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Fine structure of the midgut epithelium in two Archaeognatha, Lepismachilis notata and Machilis hrabei (Insecta), in relation to its degeneration and regeneration

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In two archaeognathans, Lepismachilis notata and Machilis hrabei, the midgut epithelium and processes of its regeneration and degeneration have been described at the ultrastructural level. In both analysed species, the midgut epithelium is composed of epithelial and regenerative cells (regenerative nests). The epithelial cells show distinct regionalization in organelles distribution with the basal, perinuclear, and apical regions being distinguished. Degeneration of epithelial cells proceeds in a necrotic way (continuous degeneration) during the entire life of adult specimens, but just before each moult degeneration intensifies. Apoptosis has been observed. Regenerative cells fulfil the role of midgut stem cells. Some of them proliferate, while the others differentiate into epithelial cells. We compared the organisation of the midgut epithelium of M. hrabei and L. notata with zygentoman species, which have just been described.
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ORIGINAL ARTICLE
Fine structure of the midgut epithelium in two
Archaeognatha, Lepismachilis notata and Machilis hrabei
(Insecta), in relation to its degeneration and regeneration
Magdalena M. Rost-Roszkowska &Petr Jansta &
Jitka Vilimova
Received: 18 February 2010 / Accepted: 8 April 2010 /Published online: 6 May 2010
#Springer-Verlag 2010
Abstract In two archaeognathans, Lepismachilis notata and
Machilis hrabei, the midgut epithelium and processes of its
regeneration and degeneration have been described at the
ultrastructural level.
In both analysed species, the midgut epithelium is
composed of epithelial and regenerative cells (regenerative
nests). The epithelial cells show distinct regionalization in
organelles distribution with the basal, perinuclear, and apical
regions being distinguished. Degeneration of epithelial cells
proceeds in a necrotic way (continuous degeneration) during
the entire life of adult specimens, but just before each moult
degeneration intensifies. Apoptosis has been observed. Re-
generative cells fulfil the role of midgut stem cells. Some of
them proliferate, while the others differentiate into epithelial
cells.
We compared the organisation of the midgut epithelium
of M. hrabei and L. notata with zygentoman species, which
have just been described.
Keywords Stem cells .Apoptosis .Degeneration .
Midgut epithelium .Archaeognatha
Introduction
The splitting of taxon Thysanura sensu stricto into two
separate orders, the more primitive Archaeognatha and more
advanced Zygentoma, is recently accepted (e.g., Grimaldi
2001; Cameron et al. 2006;Kjeretal.2006; Klug and Klaas
2006;Machida2006; Misof et al. 2007;DellAmpio et al.
2009;Regieretal.2010). The two strictly distinct and
different types of mandibles occurring in the Archaeognatha
and Zygentoma represent as well different feeding strategies
(Staniczek 2000;Koch2001). The shape and structure of the
digestive tract are correlated with the kind of food
(Billingsley 1990; Terra 1990; Billingsley and Lehane 1996).
The midgut epithelium, which is responsible for enzyme
synthesis, secretion and absorption of food, has been
examined in a number of insects. Recent studies of the
senior author have revealed the ultrastructure of the midgut
epithelium of four Zygentoma species, Thermobia domes-
tica,Lepisma saccharina (Rost et al. 2005; Rost 2006a;
Rost-Roszkowska et al. 2007), Atelura formicaria (Rost-
Roszkowska et al. 2010b), and Nicoletia phytophila (Rost-
Roszkowska et al. in press). As we know, their midgut
epithelium is composed of columnar cells and regenerative
cells, which form regenerative nests. Machida and Ando
(1981) described the development of the midgut epithelium
in archaeognathan species, but there is no information
connected with its ultrastructure. Recently, we have had an
opportunity to study in detail the midgut ultrastructure in
two archaeognathan species, Lepismachilis notata and
Machilis hrabei. Since the Archaeognatha are in a basal
position relative to the rest of the Ectognatha, information
about their midgut epithelium ultrastructure could help
clarify the evolutionary pathway of this character (Grimaldi
2009), as well as help with elucidating relationships
between the Archaeognatha and other Insecta.
M. M. Rost-Roszkowska (*)
Department of Animal Histology and Embryology,
University of Silesia,
Bankowa 9,
40-007 Katowice, Poland
e-mail: magdalena.rost-roszkowska@us.edu.pl
P. Jansta :J. Vilimova
Department of Zoology, Faculty of Science, Charles University,
Vinicna 7, 128 44,
Prague 2, Czech Republic
P. Jansta
e-mail: janstapetr@gmail.com
J. Vilimova
e-mail: vilim@natur.cuni.cz
Protoplasma (2010) 247:91101
DOI 10.1007/s00709-010-0148-2
The aim of our study was to analyse the ultrastructure of
the midgut epithelium of two archaeognathan species, L.
notate and M. hrabei, at the ultrastructural level and to
compare it with the midgut structure of zygentoman
species. It would help in preparing the proposal of
relationships between basal hexapodan groups.
Materials and methods
Materials examined
Adult specimens were collected in the Czech Republic at
two locations during June to September 2007. Specimens of
L. notata, were collected on steppes near the village of
Srbsko (protected area Czech Karst in central Bohemia),
while M. hrabei, were collected on warm but shadowed
slopes somewhat overgrown by oaks near the town of
Vranov nad Dyjí (National Park Podyjí in south-western
Moravia).
Transmission electron microscopy
After decapitation, the material was fixed in 2.5% glutar-
aldehyde in 0.1 M phosphate buffer (room temperature,
2 h) then postfixed in 2% OsO
4
in 0.1 M phosphate buffer
(temp 4°C, 2 h). After dehydration in a graded series of
ethanol (50%, 70%, 80%, 90%, 96%, and 100%, each for
15 min) and acetone (15 min), the material was embedded
in Epon 812 epoxy resin. Semi- and ultrathin sections were
cut on a Leica UCT25 ultramicrotome. Semithin sections
stained with 1% methylene blue in 0.5% borax were
observed with an OLYMPUS BX60 light microscope.
Ultrathin sections were stained with uranyl acetate and lead
citrate and analysed with a HITACHI H500 electron
transmission microscope (TEM) at 75 kV.
Results
In both species, L. notata and M. hrabei, the midgut is
divided into two distinct regions, anterior and posterior. The
anterior midgut is composed of the blind, sac-like caeca
(Fig. 1), which run towards the anteroventral part of the
body. The posterior midgut is sack-shaped (Fig. 2). Midgut
epithelium, resting on the noncellular and multilayered
basal lamina, is formed by epithelial and regenerative cells.
The latter form characteristic regenerative cell groups
(regenerative nests) (Figs. 3and 4). The epithelial cells of
the anterior midgut have cuboidal shapes, while those of the
posterior midgut are columnar. In both species, no differ-
ences in the numbers of regenerative nests between anterior
and posterior parts of the midgut were observed. The
regenerative nests of L. notata midgut epithelium are
composed of about 16 to 30 regenerative cells, while in
M. hrabei, the number of cells forming the regenerative
nests is about 20 to 40. Since ultrastructural features of
midgut epithelial cells in both species are similar, the
following description of these features applies to both,
except where specific differences between the two species
are noted.
The cytoplasm of epithelial cells possesses a character-
istic regionalization in organelles distribution. The basal
membrane forms numerous folds, while mitochondria and
single cisterns of rough endoplasmic reticulum (RER) are
observed between them (Fig. 5). In the perinuclear region
of the cytoplasm, numerous cisterns of smooth and RER, as
well as Golgi complexes, are present (Fig. 6). The apical
cytoplasm, where the apical membrane forms microvilli
protruding into the midgut lumen (Fig. 7), is filled with
mitochondria, cisterns of RER, and minor and major
secretory vesicles. A peritrophic membrane is observed in
the midgut lumen (not shown). Numerous spherites are
stored in the entire cytoplasm (Figs 6and 7). While such
structures are accumulated in the cytoplasm of all analysed
specimens of both species, only in some of them there also
appear a few lipid droplets (Fig. 8). Adjacent epithelial cells
are connected by zonula continua (smooth septate junction)
Fig. 1 Machilis hrabei. Transverse section through the anterior
midgut, which has a blind, sac-like shape (caeca, ca). Stomodaeum
(s), midgut lumen (l), dorsal (d), and ventral (v) surfaces of insects
body. Light microscope, bar = 82 μ
Fig. 2 Machilis hrabei. Transverse section through the posterior
midgut (pm), which is of a sack-like shape. Midgut lumen (l), ovary
(o). Light microscope, bar = 78 μm
Fig. 3 Lepismachilis notata. Longitudinal section through the midgut
epithelium, which rests on the basal lamina (arrow), is formed by
epithelial (e) and regenerative (r) cells. Midgut lumen (l). Light
microscope, bar = 16 μm
Fig. 4 Lepismachilis notata. Regenerative nest formed by regenera-
tive cells. Basal lamina (asterisk), external regenerative cells able for
differentiation (arrows), mitochondria (m), nuclei of regenerative cells
(n). TEM, bar = 2.93 μm
Fig. 5 Lepismachilis notata. Basal region of epithelial cytoplasm with
mitochondria (m) between folds of the basal membrane (arrows).
Basal lamina (asterisk), cisterns of RER. TEM, bar = 1.1 μm
Fig. 6 Machilis hrabei. Perinuclear cytoplasm of epithelial cells rich
in cisterns of rough (RER) and smooth (SER) endoplasmic reticulum,
Golgi complexes (d), and structures that resemble spherites (s).
Nucleus (n). TEM, bar = 1.36 μm
Fig. 7 Lepismachilis notata. Apical cytoplasm of epithelial cells with
numerous mitochondria (m), cisterns of RER, structures that resemble
spherites (s) and some vesicles (v). Microvilli (mv) of the apical
membrane, smooth septate junction (zonula continua) (arrow) between
apical membranes of adjacent epithelial cells. TEM, bar = 1.03 μm
Fig. 8 Lepismachilis notata. Lipid droplets (l) observed in epithelial
cells of some specimens of both species studied. Mitochondria (m),
nucleus (n), spherites (s). TEM, bar = 1.12 μm
Fig. 9 Lepismachilis notata. Autophagosome (au) formation. Ex-
panded cisterns of endoplasmic reticulum (arrow) surround two
smaller autophagosomes. TEM, bar = 0.31 μm
b
92 M.M. Rost-Roszkowska et al.
Fine structure of the midgut epithelium in two Archaeognatha, Lepismachilis notata and Machilis hrabei (Insecta) 93
in the apical regions (Fig. 7) and also by pleated septate and
gap junctions in their perinuclear and basal regions.
Autophagy is a process that can be observed in any of
the midgut epithelial cells. The cytoplasm with organelles is
gradually surrounded by expanding cisterns of endoplasmic
reticulum and subsequently autophagosomes are formed
(Fig. 9). The more spherites and lipid droplets that are
observed in the cytoplasm, the more autophagosomes that
are present.
The midgut epithelial cells with a great number of spherites
and autophagosomes undergo degeneration by necrosis in a
continuous manner, but necrosis intensifies just before each
moult. The cytoplasm of the necrotic cell becomes electron
lucent (Fig. 10). Its mitochondria and Golgi complexes swell
and the number of organelles decreases. Some small
vacuoles appear. The apical membrane breaks, at first
forming large and lobular evaginations and blebs into the
midgut lumen (Fig. 11). All organelles are discharged into
the midgut lumen, where they disintegrate (Fig. 12). In both
species just before each moulting, when the necrosis
intensifies, remnants of basal membranes of the epithelial
cells persist between the regenerative nests (Fig. 13).
The process of apoptosis was observed in both analysed
species. The chromatin of the apoptotic cell undergoes
condensation and the nucleus assumes a lobular shape
(Fig. 14), eventually fragmenting. In L. notata, the nucleus
proceeds to fragment in a manner resembling amitosis. The
nucleus constricts in its equatorial area, and nucleoli are
present in both fragments (Fig. 15). Eventually, two
separate nuclei are observed (Figs. 16 and 17) and the cell
undergoes apoptosis in a manner as described below.
The cytoplasm of the apoptotic cell gradually shrinks
and becomes electron dense (Fig. 18). Distinct extracellular
spaces appear between such apoptotic cell and adjacent
epithelial cells. The apoptotic cell gradually separates from
the basal lamina. Shrinkage of the cell and loss of contact
with adjacent epithelial cells cause a discharging of the
apoptotic cell into the midgut lumen, where it is digested
(Figs. 19 and 20).
In each regenerative nest, some of the regenerative cells,
located near the basal lamina and in the central region of the
nest, possess cytoplasm poor in organelles, with single
mitochondria and cisterns of endoplasmic reticulum (Figs. 4
and 21). These are able to divide intensively (Fig. 22), while
the external cells of each nest differentiate (Figs. 4and 21).
Just before the mitotic division of a regenerative cell,
numerous cisterns of RER and Golgi complexes appear in
its cytoplasm (Fig. 23). Simultaneously, the accumulation of
mitochondria just above the nucleus in cell future apical
cytoplasm is the first morphological sign of an external
regenerative cell differentiation (Figs. 21 and 24). Then the
cell elongates and its mitochondria, accumulated above the
nucleus, move towards the elongating apical region of
the cytoplasm. At the same time, the numbers of cisterns
of the rough and smooth endoplasmic reticulum and Golgi
complexes increase. The cell basal membrane starts to form
characteristic folds. The cells elongating towards the midgut
lumen causes the gradual separation of the degenerating cell
from the basal lamina. Its apical membrane begins to form
small evaginations (Fig. 25), which protrude between
differentiating and degenerating cells. The regionalization
in organelles distribution, which is characteristic for
epithelial cells, appears just after the apical membrane of
the newly formed epithelial cell contacts the midgut lumen.
Then numerous spherites and lipid droplets are gathered in
its cytoplasm. Processes of proliferation and epithelial
differentiation occur in a continuous manner; they are
intensified just before moulting, when degeneration is much
more intensive.
Discussion
Midgut epithelium, with the exception of its ends, is the
only organ in an insect body that originates from
endodermis (Machida and Ando 1981; Larink 1983). It is
responsible for digestion, secretion, absorption, storage of
toxic substances, and excretion in cases where Malpighian
tubules are lacking (Jura 1958; Krzysztofowicz et al. 1973;
Szklarzewicz and Tylek 1987). Endocrine, goblet, and
copper cells also appear in the midgut epithelium of
pterygotan insects together with epithelial and regenerative
cells (Billingsley and Lehane 1996; Uwo et al. 2002; Neves
et al. 2003; Silva-Olivares et al. 2003; Levy et al. 2004).
Fig. 10 Lepismachilis notata. Necrotic cell (nc) with electron lucent
cytoplasm and small number of organelles. Neighbouring epithelial
cell (e), midgut lumen (l), nucleus (n). TEM, bar = 2.77 μm
Fig. 11 Lepismachilis notata. Apical membrane of a necrotic cell (nc)
forms blebs (arrow) into the midgut lumen (l). TEM, bar = 1.9 μm
Fig. 12 Lepismachilis notata. After apical membrane breakage all
organelles are discharged into the midgut lumen (l). Cisterns of RER,
mitochondria (m), nucleus (n), spherites (s). TEM, bar = 2.3 μm
Fig. 13 Machilis hrabei. Remnants of basal membranes (arrows) of
epithelial cells between regenerative nests. TEM, bar = 1.67 μm
Fig. 14 Lepismachilis notata. The nucleus (n) of apoptotic cell (ac)
with a condensed chromatin achieves a lobular shape. TEM, bar =
0.97 μm
Fig. 15 Lepismachilis notata. Nucleus (n) constriction in its equato-
rial area (arrow). Nucleoli (nu) present in both lobes of the nucleus.
Cisterns of rough (RER) and smooth (SER) endoplasmic reticulum,
Golgi complexes (d), mitochondria (m). TEM, bar = 2.1 μm
Fig. 16 Lepismachilis notata. Two distinct nuclei (n) in epithelial
cell. Nucleoli (nu). TEM, bar = 1.4 μm
Fig. 17 Lepismachilis notata. Electron dense cytoplasm of apoptotic
cell (ac). Distinct nuclei (n) in apoptotic cell. Neighbouring epithelial
cell (e), cisterns of smooth endoplasmic reticulum (SER), Golgi
complexes (d), spherites (s). TEM, bar = 2.78 μm
b
94 M.M. Rost-Roszkowska et al.
Fine structure of the midgut epithelium in two Archaeognatha, Lepismachilis notata and Machilis hrabei (Insecta) 95
Evidently, however, only the epithelial and regenerative
cells have been found in the midgut epithelium of ancient
wingless hexapods (Krzysztofowicz et al. 1973; Biliński
and Klag 1979; Lauga-Reyrel 1980; Klag et al. 1981; Rost
2006a,b; Rost-Roszkowska et al. 2010a,2010b). The
condition in the Archaeognatha is identical with that in the
Zygentoma, and only the two mentioned types of cells are
developed. The regenerative cells generally might be placed
singly between epithelial cells (Rost 2006b;Rost-
Roszkowska et al. 2010a) or form special regenerative cell
groups, either regenerative nests or regenerative crypts
(Rost 2006a; Rost-Roszkowska et al. 2010b). In some
species, absence of the regenerative cells has been
described. In such cases, all midgut epithelium is composed
only of epithelial cells (Lauga-Reyrel 1980;Rost-
Roszkowska and Undrul 2008). Regenerative nests are
developed in both the Archaeognatha and Zygentoma.
The midgut epithelium of the Zygentoma species is
composed of epithelial and regenerative cells, which form
the characteristic regenerative nests (Rost et al. 2005;Rost
2006a; Rost-Roszkowska et al. 2007; Rost-Roszkowska et
al. 2010b). The present data on the midgut epithelium of the
Archaeognatha species represent the first published
information about its ultrastructure. Interestingly, previous
studies of the senior author had confirmed many differences
in the conditions of the midgut epithelium between two
closely related taxa of the entognathan Collembola, the
Poduromorpha and Symphypleona (Rost 2006b;Rost-
Roszkowska 2008; Rost-Roszkowska and Undrul 2008),
i.e., types of midgut cells developed, development of
regenerative cells, occurrence of apoptosis, manner of
necrosis, occurrence of differences in the ultrastructure
between different midgut parts. On the contrary, the midgut
epithelium of the Archaeognatha is similar to that of the
Zygentoma. Basically, it is formed by epithelial and
regenerative cells, which form distinct regenerative nests.
Only small differences in the number of regenerative nests
and number of cells forming one regenerative nest had been
Fig. 18 Lepismachilis notata. Shrinkage of the apoptotic cell (ac) causes the appearance of the distinct extracellular spaces (asterisks) between
apoptotic and neighbouring epithelial cells (e). Microvilli (mv), necrotic cell (nc). TEM, bar = 1.96 μ
Fig. 19 Lepismachilis notata. Apoptotic cell (ac) discharged into the midgut lumen (l). Midgut epithelial cells (e). TEM, bar =
1.62 μm
Fig. 20 Lepismachilis notata. Cell membranes break (arrow) and apoptotic cell (ac) is digested in the midgut lumen (l). TEM, bar = 1.67 μm
96 M.M. Rost-Roszkowska et al.
Fig. 21 Lepismachilis notata. Regenerative nest in the midgut epithelium. Accumulation of mitochondria (arrows) on the one side of each
regenerative cell. Basal lamina (asterisk), nucleus (n), nucleolus (nu). TEM, bar = 2.7 μ
Fig. 22 Machilis hrabei. Regenerative cells (r) during mitotic division. TEM, bar = 1.3 μm
Fig. 23 Lepismachilis notata. Cisterns of RER and Golgi complexes (d) in the cytoplasm of regenerative cell (r) just before its proliferation.
Nucleus (n). TEM, bar = 1.4 μm
Fig. 24 Lepismachilis notata. Accumulation of mitochondria (m) in the future apicalregion of regenerative cells (r). Nucleus (n). TEM, bar = 1 μm
Fig. 25 Lepismachilis notata. Evaginations of the apical membrane (arrow) of regenerative cells (r). Just degenerating epithelial cell (e), cisterns
of RER, Golgi complexes (d), mitochondria (m), nucleus of regenerative cell (n). TEM, bar = 0.77 μm
Fine structure of the midgut epithelium in two Archaeognatha, Lepismachilis notata and Machilis hrabei (Insecta) 97
described between T. domestica and L. saccharina. A similar
phenomenon was observed in the regenerative nests of L.
notata and M. hrabei. The ultrastructure of archaeognathan
epithelial cells is similar to that observed in Entognatha,
Zygentoma, and some pterygotan species, where character-
istic regionalization in organelles distribution has been
described (Billingsley 1990; Silva-Olivares et al. 2003;
Pigino et al. 2005;Rost2006a,b; Rost-Roszkowska 2008;
Rost-Roszkowska et al. 2010b).
Processes of degeneration, and in consequence regener-
ation, of the midgut epithelium might proceed in a cyclic
manner that is closely associated with moulting periods or
in a continuous manner during the entire life of the animal
(Garcia et al. 2001; Takeda et al. 2001; Evangelista and
Leite 2005). Previous studies of the senior author have
revealed that, in very closely related species, these
processes might proceed in a different manner (Rost
2006a). While in L. saccharina, they proceed in a cyclic
manner; in T. domestica, they occur in a continuous manner
that is not associated with moulting (Rost 2006a). It is
difficult to explain the reasons for the different modes of
midgut degeneration, but it could be an adaptation to
different temperatures in which the species live
(T. domestica 37º C, L. saccharina room temperature).
Adult specimens of the analysed archaeognathans, like
Zygentoma species, moult during their entire lives. In L.
notata and M. hrabei, however, the degeneration and
regeneration proceed in a continuous manner, but just
before each moulting, they are intensified.
Degeneration plays an important role both during embryo-
genesis and tissue and organ differentiation (Schöck and
Perrimon 2002;Proskuryakovetal.2003; Tettamanti et al.
2007a). Necrosis is defined as an incidental and passive cell
death caused by disruptive external factors (Kõműves et al.
1985; Guimarães and Linden 2004) or the type of
programmed cell death (Proskuryakov et al. 2002,2003).
Necrosis in the midgut epithelium of insects has been
described as a process in which organelles are discharged
into the midgut lumen and eventually epithelial cells undergo
lysis (Rost 2006a,b; Rost-Roszkowska 2008). An interesting
phenomenon of the degenerationthe process of apoptosis
was observed in the midgut epithelium of both analysed
archaeognathans. It is thought to be a type of programmed
cell death when an apoptotic cell is discharged into the
extracellular space (Kerr et al. 1972;Loebetal.2000;
Schöck and Perrimon 2002; Guimarães and Linden 2004).
Till now, this has been described in several insect species
(Pipan and Rakovec 1980;GregorcandBowen1997; Loeb
et al. 2000; Takeda et al. 2001;Uwoetal.2002;
Vaidyanathan and Scott 2006;Wuetal.2006;Parthasarathy
and Palli 2007,2008; Tettamanti et al. 2007a; Vilaplana et al.
2007; Park and Takeda 2008; Rodrigues et al. 2008;Rost-
Roszkowska 2008; Rost-Roszkowska et al. 2010a,2010b;
Park et al. 2009). Apoptosis in insect midgut epithelium is
regulated by juvenile hormone and 20-hydroxyecdysone
(Wu et al. 2006; Parthasarathy and Palli 2008), and induced
by many factors such as the starvation (Park et al. 2009).
Many studies connected with apoptosis detection have been
conducted with the immunostaining methods, while the
ultrastructural alterations of the apoptotic midgut cell are still
analysed (Vaidyanathan and Scott 2006; Rost-Roszkowska
et al. 2008,2010a,2010b).
However, the higher spectrum of insect species studied
can probably explain our knowledge of this presumably
rare phenomenon in the Hexapoda. Within the Zygentoma,
only in A. formicaria (Rost-Roszkowska et al. 2010b) and
N. phytophila (Rost-Roszkowska et al. in press)has
apoptosis been observed. The application of some fluores-
cent methods would probably reveal its existence also in
other Zygentoma species, where apoptosis could be rare.
Typical apoptosis appears in L. notata and M. hrabei. The
fragmentation of the nucleus is its first morphological sign.
The cytoplasm becomes electron dense. The apoptotic cell
is afterwards discharged into the midgut lumen, where it is
digested. The processes of apoptotic bodiesformation and
phagocytosis are not observed. The phagocytosis of
apoptotic bodies by adjacent epithelial cells has been
described, for example, in mosquito midgut epithelium
(Vaidyanathan and Scott 2006). This seems to be only
modestly probable in this case, however, because, as
far as we know, the epithelial cells are not capable of
phagocytosis.
Our studies revealed accumulation of a great number of
spherites of insects not possessing Malpighian tubules
(Krzysztofowicz et al. 1973;Humbert1979;Dallaiand
Burroni 1982; Szklarzewicz and Tylek 1987; Pawert et al.
1996;Piginoetal.2005;Rost2006b; Rost-Roszkowska and
Undrul 2008). In advanced insects, such crystalline struc-
tures with stored metals (Sohal and Lamb 1979) and harmful
substances are called A-type granules (Hopkin 1989). Due to
degeneration, metals accumulated in membranous structures
are shifted into the midgut lumen and are gradually
eliminated from the organism. This is one of the strategies
by which insects are able to protect themselves against the
harmful effects of metals (Sohal and Lamb 1979;Hopkin
1989). Like apoptosis, the urospherites-like structures were
recognized only in A. formicaria (Rost Rost-Roszkowska et
al. 2010b) and N. phytophila (Rost-Roszkowska et al.
in press) within the Zygentoma. Autophagy is treated as a
type of cell death that enables degradation of organelles (Lee
et al. 2002; Lockshin and Zakeri 2004; Debnath et al. 2005;
Levine and Yuan 2005;Giustietal.2007; Tettamanti et al.
2007b). Our studies show that in both L. notata and M.
hrabei, autophagy proceeds intensively, suggesting that this
is a type of elimination of, for example, toxic substances
from the organism that are accumulated in the spherites.
98 M.M. Rost-Roszkowska et al.
First, the cell gets rid of the cytoplasmic components, and
eventually, one of the irreversible pathways, apoptosis or
necrosis, is activated.
Regenerative cells of both L. notata and M. hrabei have
cytoplasm poor in organelles, and regionalization in the
organelles distribution characteristic for epithelial cells
appears gradually during their differentiation. Because of
the fact that regenerative cells are able to proliferate and
differentiate, we can treat them as stem cells of the
archaeognathan midgut epithelium. Regenerative cells of
insect midgut epithelium are suggested to be its stem cells for
many insect species (Hakim et al. 2001; Martins et al. 2006;
Rost 2006a,b; Rost-Roszkowska 2008;Fialhoetal.2009).
Our studies concerning the ultrastructure of the midgut
epithelium in primitive hexapodan groups would probably
help in elucidating relationships between the Archaeognatha
and other Insecta. Concerning the midgut anatomy of
primarily apterous insect taxa, recently, only Koch and
Dolgener (2008) have used the character of midgut, its
caeca, and have suggested to use midgut organisation in
proposing relationships of the zygentoman families. The
results of our studies would help us in preparing such
analysis of mainly primitive hexapodan relationship.
Acknowledgements We would like to express our gratitude to Prof.
Jerzy Klag (University of Silesia, Poland) and to Dr. Petr Švácha
(Entomological Institute, Czech Republic) for a critical reading of the
manuscript, to Gale Allen Kirking (EnglishEditorial Services, Brno)
for correction of English and to Mgr. Petr Dolejš(Charles University,
Czech Republic) for great help in collecting material.
The study was supported by a grant of the Ministry of Education of
Czech Republic, MSM 0021620828, to the junior authors (P.J. and J.V.).
Conflict of interest The authors declare that they have no conflict of
interest.
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Fine structure of the midgut epithelium in two Archaeognatha, Lepismachilis notata and Machilis hrabei (Insecta) 101
... However, the picture is rather variegated and marked differences have been observed even in closely related groups (Rost et al. 2005;Rost-Roszkowska 2006b;Rost-Roszkowska et al. 2007). In fact, SCs can simply differentiate into mature epithelial cells, and are thus not considered true SCs (Rost-Roszkowska 2006b), or undergo both proliferation and differentiation (Rost-Roszkowska et al. 2007, 2010b. In addition, they can be dispersed as single cells in the midgut epithelium (Rost-Roszkowska 2006b;Rost-Roszkowska et al. 2010d) or organized in nests that contain up to several dozens of cells (Rost-Roszkowska et al. 2007, 2010b, where they can be joined each other by junctions. ...
... In fact, SCs can simply differentiate into mature epithelial cells, and are thus not considered true SCs (Rost-Roszkowska 2006b), or undergo both proliferation and differentiation (Rost-Roszkowska et al. 2007, 2010b. In addition, they can be dispersed as single cells in the midgut epithelium (Rost-Roszkowska 2006b;Rost-Roszkowska et al. 2010d) or organized in nests that contain up to several dozens of cells (Rost-Roszkowska et al. 2007, 2010b, where they can be joined each other by junctions. In the latter case, inner cells usually proliferate and maintain the pool of SCs in the midgut epithelium, while outer cells undergo differentiation into CCs (Rost-Roszkowska et al. 2007, 2010b. ...
... In addition, they can be dispersed as single cells in the midgut epithelium (Rost-Roszkowska 2006b;Rost-Roszkowska et al. 2010d) or organized in nests that contain up to several dozens of cells (Rost-Roszkowska et al. 2007, 2010b, where they can be joined each other by junctions. In the latter case, inner cells usually proliferate and maintain the pool of SCs in the midgut epithelium, while outer cells undergo differentiation into CCs (Rost-Roszkowska et al. 2007, 2010b. According to this evidence, the hypothesis of a gradual increase in the evolution of SCs, i.e., starting from those Collembola species that lack SCs and regenerate epithelial cells by themselves (Jura 1958;Rost-Roszkowska and Undrul 2008), passing through single cells sparse in the epithelium, and ending up with SCs organized in nests, is questioned and further studies are necessary to fill the gap of knowledge, which is still fragmentary, on these Hexapoda. ...
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The insect midgut epithelium represents an interface between the internal and the external environment and it is the almost unique epithelial tissue by which these arthropods acquire nutrients. This epithelium is indeed able to produce digestive enzymes and to support vectorial transport of small organic nutrients, ions, and water. Moreover, it plays a key role in the defense against pathogenic microorganisms and in shaping gut microbiota. Another important midgut function is the ability to produce signaling molecules that regulate its own physiology and the activity of other organs. The two main mature cell types present in the midgut of all insects, i.e., columnar and endocrine cells, are responsible for these functions. In addition, stem cells, located at the base of the midgut epithelium, ensure the growth and renewal of the midgut during development and after injury. In insects belonging to specific orders, midgut physiology is deeply conditioned by the presence of unique cell types, i.e., goblet and copper cells, which confer peculiar features to this organ. This review reports current knowledge on the cells that form the insect midgut epithelium, focusing attention on their morphological and functional features. Notwithstanding the apparent structural simplicity of this organ, the properties of the cells make the midgut a key player in insect development and homeostasis.
... The epithelial midgut was composed of regenerative cells (RC)-which are considered as stem cells, help in the renewal of cells, and may be located singly among epithelial cells or form groups called regenerative nests or crypts-which possessed a large nucleus ( Figure 3B,C). Moreover, the normal regenerative cells were observed with normal nuclei which have a central nucleolus (n) and chromatin (CH) ( Figure 3A); these results concurred with [60,61]. Something is known about ultrastructural alterations encouraged by heavy metal pollution in the midgut tissues of the insects, according to Yingmei et al. (2001) [62] and Polidori et al. (2018) [63]. ...
... These results are in accordance with [69][70][71] who suggested that apoptosis is considered as a physiological process that enables the balance between the propagation rate and the removal of useless cells, whereas necrosis is a passive cell death resulting from acute cellular dysfunction after exposure to toxic heavy metals. The current study revealed that the damaged organelles were utilized and removed by the formation of considerable numbers of lysosomes (L) that are considered as an index of lyses due to pollution with heavy metals, as depicted in Figure 5D-F; these results agreed with [72] on the mosquito (Culex pipiens) midgut epithelium and [60] on (Archaeognatha, Lepismachilis notata, and Machilis hrabei) Coccinellidae. A lot of empty secretory vesicles (vacuoles) (ESV) without or with a few digestive enzyme secretions and spherites are presented in Figure 5A,D,E; there was a reduction of basophilic secretions towards the lumen that became very narrow (NL) due to enlargement and stretching of cells attributed to detoxification with heavy metals ( Figure 5E). ...
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... The reserve material gathered in the midgut epithelium of I. g. granulifer during oogenesis might be eventually removed from its cells by autophagy because this process plays a role in cell death (Levine and Yuan 2005;Giusti et al. 2007;Tettamanti et al. 2007;, 2010a, 2010b and enables exploiting the material or organelles gathered in the cytoplasm. Autophagy is gradually intensified (from the beginning of vitellogenesis till the end of choriogenesis), which might be connected with the increasing requisition for, for example, chorion layers formation or even for nutrition of the animal which does not feed. ...
... Autophagy is gradually intensified (from the beginning of vitellogenesis till the end of choriogenesis), which might be connected with the increasing requisition for, for example, chorion layers formation or even for nutrition of the animal which does not feed. The more autophagosomes is gathered in the cell, the more material and organelles are removed from the cell , 2010b. ...
... Three main types of cell death have been recognized in the digestive system of invertebrates: apoptosis, necrosis, and autophagy. Cell death maintains homeostasis in the event of external stressors, and can be involved in embryonic and postembryonic development of tissues and organs (Bruno et al., 2019;Caccia, Casartelli, & Tettamanti, 2019;Franzetti et al., 2012Franzetti et al., , 2016Klionsky & Emr, 2000;Kourtis & Tavernarakis, 2009;Lipovšek et al., 2018;Lipovšek, Janžekovič, & Novak, 2014;Lipovšek & Novak, 2016;Rost-Roszkowska et al., 2010, 2019Rost-Roszkowska, Chajec, Vilimova, & Tajovsky, 2016;Rost-Roszkowska, Janelt, & Poprawa, 2018;Sonakowska et al., 2016;Tettamanti, Cao, Feng, Grimaldi, & de Eguileor, 2011;Tettamanti, de Busserolles, Lecchini, Marshall, & Cortesi, 2019;Wilczek et al., 2014;Włodarczyk et al., 2019a). ...
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... T. aoutii has a pseudostratified midgut epithelium as has been described in Floridobolus penneri and Narceus gordanus (Bowen 1968), A. gigas, J. scandinavius (Sosinka et al. 2014) and Rhinocricus padbergi (Camargo-Mathias et al. 2004), while the simple prismatic or columnar midgut epithelium has been described in the majority of millipedes that have been examined (Fontanetti et al. 2015). The digestive cells are the principal cells of the midgut epithelium in millipedes and their ultrastructure resembles the ultrastructure of the digestive cells of the Hexapoda midgut epithelium (Serrão and Cruz-Landim 1996;Silva-Olivares et al. 2003;Rost-Roszkowska et al. 2010, 2016a and Chilopoda (Myriapoda) (Koch et al. The distinct regionalization in organelle distribution in T. aoutii is similar to that analyzed in other millipedes (Camargo-Mathias et al. 2004;Fonanetti et al. 2006Fonanetti et al. , 2015Sosinka et al. 2014). ...
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The midgut of millipedes is composed of a simple epithelium that rests on a basal lamina, which is surrounded by visceral muscles and hepatic cells. As the material for our studies, we chose Telodeinopus aoutii (Demange, 1971) (Kenyan millipede) (Diplopoda, Spirostreptida), which lives in the rain forests of Central Africa. This commonly reared species is easy to obtain from local breeders and easy to culture in the laboratory. During our studies, we used transmission and scanning electron microscopes and light and fluorescent microscopes. The midgut epithelium of the species examined here shares similarities to the structure of the millipedes analyzed to date. The midgut epithelium is composed of three types of cells-digestive, secretory, and regenerative cells. Evidence of three types of secretion have been observed in the midgut epithelium: merocrine, apocrine, and microapocrine secretion. The regenerative cells of the midgut epithelium in millipedes fulfill the role of midgut stem cells because of their main functions: self-renewal (the ability to divide mitotically and to maintain in an undifferentiated state) and potency (ability to differentiate into digestive cells). We also confirmed that spot desmosomes are common intercellular junctions between the regenerative and digestive cells in millipedes.
... This is seen in the contribution by Santos et al. (2017) in the current issue. The authors have been using ultrastructural analysis to address phylogenetic issues of insect evolution, such as the Archaeognatha (Rost- Roszkowska et al. 2010). In their current work, they investigate the digestive tract of Heteroptera with different feeding habits comparing zoophagous and phytophagous species and compare features of secretory, absorptive, transport, storage, and excretory cells by transmission electron microscopy. ...
... This tissue should be suitable for cytogenetic study due to continual wasting of digestive cells, followed by intensive mitotic division and differentiation of the regenerative (= stem) cells (e.g. Azevedo et al. 2009, Rost-Roszkowska et al. 2010a, 2010b. Nevertheless, midgut epithelium slides of C. lectularius contained no countable mitotic chromosomes. ...
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In the article we summarize the most common recent cytogenetic methods used in analysis of karyotypes in Heteroptera. We seek to show the pros and cons of the spreading method compared with the traditional squashing method. We discuss the suitability of gonad, midgut and embryo tissue in Cimexlectularius Linnaeus, 1758 chromosome research and production of figures of whole mitosis and meiosis, using the spreading method. The hotplate spreading technique has many advantages in comparison with the squashing technique. Chromosomal slides prepared from the testes tissue gave the best results, tissues of eggs and midgut epithelium are not suitable. Metaphase II is the only division phase in which sex chromosomes can be clearly distinguished. Chromosome number determination is easy during metaphase I and metaphase II. Spreading of gonad tissue is a suitable method for the cytogenetic analysis of holokinetic chromosomes of Cimexlectularius.
... This tissue should be suitable for cytogenetic study due to continual wasting of digestive cells, followed by intensive mitotic division and differentiation of the regenerative (= stem) cells (e.g. Azevedo et al. 2009, Rost-Roszkowska et al. 2010a, 2010b. Nevertheless, midgut epithelium slides of C. lectularius contained no countable mitotic chromosomes. ...
Article
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In the article we summarize the most common recent cytogenetic methods used in analysis of karyotypes in Heteroptera. We seek to show the pros and cons of the spreading method compared with the traditional squashing method. We discuss the suitability of gonad, midgut and embryo tissue in Cimex lectularius Linnaeus, 1758 chromosome research and production of figures of whole mitosis and meiosis, using the spreading method. The hotplate spreading technique has many advantages in comparison with the squashing technique. Chromosomal slides prepared from the testes tissue gave the best results, tissues of eggs and midgut epithelium are not suitable. Metaphase II is the only division phase in which sex chromosomes can be clearly distinguished. Chromosome number determination is easy during metaphase I and metaphase II. Spreading of gonad tissue is a suitable method for the cytogenetic analysis of holokinetic chromosomes of C. lectularius.
... Degenerating changes were also observed in crypt cells (Fig. 3D, E) which had vacuoles, translucent cytoplasm and autophagy-like structures in the cytoplasm. In some cells membranous structures, probably formed from endoplasmatic reticulum membranes (Rost-Roszkowska et al. 2010) which are similar to autophagosomes, were observed in the epithelial cells (Fig. 3E, G, H, I). After 20 days of exposure, cells in regenerative pouches were often swollen and the crypts disintegrated (Fig. 3F). ...
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