The structure of the female sand fly (Phlebotomus papatasi ...

Transactions of the Royal Society of Tropical Medicine and Hygiene (2008) 102, 161—166
available at www.sciencedirect.com
journal homepage: www.elsevierhealth.com/journals/trst
The structure of the female sand fly (Phlebotomus
papatasi) alimentary canal
Alon Warburg ∗
Department of Parasitology, The Kuvin Centre for the Study of Infectious and Tropical Diseases,
The Hebrew University-Hadassah Medical School, Ein-Kerem, Jerusalem 91120, Israel
Received 15 May 2007; received in revised form 8 October 2007; accepted 8 October 2007
Available online 26 November 2007
KEYWORDS
Phlebotomus
papatasi;
Cibarium;
Pharynx;
Midgut;
Pylorus;
Leishmania major
Summary In the sand fly vector, Leishmania parasites are confined to the alimentary canal.
During much of their development, promastigotes are attached to the wall of the gut via their
flagella. In this context, the surface of the different regions of the sand fly alimentary tract
lumen warrants scientific attention. In this paper, the various regions are described, for the
first time using scanning electron microscopy. The cibarium and the pharynx, which function as
pumping organs, are lined with cuticle. Parts of the cibarium and the pharynx bear different
types of cuticular spines and appendages. The midgut is lined with microvillar epithelium, which
secretes the peritrophic matrix following a blood meal. The wider proximal part of the hindgut
(= pylorus) is lined with transverse cuticular ridges with tentacle-like appendages. Leishmania
major promastigotes were found to anchor themselves in the midgut and the stomodaeal valve
via their flagella. The possible roles of the different internal structures and their importance
for the development of Leishmania parasites are discussed.
© 2007 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights
reserved.
1. Introduction
Phlebotomine sand flies are the proven vectors of the leishmaniases,
parasitic diseases with a wide range of clinical
symptoms that currently threaten some 350 million people
in 88 countries around the world (Desjeux, 2001).
The causative agents of leishmaniasis, protozoan parasites
belonging to the genus Leishmania (Kinetoplastida:
Trypanospmatidae), are transmitted by phlebotomine sand
∗ Tel.: +972 2 6757080; fax: +972 2 6757425.
E-mail address: warburg@cc.huji.ac.il.
flies. In the lumen of the vector’s alimentary tract, Leishmania
parasites develop as extracellular promastigotes that
are attached to the wall of the alimentary canal (Killick-
Kendrick, 1979). In the midgut, promastigotes attach by
inserting their flagella between microvilli, whereas in the
hindgut and foregut parasites attach to the cuticular surface
by forming flagellar hemidesmosomes (Killick-Kendrick
et al., 1977, 1988; Molyneux et al., 1975; Walters et al.,
1987, 1989b; Warburg et al., 1986). Thus, the topology of
the internal surfaces of the sand fly female’s alimentary
tract is directly relevant to the development of Leishmania
promastigotes within it.
Phlebotomus papatasi is the main vector of L. major in
the Middle East and many other regions (Killick-Kendrick,
0035-9203/$ — see front matter © 2007 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.trstmh.2007.10.004

162 A. Warburg
1999). The anatomy of the alimentary canal of P. papatasi
and its related musculature have been described in
detail. Like other blood-sucking nematoceran insects, the
cuticle-lined foregut of P. papatasi comprises the biting
mouthparts, the cibarium and the pharynx. The latter two
are modified into pumps flanked by the cibarial and stomodaeal
valves, which regulate blood flow into the midgut
(Adler and Theodor, 1926; Davis, 1967; Jobling, 1987). The
entire midgut is lined with a single layer of microvillar
epithelium, which secretes the peritrophic membrane following
the ingestion of blood (Rudin and Hecker, 1982;
Walters et al., 1993). The hindgut, like the foregut, is
lined with cuticle and serves as a developmental post for
some saurian Leishmania spp. as well as L. braziliensis ssp.
(Killick-Kendrick, 1979). Here I depict, for the first time,
different regions of the gut as well as the peritrophic membrane
by scanning electron microscopy (SEM) and discuss the
putative roles of the different structures during Leishmania
infections.
2. Materials and methods
2.1. Sand flies
Phlebotomus papatasi were obtained from a laboratory
colony maintained at the Department of Parasitology,
Hebrew University of Jerusalem.
2.2. Artificial Leishmania infections of sand flies
Four- to five-day-old P. papatasi females were fed on a
suspension of murine peritoneal macrophages (2 × 10 6 /ml)
infected 24 h earlier with L. major (LRC-L137) (Warburg et
al., 1986). Infected flies were maintained on 10% sucrose 2%
albumin solution for 12 d.
2.3. Preparation for SEM
To observe the pharynx and thoracic parts of the gut,
flies were anaesthetized using CO 2 and fixed in 2.5% glutaraldehyde
overnight at 4 ◦ C. Flies were rinsed in 0.2 mol/l
cacodylate buffer (three times), post-fixed in 1% osmium
tetroxide for 2 h and rinsed as above. Flies were dehydrated
in an ethanol; ethanol—xylene graded series and embedded
in paraffin (60 ◦ C). Paraffin blocks were manually sliced sagitally
using a scalpel blade, deparaffinized in several changes
of xylene for at least 24 h, transferred to 100% ethanol and
processed for SEM. To examine the midgut and hindgut, flies
were anaesthetized using CO 2 , immobilized on ice and dissected
using watchmakers’ forceps. Guts were fixed for 1 h
in cold 2.5% glutaraldehyde in cacodylate buffer (0.1 mol/l,
pH 7.2) and rinsed three times in the same buffer. Fixed
guts were slit open using sharpened entomological pins,
post-fixed for 1 h in cold 1% osmium tetroxide in the same
buffer and rinsed three times in the same buffer. The same
procedure was used for infected flies.
For SEM, specimens were passed through an ethanolfreon
series to pure freon and were air dried. Dry specimens
were mounted on SEM stubs using double-sided sticky tape,
coated with gold and viewed with a Phillips SEM 505.
3. Results
The food canal was shown to be narrow and smooth up to
the posterior part of the cibarium (Figure 1A). The cibarial
valve comprised an anterior muscle, and on the apposing
distal surface of the cibarium were several rows of cuticular
appendages (1.3—4 × 0.3 m). Some were short and
wide, resembling rose thorns, while others were elongate,
tentacle-like and appeared more flexible (Figure 1B). The
pharynx was situated posterior to the cibarium separated
Figure 1 Median sagittal section through the head of a Phlebotomus papatasi female. The cuticular lining of the cibarium and the
pharynx is smooth (A) except for the junction between them at the cibarial valve, where different types of cuticular appendages
are evident (B). The muscle controlling the flow through the valve is clearly seen in cross section (CV).

Structure of female sand fly alimentary canal 163
Figure 2 The pharynx and stomodaeal valve of a Phlebotomus papatasi female. The more proximal section of the pharynx is lined
with smooth cuticle (A). Distally are rows of leaf-like ridges bearing narrow filamentous (6 × 0.5 m) appendages (right side of B).
The dorsal side of the distal part of the pharynx is lined with triangular cuticular spikes arranged in transverse rows and pointing
obliquely backwards (C). The part of the pharynx adjacent to the stomodaeal valve is lined with several rows of transverse cuticular
ridges studded with needle-shaped appendages (C). The stomodaeal valve comprises a narrow passage into the midgut and flow is
regulated by a ring muscle (sphincter, SP) with a broader circular lobe around it. The stomodaeal valve is lined with cuticle.
from it by the cibarial valve (Figure 1A). Its narrow anterior
section comprised smooth cuticular plates with longitudinal
ridges (Figures 1A, 2A). In the wide part of the pharynx there
was a series of transverse cuticular ridges bearing different
types of projections. The more anterior rows comprised
thin leaf-like ridges with elongate filamentous (6 × 0.5 m)
appendages (Figure 2B). These were replaced posteriorly
with more robust, comb-crested teeth. The distal part of
the pharynx was lined with triangular pointed spikes, while
the part of the pharynx adjacent to the junction with the
oesophagus was lined with transverse ridges bearing needlelike
cuticular spicules similar to those found in the cibarial
valve (Figures 1B, 2C).
The midgut is composed of two parts, a narrow thoracic
section and a wide abdominal one. The anterior part
of the thoracic midgut attaches to the foregut via the
stomodaeal valve (Adler and Theodor, 1926; Davis, 1967).
The stomodaeal valve comprised a cuticle-lined sphincter,
which projected into the lumen of the thoracic midgut and
an additional circular lobe external to it (Figure 2A). In
Leishmania-infected flies, the stomodaeal valve was often
heavily colonized by parasites attached to the cuticle.
The lumen of the midgut was lined with microvillar
epithelium. The length of the microvilli varied with the
region of the gut and tended to be longer in the thoracic
midgut. Leishmania promastigotes anchored themselves to
the midgut wall by inserting their flagella between microvilli
(Figure 3A). Frequently, midgut cells were observed to be
shed into the lumen of the midgut (Figure 3B). This usually
happened after the blood was voided from the gut,
irrespective of Leishmania infections.
The peritrophic membrane of sand flies is essentially similar
to that of mosquitoes. It is secreted from vesicles in the
midgut epithelium following a blood meal and envelopes the
entire blood meal (Gemetchu, 1974; Walters et al., 1993).
When dissected from the midgut before fixation, the peritrophic
matrix appeared like a sac with a narrow opening
at its posterior end (Figure 4A). When the gut was fixed
before dissection, and the midgut (mg) wall was chipped off
using sharpened entomological pins, the peritrophic matrix
(pm) was observed to bear the impression of the columnar
epithelium of the midgut (Figure 4B,C). In Figure 4C, the
blood meal (bm) was revealed, and embedded within it were
Leishmania promastigotes.
The cuticle-lined pylorus or hind triangle contained armature
consisting of transverse rows of posteriorly-directed
protrusions. These appendages were wrinkled and appeared
soft and flexible (Figure 5).
4. Discussion
The role that cibarial and pharyngeal armatures play in
feeding for haematophagous insects such as phlebotomine
sand flies and culicine mosquitoes is not clear. It has been
suggested that spines serve to filter larger clumps, but

164 A. Warburg
Figure 3 The midgut of a Phlebotomus papatasi female is lined with microvillar (MV) epithelium, to which Leishmania promastigotes
anchor themselves (A). Frequently, epithelial cells become rounded and slough off the wall into the lumen of the gut.
Leishmania promastigotes tend to utilize gaps between cells to anchor themselves to the midgut wall (B). In the stomodaeal valve,
attachment to cuticular lining is associated with modifications of the flagellum and a generally more flaccid appearance of L. major
promastigotes (C).
Figure 4 The midgut and peritrophic matrix of a Phlebotomus papatasi female. Peritrophic matrix dissected intact from the
midgut of a P. papatasi female, 24 h post-blood-feeding (A). Phlebotomus papatasi gut fixed 48 h post-blood-feeding (B, C). The
basal side of the midgut epithelium is observed in B (mg). Where the midgut wall has been removed, the peritrophic matrix bears
the impression of the midgut cells (pm). Leishmania parasites are visible (asterisks) within the blood meal (bm) in C.

Structure of female sand fly alimentary canal 165
Figure 5 Scanning electron microscopy view of the pylorus
of Phlebotomus paptasi female sand fly. Transverse cuticular
ridges, which are crested with tentacle-like wrinkled protrusions.
such clumps do not exist in blood or plant juices. Moreover,
fluids reach the cibarium via the food canal, which is
8—10 m in diameter (drawing 25 in Jobling, 1987), allowing
the passage of single red blood cells and barring the
passage of larger clumps. In the context of larger size parasite
transmission, it has been suggested that the shape
and density of the cuticular spines can influence the capacity
to kill microfilaria. Thus, mosquito species with sparse
pharyngeal armature (Culex spp., Aedes spp.) were more
competent vectors of Wuchereria bancrofti than Anopheles
spp. that have dense armature with sharp pointed spines
that kill microfilaria (McGreevy et al., 1978). Cuticular pharyngeal
armature in mosquitoes has also been shown to
cause haemolysis (Chadee et al., 1996). However, in the
case of protozoan parasites such as Leishmania it is unlikely
that mechanical disruption plays a role in preventing
infection.
Leishmania promastigotes frequently colonize the pharynx,
cibarium and mouthparts of infected sand fly females
by attaching to the cuticular surfaces (Killick-Kendrick et
al., 1988; Shortt et al., 1926; Walters et al., 1989a, 1989b).
However, colonization of the foregut is not necessary for
transmission by bite to occur, and heavy infections in the
stomodaeal valve are sufficient to cause regurgitation of
parasites (Volf et al., 2004; Warburg and Schlein, 1986).
This mode of transmission is enhanced by a filamentous
proteophosphoglycan gel of parasite origin, which serves to
block the passage of blood into the gut (Rogers et al., 2004).
The midgut of sand flies comprises the narrow thoracic
part and the wider expandable abdominal part. The entire
midgut is of endodermal origin; it is lined with microvillar
epithelium, which both produces and secretes the digestive
enzymes and absorbs the nutrients from the digested blood
(Gemetchu, 1974; Rudin and Hecker, 1982). The peritrophic
matrix (= membrane) is secreted by the epithelium and is visible
and dissectible by 24 h post-blood-feeding (Gemetchu,
1974; Walters et al., 1993, 1995). Like that of other insects,
the peritrophic matrix of sand flies is composed of proteins,
glycoproteins and chitin (Blackburn et al., 1988;
Gemetchu, 1974; Walters et al., 1993, 1995). Dissections of
the membrane revealed a relatively flexible thin-walled sac
(Figure 4A) (Walters et al., 1993). However, its fixation in
situ maintained its true structure, showing it to be a coagulated
matrix that essentially enveloped the blood meal and
assumed the relief of the columnar midgut epithelium on
its outer surface (Figure 4B,C). The Leishmania parasites
that develop inside the blood, in the endo-peritrophic compartment,
exit the sac before it is voided (Killick-Kendrick,
1979). Exit of L. major is probably mediated by chitinase
secreted by the proliferating promastigotes, which functions
to degrade the peritrophic matrix (Schlein et al., 1991).
Establishment of leishmanial infections following the
voidance of the blood remnants depends on the ability of
parasites to attach to the midgut by inserting their flagella
between microvilli (Figure 3). This attachment is facilitated
by receptor—ligand interaction between the surface
molecule lipophosphoglycan (LPG) coating L. major promastigotes
and Pp-galectin expressed in the midgut cells of
P. papatasi following blood feeding (Kamhawi et al., 2004).
Presumably, other unidentified molecules are involved in
LPG-mediated parasite attachment to unfed guts (Pimenta
et al., 1992) and flagella-specific attachment to midgut
sections (Warburg et al., 1989). Lastly, in permissive sand
flies, attachment is mediated via lectin-like leishmanial
molecules that attach to midgut glycoproteins bearing terminal
N-acetyl-galactosamine (Myskova et al., 2007).
The pyloric armature of sand flies was suggested to
function in the disruption of the peritrophic matrix and
blood bolus during the evacuation of the gut (Christensen
et al., 1971). However, the flaccid wrinkled appearance
of the armature in P. papatasi (Figure 5) did not support
this hypothesis. Perhaps the transverse ridges, with their
tentacle-like appendages, assist in pushing the remnants of
the meal backwards during peristalsis.
Authors’ contribution
AW was responsible for all aspects of this study and is guarantor
of the paper.
Acknowledgements: The author wishes to thank Y. Schlein
for technical assistance in the preparation of scanning electron
microscopy.
Funding: None.
Conflicts of interest: None declared.
Ethical approval: Not required.
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