Parasitol Res
DOI 10.1007/s00436-006-0363-0
ORIGINAL PAPER
Fine structure of the alimentary canal of the larval blow fly
Chrysomya megacephala (Diptera: Calliphoridae)
Worachote Boonsriwong & Kom Sukontason &
Jimmy K. Olson & Roy C. Vogtsberger &
Udom Chaithong & Budsabong Kuntalue &
Radchadawan Ngern-klun & Surasak Upakut &
Kabkaew L. Sukontason
Received: 29 June 2006 / Accepted: 16 October 2006
# Springer-Verlag 2006
Abstract Morphology of the alimentary canal of the
mature third instar larva of the blow fly, Chrysomya
megacephala (F.), was examined using light, scanning,
and transmission electron microscopy. Salivary structures
consist of a single median deferent duct that bifurcates into
efferent ducts connected to paired, tubular salivary glands
comprised of closely packed conical-shaped epithelial cells
with large nuclei. The crop occurs as a large, swollen
diverticulum of the digestive tube and is lined internally
with convoluted cuticle (epicuticle and endocuticle). The
esophagus is a simple, straight tube internally lined with
cuticle and externally encompassed by muscle fibers. The
cardia is a bulb-like structure composed of anterior foregut
tissue and posterior midgut tissue from which the peritrophic membrane (PM) is produced. The midgut begins
within the cardia which is flanked posteriorly by four
tubular gastric caeca that are lined internally with four to
five layers of cuboidal epithelial cells bearing microvilli.
W. Boonsriwong : K. Sukontason (*) : U. Chaithong :
R. Ngern-klun : S. Upakut : K. L. Sukontason
Department of Parasitology, Faculty of Medicine,
Chiang Mai University,
Chiang Mai 50200, Thailand
e-mail: [email protected]
J. K. Olson
Department of Entomology, Texas A&M University,
College Station, TX 77843-2475, USA
R. C. Vogtsberger
Department of Biology, Midwestern State University,
Wichita Falls, TX 76308, USA
B. Kuntalue
Electron Microscopy Research and Service Center (EMRSC),
Faculty of Science, Chiang Mai University,
Chiang Mai 50200, Thailand
Midgut tissue is lined with simple cuboidal epithelium
whose cells are filled with numerous secretory granules and
possessed long microvilli facing the lumen. A peritrophic
membrane is contained within the midgut lumen. The larval
hindgut consists of the pylorus, Malpighian tubules, ileum,
colon, rectum, and anus, posteriorly. The pylorus is
characterized by a single layer of epithelial cells encircled
by a muscular layer and the presence of PM within the
lumen. Malpighian tubules each diverge into two tubular
structures totalling four long tubules of long chained
cuboidal cells bearing microvilli internally. The wall of
the ileum is comprised primarily of a monolayer of
cuboidal epithelial cells with large oval nuclei and more
intense muscular fibers surrounding the periphery. A
cuticular layer surrounds the lumen containing the PM.
This inner cuticle consists of a thin epicuticle that is
electron-dense; whereas, the endocuticle is much thicker
but less electron-dense. Myo-epithelial cells are dense in
the anal region, where the PM persists.
Introduction
Chrysomya megacephala (F.), the Oriental latrine fly, is a
common blow fly species of medical importance in many
parts of the world, including Thailand. Adults may feed on
food sources including nectar, animal carcasses, garbage,
and other filth materials, or even human food. Therefore, it
is possible that mechanical transfer of potential diseasecausing pathogens, such as bacteria, viruses, protozoa, and
helminth eggs, to human food may occur (Greenberg 1973;
Sukontason et al. 2000). Larvae of this species are known
to cause myiasis in several mammal species, including
humans (Zumpt 1965; Kumarasinghe et al. 2000). Another
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facet of medical importance of this blow fly is its
association with human corpses and its relevance to
forensic entomology. Many researchers have reported that
specimens of C. megacephala were found connected with
cases of human death (Lee 1996; Carvalho et al. 2000; Goff
2000; Lee et al. 2004; Sukontason et al. 2005).
As in other animals, the alimentary canal in arthropods
consists of vital organs necessary for life functions such as
digestion and absorption of nutrients, regulation of hemolymph ionic composition and pH, and detoxification, and
production of semiochemical compounds such as pheromones. To date, many researchers have reported the
ultrastructure of various alimentary organs of numerous
groups of arthropods. These include mosquitoes, Aedes
(Stegomyia) aegypti (Zieler et al. 2000; Moncayo et al.
2005), Culex quinquefasciatus (Sais et al. 2003); sand flies,
Lutzomyia longipalpis (Secundino et al. 2005), Lutzomyia
intermedia (Andrade-Coêlho et al. 2001); bot fly, Dermatobia hominis (Evangelista and Leite 2003); black fly,
Simulium pertinax (Cavados et al. 2004); fruit fly, Bactrocera dorsalis (Lee et al. 1998; Hung et al. 2000); ticks,
Haemaphysalis longicornis (Matsuo et al. 2003), Ixodes
ricinus (Jasik and Buczek 2005), Rhipicephalus (Boophilus)
microplus (Nunes et al. 2005); mite, Platytrombidium
fasciatum (Shatrov 2005); ants, Tetraponera (Billen and
Buschinger 2000), Solenopsis saevissima (Arab and
Caetano 2002), Pachycondyla villosa (Zara and Caetano
2003); reduviid bug, Triatoma infestans (Reis et al. 2003);
beetles, Dendroctonus (Silva-Olivares et al. 2003), Odontotaenius disjunctus (Nardi et al. 2006); and a moth, Hofmannophila pseudospretella (Gerard 2002). In the present study,
observations of the morphology of the alimentary canal of C.
megacephala larvae were carried out to the ultrastructural
level to clarify their morphological composition and better
understand the functional role of each associated organ.
Fig. 1 Alimentary canal of the third instar larva of C. megacephala.
The foregut is comprised of the mouth (Mo), salivary glands (SG),
esophagus (Es), and crop (Cr). The cardia (Ca) represents the junction
of the foregut and midgut. The midgut consists of the gastric caeca
(GC), followed by anterior midgut (AMG), middle midgut (MMG),
and posterior midgut (PMG). The hindgut (HG) begins with the
pylorus (Py) from which the Malpighian tubules (MT) arise, ileum (Il),
colon (Co), rectum (Re), and anus (An)
Scanning electron microscopy (SEM)
The dissected alimentary canal was transferred from the
phosphate buffer and chemically treated with 2.5% glutaraldehyde mixed in phosphate buffer solution at a pH of 7.4
at 4°C for 24 h to accomplish primary fixation. They were
then rinsed twice with phosphate buffer solution at 10-min
intervals. Rinsed specimens were treated with 1% osmium
tetroxide at room temperature for 3–4 days for postfixation. Post-fixation was followed by rinsing twice with
phosphate buffer solution and dehydrating with alcohol. To
replace the water in the specimens with alcohol, they were
subjected to the following increasing concentrations of
Materials and methods
Third instar larvae of C. megacephala were obtained from
laboratory colonies maintained at the Department of
Parasitology, Faculty of Medicine, Chiang Mai University,
Thailand. The procedure for fly rearing has been previously
described by Sukontason et al. (2004).
Dissection of alimentary canal
Approximately 20 specimens of 3-day-old larvae were
removed from the rearing box and individually dissected in
phosphate buffer with a pH of 7.4 under a binocular
dissecting microscope (Olympus®, Japan). The entire
alimentary canal was kept in phosphate buffer in preparation for the electron microscopic studies.
Fig. 2 Foregut, cardia, and details of the salivary glands of the third„
instar larva of C. megacephala. a SEM micrograph of the foregut
indicating salivary glands (SG), crop (Cr), esophagus (Es), and the
cardia (Ca). The abdominal–thoracic gland (ATG) is evident adjacent
to the beginning of the esophagus, and the gastric caeca (GC) are
visible posteriorly extending adjacent to the cardia. b SEM micrograph of salivary structures consisting of a single median deferent duct
(DD) that bifurcates into smaller efferent ducts (ED) connecting to the
paired tubular salivary glands (SG). c SEM micrograph of ruptured
salivary gland showing closely packed simple cuboidal cells. Fibrous
material in the proximal glandular lumen is probably salivary
secretion (star). d Semi-thin section through the middle of salivary
gland highlighting the conical-shaped epithelial cells projecting into
the central lumen. Arrows indicate the large, oval nuclei within cells.
e TEM micrograph through the base of salivary gland showing
smooth external surface and embedded tracheole (Tr); ×2,000
magnification. f TEM micrograph of the large, oval nucleus within
an epithelial cell of the salivary gland; ×6,300 magnification
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alcohol: 30, 50, 70, 80, 90, and 95%. The specimens were
then placed in absolute alcohol for two 12-h periods
followed by acetone for two 12-h periods. Finally, the
specimens were subjected to critical point drying, were
attached with double-stick tape to aluminum stubs, and
were coated with gold in a sputter-coating apparatus before
being viewed with a JEOL JSM-5910 scanning electron
microscope (Japan).
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Transmission electron microscopy (TEM)
Before being prepared for the TEM, the alimentary canal
was first dissected into individual organs. The methodology
for the TEM process is the same as that for the SEM
procedure up through the point where specimens are placed
in absolute alcohol for two 12-h periods. After that, organ
specimens were placed in acetone for 2 h before transferring into ratios of resin to acetone of 1:3 for 24 h, 1:1 for
24 h, and 3:1 for 24 h, sequentially. This was followed by
treatment with pure resin twice for 3 h. Each sample was
then embedded in Spurr’s resin by placing them into a
plastic block and by incubating at 70°C for 24 h. A semithin section (0.5 μm) of each sample was made with a glass
knife on an Ultramicrotome (Boeckeler®, USA). This was
followed by staining with 1% methylene blue mixed with
1% Azure II (1:1) to view under a light microscope
(Olympus®, Japan). The ultrathin sections (90 nm) were
stained with uranyl acetate and lead citrate to observe under
the ZEISS EM 10 electron microscope (Germany).
Results
The alimentary canal of the third instar larva of C.
megacephala is a single, long tubular organ system,
consisting of the anterior foregut (stomodaeum), median
midgut (mesenteron), and posterior hindgut (proctodaeum), and accessory organs projecting from the main
digestive tube. The midgut is greatly elongated into the
longest portion of the digestive tract of this species
(Fig. 1).
Foregut The foregut consists of a single tube, with
noticeable portions beginning in the anterior end being the
mouth, esophagus, and anterior portion of the cardia, with
the salivary glands and extremely prominent crop emanating from the main digestive tube (Figs. 1 and 2a). The
salivary gland is comprised of a single median deferent duct
that bifurcates into smaller efferent ducts connected to
paired, tubular salivary glands (Fig. 2b). Both the ducts and
glands have relatively smooth external surfaces (Fig. 2b).
From the ruptured base of one salivary gland, it was
observed that the wall of the gland consisted primarily of
closely-packed simple cuboidal cells. A fibrous material
that was probably salivary secretion was found proximally
oriented in the glandular lumen (Fig. 2c). The semi-thin
section around the middle of the gland revealed that the
epithelial cells were fairly conical-shaped with their apical
part projecting into the central lumen of the gland. Large,
oval nuclei were observed to be centrally located in these
cells (arrows in Fig. 2d). TEM observation of the base of
the gland provided greater detail of the smooth external
surface of the gland (Fig. 2e) and the large, oval nuclei
found within the glandular cells (Fig. 2f).
In the third instar larva of C. megacephala, the crop
laterally diverges from the esophagus as an extremely
enlarged diverticulum (Figs. 1 and 3a). In one SEM
micrograph, an area of the crop whose outer tissue layers
had peeled away revealed the highly-folded inner surface
area of the crop wall (Fig. 3b). The internal surface of the
delicate connective tissue sheath covering the crop (arrow
in Fig. 3a) that was seen under SEM shows its irregular
conformation to the highly folded crop wall (Fig. 3c). A
semi-thin section of the cuticle clearly demonstrates the
convoluted structure of the inner epicuticle (in contact with
lumen) and overlying endocuticle (Fig. 3d). Viewed with
SEM, the esophagus is a simple, straight tube (Fig. 2a)
whose external surface is longitudinally folded with faint
indication of muscular fibers (Fig. 3e). A semi-thin crosssectional view revealed that this structure is also internally
lined with a convoluted cuticular layer composed of an
inner epicuticle and overlying endocuticle invaginating into
the lumen, while circular muscles encircle these tissues
peripherally (Fig. 3f).
Posterior to the tubular esophagus is the bulb-like cardia
(Figs. 1 and 4a). Longitudinal sections of the cardia
revealed that this structure is composed of two parts: the
anterior foregut tissue and posterior midgut tissue (Fig. 4b).
Thus, this structure comprises a junction of the foregut and
midgut. The anterior foregut tissue appears as two separate,
rounded compartments (Fig. 4b). Using higher magnification, the anterior foregut tissue displayed more or less
shortened columnar epithelial cells containing numerous
dark-stained secretory granules, particularly at the internal
apices of the cells (Fig. 4c). Outer longitudinal muscles and
inner circular muscles were found wrapping the epithelium
externally. Large, oval nuclei were also obvious within the
epithelial cells (Fig. 4d). As for the posterior midgut tissue
of the cardia, it also appeared as a rounded compartment
set off by darker staining epithelial cells (Fig. 4b and e).
Within the central lumen of the cardia, an internal folded
thread-like structure was evident. This was most likely
the peritrophic membrane (PM) that is produced within the
cardia (Fig. 4b and e). Higher magnification of the
epithelial cells of this area also revealed large, oval nuclei
within them (Fig. 4f).
Midgut The larval midgut of C. megacephala is the longest
portion of the alimentary canal, lying convoluted and
twisted within the larval body cavity (Fig. 1). The midgut
proper begins with the posterior midgut tissue of the cardia
just anterior to the gastric caeca (Figs. 1 and 4a). Four long
tubular gastric caeca were observed in this species, and
their external surfaces were markedly smooth with irregular
swellings (Fig. 5a). Both inner circular and outer longitu-
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Fig. 3 Foregut of the third instar larva of C. megacephala. a SEM
micrograph of the crop (Cr). b Higher magnification of boxed region
in (a) displaying the highly-folded surface area of the crop wall.
c Higher magnification of delicate connective tissue sheath covering
crop [at arrow in (a)] showing irregular conformation to highly folded
crop wall. d Semi-thin cross-section of crop demonstrating convoluted
inner epicuticle (Ecu) and overlying endocuticle (Encu). e SEM
micrograph of esophagus displaying longitudinally folded external
surface. f Semi-thin cross-section of esophagus showing cuticle (Cu)
composed of inner epicuticle (Ecu) and overlying endocuticle (Encu),
encircling and invaginating into lumen (Lu). The cuticle, foregut
epithelium, and longitudinal muscles are encircled by a well-developed
layer of circular muscle (CM) externally
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dinal muscles were evident, wrapping the gastric caeca
externally. In a semi-thin longitudinal section of a gastric
caecum, it could be seen that the inner walls of its lumen
were comprised of approximately four to five layers of
cuboidal epithelial cells (Fig. 5b). These cells consisted
of many types, as was evident by the differential staining
of their cytoplasms and nuclei. However, most of the
nuclei of the cells are large and oval. Closer investigation
of these cells utilizing TEM revealed that the inner
margin of these gastric caeca cells consisted of microvilli
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ƒFig.
4 Cardia of third instar larva of C. megacephala. a SEM
micrograph of bulb-like cardia (Ca) between esophagus (Es) and four
gastric caeca (GC). Thread-like tracheoles (Tr) are visible on the
surface. b Longitudinal section of the cardia revealed two parts: the
anterior foregut tissue (FgT) and posterior midgut tissue (MgT).
Peritrophic membrane (PM) is seen within the lumen near the
posterior midgut tissue. c Higher magnification of anterior foregut
tissue of the cardia showing more or less columnar epithelial cells
with numerous dark-stained secretory granules (SG). Outer longitudinal muscle (LM) and inner circular muscle (CM) overlie the foregut
epithelium externally. d Epithelial cells of the anterior foregut tissue of
cardia showing large nucleus (N), secretory granules (SG). Cells were
wrapped with longitudinal muscle (LM) and circular muscle (CM), and
infrequently externally embedded tracheole (Tr) were observed.
e Higher magnification of the posterior midgut tissue of cardia
showing epithelial cells (EC), peritrophic membrane (PM), food
particle (fp), longitudinal muscle (LM), and circular muscle (CM).
f Higher magnification of the posterior midgut tissue of cardia
highlighting an epithelial cell (EC) revealing a large, oval nucleus
(N), peritrophic membrane (PM) within lumen, and Z-lines (Z) of
circular muscle (CM)
to increase surface area for absorption of nutrients in the
midgut region (Fig. 5c).
The anterior midgut emerges from the cardia and
junction of the gastric caeca. Its external appearance is
similar to that of the gastric caeca in that the surface is also
very smooth with inner circular and outer longitudinal
muscles within (Fig. 5d). Insertion of tracheal tubules into
the midgut surface is also quite prevalent in this area. Upon
further investigation of this area using semi-thin sectioning,
it is clear that the anterior midgut tissue contains peritrophic
membrane (PM) within its central lumen and is surrounded
by a single layer of cuboidal epithelial cells filled with a
considerable number of dark-stained secretory granules of
variable size (Fig. 5e). Large secretory granules were
occasionally observed outside of the cell within the gastric
lumen.
Posterior to the anterior midgut is the more dilated area
known as the middle midgut (Fig. 1). The composition of
the middle midgut resembles that of the anterior midgut in
having a PM within the lumen surrounded by a monolayer
of cuboidal epithelial cells. The PM presented as a thin,
transparent membrane (Fig. 6a); whereas, the middle
midgut epithelial cells contained numerous secretory
granules both internally and externally (Fig. 6b). In semithin cross-sectional view through the middle midgut, it was
quite obvious that the epithelial cells each contained
numerous dark-stained secretory granules inside (Fig. 6c).
Moreover, fat body cells were also evident between these
epithelial cells (Fig. 6c). An analysis of transverse sections
through these epithelial cells using both semi-thin sectioning (Fig. 6d) and TEM (Fig. 6e) revealed that these cells
project inward from their basement membranes and each
possess long microvilli covering their inner apical surfaces
signifying their significance in absorption and secretion.
Hindgut The larval hindgut of C. megacephala consists of
the pylorus, Malpighian tubules, ileum, colon, rectum, and
posterior anus (Fig. 1). In the pylorus (which anteriorly has
the Malpighian tubules projecting from it), a characteristic
single layer of flattened cuboidal epithelial cells, PM, and
muscular layer were observed (Fig. 7a). Upon examination
at a higher magnification, large secretory granules could
also be seen. These granules are similar to those found in
the midgut cells, except in much smaller amounts and only
appearing in the apical region of the pyloric cells with
relatively large nuclei (Fig. 7b).
The Malpighian tubules emerge from the midgut–
hindgut junction and are proximally paired. Each diverges
distally into two tubular structures, forming the four long
Malpighian tubules (Fig. 1). Closer examination revealed
that the Malpighian tubules consist of long chains of
cuboidal cells (Fig. 7c). In a semi-thin cross-sectional view,
it could be seen that these cells each contain a large
nucleus, numerous minute granules, and abundant microvilli at their apical surface in contact with the lumen
(Fig. 7d).
The external surface of the ileum, colon, rectum, and
anus is similar to that of the midgut in being covered with a
muscular layer and embedded with abundant tracheoles for
gaseous exchange (Fig. 7e). A highly-convoluted cuticle,
characteristic of the hindgut, was observed encircling the
lumen of the ileum (Fig. 7f). In a cross-sectional view of
the ileum, it was apparent that its wall was formed
primarily by a monolayer of cuboidal epithelial cells with
large, oval nuclei and some muscular fiber peripherally
(Fig. 8a). Peritrophic membrane was also quite obvious in
this region of the alimentary canal. A semi-thin crosssection of both the colon (Fig. 8b) and rectum (Fig. 8c)
showed extreme similarity of features to the ileum, except
that they exhibited more muscle fiber than the latter. TEM
observation through the rectal region clearly showed that
the epicuticle is a thin and electron-dense layer; whereas,
the endocuticle is less electron-dense but much thicker
(Fig. 8d). The peritrophic membrane also proved to be quite
electron-dense. Both the outer longitudinal muscles and
inner circular muscles were observed in the hindgut region.
In the anal region, myo-epithelial cells that were either
uninucleate or multinucleate comprised the largest component of the wall of the anal tube (Fig. 8e and f). Peritrophic
membrane was still quite obvious within the anal tube
(Fig. 8e).
Discussion
An overview of the gross morphology of the alimentary
canal of blow fly larvae of Calliphora vicina based entirely
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Fig. 5 Gastric caeca and anterior midgut of third instar larva of C.
megacephala. a Outer surface of gastric caecum with irregular swellings
and external wrapping of inner circular muscle (CM) and outer
longitudinal muscles (LM). b Semi-thin longitudinal section of gastric
caecum represented by ≈four to five layers of cuboidal epithelium cells
encircling the lumen (Lu). Circular muscle (CM) is seen overlying the
epithelial cells. c TEM micrograph of a cross-section of epithelial cell of
gastric caecum anchored by basement membrane (BL) and with apical
microvilli (MV) projecting inward toward lumen. An external wrapping
of longitudinal muscle (LM) is also visible; ×2,000 magnification.
d SEM micrograph of anterior midgut (AMG), passing posteriorly from
the cardia (Ca) and gastric caeca (GC). Insertion of tracheal tubules (Tr)
into the midgut surface is also evident. e Semi-thin cross-section of
anterior midgut displaying peritrophic membrane (PM) and simple
columnar epithelial cells filled with numerous dark-stained secretory
granules (SG). Muscle fibers (M) and tracheoles (Tr) are also noticeable
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Fig. 6 Middle midgut of third instar larva of C. megacephala. a SEM
micrograph of ruptured middle midgut revealing transparent peritrophic membrane (PM) within and encircling epithelial cells (EC).
b SEM micrograph of highly magnified epithelial cells containing
numerous intracellular and extracellular secretory granules (SG).
c Oblique view of semi-thin section of middle midgut with epithelial
cells (EC) containing dark-stained secretory granules and probably fat
body cells (arrows). Peritrophic membrane (PM) within lumen and
overlying muscle (M) are also seen. d Semi-thin section of middle
midgut displaying epithelial cells containing dark-stained secretory
granules and inner apical microvilli (MV). Peritrophic membrane (PM)
lies within the microvilli of cells and encapsulates food particles (fp).
Embedded tracheole (Tr), circular muscle (CM), and basement
membrane (BM) for anchoring epithelial cells were also observed in
the periphery. e TEM micrograph of epithelial cell with apparent
microvilli (MV); ×1,250 magnification
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Fig. 7 Hindgut of third instar larva of C. megacephala. a Semi-thin
cross-section of pylorus showing simple flattened cuboidal epithelial
cells (EC) and peritrophic membrane (PM) within lumen. b Higher
magnification of epithelial cells in pylorus displaying large nucleus (N)
and dark-stained secretory granules (SG) near the apical surface.
Circular muscle (CM) encircles the epithelium. c SEM micrograph of
Malpighian tubule appearing as a chain of cuboidal cells. d Semi-thin
cross-section of a Malpighian tubule consisting of large cells with
nucleus (N) and microvilli (MV) evident. e SEM micrograph of ileum
and embedded tracheole (Tr). f Higher magnification of boxed region
in e displaying the highly convoluted cuticle (Cu) encircling the lumen
of the ileum internally
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on light microscopy has been previously provided by
Greenberg (1973). Otherwise, works of this type on blow
fly larvae are lacking. Results of the current study come
from the combined use of light microscopy, SEM, and
TEM to provide a more detailed examination of the
anatomy and histology of the digestive tract of the larval
blow fly, C. megacephala.
observations of C. megacephala larvae show that the PM
occurs continuously along the alimentary canal from the
cardia to the anus. The function of the PM as a barrier
against pathogens invading the midgut epithelium or for
mechanical protection of the midgut epithelium from
damage by food particles have both been previously
described by Lehane (1997).
Foregut In Diptera, the crop is an alimentary structure that
functions in the storage and flow of the ingested food
(Stoffolano 1995). The cuticle (epi- and endocuticle) that
was clearly observed in both the crop and esophagus of the
third instar larva of C. megacephala was quite similar in
structure to that described in the adult fruit fly, B. dorsalis
by Lee et al. (1998). The gross morphology of the salivary
glands of the C. megacephala larvae observed in this study
closely resemble those depicted by Evangelista and Leite
(2005) for the first instar larva of the bot fly, D. hominis.
The similarity consists of having simple, tubular glands
opening into narrow efferent ducts on each side that
converge to form a single median deferent duct leading to
the oral cavity. At the ultrastructural level, the composition
of the salivary gland of C. megacephala larvae appears to
be comparable to that of the secretory region of the lateral
duct of the larval salivary gland of the ant, P. villosa
(Hymenoptera: Formicidae) as described by Zara and
Caetano (2003). It consists primarily of simple cuboidal
epithelium cells and contains fibrous material that is most
likely salivary secretion within its lumen (see Fig. 2c). In
fly larvae known to produce myiasis, such as Hypoderma
lineatum (Family Oestridae), secretory products containing
protease are responsible for host penetration (Lecroisey et
al. 1979).
The cardia is a distinctive organ in Diptera that
encompasses the posterior end of the foregut and anterior
end of the midgut. Longitudinal section of the organ in C.
megacephala larvae clearly showed two compartments: the
anterior foregut tissue and posterior midgut tissue (see
Fig. 4b). This is the same as can be seen in the adult stable
fly, Stomoxys calcitrans (Lehane 1997) or the fruit fly, B.
dorsalis (Lee et al. 1998). In the present study, semi-thin
and ultrathin investigations reveal that the peritrophic
membrane first appears in the posterior midgut tissue of
the cardia (see Fig. 4b and e), thereby, indicating that it is a
type II PM that actually forms in the cardia (Lehane 1997).
This correlates to the discovery of Tellam et al. (2000) who
found that intrinsic peritrophic matrix protein, peritrophin95, was synthesized in the cardia of third instar larvae of
the blow fly, Lucilia cuprina. Likewise, Hao et al. (2003)
documented the synthesis of PM constituents in the cardia
of the adult tsetse fly, Glossina morsitans morsitans. In this
same study, the cardia was also found to play a crucial role
in immunity in this tsetse fly species. The results of our
Midgut The four long blind end tubes of the gastric caeca
that were apparent in the larvae of C. megacephala in this
study resembled those seen in the third instar larva of C.
vicina (Greenberg 1973) and the flesh fly, Liosarcophaga
dux (unpublished data). However, this was in contrast with
the complete absence of gastric caeca previously reported in
the third instar larva of the bot fly, D. hominis by
Evangelista and Leite (2003). The presence of short
microvilli at the free surface of the gastric caeca cells (see
Fig. 5c) is an evidence of increased surface area for more
efficient nutrient absorption (Chapman 1998).
The presence of microvilli along the midgut epithelial
cells observed in C. megacephala larvae corresponds with
similar findings reported in other insects such as the sand
flies, L. intermedia (Andrade-Coêlho et al. 2001) and L.
longipalpis (Leite and Evangelista 2001; Secundino et al.
2005); fruit fly, Drosophila auraria (Dimitradis 1991);
mosquitoes, A. (Stegomyia) aegypti (Zieler et al. 2000;
Moncayo et al. 2005), and Anopheles darlingi (Okuda et al.
2005); tick, H. longicornis (Matsuo et al. 2003); beetle,
Dendroctonus valens (Silva-Olivares et al. 2003); bee,
Melipona quadrifasciata anthidioides (Neves et al. 2003);
or the wingless firebrat, Thermobia domestica (Rost et al.
2005). Occurrence of microvilli on cells typically indicates
regions where large amounts of absorption and/or secretion
take place.
A large number of secretory granules were evident in the
midgut cells of C. megacephala larvae. Granules were not
only observed inside the cells, but, on many occasions, were
also seen outside the cells often bound to the apical cell
membrane (see Figs. 5e and 6b–e). This suggests a secretory
role these cells must play. Secretory granules are most likely
released from their vesicles by a process known as exocytosis in which the secretory vesicles move to the inner apical
surface of the cell, fuse with the cell membrane and release
the secretory granules into the gut lumen (Chapman 1998).
Hindgut It is quite obvious that microvilli line the
entire lumen of the Malpighian tubules of C. megacephala larvae (Fig. 7d). This situation structurally
resembles that described for the fire ant, S. saevissima
(Arab and Caetano 2002) and the Malpighian papillae of
the dipluran, Campodea (Monocampa) quilisi (Pigino et
al. 2005). The presence of a cuticle comprised of a narrow
layer of epicuticle and rather thick layer of endocuticle
Parasitol Res
within the foregut and hindgut of C. megacephala larvae
resembles most other insects such as the fruit fly, B.
dorsalis (Lee et al. 1998) and the beetle, D. valens (SilvaOlivares et al. 2003).
The muscular fibers observed within the walls of the
alimentary canal of third instar larvae of C. megacephala
were most apparent in the hindgut region. Musculature was
apparent beginning in the ileum and became more pro-
Parasitol Res
ƒFig.
8 Hindgut of third instar larva of C. megacephala. a Semi-thin
cross-section of ileum showing epithelial cells (EC) with large nuclei
(N) and thin internal cuticle (Cu) surrounding lumen. Extensivelyfolded peritrophic membrane (PM) is prominent within lumen. b Semithin cross-section of colon showing epithelial cells (EC) and thin
internal cuticle (Cu) surrounding lumen. Circular muscle (CM) is
evident externally and peritrophic membrane (PM) is prominent within
the lumen. c Semi-thin partial cross-section of rectum showing thicker
epithelial cell with large nucleus (N), cuticle (Cu) lining lumen and
extensively-folded peritrophic membrane (PM) within. Some overlying
circular muscle (CM) is also apparent. d TEM micrograph of epithelial
cell of rectum at higher magnification highlighting the thin, electrondense layer of epicuticle (ECu) bordering the lumen and the thicker,
underlying endocuticle (Encu) that is less electron-dense. The peritrophic membrane (PM) was also electron-dense; ×3,150 magnification.
e Semi-thin partial cross-section at the anus showing binucleated (N)
myo-epithelial cell (M) and peritrophic membrane (PM). f Another view
of anal region displaying apparent myo-epithelial cells (M)
nounced moving posteriorly down the hindgut, with the
greatest intensity of myo-epithelial cells being found in the
anal tube (see Fig. 8e and f). These observations strongly
suggest that intense activity of muscle contractions is
performed in the hindgut region of the alimentary canal.
In conclusion, our results obtained from using a
combination of light microscopy, SEM, and TEM in this
study have provided some thorough information on the
ultrastructure of the alimentary canal of the third instar
larva of C. megacephala. The ultrastructural characteristics
of each component within the foregut, midgut, and hindgut
were found to have differences from both morphological
and functional viewpoints which help us to clarify and
better understand each particular organ in the alimentary
canal of mature larvae of this species.
Acknowledgements This work was supported by the Thailand
Research Fund (TRF). We thank Chiang Mai University for funding
the cost of reprints.
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