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Micron 43 (2012) 651–665

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Micron

j ournal homepage: www.elsevier.com/locate/micron

The evidence on the degradation processes in the midgut epithelial cells of the

larval antlion Euroleon nostras (Geoffroy in Fourcroy, 1785) (Myrmeleontidae, Neuroptera)

a,b,∗ c c d b

Saskaˇ Lipovsekˇ , Ilse Letofsky-Papst , Ferdinand Hofer , Gerd Leitinger , Dusanˇ Devetak

Faculty of Medicine, University of Maribor, Slomskovˇ trg 15, SI-2000 Maribor, Slovenia

Department of Biology, Faculty of Natural Sciences and Mathematics, University of Maribor, Koroskaˇ 160, SI-2000 Maribor, Slovenia

Research Institute for Electron Microscopy, Graz University of Technology, Steyrergasse 17, A-8010 Graz, Austria

Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Harrachgasse 21, A-8010 Graz, Austria

a r t i c l e i n f o a b s t r a c t

Article history: We analysed structural differences between midgut epithelial cells of fed instar antlions’ larvae Euroleon

Received 31 August 2011

nostras and starved ones. In starved larvae the presence of autophagolysosome-like structures was

Received in revised form

observed, which are characteristic structures associated with autophagy. The results presented here

28 November 2011

provide insight supporting the role of autophagy as a cell survival mechanism for the periods of food

Accepted 29 November 2011

deprivation. Additional structural changes in the cytoplasm were seen in the spherites. The ultrastruc-

ture and chemical composition of spherites in the midgut epithelial cells of ﬁrst, second and third instar

Keywords:

larvae were observed with light microscopy and transmission electron microscopy (TEM). A detailed

Autophagy

characterization of the elemental composition of the spherites was studied using analytical electron

Euroleon nostras

microscopy; a combination of energy dispersive X-ray spectroscopy (EDXS), electron energy-loss spec-

Larval instars

Midgut troscopy (EELS) and energy ﬁltering TEM (EFTEM) was applied. The structure and elemental composition

Spherites of the spherites changed during the period of larval life. Moreover, changes in chemical composition were

Ultrastructure found between spherites from fed and starved E. nostras. In fed ﬁrst instar larvae, the spherites contained

an organic matrix, composed of C, N and O. In this matrix, P, Cl, Ca and Fe were detected. In starved ﬁrst

instar larvae, only C, N and P were present. The spherites of fed second instar larvae were rich in organic

and inorganic elements and were composed of C, N, O, Na, Mg, P, S, Cl, K, Ca, Mn, Fe and Zn. In starved

second instar larvae, the chemical elements N, O, P, Ca and Fe were found. In fed third instar larvae, the

spherites contained C, N, O, Na, Mg, P, Cl, K, Ca, Mn, Fe, Co and Zn. In starved third larvae, C, O, Si, Ca,

and Fe were detected. Generally, the spherites are exploited in starved larvae. These results suggest that

the elemental supply of spherites may provide crucial support for physiological processes during star-

vation periods amongst E. nostras instar larvae. In some cases in fed second and fed third instar larvae,

spherites were seen in the lumen of the midgut. Such spherites could serve as reservoirs for nontoxic

waste material that cannot be metabolized.

1. Introduction prey capture during antlions’ larval stage. Larval growth has an

important impact on the individual’s ability to reproduce (Gotelli,

Antlions (Neuroptera, Myrmeleontidae) are a diverse group of 1997). The antlions feed on small arthropods that slide into the

insects dispersed worldwide in arid, sandy regions (Gepp, 2010). pit (Topoff, 1977; Grifﬁths, 1980; Gepp and Hölzel, 1989; Gepp,

The larvae of Euroleon nostras dig a conical pit in dry sand where 2010). After the arthropod slides into the pit, it is seized in the

they wait on their prey at the bottom of the sand funnel. Antlions jaws of the antlion. Digestive enzymes and toxins injected into the

detect approaching prey from a distance of a few centimetres prey kill it and dissolve its internal tissues (Koch, 1983; Nishiwaki

by sensing the vibrations that prey generates during locomo- et al., 2007). The resulting ﬂuid is then drawn into the alimentary

tory activity (Devetak et al., 2007). The potentially long wait canal of the antlion larva. E. nostras larvae pass through three

between meals in this predatory species belies the importance of stages (instars) and overwinter twice, before spinning a tough silk

cocoon, and entering the pupal stage. Adults emerge from the pupa

towards the end of July or in the ﬁrst few days of August. Larvae

∗ must acquire sufﬁcient food reserves to sustain them during

Corresponding author at: Faculty of Medicine, University of Maribor, Slomskovˇ

subsequent pupation, when they do not feed at all (Grifﬁths,

trg 15, SI-2000 Maribor, Slovenia. Tel.: +386 2 22 93 709; fax: +386 2 25 18 180.

E-mail address: [email protected] (S. Lipovsek).ˇ 1980).

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652 S. Lipovsekˇ et al. / Micron 43 (2012) 651–665

In insects, the epithelium of the midgut is composed of a major Spherites are cytoplasmic structures present in different cell

type of the cells usually named columnar cells. It also contains types in insects (Martoja and Ballan-Dufranc¸ ais, 1984; Baldwin

regenerative cells that are often collected together at the base of et al., 1996; Billingsley and Lehane, 1996; Pinheiro et al., 2008). They

the epithelium (Terra and Ferreira, 2003). As it is described by are commonly found in the insect midgut (e.g. Sohal et al., 1977;

Rost-Roszkowska et al. (2010), the midgut epithelium of differ- Cruz-Landim and Serrão, 1997) and in their Malpighian tubules

ent insects adapts to different environmental alterations and the (Cruz-Landim, 2000; Ballan-Dufranc¸ ais, 2002). Additionally, they

processes of degeneration enable its functioning. An insect’s epithe- have been described in the midgut glands of various taxa of inver-

lium cells undergo cell death in two ways: necrotic or apoptotic tebrates, e.g., spiders (Ludwig and Alberti, 1988), acari (Pigino et al.,

(Proskuryakov et al., 2002). Necrosis is incidental, passive cell death 2006; Sobotnik et al., 2008), harvestmen (Lipovsekˇ Delakorda et al.,

caused by disruptive external chemical, physical or biological fac- 2004) and molluscs (Angulo and Moya, 1989; Colville and Lim,

tors (Guimarães and Linden, 2004) or the type of programmed 2003). In the digestive glands of crustaceans, electron-dense gran-

cell death (Proskuryakov et al., 2002). Cells that die as a result ules or spherites are thought to have at least two functions: Ca and P

of necrosis typically swell and burst, spilling their contents in storage and detoxiﬁcation (Hopkin, 1989; Lowenstam and Weiner,

the surrounding. This process triggers a potentially inﬂammatory 1989; Corrêa et al., 2000). The chemical composition of spherites

response (Alberts et al., 2004). By contrast, apoptosis is an actively has been analysed in different invertebrates; various chemical ele-

regulated process, important for the removal of useless or unex- ments, e.g. C, O, N, Ca, Na, Mg, K, Cl, Si, P, S, Fe and Ba have been

ploited cells (Guimarães and Linden, 2004; Alberts et al., 2004) detected (Goyffon and Martoja, 1983; Al-Mohanna and Nott, 1989;

and has been studied as widespread process in the epithelium of Ludwig and Alberti, 1988).

insect midgut (Park et al., 2009; Rost-Roszkowska et al., 2010). It The ultrastructure of the midgut epithelial cells in E. nostras liv-

can be induced by different factors, e.g. starvation (Park et al., 2009). ing in their natural habitats has been analysed in the previous study

Early in apoptosis, dense chromosome condensation occurs along (Lipovsekˇ et al., 2011). In this study the midgut epithelium of fed

the nuclear periphery. The cell shrinks although most organelles and non-fed ﬁrst (L1), second (L2) and third (L3) instar larvae of

remain intact. Later on the nucleus and cytoplasm fragment, form- the antlion E. nostras treated in the laboratory was observed. The

ing apoptotic bodies which are phagocytosed by surrounding cells ﬁrst group of antlions was fed daily (experimental group 1; fed

(Alberts et al., 2004). larvae L1+, L2+, L3+). The second group of antlions was deprived

− −

−

Along with necrosis and apoptosis, autophagy contributes to of food (experimental group 2; starved larvae L1 , L2 , L3 ). The

the maintaining homeostasis through the disintegration of the animals were exposed to these conditions over a 30-day period.

organelles utilized by vacuoles or lysosomes (Rost-Roszkowska Using analytical electron microscopy, the chemical composition

et al., 2010). Autophagy is deﬁned as a lysosome-dependent of the spherites in each larval stage from experimental group 1

mechanism of intracellular degradation that is essential for the and experimental group 2 was analysed. The primary goal of this

turnover of cytoplasm (Xie and Klionsky, 2007). Several forms study was to gain insight into the potential impact of starvation on

of autophagy are known, e.g. macroautophagy, microautophagy the ultrastructure of the midgut epithelial cells in E. nostras. Addi-

and chaperone-mediated autophagy (Mizushima et al., 2002). tional goal was to establish whether there are any differences in the

Macroautophagy is used for the degradation of cytoplasm in spherites’ ultrastructure and their chemical composition between

processes that incorporate specialized cytosolic vesicles or vac- fed and starved instar larvae.

uoles that fuse with the lysosome. Microautophagy involves

the direct uptake of cytoplasm at the lysosome surface by

2. Materials and methods

invagination of the lysosome membrane. Chaperone-mediated

autophagy occurs at the membrane of the lysosome, and relies

2.1. Insects

on the translocation of unfolded proteins across the membrane

(Mizushima et al., 2002; Yang et al., 2005). Macroautophagy

Instar larvae of the antlion E. nostras were collected from their

represents the principal process involved in the degradation

natural environment (Maribor, Slovenia). In the laboratory, all

of cellular compounds (e.g., damaged cytoplasmic organelles

larvae were living in the same substratum as they were collected

by lysosomal enzymes), therefore, it protects cells against the

accumulation of damaged cellular components (Rideout et al.,

2004). Macroautophagy (hereafter referred to as autophagy) is

regulated during the response to the availability of nutrients

and during the development of multicellular organisms. Dur-

ing autophagy, regional sequestration of cytoplasm within an

enveloping double membrane structure creates a vacuole, termed

the autophagosome or autophagic vacuole (Rideout et al., 2004).

These autophagic structures are classiﬁed according to their

morphology. Autophagosomes exhibit double membranes and

uncompacted cytoplasmic material, and the degradation of the

cytoplasmic substrates is initiated when the autophagosome fuses

with degradative compartments of the endosomal–lysosomal sys-

tem (Schmid and Münz, 2007). The outer membrane of the

autophagosome subsequently fuses with a lysosome, exposing

the inner single membrane of the autophagosome to lysosomal

hydrolases. When the autophagosome fuses with the lysosome,

an autophagolysosome is formed. After this fusion, the degrada-

tive lysosomal enzymes break down the contents in the vacuole.

Autophagolysosomes are double- or single-membrane struc-

Fig. 1. Semi-thin section of the midgut in E. nostras fed ﬁrst instar larvae. The apical

tures containing densely compacted amorphous or multilamellar

plasma membrane of each epithelial cell is differentiated into microvilli (MV). In

material that is electron-dense (Mizushima et al., 2002; Xie and the apical cytoplasm and perinuclear cytoplasm many spherites (S) are seen. L, lipid

␮

Klionsky, 2007). droplet; LU, lumen of the midgut; N, nucleus. Scale bar: 10 m.

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S. Lipovsekˇ et al. / Micron 43 (2012) 651–665 653

Figs. 2–5. (2) Ultra-thin section of the midgut in E. nostras fed ﬁrst instar larvae. Apical cytoplasm of the epithelial cell contains numerous mitochondria (M) and spherites

(S). G, glycogen. Scale bar: 2 ␮m. (3) Ultra-thin section of the midgut in E. nostras starved ﬁrst instar larvae. Scale bar: 1 ␮m. Beside the spherites (S) and the mitochondria (M),

␮

electron dense (ED) structures were seen individually in the midgut epithelial cells. G, glycogen. Scale bar: 1 m. (4) Ultra-thin section of the midgut in E. nostras fed second

instar larvae. Perinuclear cytoplasm of the epithelial cell is rich in the mitochondria (M) and concentrically layered spherites (S). Scale bar: 2 ␮m. (5) Ultra-thin section of

the midgut in E. nostras starved second instar larvae. Some spherites (DS) were in an advanced phase of the degeneration. One of them is shown at better resolution in this

␮

ﬁgure. M, mitochondrium; N, nucleus; S, spherites. Scale bar: 1 m.

together with the sand from their natural environment. Dur- nostras was ﬁxed in 2.45% glutaraldehyde and 2.45% paraformalde-

ing the experiment the room temperature was the same as in hyde in a 0.1 M sodium cacodylate buffer (pH 7.4) at room

◦ ◦

their natural habitat (24 C). The ﬁrst group of animals was fed temperature for 3 h and at 4 C for 14 h. The tissue was washed

daily and each antlion received one ant per day. In the second in 0.1 M sodium cacodylate buffer at room temperature for 3 h,

group of antlions, feeding was stopped. The animals were under postﬁxed with 2% OsO4 at room temperature for 2 h and dehy-

such conditions for 30 days in September 2008. After this period, drated in a graded series of ethanol (50%, 70%, 90%, 96%, 100%,

the midgut epithelial cells and their spherites from the animals each for 30 min at room temperature). Finally, the samples were

treated in the laboratory were observed. In each experimental embedded in TAAB epoxy resin (Agar Scientiﬁc Ltd., Essex, Eng-

group consisting of ten individuals of fed and starved ﬁrst, sec- land). Semithin sections were stained with 0.5% toluidine blue

ond and third larval antlions, the midgut epithelial cells and their in aqueous solution. For electron microscopy, ultrathin sections

spherites showed, in general, a quite comparable size and ultra- (70 nm) were obtained and transferred to copper grids. After

structure. staining with uranyl acetate and lead citrate, the ultrathin sec-

tions were examined with a Zeiss EM 902 transmission electron

microscope.

2.2. Light microscopy and transmission electron microscopy

The structure of the midgut epithelial cells and the structure 2.3. Elemental analysis

of the spherites were studied by light microscopy (n per indi-

vidual = 100) and conventional transmission electron microscopy Chemical composition of the spherites was analysed by

(TEM). For light microscopy and TEM, the midgut of the antlions E. a combination of electron energy-loss spectroscopy (EELS),

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654 S. Lipovsekˇ et al. / Micron 43 (2012) 651–665

Figs. 6–9. (6) The degenerated spherite is composed of the material of various electron density. In the cytoplasm individuar cisternae of rough endoplasmic reticulum are

seen (RER). Scale bar: 400 nm. (7) Ultra-thin section of the midgut in E. nostras starved second instar larvae. The majority of the spherites seem to be in process of inner

degeneration (DS). The cytoplasm contains numerous small vacuoles (asterisk). Scale bar: 400 nm. (8) Ultra-thin section of the midgut in E. nostras starved second instar

larvae. The nucleus (N) of the degenerated cell, containing a condensed chromatin near the nuclear envelope, achieves lobular shape. Some spherites and mitochondria are

␮

present in the cytoplasm. Numerous vacuoles (asterisk) are present in the cytoplasm. L, lipid droplet. Scale bar: 2 m. (9) Ultra-thin section of the midgut in E. nostras fed

second instar larvae. Oval nucleus (N) is surrounded by many lipid droplets (L). Scale bar: 2 ␮m.

Table 1

Chemical elements in spherites of the midgut epithelial cells of fed and starved larval instars of Euroleon nostras. The presence of chemical elements is shown by + and their

−

absence by . Fed instar larvae (L1+: fed ﬁrst instar larvae; L2+: fed second instar larvae; L3+: fed third instar larvae): each antlion received one ant per day during the

− −

experimental period of 30 days. Starved instar larvae (L1 : starved ﬁrst instar larvae; L2 : starved second instar larvae; L3−: starved third instar larvae): food was omitted

during the experimental period of 30 days. For comparison, data about the chemical composition of spherites from E. nostras living in natural habitat (NH) are added for each

instar larvae.

Larval instar C N O Na Mg Al Si P S Cl K Ca Mn Fe Co Zn

1st

Fed (L1+) + + + − − − − + − + − + − + − −

Non-fed (L1−) + + − − − − − + − − − − − − − −

NH + + + − − − − − − − − − − − − −

2nd

Fed (L2+) + + + + + − − + + + + + + + − +

Non-fed (L2−) − + + − − − − + − − − + − + − −

NH + + + − − − + + − − − + − + − −

3rd

Fed (L3+) + + + + + − − + − + + + + + + +

Non-fed (L3−) + − + − − − + − − − − + − + − −

NH + + + − − + + + − − − + − + − −

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S. Lipovsekˇ et al. / Micron 43 (2012) 651–665 655

Figs. 10 and 11. (10) Ultra-thin section of the midgut in E. nostras starved second Figs. 12 and 13. (12) Ultra-thin section of the midgut in E. nostras starved third instar

instar larvae. Epithelial cell Type 1 (EC1) contains numerous spherites (S), mitochon- larvae. Degenerated spherites (DS) are surrounded by rough endoplasmic reticulum

dria (M). In the cytoplasm of EC1 numerous vacuoles (black asterisk) are seen. In the (ER). White asterisk: electron-dense granula. Scale bar: 400 nm. (13) Ultra-thin sec-

epithelial cell Type 2 (EC2) electron-dense granulae (white asterisk) are found. MV, tion of the midgut in E. nostras starved third instar larvae. DS, degenerated spherite

microvilli. Scale bar: 2 ␮m. (11) Ultra-thin section of the midgut in E. nostras starved with amorphous material; L, lipid; MVB, multivesicular body. Scale bar: 400 nm.

second instar larvae. Around spherite (S) many vacuoles (black asterisk) are seen.

Scale bar: 1 ␮m.

in front of the edge and one image at the ionization edge of the ele-

ment of interest were acquired. An extrapolated background image

energy-ﬁltering TEM (EFTEM) and energy dispersive X-ray spec- −r

was calculated using the power-law model I = A·E , where I is the

troscopy (EDXS). The elemental analysis of the spherites was

intensity, E is the energy loss, and A and r are two ﬁtting param-

carried out on unstained ultrathin sections of the midgut. The

eters (Egerton, 1996). The background image is subtracted from

sections (75 nm) were collected on nickel grids covered with a per-

the ionization-edge image, thus giving a net image, which is the

forated carbon support ﬁlm and studied in a Philips CM 20/STEM

elemental map. This method is used for the elements P and O. For

electron microscope operated at 200 kV (LaB6 cathode), equipped

Ca, the “two window method” resulted in a better signal-to-noise

with a Gatan imaging ﬁlter (GIF) and a Noran EDX system with a

ratio. To get what is called a jump ratio image, the ionization edge

HPGe-detector. EEL-spectra and the elemental distribution images

is divided by a pre-edge image.

were recorded with the slow-scan CCD camera integrated into the

GIF. To collect information on light elements, EEL-spectra were

3. Results

acquired with the GIF operated in spectrum mode. The elemental

maps were obtained by recording an energy-ﬁltered TEM (EFTEM)

3.1. The midgut

image at the energy of an element-speciﬁc ionization edge. This

image contains a non-speciﬁc background, which must be removed In fed and starved larvae of the antlion E. nostras the midgut

in order to get the true elemental map (Hofer et al., 1995). For the is composed of the epithelial cells (Fig. 1), the basal lamina and

“three window method”, two energy-ﬁltered background images the muscle cells. In the midgut epithelium two different types of

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656 S. Lipovsekˇ et al. / Micron 43 (2012) 651–665

the cells were seen, described as epithelial cells Type 1 (EC1) and

epithelial cells Type 2 (EC2) (Lipovsekˇ et al., 2011).

3.1.1. The midgut epithelial cells in fed animals

Structural characteristics of the midgut epithelial cells observed

in all fed instar larvae E. nostras were the same as their characteris-

tics in E. nostras living in their natural habitat (Lipovsekˇ et al., 2011).

In short, apical plasma membrane of both cell types (EC1 and EC2)

is differentiated in numerous microvilli (Figs. 1 and 10). Epithelial

cells Type 1 are large columnar cells with electron-lucent cyto-

plasm. The latter is characterized by a speciﬁc regionalization in the

distribution of the organelles. In the apical cytoplasm (Figs. 1 and 2)

many mitochondria and cisternae of rough endoplasmic reticulum

(RER) are seen (Fig. 3). Characteristic structures of the apical cyto-

plasm of the midgut epithelial cells are the spherites (Figs. 2 and 4),

which are also present in the perinuclear region of the cytoplasm

(Figs. 1 and 4). In the perinuclear region of the cytoplasm the

mitochondria, abundant RER and Golgi complexes are present. In

the basal region of the cytoplasm the mitochondria, single cister-

nae of RER are seen. Lipid droplets are found in the perinuclear

region of the cytoplasm (Fig. 9) and the basal one. Glycogen is

distributed in all regions of the cytoplasm. The nucleus of EC1 is

round or oval (Fig. 9). Narrow columnar EC2 are located individually

between the EC1. They have electron-dense cytoplasm contain-

ing mitochondria, rER, Golgi apparatus, electron-dense vesicles and

oval nucleus. Spherites are not as numerous as in the cytoplasm of

the EC1.

3.1.2. The midgut epithelial cells in non-fed animals

The degeneration of the midgut epithelium was observed in EC1

in non-fed second and third instar larvae of E. nostras. The majority

of the EC1 of antlions starved for 30 days did not show signiﬁcant

structural changes from those of fed antlions. But in some of EC1

of starved E. nostras the RER was not seen, but the cytoplasm con-

tained numerous small vacuoles (Figs. 7, 8, 10 and 11). In those

cells, the heterochromatin was more condensed than that in the

fed antlions. The condensation was present on the periphery of

the nucleus (Fig. 8). Some spherites seemed to be in an advanced

phase of inner degeneration (Figs. 5–8 and 12). In very close vicin-

ity of some spherites electron-dense structures (Figs. 11 and 12)

were observed. In some EC1 cells individual multivesicular bodies

(Fig. 13) and/or numerous autophagolysosomes (Fig. 14) were seen.

The remnants of the spherites and organelles discharged from the

Figs. 14 and 15. (14) Ultra-thin section of the midgut in E. nostras starved third

epithelial cells are seen in the lumen of the midgut (Fig. 15). instar larvae. Electron-dense autophagolysosomes in the cytoplasm of EC1. M, mito-

chondrium. Scale bar: 400 nm. (13) Ultra-thin section of the midgut in E. nostras

starved third instar larvae. Degenerated spherites (DS) and remnants of organelles

3.2. The spherites

are present in the lumen (LU) of the midgut. Scale bar: 1 ␮m.

Study of the midgut of E. nostras instar larvae showed numerous

made by analytical methods of electron microscopy are presented

spherites, intracellular membrane-bound structures, in the cyto-

in Table 1.

plasm of epithelial cells (Fig. 16a–f). Spherites were present in all

the experimental antlions. Ultrastructure and chemical composi-

3.2.1. The ﬁrst instar larvae

tion of the spherites in both types of epithelial cells are similar

In ﬁrst instar larvae, spherites were never seen in the lumen of

therefore the following description of the features applies to both

the midgut.

cell types.

The ultrastructure and chemical composition of the spherites

were studied for all three instar larval stages, in fed and starved 3.2.1.1. Spherites of fed larvae. The spherites of fed ﬁrst instar lar-

experimental insects. The ultrastructure and chemical composi- vae were round, composed of ﬁnely ﬂocculated electron-lucent

tion of the spherites changed during the period of larval life, and material, enwrapped by a membrane (Fig. 16a). Some spherites

spherites with different structural characteristics were present in were round and composed of a few electron-dense and electron-

various larval stages. In the spherites, similar structural changes lucent laminated structures (Fig. 17a). Using analytical electron

between different larval stages were also observed in E. nostras microscopy, it was established that these spherites contained an

from the natural habitat (Lipovsekˇ et al., 2011). In this study, addi- organic matrix, composed of C, O and N. The chemical elements

tional differences in the spherites’ ultrastructure and elemental C, Fe and Cl were identiﬁed using EDX-spectroscopy; Ca, P, O

composition were also present when spherites from fed and non- and N were conﬁrmed by elemental maps. N, P, Ca are seen in

fed antlions were compared. The results of the chemical analyses Fig. 17b–d.

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S. Lipovsekˇ et al. / Micron 43 (2012) 651–665 657

Fig. 16. Ultra-thin sections of the midgut in the antlion E. nostras representing spherites (S) from different E. nostras instar larvae. (a) Spherites in fed ﬁrst instar larvae (L1+).

(b) Spherites in starved ﬁrst instar larvae (L1−). (c) Spherites in fed second instar larvae (L2+). Well developed RER is seen in the surrounding of the spherites. (d) Spherites

in starved second instar larvae (L2−). Please, see many vacuoles in the cytoplasm of the cell. (e) Spherites in fed third instar larvae (L3+). (f) Spherites in starved third instar

−

larvae (L3 ). M, mitochondria; MS, the membrane of the spherite.

3.2.1.2. Spherites of non-fed larvae. The spherites of starved ﬁrst 3.2.2.1. Spherites of fed larvae. The majority of spherites were

instar larvae were round, composed of ﬁnely ﬂocculated electron- round structures, with concentric mineralized layers appearing as

lucent material, enwrapped by a membrane (Figs. 16b and 18a). alternating electron-dense and electron-lucent regions in the tissue

Spherites composed of concentric layers were not seen in prepared for both transmission electron microscopy (TEM; Fig. 16c)

the midgut epithelial cells of starved ﬁrst instar larvae. In and analytical TEM (Fig. 19). Many of these were characterized by

the spherites of starved larvae, only C, N and P were found an electron-dense (e.g. Fig. 16c) or electron-lucent central compact

(Fig. 18b–d). core (Fig. 19a). The chemical composition of these spherites was

more complex than the chemical composition of those from the

3.2.2. The second instar larvae ﬁrst instar larvae. These spherites contain C, N, O, Na, Mg, P, Cl, K,

In second instar larvae, the midgut epithelial cells contained Ca, Mn, Fe and Zn, as was revealed by EDXS (Fig. 19b). Using ele-

concentrically layered spherites (Fig. 16c and d). In fed larvae, some mental maps, O, Fe, Ca and P were detected. The presence of P and

spherites were seen in the lumen of the midgut. Ca can be seen in Fig. 4c and d. As we can see in Fig. 16c, some

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658 S. Lipovsekˇ et al. / Micron 43 (2012) 651–665

Fig. 17. Ultra-thin section of the midgut in E. nostras fed ﬁrst instar larvae. (a) Bright ﬁeld TEM image of a spherite. (b) N elemental map, recorded with N K ionization map.

(c) P elemental map, recorded with the P L ionization edge at 132 eV. (d) Ca jump ratio image, recorded with the Ca L ionization edge at 346 eV. The bright areas indicate N,

P and Ca enrichment, which corresponds to the electron-dense layers in (a).

spherites had an appearance similar to that from fed ﬁrst instar concentric layers of homogeneously distributed, electron-dense

larvae (Fig. 16a). and electron-lucent material (Figs. 16e and 21a). The EDXS

of these spherites revealed that they contained C, N, O,

Na, Mg, P, Cl, K, Ca, Mn, Fe, Co, Zn. In the EDX-spectrum

3.2.2.2. Spherites of non-fed larvae. The spherites of starved second

(Fig. 21f), C, O, Zn, Mg, Cl, Ca can be seen. The presence

instar larvae were round, and composed of lamellae (Fig. 16d). The

of P, Cl, C, Ca, O and Fe appears on the EELS-spectrum

majority of these spherites looked less dense and more empty than

(Fig. 21e).

the spherites of fed larvae. The material acquired a ﬁbrillary appear-

ance (or structure) (Fig. 20a). The EDXS analyses showed C, O, and

Ca in these spherites (Fig. 20b). Using EFTEM, P and Ca were found

3.2.3.2. Spherites of non-fed larvae. In contrast to the spherites

(Fig. 20c and d).

of fed larvae, the spherites of starved larvae were composed

of only a few electron-dense layers, with electron-lucent

3.2.3. The third instar larvae “empty” rings between them; therefore, they were never

In some cases, in fed third instar larvae, spherites were present “fully” formed (Fig. 16f). The material was not closely packed,

in the lumen of the midgut. so the spherites looked more empty (Figs. 16f and 22a).

Using elemental maps, Fe, Si and Ca were detected

(Fig. 22b–d). EDXS and EELS showed the presence of C, O, Si

3.2.3.1. Spherites of fed larvae. The spherites of fed larvae were

(Fig. 22e and f).

compact, laminated structures consisting of densely packed

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Fig. 18. Ultra-thin section of the midgut in E. nostras starved ﬁrst instar larvae. (a) Bright ﬁeld TEM image of a spherite. (b) N elemental map, recorded with N K ionization

map. (c) P elemental map, recorded with the P L ionization edge at 132 eV. (d) Ca jump ratio image, recorded with the Ca L ionization edge at 346 eV. The bright areas indicate

N, P and Ca enrichment, which corresponds to the electron-dense layers in (a).

4. Discussion E. nostras in some epithelial cells Type 1 (EC1) the heterochro-

matin was condensed. The condensation of the chromatin near

In this study, we documented some structural changes in the the nuclear envelope could be the consequence of the apopto-

midgut epithelial cells of E. nostras associated with induction by the sis (Alberts et al., 2004). But in the case of E. nostras we did not

starvation of instar larvae in the laboratory. Antlions’ larvae were observe any apoptotic bodies or any other changes which are typ-

collected from their natural environment and after that the exper- ical for the process of apoptosis. In some EC1 in starved E. nostras,

iment was conducted in laboratory conditions. They were kept in the RER was absent and numerous small vacuoles were present

the same sand and at the same temperature as in their natural habi- in the cytoplasm. Similar observation were described for some

tat; there were no extreme changes of their living conditions in columnar cells of the starved cockroaches Periplaneta americana by

the laboratory. Therefore we hope that we could conclude that all Park et al. (2009); those cells lost RER and exhibited many small

observed changes are connected with the experimental conditions. vacuoles in their non-dark cytoplasm. The small vacuoles were

Whilst representative control cells from fed larvae showed nor- probably degenerated RER, as it was explained by Park et al. (2009).

mal morphology without any ultrastructural changes, we found But in the midgut cells of P. americana starvation induced apopto-

that the starvation could induce morphological changes with sis, as it was shown using histochemical methods and methods of

ultrastructural features of autophagy using transmission electron transmission electron microscopy by Park et al. (2009). Apoptosis

microscopy. was previously described for the intestinal cells of mammals by

In fed antlions, the epithelial cells showed a round or oval Chappell et al. (2003). It was observed and detected using terminal

nucleus, normally, structured mitochondria as well as a rough deoxynucleotidyl-transferasemediated nick-end labelling (TUNEL)

endoplasmic reticulum (RER) and Golgi apparatus. In non-fed assays (Chappell et al., 2003).

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660 S. Lipovsekˇ et al. / Micron 43 (2012) 651–665

Fig. 19. Ultra-thin section of the midgut in E. nostras fed second instar larvae. (a) Bright ﬁeld TEM image of a spherite. (b) Overview EDX spectrum of the spherite in Fig. 4a,

showing C, O, Ca. Ni signal in the EDX spectrum comes from Ni-TEM grid; the Os signal comes from the OsO4. (c) P elemental map, recorded with the P L ionization edge at

132 eV. (d) Ca jump ratio image, recorded with the Ca L ionization edge at 346 eV. The bright areas indicate P and Ca enrichment, which correspond to the electron-dense

layers in (a).

However, in E. nostras in some EC1 after starvation autophagic spherites. The general structure of the spherites in fed second and

structures were observed. These autophagic structures were recog- third antlions reared in the laboratory was comparable to the gen-

nized according to their morphological characteristics (Mizushima eral structure of spherites from second and third instar larvae of

et al., 2002; Xie and Klionsky, 2007) as autophagolysosomes. antlions living in their natural conditions (Lipovsekˇ et al., 2011) and

Additionally, the cytoplasm of some EC1 contained multivesicu- to the majority of spherites described in other invertebrates (e.g.

lar bodies and numerous lipid droplets. In fed antlions, autophagic Kalender and Kalender, 1996; Ballan-Dufranc¸ ais, 2002; Kalender

structures were very rarely observed. Once the occurrence of et al., 2002; Marigómez et al., 2002; Colville and Lim, 2003; Schill

autophagy has been detected in non-fed E. nostras by this method, and Köhler, 2004; Pigino et al., 2006; Pinheiro et al., 2008). Accord-

these autophagic structures can be conﬁrmed using additional ing to Hopkin (1989) all spherites found in E. nostras are Type A

methods, e.g., immunocytochemistry. Additional studies are neces- granule consisting of concentric layers of calcium and magnesium

sary to further elucidate the mechanisms that underlie the effects of phosphates which may contain other class A and borderline metals

starvation on the midgut epithelial cells in E. nostras. In the future, such as manganese and zinc.

we must study the ultrastructure of the midgut epithelial cells in In ﬁrst instar larvae of E. nostras, the spherites are structured

the larvae living from the beginning of their lives in laboratory differently from those in second and third instar larvae. They are

conditions. composed of amorphous ﬂocculent material, enclosed by the mem-

Additionally, comparing the results of both experimental brane. Similar spherites, with an interior composed of amorphous

antlions (fed antlions and non-fed ones) differences were observed material, were described in the digestive cells amongst nurse work-

in the ultrastructure and the chemical composition of their ers of Trigona spinipes (Serrão and Da Cruz-Landim, 1996).

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S. Lipovsekˇ et al. / Micron 43 (2012) 651–665 661

Fig. 20. Ultra-thin section of the midgut in E. nostras starved second instar larvae. (a) Bright ﬁeld TEM image of a spherite. (b) Overview EDX spectrum of the spherite in

Fig. 5a, showing C, O, Na, Mg, P, Cl, K, Ca, Mn, Fe and Zn. The Ni signal in the EDX spectrum comes from Ni-TEM grid. (c) P elemental map, recorded with the P L ionization edge

at 132 eV. (d) Ca jump ratio image, recorded with the Ca L ionization edge at 346 eV. The bright areas indicate P and Ca enrichment, which correspond to the electron-dense

layers in (a).

In starved larvae, the reduced number of chemical elements N; adenosine triphosphate (ATP) is composed of P, O, C, H, O and N

could be a consequence of the antlions’ starvation. Larval growth (Cooper and Hausman, 2009).

has an important impact on the individual’s ability to reproduce In spherites of fed ﬁrst instar larvae E. nostras, more chemical

(Gotelli, 1997), and the spherites, especially in the starved antlions, elements were found in comparison to spherites from ﬁrst instar

might represent an important source of organic and inorganic larvae living in natural habitat. In the latter, the spherites contained

elements, which could be utilized on demand for various vital pro- only C, O and N (see Table 1). Comparing the spherites from fed

cesses in the midgut epithelial cells. Some elements seem to be and starved antlion second instar larvae and the spherites from

used in various biochemical reactions, e.g. synthesis processes dur- larvae living in natural habitat, we can conclude that the chemical

ing the growth and metamorphosis of instar larvae, since they are composition of spherites is richest amongst fed instar larvae. This

not present in the starved larvae of E. nostras. Certain combina- was also the case in third larvae. In second larvae, the spherites

2−

tions of atoms, such as the phosphate ( PO3 ) and amino ( NH2) contained the majority of chemical elements (lacking only Si) also

groups occur repeatedly in organic molecules. Generally, cells con- found in second instar larve from natural habitats (C, N, O, Si, P, Ca,

tain 4 major groups of small organic molecules: the sugars, the fatty and Fe; Lipovsekˇ et al., 2011) and additionally Na, Mg, S, Cl, K, Mn,

acids, the amino acids and the nucleotides. These small molecules and Zn. In third instar larvae, besides the C, N, O, P, Ca and Fe found

form the monomeric subunits for most of the macromolecules and in spherites from E. nostras living in natural conditions, Na, Mg, Cl,

other structures of the cell. Some, like the sugars and the fatty acids, K, Mn, Co and Zn were additionally detected in fed E. nostras larvae.

are also energy sources; e.g., C, O and H are the main chemical ele- It was expected that the abundance of chemical elements would be

ments in fatty acids; the amino acids are composed of C, O, H and highest in fed antlion larvae because of the constant nourishment

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662 S. Lipovsekˇ et al. / Micron 43 (2012) 651–665

Fig. 21. Ultra-thin section of the midgut in E. nostras fed third instar larvae. (a) Bright ﬁeld TEM image of a spherite. (b) Fe M jump ratio image. (c) P elemental map, recorded

with the P L ionization edge at 132 eV. (d) Ca jump ratio image, recorded with the Ca L ionization edge at 346 eV. (e) EELS-spectrum showing the presence of P, Cl, C, Ca, O and

Fe. (f) Overview EDX spectrum of the spherite in (a), showing C, O, Zn, Mg, Cl and Ca. Ni signal in the EDX spectrum comes from Ni-TEM grid; Os signal comes from the OsO4.

of the insects. Chemical compounds, accumulated in the spherites is assumed that the presence of spherites in the cytoplasm of the

of antlion larvae could be used for the growth of the larvae and are cell is associated with food ingestion or insect age; the richer in

essential for the growth of the pupae. It is known that the pupae do metals the food, or the older the larvae, the greater is the quantity

not feed (Grifﬁths, 1980). In second and third larvae the spherites of spherites (Sohal et al., 1977). According to the results, we can

contain 13 chemical elements (see Table 1) and in this respect have conﬁrm this observation for E. nostras, since the number of different

been recognized as the richest ones so far detected in E. nostras. It chemical elements was highest in older fed antlions (Table 1).

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S. Lipovsekˇ et al. / Micron 43 (2012) 651–665 663

Fig. 22. Ultra-thin section of the midgut in E. nostras starved third instar larvae. (a) Bright ﬁeld TEM image of a spherite. (b) Fe M jump ratio image. (c) Si L jump ratio image.

(d) Ca jump ratio image, recorded with the Ca L ionization edge at 346 eV. (e) EELS-spectrum showing the presence of Si, C and O. (f) Overview EDX spectrum of the spherite

in (a), showing C, O and Si. Ni signal in the EDX spectrum comes from Ni-TEM grid; Os signal comes from the OsO4.

5. Conclusions important protective mechanism for cell survival for the periods

of food deprivation.

1. In E. nostras, the presence of autophagic structures in the cyto- 2. The inﬂuence of starvation on the ultrastructure and chemical

plasm of the midgut epithelial cells suggests that autophagy composition of spherites in the midgut of the instar antlion lar-

might be induced in these cells by starvation and this could be an vae E. nostras was studied using the following methods:

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664 S. Lipovsekˇ et al. / Micron 43 (2012) 651–665

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