A chemotactic response facilitates mosquito salivary gland infection by malaria sporozoites
Biochemical and Biophysical Parasitology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12735 Twinbrook Parkway, Bethesda, MD 20892-8132, USA
* Author for correspondence (e-mail: jdvorak{at}niaid.nih.gov)
Accepted 15 June 2006
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Summary |
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Key words: malaria, sporozoite, salivary gland, chemotaxis, mosquito
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Introduction |
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Histologically, sporozoites have been found throughout the mosquito
hemocoel (Garnham, 1966;
Golenda et al., 1990
;
Mayer, 1920
;
Mülens, 1921
;
Wenyon, 1926
). These data
imply that sporozoite migration to salivary glands may be a passive process
involving normal hemolymph circulation. However, it is still unclear whether
sporozoites randomly delivered to salivary glands are able to detect the
presence of the salivary glands during their circulation through the
hemolymph. Several histological studies have shown that sporozoites are most
frequently associated with the salivary glands and occasionally in spaces
between but not directly on thoracic muscles
(Golenda et al., 1990
;
Mayer, 1920
;
Mülens, 1921
;
Oelerich, 1967
). These
findings were confirmed and quantified using an enzyme-linked immunosorbent
assay for a circumsporozoite antigen, which covers the entire surface of
sporozoites (Robert et al.,
1988
). Therefore, the suggestion by Oelerich
(1967
) that once in the
vicinity of the salivary glands, the location and invasion of the salivary
glands may involve short-range chemotactic interactions is relevant.
Chemotaxis is used by many organisms, such as bacteria and leukocytes, to
detect environmental stimuli (Devreotes and
Zigmond, 1988
; Taylor et al.,
1999
). Although the involvement of chemotaxis has not been
demonstrated, it is possible that the successful invasion of salivary glands
by sporozoites is facilitated by a chemotactic attraction at the surface of
the salivary glands to aid physical contact with a ligandreceptor
binding substance.
Sporozoite motility was first observed by Vanderberg
(1974). He showed that
Plasmodium berghei sporozoites in medium exhibit a unidirectional
circular gliding motion. However, the experimental setup was not designed to
reveal any net forward motion. The recent development of green fluorescent
protein (GFP)-expressing P. berghei sporozoites
(Natarajan et al., 2001
) has
resulted in improved methods to study sporozoite motility. For example,
sporozoite motility within salivary gland ducts
(Frischknecht et al., 2004
) and
skin (Vanderberg and Frevert,
2004
) have been demonstrated. However, sporozoite motility in the
hemocoel in proximity to the salivary glands has not been observed because the
thorax containing the salivary glands is covered with a thick, optically
opaque chitinized cuticle. Therefore, we used GFP-expressing sporozoites
suspended in Matrigel to establish and maintain a three-dimensional chemical
gradient (Lehmann et al.,
1995
), low-light-level video microscopy, precise temperature
control and computer-based data acquisition for an in vitro assay to
determine whether sporozoites display a chemotactic response to salivary gland
products. The results of our study are reported here.
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Materials and methods |
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Sporozoite preparation
We isolated sporozoites at 1419 days post-infection by two methods.
To determine if sporozoite motility is similar in both medium alone and in
Matrigel, we isolated sporozoites in infected salivary glands as described
previously (Vanderberg, 1974)
except that we used a 10 µl Hamilton syringe to disrupt the salivary
glands. Liberated sporozoites were suspended in assay medium consisting of
Medium 199 (Biosource, Rockville, MD, USA) containing 1.5% bovine serum
albumin (Sigma-Aldrich Co., St Louis, MO, USA) and 3 mg ml1
glucose, and either placed on a microslide and sealed with a coverglass or
mixed with Matrigel.
We also used the centrifugation method to purify sporozoites from whole
mosquito bodies (Ozaki et al.,
1984). Infected mosquitoes were anesthetized with CO2,
their legs and wings removed and their neck incised. The mosquito body parts
were suspended in approximately 200 µl of assay medium and layered over an
approximately 100 µl volume of glass wool in a 0.5 ml microcentrifuge tube,
which had an 900 µm diameter hole in its apex made with a hot 20-gauge
needle. The tube was placed inside a 1.5 ml microcentrifuge tube and the
assembly was centrifuged at 16 000 g for 3 min. Purified
sporozoites were collected as a pellet from the bottom of the 1.5 ml
microcentrifuge tube. The pellet was disbursed in assay medium and mixed with
fluorescent polystyrene microsphere beads (Fluorespheres, 10 µm in
diameter, normalized 1/8 brightness, Molecular Probes, Eugene, OR, USA) for
some assays.
To isolate salivary gland-derived sporozoites, we removed infected salivary glands from the mosquito body, suspended them in 15 µl of assay medium, and disrupted them by passage through a 10 µl Hamilton syringe. To isolate sporozoites from hemocoels and oocysts, we used the mosquito body but excluded the salivary glands. All sporozoites harvested either from isolated salivary glands or from hemocoels and oocysts were concentrated by centrifugation as described above.
The osmolarities of all assay solution components were measured in a precalibrated vapor pressure osmometer (Model 5500, Wescor, Logan, UT, USA).
Preparation of mosquito organ homogenates
Eight pairs of salivary glands harvested from 310 day-old An.
stephensi mosquitoes were suspended in 10 µl of assay medium and
homogenized by sonication (30 times, 35 s total) at an output power of 8 using
a Model 450 Sonifier (Branson Ultrasonics Corp., Danbury, CT, USA). As a
control, some salivary gland homogenates were heated in a water bath at
56°C for 30 min. This procedure was also used to produce midgut and
Malpighian tubule homogenates.
Data collection and analyses
To observe sporozoites in the hemolymph circulation, live mosquitoes were
attached to a microscope slide using superglue (Axis Electronics, Inc.,
Damascus, MD, USA). For chemotaxis assays, we used custom-built chambers
(Fig. 1A) to observe and
quantify sporozoite movement. Two 9 mmx18 mm No. 1 coverglasses were
adhered to a 70 mmx25 mm glass slide with a thin layer of silicon oil,
leaving an approximately 5 mm wide gap between them. An 18 mmx18 mm No.
1 coverglass adhered with silicon oil bridging the two 9 mmx18 mm
coverglasses was used to form a 150 µm thick observation chamber. The
chamber capacity was approximately 3.5 µl. Unless otherwise noted, both
sporozoites and mosquito organ homogenates in assay medium were mixed quickly
at 4°C with Matrigel (Beckton Dickinson Labware, Bedford, MA, USA)
individually at a final ratio of 67% assay medium to 33% Matrigel, and placed
in the chamber. To prevent evaporation and exclude air bubbles from the
chamber, silicon oil was added to fill the void remaining in the chamber,
which was further sealed with high vacuum grease (Dow Corning, Midland, MI,
USA).
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The microscope slide or the chamber was placed on a custom-built thermostated stage incorporating two Peltier elements. Temperature was maintained at 2021°C with the combination of a Model 5000 Microincubator (20/20 Technology, Inc., Wilmington, NC, USA) and a Hoefer Model RCB-300 refrigerated circulating bath (Hoefer Scientific Instruments, San Francisco, CA, USA) and monitored by a YSI Model 408 thermistor and a YSI Model 4000A Precision Thermometer (Yellow Springs Instrument Co., Inc., Yellow Springs, OH, USA).
A Leica DM IRE2 inverted light microscope (Leica Microsystems, Inc., Bannockburn, IL, USA) equipped with a Märzhäuser motorized stage (Märzhäuser, Wetzler, Germany), 20x and 100x HC PL FLUORTAR objectives and a GFP fluorescence filter module (Model XF100-2, Omega Optical, Brattleboro, VT, USA) was used to visualize the sporozoites. Microscope images were projected into a Hamamatsu Model ICCD-2400 low-light-level intensified video camera (Hamamatsu, Bridgewater, NJ, USA), captured using a DT3155 image capture card (Data Translation, Marlboro, MA, USA) and stored as AVI files. To avoid radiation-induced damage to the GFP-expressing sporozoites, a mercury lamp was not used; the preparations were illuminated with a halogen light source operating at 2.5 to 7.2 V.
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Equally distributed distributions were tested for statistical significance using a two-sample t-test. If the samples were not equally distributed as determined by an F-test, we used a two-sample t-test with Welch's correction.
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Results |
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Sporozoites motility is modulated by vehicle
Sporozoites isolated and suspended in medium as described previously
(Vanderberg, 1974) showed
circular gliding motion at a velocity of 2 µm s1 and
attached waving motion (Fig. 3
and Movie 2 in supplementary material). In contrast to the previous report, we
found that the proportions of sporozoites circling clockwise or anticlockwise
were essentially identical. However, neither circular gliding nor attached
waving motion resulted in any net forward motion.
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To determine if forward motility could be promoted in a three-dimensional environment, we isolated sporozoites by the same method and suspended them in Matrigel in our custom-built chamber. In this environment, sporozoites exhibited a corkscrew-like forward motion with frequent random directional changes (Fig. 4A). Since sporozoites exhibited directional motility in Matrigel in addition to circular gliding motility, we mixed Matrigel with sample for all experiments to analyze sporozoite motility.
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Sporozoite movement is modulated by the presence of salivary gland homogenate
To determine whether salivary gland homogenate (SGH) influenced the
direction of sporozoite movement, we quantified motility in the presence of
either fresh or heated SGH. Sporozoites collected from whole bodies of
infected mosquitoes were suspended in Matrigel together with 10 µm diameter
fluorescent beads as fiducial points and either unheated or heated SGH. In a
typical experiment, a total of 21 out of 22 sporozoites (95.5%) moved toward
unheated SGH (Fig. 7A, Movie 3
in supplementary material) with =77.5±15.7° (N=21,
Table 1). In contrast,
sporozoites in the presence of heated SGH moved randomly
(Fig. 7B) with
=58.1±127.1° (N=20,
Table 1). Sporozoite movement
in the presence of SGH was relatively uniform with time, compared to
sporozoites in the absence of SGH (Fig.
4A,B). The difference in
between sporozoites in the
presence of SGH vs heated SGH was significant (P<0.001,
two-sample t-test with Welch's correction), demonstrating that the
majority of the sporozoites isolated from whole mosquito bodies were attracted
to unheated SGH. Mosquito organ extracts of midguts and Malpighian tubules did
not attract sporozoites (data not shown). The fluorescent beads remained
stationary in all cases, demonstrating that no passive fluid streaming
occurred. To further confirm the lack of passive fluid movement, the
osmolarities of all solutions were determined
(Table 2). There was no
significant difference in osmolarity between SGH and sporozoites suspended in
Matrigel (P>0.4, two-sample t-test), demonstrating that
solution osmolarity differences were not responsible for the directional
motility of sporozoites.
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Mosquito isolation site modulates sporozoite movement
Sporozoites in whole mosquito bodies on days 1419 post-infection
were present in both salivary glands and hemolymph plus oocysts (data not
shown). To determine whether the attraction of sporozoites to SGH is dependent
upon the site from which the sporozoites were isolated, we compared the
motility of salivary gland-derived sporozoites to sporozoites derived from
hemocoels and oocysts. Salivary gland-derived sporozoites moved randomly in
the presence of SGH (Fig. 8A)
with =32.5±97.4° (N=12,
Table 1). In contrast, 15
hemocoel- and oocyst-derived sporozoites out of 19 (78.9%) were attracted to
salivary gland homogenate (Fig.
8B) with
=71.8±34.0° (N=15,
Table 1). The difference in
between salivary gland-derived vs hemocoel- and
oocyst-derived sporozoites was significant (P<0.005, two-sample
t-test with Welch's correction).
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Discussion |
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We demonstrate that sporozoites do not exhibit directed movement toward
heated salivary gland homogenate. These data imply that chemoattractant
factors may be denatured or inactivated by heating (56°C for 30 min),
suggesting that they may be high molecular mass protein(s) or
carbohydrate-binding protein(s). High molecular mass protein(s) are usually
denatured (Shin et al., 2002)
and carbohydrate-binding proteins lose sugar-binding activity
(Gaikwad et al., 2002
) when
heated under the conditions we used. High molecular mass glycoproteins could
be secreted from the basal lamina into the extracellular space
(Mariassy et al., 1989
). Also,
it has been shown that P. gallinaceum sporozoites recognize lectins
when they invade the salivary glands of Aedes aegypti mosquitoes
(Barreau et al., 1995
).
Consequently, these sporozoites may chemotactically detect secreted
carbohydrate-binding proteins and a similar mechanism may exist for P.
berghei sporozoites.
We also demonstrate that there is a marked difference in chemoattraction
between salivary gland-derived sporozoites and oocyst- and hemolymph-derived
sporozoites. Salivary gland-derived sporozoites were not attracted to salivary
gland homogenates. In contrast, the majority (80%) of oocyst- and
hemolymph-derived sporozoites are attracted to salivary gland homogenate.
These data can be explained by the fact that once sporozoites enter salivary
glands they no longer need to locate the salivary glands and may lose their
chemotactic ability quickly after invasion, possibly through the saturation of
chemoattractant receptors. The remaining 20% did not show directed
motility. This is probably the consequence of the heterogeneous maturation
state of the oocyst- and hemolymph-derived sporozoite population. It has been
shown that sporozoite mobility, infectivity of the vertebrate host, and the
ability to invade salivary glands change during their maturation
(Touray et al., 1992
;
Vanderberg, 1974
;
Vanderberg, 1975
) in a
time-dependent manner (Al-Olayan et al.,
2002
). The subset of the population that did not display directed
motility in the presence of salivary gland homogenate is probably immature
with respect to the development of a chemotactic response.
In conclusion, we demonstrate that sporozoites locate mosquito salivary glands by chemotaxis, suggesting the possibility that chemical component(s) can be identified and synthesized to block or suppress mosquito salivary gland invasion as a malaria transmission blocking strategy.
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Acknowledgments |
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Footnotes |
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References |
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Al-Olayan, E. M., Beetsma, A. L., Butcher, G. A., Sinden, R. E.
and Hurd, H. (2002). Complete development of mosquito
phases of the malaria parasite in vitro. Science
295,677
-679.
Barreau, C., Touray, M., Pimenta, P. F., Miller, L. H. and Vernick, K. D. (1995). Plasmodium gallinaceum: sporozoite invasion of Aedes aegypti salivary glands is inhibited by anti-gland antibodies and by lectins. Exp. Parasitol. 81,332 -343.[CrossRef][Medline]
Devreotes, P. N. and Zigmond, S. H. (1988). Chemotaxis in eukaryotic cells: a focus on leukocytes and Dictyostelium.Annu. Rev. Cell Biol. 4,649 -686.[CrossRef][Medline]
Frischknecht, F., Baldacci, P., Martin, B., Zimmer, C., Thiberge, S., Olivo-Marin, J. C., Shorte, S. L. and Menard, R. (2004). Imaging movement of malaria parasites during transmission by Anopheles mosquitoes. Cell Microbiol. 6, 687-694.[CrossRef][Medline]
Gaikwad, S. M., Gurjar, M. M. and Khan, M. I.
(2002). Artocarpus hirsuta lectin. Differential modes of
chemical and thermal denaturation. Eur. J. Biochem.
269,1413
-1417.
Garnham, P. C. C. (1966). Malaria Parasites and Other Hemosporidia. London: Blackwell.
Golenda, C. F., Starkweather, W. H. and Wirtz, R. A. (1990). The distribution of circumsporozoite protein (CS) in Anopheles stephensi mosquitoes infected with Plasmodium falciparum malaria. J. Histochem. Cytochem. 38,475 -481.[Abstract]
Kirk, K., Horner, H. A. and Kirk, J. (1996). Glucose uptake in Plasmodium falciparum-infected erythrocytes is an equilibrative not an active process. Mol. Biochem. Parasitol. 82,195 -205.[CrossRef][Medline]
Lehmann, T., Cupp, S. M. and Cupp, W. E. (1995). Chemical guidance of Onchocerca lienalis microfilariae to the thorax of Simulium vittatum.Parasitology 110,329 -337.[Medline]
Mariassy, A. T., McCray, M. N., Lauredo, I. T., Abraham, W. M. and Wanner, A. (1989). Lectin-detectable effects of localized pneumonia on airway mucous cell populations: role of cyclooxygenase metabolites. Exp. Lung Res. 15,113 -137.[Medline]
Mayer, M. (1920). Über die Wanderung der Malariasichelkeime in den Stechmücken und die Möglichkeit der Uberwinterung in diesen. Medizinische Klinik 16,1290 -1291.
Mülens, P. (1921). Das Verhalten der Malaria Sporozoiten in der Anophelesmücke. Archiv. für Schiffs-und Tropen-Hygiene 25,58 -61.
Natarajan, R., Thathy, V., Mota, M. M., Hafalla, J. C., Menard, R. and Vernick, K. D. (2001). Fluorescent Plasmodium berghei sporozoites and pre-erythrocytic stages: a new tool to study mosquito and mammalian host interactions with malaria parasites. Cell Microbiol. 3,371 -379.[CrossRef][Medline]
Oelerich, S. (1967). Vergleichende Untersuchungen über das Auftreten von Malaria-sporozoiten in den Speicheldrüsen und in den übgrigen Organen der Mucke. Zeitschr. Tropen. Med. Parasitol. 18, 285.
Ozaki, L. S., Gwadz, R. W. and Godson, G. N. (1984). Simple centrifugation method for rapid separation of sporozoites from mosquitoes. J. Parasitol. 70,831 -833.[Medline]
Robert, V., Verhave, J. P., Ponnudurai, T., Louwe, L., Scholtens, P. and Carnevale, P. (1988). Study of the distribution of circumsporozoite antigen in Anopheles gambiae infected with Plasmodium falciparum, using the enzyme-linked immunosorbent assay. Trans. R. Soc. Trop. Med. Hyg. 82,389 -391.[CrossRef][Medline]
Shin, I., Wachtel, E., Roth, E., Bon, C., Silman, I. and Weiner,
L. (2002). Thermal denaturation of Bungarus
fasciatus acetylcholinesterase: Is aggregation a driving force in protein
unfolding? Protein Sci.
11,2022
-2032.
Taylor, B. L., Zhulin, I. B. and Johnson, M. S. (1999). Aerotaxis and other energy-sensing behavior in bacteria. Annu. Rev. Microbiol. 53,103 -128.[CrossRef][Medline]
Touray, M. G., Warburg, A., Laughinghouse, A., Krettli, A. U.
and Miller, L. H. (1992). Developmentally regulated
infectivity of malaria sporozoites for mosquito salivary glands and the
vertebrate host. J. Exp. Med.
175,1607
-1612.
Vanderberg, J. P. (1974). Studies on the motility of Plasmodium sporozoites. J. Protozool. 21,527 -537.[Medline]
Vanderberg, J. P. (1975). Development of infectivity by the Plasmodium berghei sporozoite. J. Parasitol. 61,43 -50.[Medline]
Vanderberg, J. P. and Frevert, U. (2004). Intravital microscopy demonstrating antibody-mediated immobilisation of Plasmodium berghei sporozoites injected into skin by mosquitoes. Int. J. Parasitol. 34,991 -996.[CrossRef][Medline]
Wenyon, C. M. (1926). Protozoology. In A Manual for Medical Men, Vetenarians and Zoologists. London. Balliére, Tindall and Cox.
Wernsdorfer, W. (1980). The Importance of Malaria in the World. In Malaria. Vol.1 (ed. J. Kreier), pp. 1-93. New York: Academic Press.
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