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FEMSRE-0301-0047R1.R1.
Exploi ing he po en ial o Insec s o in i o
pa hogenici y es ing o Mic obial pa hogens.
Ke in Ka anagh and Eme P. Ree es
Na ional Ins i u e o Cellula Bio echnology, Depa men o Biology, Na ional
Uni e si y o I eland, Maynoo h, Co. Kilda e, I eland.
Keywo ds: Humo al immuni y; Inna e immune esponse; Insec ; in i o pa hogenici y
es ing.
Co esponding au ho : D . K. Ka anagh, NICB, Depa men o Biology, NUI
Maynoo h, Co. Kilda e, I eland.
T: 353 - 1- 708 3859. Fax: 353 - 1- 708 3845. Email: [email p o ec ed].
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CONTENTS
1.0. Abs ac
2.0. In oduc ion
3.0. The Insec Immune Sys em
3.1 The Cu icle
3.2. The Haemolymph
4.0. Cellula Elemen s in he Haemolymph
4.1. Phagocy osis
4.2. Nodulisa ion
4.3. Encapsula ion
5.0. Humo al Immuni y
5.1. Clo ing Mechanisms
5.2. Melanisa ion.
5.3. An i-mic obial Pep ides.
6.0. The use o Galle ia mellonella as a model o mic obial i ulence es ing.
6.1. Inocula ion o G. mellonella la ae.
7.0. Conclusion
8.0. Re e ences.
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1.0 ABSTRACT
Con en ional assays o quan i ying he i ulence o mic obial pa hogens and
mu an s ha e adi ionally elied upon he use o a ange o mammalian species. A
numbe o wo ke s ha e demons a ed ha insec s can be used o e alua ing mic obial
pa hogenici y and p o ide esul s compa able o hose ha can be ob ained wi h
mammals since one componen o he e eb a e immune sys em, he inna e immune
esponse, emains simila o ha ound in insec s. La ae o he G ea e Wax Mo h
Galle ia mellonella ha e been used o e alua e he i ulence o a ange o bac e ial and
ungal pa hogens and a co ela ion wi h he i ulence o hese mic obes in mice has been
es ablished. This e iew highligh s he simila i ies o he e eb a e and insec inna e
immune esponses o in ec ion and iden i ies he po en ial use o insec s o he in i o
e alua ion o he mic obial pa hogenici y.
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2.0 INTRODUCTION
Insec s a e one o he mos success ul and geog aphically widesp ead g oups o
animals on Ea h. They a e ound in almos e e y habi a - he main excep ion being he
oceans, and ha e succeeded in colonising niches inaccessible o, o unusable by, o he
animal li e o ms. Conse a i e es ima es sugges ha he e a e 750,000 species o insec
bu in eali y his igu e may be close o 1,000,000 - making hem he mos di e se
animal li e o m [1]. They a e also he mos abundan wi h as many as 1018 indi iduals a
any one ime [2].
F om an e olu iona y pe spec i e insec s and e eb a es di e ged app oxima ely
500 million yea s ago howe e many aspec s o hei physiology emain simila [3]. The
inna e immune sys em, unlike he adap i e immune sys em [4, 5] o insec s and
mammals, sha e a high deg ee o s uc u al and unc ional homology [1, 6]. In pa icula ,
a numbe o ea u es o he inna e immune esponse a e common o mammals and insec s
[7, 8] and analysis o insec esponses o pa hogens can p o ide an indica ion o he
e eb a e esponse o in ec ion. [7, 9]. Since he inna e immune esponse is he main line
o de ence in e eb a es agains many mic obial pa hogens [10], much e o has been
ocused on examining he mammalian and insec esponses o mic obial in ec ion and a
s ong co ela ion be ween bo h sys ems has been demons a ed [6].
The s udy o insec immunology has been desc ibed as he 'poo ela ion' o
immunology since i was assumed ha he as numbe s o insec s and hei apid a es o
ep oduc ion ob ia ed he need o a sophis ica ed and inely uned immune sys em.
Howe e , we a e now beginning o app ecia e he e icacy o he insec esponse o
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in ec ion [6]. Close s udy o he insec immune esponse demons a es a highly adap ed
and e ec i e sys em ha is capable o dealing wi h a wide ange o bac e ial, ungal and
p o ozoan pa hogens and wi h mic obial loads ha would p o e a al o e eb a es [1, 3].
In ecen yea s he e has been a ecogni ion o he homology be ween he insec and
mammalian inna e immune sys ems and ha a knowledge o he insec esponse o
in ec ion could p o ide aluable in o ma ion in o he unc ioning o he mammalian
inna e sys em [6]. The e may also be an ul e io mo i e in lea ning mo e abou he
insec immune esponse since his could be used o design mo e e ec i e o no el
insec icides ha unc ion by inhibi ing he insec immune esponse o mic obial
pa hogens [11].
3.0 THE INSECT IMMUNE SYSTEM
3.1 The cu icle
The i s line o de ence in insec s agains he majo i y o pa hogens is he cu icle,
which se es a unc ion analogous o he skin in mammals. The cu icle is a s uc u ally
and chemically complex ba ie designed o p e en o e a d he en y o pa hogens in o
he haemoceol ( he body ca i y) [12]. The ou e laye o he cu icle ( he epicu icle) is
co e ed in a waxy laye con aining lipids, a y acids and s e ols, which may display an i-
mic obial p ope ies [13]. The cu icle i sel consis s o chi in ib ils embedded in a

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p o ein ma ix. The in ac cu icle p e en s en y o mic obial pa hogens bu once i is
up u ed by inju y o deg ada ion he e is an inc eased chance o in ec ion [14]. The
lesion may be plugged and subsequen ly epai ed o es o e he s uc u al and unc ional
in eg i y o he cu icle. Inju y o he cu icle ac i a es he humo al immune esponse,
which leads o he p oduc ion o cec opins and a acins, which display an i-bac e ial
ac i i y.
3.2 The Haemolymph
The insec body ca i y o haemocoel con ains haemolymph, which se es a
unc ion analogous o blood in mammals in ha i anspo s nu ien s, was e p oduc s
and signal molecules [15] al hough i plays no ole in espi a ion. In addi ion,
haemolymph con ains cells and an i-mic obial pep ides capable o immobilising and
killing in ading mic oo ganisms [2, 16]. The olume o haemolymph wi hin an insec
a ies be ween species and e en wi hin a species depending upon he de elopmen al
s age o he indi idual insec [1]. The insec immune esponse o mic oo ganisms has
been shown o in ol e a change in he ci cula ing haemocy e popula ion and syn hesis o
new haemolymph p o eins [17]. The haemolymph is he main si e o he immune
esponse o mic oo ganisms. The inna e immune esponse consis s o cellula and
humo al mechanisms ha a e igh ly in e connec ed.
4.0 CELLULAR ELEMENTS IN HAEMOLYMPH.
The insec haemolymph con ains haemocy es, which unc ion in a simila manne
o phagocy es o mammals. The majo i y o haemocy es ci cula e eely wi hin he
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haemolymph bu a signi ican numbe (up o 30% in some insec species) can be ound
associa ed wi h in e nal o gans such as he a body, achea o diges i e sys em [1]. The
haemocy e densi y a ies upon in ec ion and in he case o Galle ia mellonella in ec ed
wi h S ine ema nema odes he e is a dec ease in haemocy e numbe s by 80% in he i s 4
hou s ollowed by a g adual inc ease o e he nex 12 hou s o 160% o he con ol.
Subsequen ly, numbe s decline o less han 5% o he con ol haemocy e densi y [15].
The ini ial decline in haemocy e numbe s has been a ibu ed o he o ma ion o clumps
consis ing o haemocy es and in ading mic obe [18]. The subsequen ise in hei
numbe s is hough o be due o he elease o haemocy es bound o in e nal o gans.
A leas six ypes o haemocy es ha e been iden i ied in lepidop e ous (e.g.
Galle ia mellonella) al hough mo e ypes may exis in o he species [3] (Figu e 1). P ice
and Radcli e [19] classi ied haemocy es as p ohaemocy es, plasma ocy es, g anulocy es
(g anula cells), coagulocy es, sphe ulocy es and oenocy oids. Al hough a la e s udy by
B ehelin [20] p o ided an al e na i e classi ica ion con aining nine g oups. P ohemocy es
(6-13µM in diame e ) a e small ounded cells wi h la ge nuclei, which di ide and may
di e en ia e in o o he cell ypes (Figu e 1). Plasma ocy es (40-50µM) and g anulocy es
(45µM) a e he p edominan phagocy ic cells. Plasma ocy es con ain lysosomal enzymes
and a e he mos abundan cell ype. G anulocy es possess a ela i ely small nucleus and
g anule ich cy oplasm. Sphe ulocy es a e o al o ound cells (25µM) wi h a ying
numbe s o small sphe ical inclusions. Oenocy oids a e la ge, binuclea e, nonphagocy ic
cells which may con ain p ophenoloxidase. Coagulocy es ha e also been e med hyaline
hemocy es and a e in ol ed in he clo ing p ocess. Adipohemocy es a e cha ac e ised by
he p esence o a d ople s (Figu e 1).
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The plasma ocy es and g anulocy es pa icipa e in phagocy osis, nodule o ma ion
and encapsula ion [21] which a e impo an elemen s o he insec 's cellula de ence
agains bac e ia and unicellula ungi [22]. Wi hin D osophila melanogas e ,
plasma ocy es pa icipa e, o some ex en , in he syn hesis o an imic obial pep ides
du ing he humo al esponse [23, 24] and assume he unc ion o phagocy osis o mic o-
o ganisms [25] while lamellocy es and c ys al cells play espec i e oles in encapsula ion
and melaniza ion o la ge in ude s [26].
4.1 Phagocy osis
In mammalian cells phagocy osis equi es ecogni ion wi h subsequen
engul men o pa icles such as pa hogens ha accumula e du ing in ec ion and
in lamma ion [27] and is mainly pe o med by mig a ing p o essional phagocy es de i ed
om he myeloid cell line. They a e he neu ophils and monocy es ha ci cula e in he
blood, and issue esiding mac ophages [28]. Phagocy osis equi es sequen ial signal
ansduc ion e en s, which lead o he ec ui men o he phagocy e o he si e o in ec ion
ollowed by ecogni ion o he pa icle as o eign [29] and he subsequen inges ion
wi hin a phagosome.
While he p ocess o phagocy osis in insec s is no ully unde s ood ecep o s on
he su ace o plasma ocy es and g anulocy es a e simila o ecep o s on mammalian
phagocy es [2]. The insec p o eins mal olio and dSR-C1 show a high deg ee o
homology o mouse na u al esis ance associa ed mac ophage p o ein-1 (NRAMP-1).
D osophila melanogas e p o eins pe oxodasin and C oquemo [30, 31], ollow he
classical dis ibu ion o mac ophages as desc ibed by Tepass and colleagues [32] and
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may media e he b eakdown o apop o ic cells, simila ly o mu ine mac ophage p o eins
[2].
The p ocess o phagocy osis in insec s and mammals appea s o be e y simila .
In bo h cases he e is he binding o opsonic ligands o he su ace o he pa icle which is
hen ollowed by ecogni ion by speci ic ecep o s. An in acellula cascade esul s in he
in e nalisa ion o he o eign body. I was o iginally hough ha only plasma ocy es
we e in ol ed in phagocy osis o o eign ma e ial in G. mellonella howe e ecen wo k
has demons a ed ha g anula cells a e also in ol ed [21]. Ac i a ion o he
p ophenoloxidase (PPO) cascade is equi ed o g anula cells o bind o non-sel ma e
and conduc phagocy osis while calcium is equi ed o he adhe ence o plasma ocy es.
Phagocy osis is a lec in-media ed p ocess and lec ins a e ound in he insec haemolymph
along wi h lysozyme - an an i-mic obial pep ide usually associa ed wi h he humo al
esponse. Lysozyme has been ound wi hin haemocy es and he in a-haemolymph le els
o lysozyme and lec in inc ease upon in ec ion indica ing ha hese ac syne gis ically
upon he p ocess o phagocy osis [33]. Upon in asion wi h g am-nega i e bac e ia N-
ace ylglucosamine (GlcNA) speci ic lec ins (BDL-2 lec ins) ecognise and bind o
pep igoglycans on he bac e ial cell su ace. These bind o plasma ocy es and acili a e
phagocy osis. A he same ime lysozyme deg ades he pep idoglycan laye eleasing
suga s and exposing echnoic acid and lipomannans which a e ecognised by BDL-1
lec ins. This p ocess gi es he insec he abili y o ecognise and engul a ange o
bac e ia despi e he changing na u e o he exposed bac e ial su ace [33]. This also
indica es how he cellula and humo al a ms o he inna e immune sys ems co-ope a e in
comba ing in ec ion.
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5.3 An i-mic obial pep ides.
Al hough he cellula and humo al esponses so a desc ibed a e e ec i e in
comba ing mic obial in asion hey a e unable o o ally clea he haemocoel i a la ge
numbe o mic oo ganisms en e . The las line o de ence is he syn hesis o a ange o
an i-mic obial pep ides, which a e eleased in o he haemolymph whe e hey a ack
elemen s o he bac e ial o ungal cell wall [1]. These pep ides play a c ucial ole in
comba ing in ec ion and simila classes o p o eins a e ound in e eb a es, in e eb a es
and plan s [2, 6]. (Table 2).
The main si es o syn hesis o an i-mic obial pep ides in he insec a e he a
body, haemocy es, he diges i e ac , sali a y glands and he ep oduc i e ac . The a
body unc ions as a biosyn he ic o gan, is analogous o he li e in mammals and is also a
si e o binding o many haemocy es [65]. A numbe o pep ides a e p oduced and all a e
amphipa ic basic molecules ha ac in a de e gen -like manne on cell memb anes
causing he dea h o he mic oo ganism by lysis. An i-mic obial pep ides a e syn hesised
as p e-p op o eins a a a e up o 100 imes as e han IgM in mammals [65]. Thei small
size allows di usion h ough he haemolymph o coun e ac in ading pa hogens.
In humans he an imic obial neu ophil p o eins a e loca ed wi hin in acellula
g anules which a e eleased in o newly o med phagocy ic acuoles. The p o eins and
pep ides s o ed in he g anules a e o wo kinds: 1) hose wi h cy o oxic p ope ies,
including bac e icidal/pe meabili y-inc easing p o ein, azu ocidin and de ensins, and 2) a
ange o enzymes, capable o con ibu ing o he des uc ion o killed bac e ia by
diges ing hei mac omolecules [66, 67]. Among hese a e lysozyme, p o einases, some

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wi h independen an imic obial ac i i y (elas ase and ca hepsin G), nucleases, and
saccha idases (Table 2). Enzymes deg ading bac e ial phospholipids [68] and
lipopolysaccha ides (LPS) [69] a e also known o be g anule associa ed.
A acins display a ela i ely na ow spec um o an i-bac e ial and an i- ungal ac i i y
and a e belie ed o ac on he ou e memb anes o mic obial cells [17]. I appea s ha he
p ima y unc ion o a acins may be o acili a e he ac ion o lysozyme and cec opins
he eby allowing he h ee immune p o eins o wo k in conso .
P oline- ich pep ides, Glycine- ich pep ides and Dip e icins: P oline ich pep ides a e
small, 15-34 esidues and be ween 2 - 4 kDa [7]. These we e i s isola ed in la ae o
Pho mia e ano ae [70]. O he examples o such pep ides include abaecin and he
apidaecins om honey bees and o he hymenop e a and d osocin om D osophila.
These pep ides appea o unc ion by inc easing memb ane pe meabili y o bac e ia and
lyse g am-nega i e bac e ia. Glycine- ich pep ides a e 9-30 kDa and a e ac i e agains
g am-nega i e bac e ia. Dip e icins a e only ound in dip e in species and a e induced by
and ac i e agains g am-nega i e bac e ia.
Lysozyme is a 14.4kDa (pI>10) ca ionic p o ein wi h he abili y o kill a wide
ange o G am-posi i e bac e ia, by i ue o i s abili y o hyd olyze cell wall
componen s. I is p esen in bo h azu ophilic and speci ic g anules o human neu ophils
and is also ound in he g anules o monocy es and mac ophages, in blood plasma, ea s,
sali a and ai way sec e ions. Lysozyme is ex emely ac i e agains such bac e ia as
Bacillus sub ilis, B. mega e ium [71, 72] and Mic ococcus lysodeik icus, indeed, he
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suscep ibili y o his la e o ganism o lysozyme o ms he basis o a labo a o y assay o
his enzyme [73]. Bac e ial cell walls consis in gene al o linea polysaccha ide chains
con aining epea ing uni s o N-ace ylglucosamine and N-ace ylmu amic acid esidues in
β-1-4 linkage. Lysozyme hyd olyses β-(1,4) glycosidic bonds in pep idoglucan o
bac e ial cell wall, is p o einaceous in na u e wi h insec lysozyme possessing a high
deg ee o simila i y wi h mammalian lysozymes [2]. Lysozymes in insec s a e 14kDa
p o eins [2], may be ound in haemos a ic cells [1] and we e he i s an i-bac e ial ac o
pu i ied om insec haemolymph [74]. Lysozyme has been loca ed in he gu o se e al
insec s, in haemocy es o Spodop e a e idania and Locus a and in haemocy e cell lines
[3]. While lysozyme displays an i-bac e ial ac i i y i appea s o wo k in combina ion
wi h cec opins and a acins [17].
LPS-binding p o eins. In insec s, bac e ial LPS-binding p o ein acili a es he clea ance
o bac e ia by p omo ing nodule o ma ion. Smoo h s ains o E. coli a e clea ed slowly
om la ae o Bomyx mo i since hey possess 0-speci c polysaccha ides which p o ec
he lipid A binding si e. In con as ough s ains a e clea ed wi hin 30 minu es by nodule
o ma ion and ha e no polysaccha ides p o ec ing he ele an binding si e [75].
T ans e in: In insec s ans e in has an i on binding domain in he N- e minal egion
and may unc ion by seques e ing i on om pa hogens hus inhibi ing hei g ow h [65].
Human lac o e in is a membe o he ans e in amily and displays an imic obial
p ope ies agains G am-posi i e and G am-nega i e bac e ia [76] by limi ing he
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a ailabili y o en i onmen al i on [77]. Howe e , since i on-sa u a ed lac o e in is also
able o kill ce ain bac e ia, mechanisms o he han i on deple ion a e in ol ed [78].
De ensins: Ano he impo an g oup o an imic obial pep ides is he g oup o be a-shee
de ensins ha comp ise ou membe s in humans: HNPI-HNP4 [79]. De ensins in insec s
a e an i-mic obial ca ionic pep ides o 4 kDa and 40 esidues long which play an
impo an ole in inna e and adap i e immuni y [80]. In insec s, de ensins show ac i i y
agains g am-posi i e bac e ia and some g am-nega i e species [2]. De ensins a e
cys eine ich ca ionic pep ides con aining h ee o ou disulphide b idges and ep esen
an ea ly de ence agains in ading mic oo ganisms. De ensins may be p oduced wi hin 3
hou s o in ec ion and hei le el declines 12-36 hou s pos -in ec ion sugges ing a
co ela ion be ween exp ession and p esence o bac e ia. De ensins ac on he
cy oplasmic memb ane o bac e ia and lyse cells by o ming ol age-dependen ion
channels, which lead o leakage o po assium and o he ions [7]. The widesp ead
occu ence o de ensins in highe animals and mo e dis an de ensin ela i es in plan s
[81] and insec s [82] is consis en wi h an ea ly e olu iona y o igin [79, 83]. In i o
s udies e eal he mic obicidal ac i i y o de ensins agains a a ie y o bac e ia,
including S aphylococcus au eus, Pseudomonas ae uginosa and Esche ichia coli, many
ungi, and some i uses [84 -86].
Cec opins a e ac i e agains g am posi i e and nega i e bac e ia [87] and a e
app oxima ely 4kDa wi h 35 - 39 esidues. Cec opins a e amphipa hic molecules ha
pene a e bac e ial cell walls esul ing in po e o ma ion and subsequen ion leakage [65].
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6.0 THE USE OF Galle ia mellonella AS A MODEL FOR MICROBIAL
VIRULENCE TESTING
The use o mammals o e alua ing he i ulence o mic obial pa hogens has
p o ided much in o ma ion on he unc ioning o he immune sys em bu such es s can
be ime consuming, labou in ensi e and expensi e in e ms o pu chasing animals and
he need o p o ide eed and housing acili ies. In addi ion, he e is a legal equi emen o
ob ain pe mission o wo k wi h animals and o pe o m such p ocedu es. O e he las
wo decades he e has been a g owing in e na ional awa eness o he need o educe he
use o mammals o ou ine p oduc and mic obial i ulence es ing [88]. Al e na i e
sys ems ha could p o ide compa able da a wi hou he need o use mammals o in i o
es ing would be use ul and desi able o e alua ing mic obial pa hogenici y. The use o
cell, issue and o gan cul u es has been adop ed in many ins ances bu he numbe o
animals used o such es s emains high. An al e na i e sys em ha would be mo e cos
e ec i e han mammalian es ing, e hically accep able and p o ides compa able da a
would be o g ea bene i .
The s udy o he esponse o insec s o in ec ion was pionee ed by Pas eu in he
nine een h cen u y who used insec s o demons a e he mic obial o igin o disease in Silk
mo hs. Mo e ecen ly he esponse o insec s o en omopa hogenic nema odes and hei
endosymbio ic bac e ia has been s udied in an e o o op imise he use o nema odes o
he con ol o insec pes s in he ield [87]. The la ae o G. mellonella ha e also been
used o e alua e he pa hogenici y o P o eus mi abilis [16], Esche ichia coli and
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Bacillus ce eus [22]. D. melanogas e and Spodop e a li o alis ha e been used o
de e mine he oxici y o seconda y me aboli es o Penicillium species [89].
Gi en he ole o he inna e immune esponse in p o ec ing mammals om
mic obial in ec ion [10, 90] and he high deg ee o simila i y ha exis s be ween he
mammalian and insec inna e immune esponses [2, 6], s udying he insec esponse o
in ec ion may p o ide compa able da a o hose which may be ob ained using mammals.
The G ea e Wax Mo h, G. mellonella, is being used inc easingly as a model o
assessing he i ulence o a ange o mic oo ganisms. G. mellonella belongs o he o de
Lepidop e a and he amily Py alidade. G. mellonella is a pes o beehi es eeding upon
pollen and des oying he combs o weak o diseased hi es. The la ae a e dull whi e in
colou , abou 3 cm in leng h, weigh app oxima ely 0.3 - 0.5g and unde go a
me amo phosis o gi e a g ey mo h. La ae o G. mellonella can be pu chased om a
a ie y o comme cial sou ces and can be main ained o a ac ion o he cos associa ed
wi h mammals used in con en ional in i o pa hogenici y es ing.
The abili y o G. mellonella la ae o de ec di e ences in he pa hogenici y o
lipopolysaccha ide-de icien mu an s o Pseudomonas ae uginosa has been demons a ed
[52], and a good co ela ion exis s be ween he i ulence o P. ae uginosa in Galle ia
la ae and in mice [91]. La ae o G. mellonella ha e been used o assess he i ulence o
Bacillus hu ingiensis and Bacillus ce eus and s ong ag eemen has been es ablished
be ween he esul s ob ained in insec s and mice [92]. In an examina ion o he esponses
o plan s, nema odes, insec s and mice o a P. ae uginosa PA14 poN mu an [93] i was
de e mined ha i ulence in nema odes was educed bu , in e es ingly, ha i ulence in
plan s, insec s and mice was unimpai ed sugges ing ha poN does no egula e i ulence

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genes equi ed o in ec ion in a ange o hos s. The insec in his case was G. mellonella
and he esul sugges s ha simila i ulence genes may be equi ed o in ec ion in
insec s and mice. La ae o G. mellonella ha e ecen ly been employed o de e mine he
ela i e i ulence o C. albicans isola es and o di e en ia e be ween pa hogenic and non-
pa hogenic yeas species [94]. A posi i e co ela ion be ween he i ulence o C. albicans
mu an s when es ed in G. mellonella and in BalbC mice has been es ablished [95]. The
abo e examples demons a e he po en ial o using G. mellonella as a model o
e alua ing mic obial pa hogenici y and show how compa able da a may be ob ained
using species as di e se as G. mellonella and mice.
6.1 Inocula ion o G. mellonella la ae.
La ae o G. mellonella a e easy o inocula e ia injec ion in o he haemocoel
h ough he las le p o-leg [94]. The base o he p o-leg can be opened by applying
gen le p essu e o he sides o he leg and his ape u e will e-seal a e emo al o he
sy inge needle wi hou lea ing a sca . Inocula ion o la ae wi h es mic oo ganisms
mus be accompanied by inocula ion o la ae wi h he bu e used o e-suspend he es
mic oo ganisms o ensu e ha his has no a ec on la al iabili y. A numbe o wo ke s
also sugges he 'mock-inocula ion' o a numbe o la ae pe expe imen o ensu e ha
he handling and inocula ion p ocedu es a e no dele e ious o he heal h o he la ae [87,
94]. La ae can be s o ed a 15oC p io o use and, once inocula ed, may be main ained a
empe a u es up o 37oC as long as app op ia e con ols a e implemen ed o quan i y he
23
e ec o empe a u e on su i al. La ae should be handled wi h ca e as ough handling
a ec s su i al and also leads o he exp ession o s ess p o eins.
End poin s ha may be used wi h G. mellonella la ae include he pe cen age
su i al a di e en ime poin s ollowing inocula ion [94], he luc ua ions in he
haemocy e densi y [15, 18] and he exp ession o an i-mic obial p o eins in esponse o
in ec ion [2]. La al dea h is he easies pa ame e o measu e whe eas he o he end-
poin s a e mo e labou in ensi e bu may gi e insigh s in o he immune esponse(s) o he
insec o challenge wi h sub-le hal doses o mic o-o ganisms o o inocula ion wi h
pa hogens o low o a enua ed i ulence.
7.0 CONCLUSION.
The inna e immune esponse in insec s sha es many common ea u es wi h ha in
mammals [6] and as a esul an inc easing numbe o wo ke s a e e alua ing mic obial
i ulence in insec s as well as in mammals. While he insec immune esponse is no as
complex as he mammalian esponse and lacks he acqui ed esponses o he mammalian
sys em, he ac ha la ge elemen s o he inna e immune esponse a e simila in bo h
mammals and insec s [2, 6, 10] opens he possibili y o using insec s o model he inna e
immune esponse o pa hogens in o de o ine- une subsequen expe imen a ion in
mammals. The use o insec s may allow he compa ison o he e ec o speci ic
24
pa hogens in insec s and mice bu i does no acili a e he s udy o many o he disease
p ocesses ha may be speci ic o mammalian issue. Consequen ly, he use o insec s may
be limi ed o quan i ying he al e a ions in i ulence o mic obial cells o mu an s a he
han elucida ing he p ocesses in ol ed in disease p og ession and dissemina ion in
mammals. While i is ex emely unlikely ha he use o insec s will eplace he need o
mammals o in i o pa hogenici y es ing i does o e he possibili y o e ining
mammalian expe imen a ion so ha he leas numbe o mammals a e used wi h
addi ional da a being supplied by s udying he inna e immune esponses o insec s.
25
8.0 REFERENCES.
[1] Ra cli e, N. (1985). In e eb a e immuni y - a p ime o he non-specialis .
Immunol. Le s. 10, 253-270.
[2] Vilmos, P. and Ku ucz, E. (1998). Insec immuni y: e olu iona y oo s o he
mammalian inna e immune sys em. Immunol. Le s. 62, 59-66.
[3] Boman, H.G. and Hul ma k, D. (1987). Cell- ee immuni y in insec s. Ann. Re .
Mic obiol. 41, 103-126.
[4] Klein, J., (1997). Homology be ween immune esponses in e eb a es and
in e eb a es: does i exis ? Scand. J. Immunol. 46, 558-564.
[5] A ala-Cha es M and Sequei a T. (2000). Is he e any adap i e immuni y in
in e eb a es? Aquacul u e. 191, 247-258.
[6] Salze , M. (2001). Ve eb a e inna e immuni y esembles a mosaic o in e eb a e
immune esponses. TRENDS Immunol. 22, 285-288.
[7] Ho man, J. (1995). Inna e immuni y o insec s. Cu . Opin. Immunol. 7,4-10.
[8] Fallon, A. and Sun, D. (2001). Explo a ion o mosqui o immuni y using cells in
cul u e. Insec. Biochem. Molec. Biol. 29, 965-972.
[9] Kimb ell, DA. and Beu le , B. (2001). The e olu ion and gene ics o inna e
immuni y. Na . Re . Gene . 2, 256-267.
[10] Le y, J.A. 2001. The impo ance o he inna e immune sys em in con olling HIV
in ec ion and disease. TRENDS Immunol. 22, 312-316.
[11] Cla kson, J.M. and Cha nley, A.K. (1996). New insigh s in o he mechanisms o
ungal pa hogenesis in insec s. T ends Mic obiol. 4, 197-203.
[12] Cla kson, J., Sc een, S., Bailey, A., Cobb, B. and Cha nley, K. (1998). Fungal
pa hogenesis in insec s. Chap e 6 (p83-93) in Molecula a iabili y o ungal pa hogens.
Eds. B idge, P., Cou eaudie , Y. and Cla kson, J.). Publ: CAB In e na ional.
[13] Lecuona, R., Clemen , J.M., Riba, G., Joulie, C. and Jua ez, P. (1997). Spo e
ge mina ion and hyphal g ow h o Beau e ia sp. on insec lipids. J. Econ. En omol. 90,
119-123.
[14] Tee o -Ba sch, A. and Robe s, D.W. (1983). En omogenous Fusa ium species.
Mycopa hologia 43: 423-429.
32
[81] B oekae , W.F., Te as, F.R., Cammue, B.P. and Osbo n, R.W. (1995). Plan
de ensins: no el an imic obial pep ides as componen s o he hos de ence sys em. Plan
Physiol. 108, 1353-1358.
[82] Ho man, J.A. and Reichha , J.M. (1997). D osophila immuni y. T ends Cell Biol.
7, 309-316.
[83] Ganz, T. and Leh e , R.I. (1995). De ensins. Pha macol. The . 66, 191-205.
[84] Ganz, T., Sels ed, M.E., Szkla ek, D. Ha wig, S.S., Dahe , K., Bain on, D.P. and
Leh e , R.I. (1985). De ensins. Na u al pep ide an iobio ics o human neu ophils. J. Clin.
In es . 76, 1427-1435.
[85] Leh e , R.I., Ba on, A., Dahe , K.A., Ha wig, S.S., Ganz, T. and Sels ed, M.E.
(1989). In e ac ion o human de ensins wi h Esche ichia coli. Mechanism o bac e icidal
ac i i y. J. Clin. In es . 84, 553-561.
[86] Leh e , R.I., Lich ens ein, A.K. and Ganz, T. (1993). De ensins: an imic obial and
cy o oxic pep ides o mammalian cells. Annu. Re . Immunol. 11, 105-128.
[87] Dunphy, G. and Webs e , J. (1984). In e ac ion o Xeno habdus nema ophilus subsp.
nema ophilus wi h he haemolymph o Galle ia mellonella. J. Insec . Physiol. 30, 883-
889.
[88] Balls, M. (1999). Science wi hou guinea pigs. RTD In o m. 24, 26-28.
[89] Pa e son, R.P., Simmonds, M.S. and Blaney, W.M. (1987). Mycopes icidal e ec s o
cha ac e ised ex ac s o Penicillium isola es and pu i ied seconda y me aboli es on
D osophila melanogas e and Spodop e a li o alis. J. In e . Pa hol. 50, 124-133.
[90] Romani, L. 1999. Immuni y o Candida albicans: Th1, Th2 cells and beyond. Cu
Opin Mic obiol. 2:363-7.
[91] Jande , G., Rahme, L. and Ausbel, F. (2000). Posi i e co ela ion be ween i ulence
o Pseudomonas ae uginosa mu an s in mice and insec s. J. Bac e iol. 182, 3843-3845.
[92] Salami ou, S., Ramisse, F., B ehelin, M., Bou gue , D., Gilois, N., Gomine , M.,
He nandez, E. and Le eclus, D. (2000). The plcR egulon is in ol ed in he
oppo unis ic p ope ies o Bacillus hu ingiensis and Bacillus ce eus in mice and insec s.
Mic obiol. 146, 2825-2832.

33
[93] Hend ickson, E., Plo niko a, J., Mahajan-Miklos, S., Rahme, L. and Ausbel, F.
(2001). Di e en ial oles o he Pseudomonas ae uginosa PA14 poN gene in
pa hogenici y in plan s, nema odes, insec s and mice. J. Bac e iol. 183, 7126-7134.
[94] Co e , G., Doyle, S. and Ka anagh, K.(2000) De elopmen o an insec model o
he in i o pa hogenici y es ing o yeas s. FEMS Immunol. Med. Mic obiol. 27: 163 -
169.
[95] B ennan M, Thomas DY, Whi eway, M and Ka anagh K. (2002) Co ela ion
be ween i ulence o Candida albicans mu an s in mice and Galle ia mellonella la ae.
FEMS Immunol. Med. Mic obiol. 34, 153-157.
34
LEGENDS TO FIGURES
Figu e 1. Haemocy e ypes in ol ed in cellula immune esponse.
Linea ma u a ion p ocess in Lepidop e a (closed a ow) o di e en ia ion o
p ohemocy es in o di e en cell ypes (open a ows).
Figu e 2. Diag amma ic ep esen a ion o ac i a ion o he espi a o y bu s and possible
oxidan gene a ion du ing phagocy osis by neu ophils.
Mic oo ganisms a e phagocy osed and encapsula ed wi hin a phagocy ic acuole.
NADPH oxidase is ac i a ed and accep s elec ons om NADPH o educe O2 o O2
-
(supe oxide). The cy oplasmic g anules mig a e o he phagocy ic acuole and discha ge
hei con en s. Myelope oxidase (MPO) en e s he acuole and u ilises H2O2 in he
p esence o chlo ide, o gene a e hypochlo ous acid (HOCl). Hyd oxyl adical (.OH)
o ma ion wi hin he acuole may occu by a numbe o mechanisms. O2
- in e ac s wi h
ni ic oxide (NO.) o gene a e pe oxyni i e (ONOO-). The o he g anule enzymes (e.g.
de ensins, p o eases) can a ack he phagocy osed mic oo ganism.
Figu e 3. A diag amma ic ep esen a ion o he simila i ies be ween D osophilia and
human Toll cascades.
Signalling h ough Toll and Cac us p o eins esul ing in ac i a ion o Do sal o
Di , pa allels signalling induced by Toll/I-κB and ac i a ion o NF-κB. In insec s,
mic obicidal pep ide syn hesis is con olled by spae zle, he ex acellula Toll ligand,
in ol ing he gene casse e spae zle/Toll/cac us/Di (illus a ed by dashed line).TL 2 and
TL 4 a e he bes s udied in humans as he co- ecep o o LPS, he o he co- ecep o
being CD14 leading o he exp ession o p o-in lamma o y cy okines. Abb e ia ions
used: LBP; lipopolysaccha ide binding p o ein, I-κB; inhibi o y κB, NF-κB; nuclea
ac o κB.
Table 1. P ope ies o eac i e oxygen species.
35
P oposed ROI and RNI p oduc ion in mammalian phagocy es (*) and in cells o he
insec haemolymph (+).
Table 2. A compa ison o humo al and cellula PPRs, and an i-mic obial pep ides and
enzymes in humans and insec s.
An ibac e ial ac i i y (AB) and an i ungal ac i i y (AF)
36
Table 1. Ka anagh and Ree es.
Supe oxide: O2 + e- + H+ -> O2
- + H+
* + Supe oxide is bo h a one-elec on educ an and a one-
elec on oxidan , wi h limi ed le els o biological
ac i i y. Does no ha e di ec oxic e ec s on a ge s,
bu a he exe s i s oxici y by con e sion o o he ROI.
Hyd ogen pe oxide: O2
- + O2
- + 2H+ -> O2 + H2O2
* + Reac s wi h heme p o eins and pe oxidases o ini ia e
adical eac ions and lipid pe oxida ion. Memb ane
pe meable. Reac s wi h educed i on and coppe sal s o
supe oxide o gene a e hyd oxyl adicals.
Hyd oxyl adical: Fe2
+ + H2O2 -> Fe3
+ + OH- + .OH
*Ex emely eac i e wi h mos biological molecules.
In ol ed in mic obicidal and cy o oxic eac ions,
causes DNA modi ica ions and b eaks.
Hypochlo ous acid: H2O2 + Cl- + H+ (p esence o MPO) -> H2O + HOCl
*Hypochlo ous acid induced dea h occu s e y apidly
and is 100 o 1000 imes mo e e ec i e han H2O2.
S ong non adical oxidan o a wide ange o biological
compounds. P e e ed subs a es hiols and hioes e s.
Single oxygen: HOCl -> H+ + OCL-
* OCL- + H2O2 -> Cl- + H2O + 1O2
Elec onically exci ed s a e o oxygen. Reac i i y wi h a
ange o biological molecules. Toxici y by inac i a ion
o memb ane espi a o y chain enzymes has been
epo ed.
Ni ic oxide: Reac s e y apidly wi h supe oxide o
* + gene a e he highly oxic pe oxyni i e. Inac i a es
i on/sulphu cen es.
Pe oxyni i e: NO. + O2
- -> ONOO-
*S ong li ed, uns able s ong oxidan wi h p ope ies
simila o hyd oxyl adical. May be ans o med in an
acid milieu o pe oxyni i e acid and hen o hyd oxyl
adical.
Table 2. Ka anagh and Ree es.
Ve eb a es In e eb a es
Humo al PRRs Mac ophage mannose LPS Binding P o ein.
ecep o (175kDa).
-Me -Leu Phe ecep o
(binds o N- o myl pep ide).
c- ype lec ins Lec ins.
37
C2- ype immunoglobulin Hemolin.
domain.
β-1,3 glucan binding
p o ein.
G am (- e) bac e ial
ecogni ion p o ein.
Pep idoglycan
ecogni ion p o ein.
Complemen /
α2 mac oglobulin. α TEPI.
on Willeb and pla ele
agg ega ion ac o . Hemocy in.
Sca enge ecep o .
Cellula PRRs Toll like ecep o s. Toll
Toll 3-8
18 wheele
immune de iciency
(imd)
In eg ins (CD11b/(CD18) and In eg ins (α, β)
LFA-1. he e odime ic p o eins
Ca ionic P o eins Elas ase (29-31kDa) AB, AF A ac in/
Ca hepsin G (25-29kDa) AB, AF Sa co oxin (20-28kDa) AB
BPI (55-60kDa) AB
Lac o e in (78kDa) AB
P o einase 3
Azu ocidine (29kDa) AB, AF
Lysozyme (14.4kDa) AB, AF Lysozyme AB, AF
MPO/H2O2 (150kDa) AB, AF
Me allop o einases Collagenase Me allop o einase (297,
198 & 95kDa)
Gela inase
Pep ides De ensins (4kDa) AB, AF De ensins AB
Cep opins (4kDa) AB,AF
Dip e icins (9kDa) AB
D osocin AB
Me chnikowin AB, AF
P oline ich an imic obial
pep ides AB
D osomycin AF
AFP AF