1. Journal of Invertebrate Pathology 87 (2004) 123–128
www.elsevier.com/locate/yjipa
0022-2011/$ - see front matter  2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.jip.2004.09.001
Leishmania parasites (Kinetoplastida: Trypanosomatidae) reversibly
inhibit visceral muscle contractions in hemimetabolous
and holometabolous insects
Rajeev Vaidyanathan¤
Department of Parasitology, Hadassah Medical School, Hebrew University, Ein Kerem, P.O. Box 12272, Jerusalem 91120, Israel
Received 30 June 2004; accepted 1 September 2004
Available online 5 November 2004
Abstract
Female sand Xies can acquire protozoan parasites in the genus Leishmania when feeding on an infected vertebrate host. The para-
sites complete a complex growth cycle in the sand Xy gut until they are transmitted by bite to another host. Recently, a myoinhibitory
peptide was isolated from Leishmania major promastigotes. This peptide caused signiWcant gut distension and reversible, dose-depen-
dent inhibition of spontaneous hindgut contractions in the enzootic sand Xy vector, Phlebotomus papatasi. The current study further
characterizes myoinhibitory activity in L. major and other kinetoplastid parasites, using the P. papatasi hindgut and other insect
organ preparations. Myoinhibitory activity was greatest in cultured promastigotes and in culture medium in late log-phase and early
stationary-phase, coinciding with development of infective Leishmania morphotypes in the sand Xy midgut. L. major promastigote
lysates inhibited spontaneous contractions of visceral muscle preparations from hemimetabolous (Blattaria and Hemiptera) and
holometabolous (Diptera) insects. Inhibition of visceral muscle contractions in three insect orders indicates a conserved mode of
action. Myoinhibitory activity was detected also in Leishmania braziliensis braziliensis, a Sudanese strain of Leishmania donovani, and
the kinetoplastid parasite Leptomonas seymouri. Protozoan-induced myoinhibition mimics the eVect of insect myotropins. Inhibiting
host gut contractions protects Leishmania parasites from being excreted after blood meal and peritrophic matrix digestion, allowing
development and transmission of infective forms.
 2004 Elsevier Inc. All rights reserved.
Keywords: Host–parasite interactions; Vector competence; Sand Xy; Leishmania; Leptomonas; Herpetomonas; Myoinhibition
1. Introduction
The sand Xy Phlebotomus papatasi (Diptera: Psychod-
idae) is the sole vector of Leishmania major (Kinetop-
lastida: Trypanosomatidae) to humans in arid foci of
enzootic cutaneous leishmaniasis (Peters and Killick-
Kendrick, 1987; Schlein et al., 1982). Female sand Xies
ingest macrophages containing L. major amastigotes
when they bite an infected vertebrate host. Blood is
encased in a chitinous peritrophic matrix (PM) in the
gut, wherein amastigotes divide repeatedly and trans-
form into a uniform population of promastigotes
(Blackburn et al., 1988). The PM disintegrates after
blood meal digestion, and free parasites may be excreted
along with PM and blood meal remnants.
Several mechanisms facilitate parasite persistence in
the gut after blood digestion. Promastigotes may anchor
their Xagella between midgut microvilli (Adler and The-
odor, 1926; Killick-Kendrick et al., 1974; Warburg et al.,
1986, 1989). Polymorphisms in Leishmania lipophospho-
glycan (LPG), the main surface glycoconjugate, mediate
*
Fax: +1 530 752 1537, Present address: Department of Entomolo-
gy, Briggs Hall, Shields Avenue, University of California, Davis, CA
95616, United States..
E-mail address: rvaidyanathan@ucdavis.edu.

2. 124 R. Vaidyanathan / Journal of Invertebrate Pathology 87 (2004) 123–128
promastigote binding, release, and anterior migration
(Pimenta et al., 1992; Sacks and Kamhawi, 2001; Sacks
et al., 1994, 1995). However, a large population of unat-
tached parasites persists in the gut lumen (Walters et al.,
1987, 1989), and recently a mechanism was proposed for
how these parasites might remain in the gut. A 12 kDa
peptide isolated from L. major promastigotes was shown
to reversibly inhibit hindgut contractions and cause sig-
niWcant gut distension in P. papatasi, thereby protecting
unattached parasites from expulsion after blood diges-
tion (Vaidyanathan, in press).
The current study further characterizes myoinhibi-
tory activity in L. major and other kinetoplastid para-
sites. Stage-speciWc expression of L. major myoinhibitory
activity in cultured promastigotes and conditioned cul-
ture medium was detected using the P. papatasi hindgut
assay. Contractions of hindgut, dorsal blood vessel and
oviduct preparations from other insect species were also
inhibited by L. major promastigote lysate. Four other
species of Leishmania and three other species of obligate
gut parasites of insects were tested for their ability to
inhibit P. papatasi hindgut contractions.
2. Materials and methods
2.1. Parasite cultures
Leishmania major MHOM/IL/86/Blum (Jordan Val-
ley strain), Leishmania braziliensis braziliensis MHOM/
BR/75/M2903, Leishmania infantum MCAN/IL/L760,
Leishmania tropica MHOM/IL/L590, Leishmania dono-
vani (Khartoum and DD8 Indian strains), Herpetomo-
nas muscarum (a kinetoplastid parasite of house Xies),
and Leptomonas seymouri (a kinetoplastid parasite of
lygaeid bugs) were obtained from the W.H.O. Leish-
mania Reference Center, the Kuvin Centre for the
Study of Infectious and Tropical Diseases, Jerusalem.
All reagents and protease inhibitors were purchased
from Sigma (Rehovot, Israel), unless otherwise speci-
Wed. Parasites were grown in Dulbecco’s modiWed
Eagle’s medium (DMEM) (Biological Industries [B.I.],
Beit Haemek, Israel) with high glucose content, 10%
heat-inactivated fetal calf serum (FCS) (B.I.), 4 mM L-
glutamine, 2 mM adenosine, and 2% (v/v) Wlter-steril-
ized human urine. Cultures were grown at 28 °C and
passaged every 4 days. Cultures of Crithidia fasciculata,
a kinetoplastid gut parasite of mosquitoes, were grown
at 28 °C in brain–heart infusion (B.I.) and passaged
daily.
Homogenate preparation was the same for all para-
sites. Late log-phase promastigote cultures with high
parasite density (107
–108
parasites/ml) were spun at
2000g for 10 min at 8 °C and washed twice with ice-cold
Aedes aegypti buVered saline (ABS, Wnal concentra-
tions: 0.6 mM MgCl2, 4.0 mM KCl, 1.8 mM NaHCO3,
150 mM NaCl, 25 mM Hepes–NaOH, and 1.7 mM
CaCl2, pH 7.4). A protease inhibitor cocktail was added
to wet parasite volume to inhibit autolysis (Wnal
concentrations: 1.0 mM AEBSF, 0.5 mM EDTA, 65 M
bestatin, 7 M E-64, 0.5 M leupeptin, and 0.15 M
aprotinin). To lyse parasites, cell pellets were freeze–
thawed three times in liquid N2 and at 30 °C. Crude
homogenates were spun at 12,000g for 30 min.
Concentrations of supernatant proteins were assayed
by the Bradford method (Bradford, 1976). Based on
previous results, only lysate supernatant was used for
bioassays.
2.2. Stage-speciWc expression of myoinhibitory activity
Protein samples from culture medium used for L.
major growth were precipitated with 85% of saturation
(NH4)2SO4 at 0 °C. Both precipitate and supernatant
were dialyzed (3500 Da MWCO, Spectra/Por Mem-
brane, Spectrum Labs, Jerusalem) for 24 h at 4 °C
against ABS decreased to 50 mM NaCl, and tested using
the sand Xy hindgut bioassay (below). Parasites and
medium (four replicates) were harvested from day 1 to 7
of growth, and daily parasite density was calculated.
Cultures were centrifuged at 2000g for 10 min at 8°C
and washed twice with ABS. Parasites were lysed as
described above, and crude homogenates were stored at
¡70 °C until bioassayed for stage-speciWc expression of
the paralytic factor. For culture medium and parasite
lysates, a range of 10–100 g/ml proteins were tested, and
activity was standardized as percent inhibition per
microgram proteins.
2.3. Experimental insects
Colonies of the sand Xies P. papatasi (originated from
Kfar Adumim, 10 km east of Jerusalem) and Lutzomyia
longipalpis (Bahia strain) were maintained according to
Modi and Tesh (1983). Insectary conditions were tem-
perature of 26 §1 °C, relative humidity 80% and a 17:7
light:dark cycle. Two- to six-day-old sugar fed sand Xies
were used for all experiments. The Rockefeller strain of
A. aegypti (Diptera: Culicidae) was maintained under
the same conditions.
The blood-feeding bug Rhodnius prolixus (Hemiptera:
Reduviidae) was a gift from J.M.C. Ribeiro and main-
tained on rabbit blood under the same conditions; sec-
ond instars were used in all experiments. Cockroaches
(Blattaria: Periplaneta americana and Supella longipalpa)
were kept at room temperature, with pulverized cat food
and water. The sheep blowXy Lucilia sericata (Diptera:
Calliphoridae) was donated by K.Y. Mumcuoglu and
maintained on 10% sucrose and beef liver for oviposi-
tion. Flies (Diptera) in the families Sarcophagidae, Psy-
chodidae, and Muscidae were caught at natural resting
sites and maintained on 10% sucrose.

3. R. Vaidyanathan / Journal of Invertebrate Pathology 87 (2004) 123–128 125
2.4. Insect bioassays
Whole guts from male and female P. papatasi were
dissected into a watch glass with 90–99 l oxygenated
ABS warmed to 30 °C and allowed to recover until
hindgut contractions stabilized. Hindgut contractions
were counted for 5 min increments. Samples of proteins
from culture medium or from parasite lysate were
added 5 min after the hindgut stabilized and contracted
regularly. Values for activity were standardized as
percent inhibition of P. papatasi hindgut contractions
(relative to 5 min equilibration) per microgram pro-
teins.
Organs of other insects were dissected into either
Cockroach buVered saline (CBS, Wnal concentrations:
150mM NaCl, 5.0mM KCl, 5.0mM CaCl2, 3.0mM
MgCl2, 4.0mM Hepes–NaOH, and 40mM glucose, pH
7.2) (Lange et al., 1993) or an insect Ringer’s solution
(Wnal concentrations: 154mM NaCl, 2.7mM KCl,
1.8mM CaCl2, 12.0mM Hepes–NaOH, 22.0mM glucose,
pH 7.0) (Cook et al., 1975; Duve et al., 1999). BuVer vol-
ume depended on the size of the preparation. As with the
sand Xy hindgut bioassay, other insect tissues were
allowed to equilibrate and their contractions counted for
1–5min before addition of L. major lysate. Contractions
were counted further for 1–5min intervals. Inhibition was
calculated as the number of contractions after addition of
lysate proteins relative to the number of contractions dur-
ing the equilibration period. A range (1.2–24 g/ml) of
protein concentrations was tested, and activity was stan-
dardized as percent inhibition per microgram protein.
Reversibility of inhibition was tested using prepara-
tions of P. americana hindgut, Sarcophaga dorsal blood
vessel, and A. aegypti oviduct. Treated preparations were
rinsed twice in CBS or ABS and returned to warm,
untreated buVer.
3. Results and discussion
3.1. Stage-speciWc expression
A previous study showed that a unique peptide was
produced by L. major promastigotes in vitro, which
reversibly inhibited P. papatasi hindgut contractions and
resulted in gut distension (Vaidyanathan, in press). In
the current study, myoinhibitory activity was detected in
L. major lysate proteins on days 2–7 of growth, with
peak activity detected at late log-phase, early stationary-
phase (Fig. 1). By day 7, myoinhibitory activity had
decreased by 75% relative to peak activity on day 5.
Three days after blood feeding, the blood bolus has been
digested and voided. Maximal activity from cultured
promastigotes coincided with presence of free forms in
the sand Xy midgut lumen when nectomonads are vul-
nerable to peristaltic ejection.
Myoinhibitory activity in culture medium was detected
at the same time as its appearance in parasites (Fig. 1).
Peak activity in culture medium was detected at station-
ary-phase. Activity in the culture medium was higher than
in parasite lysate for days 6 and 7. Activity decreased by
day 7, the second day of stationary phase, a decrease of
20% relative to peak activity on day 5. Activity in both
sources was abolished by trypsin or proteinase-K, indicat-
ing that the active factor was a protein (results not
shown). During blood meal digestion and excretion, pro-
cyclic promastigotes transform into nectomonad forms in
the anterior abdominal midgut. Nectomonads may
migrate anteriorly to the thoracic midgut and stomodeal
valve, remain in the midgut lumen, or interdigitate their
Xagella between midgut microvilli. By day 7, slender nec-
tomonads transform into shorter haptomonads which
invade the Xy pharynx and esophagus (Schlein, 1993).
Most parasites have not migrated forward and remain
vulnerable to expulsion from the midgut. Although obser-
vations from cultured parasites might not reXect the situa-
tion in a sand Xy, if parasites and secreted proteins act
similarly in the gut, then myoinhibition would function
even after parasites are no longer secreting (or secreting
very little), ensuring that free forms in the lumen are not
voided. Moreover, maximal myoinhibitory activity after
blood digestion coincides with low midgut trypsin and
chymotrypsin levels, thereby avoiding myoinhibitory pep-
tide inactivation during proteolysis of blood (Borovsky
and Schlein, 1987; Ramalho-Ortigão et al., 2003).
In addition, the parasite population in sand Xies
includes several morphotypes not found in culture. Flies
remain infected for life, and some morphotypes might
continue secreting constantly.
3.2. Myoinhibition in diVerent insect preparations
Leishmania major lysate proteins were evaluated
for myoinhibitory activity on hindgut preparations of
Fig. 1. Inhibition of sand Xy hindgut contractions 5 min after applica-
tion of L. major proteins (10–100 g/ml) from parasites and precipi-
tated culture medium, harvested at 1–7 days of culture growth. Each
bar represents the mean of four trials.
0
1
2
3
4
5
6
7
8
1 2 3 4 5 6 7
Days
Percentinhibition
permicrogramproteins
0.0E+00
5.0E+06
1.0E+07
1.5E+07
2.0E+07
2.5E+07
Parasitesperml
Inhibition from parasite lysate
Inhibition from culture medium
Parasites per ml

4. 126 R. Vaidyanathan / Journal of Invertebrate Pathology 87 (2004) 123–128
P. americana, Psychoda sp., A. aegypti, L. sericata, S.
longipalpa, and R. prolixus. Hindguts of second instar P.
americana stopped contracting immediately upon appli-
cation of 65 g/ml L. major proteins. P. americana hind-
guts rinsed in CBS and returned to fresh CBS resumed
contracting (Fig. 2). Between 1 and 13% inhibition per
microgram L. major proteins (1.2–24 g/ml) was detected
against hindgut preparations of all insects tested (Fig. 3).
Highest activity per microgram lysate proteins was seen
in hindgut preparations of the sand Xies P. papatasi
(13%) and L. longipalpis (8.3%), both enzootic vectors of
Leishmania parasites. P. papatasi is the enzootic vector
of L. major; L. longipalpis, a South American sand Xy
species, transmits L. b. braziliensis, a peripylarian para-
site which has a stage of development in the hindgut
before establishing a midgut infection (Lainson and
Shaw, 1979).
To test for general inhibition against spontaneous vis-
ceral muscle contractions, L. major lysate proteins were
applied to dorsal blood vessels of Sarcophaga sp. and oviducts of A. aegypti. L. major proteins at a concentra-
tion of 24 g/ml immediately inhibited spontaneous con-
tractions of oviduct (59%) and dorsal blood vessel (22%)
preparations (Fig. 4). Surprisingly, in assays with A.
aegypti, inhibition of spontaneous oviduct contractions
was greater than inhibition of hindgut contractions.
The protein’s mode of action might be deduced from
these bioassays with insect visceral muscle. The myoin-
hibitory protein likely exerts its eVect through a com-
mon pathway of receptor–ligand interactions. The
enteric nervous system in arthropods releases neuropep-
tides which regulate visceral muscle activity (Coast and
Webster, 1998). Insect visceral muscle contraction and
relaxation are mediated by Ca2+
(Cook et al., 1975; Wil-
cox and Lange, 1995). Myosuppressins and other
myoinhibiting peptides in insects function by blocking
voltage-gated and ligand-gated Ca2+
channels in the
plasma membrane (Orchard et al., 1997). The Wrst
inhibitory neuropeptide isolated from an insect was
leucomyosuppressin, which inhibited spontaneous
contractions of the cockroach hindgut (Holman et al.,
1986). The tetrapeptides Phe–Met–Arg–Phe–amide
(FMRF-amide) and Phe–Leu–Arg–Phe–amide (FLRF-
amide) stimulate and inhibit cardiac and skeletal muscle
contractions in invertebrates (Cuthbert and Evans,
1989). FLRF-amides isolated from Locusta migratoria
inhibit locust heart rhythm, reduce spontaneous oviduct
contractions, and relax basal tone and amplitude of
hindgut contractions (Schoofs et al., 1993). Many other
insect neuropeptides interfere with contractions of gut
muscle and of dorsal blood vessel (Bendena et al., 1997;
Vilaplana et al., 1999).
Leishmania major lysates inhibited hindgut contrac-
tions of eight insect species and oviduct and dorsal blood
vessel contractions in Diptera. This conserved activity in
a spectrum of insects and insect organs may operate by
interfering with Ca2+
Xux in target tissues by blocking
Fig. 2. Reversible inhibition of P. americana hindgut contractions with
application of 65 g/ml L. major lysate proteins. Lysate was added to
hindgut preparations at 7 min, and allowed to act for 4 min. The hind-
gut was rinsed with CBS and returned to fresh saline at 11 min. Mean
values for Wve assays are shown.
Fig. 3. Means and standard deviations of percent inhibition by L.
major lysate proteins on isolated hindguts of diVerent insect species.
Protein concentrations varied among samples (1.2–24 g/ml), so all
values are standardized by percent inhibition per microgram lysate
proteins.
0
2
4
6
8
10
12
14
P. papatasi L. longipalpis Psychoda R. prolixus L. sericata S. longipalpa A. aegypti
Percentinhibitionpermicrogramproteins
Fig. 4. Reversible inhibition of spontaneous contractions of A. aegypti
oviduct (OVI) and Sarcophaga dorsal blood vessel (DBV) with appli-
cation of 24 g/ml L. major lysate proteins. Lysate was added to dis-
sected Xies at 6 min, and allowed to act for 4 min. Dissections were
rinsed with ABS at 10 min and allowed to recover. Means and stan-
dard deviations for three assays are shown.
0
25
50
75
100
1 3 5 7 9 11 13 15
Minutes
Percentinhibitionpermicrog.proteins
DBV
OVI

5. R. Vaidyanathan / Journal of Invertebrate Pathology 87 (2004) 123–128 127
Ca2+
channels and decreasing Ca2+
-dependent action
potentials, a mechanism shared by myotropic neuropep-
tides of insects. Further work is necessary to identify the
exact mode of action.
3.3. Myoinhibitory activity in diVerent kinetoplastid
parasites
Pathogenic eVects of trypanosomatids in their insect
hosts are well-documented (Schaub, 1992, 1994). Lysate
proteins (100 g/ml) of L. seymouri, L. donovani, L. b.
braziliensis, and L. major inhibited P. papatasi hindgut
contractions in increasing order of eVect (Fig. 5). The
Sudanese strain of L. donovani inhibited 20% of P.
papatasi hindgut contractions, and this eVect was
reversible (results not shown). The Indian strain had no
eVect. Growth media composition may profoundly
aVect enzyme secretion by trypanosomatid parasites
(Schlein and Jacobson, 1992). EVect of growth medium
likely explains diVerences in myoinhibitory expression.
Cultures of DMEM supplemented with 10% FCS were
used for all parasite species, and this could have inhib-
ited or masked paralytic peptide expression in diVerent
protozoa. Another explanation may be that diVerent
parasites have adapted to diVerent barriers in their
respective hosts, and testing these species with P. papat-
asi does not reXect their potential for persistence and
transmission. Further studies should include culturing
L. b. braziliensis in diVerent media and testing parasite
lysate on hindgut contractions of the enzootic vector,
L. longipalpis.
Herpetomonas muscarum lysate proteins (60 g/ml)
were tested on spontaneous contractions of hindgut and
oviduct in the natural host Musca domestica, but no
eVect was detected (results not shown). Lysate proteins
of C. fasciculata and H. muscarum were controls for
other insect hindgut, oviduct, and dorsal blood vessel
preparations. They did not inhibit muscle contractions
in any insect assay. These two protozoan species are
obligate gut parasites of mosquitoes and house Xies,
respectively. Both parasites are monoxenous; they exist
only in their insect hosts and are not transmitted to ver-
tebrates, unlike Leishmania spp. which alternate between
insect and vertebrate hosts. These parasites bind to the
insect gut and are not free in the lumen. Thus, they likely
employ mechanical adaptations such as Xagellar hemi-
desmosomes or interdigitating Xagella between micro-
villi (Brooker, 1971; Romeiro et al., 2003) to persist in
the insect gut.
Leptomonas seymouri was the only non-Leishmania
kinetoplastid in which myoinhibitory activity was
detected (»5%). L. seymouri is an obligate monoxenous
parasite of the cotton stainer bug Dysdercus suturellus
(Hemiptera) (Wallace, 1977). There is little information
on D. suturellus gut physiology and this protozoan, and
it is diYcult to speculate how myoinhibition of host gut
contractions may beneWt the parasites (Romeiro et al.,
2003).
Acknowledgments
I am grateful for the guidance and support of Y.
Schlein, J. Shlomai, and A. Warburg at the Kuvin Centre
for the Study of Infectious and Tropical Diseases, Had-
assah Medical School, Jerusalem. L. Schnur provided
seed cultures for all parasites. R.L. Jacobson helped in
culturing parasites.
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