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Arthropod Structure & Development 38 (2009) 377–389
Contents lists available at ScienceDirect
Arthropod Structure & Development
journal homepage: www.elsevier.com/locate/asd
The larval alimentary canal of the Antarctic insect, Belgica antarctica
James B. Nardi a, *, Lou Ann Miller b, Charles Mark Bee c, Richard E. Lee, Jr. d, David L. Denlinger e
a
Department of Entomology, University of Illinois, 320 Morrill Hall, 505 S. Goodwin Avenue, Urbana, IL 61801, USA
Center for Microscopy and Imaging, College of Veterinary Medicine, University of Illinois, 2001 S. Lincoln Avenue, Urbana, IL 61801, USA
c
Imaging Technology Group, Beckman Institute for Advanced Science and Technology, University of Illinois, 405N Mathews Avenue, Urbana, IL 61801, USA
d
Department of Zoology, Miami University, Oxford, OH 45056, USA
e
Department of Entomology, Ohio State University, 400 Aronoff Laboratory, 318 West 12th Avenue, Columbus, OH 43210, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 October 2008
Accepted 17 April 2009
On the Antarctica continent the wingless midge, Belgica antarctica (Diptera, Chironomidae) occurs
further south than any other insect. The digestive tract of the larval stage of Belgica that inhabits this
extreme environment and feeds in detritus of penguin rookeries has been described for the first time.
Ingested food passes through a foregut lumen and into a stomodeal valve representing an intussusception of the foregut into the midgut. A sharp discontinuity in microvillar length occurs at an interface
separating relatively long microvilli of the stomodeal midgut region, the site where peritrophic
membrane originates, from the midgut epithelium lying posterior to this stomodeal region. Although
shapes of cells along the length of this non-stomodeal midgut epithelium are similar, the lengths of their
microvilli increase over two orders of magnitude from anterior midgut to posterior midgut. Infoldings of
the basal membranes also account for a greatly expanded interface between midgut cells and the
hemocoel. The epithelial cells of the hindgut seem to be specialized for exchange of water with their
environment, with the anterior two-thirds of the hindgut showing highly convoluted luminal
membranes and the posterior third having a highly convoluted basal surface. The lumen of the middle
third of the hindgut has a dense population of resident bacteria. Regenerative cells are scattered
throughout the larval midgut epithelium. These presumably represent stem cells for the adult midgut,
while a ring of cells, marked by a discontinuity in nuclear size at the midgut-hindgut interface,
presumably represents stem cells for the adult hindgut.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Alimentary canal
Midge
Antarctica
Stomodeal valve
Microvilli
Regenerative cells
1. Introduction
Despite the dominance of insects and other terrestrial arthropods throughout the world, only a few species are found in
Antarctica. Most are collembolans and mites, with the Class Insecta
represented by only two endemic species of midges (Diptera:
Chironomidae) (Convey and Block, 1996). Of these, the range of the
more abundant midge Belgica antarctica extends further south on
the Antarctic Peninsula than that of any other insect.
B. antarctica has a patchy distribution on the Peninsula, but it is
particularly abundant near penguin rookeries on the small, offshore islands near Palmer Station. In this habitat, midge larvae are
subjected to a range of environmental stresses including desiccation, freezing, anoxia, pH fluctuation from the nitrogenous run-off,
and inundation from seawater as well as fresh water from
* Corresponding author. Tel.: þ1 217 333 6590; fax: þ1 217 244 3499
E-mail address: [email protected] (J.B. Nardi).
1467-8039/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.asd.2009.04.003
precipitation and ice melt. Numerous physiological adaptations are
apparent in the larvae: heat shock proteins are continuously upregulated (Rinehart et al., 2006), agents to counter oxidative stress
are present in abundance (Lopez-Martinez et al., 2008), loss of
a high percentage of body water is tolerated and in fact contributes
to enhanced freeze tolerance (Hayward et al., 2007; Elnitsky et al.,
2008). Dramatic changes in metabolites (Michaud et al., 2008) and
gene expression (Lopez-Martinez et al., 2009) accompany these
physiological responses to environmental stress.
In addition to these distinctive biochemical features of cells of
B. antarctica, it is quite possible that structural features of this
insect may also deviate somewhat from the patterns observed in
insects at lower latitudes. The harsh environment of Antarctica
obviously places great demands on the resiliency of cells that are
exposed to periodic desiccation and freezing. To promote
formation of ice crystals within extracellular spaces rather than
within cells, insect cells must rapidly exchange water with their
extracellular environments. Expansion of luminal and/or basal
surface areas of epithelial cells would facilitate this rapid
exchange.
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Special emphasis in this manuscript has been placed on the
cellular architecture of the three well-delineated epithelial regions
of the insect alimentary canal – foregut, midgut, and hindgut.
Secretion from the midge’s large salivary gland cells aggregates
detritus particles and facilitates uptake of detritus into the gut
(Oliver, 1971). After passage of ingested food through the midge
larva’s short and narrow foregut, digestion and absorption of
ingested material occurs across the peritrophic membrane and
microvilli that line the lumen of the midgut. Subsequently digested
food passes into the hindgut where ions, small molecules and water
are absorbed across a cuticular lining.
Comparisons of internal gut morphology of arthropods
complement existing studies of external morphological features
and molecular characters on which traditional phylogenetic
relationships, representing genetic differences among organisms,
are based. Although the alimentary canals of other species in the
family Chironomidae have not been examined at the cellular
level of organization examined in this manuscript, a comprehensive comparison of internal morphological characters among
related species would reveal differences that presumably are
based on environmental adaptations. In this study we explore
the possibility that the extreme environment in which these
midge larvae live shapes the architecture of their gut epithelial
cells.
2. Materials and methods
Larvae of B. antarctica were collected from sites near penguin
rookeries on Cormorant and Humble Islands, near Palmer Station,
Antarctica (64 460 S, 64 040 W) in January and February 2007. The
high nitrogen run-off from penguin rookeries provides the
nutrient base for the microbes, algae (Prasiola crispa), lichens and
moss with which midge larvae are usually associated. Larval
development is restricted to the brief austral summer, from late
December until mid-February; and larvae remain immobilized in
the frozen substrate for the remainder of the year. Two years are
required for the larva to complete its development (Usher and
Edwards, 1984), and adult life is compressed into a 1–2 week
period in late December/early January (Sugg et al., 1983). Larvae
collected in Antarctica were shipped to the laboratory at Ohio
State University where they were maintained with their substrate
at 4 C.
Digestive tracts and salivary glands from 32 individuals were
dissected with fine watchmaker’s forceps in Grace’s insect culture
medium. Tissues were immediately fixed at 4 C in a primary
fixative of 2.5% glutaraldehyde (v/v) and 0.5% paraformaldehyde
(w/v) dissolved in a rinse buffer of 0.1 M cacodylate (pH 7.4)
containing 0.18 mM CaCl2 and 0.58 mM sucrose. After 3 h in this
fixative, tissues were washed three times with rinse buffer before
being transferred to secondary fixative (2% osmium tetroxide
dissolved in rinse buffer, w/v) for 4 h. After thoroughly washing
with rinse buffer, tissues were gradually dehydrated in a graded
ethanol series (10–100%, v/v). From absolute ethanol, tissues were
transferred to propylene oxide and infiltrated with mixtures of
propylene oxide and resin before being embedded in pure LX112
resin.
Semithin sections for light microscopy were mounted on glass
slides and stained with 0.5% toluidine blue in 1% borax (w/v).
Ultrathin sections were mounted on grids and stained briefly with
saturated aqueous uranyl acetate and Luft’s lead citrate to enhance
contrast. Images were taken with a Hitachi 600 transmission
electron microscope operating at 75 kV.
Whole mounts were prepared by fixing tissues at room
temperature for 30 min in a 4% solution of paraformaldehyde
(w/v) dissolved in phosphate-buffered saline (PBS). After
Fig. 1. (a) Whole mount of larval gut with the three major divisions of the gut
demarcated with arrows: fm ¼ foregut–midgut boundary; mh ¼ midgut–hindgut
boundary. The stomodeal valve lies immediately posterior to fm (see Fig. 4). The
sclerotized head capsule is attached at the anterior end and four Malpighian
tubules attach at the midgut–hindgut boundary. (b) Lateral view of the larva.
Dorsal is to the right. Beneath the relatively translucent integument of the three
thoracic segments (arrows point to prothoracic and metathoracic segments), lie
imaginal discs for the three pairs of legs. Dorsal to these leg discs in the mesothoracic and metathoracic segments are wing and haltere discs, respectively. Scale
bar ¼ 1.0 mm.
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Fig. 2. Whole mount of Belgica salivary gland as viewed with Nomarski optics (a) and after labeling DNA with DAPI (b). The salivary duct is located at the arrow. Scale
bar ¼ 50 mm.
several rinses in PBS, nuclei of cells were labeled with 1:1000
dilution of 40 ,6-diamidino-2-phenylindole (DAPI, 1 mg/ml
water) after first permeabilizing cells for 30 min in a solution of
0.1% Triton X-100 in PBS (v/v). Tissues were mounted under
cover glasses in a solution of 70% glycerin in 0.1 M Tris at pH
9.0 (v/v).
3. Results
Malpighian tubules that converge with the alimentary canal at
the junction of midgut and hindgut (Fig. 1, mh). A prominent
stomodeal valve occupies the interface between foregut and
midgut (Fig. 1, fm). The foregut occupies only about 5% of the
total length of the alimentary canal, while the endodermal
midgut that occupies the region between fm and mh in Fig. 1
clearly represents more than half the length of the alimentary
canal.
3.1. Global organization of the Belgica larval gut
3.2. Salivary glands and foregut
The larval gut is a straight alimentary canal that is associated
with a pair of salivary glands at its anterior end and four
Conspicuous salivary glands occupy the anterior end of the
larval midge. These glands are both polyploid and polytene (Fig. 2).
Fig. 3. Cuticles of foregut and epidermal epithelia have different stratification. At higher magnification, the internal folded foregut cuticle (a) is compared with the cuticle of the
external epidermis (b). A bracket extends across the foregut cuticle. The gut lumen (*) is surrounded by relatively thin cuticle compared to the thicker epidermal cuticle in (b)
epithelial cells (e), pigmented fat body (f). In (c) the convoluted integument of the foregut (arrowhead) is surrounded by muscles (arrows), tracheoles (t), neural tissue (n) and
salivary gland (g). Scale bars: (a) 1.0 mm, (b) 5 mm, and (c) 20 mm.
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Like glands found in other larval members of the Chironomidae,
these salivary glands secrete silken threads that entrap food
particles.
On the foregut’s apical surface, the cuticle lining the narrow
foregut lumen is about 0.3–0.5 mm thick (Fig. 3a). Unlike the thicker,
contiguous cuticle of the larva’s exoskeleton with its inner electrondense layer (Fig. 3b), this foregut cuticle is highly convoluted and its
outermost layer is electron-dense. Conspicuous muscle layers
surround the foregut. The large salivary glands and larval brain in
turn surround these muscles on the basal surface of the larval
foregut (Fig. 3c).
3.3. Stomodeal valve at foregut–midgut interface
After being channeled through a foregut lumen lined by
a convoluted cuticle, the contents of the gut pass through
a conspicuous stomodeal valve into the endoperitrophic space of
the midgut epithelium. The stomodeal valve represents an
intussusception of the foregut epithelium into the midgut (Figs.
4a–d and 5a–c). This folding of the foregut epithelium and its
cuticle creates a caecum that is lined centrally by foregut cuticle
and peripherally by midgut microvilli. The interface of foregut
and midgut lies at the anterior end of the caecum. At this
junction of foregut and midgut, a peritrophic membrane originates and lines the lumen of the more posterior midgut
epithelia.
3.4. Spatial differentiation of the midgut epithelium
An abrupt epithelial discontinuity marked by disparity in
midgut microvillar length occurs at the interface between the
stomodeal region and the more posterior midgut epithelium
(Fig. 4c and 5d). Certain cells at this interface are specialized for
secretion (Fig. 5d–f) and possibly are endocrine cells. These cells
at the posterior edge of the stomodeal valve were the only cells of
the midgut observed to contain conspicuous secretory granules
Fig. 4. The stomodeal valve represents an intussusception of posterior foregut epithelium (fe) into anterior midgut epithelium (me). (a) The folded foregut epithelium is surrounded
by the anterior midgut. Anterior is to the right. (b, c) Longitudinal sections show inner lumen cuticle (ic) and outer lumen cuticle (oc) of the foregut. Between these two cuticles lie
two foregut epithelial layers and an enteric muscle layer (m). Midgut epithelium is the outermost layer of the valve. (c) Represents the region delimited by the rectangle in (b). The
morphological discontinuity is indicated by the arrow. The peritrophic membrane (p) lies in the lumen separating foregut and midgut. (d) The concentric arrangement of three
epithelia, two lumina and one muscle layer is shown in this transverse section of the valve. From periphery to center: (1) midgut epithelium with microvilli lining the lumen in
which the peritrophic membrane forms; (2) foregut epithelium faces this lumen and (3) enteric muscles (m) occupy the space between this foregut epithelium and the foregut
epithelium facing the innermost lumen. Scale bars: (a, b, d) 50 mm; (c) 20 mm.
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Fig. 5. At higher resolution, longitudinal sections of the stomodeal valve reveal details of peritrophic membrane formation and the presence of special secretory cells. (a) At the
interface between the foregut epithelium and midgut epithelium lies a confluence of muscle (m), foregut epithelium covered by cuticle (fg) and secretory microvilli (arrows) of
midgut epithelium. Anterior is at the bottom. (b, c) Copious secretion of peritrophic membrane material (*) occurs from the microvilli of midgut epithelial cells at the anterior end of
the stomodeal valve. Note the high density of mitochondria in the adjacent midgut cells. In (b) the newly formed peritrophic membrane (arrow) lies between the foregut (fg) cuticle
and the tips of the microvilli. (d) Distinctive secretory cells (arrow) lie within the midgut epithelium of the stomodeal valve at the border between midgut cells with microvilli and
midgut cells without obvious microvilli (See Fig. 4c). The gut lumen is at upper left. (e, f) Close-ups of the secretory cell showing the nucleus (n), rough enoplasmic reticulum
(arrowhead) and the high density of secretory granules (*). Scale bars: (a) 10 mm; (b, c) 2 mm; (d) 5 mm; (e) 1 mm; and (f) 0.2 mm.
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(Fig. 5e and f). Posterior to the stomodeal valve, a striking
gradient of microvillar length occurs along the antero-posterior
(AP) axis of the midgut epithelium (Fig. 6), with short (w0.1 to
0.2 mm) microvilli occupying anterior regions of the midgut and
extremely long, straight and densely packed (w10 mm, Figs. 7 and
9) microvilli occupying posterior regions of the midgut. This
morphological gradient is evident in longitudinal sections of the
midgut (Fig. 6a–c) as well as the series of transverse sections from
different locations along the AP axis of the Belgica gut (Figs. 6d–f
and 7–9).
Underlying the antero-posterior topography revealed by the
microvilli of the midgut is a parallel topography, at the base of the
microvilli, reflected by contours of the apical surfaces of the midgut
cells. These surfaces are most convoluted in regions of the midgut
with the shortest microvilli and are least convoluted in regions of
the midgut with the longest microvilli (Fig. 6d–f).
Rough endoplasmic reticulum (RER) is present throughout all
regions of the midgut (Figs. 7d, 8d, and 9d). Stacks of large
flattened sacs of endoplasmic reticulum are especially evident in
the posterior region of the midgut epithelium. Some smooth
endoplasmic reticulum (SER) is interspersed among the RER of
the anterior third of the midgut, with little if any SER is observed
in the middle third or the posterior third of the midgut. However,
clearly defined Golgi complexes are not evident in any of the
midgut cells.
The lumen of the middle third of the Belgica midgut is lined
with microvilli of intermediate length (w1 mm) that are associated with electron-dense particles of uniform size (w0.05 mm).
These particles lie within the ectoperitrophic space and show
a strong affinity for the microvilli (Fig. 8a–c). Within the cytoplasm of the underlying midgut cells, electron-dense particles of
identical size are concentrated in autophagic vacuoles, each
delimited by a plasma membrane, and are presumed to enter
these epithelial cells by endocytosis. Numerous coated vesicles at
the base of microvilli on the luminal surfaces of these midgut
cells (Fig. 8c) offer an obvious route for the cellular uptake of
these electron-dense particles from the ectoperitrophic space of
the gut lumen.
Infoldings of the basal membranes of epithelial cells are most
prominent in the anterior and the posterior regions of the midgut
(Figs. 7c, 8e, and 9c); mitochondria are most conspicuous along the
basal surface of the anterior midgut (Fig. 7a, c); and infoldings of
the basal plasma membrane contain numerous mitochondria and
electron-lucent vacuoles (Figs. 7c and 9c).
Regenerative cells are scattered throughout the larval midgut
epithelia and presumably represent imaginal stem cells that
replace the larval epithelium at metamorphosis. These cells are
located basally in the larval epithelium and are densely packed
with ribosomes and endoplasmic reticulum (Fig. 10).
3.5. Contents of the midgut lumen
3.5.1. Endoperitrophic space
Within the gut lumen, the peritrophic membrane separates an
inner endoperitrophic space from an outer ectoperitrophic space
(Terra and Ferreira, 1994; Fig. 6). The anterior endoperitrophic
space of the midgut is packed with relatively intact multicellular
microbial organisms. Gradual digestion of microbes is reflected in
the disappearance of pigmentation from the gut lumen in the
posterior third of the midgut (Fig. 1a). Within the confines of the
midgut’s peritrophic membrane, microbial cells arranged singly or
in various aggregates can be readily discerned. These cells can be
identified as predominantly nonbacterial microbes – i.e., algae,
fungi, lichens, protists (Fig. 11a–c).
3.5.2. Ectoperitrophic space
The surrounding ectoperitrophic space is lined by midgut
epithelium with microvilli whose lengths are graded along the
Fig. 6. Longitudinal (a–c) and transverse (d–f) sections of midgut epithelium posterior to the stomodeal valve show regional differences along the antero-posterior axis of the gut.
Three equally spaced regions along this axis are illustrated: (a, d) anterior region; (b, e) middle region; (c, f) posterior region. In each image the microvillar surface and gut lumen are
at top. The peritrophic membrane (arrow) is visible in (a, b, d, and e). Scale bars ¼ 20 mm.
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Fig. 7. These are ultrastructural images of the anterior region of the midgut epithelium. Note the high concentration of mitochondria along the basal surface of the epithelium in
(a) as well as the dark cell (arrow) at the basal surface of this epithelium. Lumen is marked with an asterisk (*). (b) The epithelial surface facing the lumen (*) is covered by short
microvilli. (c) Mitochondria (arrows) are localized among membrane infoldings that lie adjacent to the basal lamina and enteric muscles (arrowhead). (d) Perinuclear region of
midgut cell. Rough endoplasmic reticulum (ER) marked with arrowhead. Smooth ER marked with double arrowhead. Arrows point to mitochondria. n ¼ nucleus. Scale bars:
(a) 5 mm; (b, c) 2 mm; and (d) 1 mm.
antero-posterior axis. Within one of the 10 whole mounts of
larval guts prepared, gregarines with distinctive appendages
were found in this midgut zone (between the peritrophic matrix
and midgut epithelial surface) (Fig. 11d and e). Considering the
isolation of these gregarines from other related host species,
these protists most likely represent a distinct species of dipteran
parasite.
3.6. Spatial differentiation of the hindgut
3.6.1. Ring of undifferentiated, presumptive adult
hindgut epithelium
In the images of the Belgica gut labeled with DAPI (Fig. 12a),
a discontinuity in nuclear labeling is observed at the midgut–
hindgut boundary. At the anterior-most region of the larval
hindgut, an imaginal ring of undifferentiated presumptive adult
hindgut cells appears in whole mounts as a zone of cells with small
nuclei among the polyploid nuclei in cells of the larval hindgut
epithelium.
Immediately posterior to the imaginal ring of epithelial cells,
the anterior third of the hindgut is lined by a smooth cuticle
approximately 0.25 mm thick. This hindgut cuticle, like the
foregut cuticle, is contiguous with the exoskeleton. Also, like the
foregut cuticle, the hindgut cuticle secretes a well-delineated,
electron-dense outermost cuticular layer. The arrangement of
these different layers secreted by hindgut epithelial cells is
distinct from the arrangement of the cuticular layers secreted by
epidermal epithelia (Fig. 3b). The anterior hindgut cuticle is
secreted by attenuated, highly folded extensions of the apical
surfaces of the hindgut epithelium containing conspicuous
mitochondria and separated by large vacuoles that lack electrondensity (Fig. 12b and c).
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Fig. 8. Different magnifications of the middle region of the midgut are illustrated in (a–e). In images a and c, the midgut lumen is at the top; in e, it is down. Electron-dense
particles occupy the space between the peritrophic membrane (arrow in a) and the microvillar surface. Comparable particles are observed in autophagic vacuoles of midgut cells
(arrowheads in b). (c) Higher resolution images suggest that these particles are taken up by endocytosis (arrows) at the microvillar surface. (d) Perinuclear region of midgut cell.
Arrowheads indicate rough endoplasmic reticulum; arrows point to mitochondria; n ¼ nucleus; me ¼ membrane between two cells. (e) Basal lamina (arrowhead) and muscles
(m) cover the basal surface of this epithelium. Infoldings of the basal surface of plasma membrane are not conspicuous. Scale bars: (a) 5 mm; (b, c) 1.0 mm; (d) 1 mm; and (e)
2 mm.
While apical ends of epithelial cells in the anterior third of the
hindgut have conspicuous large vacuoles, the central region of the
hindgut is occupied by epithelial cells that lack vacuoles but that
have extremely convoluted apical membranes characteristic of
epithelial cells involved in active transport of ions and water
(Fig. 12d and e). Also in the central region of the hindgut, the
presence of the electron-dense bacteria observed in high-resolution images is reflected in the pigmentation of the central portion of
the hindgut as viewed in whole mounts of larval guts (Fig. 1a).
In the posterior third of the hindgut or rectum, the highly
convoluted cuticle lining the lumen mirrors the structure of the
foregut. The apical surfaces of the epithelial cells of the rectum
lack infolded membranes associated with mitochondria and are
apparently not specialized for transport of ions and water. The
basal surface of this region, by contrast, is highly infolded.
Association of this basal surface with numerous muscles suggests
this posterior-most hindgut epithelium serves a mechanical
function. In this most posterior portion of the hindgut, the lumen
is devoid of both resident microbes as well as ingested microbes
(Fig. 12f).
3.7. Muscles associated with the gut epithelia
The basal surface of the hindgut epithelium, like the foregut
epithelium, is closely apposed to a uniformly thick layer of circumferential muscles (w10 mm) (Figs. 3c, 12e, f). By contrast, muscles
associated with the midgut epithelium are sparsely but regularly
distributed over the midgut’s basal surface (Figs. 6–9). Relatively
widely dispersed muscle cells lie on the basal surface of the midgut
epithelium and are embedded in the matrix of the epithelial basal
lamina. These represent the longitudinal muscles of the gut. Sparsely
distributed circumferential muscles are also present.
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Fig. 9. In a–c, the midgut lumen is to the left. Different magnifications of the posterior region of the midgut are illustrated. (a) Note numerous clear vacuoles (arrows) that lie
between the luminal microvilli and the basal lamina (lower right). (b) The long microvilli are densely packed and extend approximately 10 mm into the lumen. (c) The basal
membranes of cells in this posterior region are highly folded. Basal lamina is indicated with arrowheads. Muscle ¼ m. (d) Perinuclear region of midgut cell. Arrowheads indicate
rough endoplasmic reticulum; arrows point to mitochondria. Scale bars: (a) 10 mm; (b) 5 mm; (c) 2 mm; and (d) 1 mm.
4. Discussion
The study of insect diversity and evolution has been
advanced by extensive surveys of molecular phylogeny and
morphology of external integuments; however, knowledge and
appreciation of insect diversity remain incomplete without
adequate knowledge of the diversity of internal anatomy/physiology of insects and how this diversity is influenced by
environment.
With the paucity of information on the cellular architecture of
insect guts, however, associating particular gut epithelial structures with adaptation to particular diets and/or environments
remains in a rudimentary state. Conventional descriptions of gut
epithelial diversity (Lehane, 1998; Noble-Nesbitt, 1998; Terra
et al., 1988) clearly do not consider the marked regional differentiation of microvilli on midgut cells of B. antarctica to be
a common feature of epithelial cells of insect guts. Establishing
how general or how unique internal features are among insects,
however, awaits additional structural studies on other related
insect species.
4.1. Differentiation of foregut–midgut boundary: structure
of the peritrophic membrane and stomodeal valve
A specialized luminal region at the foregut–midgut boundary
can be visualized even in whole mounts of the alimentary canal
(Fig. 1a). In cross-sections and longitudinal sections of the
alimentary canal at this boundary region, an inner concentric
ring of folded foregut epithelium and associated muscle layers
lies within the anterior midgut epithelium (Figs. 4, 5). Among
the Diptera, the degree of specialization of foregut and midgut
epithelial cells varies among the suborders. The most complex
specialization of the foregut–midgut interface is found among
the muscoid flies, in which specialized anterior midgut
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epithelium is closely apposed to the stomodeal valve to form the
distinctive cardia (Eisemann et al., 2001; Binnington, 1988); but
the simplest specialization of the foregut–midgut interface is
observed in the suborder Nematocera, of which Belgica is
a member. In these flies, the foregut has been described as
forming a short intussusception into the anterior midgut
referred to as the stomodeal valve (King, 1991; Wigglesworth,
1930). For Belgica, however, this intussusception of foregut as
a percentage of total foregut surrounded by midgut is higher
than that reported by Volf et al. (2004) for four other nematoceran Diptera in the families Culicidae (Culex pipiens) and
Psychodidae (Lutzomyia longipalpis, Phlebotomus duboscqi, Phlebotomus papatasi).
Peritrophic structures for many insect species have often been
described as chitinous, reticulated membranes with chitin microfibrils in a hexagonal or orthogonal arrangement (Lehane, 1998).
These peritrophic structures consist of chitin networks embedded
in protein–carbohydrate matrices (Wang and Granados, 2001;
Tellam et al., 1999).
The origin and consistency of peritrophic membranes, gels and
matrices differ among the insects (Terra, 2001; Binnington et al.,
1998). The peritrophic structures of some insects, such as lepidopteran larvae, have traditionally been described as arising from
cells along the length of the midgut epithelium (type I peritrophic
matrix). Recent studies involving labeling of lepidopteran peritrophic proteins have indicated that while one peritrophic protein
(i.e., invertebrate intestinal mucin) is secreted by epithelial cells
throughout the length of the midgut (Harper and Granados, 1999),
certain peritrophic proteins recognized by an antibody raised
against the peritrophic membrane of Heliothis virescens are
produced by specialized cells near the foregut–midgut interface
(Ryerse et al., 1992).
At the junction of foregut and midgut epithelia in Diptera, the
peritrophic structure (type II) arises from microvilli of midgut
epithelia (Eisemann et al., 2001) and lines the lumen of the more
posterior midgut epithelia. In Belgica, the midgut epithelial cells of
the stomodeal valve are the only cells observed to produce
a copious secretion associated with a newly formed structure that
represents a type II peritrophic membrane.
4.2. Regional differentiation of midgut epithelium
Fig. 10. Regenerative cells (asterisks) of midgut epithelium are scattered throughout
the larval epithelium. Cells from different regions along the antero-posterior axis are
shown. (a) Anterior third. (b) Middle third. (c) Posterior third. Arrowheads point to
basal laminae; m ¼ muscles; n ¼ nuclei of regenerative cells. Scale bars ¼ 2.0 mm.
At least in some insects, the midgut is differentiated both
structurally and functionally along its length (Lehane, 1998; Marana
et al., 1997; Ferreira et al., 1990; Terra et al., 1988; Dow, 1981). The
marked and graded differences in microvillar lengths observed for
Belgica midgut epithelial cells, however, represent an extreme
example of such regional differentiation (Fig. 6).
Extensive infolding of the basal epithelial surface of midgut and
hindgut cells, however, as frequently observed in other insects, is
only evident in certain regions (anterior third and posterior third)
of the Belgica midgut and the posterior third (rectum) of its hindgut
(Villaro et al., 1999; Lehane, 1998; Marana et al., 1997). See Figs. 7c,
8e, 9c and 12f.
The high concentrations of electron-dense particles that occupy
the ectoperitrophic space of the central region of the midgut are not
observed elsewhere in the alimentary canal. The presence of
comparable particles in autophagic vacuoles of these midgut cells
suggests that these particles are taken up by cells rather than
secreted by gut cells. The high concentration of particles in the gut
lumen also implies that their movement proceeds toward the low
concentration of particles observed within the midgut epithelial
cells.
Although regenerative cells have not been observed in
midgut epithelia of certain immature arthropods such as larval
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J.B. Nardi et al. / Arthropod Structure & Development 38 (2009) 377–389
387
Fig. 11. The contents of the midgut lumen are described in both the endoperitrophic space (a–c) and the ectoperitrophic space (d, e). The multicellular microbial forms observed in
these thin sections (a, b, c) of the midgut’s endoperitrophic space are fungal and algal. (d, e). An unidentified but distinctive gregarine protist inhabits the ectoperitrophic space. Note
the spiral appendages (arrows) visible on these gregarines. p ¼ peritrophic membrane; m ¼ midgut epithelium; en ¼ endoperitrophic space. Scale bars: (a, b) 20 mm; (c) 3 mm;
(d) 50 mm; and (e) 20 mm.
Musca domestica (Terra et al., 1988) and immature Allacma fusca
(Rost-Roszkowska et al., 2007a), regenerative cells (presumptive
imaginal stem cells) are observed scattered throughout the
monolayer of the Belgica midgut epithelium as they are in the
gut epithelia of many other insects (Rost-Roszkowska et al.,
2007a,b; Rost, 2006; Ohlstein and Spradling, 2005; Evangelista
and Leite, 2003; Lehane, 1998; Hecker, 1977). In addition to
high-resolution images of Belgica regenerative cells (Fig. 10), the
dark cell on the basal surface of the anterior midgut epithelium
in Fig. 7a, probably also represents such a regenerative cell.
4.3. Differentiation of hindgut epithelium
A discontinuity in nuclear size at the midgut–hindgut interface is evident in whole mounts of Belgica guts (Fig. 12a). A ring
of epithelial cells with nuclei that are smaller than those nuclei
located both anterior and posterior to the ring presumably
represents the stem cells or presumptive imaginal cells for the
adult hindgut (Takashima et al., 2008; Murakami and Shiotsuki,
2001).
The rectal pads and/or papillae of many terrestrial insects
have been shown to be involved in uptake of water and ions.
Rectal epithelial cells (posterior hindgut) of these terrestrial
insects have conspicuous folding of their apical plasma
membranes. However, the ilea or anterior hindguts of aquatic
insects such as Cenocorixa and Ephydrella as well as some
terrestrial insects such as Formica, are adapted for ion and/or
water transport (Villaro et al., 1999; Marshall and Wright, 1974;
Jarial and Scudder, 1970). Insects living in aquatic or semi-aquatic
environments presumably have different absorptive activities,
being more concerned with absorption of inorganic ions rather
than with water absorption.
Based on examination of other insect hindguts (Noble-Nesbitt,
1998; Jarial and Scudder, 1970), numerous vacuoles are correlated
with an absorptive function for these epithelial cells. The anterior
two-thirds of the Belgica hindgut or ileum seems to be adapted for
absorption of ions and water in the larva’s semi-aquatic environment (Murakami and Shiotsuki, 2001; Heuser et al., 1993). Unlike
the rectal pads or papillae of terrestrial insects, the posterior third
of the hindgut or rectum epithelium of Belgica shows no special
adaptations on its apical surface for transport of ions and water;
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J.B. Nardi et al. / Arthropod Structure & Development 38 (2009) 377–389
Fig. 12. Images of the hindgut epithelium are presented as DAPI-labeled whole mounts (a) and thin sections (b–f). Fig. 10a shows the midgut (mg)–hindgut interface of the larval gut
that has been labeled with the specific DNA stain DAPI. At its anterior-most region, the nuclei of the hindgut epithelial cells are dwarfed by the polyploid nuclei of the Malpighian
tubules (arrows) and more posterior hindgut cells (*). (b) The anterior third of the hindgut consists of cells with polyploid nuclei (n) whose apical ends are occupied by numerous
vesicles and covered by a thin cuticle (arrow). A thin basal lamina (bl) is located on its basal surface. (c) Convoluted membranes, electron-lucent vesicles and mitochondria are
concentrated at apical surfaces of this anterior third of the hindgut epithelium. The cuticle is indicated with an arrow. (d, e) Bacteria inhabit the lumen of the middle third of the
hindgut. The apical ends of these epithelial cells are covered by a thin cuticle and have extremely convoluted membranes (arrows). Basally the epithelium has a thick muscle layer
(m). (f) The cuticle lining the lumen of the posterior third of the hindgut is convoluted (arrow). The basal lamina of epithelial cells has conspicuous infoldings (arrowheads);
m ¼ muscles. Scale bars: (a) 50 mm; (b) 2 mm; (c) 1.0 mm; (d) 2 mm; (e) 20 mm; and (f) 2 mm.
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J.B. Nardi et al. / Arthropod Structure & Development 38 (2009) 377–389
this portion of the hindgut is surrounded by a thick layer of muscles
and apparently has a mechanical rather than a transport function
(Fig. 12c–f).
Acknowledgments
We are grateful to the staff at Palmer Station for logistical assistance in Antarctica. The thoughtful suggestions of two anonymous
reviewers helped improve the quality of this manuscript. This
research was supported in part by NSF grants OPP-0337656 and OPP0413786.
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