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Journal of Invertebrate Pathology 76, 257–262 (2000)doi:10.1006/jipa.2000.4980, available online at http://www.idealibrary.com on

Immune Defense Mechanisms of Culex quinquefasciatus(Diptera: Culicidae) against Candida albicans Infection

Jose B. Da Silva,* Cleide M. R. De Albuquerque,* Eva Cristina De Araujo,*Christina A. Peixoto,† and Hilary Hurd‡

*Mestrado em Biologia Animal, Departamento de Zoologia Universidade Federal de Pernambuco, Recife PE, Brazil; †Centro de PesquisasAggeu Magalhaes, Departamento de Patologia e Biologia Celular, Recife PE, Brazil; and ‡Centre for Applied Entomology and

Parasitology, School of Life Sciences, Keele University, Keele, United Kingdom

E-mail: bia37@bio.keele.ac.uk

Received January 6, 2000; accepted September 13, 2000

defenses, active against almost all non-self-material,

Mosquitoes have an efficient defense system against

infection. The cellular immune defense mechanisminitiated by the mosquito Culex quinquefasciatus in-fected with the fungus Candida albicans was investi-gated in this study. Differences in the hemocytecounts in hemolymph perfused from uninoculated, sa-line-inoculated, and C. albicans-infected mosquitoeswere compared using a light microscope. Phagocytosiswas also investigated using electron microscopy. Fourtypes of hemocytes were identified in control mosqui-toes: prohemocytes (9.8%), plasmatocytes (38.8%),granular cells (44.2%), and oenocytoids (7.3%). Be-tween 3 and 18 h postinoculation the total hemocytecount was significantly higher in infected, comparedto uninfected, mosquitoes. Differential hemocytecounts from infected mosquitoes at 3, 6, and 18 h afterinoculation showed that the relative proportion ofplasmatocytes (48.6, 50.7, 45%) was higher and, con-comitantly, the proportion of granular cells was lower(38, 36.8, 35%, respectively). Yeast cells were phagocy-tosed and limited growth was observed within theplasmatocytes. Melanized nodules were found at-tached to different insect tissues at 24 to 72 h followinginfection. These results suggest that phagocytosis, fol-lowed by nodule formation, was capable of clearingthe hemolymph of yeast cells. © 2000 Academic Press

Key Words: hemocytes; cellular defense; phagocyto-is; nodulation; Culex quinquefasciatus; Candida albi-ans.

INTRODUCTION

Insects have developed resistance and immunemechanisms that enable them to survive exposure to ahuge variety of microbial and parasitic pathogens.These mechanisms include anatomical and physiolog-ical barriers, cellular defense reactions, and innate orinduced humoral responses. Some of these are general

257

but some potential pathogens elicit specific responsesand thus an insect may utilize different mechanisms indifferent circumstances (Dimopoulus et al., 1997).

Despite this, mosquitoes act as efficient vectors formany viral, bacterial, and parasitic organisms. Vecto-rial capacity for individual pathogens varies widelybetween species and this variation may be a reflectionof innate differences in immune mechanisms. Culexquinquefasciatus is a culicid mosquito that transmitsseveral pathogens, including Wuchereria bancrofti andarboviruses, such as St. Louis encephalitis (SLE) inWestern United States and Oropouch virus in thenorth of Brazil. However, it is unable to act as a vectorfor Plasmodium spp. We are studying the immuneresponse of this mosquito to several pathogens andparasites in an attempt to monitor the efficiency andspecificity of its response to different organisms.

The majority of studies of insect immune systemshave concentrated on humoral immune responses tobacteria (Cociancich et al., 1994). Antifungal activityhas been less well studied. A Candida albicans innocu-lum provides a convenient model with which to moni-tor antifungal defense mechanisms mounted by C.quinquefasciatus. We have observed that the yeast elic-its both humoral and cellular responses that contributeto the clearance of the organism from the hemocoel.Here we report our findings concerning the cellularresponse.

Insect cellular defenses are dependent upon peptidesand protein factors, biogenic amines, and eicosanoids(Pech and Strand, 1995; Baines et al., 1992; Stanley-Samuelson et al., 1991). Responses result in phagocy-tosis and nodule formation mediated by the insect he-mocytes (Ratcliffe and Rowley, 1979). Nodules areformed by aggregation of hemocytes that entrap themicroorganism in an extracellular matrix. This massmay then undergo melanization that kills the trapped

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microorganism. Despite mosquitoes having a low num-

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258 DA SILVA ET AL.

ber of circulating hemocytes, these cells seem to play acritical role in immune reactions against parasites thatthey transmit (Christensen and Severson, 1993). Inresponse to Dirofilariae immitis infection, the hemo-cyte population of Aedes aegypti increased from Day 1to Day 4 post-microfilariae inoculation (Christensen etal., 1989). Ultrastructural studies have shown thatthese hemocytes are involved in melanotic encapsula-tion of the parasite. Similar reactions were observed inAedes trivittatus during encapsulation of Dirofilariaimmitis microfilariae and in Anopheles quadrimacula-tus infected with Brugia pahangi (Forton et al., 1985;Chen and Laurence, 1985).

Limited data are available on the immune mecha-nisms of C. quinquefasciatus. This study aimed to mon-itor how the hemocyte population changed with respectto C. albicans infection and to follow the fate of thesefungi after inoculation into the hemocoel.

MATERIALS AND METHODS

Mosquitoes

Three- to 4-day-old adult females obtained from pu-pae collected in open breeding areas at Olinda, Brazil,were used throughout this study. Pupae were hatchedin an insectary and maintained at 27°C and 60–80%relative humidity, on a diet of 10% sucrose solution.

Fungus Strain and Media

The C. albicans (strain DAUFPE 1007) used in thistudy was kindly provided by Institute de AntibioticsFPE. Fungi were grown at 37°C in Saboraud me-ium.

nfection

Adults were immunized by intrathoracic injection ofml of log-phase culture C. albicans (1.5 3 103 colony-

orming units (CFU). Cells were harvested at an A 600 of0.4, centrifuged, and resuspended in 10 mM sterilesodium phosphate buffer before inoculation. Mosqui-toes were immobilized on ice and inoculated by using afinely drawn glass capillary inserted into the thorax.

Clearance of Yeast Cells

The number of viable colonies was monitored imme-diately and 3, 6, 18, 24, and 48 h after inoculation.Perfused hemolymph (see below) was plated on toSaboraud agar medium and incubated at 37°C, and thenumber of visible colonies was counted 24 h later.

Hemocyte Studies

Adults were anesthetised by chilling at 4°C for 1–2min. One microliter of anticoagulant II solution (Mead

using a finely drawn glass capillary, and hemolymphwas perfused from the wound by the application ofgentle pressure (Chalk, 1992) at different timepoints following inoculation (30 min, 3, 6, 18, and24 h). Perfused hemolymph was placed on a cleanmicroscope slide; hemocytes in the perfusate wereallowed to settle for 5 min and then were fixed withmethanol. Slides were stained with Giemsa stain (3drops in 1 ml of water for 3 min) pH 7.2, washed intap water, dried, and examined at 31000 under alight microscope. Hemocytes were assigned to one offour groups, according to their appearance afterstaining, and the proportion of each was noted. Allhemocytes collected on each slide were counted andused to determine total hemocyte numbers. Baselinepopulation levels of hemocytes in un-inoculated mos-quitoes were determined for females at 24, 48 and72 h following adult emergence. Three replicatestudies were conducted. According to Chalk (1992)approximately 50% of mosquito hemolymph is col-lected using this perfusion technique. We thus as-sumed that counts made in this study representedapproximately 50% of the hemocyte population thatwas freely circulating and available for perfusion.

Differential and total hemocyte counts were assessedand compared by analysis of variance using Turkey’smultiple comparison. A confidence level of P 5 0.05

as used as the level of significance. For each group–10 mosquitoes were examined.

hagocytosis and Nodulation Assays

In an attempt to determine the ability of the hemo-ytes to undertake phagocytosis, ultrastructural stud-es were performed. Hemolymph from 50 females in-ected with C. albicans was perfused directly in a pre-xation solution of 2.5% glutaraldehyde and 2%ormaldehyde (freshly prepared from paraformalde-yde) in 0.1 M cacodylate buffer, pH 7.2. Perfusedemolymph was pooled and centrifuged at 500g for 5in. The pellet was resuspended and rinsed twice in

he same buffer. The hemocyte pellet was postfixed in% solution of osmium tetraoxide, 0.8% potassium fer-icyanide in cacodylate buffer, dehydrated in acetonend embedded in Spurr’s epoxy resin, which has aower viscosity than Epox. Ultrathin sections wereounterstained with uranyl acetate and lead citratend examined with a Zeiss EM 109 transmission mi-roscope.Nodulation was assessed at 6, 24, 48, and 72 h after

njection. The abdomen was dissected and nodules at-ached to different tissues were counted under a stereoicroscope (Olympus BX-50). Melanized, darkened

odules were noted.

No nodules were found in untreated mosquitoes (n 5

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259MOSQUITO CELLULAR RESPONSE TO FUNGI

RESULTS

Yeast cells were rapidly cleared from the hemolymphfollowing inoculation (PI). A mean number of 972 666.5 (SD) yeast cells/insect were recovered immedi-ately following inoculation (n 5 9). There was a 66%reduction 6 h PI (n 5 9) and by 24 h PI this decreasehad reached approximately 98% (n 5 9) (see Fig. 1). Aconcomitant increase in the total hemocyte populationwas observed together with changes in differential he-mocyte counts of plasmatocytes and granular cells fol-lowing infection (Table 1). Numerous nodules were ob-served 24 to 72 h after C. albicans inoculation. Fromthe total nodules counted (n 5 1,021) in 15 insects,73.7% was found in the tegument followed by 8.6% inthe fat body and 4.9% in the Malpighian tubules. Nod-ule sizes ranged from aggregates of 4–5 up to 250 cells.Melanization was mainly observed in the largest nod-ules and less frequently seen among the smaller ones(Fig. 3B). Due to the intense melanization response itwas not possible to distinguish hemocytes from yeastcells within the nodules; thus, we were unable to de-termine the cell type responsible for their formation.

TABDifferential Hemocyte Counts at Differe

Hours

PR PL

CNT CPT INF CNT CPT IN

0.5 10.5 6 2.3 9.7 6 3.1 8.1 6 3.3 38.3 6 6.0 41.0 6 4.9 39.63 8.1 6 2.1 7.6 6 2.8 7.1 6 2.7 41.5 6 7.0 37.0 6 6.0 48.66 11.4 6 2.7 5.3 6 1.9 8.8 6 4.3 39.8 6 6.1 34.4 6 5.7 50.7

18 6.6 6 1.9 6.5 6 1.8 9.0 6 3.3 37.4 6 6.3 35.0 6 5.0 45.024 12.3 6 4.0 8.8 6 2.1 11.2 6 8.0 36.8 6 7.4 35.5 6 6.8 43.4

Note. Data are given as means 6 standard deviation (n 5 9 differeespective sham-injected controls at 3, 6, and 18 h postinoculationifferences (F 5 95.97, P , 0.05). PR, prohemocyte; PL, plasmatocham inoculated; INF, infected.

FIG. 1. The changing response of Culex quinquefasciatus to Can-dida albicans infection over time postinfection. (A) Mean number ofyeast cell colony-forming units (CFU). (B) Mean number of hemo-cytes per mosquito. (C) Mean number of nodules per mosquito. Barsrepresent standard error.

5) or in treated ones at 6 h PI (n 5 5).

Effect of C. albicans Infection on Total andDifferential Hemocyte Counts

The mean number of total hemocytes in untreatedfemales was 195.4 6 22 (SD)/mosquito. We wereclearly able to distinguish four types of hemocytes,based on their morphology after Geimsa staining (Fig.2). Prohemocytes were spherical, ranging from 6.0 to10.0 mm in diameter and with a nucleus occupyingfrom 70 to 90% of the total cell volume. In untreatedmosquitoes (n 5 30), prohemocytes accounted for 9.8%of the total hemocytes. The predominant cells weregranular cells (44.2%), with diameters ranging from 20to 36 mm. They were round or slightly oval, with asmall, central, nucleus occupying 5–30% of the totalcell volume and numerous granules in the cytoplasm.Plasmatocytes were also very abundant in C. quinque-fasciatus hemolymph (38.8%). Their shapes were var-ied and round; oval or spindle-shape cells were ob-served. They frequently formed cytoplasmic extensionsin a star-like shape and their size ranged from 18 to 27mm in diameter. Oenocytoids were round or oval cellswith a small nucleus and fewer granules in the cyto-plasm, compared to granular cells, and cytoplasm oc-casionally containing vacuoles. Their size varied be-tween 7.0 and 10 mm in diameter. Oenocytoids wereless frequently observed (7.2%).

Mean total hemocyte numbers (6SD) in sham-inoc-ulated females showed little change from 30 min to24 h PI (206.4 6 27.4) and were not significantly dif-ferent from un-inoculated controls. However, the totalhemocyte counts in mosquitoes inoculated with C. al-bicans increased gradually to 374.5 6 88.1/mosquito at

h postinoculation and then decreased in a similaranner until 24 h postinoculation, where a similar

umber of hemocytes were found in controls (195.4 62) and infected mosquitoes (222.6 6 54.8) (Fig. 1).opulations were significantly elevated from 3 to 18 h,

ANOVA F 5 6.56; F ratio 5 12.3, P , 0.05) in

1Times after Candida albicans Infection

GR OE

CNT CPT INF CNT CPT INF

.9 44.5 6 6.1 46.0 6 3.2 46.3 6 8.2 6.7 6 2.1 3.3 6 1.3 6.1 6 4.0

.2 42.5 6 6.5 46.2 6 5.2 38.0 6 5.5 7.9 6 3.2 8.5 6 0.9 6.3 6 1.5

.5 43.6 6 5.0 53.0 6 9.6 36.8 6 6.7 5.2 6 1.8 6.1 6 2.5 3.7 6 1.1

.3 47.2 6 7.2 52.5 6 7.9 35.0 6 4.0 8.8 6 2.9 6.0 6 2.1 11.0 6 3.20 42.9 6 8.1 50.7 6 11 39.1 6 12 8.0 6 2.5 5.0 6 1.0 6.3 6 3.6

mosquitoes). Plasmatocyte numbers differed significantly from their5 18.49, P , 0.05). Granulocytes exhibited similar significant

; GR, granular cell; OE, oenocytoids; CNT, untreated control; CPT,

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260 DA SILVA ET AL.

immune activated mosquitoes as compared to un-inoc-ulated and saline-inoculated controls.

Changes in differential hemocyte counts were foundfollowing infection (Table 1). After 6 h, the proportionof plasmatocytes had risen significantly by approxi-mately 16% in infected mosquitoes compared to sham-inoculated females. A similar, significant decrease inthe proportion of granular cells was observed whensham-inoculated and infected mosquitoes were com-pared.

Phagocytosis

Examination of C. quinquefasciatus hemolymph 6 hpostinoculation revealed that plasmatocytes were in-volved in phagocytosis. Up to six fungal cells werefound in one single plasmatocyte. The C. albicans cellswere covered with a melanotic capsule of regular thick-ness that completely surrounded the cell wall and var-ied in thickness between cells (see Fig. 3A). Occasion-ally, melanotic material was also observed free in thehemolymph. Once inside the plasmatocyte the elec-tron-dense material started to exhibit fragmentation.

DISCUSSION

The results of this study provide evidence that themosquito C. quinquefasciatus exhibits an efficient de-ense against C. albicans. The reduction in numbers ofFUs in the hemolymph, the concomitant rise in he-ocyte numbers, and the observation of phagocytosis

nd nodule formation support the hypothesis that an

FIG. 2. Light microscopic micrographs of Culex quinquefasciatusstained with Giemsa stain. (A) Prohemocyte, (B) plasmatocyte, (C) g

ffective cellular immune response occurs in responseo this fungal pathogen.

Four different hemocytes were identified in the he-olymph of C. quinquefasciatus under our study con-

itions. We recognize that additional light microscopi-al examinations and ultrastructural studies would beequired for a definitive classification. Nevertheless,or the purpose of obtaining our objective of monitoringhanges in hemocyte population in response to fungalnfection our descriptions proved adequate. Kaaya andatcliffe (1982), observing live cells by phase-contrasticroscopy, described the occurrence of five types of

emocytes in C. quinquefasciatus. They found the fifthype, the adipocyte, in very low abundance. It is likelyhat the methanol used for cell fixation in our workissolved the lipids in these cells, resulting in cellupture in our preparations.Many insects have been reported to rapidly and

ffectively clear foreign bodies from the hemolymphfor example, Orgyia leucostigma (Guzo and Stoltz,987), Aedes aegypti (Christensen et al., 1989), Gal-eria mellonella (Wiesner and Gotz, 1993), and Dro-ophila sp. (Elsin and Prevost, 1998). Our observa-ions are consistent with others, in that infection isollowed by a significant increase in the total hemo-yte count and a selective increase in the proportionf plasmatocytes. Plasmatocytes are described ashagocytic cells in several insect species (Ratcliffend Rowley, 1979; Anggraeni and Ratcliffe, 1991;iesner and Gotz, 1993). This is likely to be the case

or C. quinquefasciatus.

mocytes obtained from whole hemolymph of untreated females andular cell, (D) oenocytoids.

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261MOSQUITO CELLULAR RESPONSE TO FUNGI

Spodoptera exigua infected with C. albicans rapidlyengulfed the yeast cells and exhibited a decrease inhemocyte spreading but recovered by 36 h postchal-lenge (Hung et al., 1993). As with our study, limitedgrowth of C. albicans occurred within the S. exiguaephagocytic cells and nodule formation was observed at24 h. Hung et al. (1993) observed that yeast infectiondid not affect adult emergence rate and that phagocytichemocytes and the process of nodule formation wereprotective.

In addition to plasmatocyte increase, a reduction inthe number of C. quinquefasciatus granular cells wasobserved in this study. Granular cells can easily beruptured (Ratcliffe and Rowley, 1979); thus, the possi-

FIG. 3. Phagocytic response of adult female Culex quinquefas-ciatus to Candida albicans infection (A) Plasmatocyte containingeveral yeast cells, viewed under electron microscopy 6 h postinocu-ation. Two forms of C. albicans cells inside the phagocytic vacuolesan be observed; a melanotic capsule (MC) encloses one cell whichas amorphous material (AM) between the fungus and the vacuole;onmelanized C. albicans with large spaces inside the phagocyticesicle (small arrow) are also shown. Note replicating C. albicansnside a phagocytic vacuole (large arrow). (B) Light microscope pic-ure (1003) of yeast cells aggregation and nodulation reaction to C.lbicans infection in the mosquito C. quinquefasciatus. Arrow pointso one of several visible nodules observed on the mosquito tegument8 h postchallenge. No hemocytes are visible.

these cells in our preparations cannot be ruled out.However, recorded changes in the proportions of celltypes are unlikely to be incorrect as hemolymph per-fusates were treated in the same way throughout. Inthe majority of insects, the role of the granular cells isto entrap the invader by releasing its granular inclu-sions before plasmatocytes form multicellular layersaround the aggregates of disintegrated granular cellsand pathogen (Wiesner and Gotz, 1993). We suggestthat this may be happening in response to C. albicansinfection. As no significant difference in the hemocytecount was observed when C. quinquefasciatus was sim-ply wounded, it is possible that a signal is producedeither by the fungi itself, or by the cells initially re-sponding to it (Guzo and Stoltz, 1987).

The humoral encapsulation reactions of larvae ofCulex sp., against fungi, were studied in vitro by Veyand Gotz (1975). They observed an abundant granulardeposition around the spores but no cellular participa-tion. This material gradually darkened and becamemore regular in appearance, although it conserved itsgranular aspect. We too were unable to identify hemo-cytes associated with nodules.

In the present study, aggregation and nodule forma-tion were initiated only after a clearly defined lag pe-riod, lasting 18–24 h. This supports the hypothesisthat nodule formation may be a secondary immunemechanism, working when phagocytosis is not suffi-cient to clear the pathogen (Ratcliffe and Rowley,1979). However, it is clear that, unlike some Diptera,plasmatocytes play a major role in rapidly clearing themajority of C. albicans from the hemocoel of C. quin-uefasciatus. Although we have some evidence that theroduction of peptides is induced in response to theseeast cells (unpublished observations) we believe thatellular mechanisms are the principal line of defensegainst fungi in C. quinquefasciatus.

ACKNOWLEDGMENTS

The authors thank Dr. Leda Regis who generously arranged for usto use the facilities at Centro de Pesquisas Aggeu Magalhaes/FIOCRUZ and Dr. Rod Chalk for comments on the manuscript. Thiswork was supported by The Wellcome Trust and CAPES/FACEPEprovided studentships.

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