Mosquito Phenoloxidase and Defensin Colocalize in Melanization Innate Immune Responses
Department of Animal Health & Biomedical Sciences, University of WisconsinMadison, Madison, Wisconsin
Correspondence to: Bruce M. Christensen, Department of Animal Health & Biomedical Sciences, University of WisconsinMadison, 1656 Linden Dr., Madison, WI 53706. E-mail: Christensen{at}svm.vetmed.wisc.edu
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Summary |
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(J Histochem Cytochem 53:689698, 2005)
Key Words: phenoloxidase defensin mosquito Aedes aegypti melanization innate immunity hemocyte antimicrobial peptide
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Introduction |
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The mosquito immune response elicited by invading organisms is robust and involves humoral and cellular components. The humoral component includes the phenoloxidase cascade system of parasite melanization and wound healing (Zhao et al. 1995; Lai et al. 2002
), inducible antimicrobial peptides (Lowenberger 2001
; Vizioli et al. 2001a
), and reactive oxygen and nitrogen intermediates (Luckhart et al. 1998
; Lanz-Mendoza et al. 2002
; Kumar et al. 2003
). The cellular component includes phagocytosis (Hernández-Martínez et al. 2002
; Hillyer et al. 2003a
,b
,2004
,2005
) and encapsulation (Forton et al. 1985
; Christensen and Forton 1986
) by hemocytes. Of these, two areas of intense study are phenoloxidase-based melanization and the antimicrobial peptides. Melanization involves a complex series of reactions that include the hydroxylation of tyrosine by phenoloxidase to form 3,4-dihydroxyphenylalanine (dopa) and its sequential conversion to melanin by phenoloxidase and other enzymes (Christensen et al. 2005
). This process is used by certain mosquito species and strains as a resistance mechanism against filarial nematodes and malaria (Plasmodium) by sequestering parasites inside a hardened proteinaceous capsule (Collins et al. 1986
; Beerntsen et al. 1989
). Antimicrobial peptides have been studied mostly for their antibacterial activity. They have also been shown or suggested to have anti-parasitic activity and have been hypothesized to function by triggering lysis (Lowenberger et al. 1996
,1999a
; Vizioli et al. 2001a
,b
), although this notion has been recently challenged (Bartholomay et al. 2004
).
The yellow fever mosquito, Aedes aegypti, is the natural vector of dengue virus and yellow fever virus (World Health Organization 1996; Roberts 2002
). Aside from diseases naturally transmitted by Ae. aegypti, this mosquito is often used as a laboratory model because it is easy to rear, its biology and genetics are well understood, and it serves as a model for studies relating to malaria and filarial nematode transmission (Beerntsen et al. 2000
). Furthermore, we have previously shown that Ae. aegypti mount strong immune responses against bacteria and that different bacterial species elicit different immune responses (Hillyer et al. 2003b
,2004
,2005
). For example, Gram(-) Escherichia coli are primarily phagocytosed and Gram(+) Micrococcus luteus are primarily melanized (Hillyer et al. 2003b
), but the factors triggering phagocytic vs melanization immune responses against bacteria are independent of Gram type (Hillyer et al. 2004
). In the current study, we used immunocytochemistry and transmission electron microscopy (TEM) to localize the melanization rate-limiting enzyme phenoloxidase and the antimicrobial peptide defensin in immune reactions against bacteria.
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Materials and Methods |
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Conjugation of Proteins
Protein A (Sigma) and mouse anti-rabbit IgG (Pierce Biotechnology; Rockford, IL) were conjugated to colloidal gold particles by hydrophobic bonding. Initially, concentration and pH adsorption isotherms were done to determine the optimal conditions for conjugation (Albrecht et al. 1993). Briefly, 200 µl of colloidal gold at various pHs (ranging from 5 to 9) was added to 20 µl of Protein A or mouse anti-rabbit IgG at varying concentrations (ranging from 200 µg/ml to 25 µg/ml, in water) and mixed. After 20 min, 100 µl of a saturated NaCl solution was added and mixed. After another 20 min the color of the conjugate was assessed: red/pink was indicative of a stable conjugate and blue/purple was indicative of an unstable conjugate. The colloidal gold pH and protein concentration combination that required the minimum amount of protein to stabilize the colloidal gold, plus 10% excess protein, was used to make the final conjugates. For the final conjugation of mouse anti-rabbit IgG to 12.7 nm colloidal gold, 18 ml of colloidal gold (pH 7.3) was added to 1.8 ml of 75 µg/ml mouse anti-rabbit IgG (in water). For the final conjugation of Protein A to 14.4 nm colloidal gold, 10 ml of colloidal gold (pH 5.9) was added to 1 ml of 50 µg/ml Protein A (in water). Both solutions were individually mixed and allowed to stand for 30 min. The conjugates were then spun at 10,600 x g for 8 min, the supernatant removed, and the loose pellet resuspended in storage buffer (20 mM Tris base, 20 mM NaN3, 150 mM NaCl, 0.1% BSA, pH 7.2) to one tenth the original volume.
Mosquito Rearing, Immune Challenges, and Sample Collections
Because only female mosquitoes take blood meals and, hence, are the only mosquitoes capable of transmitting diseases through infective bites, the current study focused exclusively on the immune response elicited in females. Female Ae. aegypti, Liverpool strain, were reared and maintained inside an environmental chamber at 26.5 ± 0.3C and 80 ± 5% relative humidity and adults fed ad libitum on 0.3 M sucrose-soaked cotton pads as described (Hillyer et al. 2004). E. coli K12 and M. luteus bacteria were grown separately and overnight in LuriaBertani's rich nutrient medium (10 g tryptone, 5 g yeast extract, 10 g NaCl in 1 liter H2O; Fisher Scientific, Pittsburgh, PA) inside a 37C shaking incubator until stationary phase had been reached.
To immune challenge mosquitoes, cultured bacteria at stationary phase were centrifuged at 2655 x g for 5 min and the supernatant withdrawn. A 0.15-mm steel probe was dipped in the bacterial pellet and inserted through the neck membrane of 15-day-old adult female mosquitoes (Hillyer et al. 2003b). At 1 hr and 24 hr postchallenge, hemolymph was collected by volume displacement (perfusion) directly into formaldehyde (Electron Microscopy Sciences; Hatfield, PA) and glutaraldehyde (Electron Microscopy Sciences) fixative as described (Hillyer et al. 2003a
). One min after completing mosquito hemolymph collection, the supernatant was transferred to a new tube, allowed to fix for an additional 1 hr, and centrifuged for 20 min at 210 x g. The supernatant was removed and the cellular pellet was embedded in a plug of low-melting-temperature agarose. The agarose plug was then fixed for 20 min, rinsed with buffer, cut into 1.5-mm cubes, dehydrated through 70% ethanol, and embedded in LR-White resin (Polysciences; Warrington, PA). Each sample was comprised of the pooled hemolymph of 50100 mosquitoes of the same age. A total of six sets of perfusions were done, each yielding at least two samples after cutting the agarose plugs. Furthermore, samples were prepared for 1-, 4-, and 5-day-old mosquitoes at 24-hr postchallenge and for 5-day-old mosquitoes at 1-hr postchallenge.
Labeling and Transmission Electron Microscopy
All labeling experiments were done at room temperature, and all solutions and rinses were made in storage buffer (20 mM Tris base, 20 mM NaN3, 150 mM NaCl, 0.1% BSA, pH 7.2) unless otherwise noted. One hundred-nm-thick sections were cut using a diamond knife on a Reichert Om U3 ultramicrotome (Reichert; Vienna, Austria) and transferred to 200-mesh pioloform-coated nickel grids. Sections were allowed to dry for 1030 min and rehydrated for 5 min. Sections were then blocked (5% fetal calf serum when using IgG conjugate or 1% BSA when using Protein A conjugate) for 30 min, rinsed three times for 3 min each, incubated in primary antibody [rabbit anti-defensin (Bartholomay et al. 2004) or rabbit anti-phenoloxidase (Lai et al. 2002
)] for 2 hr, rinsed three times for 3 min each, incubated in Protein Acolloidal gold or mouse anti-rabbit IgGcolloidal gold for 2 hr, rinsed three times for 3 min each, rinsed three times for 3 min each in water, and allowed to dry. Sections were then counterstained for 5 min in 1% uranyl acetate and for 3 min in Reynold's lead, and viewed with a Philips CM120 TEM (Philips Electron Optics; Eindhoven, The Netherlands) as described (Hillyer and Albrecht 1998
).
A total of 10 experiments were done, each including multiple samples, and four of the samples were independently labeled in separate trials at least twice. For all labeling experiments, serial sections for all samples were labeled for both defensin and phenoloxidase. Out of the 10 labeling experiments, 6 were done using mouse anti-rabbit IgGcolloidal gold and 4 with Protein Acolloidal gold. These experiments yielded similar results, with the exception that experiments using IgGcolloidal gold resulted in higher labeling intensities. Controls included labeling cultured bacteria (bacteria never injected into mosquitoes), omission of the primary antibody, and the use of an irrelevant antibody. Control samples were run on three of the labeling experiments with identical results. Finally, labeling experiments for samples collected at 1 hr and 24 hr postchallenge were done within days of each other and with the same reagents. Because they were not labeled on the same day, colloidal gold particles on bacterial melanotic capsules and on other regions of the section were counted, and statistical analysis (Student's t-test) was done to compare the background labeling of both samples and the specific labeling of both samples. Student's t-test was also used to compare labeling of bacteria in experimental samples vs background labeling of bacteria in control samples. Differences were deemed significant at p<0.05.
When observing hemolymph samples from bacteria-challenged mosquitoes collected by perfusion, the biological structures present are hemocytes, fat body, cellular debris from lysed cells (predominantly nuclei, mitochondria, and membranes), and bacteria (melanized and unmelanized) (Hillyer and Christensen 2002; Hillyer et al. 2003a
). For all labeling experiments, all structures were examined for the presence of colloidal gold particles.
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Results |
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Localization of Defensin
Defensin is one of the most studied antimicrobial peptides in mosquitoes. Because it has been widely speculated that defensin functions by lysing bacteria, we originally expected to see defensin bound to unmelanized bacteria. This was not the case. Similar to phenoloxidase, at 24 hr postchallenge, defensin localized to E. coli and M. luteus melanotic capsules (Figure 2). Defensin was commonly present in fully melanized bacteria (Figures 2A2C), but in a small percentage of cases defensin localized to bacteria that appeared to be in the early stages of melanization (Figure 2D). Within melanized bacteria, defensin was most often found on the melanized capsule (Figure 2A), but it was also observed in the melanized capsule and the cytosol (Figure 2B) or mostly in the cytosol (Figure 2C).
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Because defensin is not detectable in naïve mosquitoes and becomes measurable for the first time several hours following challenge (Lowenberger et al. 1999b; Bartholomay et al. 2004
), we labeled, in parallel, samples collected at 1 hr and 24 hr following bacterial challenge. Defensin was present in melanotic capsules at both 1 hr and 24 hr postchallenge (Figure 3). The overall labeling pattern was similar for both timepoints. However, the intensity of labeling at 24 hr postchallenge was 3.6 times higher than at 1 hr postchallenge (Figure 3). When comparing samples labeled at 1 hr postchallenge vs 24 hr postchallenge, background for both was negligible and statistically similar (p=0.1), and specific labeling was statistically different (p=0.01).
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Discussion |
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In Ae. aegypti, developmental Northern blots showed that the gene coding the antimicrobial peptide defensin is not transcribed in the larval stages or adult mosquitoes (Lowenberger et al. 1999b). However, high levels of transcript are detected in callow (white) pupae but not in black pupae, an interesting finding given that the development from callow pupae to black pupae usually takes 12 hr (Lowenberger et al. 1999b
). Previously, it was suggested that defensin upregulation in this developmental stage is likely 1. to lyse bacteria entering through the delicate early pupal cuticle prior to sclerotization, 2. to lyse bacteria released during histolysis of specific tissues during metamorphosis, or 3. because defensin may have a dual role in defense and development (Lowenberger et al. 1999b
). Upon further review, it is unlikely that in these instances defensin acts in defense because similar events occur between larval molts and between pupaladult ecdysis, a time when transcription of the defensin gene is unchanged. However, the timing of defensin transcription correlates with the time of extensive melanization during cuticular sclerotization. These observations and our data showing the colocalization of defensin and phenoloxidase in the antibacterial immune melanization response in adults suggest that there may be a link between defensin and the melanization cascade. For example, it is possible that one function of defensin may be the initiation of phenoloxidase-based melanization reactions, explaining its necessity during pupal cuticular sclerotization. However, we cannot discount the possibility that hemolymph proteins are randomly incorporated into melanization reactions, and that the high concentration of defensin in the hemolymph at 24 hr postchallenge (45 µM; Lowenberger et al. 1995
) explains its presence in melanization immune responses.
Furthermore, in the current study, the cellular immunolocalization of phenoloxidase was as expected: in bacteria-challenged mosquitoes, phenoloxidase was found exclusively in oenocytoids. This is in accord with our previous studies in Ae. aegypti and Ar. subalbatus that showed that oenocytoids are the only hemocytes containing phenoloxidase in naïve, and naïve and bacteria-challenged mosquitoes, respectively (Hillyer and Christensen 2002; Hillyer et al. 2003a
). However, we were unable to determine the source of defensin. Northern analyses have shown that defensin is produced in the fat body (Lowenberger et al. 1999a
). Hence, we expected to find defensin in adipohemocytes: a population of cells also collected during mosquito perfusions that, because of morphological similarities, have been hypothesized to be cells sloughed off the fat body during hemolymph collection (Hillyer and Christensen 2002
; Hillyer et al. 2003a
). Possibilities for the lack of labeling in adipohemocytes include that they are different from fat body cells, or that defensin is produced in specific regions of the fat body, and that these regions are different from the ones adipohemocytes originate from.
In summary, these data show that defensin and phenoloxidase colocalize in melanization immune responses and suggest that the antimicrobial peptide defensin may be involved in the mosquito melanization response against bacteria. These data argue for further studies into the potential role of defensin in melanization innate immune responses in mosquitoes.
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Acknowledgments |
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We thank S.L. Schmidt for mosquito rearing. We also thank C.C. Chen and J. Vizioli for providing the anti-phenoloxidase and anti-defensin antibodies, respectively. Useful discussions with A.J. Nappi are also greatly appreciated.
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Footnotes |
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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Albrecht RM, Simmons SR, Pawley JB (1993) Correlative video-enhanced light microscopy, high voltage transmission electron microscopy, and field emission scanning electron microscopy for the localization of colloidal gold labels. In Beesley JE, ed. Immunocytochemistry: A Practical Approach. New York, Oxford University Press, 151176
Bartholomay LC, Fuchs JF, Cheng LL, Beck ET, Vizioli J, Lowenberger C, Christensen BM (2004) Reassessing the role of defensin in the innate immune response of the mosquito, Aedes aegypti. Insect Mol Biol 13:125132[CrossRef][Medline]
Beaty BJ (2000) Genetic manipulation of vectors: a potential novel approach for control of vector-borne diseases. Proc Natl Acad Sci USA 97:1029510297
Beerntsen BT, James AA, Christensen BM (2000) Genetics of mosquito vector competence. Microbiol Mol Biol Rev 64:115137
Beerntsen BT, Luckhart S, Christensen BM (1989) Brugia malayi and Brugia pahangi: inherent difference in immune activation in the mosquitoes Armigeres subalbatus and Aedes aegypti. J Parasitol 75:7681[Medline]
Blandin S, Shiao SH, Moita LF, Janse CJ, Waters AP, Kafatos FC, Levashina EA (2004) Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae. Cell 116:661670[CrossRef][Medline]
Brasseur P, Kouamouo J, Moyou-Somo R, Druilhe P (1992) Multi-drug resistant falciparum malaria in Cameroon in 19871988. II. Mefloquine resistance confirmed in vivo and in vitro and its correlation with quinine resistance. Am J Trop Med Hyg 46:814[Medline]
Christensen BM, Forton KF (1986) Hemocyte-mediated melanization of microfilariae in Aedes aegypti. J Parasitol 72:220225[Medline]
Christensen BM, Li J, Chen CC, Nappi AJ (2005) Melanization immune responses in mosquito vectors. Trends Parasitol 21:192199[CrossRef][Medline]
Collins FH, Sakai RK, Vernick KD, Paskewitz S, Seeley DC, Miller LH, Collins WE, et al. (1986) Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae. Science 234:607610[Medline]
Forton KF, Christensen BM, Sutherland DR (1985) Ultrastructure of the melanization response of Aedes trivittatus against inoculated Dirofilaria immitis microfilariae. J Parasitol 71:331341[Medline]
Hernández-Martínez S, Lanz H, Rodríguez MH, González-Ceron L, Tsutsumi V (2002) Cellular-mediated reactions to foreign organisms inoculated into the hemocoel of Anopheles albimanus (Diptera: Culicidae). J Med Entomol 39:6169[Medline]
Hillyer JF, Albrecht RM (1998) Correlative instrumental neutron activation analysis, light microscopy, transmission electron microscopy, and X-ray microanalysis for qualitative and quantitative detection of colloidal gold spheres in biological specimens. Microsc Microanal 4:481490[Medline]
Hillyer JF, Christensen BM (2002) Characterization of hemocytes from the yellow fever mosquito, Aedes aegypti. Histochem Cell Biol 117:431440[CrossRef][Medline]
Hillyer JF, Schmidt SL, Christensen BM (2003a) Hemocyte-mediated phagocytosis and melanization in the mosquito Armigeres subalbatus following immune challenge by bacteria. Cell Tissue Res 313:117127[CrossRef][Medline]
Hillyer JF, Schmidt SL, Christensen BM (2003b) Rapid phagocytosis and melanization of bacteria and Plasmodium sporozoites by hemocytes of the mosquito Aedes aegypti. J Parasitol 89:6269[Medline]
Hillyer JF, Schmidt SL, Christensen BM (2004) The antibacterial innate immune response by the mosquito Aedes aegypti is mediated by hemocytes and independent of Gram type and pathogenicity. Microbes Infect 6:448459[CrossRef][Medline]
Hillyer JF, Schmidt SL, Fuchs JF, Boyle JP, Christensen BM (2005) Age-associated mortality in immune challenged mosquitoes (Aedes aegypti) correlates with a decrease in haemocyte numbers. Cell Microbiol 7:3951[CrossRef][Medline]
Infanger LC, Rocheleau TA, Bartholomay LC, Johnson JK, Fuchs JF, Higgs S, Chen CC, et al. (2004) The role of phenylalanine hydroxylase in melanotic encapsulation of filarial worms in two species of mosquitoes. Insect Biochem Mol Biol 34:13291338[CrossRef][Medline]
Kumar S, Christophides GK, Cantera R, Charles B, Han YS, Meister S, Dimopoulos G, et al. (2003) The role of reactive oxygen species on Plasmodium melanotic encapsulation in Anopheles gambiae. Proc Natl Acad Sci USA 100:1413914144
Lai SC, Chen CC, Hou RF (2002) Immunolocalization of prophenoloxidase in the process of wound healing in the mosquito Armigeres subalbatus (Diptera: Culicidae). J Med Entomol 39:266274[Medline]
Lanz-Mendoza H, Hernandez-Martinez S, Ku-Lopez M, Rodriguez Mdel C, Herrera-Ortiz A, Rodriguez MH (2002) Superoxide anion in Anopheles albimanus hemolymph and midgut is toxic to Plasmodium berghei ookinetes. J Parasitol 88:702706[Medline]
Lowenberger C (2001) Innate immune response of Aedes aegypti. Insect Biochem Mol Biol 31:219229[CrossRef][Medline]
Lowenberger C, Bulet P, Charlet M, Hetru C, Hodgeman B, Christensen BM, Hoffmann JA (1995) Insect immunity: isolation of three novel inducible antibacterial defensins from the vector mosquito, Aedes aegypti. Insect Biochem Mol Biol 25:867873[CrossRef][Medline]
Lowenberger CA, Ferdig MT, Bulet P, Khalili S, Hoffmann JA, Christensen BM (1996) Aedes aegypti: induced antibacterial proteins reduce the establishment and development of Brugia malayi. Exp Parasitol 83:191201[CrossRef][Medline]
Lowenberger CA, Kamal S, Chiles J, Paskewitz S, Bulet P, Hoffmann JA, Christensen BM (1999a) MosquitoPlasmodium interactions in response to immune activation of the vector. Exp Parasitol 91:5969[CrossRef][Medline]
Lowenberger CA, Smartt CT, Bulet P, Ferdig MT, Severson DW, Hoffmann JA, Christensen BM (1999b) Insect immunity: molecular cloning, expression, and characterization of cDNAs and genomic DNA encoding three isoforms of insect defensin in Aedes aegypti. Insect Mol Biol 8:107118[CrossRef][Medline]
Luckhart S, Vodovotz Y, Cui L, Rosenberg R (1998) The mosquito Anopheles stephensi limits malaria parasite development with inducible synthesis of nitric oxide. Proc Natl Acad Sci USA 95:57005705
Meshnick SR (2002) Artemisinin: mechanisms of action, resistance and toxicity. Int J Parasitol 32:16551660[CrossRef][Medline]
Roberts L (2002) Mosquitoes and disease. Science 298:8283
Shiao SH, Higgs S, Adelman Z, Christensen BM, Liu SH, Chen CC (2001) Effect of prophenoloxidase expression knockout on the melanization of microfilariae in the mosquito Armigeres subalbatus. Insect Mol Biol 10:315321[CrossRef][Medline]
Vizioli J, Bulet P, Hoffmann JA, Kafatos FC, Muller HM, Dimopoulos G (2001a) Gambicin: a novel immune responsive antimicrobial peptide from the malaria vector Anopheles gambiae. Proc Natl Acad Sci USA 98:1263012635
Vizioli J, Richman AM, Uttenweiler-Joseph S, Blass C, Bulet P (2001b) The defensin peptide of the malaria vector mosquito Anopheles gambiae: antimicrobial activities and expression in adult mosquitoes. Insect Biochem Mol Biol 31:241248[CrossRef][Medline]
Wang X, Fuchs JF, Infanger LC, Rocheleau TA, Hillyer JF, Chen CC, Christensen BM (2005) Mosquito innate immunity: involvement of beta 1,3-glucan recognition protein in melanotic encapsulation immune responses in Armigeres subalbatus. Mol Biochem Parasitol 139:6573[CrossRef][Medline]
Wellems TE (2002) Plasmodium chloroquine resistance and the search for a replacement antimalarial drug. Science 298:124126
World Health Organization (1996) The World Health Report 1996: Fighting Disease, Fostering Development. France, WHO
Zhao X, Ferdig MT, Li J, Christensen BM (1995) Biochemical pathway of melanotic encapsulation of Brugia malayi in the mosquito, Armigeres subalbatus. Dev Comp Immunol 19:205215[CrossRef][Medline]