The antibacterial immune response of the medfly, Ceratitis capitata

J. Insect Physiol. Vol. 34, No. 2, pp. 91-96, 1988 Printed in Great Britain. All rights reserved

0022-1910/88 $3.00+0.00 Copyright © 1988 Pergamon Journals Ltd

THE ANTIBACTERIAL IMMUNE RESPONSE OF THE MEDFLY, C E R A T I T I S C A P I T A T A JOHN H. POSTLETHWAIT,STEPHEN H. SAUL and JUANITA A. POSTLETHWAIT Department of Biology, University of Oregon, Eugene, OR 97403 and Department of Entomology, University of Hawaii at Manoa, Honolulu, HI 96822, U.S.A. (Received 25 March 1987; revised 28 July 1987) Abstract--Haemolymph withdrawn from Ceratitis capitata larvae and adult males inoculated a day earlier with Enterobacter cloacae contains potent antibacterial factors, while haemolymph from untreated controls has none. A sterile wound can also induce the antibacterial immune response, but it is augmented substantially by bacteria. The response is detectable within 3 h and lasts for at least 8 days. Ceratitus larvae and adult males produce about four factors with isoelectric points between 8.5 and 10 and larvae make an additional factor at about pI 7. The fat body of both larvae and adults produces antibacterial factors. Key Word Index: Tephritid fruit flies, Ceratitis capitata, Dacus dorsalis, Dacus cucurbitae, bacterial infection, insect defence system INTRODUCTION

play an important role in the life history of tropical fruit flies, it may be that their immune systems differ in significant ways from that of Drosophila. In this paper we demonstrate that Ceratitus has a strong humoral antibacterial immune response that is rapidly induced and lasts for days.

The ability to combat pathogens vigorously is one of the factors that contributes to the enormous evolutionary success of insects. Immune systems of insects can recognize and eliminate bacteria, fungi, and metazoan parasites (Whitcomb et al., 1974). Although insect immune systems lack important features of vertebrate immunity, such as lymphocytes and immunoglobulins (Boman and Hultmark, 1981; G6tz and Boman, 1984; Dunn, 1986), both cellular and humoral factors play a role in the insect defence system. Three types of circulating antibacterial proteins have been described in moths (Boman, 1986), and antibacterial proteins have been identified in flies (Boman et al., 1972; Kubo et al., 1984; Okada and Natori, 1983, 1985a, b; Keppi et al., 1986; Robertson and Postlethwait, 1986; Flyg et al., 1987). Injections of bacteria into Drosophila melanogaster adult males causes about eight polypeptides to be newly synthesized and secreted into the haemolymph, and at least three of these have isoelectric points identical to those of proteins that block bacterial growth (Robertson and Postlethwait, 1986). In the experiments reported here we investigated the antibacterial immune response of the Mediterranean fruit fly (or Medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Both Drosophila and Ceratitus adults are strongly attracted by fermenting substances, but the larvae of Drosophila species live in the fermenting medium and pupate in air, while the larvae of Tephritidae feed on the living fruit and associated bacteria and fungi, and then burrow into the soil to pupate. While Drosophila feeds on yeast, at least some Australian populations of Tephritid fruit flies are regulated by the supply of specific leaf-surface bacteria on which they feed (Drew et al., 1983; Courtice and Drew, 1984). Bacteria of the genus Enterobacter obtained from the guts of Tephritid adults caught in the wild are highly attractive to these flies, and provide a complete diet. Since fruit-surface bacteria

MATERIALS AND METHODS

Stocks Ceratitis capitata, Dacus cucurbitae, Dacus dorsalis and Drosophila melanogaster (Oregon R) were cultured under standard conditions (Steiner and Mitchell, 1966). Bacteria stocks were Enterobacter cloacae, strain beta-12, naladixic acid resistant and Escherichia coli, strain D31, streptomycin resistant (Boman et al., 1974). Surgical procedures Two- to five-day-old flies or wandering-stage larvae were injected in the abdomen with 0.5/~1 of a suspension of stationary phase E. cloacae cells grown in LB broth and diluted 1:5 with 0.9% sterile NaCI. Haemolymph was collected by injecting 0.5/~1 of 0.9% sterile NaC1 containing phenylthiourea (10% saturated) and aprotinin (2.3 TIU/ml, Sigma) into the abdominal body cavity, and recovering the solution in a glass capillary. Haemolymph samples were analyzed directly after collection. For organ-culture experiments, animals were surface sterilized and dissected in sterile Schneider's medium (Schneider and Presley, 1978). Organs from three animals were pooled and cultured in 20/~1 Schneider's medium. Antibacterial assays In some experiments, antibacterial activity was tested by the inhibition zone assay. In this test, factors diffuse from a well cut in an agar plate that contains E. coli strain D31 bacteria. Any antibacterial factors in the test solution inhibit bacterial growth in a halo surrounding the well. The diameter of the clear plaque is a logarithmic function of concentration 91

JOHN H. POSTLETHWAITel al.

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(Hultmark et al., 1982). In other experiments, haemolymph proteins were separated by isoelectric focusing on LKB PAG plates at pH 3.5-9.5 according to the manufacturer's instructions. To detect antibacterial activity after isoelectric focusing, the electrophoretic plates were overlaid with phosphate buffered LB agar seeded with E. coli strain D31. The antibacterial factors diffused from the isoelectric focusing plate into the bacteria-containing overlay thus blocking bacterial growth and revealing bands of activity (Hultmark et al., 1980). RESULTS

Ceratitus has a potent inducible antibacterial immune response Initial experiments were conducted to find if Ceratitus can mount an inducible immune response. We inoculated jumping-stage third-instar larvae or young adult males with bacteria and collected animals of the same stage as untreated controls. Twentyfour hours later we collected haemolymph for testing by the inhibition zone assay. A typical test plate is shown in Fig. 1. Haemolymph taken from laboratory-grown Ceratitus larvae or adults contained no factors that blocked the growth of bacteria. In contrast, haemolymph from animals inoculated a day earlier with bacteria possessed potent antibacterial activity. The results show that Ceratitus has an inducible antibacterial immune response. To determine the potency of the response we inoculated larval and adult male Ceratitus and larval and adult male Drosophila, exsanguinated whole animals a day later, and then applied serial dilutions of the haemolymph samples to inhibition zone assay plates. After incubation, the diameters of plaques surrounding the test wells were measured and plotted in Fig. 2. On a per animal basis, Ceratitus adult males had 4 times as much activity as Drosophila adult males, and Ceratitus larvae had 4 times as much activity as Ceratitus adult males. Since Ceratitus males are about 8 times as large as Drosophila males, antibacterial activity is about half as concentrated in Ceratitus haemolymph compared to Drosophila haemolymph under the conditions used in this experiment.

The dilution curve of Fig. 2 shows that the log of haemolymph concentration was directly related to the diameter of the cleared spot. This allows us to define one activity unit as the amount of activity needed to clear a I mm zone around the 2.4 mm dia well in the agar test plate. The rest of the graphs in this section will be given in terms of these arbitrary concentration units.

Induction parameters To begin to understand the mechanisms that induce the antibacterial immune response of Ceratitis, we investigated several aspects of the induction process. While the experiments reported above showed that bacteria inoculations stimulate the immune response, they left open the possibility that the animals responded not to the bacteria in the injection, but rather to the wound of the injection itself. To test this possibility, we sterilized the abdominal surface of adult males with alcohol and injected them with either sterile Drosophila Ringers (Ephrussi and Beadle, 1936), sterile 0.9% NaC1, or stabbed them with a sterile glass injection needle without injecting any fluid. Untreated males and males inoculated with bacteria served as controls. Figure 3 shows that sterile wounds induced the antibacterial system in some flies, although the response was augmented by bacteria. To determine the number of bacteria that are needed to raise the antibacterial activity above that of wounded controls, we made serial dilutions of an overnight bacterial culture, injected equal volumes into Ceratitus adult males, and tested their haemolymph 24 h later. Figure 4 shows that somewhere between 300 and 2000 bacteria per fly were needed to raise the activity level above that caused by a sterile wound. Increasing doses up to a million bacteria per animal resulted in gradually increasing responses. At even the highest dose inoculated, animals survived for at least a week after injection. To learn how long it takes Ceratitus to mount an immune response, we withdrew haemolymph at varilO 9 8

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Fig. 2. Antibacterial activity plotted against amount of blood loaded in well. All of the haemolymph collected from individual Ceratitus adult males or larvae or Drosophila adult males was loaded directly or diluted and tested by the inhibition zone assay. The diameter of the plaque is plotted vs the number of animals from which the blood came on a log scale. The diameter of the plaque is related to the concentration of antibacterial activity.

0

leman A

D

E

F

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Fig. 3. Antibacterial activity in inoculated and wounded animals. Animals were left untreated (A42), stabbed with a sterile injection needle (D-F), injected with sterile ringers (G-I) or 0.9% NaC1 (J-L) or inoculated with bacteria (M-O). The results show that sterile wounding induces the antibacterial immune response in Ceratitus adult males.

Adult

Larva

Control

Inoculated

Fig. 1. The inhibition zone assay for antibacterial activity. H a e m o l y m p h from untreated or bacteriainoculated larvae or adults was tested by the inhibition zone assay in wells cut in an agar plate seeded with streptomycin-resistant Escherichia coli. After the plate was incubated to allow for bacterial growth, bacteria-free plaques surround wells with antibacterial activity. Haemolymph from one animal was placed in each well. The results show that Ceratitus possesses an inducible antibacterial immune response.

control #

inoculated 6

3

6

1

1/2

A B

C

D Fig. 6. Isoelectric focusing of Ceratitus antibacterial factors. The haemolymph from 6 untreated control adult male flies or 6, 3, 1, or 2~ inoculated male flies was loaded on the gel. After isoelectric focusing, the gel was overlaid with bacteria. The clear plaques signal the location of an antibacterial factor.

93

adult larva

F

A B C D

E

Fig. 7. Isoelectric focusing patterns of antibacterial factors in adult and larval haemolymph. Three larvae or 3 adult male Ceratitus were inoculated and the next day their haemolymph proteins were separated by isoelectric focusing and the gel treated with a bacterial overlay.

Dro

Med

Mel

Dor

AB7.1

AB8.7 AB9.0 AB9.2

Fig. 8. Isoelectric focusing of antibacterial factors from adult males of four fly species. Haemolymph from 30 Drosophila melanogaster (Dro), 3 Ceratitis capitata (Med), 3 Dacus cucurbitae (Mel), or 3 D. dorsalis (Dor) was subjected to isoelectric focusing and the gel was overlaid with bacteria. The numbers at the side indicate the isoelectric points of the antibacterial proteins (ABs) from Drosophila.

94

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Fig. 4. Antibacterial activity plotted against number of bacteria injected. Adult male Ceratitus were inoculated with different dilutions of an overnight culture on Enterobacter. The next day their haemolymph was withdrawn and serially diluted and tested on the inhibition zone assay. Results are plotted in terms of activity units, defined as the amount of activity needed to clear a I mm ring around the 2.4 mm well. The results indicate that more bacteria induce a greater response. ous times after inoculation. The first response was detectable 3 h after inoculation and then antibacterial activity increased steadily for the next day (Fig. 5). Antibacterial activity was still strong 8 days after inoculation, the latest day tested. The rapid response suggests that extensive cell growth and differentiation are not required for immunity, but rather that antibacterial activity appears in the blood relatively directly (for example, by stimulating cells to synthesize and secrete antibacterial factors into the blood). Biochemical nature o f Ceratitus antibacterial immune response To determine the biochemical nature of the humoral immune response, we separated active antibacterial factors on isoelectric focusing gels and overlaid the gel with agar in which bacteria were embedded. Antibacterial proteins diffusing up into the agar overlay blocked bacterial growth. The results (Fig. 6) showed four different molecular forms with antibacterial activity (called A, B, C, and D), 50.0

37.5

2 5 . 0 --

1 2 . 5 --

0.0

i/~

hours

days

Fig. 5. Time-course of antibacterial activity. A group of adult male Ceratitus was inoculated and haemolymph was taken from two flies at each time point and pooled. Serial dilutions of haemolymph at each time point were tested in the inhibition zone assay.

95

having pIs between 8.5 and 9.5. Dilution of the blood showed that the different forms have different levels of activity in the order from strongest to weakest: B - C - A - D . Whether these forms represent the products of different genes, or are differently charged forms of the same gene product(s) remains to be tested. Since both larvae and adults respond to bacteria by producing antibacterial proteins, we wondered whether the same factors are produced at both life stages. Isoelectric focussing experiments (Fig. 7) showed that larvae possessed two factors not found in adults--band E, which is more basic than band D, and a band with a neutral pI, called F. This result suggests that there may be stage-specific expression of antibacterial response genes in Ceratitus as there is in Drosophila (J. Postlethwait, D. Kolstoe, I. Bach and K. Lodmell, unpubl.) and Manduca (Hurlbert et al., 1985). To test whether the fruit fly pests Dacus dorsalis Handel (the Oriental fruit fly) and Dacus cucurbitae Coquillett (the melon fly) possess an antibacterial system similar to Ceratitus and Drosophila, we inoculated adult male flies of all four species, collected haemolymph the next day, and separated the antibacterial factors by isoelectric focussing (Fig. 8). All four flies showed a potent antibacterial system including several antibacterial proteins with basic isoelectric points between 8 and 9.2. Nevertheless, the pattern of antibacterial activity was characteristic for each fly species. We conclude that Drosophila and Ceratitus will provide models with general applicability to other species of fruit fly that are serious agricultural pests. The source o f antibacterial factors To determine the cellular origin of the antibacterial immune response in Ceratitus, we inoculated larvae or adults and after 24 h cultured in Schneider's medium fat body, Malphigian tubules or brain from larvae and fat body, Malphighian tubules, testes, or muscle from adults. After incubating the organs for 7 h, we tested the culture medium for antibacterial activity in the inhibition zone assay. Antibacterial activity was detected only in the medium in which fat body was cultured; cultures of muscle, Malphighian tubules, brain and testes gave no detectable antibacterial activity. While the response was strongest with fat body from inoculated larvae and adults, none the less, medium from uninoculated control fat body also gave clearly detectable activity in some cases. In three separate experiments, cultures of fat body from inoculated animals gave 20, 14, and 1.7 units of activity for larvae and 0.0, 1.3 and 1.3 units for adults. The values for culture medium from uninoculated control of fat body was 4.0, 0.0 and 3.8 units for larvae and 0.0, 1.0 and 0.0 for adults. We conclude not only that the fat body is the origin of antibacterial factors in both larvae and adults, but that the wounding associated with dissecting out the organs is sufficient to stimulate the response. DISCUSSION These experiments demonstrate the presence of an antibacterial immune response in Ceratitus and two

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Boman H. G. and Hultmark D. (1981) Cell-free immunity in insects. Trends Biochem. Sci. 6, 306-309. Boman H. G., Nilsson I. and Rasmuson B. (1972) Inducible antibacterial defense system in Drosophila. Nature 237, 232-235. Boman H. G., Nilsson-Faye I., Paul K. and Rasmuson T. (1974) Insect immunity. I. Characteristics of an inducible cell-free antibacterial reaction in bemolymph of Samia cynthia. Infect. Immun. 10, 136. Courtice A. C. and Drew R. A. I. (1984) Bacterial regulation of abundance in tropical fruit flies (Diptera:Tephritidae). Aust. Zool. 21, 251-268. Drew R. A. I., Courtice A. C. and Teakle D. S. (1983) Bacteria as a natural source of food for adult fruit flies. (Diptera:Tephritidae). Oecologia 60, 279-284. Dunn P. E. (1985) Biochemical aspects of insect immunology. A. Rev. Ent. 31, 321-339. Ephrussi B. and Beadle G. (1936) A technique of transplantation for Drosophila. Am. Nat. 70, 218-225. Flyg C., Dalhammar G., Rasmuson B. and Boman H. (1987) Insect immunity: inducible antibacterial activity in Drosophila. Insect Biochem. 17, 153-160. Grtz P. and Boman H. G. (1984) Insect immunity. In Comprehensive Insect Physiology, Biochemistry, and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 3, pp. 454-485. Pergamon Press, Oxford. Hultmark D., Steiner H., Rasmuson T. and Boman H. (1980) Insect immunity: Purification and properties of three inducible bactericidal proteins from bemolymph of immunized pupae of Hylophora cecropia. Eur. J. Biochem. 106, 7-16. Hultmark D., Engstrom E., Bennich H., Kapar R. and Boman H. (1982) Insect immunity: Isolation and structure of Cecropia D and four minor antibacterial components from Cecropia pupae. Eur. J. Biochem. 127, 207-217. Hurlbert R., Karlinsey J. and Spence K. (1985) Differential synthesis of bacteria induced proteins of Manduca sexta larvae and pupae. J. Insect Physiol. 31, 205-215. Keppi E., Zachary D., Robertson M., Hoffmann D. and Hoffmann J. A. (1986) Induced antibacterial proteins in the hemolymph of Phormia terranovae (Diptera): Purification and possible origin of one of these proteins. Insect Biochem. 16, 395~,02. Kubo T., Komano H., Okada M. and Natori S. (1984) Identification of hemagglutinating protein and bactiricidal activity in the bemolymph of adult Sarcophaga peregrina on injury of the body wall. Dev. Comp. Imman. 8, 283-291. Okada M. and Natori S. (1983) Purification and characterization of an antibacterial protein from haemolymph of Sarcophaga peregrina (flesh-fly) larvae. Biochem. J. 211, 727-734. Okada M. and Natori S. (1985a) Ionophore activity of sarcotoxin I, a bactericidal protein of Sarcophaga peregrina. Biochem. J. 229, 453-458. Okada M. and Natori S. (1985b) Primary structure of sarcotoxin I, an antibacterial protein induced in the hemolymph of Sarcophaga peregrina. J. biol. Chem. 260, 7174-7177. Robertson M. and Postlethwait J. H. (1986) The humoral antibacterial response of Drosophila adults. Dev. Comp. Immun. 10, 167-179. Acknowledgements--This material is based upon work sup- Schneider I. and Presley M. (1978) Drosophila cell and ported by the Cooperative State Research Service, U.S. tissue culture. In The Genetics and Biology of Drosophila Department of Agriculture under Agreement No. GAM (Ed. by Ashburner M. and Wright T. R. F.), Vol. 2a, 8503043. We thank K. Lodmell for technical assistance and pp. 265-315. Academic Press, N.Y. Dr T. Landon and M. Robertson for comments on the Steiner L. F. and Mitchell LS. (1966) Tephritid fruit flies. manuscript. In Insect Colonization and Mass Production (Ed. by Smith C. N.), pp. 555-583. Academic Press, NY. Whitcomb R. F., Shapiro M. and Granados R. R. (1974) REFERENCES Insect defense mechanisms against microorganisms and parasitoids. In The Physiology of Insecta (Ed. by RockBoman H. G. (1986) Antibacterial immune proteins in stein M.), Vol. 5, pp. 447-536. Academic Press, New York. insects. Syrup. Zool. Soc. Lond. 56, 45-58.

other pest species of fruit fly. The Ceratitus antibacterial response is similar to that of other flies in several respects. First, it is inducible: haemolymph of a naive animal lacks antibacterial factors, while haemolymph of an animal inoculated with bacteria contains substances that block bacterial growth. Second, animals can respond to a sterile wound. This would seem to be advantageous since in nature, any wound is likely to introduce bacteria, and an animal that was genetically able to respond immediately to a wound without having to wait for bacteria to grow to a detectable level might have an advantage in surviving an infection. Third, the response is rapid, and lasts for days. The 3 h that intervene between inoculation and a detectable response are much more rapid than the vertebrate antibody-producing immune response, and so it is unlikely to require several steps of cell-cell interaction or proliferation of clones of cells. Fourth, the response does not seem to be specific for the species of infecting bacterium: inoculations were with Enterobacter but tests of activity were against Escherichia. And finally, the molecular nature of the antibacterial factors is similar to that of Drosophila, with several proteins in the range of pI 9 and another at about pI 7. These extensive similarities between the antibacterial immune response of Ceratitus and Drosophila suggest that Ceratitus has not evolved a special mechanism for protecting itself from the bacteria that it depends on for food (Drew et al., 1983; Courtice and Drew, 1984). Rather, Drosophila and Ceratitus use very similar mechanisms to combat bacterial infections, suggesting that the two species may be exposed to the same kinds of bacterial attack in the wild. The speed and magnitude with which Ceratitus responds to an invasion of bacteria, or even to just a breach of its cuticular line of defence by a wound suggest that the antibacterial system may play an important role in allowing fruit flies to combat bacterial infection in nature. Presumably flies that were genetically unable to m o u n t a rapid potent response would soon die of bacterial infections in the wild. The recent discovery of induced mutations in Drosophila that die when inoculated with a bacterial dose that is harmless to wild-type flies (D. Grace and J. Postlethwait, unpubl.) confirms this prediction. These discoveries suggest a powerful new approach to regulation of fruit fly pests that relies on inhibiting the insect antibacterial immune response. A strategy causing insect agricultural pests or vectors of diseases to acquire an immune deficiency syndrome and die could stimulate agriculture and improve human health. Further research on the mechanisms of the antibacterial immune system is needed before such strategies can be formulated.