm in diameter, were distributed randomly in the cytoplasm of Naegleria with ultrastructural features typical of trophozoites. No evidence was obtained that the contents of the black bodies were synthesized in the rough endoplasmic reticulum (ER) and packaged by membranous components, which could be a primitive “Golgi complex” in these amoebae. Examination of cells in various stages of encystment indicated that at least some of the cyst wall material was synthesized and packaged by the rough ER. After condensation into amorphous granules in the cisternae, the cyst wall material appeared in vesicles of the rough ER; these were frequently seen in close proximity to the cell membrane in the vicinity of developing cyst wall. Amorphous granules (～100 nm in diameter), which had variable densities and did not appear to be membrane bound, were seen in the cytoplasm of encysting cells. The substance of these granules also seemed to be incorporated into the cyst wall. The membrane-bound black bodies appeared to be destroyed in lysosomal elements during encystment. The membrane-bound black bodies were concluded to be characteristic of trophozoites and unrelated to encytment of Naegleria.     

Allomyces neo-moniliformis gametogenesis

Summary InAllomyces neo-moniliformis meiosis takes place during resting sporangium germination. The meiospores are characteristically binucleate and biflagellate as described byEmerson (1938) andTeter (1944). A variation in the number of nuclei and flagella per meiospore from two is correlated with germination of the resting sporangia under reduced oxygen tension. The meiospores are extremely poor swimmers and are typically amoeboid. At encystment the gamma bodies of the cell are mobilized and appear involved in cyst wall synthesis. A single mitotic division of each nucleus gives rise to four nuclei. Gamete cleavage is as described for spore cleavage inBlastocladiella (Lessie andLovett 1968). The assembly of the nuclear cap and side body complex of the spore are extremely late processes in gametogenesis. The gametes are released when the single papilla dissolves. The gametes fuse in pairs and after zygote formation the cell is uninucleate with two flagella. The biflagellate zygote is an active swimming cell. The presence of homothallism or hetero-thallism inA. neo-moniliformis is discussed.     

Light and Electron Microscopic Observations on Encystment of Acanthamoeba palestinensis, Reich

SYNOPSIS. The ultrastructural changes occurring during encystment of Acanthamoeba palestinensis have been investigated. The cyst wall consists of endocyst and exocyst, both having the same fine structure. At irregular intervals in the cyst wall ostioles occupied by opercula are present. The nuclear membrane forms bulb-shaped projections and releases vesicles bounded by double membranes into the cytoplasm. Dense nucleolus-like bodies of different sizes and variable numbers are found in the nucleus of every cyst. The importance of the cyst structure as a taxonomic criterion is discussed.     

Fine structure of excystment of the quadriflagellate algaPolytomella agilis

Summary Populations of mature resting cysts of the algaPolytomella agilis were purified from asynchronously encysting cultures and incubated in fresh culture medium to promote excystment. Up to 90 percent of the cysts germinated, with approximately 50 percent excysting between 3 and 7 hours of incubation. Each germinating cyst releases a single, fully differentiated, swimming cell. The entire excystment process of individual cysts was followed by light microscopy to establish the time course of release and cells at comparable stages of excystment were examined by electron microscopy. During the first 3 hours of incubation the cysts increase in size, presumably due to uptake of water, and a polarity is established in the cytoplasm which makes it possible to identify the site of subsequent release. Release involves a selective degradation of a portion of the cyst wall at this site followed by a physical rupturing of the weakened area. Details of the structural alterations in the wall and cytoplasm are described. The cytoplasmic organelles observed to dedifferentiate during encystment (preceding paper) are completely redifferentiated during excystment. The emergent cell is flagellated and possesses the elongate form typical of the swimming cell.This work was supported by grant A6353 from the National Research Council of Canada to D. L.Brown and by the Inland Waters Directorate of Environment Canada.     

The formation of the cyst wall of the metacercaria of Fasciola hepatica L.

Summary The several concentric layers of the cyst wall of Fasciola hepatica are formed from precursors synthesised in the cystogenic cells of the cercaria during its development in the redia. A cinematographic analysis shows that the separate components are released in succession during encystment.The outer portion of the wall consists of two layers: a tanned protein and a carbohydrate-protein complex. The granular precursors of these are formed in separate groups of cells and migrate from these cells into the superficial epithelium (embryonic epithelium) during development. They are released to form the outer wall by the bursting of the embryonic epithelium at the beginning of encystment. This process is rapid and is completed in a few minutes.A pause follows the separation of the outer wall during which a further polysaccharide layer is released and the cells, which contain the rod-like scrolls of sheets of the laminated component of the inner wall, migrate from within the cercaria through gaps in the superficial musculature on to the cercarial surface to form a new epithelium replacing that previously shed.The cercaria now begins a series of complex oscillatory movements within the enveloping outer cyst wall during which the scrolls are secreted into the space underneath the outer wall, unroll and are compacted by the animal's movements into the lamellar inner wall.The rodlets are enclosed in vacuoles and their secretion is effected by the fusion of the vacuolar membrane with the plasma membrane without destroying the integrity of the cells, which remain to constitute the epithelium of the juvenile fluke when this emerges later.     

EXOCYTOSIS OF LATEX BEADS DURING THE ENCYSTMENT OF ACANTHAMOEBA

Cells of Acanthamoeba castellanii (Neff) are known to form mature cysts characterized by a cellulose-containing cell wall when transferred to a nonnutrient medium. Amebas which engulfed latex beads before encystment formed mature cysts essentially devoid of bead material. The encystment of bead-containing cells appeared to be similar to that of control cells since no important differences between the two were observed with respect to cellular levels of glycogen or protein, cellulose synthetase activity, the amount of cyst wall polysaccharide formed, or the percentage of cysts formed. Actinomycin D and cycloheximide inhibited encystment as well as bead expulsion. Ultrastructural analysis revealed that the beads, which initially were contained in phagocytic vesicles, were released from the cell by fusion of vesicular membranes with the plasma membrane. Exocytosis was observed in cells after 3 hr of encystment, with most of the beads being lost before cyst wall formation. Each bead-containing vesicle involved in expulsion was conspicuously demarcated by an area of concentrated cytoplasm, which was more homogeneously granular than the surrounding cytoplasm. Beads were not observed in the cytoplasm of mature cysts but were occasionally found in the cyst wall.     

Formation of the Giardia Cyst Wall: Studies on Extracellular Assembly Using Immunogold Labeling and High Resolution Field Emission SEM

Encystment of the intestinal protozoan, Giardia, is a key step in the life cycle that enables this parasite to be transmitted from host to host via either fecal oral, waterborne, or foodborne transmission. The process of encystment was studied by localizing cyst wall specific antigens with immunofluorescence for light microscopy and immunogold staining for field emission scanning electron microscopy. Chronological sampling of Giardia cultures stimulated with endogenous bile permitted identification of an intracellular and extracellular phase in cyst wall formation, a process which required a total of 14-16 h. The intracellular phase lasted for 8-10 h, while the extracellular phase, involved the appearance of cyst wall antigen on the trophozoite membrane, and the assembly of the filamentous layer, a process requiring an additional 4-6 h for completion of mature cysts. The extracellular phase was initiated with the appearance of cyst wall antigen on small protrusions of the trophozoite membrane (-15 nm), which became enlarged with time to caplike structures ranging up to 100 nm in diameter. Caplike structures involved with filament growth were detected over the entire surface of the trophozoite including the adhesive disc and flagella. Encysting cells rounded up, lost attachment to the substratum, and became enclosed in a layer of filaments. Late stages in encystment included a “tailed” cyst in which flagella were not fully retracted into the cyst. Clusters of cysts were seen in which filaments at the surface of one cyst were connected with the surface of adjacent cysts or the “tailed” processes of adjacent cysts, suggesting that the growth of cyst wall filaments may be at the terminal end. In conclusion, the process of encystment has been shown to consist of two morphologically different stages (intracellular and extracellular) which requires 16 h for completion. Further investigation of the extracellular stage with regard to assembly of the filamentous layer of the cyst wall may lead to innovative methods for interfering with production of an intact functional cyst wall, and thereby, regulation of viable Giardia cyst release from the host.     

Ultrastructure of the Amoeboflagellate Tetramitus rostratus1

The life-cycle of the amoeboflagellate Tetramitus rostratus includes amoeboid, cyst, and flagellate stages. The ultrastructure of these three stages is illustrated, with particular emphasis on flagellate morphology. Amoeba morphology is typical of that of limax amoebas. Cysts, forming from trophic amoebas, are enclosed by a wall made up of two layers: ectocyst (ca. 70 nm), and endocyst (200 nm). The wall apparently forms from precursor material present in vesicles in the pre-cyst stage cytoplasm. Flagellate morphology is characterized by a well-defined top-shaped profile, maintained by microtubules under the plasma membrane. The flagellar apparatus or mastigont consists of four flagella, their basal bodies, sheaves of microtubules associated with two of the basal bodies, and several rhizoplasts (periodicity 20 nm). A deep, microtubule-supported, ventral invagination appears to function as a gullet. A small number of mitotic stages observed in amoeboid and flagellate individuals suggests similarity in the division process in both stages: intranuclear mitotic apparatus, nucleolus persisting through mitosis, no centrioles or basal bodies functioning as centrioles, difficulty in resolving chromosomes. The text compares ultrastructures of several amoeboflagellate organisms and evaluates the phylogenetic significance of those features common to different species. On the basis of this study, Tetramitus most closely resembles Naegleria spp.     

Characterisation of the water expulsion vacuole inPhytophthora nicotianae zoospores

Summary The water expulsion vacuole (WEV) in zoospores ofPhytophthora nicotianae and other members of the Oomycetes is believed to function in cell osmoregulation. We have used videomicroscopy to analyse the behaviour of the WEV during zoospore development, motility and encystment inP. nicotianae. After cleavage of multinucleate sporangia, the WEV begins to pulse slowly but soon attains a rate similar to that seen in motile zoospores. In zoospores, the WEV has a mean cycle time of 5.7 ± 0.71 s. The WEV continues to pulse at this rate until approximately 4 min after the onset of encystment. At this stage, pulsing slows progressively until it becomes undetectable. The commencement of WEV operation in sporangia coincides with the reduction of zoospore volume prior to release from the sporangium. Disappearance of the WEV during encystment occurs as formation of a cell wall allows the generation of turgor pressure in the cyst. As in other organisms, the WEV inP. nicotianae zoospores consists of a central bladder surrounded by a vesicular and tubular spongiome. Immunolabelling with a monoclonal antibody directed towards vacuolar H⁺-ATPase reveals that this enzyme is confined to membranes of the spongiome and is absent from the bladder membrane or zoospore plasma membrane. An antibody directed towards plasma membrane H⁺-ATPase shows the presence of this ATPase in both the bladder membrane and the plasma membrane over the cell body but not the flagella. Analysis of ATPase activity in microsomal fractions fromP. nicotianae zoospores has provided information on the biochemical properties of the ATPases in these cells and has shown that they are similar to those in true fungi. Inhibition of the vacuolar H⁺-ATPase by potassium nitrate causes a reduction in the pulse rate of the WEV in zoospores and leads to premature encystment. These results give support to the idea that the vacuolar H⁺-ATPase plays an important role in water accumulation by the spongiome in oomycete zoospores, as it does in other protists.Abbreviations BMM butyl methylmethacrylate - F fix 4% formaldehyde fixation - GF fix 4% formaldehyde and 0.2% glutaraldehyde fixation - V-ATPase vacuolar H⁺-ATPase - WEV water expulsion vacuole     

The ultrastructure of cell wall formation and of gamma particles during encystment of Allomyces macrogynus zoospores

Structural changes during cell wall formation by populations of semisynchronously germinating zoospores were studied in the water mold Allomyces macrogynus. Fluorescence microscopy using Calcofluor white ST (which binds to -1,4-linked glycans) demonstrated that Calcofluor-specific material was deposited around most cells between 2–10 min after the induction of encystment (beginning when a wall-less zoospore retracts its flagellum and rounds up). During the first 15 min of encystment there was a progressive increase in fluorescence intensity. Ultrastructural analysis of encysting cells showed that within 2–10 min after the induction of encystment small vesicles 35–70 nm diameter were present near the spore surface, and some were in the process of fusing with the plasma membrane. The fusion of vesicles with the zoospore membrane was concomitant with the appearance of electron-opaque fibrillar material outside the plasma membrane. Vesicles similar to those near the spore surface were found within the gamma () particles of encysting cells. These particles had a crystalline inclusion within the electron-opaque matrix. During the period of initial cyst cell wall formation numerous vesicles appeared to arise at the crystal-matrix interface. Approximately 15–20 min was required for the cell wall to be formed. We suggest that the initial response of the zoospore to induction of encystment is the formation of a cell wall mediated by the fusion of cytoplasmic vesicles with the plasma membrane.Non-Standard Abbreviations GlcNac N-Acetylglucosamine - DS sterile dilute salts solution - PYG peptone-yeast extract-glucose broth     

Morphological Study of the Encystment and Excystment of Vermamoeba vermiformis Revealed Original Traits

Free‐living amoebae are ubiquitous protozoa commonly found in water. Among them, Acanthamoeba and Vermamoeba (formerly Hartmannella) are the most represented genera. In case of stress, such as nutrient deprivation or osmotic stress, these amoebae initiate a differentiation process, named encystment. It leads to the cyst form, which is a resistant form enabling amoebae to survive in harsh conditions and resist disinfection treatments. Encystment has been thoroughly described in Acanthamoeba but poorly in Vermamoeba. Our study was aimed to follow the encystment/excystment processes by microscopic observations. We show that encystment is quite rapid, as mature cysts were obtained in 9 h, and that cyst wall is composed of two layers. A video shows that a locomotive form is likely involved in clustering cysts together during encystment. As for Acanthamoeba, autophagy is likely active during this process. Specific vesicles, possibly involved in ribophagy, were observed within the cytoplasm. Remarkably, mitochondria rearranged around the nucleus within the cyst, suggesting high needs in energy. Unlike Acanthamoeba and Naegleria, no ostioles were observed in the cyst wall suggesting that excystment is original. During excystment, large vesicles, likely filled with hydrolases, were found in close proximity to cyst wall and digest it. Trophozoite moves inside its cyst wall before exiting during excystment. In conclusion, Vermamoeba encystment/excystment displays original trends as compare to Acanthamoeba.     

Encystment ofBodo caudatus

Summary Before formation of the cyst wall, the food vacuoles are lost, the cell rounds up and the flagella lie close against the body in a flagellar groove. At this early stage, the contractile vacuole is very active, the Golgi apparatus is prominent and the basophilic cytoplasm is composed of closely packed ribosomes. As the cyst wall is secreted, layer by layer, the large Golgi apparatus is replaced by several smaller membrane stacks and mitochondrial changes occur involving local loss and modification of the cristae. Some parts of the mitochondrion undergo degenerative changes and may become surrounded by bacilliform bodies. These same bodies are also associated with small particles of sequestered cytoplasm which are present throughout the encystment process and are believed to be autophagic vacuoles. As the cyst wall thickens, cell shrinkage is manifest as a number of membrane invaginations. The final cyst wall is of uneven thickness and possesses a single operculum which is visible only by electron microscopy. Probable cyst wall precursor is found in small vesicles scattered throughout the cytoplasm.     

The encystment of a freshwater dinoflagellate: A light and electron-microscopical study

The process of encystment, or resting spore formation, in a freshwater dinoflagellate (Woloszynskia tylota nov. comb.) has been studied with both light and electron microscopy. The main features of the process are as follows: (i) the replacement of the theca by a thin, amorphous outer wall, which gradually thickens by the deposition of material on its inner face; (ii) the appearance of a layer of closely-packed lipid droplets at the cytoplasmic margin of the mature cyst, resembling a granular ‘inner wall’ in the light microscope; (iii) the reduction in size or disappearance of cytoplasmic structures such as chloroplasts, Golgi bodies and pusule; and (iv) the enlargement of a central ‘accumulation body’ and cytoplasmic vacuoles containing crystals. Comparisons are made with light-microscope studies of encystment of other dinoflagellates, with ultrastructural studies of non-motile division stages, with zooxanthellae and with fossil dinoflagellate cysts or hystrichospheres.     

The differentiation of cellular structure during encystment in the soil hypotrichous ciliate Australocirrus cf. australis (Protista,Ciliophora)

Ciliates are able to form resting cysts as a survival strategy in response to stressful environmental factors. Studies on the characteristics of cellular structure during encystment may provide useful information for further understanding of the regulatory mechanism of cellular patterns and supply new clues regarding the phylogeny of ciliates. Scanning and transmission electron microscopies were used to observe the ultrastructure of cells during encystment of the soil ciliate Australocirrus cf. australis. The dedifferentiation of ciliature was revealed for the first time. Ciliary shafts first shortened, and the remaining ciliature, including basal bodies and the fibrillar cirral basket, retracted into the cytoplasm and was surrounded by the autophagic vacuoles and then gradually digested. A large number of autophagic vacuoles were observed in mature resting cysts. Autophagy might not only be necessary for the differentiation of cellular structures during encystment but might also be important to sustain the basic life activities in the resting stage. Australocirrus cf. australis formed a kinetosome-resorbing cyst and contained four layers in the cyst wall: the ectocyst, mesocyst, endocyst and granular layer. The ciliature resorbing state and the number of layers in the cyst wall were consistent with those found in other oxytrichous ciliates. However, the phenomenon wherein the two macronuclear nodules are not fused during encystment is not commonly observed among oxytrichids. Additionally, the octahedral granules in the mesocyst of this species exhibit different morphology from the congeners.     

The gamma body: A vesicle generating structure

Gamma bodies, which are present in the sporangia and gametangia of Allomyces and in its spores, are interpreted as constituting vesicle generating structures. During spore cleavage the mobilization–decay of the gamma bodies leads to vesicle formation; the vesicles appear to fuse to form the axonemal and plasma membrane of the spore. Vesicle formation by the gamma bodies during spore cleavage can be perturbed by phosphate buffer which leads to the formation of myelin–figure arrays of membranes, or by colchicine and benomyl which give rise to large vacuolar structures after gamma body decay. During the motile period of the spores of Allomyces , mobilization of the gamma bodies leads to vacuole formation and the resulting vacuoles fuse with the plasma membrane of the spore and by this means maintain the osmotic balance of the spore. During spore encystment the gamma body decays and forms vesicles which fuse with the plasma membrane of the cyst; these vesicles appear to be instrumental in chitin wall synthesis.     

The ultrastructure of zoospores ofAphanomyces euteiches and of their encystment and subsequent germination

Summary The ultrastructure ofAphanomyces euteiches during the periods of zoospore motility, encystment, and germination has been studied. The motile spore has two heterokont flagella inserted laterally into the groove of the zoospore body where each is attached to a kinetosome. The kinetosomes and flagella are anchored into the zoospore body by rootlets comprised of two rows of microtubules with up to 12 microtubules in the outer row and are attached by fine threads to a striate fiber bundle. Secondary microtubules are attached at right angles at regular intervals along the rootlets. An unidentified body, 1.25m in diameter, containing helical fibers 16 nm in diameter is present in each zoospore. This body is situated near the two kinetosomes on the side of the pyriform nucleus opposite the contractile vacuole. The Golgi complex is between the nucleus and the contractile vacuole. The latter is surrounded by a 0.5–1.0m wide zone of Golgi proliferated vesicles. Ribosomes are generally absent from this region. Endoplasmic reticulum containing tubules within the expanded cisternae are also present. Vesicles with striated electron opaque inclusions and vesicles containing a granular cortex and center that developed in previous stages of zoosporogenesis were also present. During encystment of the zoospore the latter vesicles disappear. The two flagella are shed at this time leaving a membrane-bounded granular knob protruding from each of the kinetosome terminal plates. The contractile vacuole becomes disorganized and the zoospore assumes a spherical shape. Cyst wall deposition begins immediately and is completed in 30 minutes. The spore begins to germinate 1 hour following initiation of encystment with the appearance of a bulge in the cyst wall which elongates into a germ tube. Mitotic nuclear division follows.Research supported by the College of Agricultural and Life Sciences Station Project No. 1281.Research assistant and Professor. The advice and assistance of G. A. deZoeten, G. R.Gaard, and S.Vicen are most gratefully acknowledged.     

Liberation and Development of Allomyces arbuscula Mitospores Viewed by Scanning Electron Microscopy

Scanning electron microscopy has been employed to examine events in the release and development of mitospores of the aquatic fungus, Allomyces arbuscula. Among the salient features of spore release from the mitosporangium is the digestion of the inner matrix of the exit papillum. Hydrolysis appears to begin at the outer layer of the papillum plug matrix and probably results from activation of localized hydrolytic enzymes. The plug clearly consists of at least two different component layers. Elaboration of mitospores from the mitosporangium is depicted in several micrographs. Motile spores were induced to begin development, and the sequence of surface changes associated with the encystment process was studied. Time course studies show the retraction of the flagellum, the change from elipsoidal to spherical shape, and the deposition of the cell wall. Early in encystment, small vesicles accumulate on the surface of the plasma membrane. These enlarge and fuse to form the mature cyst wall. This surface view of cell wall deposition appears to support the possible role of gamma particles in cell wall synthesis during encystment.     

The Ultrastructure of Polytomella agilis*

SYNOPSIS Motile cells and cysts of Polytomella agilis, obtained over the entire growth cycle, were examined by electron microscopy. In typical late log phase cells there is a concentric arrangement of the internal organelles around the centrally located nucleus. Lying just beneath the plasma membrane is a peripheral band of elongate mitochondria. Numerous well defined Golgi bodies are also distributed around the nucleus. Vesicles associated with the Golgi body increase in size with distance from the secretory edges of the organelle. Cytoplasmic membranes with associated ribosomes are found between the mitochondrial and Golgi regions. A layer of slender membrane-limited structures is located near the mitochondrial layer. These organelles, which resemble proplastids, become highly branched during late log and early stationary phase, reaching maximum development in late stationary and early pre-cyst stages. Large storage granules of varying density are found within the cell. The PAS-positive granules have been isolated and shown to contain starch. There is an increase in the amount of this storage material as the cells enter the stationary phase. The remainder of the cytoplasmic matrix is finely granular and contains numerous free ribosomes except in the region of the anterior papilla. Four flagella arise from basal bodies at the anterior end of the cell. The cyst is characterized by a thick multi-layered cell wall whose electron density obscures the limiting plasma membrane. Large storage granules are located close to and often in contact with the periphery of the cell, suggesting their involvement in the process of cell wall deposition. Altho mitochondria can still be seen in the mature cyst, other cytoplasmic organelles often appear atypical. The mature cyst has an irregular profile possibly due to shrinkage from dehydration.