Content uploaded by Jitka Vilimova
Author content
All content in this area was uploaded by Jitka Vilimova on Feb 19, 2015
Content may be subject to copyright.
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;Dell’Ampio 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) 247:91–101
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 cell’s 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 apical”region 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 degeneration—the 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 bodies’formation 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.
References
Biliński S, Klag J (1979) The ultrastructure of midgut in Acerentomon
gallicum (Jonescu) (Protura). Folia Biol (Krakow) 27:3–7
Billingsley PF (1990) The midgut ultrastructure of hematophagous
insects. Annu Rev Entomol 35:219–248
Billingsley PF, Lehane MJ (1996) Structure and ultrastructure of the
insect midgut. In: Lehane MJ, Billingsley PF (eds) Biology of the
insect midgut. Chapman & Hall, London, pp 3–30
Cameron SL, Beckenbach AT, Dowton M, Whiting M (2006)
Evidence from mitochondrial genomics on interordinal relation-
ships in insects. Arthropod Syst Phylogeny 64:45–52
Chapman RF (1998) The insects: structure and function, 4th edn.
Cambridge Univ, Cambridge
Dallai R, Burroni D (1982) Fine structure of the pyloric region and
Malpighian papillae of Diplura. Mem Soc Entomol Ital 60:125–
135
Debnath J, Baehrecke EH, Kroemer G (2005) Does autophagy
contribute to cell death? Autophagy 1:66–74
Dell’Ampio E, Szucsich NU, Carapelli A, Frati F, Steiner G,
Steinacher A, Pass G (2009) Testing for misleading effects in
the phylogenetic reconstruction of ancient lineages of hexapods:
influence of character dependence and character choice in
analyses of 28 S rRNA sequences. Zool Scr 38:155–170
Evangelista LG, Leite ACR (2005) Optical and ultrastructural studies
of midgut and salivary glands of first instar of Dermatobia
hominis (Diptera: Oestridae). J Med Entomol 42:218–223
Fialho M, Zanuncio JC, Neves CA, Ramalho FS, Serrão JE (2009)
Ultrastructure of the digestive cells in the midgut of the predator
Brontocoris tabidus (Heteroptera: Pentatomidae) after different
feeding periods on prey and plants. Ann Entomol Soc Am
102:119–127
Garcia JJ, Li G, Wang P, Zhong J, Granados RR (2001) Primary and
continuous midgut cell cultures from Pseudaletia unipuncta
(Lepidoptera, Noctuidae). In Vitro Cell Dev Biol Anim 37:353–359
Giusti F, Dallai L, Beani L, Manfredini F, Dallai R (2007) The midgut
ultrastructure of the endoparasite Xenos vesparum (Rossi)
(Insecta, Strepsiptera) during post-embryonic development and
stable carbon isotopic analyses of the nutrient uptake. Arthropod
Struct Dev 36:183–197
Gregorc A, Bowen ID (1997) Programmed cell death in the honey-bee
(Apis mellifera L.) larvae midgut. Cell Biol Int 21:151–158
Grimaldi D (2001) Insect evolutionary history from Handlirsch to
Hennig, and beyond. J Paleontol 75:1152–1160
Grimaldi DA 400 million years on six legs: on the origin and early
evolution of Hexapoda. Arthropod Struct Dev (2009), doi:
10.1016/j.asd.2009.10.008
Guimarães CA, Linden R (2004) Programmed cell death. Apoptosis
and alternative deathstyles. Eur J Biochem 271:1638–1650
Hakim RS, Baldwin KM, Loeb M (2001) The role of stem cells in
midgut growth and regeneration. In Vitro Cell Dev Biol Anim
37:338–342
Hopkin SP (1989) Ecophysiology of metals in terrestrial invertebrates.
Elsevier Applied Science, New York
Humbert W (1979) The midgut of Tomocerus minor Lubbock
(Insecta, Collembola): ultrastructure, cytochemistry, ageing and
renewal during a moulting cycle. Cell Tissue Res 196:39–57
Jura Cz (1958) The alimentary canal of the Tetrodontophora bielanensis
(Waga) (Collembola). Polskie Pismo Entomol 27:85–89
Kerr JFR, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological
phenomenon with wide-ranging implications in tissues kinetics.
Brit J Cancer 26:239–257
Kjer KM, Carle FL, Litman J, Ware J (2006) A molecular phylogeny
of Hexapoda. Arthropod Syst Phylogeny 64:35–44
Klag J, Książkiewicz M, Rościszewska E (1981) The ultrastructure of
the midgut in Xenylla grisea (Collembola). Acta Biol Cracov Ser
Zool 23:47–52
Klug R, Klaas KD (2006) The potential value of the mid-abdominal
musculature and nervous system in the reconstruction of
interordinal relationships in lower Neoptera. Arthropod Syst
Phylogeny 65:73–100
Koch M (2001) Mandibular mechanisms and the evolution of
Hexapods. Ann Soc Entomol Fr (N.S.) 37: 129–174.
Koch M, Dolgener N (2008) Comparative anatomy and phylogeny of
the Zygentoma (Insecta). J Morphol 269:1476
Kõműves LG, Sass M, Kovács J (1985) Autophagocytosis in the
larval midgut cells of Pieris brassicae during metamorphosis.
Induction by 20-hydroxyecdysone and the effect of puromycin
and cycloheximide. Cell Tissue Res 240:215–221
Krzysztofowicz A, Cz J, Biliński S (1973) Ultrastructure of midgut
epithelium cells of Tetrodontophora bielanensis (Waga) (Col-
lembola). Acta Biol Cracov Ser Zool 20:257–265
Larink O (1983) Embryonic and postembryonic development of
Machilidae and Lepismatidae (Insecta: Archaeognatha et Zygen-
toma). Entomol Gen 8:119–133
Lauga-Reyrel F (1980) Aspect histophysiologique de l’écomorphose:
étude ultrastructurale du mesenteron chez Hypogastrura tullbergi
Fine structure of the midgut epithelium in two Archaeognatha, Lepismachilis notata and Machilis hrabei (Insecta) 99
(Collemboles). Trav Lab Ecobiol Arthropodes Edaphiques,
Toulouse 2:1–11
Lee CY, Cooksey BAK, Baehrecke EH (2002) Steroid regulation of
midgut cell death during Drosophila development. Dev Biol
250:101–111
Levine B, Yuan J (2005) Autophagy in cell death: an innocent
convict? J Clin Invest 115:2679–2688
Levy SM, Falleiros AMF, Gregório EA, Arrebola NR, Toledo LA
(2004) The larval midgut of Anticarsia gemmatalis (Hübner)
(Lepidoptera: Noctuidae): light and electron microscopy studies
of the epithelial cells. Br J Biol 64:633–638
Lockshin RA, Zakeri Z (2004) Apoptosis, autophagy and more. Int J
Biochem Cell Biol 36:2405–2419
Loeb MJ, Hakim RS, Martin P, Narang N, Goto S, Takeda M (2000)
Apoptosis in cultured midgut cells from Heliothis virescens
larvae exposed to various conditions. Arch Insect Biochem
Physiol 45:12–23
Machida R (2006) Evidence from embryology for reconstructing the
relationships of hexapod basal clades. Arthropod Syst Phylogeny
64:95–104
Machida R, Ando H (1981) Formation of midgut epithelium in the
jumping bristletail Pedetontus unimaculatus (Machida) (Archae-
ognatha, Machilidae). Int J Insect Morphol Embryol 10:297–307
Martins GF, Neves CA, Campos LAO, Serrão JE (2006) The
regenerative cells during the metamorphosis in the midgut of
bees. Micron 37:161–168
Misof B, Niehuis O, Bischoff I, Rickert A, Erpenbeck D, Staniczek A
(2007) Towards an 18 S phylogeny of hexapods: accounting for
group-specific character covariance in optimized mixed nucleo-
tide/doublet models. Zoology 110:409–429
Neves CA, Gitirana LB, Serrão JE (2003) Ultrastructural study of the
metamorphosis in the midgut of Melipona quadrifasciata anthi-
dioides (Apidae, Meliporuni) Worker. Sociobiology 41:443–459
Park MS, Takeda M (2008) Starvation suppresses cell proliferation
that rebounds after refeeding in the midgut of the American
cockroach, Periplaneta americana. J Insect Physiol 54:386–392
Park MS, Park P, Takeda M (2009) Starvation induces apoptosis in the
midgut nidi of Periplaneta americana: a histochemical and
ultrastructural study. Cell Tissue Res 335:631–638
Parthasarathy R, Palli SR (2007) Developmental and hormonal
regulation of midgut remodeling in a lepidopteran insect, Helio-
this virescens. Mech Dev 124:23–34
Parthasarathy R, Palli SR (2008) Proliferation and differentiation
of intestinal stem cells during metamorphosis of the red
flour beetle, Tribolium castaneum. Dev Dynam 237:893–908
Pawert M, Triebskorn R, Gräff S, Berkus M, Schulz J, Köhler H-R
(1996) Cellular alteration in collembolan midgut cells as a
marker of heavy metals exposure: ultrastructure and intracellular
metal distribution. Sci Total Environ 181:187–200
Pigino G, Migliorini M, Paccagnini E, Bernini F, Leonzio C (2005)
Fine structure of the midgut and Malpighian papillae in
Campodea (Monocampa)quilisi Silvestri, 1932 (Hexapoda,
Diplura) with special reference to the metal composition and
physiological significance of midgut intracellular electron-dense
granules. Tissue Cell 37:223–232
Pipan N, Rakovec V (1980) Cell death in the midgut epithelium of the
worker honey bee (Apis mellifera carnica) during metamorpho-
sis. Zoomorphology 94:217–224
Proskuryakov SY, Gabli VL, Konoplyannikov AG (2002) Necrosis is
an active and controlled form of programmed cell death.
Biochem (Moscow) 67:387–408
Proskuryakov SY, Konoplyannikov AG, Gabli VL (2003) Necrosis: a
specific form of programmed cell death? Exp Cell Res 283:1–16
Regier JC, Shultz JW, Zwick A, Hussey A, Ball B, Wetzer R, Martin
JW, Cunningham CW (2010) Arthropod relationships revealed
by phylogenomic analysis of nuclear protein-coding sequences.
Nature. doi:10.1038/nature08742
Rodrigues A, Cunha L, Amaral A, Medeiros J, Garcia P (2008)
Bioavailability of heavy metals and their effects on the midgut
cells of a phytophagous insect inhabiting volcanic environments.
Sci Total Environ 406:116–122
Rost MM (2006a) Comparative studies on regeneration of the midgut
epithelium in Lepisma saccharina L. and Thermobia domestica
Packard (Insecta, Zygentoma). Ann Entomol Soc Am 99:910–
916
Rost MM (2006b) Ultrastructural changes in the midgut epithelium in
Podura aquatica L. (Insecta, Collembola, Arthropleona) during
regeneration. Arthropod Struct Dev 35:69–76
Rost MM, Kuczera M, Malinowska J, Polak M, Sidor B (2005)
Midgut epithelium formation in Thermobia domestica (Pack-
ard) (Insecta, Zygentoma). Tissue Cell 37:135–143
Rost-Roszkowska MM (2008) Degeneration of the midgut
epithelium in Allacma fusca L. (Insecta, Collembola, Sym-
phypleona): apoptosis and necrosis. Zool Sci 25:753–759
Rost-Roszkowska MM, Undrul A (2008) Fine structure and differen-
tiation of the midgut epithelium of Allacma fusca (Insecta,
Collembola, Symphypleona). Zool Stud 47:200–206
Rost-Roszkowska MM, Piłka M, Szymska R, Klag J (2007) Ultrastruc-
tural studies of midgut epithelium formation in Lepisma saccharina
L. (Insecta, Zygentoma). J Morphol 268:224–231
Rost-Roszkowska MM, Poprawa I, Klag J, Migula P, Mesjasz-
Przybyłowicz J, Przybyłowicz W (2008) Degeneration of the
midgut epithelium in Epilachna cf nylanderi (Insecta, Cocci-
nellidae): apoptosis, autophagy and necrosis. Can J Zool
86:1179–1188
Rost-Roszkowska MM, Machida R, Fukui M (2010a) The role of cell
death in the midgut epithelium in Filientomon takanawanum
(Protura). Tissue Cell 42:24–31
Rost-Roszkowska MM, Vilimova J, Chajec Ł(2010b) Fine structure
of the midgut epithelium in Atelura formicaria (Hexapoda,
Zygentoma, Ateluridae), with special reference to its regeneration
and degeneration. Zool Stud 49:10–18
Rost-Roszkowska MM, Vilimova J, Chajec Ł(in press) Fine structure
of the midgut epithelium of Nicoletia phytophila Gervais, 1844
(Zygentoma: Nicoletiidae: Nicoletiinae) with the special empha-
sis on its degeneration. Folia Biol Cracov.
Schöck F, Perrimon N (2002) Molecular mechanisms of epithelial
morphogenesis. Annu Rev Cell Dev Biol 18:463–493
Silva-Olivares A, Diaz E, Shibayama M, Tsutsmi V, Cisneros R,
Zuniga G (2003) Ultrastructural study of the midgut and
hindgut in eight species of the genus Dendroctonus Erichson
(Coleoptera: Scolytidae). Ann Entomol Soc Am 96:883–900
Sohal RS, Lamb RE (1979) Storage-excretion of metallic cations in
the adult housefly Musca domestica. J Insect Physiol 25:119–124
Staniczek AH (2000) The mandible of silverfish (Insecta: Zygentoma)
and mayflies (Ephemeroptera): its morphology and phylogenetic
significance. Zool Anz 239:147–178
Szklarzewicz T, Tylek W (1987) Ultrastructure of midgut epithelial
cells of Campodea sp. (Diplura). Acta Biol Cracov Ser Zool
29:127–131
Takeda M, Sakai T, Fujisawa Y, Narita M, Iwabuchi K, Loeb MJ (2001)
Cockroach midgut peptides that regulate cell proliferation, differen-
tiation and death in vitro. In Vitro Cell Dev Biol Anim 37:343–347
Terra WR (1990) Evolution of digestive systems of insects. Annu Rev
Entomol 35:181–200
Tettamanti G, Grimaldi A, Casartelli M, Ambrosetti E, Ponti B, Congiu T,
Ferrarese R, Rivas-Pena ML, Pennacchio F, de Eguileor M (2007a)
Programmed cell death and stem cell differentiation are responsible
for midgut replacement in Heliothis virescens during prepupal instar.
Cell Tissue Res 330:345–359
100 M.M. Rost-Roszkowska et al.
Tettamanti G, Grimaldi A, Pennacchio F, de Eguileor M (2007b) Lepidop-
teran larval midgut during prepupal instar. Autophagy 3:630–631
Uwo MF, Vi-Tei K, Park P, Takeda M (2002) Replacement of midgut
epithelium in the greater wax moth Galleria mellonella during
larval—pupal moult. Cell Tissue Res 308:319–331
Vaidyanathan R, Scott TW (2006) Apoptosis in mosquito midgut
epithelia associated with West Nile virus infection. Apoptosis
11:1643–1651
Vilaplana L, Pascual N, Perera N, Bellés X (2007) Molecular
characterization of an inhibitor of apoptosis in the Egyptian
armyworm, Spodoptera littoralis, and midgut cell death during
metamorphosis. Insect Biochem Mol Biol 37:1241–1248
Wu Y, Parthasarathy R, Bai H, Palli SR (2006) Mechanisms of midgut
remodeling: juvenile hormone analog methoprene blocks midgut
metamorphosis by modulating ecdysone action. Mech Dev
123:530–547
Fine structure of the midgut epithelium in two Archaeognatha, Lepismachilis notata and Machilis hrabei (Insecta) 101