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ABSTRACT Hyaluronidase activity in the salivary gland homogenates of Simulium vittatum. (Zetterstedt) is described, and its optimal pH determined. Salivary ...

Simulium vittatum (Diptera: Simuliidae) and Lutzomyia longipalpis (Diptera: Psychodidae) Salivary Gland Hyaluronidase Activity JOSE´ M. C. RIBEIRO,1 ROSANE CHARLAB,1 EDGAR D. ROWTON,2



J. Med. Entomol. 37(5): 743Ð747 (2000)

ABSTRACT Hyaluronidase activity in the salivary gland homogenates of Simulium vittatum (Zetterstedt) is described, and its optimal pH determined. Salivary activity was reduced signiÞcantly after a blood meal, indicating that it was secreted after blood feeding. Phlebotomus papatasi (Scopoli) also exhibited salivary hyaluronidase activity. These results indicate that hematophagous poolfeeding insects may secrete this enzyme to help the spread of salivary antihemostatic agents in the vicinity of the feeding lesion, and perhaps to increase the size of the feeding lesion itself. Additionally, this enzyme may affect local host immune reactions and promote arboviral transmission. KEY WORDS Simulium vittatum, Phlebotomus papatasi, salivary glands, hyaluronidase, hematophagy, blood feeding

HYALURONIC ACID IS a mucopolysaccharide consisting of repeating units of glucuronic acid and N-acetyl-glucosamine, achieving a molecular weight of many hundreds of thousands to over one million daltons. It is abundant in connective tissues, such as the subcutaneous structures of the vertebrate integument, where it confers most of the gel-like structure to this tissue, including its permeability characteristics (Csoka et al. 1997). Perhaps to increase their success in invading their hosts, some pathogenic bacteria produce a virulence factor originally described as “spreading factor” found also in mammalian testes (Duran-Reynals 1928), and later identiÞed as a hyaluronidase (Chain et al. 1940). Although hyaluronidases are found in blood sucking leeches (Budds et al. 1987) and in Ancylostoma worms (Hotez et al. 1992), in addition to being common in arthropod venoms (Csoka et al. 1997), their occurrence in blood-sucking arthropods is not well documented. They are present in the saliva of a tick (Neitz et al. 1978), and in the salivary glands of Lutzomyia longipalpis (Lutz & Neiva), from where a cDNA with high similarity to hyaluronidases also was found (Charlab et al. 1999). Sand ßies and ticks feed from skin hemorrhages, rather than from canulating blood vessels, as, for example, do most triatomine bugs and mosquitoes (Lavoipierre 1964). Hyaluronidase may help diffusion of antihemostatic agents into the vicinity of the feeding wound or help to enlarge the size of the feeding hematoma. 1 Section of Medical Entomology, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 4, Room 126, 4 Center Drive, MSC-0425, Bethesda, MD 20892Ð 0425. 2 Department of Entomology, Walter Reed Army Institute of Research, Washington, DC 20307Ð5100. 3 Department of Entomology, 301 Funchess Hall, Auburn University, AL 36849 Ð5413.

In the current article we demonstrate the existence of hyaluronidase activity in the salivary glands of the black ßy Simulium vittatum (Zetterstedt) and the sand ßy Phlebotomus papatasi (Scopoli). Because sand ßies and black ßies are vectors of arboviruses, we discuss the implications of a salivary hyaluronidase for the transmission of vesicular stomatitis virus, particularly by the phenomenon of co-feeding transmission (Mead et al. 2000), and the modulation of the local host immune response.

Materials and Methods Hyaluronic acid (HA, from human umbilical cord, Sigma catalog H-1504, Sigma, St. Louis, MO) was diluted to 10 mg/ml with water, shaken for 4 h at room temperature, and stored at Ð20⬚C. Water was of 18 M⍀ quality and obtained with a MilliQ apparatus from Millipore (Bedford, MA). Black ßies were reared using standard procedures (Bernardo et al. 1986). Adult S. vittatum females were maintained on a sucrose diet ad libitum for 5Ð 8 d and then transferred to a distilled water diet for 1 d before blood-feeding. Cages containing 5Ð10 ßies were offered a blood meal by exposure to the volar surface of a human arm. Protocols were approved by the institutional review board of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. Flies were dissected 5Ð20 min after engorgement. Before salivary gland recovery during dissection, the midgut was removed and examined for fresh blood. If more than one-half of this organ was distended with blood, then the salivary glands were dissected immediately in chilled HEPES saline (10 mM Hepes, 150 mM NaCl, pH 7.4). Six glands were placed in each Nunc vial containing 10 ␮l of HEPES saline and then snap-frozen at ⫺70⬚C.



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Fig. 1. Time course of hyaluronic acid hydrolyzed by salivary homogenates of the black ßy S. vittatum. The reaction mixture consisted of Þve homogenized salivary glands in 1 ml of 50 mM sodium phosphate buffer pH 7.0 containing 50 ␮g/ml hyaluronic acid and 0. 1 mg/ml bovine serum albumin. Aliquots of 50 ␮l were used to measure residual HA at the indicated time points. The inset shows a standard curve of HA. Symbols and bars represent the mean ⫾ SEM of triplicate measurements. Some SEM are smaller than the symbol sizes.

Sand ßies were reared at the Walter Reed Army Medical Research Institute on a fermented mixture of rabbit chow and rabbit feces as described previously (Modi and Tesh 1983). Adult sand ßies were kept with free access to a 10% solution of sucrose unless otherwise speciÞed. Salivary glands of adult female sand ßies were dissected and transferred to 1.5-ml polypropylene vials, usually in groups of 20 pairs of glands in 20 ␮l of HEPES saline. Salivary glands were kept at

Ð75⬚C until needed, when they were disrupted by sonication using a Branson SoniÞer 450 homogenizer (Danbury, CT), (Ribeiro et al. 1999). Aedes aegypti (L.) (Liverpool strain) and Anopheles gambiae (Giles) (G3 strain) were reared in the insectary of the Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases. Adult mosquitoes were kept on a diet of diluted (10%) Karo syrup (Best Foods, Englewood Cliffs, NJ). Salivary

Fig. 2. Hydrolysis of hyaluronic acid by different concentrations of salivary homogenate from S. vittatum obtained before or after a blood meal. Experiments represent the average and SEM of Þve pools of salivary glands (each containing 10 or more salivary glands) for unfed ßies, and seven pools for blood-fed ßies, incubated for 60 min at 37⬚C. Other reaction mixture conditions are similar to those described in Fig. 1.

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Fig. 3. pH dependence of the salivary hyaluronidase of Simulium vittatum. (A) Hydrolysis as a function of different pH values and different amounts of salivary gland homogenates. (B) Hyaluronic acid clearance in the presence of 0.25 homogenized salivary gland, when incubated for 60 min at 37⬚C. At pH 4 and 5, 50 mM sodium acetate was used. At other pH values, 50 mM sodium phosphate was used. Other conditions are as in Fig. 1.

glands from 3- to 7-d-old adult female mosquitoes were dissected and stored as described above for sand ßies. Hyaluronidase assays were performed by the turbidimetric method resulting from the interaction of the cationic detergent cetylpyridinium chloride (CTACl) with HA (Ramanaiah et al. 1990), modiÞed for 96-well microtiter plates (Charlab et al. 1999). The reaction mixture, with a Þnal volume of 50 ␮l, consisted of 50 mM sodium phosphate pH 7.0 (or 50 mM of another buffer as indicated) containing 50 ␮g/ml HA and the indicated amount of homogenized salivary gland pairs. Bovine serum albumin (BSA) (0.1 mg/ml Þnal concentration) was added to prevent adsorption and possible inactivation of salivary enzymes, and as a

hyaluronidase activator (Maingonnat et al. 1999). All concentrations indicated are Þnal concentrations. The reaction was started by addition of substrate, incubated at 37⬚C for the indicated amount of time, and stopped by the addition of 100 ␮l of 10 mg/ml CTACl dissolved in 1% NaOH immediately before the assay. The plate was stirred for 15 s, and the absorbance at 405 nm measured after 3 min using a ThermoMax plate reader (Molecular Devices, Menlo Park, CA). The readings were stable 3Ð5 min after CTACl was added. Blanks not containing HA, as well as not containing enzyme, were run together with every assay, as were different concentrations of HA to create a standard curve (with 0, 10, 20, 30, 40, and 50 ␮g/ml HA). The CTACl reagent develops a yellow coloration after dis-



Vol. 37, no. 5

solution in NaOH, and for this reason it must be prepared immediately before each assay. Statistical tests (MannÐWhitney rank sum tests) were performed using SigmaStat (Jandel ScientiÞc 1995).

Results and Discussion To test for the ability of S. vittatum salivary gland homogenates to hydrolyze HA, homogenates were incubated with HA and the remaining HA measured at discrete time points after the initiation of the reaction. The standard curve for measuring HA is linear in the range of 0.5Ð2.5 ␮g of HA (Fig. 1, inset). When salivary homogenate was incubated with HA, a lag was observed in the resulting curve, after which the reaction proceeded linearly within the conditions of the assay (Fig. 1). This lag phase was observed in several experiments with S. vitattum and P. papatasi (see below), and possibly results from the initial cleavage of HA into relatively large pieces that continue to give turbidity when mixed with the cationic detergent. Because of the lag observed in the hyaluronidase reaction of S. vittatum, several time measurements of the enzyme activity were taken to properly estimate enzyme activity, using only the linear portion of the curve. Alternatively, for a more convenient single time of incubation, several concentrations of the enzyme source could be used. In this method, one unit of enzyme activity was quantiÞed as the amount of enzyme necessary to clear 50% of the measured HA within the speciÞed assay conditions of concentrations and temperature of incubation. Accordingly, the enzyme activity was measured in salivary homogenates of S. vittatum maintained on distilled water for 24 h or from ßies fully blood fed within 5Ð20 min (Fig. 2). Salivary hyaluronidase activity was reduced signiÞcantly from 4.9 ⫾ 1.1 to 1.7 ⫾ 0.3 U per gland (MannÐWhitney rank sum test P ⫽ 0.018) after a blood meal, indicating that most likely the activity was secreted from the glands during the blood meal. The salivary hyaluronidase activity in S. vitattum had an optimum activity at pH 6.0, substantial activity was observed at pH 5.0 and 7.0. No activity was detected at pH 4.0 or pH 8.0 (Fig. 3). This result contrasts with those observed for other hyaluronidases, which have optima at or below pH 5 (Yang and Srivastava 1975, Harrison 1988, Maingonnat et al. 1999). To explore further the range of salivary hyaluronidase in blood sucking Diptera, salivary homogenates of P. papatasi, Ae. aegypti, and An. gambiae were incubated with HA. Homogenates of P. papatasi hydrolyzed HA efÞciently, using ⬍1⁄10 of a salivary gland per assay (Fig. 4). However, salivary homogenates of the mosquitoes Ae. aegypti and An. gambiae (at one pair of salivary homogenate per 50 ␮l assay) failed to hydrolyze HA (results not shown). Because sand ßies, black ßies, ticks, and leeches mostly feed from hemorrhages rather than from blood vessels (Lavoipierre 1964), it therefore appeared that salivary hyaluronidase may be of selective advantage in the exclusive pool mode of blood feeding.

Fig. 4. Salivary hyaluronidase activity of Phlebotomus papatasi. (A) Time course of the hyaluronidase reaction using 0.3 pairs of salivary gland homogenate per assay point (B) Hyaluronidase activity as a function of added salivary gland homogenate. Results shown are the average of a duplicate experiment. Reaction media was as in Fig. 1.

Because hyaluronidase may help invasion of pathogens, black ßy and sand ßy salivary hyaluronidase may facilitate movement of certain arboviruses through the intercellular matrix of the skin, thereby promoting co-feeding transmission of viruses such as vesicular stomatitis viruses (VSV). Although the transmission dynamics of VSV is not very well understood, it has been proposed that these viruses are maintained enzootically by sand ßies (Tesh et al. 1974), with sporadic epizootics of the New Jersey serotype occurring as a result of biological transmission by black ßies, including S. vittatum (Cupp et al. 1992). VSV does not produce a measurable viremia in the vertebrate host, and it was believed that it was not arthropod-transmitted (Letchworth et al. 1999). However, uninfected

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S. vittatum females can acquire virus when feeding simultaneously or up to 48 h after ingesting blood from a nonviremic host that has been fed on by VSV-infected ßies (Mead et al. 2000). Therefore, the presence of a salivary hyaluronidase in both black ßies and sand ßies indicates that this molecule may facilitate VSV transmission by depolymerizing hyaluronic acid to create interstitial gaps between cells and allow dispersal of the virus subcutaneously. Hyaluronidase also down-regulates polyinosinic-polycytidylic acid stimulation of gamma-interferon (Romano et al. 1983), an effect that would make cells more susceptible to infection with VSV contributing to establishment of the virus at the bite site. Low molecular weight species of hyaluronic acid generated during inßammation induce chemokine (McKee et al. 1996) and nitric-oxide synthase (iNOS) (McKee et al. 1997) gene expression in macrophages. Hyaluronic acid in the native, higher molecular weight size range does not have these effects. These studies suggest a role for hyaluronic acid fragments in macrophage activation. In this context, it would be interesting to investigate whether salivary hyaluronidase contributes to the host inßammatory response to vector saliva and/or insect-borne pathogens. Acknowledgments We thank Frank Ramberg (University of Arizona, Tucson, AZ) for providing S. vittatum females and Andre Laughinghouse (Laboratory of Parasitic Diseases, NIH) for rearing mosquitoes.

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