INFECTION AND IMMUNITY, July 1974, p. 136-145Copyright 0 1974 American Society for Microbiology

Vol. 10, No. 1Printed in U.S.A.

Insect ImmunityI. Characteristics of an Inducible Cell-Free Antibacterial Reaction in

Hemolymph of Samia cynthia PupaeHANS G. BOMAN, INGRID NILSSON-FAYE, KERSTIN PAUL, AND TORGNY RASMUSON, JR.

Department of Microbiology, University of Umed, S-901 87 Umed, Sweden

Received for publication 5 March 1974

Pupae of the silk moth, Samia cynthia, were found to contain an inducibleantibacterial activity in their hemolymph. This immunity response was provokedby primary infections with either Escherichia coli K-12 or Enterobacter cloacae.In both cases the antibacterial activity was directed chiefly towards E. coli.During standard conditions, 1% of hemolymph could kill 10' to 10' viable E. coli,strain D31, within 5 min. A lower level of antibacterial activity was induced byinjections of a sterile salt solution. The killing of strain D31 followed single-hitkinetics, and increasing rate constants were obtained for increasing amounts ofhemolymph. The reaction was sensitive to pretreatment with trypsin and it wasprotected by reducing agents. The activity was inhibited by microgram quan-tities of lipopolysaccharide (LPS) prepared from certain LPS mutants of E. coliK-12. A comparison of the susceptibility showed that "heptose-less" LPS mu-tants were more sensitive to killing than other strains. During standard conditionshemolymph will lyse both E. coli and Micrococcus lysodeikticus. Lysis of E. colifollowed a multi-hit kinetics and it was inhibited by LPS, whereas lysis of M.lysodeikticus was unaffected by LPS. Hemolymph was fractionated on SephadexG-200, and the lytic activities were recovered in partly overlapping peaks.Reconstitution with pooled fractions gave synergistic effects with the killingassay.

Estimates indicate that the number of insectspecies may be close to a million and that thetotal insect population of the world may be aslarge as 1018 (38). For this reason alone, insectimmunity is a problem in its own right. Inaddition, studies of insect immunity may bemotivated by the facts that: (i) insects areknown to lack immunoglobulins and organtransplantations are unopposed by defensemechanisms like those in mammals (4, 9, 17);(ii) insects are known to transmit helminths,protozoa, bacteria, and viruses which causesevere human diseases (19); and (iii) microbialcontrol of pests is currently tried as an alterna-tive to chemical insecticides (8, 26). For each ofthese aspects, knowledge of insect immunitywould further both understanding and applica-tions.

Despite an extensive early literature (9, 17,33), relatively little is known about the molecu-lar mechanism behind immunity phenomena ininsects. Within the last decade, some authorshave studied the antibacterial activity of insecthemolymph (2, 10, 11, 15, 18, 21, 24, 27). Herewe characterize an inducible antibacterial ac-

tivity, which develops in pupae of the silk mothSamia cynthia after primary infections witheither Escherichia coli or Enterobacter cloacae.These bacteria are known to be associated withother silk moths and with the wax moth (33).Larvae of the latter animal have also been usedfor several studies of immunity (10, 11, 18, 21,24, 27, 31, 36). Pupae of large Lepidoptera seemto have received little or no attention, which isodd since the very stable diapause of animalslike S. cynthia make them excellent for labora-tory studies. In addition, the molting of the gutmay in some species make the immune systemof special importance in pupae (31).

MATERIALS AND METHODSBacteria. The main test organism used was E. coli

K-12, strain D31, which carries the amino acidmarkers given in Table 1 as well as A prophage. Thestrain is resistant to streptomycin and to ampicillindue to mutations in the strA and ampA genes,respectively. In addition, D31 carries two less-knownmutations, one closely linked to ampA, and one whichdeleted part of the glucose, galactose, and rhamnosefrom the core of the lipopolysaccharide (LPS) (25).Together they enhance the ampicillin resistance on

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TABLE 1. Origin, requirements, and antibiotic response of the main bacterial strains used

Amino acid Antibiotic responseOrganism Strain

r ment Reference or originrequirements Growth on No growth on

E. coli K-12a D31 Pro, Trp, His 80 Amp; 100 Str 30 Nal; 20 Kan (4)E. cloacae o11 None 100 Str; 10 Pen 80 Amp; 30 Nal Materials and MethodsE. cloacae ,12 None 30 Nal; 10 Pen 100 Str; 80 Amp Materials and MethodsP. aeruginosa OT97 His, Trp 20 Kan; 100 Str 80 Amp; 30 Nal From LoutitB. thuringiensis Btll Ser, Thr, Asp, Ile, 100 Str 80 Amp; 10 Pen Materials and Methods

His, Val, Met,Glu

a Other E. coli strains used are also resistant to streptomycin and sensitive to Nal and Kan. They are relatedto D31 as described elsewhere (5, 7). For selective growth on LA plates the respective antibiotic was added to thefinal concentration in micrograms per milliliter as indicated by numbers before the abbreviation. Except forBtll two alternative selections are given and abbreviations are as follows: D-ampicillin (Amp); streptomycin(Str); benzyl-penicillin (Pen); nalidixic acid (Nal); kanamycin (Kan). Amino acids are abbreviated as follows:aspartic acid (Asp); glutamic acid (Glu); histidine (His); isoleucine (Ile); methionine (Met); proline (Pro);serine (Ser); threonine (Thr); tryptophan (Trp); valine (Val).

plates to 80 mg/ml. Besides D31 we used also someother related LPS mutants for which pedigrees andother details are given in two reviews (5, 7). (Thesestrains have been deposited at the E. coli GeneticStock Center, Department of Microbiology, YaleUniversity School of Medicine, New Haven, Conn.,where they can be requested.) For the present experi-ments LPS was prepared by the method of Galanos etal. (13).The other bacteria used (Table 1) were obtained as

follows. The parental strain jt1 of E. cloacae wasisolated from a pile of waste bark near UmeA. Themutants 311 and 012 were isolated on LA plates (25)with streptomycin or nalidixic acid, respectively. Theparental strain of Bacillus thuringiensis var. gelechiaeAUCT is a non-crystal-forming strain obtained fromSebasta (32) but originating from Institute Pasteur.From the parent we isolated a clone able to grow on aglucose salt medium supplemented with the eightamino acids given in Table 1. This clone was used forthe isolation of a streptomycin-resistant mutant des-ignated Btll. Bacillus subtilis was a wild-type strainkept in our stock collection.

All strains were grown exponentially in a richmedium to a cell density of about 4 x 10' cells per mlby using conditions described before (25). The bacte-ria were harvested by filtration, washed twice on thefilter with 0.9% NaCl, and resuspended in NaCl toabout the same cell concentration (without any chill-ing). The bacteria were used as soon as possible afterharvest; just before an assay they were diluted 50 to100 times in the standard buffer (see below). Exceptfor B. subtilis the bacterial strains used were resistantto one or more antibiotics, properties which were usedfor selective plating with the antibiotic concentrationsgiven in Table 1. This procedure eliminates the lowlevel of bacterial contamination which could comefrom our semisterile standard assay (see below).

Standard assay for antibacterial activity. Unlessnoted otherwise, the standard buffer was 0.1 Msodium or potassium phosphate, pH 6.4, with 2 x 10'M dithiothreitol (DTT, "Cleland's reagent," SigmaChemical Co., St. Louis, Mo.). The final reaction

mixture contained normally 5 or 10 gliters of 10 to20-times-diluted S. cynthia hemolymph and 100 uli-ters of test bacteria (normally strain D31) diluted inthe standard buffer to give a final concentration ofabout 5 x 105 cells per 0.1 ml. (Reactions were carriedout in 1.5-ml conical tubes of polypropylene, carefullywashed and reused.) Using disposable capillary pi-pettes ("Microcaps," Drummond Scientific Co.,Broomall, Pa.), 5-uliter samples were withdrawn fromthe reaction mixture at different times and emptiedinto 1 ml of ice-cold 0.9% NaCl. This dilution andchilling effectively stopped the killing reaction. Thesamples were further diluted, transferred to 3 ml ofsoft agar at 45 C, and spread on LA plates which forstrain D31 were supplemented with 80 Ag of ampicil-lin or streptomycin per ml at a concentration of 100jgg/ml (25). After about 30 min a second layer of softagar was spread on each plate, a procedure whichallowed an accurate counting of up to 1000 coloniesper plate. Incubation conditions for plates were 15 to18 h at 37 C.

Lysis of bacteria. The substrate was either freshlygrown E. coli strain D31 or dried Micrococcuslysodeikticus obtained from Mann Research Labora-tories (catalogue no. 935). The cells were suspended inthe standard buffer, giving an absorbance of 0.3 to 0.5at 570 nm. Lysis was followed in 1-ml samples byusing continuous recording with a Beckman double-beam spectrophotometer, Acta CII. The referencecontained 1 ml of the same cell suspension. Thehemolymph added to the reaction mixture had asigniflcant absorption at 450 nm. At this wave lengthwe therefore added an equal volume of hemolymphfrom untreated pupae to the reference cell.

Insects. Most experiments were performed withchilled diapausing female pupae of S. cynthia ob-tained from C. M. Williams, Harvard University, butoriginating from American dealers. One batch ofpupae was raised in UmeA on the synthetic dietrecommended by Riddiford (28). The larvae werereared under short-day conditions (1OL: 14D) to pro-voke pupael diapause (C. M. Williams and L. M.Riddiford, personal communication). Temperature

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BOMAN ET AL.

was always around 25 C and humidity was 60 to 80%.The weight of the American female pupae was 2 to 3 g,whereas those raised in UmeA were only half the size.Pupae in diapause were stored in a refrigerator at 8 Cfor at least 10 weeks. During experiments all animalswere kept under long-day conditions (15L: 9D) at25 C and 60 to 80% humidity to terminate the dia-pause and cause initiation of adult development.

Small samples of S. cynthia hemolymph (5 to 25uliters) were taken up in capillaries directly fromincisions (2 to 4 mm) in the wings. Exhaustivebleedings were done as recommended by Schneider-man (30) by using a few crystals of 1-phenyl-2-thi-ourea (Eastman Kodak Co.. Rochester, N.Y.) toinhibit the phenolase. Such samples were centrifugedat 500 x g for 15 min before use.

Vaccination of insects. Pupae of S. cynthia werenormally vaccinated by injecting into the thorax 4 x105 E. coli, strain D31, or in a few experiments E.cloacae strains ,B11 or 012. An "AGLA" micrometersyringe (Wellcome Reagents Ltd., London) was usedand the volume was always 50 Aliters. Control animalswere given an equal dose of"W-saline." a salt mixturephysiological to Lepidoptera (35) (made up from fourparts of NaCl, 40 parts of KCI, 18 parts of MgCl2, andthree parts of CaCl2, the molarity of each of thesolutions being 0.15 M). Bacteria used for injectionwere diluted in W-saline, and care was taken thateach animal in an experiment was given an equalnumber of injections. At least 2 days were allowed forimmunity to develop.

RESULTS

Induction of S. cynthia antibacterialactivity. For Drosophila we have previouslyshown that a primary inf'ection (referred to as"vaccination") with E. cloacae induced a de-fense system which protected the flies against asecondary infection with Pseudomonasaeruginosa, an organism which otherwise wouldkill the flies (6). To explore the biochemistrybehind this protection it was important toreproduce the Drosophila experiment with alarger insect. We therefore took pupae of' S.cynthia, injected one group with E. cloacae(dose of about 3 x 106 viable bacteria per pupa)and the other group with a similar volume ofW-saline. Four days later all pupae were chal-lenged by an injection of' P. aeruginosa (dose ofabout 106 viable bacteria per pupa). Figure 1shows that this secondary infection was rapidlyeliminated from the hemolymph of pupae vac-cinated with E. cloacae, whereas in the controlpupae P. aeruginosa grew up to more than 109cells per ml of hemolymph. We also followed theconcentration of E. cloacae in the hemolymph,and Fig. 1 shows that the primary infection("vaccine") persisted longer than P. aeruginosa.This is in agreement with our earlier findings (6)and indicates that for reasons not yet known,

10

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0-J

9

8

7

6

5

4

3

2

P. aerugiosasec. infection -

0 2 3 4 5 678------0 1 2 3 4 5 6 7 8

Time (days)FIG. 1. Vaccination of S. cvnthia pupae with E.

cloacae and test for immunity bv a secondary infec-tion with P. aeruginosa. At the start of the experimentthree pupae were injected with E. cloacae (0), threecontrol pupae were given W-saline. On day 4 all pupaewere giuen injections uwith about 106 viable P.aeruginosa (control pupae, E; vaccinated pupae, 0).At the times indicated 5-gliter samples of hemolyvmphwere withdrawn from the pupae and assaved forviable bacteria by plating on media containing theselective antibiotics gilen in Table 1. Each individualpupa is represented by one point and the dotted linein bottom part of the figure indicates th,e lowest levelof bacteria that could be detected.

the primary infecting organism became lesssusceptible to host defense than was the strainused for secondary infection.

Similar experiments have shown that theantibacterial activity in the hemolymph couldbe induced also by infections with E. coli strainD31 despite the fact that the organism wasrapidly eliminated from the hemolymph. Induc-tion of immunity could occur in developingpupae of both sexes as well as in diapausingpupae. However, no significant immunizationwas found with living P. aeruginosa or B.thuringiensis, which both gave fatal infections.In vitro assay of antibacterial activity with

E. coli strain D31. As test organism we selectedE. coli strain D31, an ampicillin- and strep-tomycin-resistant LPS mutant, hoping that ourprevious knowledge of the strain (5, 7, 25) mightbe helpful for understanding the antibacterialactivity. Our aim was to find conditions underwhich in vitro killing of strain D31 could be usedas an assay. Preliminary experiments showed

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that reactions performed at room temperatureand pH 6.4 with reducing agents like DTTrequired only 1% hemolymph to give 99% killingin less than 5 min. By using the procedure givenin details in Materials and Methods we com-pared the antibacterial activity of pupaetreated in different ways.

Significant antibacterial activities were in-duced both by living bacteria (E. coli strainD31) and by a sterile physiological salt solution(W-saline) (Fig. 2). The response obtained withbacteria was always larger and lasted longerthan the corresponding effect of W-saline. Im-mune responses with salt solutions have beenreported for other systems (15, 24) and can beexplained as wound effects created by theinjections. This interpretation is consistantwith the observation that in vivo W-saline gaveno protection against infections (cf. Fig. 1).However, further experiments are needed toinvestigate whether the difference in Fig. 2 isonly quantitative or also qualitative.

106

E0

'a.0

z

.0

to

0

E

z

105

104

103

102

0 1 3 6

Incubation time (min)

FIG. 2. In vitro assay of the antibacterial activit,yof 1% of hemolymph from S. cynthia. The testorganism was E. coli, strain D31, and the reactionconditions are given in detail in Materials andMethods. Symbols: pupa vaccinated with living E.coli strain D31, 0; pupa injected with an equalvolume of sterile W-saline, A; untreated pupa, 0.

The left part of Fig. 3 shows that at the onsetof the reaction the rate of killing followed anapparent single-hit kinetics. However, with lowconcentrations of hemolymph the rate of thereaction decreased with increasing times. Withincreasing amounts of hemolymph in the reac-tion mixture there was an increase in the killingrate. Assuming single-hit kinetics, we have inthe right part of Fig. 3 plotted the rate constantsobtained from the data in the left part versusthe amount of hemolymph in the reactionmixture. With an initial concentration of 3.4 x105 viable bacteria per 0.1 ml, there was anear-linear relationship between the reactionconstants, although the line does not extrapo-late to zero.

Properties of the antibacterial reaction.The assay for antibacterial activity uses as"substrate" whole living bacteria. Although thereaction may involve one or more insect en-zymes, the sensitivity of the "substrate" makesit impossible to use many of the biochemicalreagents frequently employed for the character-ization of enzymatic reactions. However, inorder to optimize the sensitivity of the assayand possibly to discriminate between differentreactions in a multicomponent system, we in-vestigated the factors listed in Table 2. Theresults indicate that the defense system wasnoncellular, that at least one component waslost during a dialysis, that a reducing agent likeDTT offered some protection, and that at leastone component was sensitive to pretreatmentwith trypsin. A comparison of LPS obtainedfrom three interrelated strains showed that LPStypes, which have lost parts of the core polysac-charide (25), were better inhibitors than intactLPS from wild-type E. coli K-12.

Species and strain specificities of the kill-ing reaction. Mammalian immunity is charac-terized by a narrow specificity in the defensemechanism induced. Although previous inves-tigators have emphasized that insect immunitydoes not have a similar specificity (9, 17),referees of an earlier manuscript demanded thatwe investigate this point in detail. We havetherefore compared the killing of four differentbacteria by using 5% of hemolymph from twodifferent pupae, one vaccinated with E. colistrain D31 and the other with E. cloacae strain,B12. Figure 4 shows that in both of the pupaethe antibacterial activity was directed primarilyagainst E. coli. The killing of the three otherbacteria was hardly significant. However, weknow from in vivo experiments like that in Fig.1 that both P. aeruginosa and E. cloacae areeliminated in vaccinated pupae. Since the ratiobetween hemolymph and injected bacteria is

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105 a0E

007

' 0 w006

eo.o

.62%~~~~~~~~~~

103 c~~~~~~~~~~~~~~~

0 2 3 4. 0 0.4 0.8 12 16 20

Incubalson lime(min) Haemolymph in reachion(%/)

FIG. 3. Antibacterial activity obtained with differ-

ent concerntrations of hemolymph from pupa vac-

cinated with living E. coli strain D31. Left, Number of

viable bacteria (strain D31) at different times; right,

calculated rate constants versus final concentration of

hemolymph in reaction mixture. For the reaction with

0.960hemolymph the point at 4 mm is not shown. A

control with bacteria and no hemolymph is included

in the left part (open circles).

vastly different in vivo, we performed an experi-ment with 95% hemolymph. During such in

vitro conditions both P. aeruginosa and E.

cloacae were slowly killed. However, further

investigations are required to determine

whether the humoral immunity here studied

can fully account for the in vivo protection

against P. aeruginosa.

The only gram-positive bacterium includedin Fig. 4, B. thuringiensis, was neither killed in

vitro nor in vivo. We have therefore also testedB. subtilis and found that with 5% of hemo-

lymph there was 95% killing within 5 mn. Thisreaction was unaffected by LPS from strainD31.

Table 2 shows that the inhibitory power of

LPS was affected by the carbohydrate composi-

tion of the polysaccharide part. We have there-

fore compared a number of our LPS mutants (7,

25) with respect to their susceptibility to killing

by 1% of hemolymph (Fig. 5). Together withother results it indicates that our E. coli K-12

strains could be classified as follows. The leastsusceptible group contained strains with a nor-mal LPS, the parental strain D21, as well as our

envA mutant D22, which is highly permeable toa number of antibiotics (5). A group of moder-

ately sensitive strains, which have lost some

carbohydrates in their LPS, included our com-mon test organism, strain D31 as well as the

lpsA mutant D21e7. The most susceptible groupcontained our two "heptose-less" mutants,D31m4 and D21f2, both of which have lost allheptose, glucose, galactose, and rhamnose in

their LPS (7, 25). We have recently shown thatLPS mutants have defects in the barrier func-tion of their outer membranes (7). The differ-ence illustrated in Fig. 5 could therefore be dueto an LPS dependent difference in the uptake ofthe antibacterial activity.

Lytic action of hemolymph. Earlier inves-tigators have reported lytic activities both ininsect hemolymph (10, 15, 24, 27) and in the gut(39). We therefore investigated whether hemo-lymph from S. cynthia pupae vaccinated withE. coli, strain D31, could also lyse strain D31and M. lysodeikticus. In both cases we foundlytic action. With E. coli and low concentra-tions of hemolymph, there was a lag period be-fore any detectible lysis occurred (Fig. 6). Thisis consistent with a multi-hit kinetics and withthe interpretation that a number of chemicalbonds have to be split before the bacteria startlysing.The lysis of E. coli did not normally go to

completeness. The addition of more hemo-lymph (indicated by the arrows) reinitiatedboth the killing and the lytic reactions (Fig. 7).

TABLE 2. Properties of S. cynthia antibacterialactivity

Relative killing ofTreatment of hemolymph strain D31a

After 1 min After 5 min

Centrifugation (600 g/10 min) 1 1Dialysis (4 h) ............... 1 4 x 10'Preincubation without DTT

(45 min) ................. 8 x 102 7 x 102Pretreatment with trypsin

(15 min) ................. 1 x 10-2 1 x103Trypsin added after 1 min ... 0.2D21 LPS (1 ,g/ml) ......... 0.3 0.2D31 LPS (1 Ag/ml) ......... 0.2 6 x 10-2D31m4 LPS (1 Ag/ml) ....... 0.2 6 x 10- 2

a Relative killing was defined as the ratio betweenthe number of viable bacteria in the control and thenumber of viable bacteria in the treated sample usingstandard assay conditions. All viable count valueswere interpolated from time curves as in Fig. 2.Dialysis was against water with 2 x 10' M DTT. Inthe pretreatment experiment trypsin concentration inthe reaction was 100 Ag/ml; when added after 1 min itwas 1 mg/ml. In the latter experiment relative killingafter 1.5 min was 1. LPS was added to the hemolymph15 min before the start of the reaction. LPS fromstrain D21 contains in the outer part of the core poly-saccharide, heptose, glucose, galactose, and rham-nose. D31 LPS has lost part of the glucose, galactose,and rhamnose. D31m4 is a "heptose-less" mutantwith an LPS lacking heptose, glucose, galactose,and rhamnose (25).

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10-1 .0-

TEST BACTERIAe \ E. coli strain D31 .

Ent. cloacae o c

P. 10-B. thuringiensis *

10'\3 10-3

1 2 3 4 1 2 3 4

Incubation time (min)

FIG. 4. Susceptibility of different bacteria to theantibacterial activity in hemolymph (5%) from pupaevaccinated with E. coli strain D31 (left) and E.cloacae strain ,11 (right). The test organisms were E.coli strain D31 (0), E. cloacae strain ,11 (0), P.aeruginosa strain OT97 (-), and B. thuringiensisstrain Btll (A).

go

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FIG. 5.ent LPS r

activity o(0) has Ltaining hstrain D3and galacglucose atD31m4 (Cand rham

0.

EC

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00.12.

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0.30.0

0 1 2 3 4 S

I ncubat ion time (min)FIG. 6. A continuous recording of the lysis of E.

coli strain D31 with hemolymph in the concentra-tions indicated in the figure. Reactions were carriedout in a Beckman spectrophotometer, Acta CII (27 to28 C) by using the same conditions as for the killingreaction. The initial concentration of bacteria wasabout 2 x 107 cells per ml, which gave an extinction of0.430. The reference was a similar bacterial suspen-sion to which was added hemolymph from an un-treated pupa.

ei This pattern of kinetics is consistent with eithera rate-limiting component being consumed or

D31 with product inhibition of an enzymatic reac-tion. The killing reaction proceeded faster thanthe lysis and there was no activity in anuntreated pupa (Fig. 7).

Since the lytic action can be expected to setfree significant amounts of LPS, we examinedthe reaction for LPS inhibition. Lysis of E. coli

D31 m4 was susceptible to LPS inhibition, whereas lysisof M. lysodeikticus was insensitive to the samehigh concentration of LPS (Fig. 8). The results

.______________________________ imply that the lyses of the two bacteria were dueto separate enzymes.

0 1 2 3 4 5 6 7 Gross fractionation of S. cynthiahemolymph. If the antibacterial activity inhemolymph were due to a multicomponent

Comparison of the susceptibility of differ- system, it would ultimately have to be sepa-nutants of E. coli K-12 to the antibacterial rated and characterized by reconstitution ex-

of 1% of S. cynthia hemolymph. Strain D21 periments. Should the complexity be large, one

,PS with an outer core polysaccharide con- might expect separation on ion exchanger to

eptose, glucose, galactose, and rhamnose; give loss of activity. On the other hand, some

1 (0) has lost part of its glucose, rhamnose, . .ltose; strain D21e7 (4O) has lost part of its actlvity could be expected after a moderately

nd all of its galactose and rhamnose; strain resolving fractionation by gel filtration. So far

O) has lost all its heptose, glucose, galactose, these predictions have come true. We lostnose. almost all activity during separation attempts

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0

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to.0

W

z

it

4,.0'anU4,

Ez

107

106

105

1040 5 10 15 2(

Incubation time (min)FIG. 7. Simultaneous recording of Iysis

and the killing (broken line) of E. coli susing 1% of hemolymph from a pupa vaccistrain D31 (0) and from a control pupa wpretreatment (0). After 10 min (as indicaarrows), a fresh sample of hemolymph wa

the reaction mixtures. In this experiment, twithdrawn were 1 jliter (in other expE,uliters), which increased the pipetting errence and other conditions were as in Fig. f

0

c

o 0.05in

_

qs 0.1

0 2 3 4 5 0 20 40 60

Incubalion time (min)

FIG. 8. Lysis of E. coli (left) and of M. lysodeik-ticus (right) at two different concentrations of hemo-lymph (0.8%, 0; 1.6%, 5). To the higher concentra-tion was added LPS from strain D31 (M)-to a finalconcentration of 50 qg/ml. The reference used con-tained onlv bacteria.

on anion or cation exchangers with celluloseskeletons. However, Sephadex G-200 resolvedhemolymph into two main peaks, which werewell separated (Fig. 9). In an intermediary zone,we could identify the lysing activities against E.coli and M. lysodeikticus, respectively.

In comparable fractionations we assayed alsofor the killing of strain D31. For those experi-ments the eluant from the same column as inFig. 9 was pooled approximately as follows.

Sample A with the yellow pigment of thehemolymph was 20 to 40 ml; sample B was 41 to57 ml; sample C with most of the antibacterial

1.0 activity was 58 to 68 ml; sample D was 69 to 770.5 E ml; and sample E was 78 to 93 ml. With these

c fractions we made several reconstitution experi-0.2 0 ments. A synergistic effect could be demon-0.1 strated by combining samples B and E (Fig.

e 10). Fraction A had neither any activity alone0.05 t nor in combination with other fractions. Differ-

C ent combinations with the other fractions gave0.02 n~ in most cases some synergistic effects, but the

0 magnitude fluctuated from experiment to ex-< periment. These variations could have been due

both to difficulties in making reproducible cutsin the eluate and to a marked instability of theisolated fractions.

DISCUSSIONIs the antibacterial activity of insect

(solid line) origin? Up to now the following experimentstrain D31, imply that the activity studied was produced bynated with the pupae and not the living bacteria used as)ithout anY vaccine: (i) E. coli strain D31 or our E. cloacae.ted by the mutants can be used either as vaccine or as testts added to organism in vivo (6); (ii) no killing was observederimentss when these strains used were grown in mixed*ors. Refer- cultures; (iii) when injected into pupae, E. coli6ors. Referstrain D31 was rapidly eliminated from the

hemolymph. However, pretreatment of thepupae with actinomycin D allowed strain D31 to

o In grow up in the hemolymph (manuscript inpreparation). This drug does not affect E. colisince it cannot normally penetrate the outer

0l membrane of gram-negative bacteria. Since in

n ~the pupae actinomycin D will block RNA syn-

02va,C)a

6

A2oL y, o y

o

0 10 20 30 40 50 60 70 80 90o

FIG. 9. Fractionation of 1.0 ml of S. cynthia hemo-lymph obtained from two pupae vaccinated with E.coli strain D31. The column (44 by 1.35 cm) withSephadex G-200 (Pharmacia, Uppsala, Sweden) wasequilibrated with 0.1 M potassium phosphate buffer,pH 6.4, with 2 x 10- M ,3-mercaptoethanol. Theeluant from the column was assayed for absorbance at280 nm (0) for lysis of E. coli strain D31 (U, and forlysis of M. lysodeikticus (A). Both determinations ofbacterial lyses were made after incubation of 0.1 ml ofsample ar.d 1 ml of cell suspension at 37 C for 45 min.

0%-I --0 Normal

0° Pu pa

A450,

Vacc. pupae \,.

142 INFECT. IMMUNITY

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to

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10-3

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FIG. 10. Antibacterial activitystrain D31 in single fractions (A, A

E, 1) and the combined fractiobtained from a column experimshown in Fig. 9. The fractions weri

the text. For C there was an axshown.

thesis, the expression of immuthis biosynthetic activity. Estype of experiment makes it v4

antibacterial factors here studorigin.

S. cynthia system and thepupae of some insect species, tof the immunity may be to d4against invasions of the naticould occur during moltings.risks may come from transitiorsystem (37). If so, this wouldlack of specificity and the factterial activity here recordedcantly higher than previouslyvae of Drosophila (2, 3) or G(9, 18).For several reasons it is

compare our results with othetigators have rarely document(bacterial assays give proportiresults can therefore not be ev

tively. Our Fig. 3 shows thatstrain D31 there was proporti2% of hemolymph. Lysis as rec

8 is only suitable as a semiquantitative assaybut has the advantages of being fast and con-

E venient. Our results with different strains (Fig.A 4 and 5) clearly show that comparative results

can be obtained only if the same bacterial strainis used as test organism. The fact that strain

D , D31 is well characterized (5) together with itshigh susceptibility makes it a suitable testorganism. We suggest that it would be a usefulstandard reference for comparing results ob-tained in different laboratories.We have so far assayed immunity in more

than 100 individual pupae of S. cynthia, treatedin a variety of ways. In vaccinated pupae wecould normally predict the killing of strain D31by a factor of 2 to 10. This reproducibility maybe satisfactory considering the fact that theanimals are not inbred. We have encounteredcertain problems in storing hemolymph andsometimes lost most of the activity even duringa rapid freezing. Despite these complicationsthe high antibacterial activity found in vacci-nated pupae of S. cynthia makes them a very

10 1 5 good material for the study of insect immunity.

rn e ( m i n )Mechanism of the bacterial killing. Earlier

literature contains different judgements aboutv against E. coli the role of agglutination and phagocytosis ini; B,E3; C, U; D, V; insects (15, 16, 18, 29, 36), and a hemagglutinat-ons B+E (0), all ing protein has been isolated from a marinee pooled asgiven in invertebrate (23). By using hemolymph frome pooledaspgient int vaccinated pupae, we have so far not detected

any microscopic evidence for agglutination.Table 2 shows that a large dose of trypsin (1

nity must require mg/ml) added after 1 min only gave a small;pecially the last decrease in the relative killing after 5 min.Dry likely that the There was no detectible reversing of the killingied were of insect reaction. These experiments therefore make it

unlikely that agglutination contributes to theassays used. In rate of killing observed.

,he main function The insect lysozymes induced by heat-killedefend the animal bacteria (27) may either be part of the defenseural flora, which against gram-positive bacteria or function asEspecially large chitinases in the repair of the exoskeleton (22).

as of the digestive The fact that gram-negative bacteria normallyexplain both the are resistant to egg-white lysozyme makes itthat the antibac- unlikely that closely related enzymes (27) couldmay be signifi- be the only factor responsible for the killing ofreported for lar- E. coli. Moreover, we have shown that the

alleria mellonella killing and lysis of E. coli was inhibited by LPS,whereas the activity against B. subtilis and M.

very difficult to lysodeikticus (Table 2 and Fig. 8) was unaf-Drs. Earlier inves- fected by LPS. It is therefore unlikely thated that their anti- lysozymes of the type purified from Bombyxonality and their mori and G. mellonella (27) can fully accountaluated quantita- for the immunity in S. cynthia.for the killing of In all our killing experiments with strain D31,ionality with 1 to we initially observed single-hit kinetics (Fig. 2corded in Fig. 6 to and 3). This is contrary to the lysis of strain

B+E

C

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BOMAN ET AL.

D31, where low hemolymph concentrations gavea lag (Fig. 6) compatible with a multi-hitmechanism. We have so far been unable toseparate the killing and the lytic activitiesagainst strain D31, and the lysis could thereforebe either a late stage of a repeated killingreaction or a late step in a sequence of reactionsleading to the elimination of the bacteria.Sense of the LPS inhibition. We have here

shown two effects of LPS on the antibacterialdefense system of S. cynthia. (i) When hemo-lymph was preincubated with LPS with areduced core polysaccharide, there was a pro-nounced inhibition of the bacterial killing(Table 2). (ii) Bacterial strains with such LPSwere more susceptible to killing than the paren-tal strain with wild-type LPS (Fig. 5). Both ofthese effects are consistent with the hypothesisthat one or more components of the defensesystem have a strong affinity for either lipid Aor the innermost core of the LPS molecule.From a biological point of view it may look

senseless for a host to have a defense systeminhibited by LPS. However, Andersson et al. (1)have shown that LPS in vitro can induce thesynthesis of immunoglobulins in mammalianlymphocytes. Chadwick and Vilk (11) havereported that large doses of crude LPS in waxmoth larvae can induce an immunity lastingabout 48 h. In mammals LPS is also known tohave an anticomplementary activity (14), andthis fact has been used for tracing complement-like systems in some in vertebrates (12, 20).Although some of our results could indicate thatS. cynthia has a complement-like system, moredata are required to justify such a conclusion.We have sometimes encountered difficulties

in reproducing Chadwick and Vilk's immuniza-tion with LPS (11) by using S. cynthia pupaeand our own LPS samples prepared by themethod of Galanos et al. (13). Disregardingfactors like possible species differences (21), thediscrepancy could be resolved by one or more ofthe following assumptions. (i) The active signalin Chadwick and Vilk's experiments was eitheran impurity or a degradation product of LPS.(ii) As suggested for mammals (34), two signalsmay be needed and LPS (or lipid A) may beonly one of the signals. (iii) The moleculeinhibited by LPS could be part of a feedbacksignal which turns on the gene system needed.The last assumption is in consonance with theobservation that a low dose of LPS (0.1 ,ug) inwax moth larvae could induce a large increasein hem6cytes (31). Clearly, separation and char-acterization of the components of the hemo-lymph as well as an effective immunizationwith "dead" material are required for a better

understanding of the antibacterial activity andthe LPS effect.

ACKNOWLEDGMENTS

We thank Carroll Williams for showing us how insectpupae can be used as "one-test-tube animals," for helpfuldiscussions, and for most generously supplying us with pupaeof S. cynthia. Some preliminary experiments were carried outat The Biological Laboratories, Harvard University, whileH.G.B. was the holder of an Eleanor Roosevelt Fellowshipfrom The International Union Against Cancer. The work inUmea was supported by grants from The Swedish NaturalScience Research Council (Dnr 2453) and The SwedishCancer Society (project no. 157).

LITERATURE CITED

1. Andersson, J., 0. Sjoberg, and G. Moller. 1972. Inductionof immunoglobulin and antibody synthesis in vitro bylipopolysaccharides. Eur. J. Immunol. 2:349-353.

2. Bakula, M. 1970. Antibacterial compounds in the cell-free haemolymph of Drosophila melanogaster. J. InsectPhysiol. 16:185-197.

3. Bakula, M. 1971. The isolation of intracellular antibacte-rial activity from Drosophila melanogaster larvae. J.Insect Physiol. 17:313-319.

4. Bernheimer, A. W., E. Caspari, and A. D. Kaiser. 1952.Studies on antibody formation in caterpillars. J. Exp.Zool. 119:23-35.

5. Boman, H. G., S. Jonsson, D. Monner, S. Normark, andG. D. Bloom. 1971. Cell-surface alterations in Esche-richia coli K-12 with chromosomal mutations changingampicillin resistance. Ann. N.Y. Acad. Sci.182:342-357.

6. Boman, H. G., I. Nilsson, and B. Rasmuson. 1972.Inducible antibacterial defence system in Drosophila.Nature (London) 237:232-235.

7. Boman, H. G., K. Nordstrom, and S. Normark. 1974.Penicillin resistance in Escherichia coli K12: syner-gism between penicillinases and a barrier in the outerpart of the envelope. Ann. N.Y. Acad. Sci. 235:569.

8. Bulla, L. A. (ed.). 1973. Regulation of insect populationsby microorganisms. Ann. N.Y. Acad. Sci. 217:1-243.

9. Chadwick, J. S. 1967. Serological responses of insects.Fed. Proc. 26:1675-1679.

10. Chadwick, J. S. 1970. Relation of lysozyme concentrationto acquired immunity against Pseudomonas aeruginosain Galleria mellonella. J. Invertebr. Pathol. 15:455-456.

11. Chadwick, J. S., and E. Vilk. 1969. Endotoxin fromseveral bacterial species as immunizing agents againstPseudomonas aeruginosa in Galleria mellonella. J.Invertebr. Pathol. 13:410-415.

12. Day, N. K. B., H. Gewurz, R. Johannsen, J. Finstad, andR. A. Good. 1970. Complement and complement-likeactivity in lower vertebrates and invertebrates. J. Exp.Med. 132:941-950.

13. Galanos, C., 0. Liideritz, and 0. Westphal. 1969. A newmethod for the extraction of R lipopolysaccharides.Eur. J. Biochem. 9:245-249.

14. Galanos, C., E. T. Rietschel, 0. Luderitz, and 0. West-phal. 1971. Interaction of lipopolysaccharides and lipidA with complement. Eur. J. Biochem. 19:143-552.

15. Gingrich, R. E. 1964. Acquired humoral immune responseof the large milkweed bug, Oncopeltus fasciatus (Dal-las) to injected materials. J. Insect Physiol. 10:179-194.

16. Harshbarger, J. C., and R. M. Faust. 1973. Environmen-tal factors internal to the host that affect the success ofmicrobial insecticides. Ann. N.Y. Acad. Sci.217:131-140.

17. Heimpel, A. M., and J. C. Harshbarger. 1965. Sympo-sium on microbial insecticides. V. Immunity in insects.Bacteriol. Rev. 29:397-405.

144 INFECT.IMMUNITY

on March 2, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

INSECT ANTIBACTERIAL FACTORS

18. Hink, W. F., and J. D. Briggs. 1968. Bactericidal factorsin haemolymph from normal and immune wax mothlarvae, Galleria mellonella. J. Insect Physiol.14:1025-1034.

19. James, M. T., and R. F. Harwood. 1969. Herms' medicalentomology. McMillan Publ. Co., New York.

20. Johannsen, R., R. S. Anderson, R. A. Good, and N. K.Day. 1973. A comparative study of the bactericidalactivity of horseshoe crab (Limulus polvphemus) he-molymph and vertebrate serum. J. Invertebr. Pathol.22:372-376.

21. Kamp, H. 1968. Untersuchungen zur humoralenImmunitat bei Pyrrhocoris apterus L. und Galleriamellonella F. Z. Vergl. Physiol. 58:441-464.

22. Landureau, J. C., and P. Jolles. 1970. Lytic enzyme

produced in vitro by insect cells: lysozyme or chitin-ase? Nature (London) 225:968-969.

23. Marchalonis, J. J., and G. M. Edelman. 1968. Isolationand characterization of a hemagglutinin from Limuluspolyphemus. J. Mol. Biol. 32:453-465.

24. Mohrig, W., and B. Messner. 1968. Immunreaktionen beiInsekten. I. Lysozym als grundlegender antibakteriellerFaktor im humoralen Abwehrmechanismus der Insek-ten. Biol. Zentralbl. 87:439-470.

25. Monner, D. A., S. Jonsson, and H. G. Boman. 1971.Ampicillin-resistant mutants of Escherichia coli K-12with lipopolysaccharide alterations affecting matingability and susceptibility to sex-specific bacterio-phages. J. Bacteriol. 107:420-432.

26. Norris, J. R. 1971. Microbes as biological control agents,p. 197-229. In D. E. Hughes and A. H. Rose (ed.),Microbes and biological productivity. Cambridge Uni-versity Press, Cambridge.

27. Powning, R. F., and W. J. Davidson. 1973. Studies on

insect bacteriolytic enzymes I. Lysozyme in hae-molymph of Galleria, mellonella and Bombyx mori.Comp. Biochem. Physiol. 45B:669-686.

28. Riddiford, L. M. 1968. Artificial diet for Cecropia and

other saturmiid silkworms. Science 160:1461-1462.29. Ryan, M., and W. L. Nicholas. 1972. The reaction of the

cockroach Periplaneta americana to the injection offoreign particulate material. J. Invertebr. Pathol.19:299-307.

30. Schneiderman, H. G. 1967. Insect surgery, p. 753-766.In F. H. Wilt and N. K. Wessels (ed.), Methods indevelopmental biology. Thomas Y. Crowell Co., NewYork.

31. Schwalbe, C. P., and G. M. Boush. 1971. Clearance of51Cr-labeled endotoxin from hemolymph of activelyimmunized Galleria mellonella. J. Invertebr. Pathol.18:85-88.

32. Sebasta, K., K. Horska, and J. Vankova. 1969. Isolationand properties of the insecticidal exotoxin of Bacillusthuringiensis var. gelechiae AUCT. Collection Czecho-slov. Chem. Commun. 34:891-900.

33. Steinhaus, E. A. 1967. Insect microbiology. Hafner Pub-lishing Company, New York.

34. Watson, J., E. Trenkner, and M. Cohn. 1973. The use ofbacterial lipopolysaccharides to show that two signalsare required for the induction of antibody synthesis. J.Exp. Med. 138:699-714.

35. Weevers, R. de G. 1966. A lepidopteran saline: effects ofinorganic cation concentrations on sensory reflex andmotor responses in a herbivorous insect. J. Exp. Biol.44:163-175.

36. Werner, R. A., and J. C. Jones. 1969. Phagocytic hae-mocytes in unfixed Galleria mellonella larvae. J.Insect Physiol. 15:425-437.

37. Wigglesworth, V. B. 1972. The principles of insect physi-ology, p. 436-437, 7th ed. Chapman and Hall, London.

38. Williams, C. B. 1960. The range and pattern of insectabundance. Amer. Natur. 94:137-151.

39. Zacharuk, R. Y. 1973. Ultrastructural changes in themidgut epithelium of an elaterid larva (Coleoptera)infected enterically with Pseudomonas aeruginosa.Can. J. Microbiol. 19:811-821.

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