ARTICLE IN PRESS

Journal of Insect Physiology 51 (2005) 575–586

0022-1910/$ - se

doi:10.1016/j.jin

�CorrespondMacdonald Ca

Ste Anne de Be

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Cyclic AMP affects the haemocyte responses of larval Galleriamellonella to selected antigens

David Marina, Gary B. Dunphya,�, Craig A. Mandatob

aDepartment of Natural Resource Sciences, Macdonald Campus of McGill University, 21,111 Lakeshore Road, Ste Anne de Bellevue, Que.,

Canada H9X 3V9bDepartment of Anatomy and Cell Biology, McGill University, Montreal, Que., Canada H3A 2B2

Received 26 July 2004; received in revised form 1 February 2005; accepted 10 February 2005

Abstract

Signal transduction of the innate immediate responses of insect haemocytes to foreign matter is rarely considered. Herein using a

combination of adenylate cyclase inhibitors and activators and phosphodiesterase inhibitors we determined that cyclic adenosine

monophosphate (cAMP) at high levels normally impairs non-self response. Haemocyte contact with glass and bacteria lowered

cAMP in vitro. Inactive phosphodiesterases, including type 4, impaired haemocyte reactions in vitro. Using the drugs in vivo to

modulate adenylate cyclase and phosphodiesterases altered the total and types of haemocytes. Adenylate cyclase inhibitors and

etazolate (a type 4 phosphodiesterase inhibitor) alone produced changes in the haemograms similar to those caused by Bacillus

subtilis. Sequential injections of an enzyme modulator followed by B. subtilis impaired bacterial removal due (1) in the case of

enzyme inhibitors, to the removal of haemocytes prior to bacterial challenge and (2) in the case of forskolin and IBMX to the shut-

down of the haemocytes. Activating adenylate cyclase or inhibiting phosphodiesterase impaired bacterial removal when co-injecting

the compounds and bacteria.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: Adenylate cyclase; Phosphodiesterase; cAMP; Haemocyte

1. Introduction

Haemocytes of the larval stage of the greater waxmoth, Galleria mellonella, like other lepidopterans,respond to foreign materials by phagocytosis, nodula-tion and encapsulation. The granular cells and plasma-tocytes are the main haemocyte types participating inthese reactions (Tojo et al., 2000). Nodulation, once theparticulate antigen level exceeds a threshold, is initiatedas a biphasic response in which foreign materials adhereto proteins discharged about the granular cells; theresulting aggregates are ultimately encased by the

e front matter r 2005 Elsevier Ltd. All rights reserved.

sphys.2005.02.010

ing author. Department of Natural Resource Sciences,

mpus of McGill University, 21,111 Lakeshore Road,

llevue, Que., Canada H9X 1C0. Tel.: +1 514 398 7903;

7990.

ess: dunphy@nrs.mcgill.ca (G.B. Dunphy).

plasmatocytes (Ratcliffe et al., 1985). Encapsulation issimilar to nodulation except the particles are too large tobe contained by nodulation (Gillespie et al., 1997). Bothreactions involve haemocytes adhering to foreignmaterials including glass (Yokoo et al., 1995) andmicro-organisms (Dunphy and Webster, 1984), thusantigen-haemocyte binding provides an assay system fordefining non-self signal transduction systems (Zakarianet al., 2003).These types of immediate innate immunity are

initiated by the antigens binding to either patternrecognition receptors and/or promiscuous, non-patternrecognition receptors (Dettloff et al., 2001). Theinformation is then transferred into the haemocytes bysignal transduction. In Ceratitis capitata haemocyticphagocytosis of micro-organisms involves tyrosinekinases (Marmaras et al., 1994; Charalambidis et al.,1995) and focal tyrosine kinase systems (Foukas et al.,

ARTICLE IN PRESSD. Marin et al. / Journal of Insect Physiology 51 (2005) 575–586576

1998). However, in the malignant haemocyte cultures(mbl 2) of Drosophila melanogaster and haemocytes ofHylaophora cecropia protein kinase C and tyrosinekinases participate (Lanz-Mendozoa et al., 1996). In thecase of the former cell line soluble antigens andparticulates may elicit different haemocyte responsesbecause cecropin gene induction for downstream reac-tions after initial antigen-haemocyte contact leading tohumoral immunity is mediated by G-proteins and notby protein kinase C or eicosanoids (Samakoulis et al.,1992). Eicosanoids such as arachidonic acid andprostaglandin E2, downstream products of signalling,enhance antibacterial contact by haemocytes of insectsfrom different orders (Miller and Stanley, 2001; Parkand Kim, 2000; Park et al., 2003); haemocyte adhesionto glass (Mandato et al., 1997) may contribute toparasitoid encapsulation in D. melanogaster (Carton etal., 2002). Signal transduction in terms of haemocyteattachment to objects larger than bacteria has seldombeen reported. Active phospholipase A2 and cycloox-ygenase are necessary for G. mellonella haemocytespreading (Mandato et al., 1997). Ca2+- and lipid-dependent PKC isoforms in the active state (the state inresting haemocytes) impair the adhesion of G. mellonella

plasmatocytes and granular cells to glass (Zakarian etal., 2003). Similarly, active protein kinase A, the normalstate for non-reacting haemocytes, impairs G. mellonella

haemocyte adhesion to glass, and bacterial phagocytosisin vitro and bacterial removal from the haemolymph invivo (Brooks and Dunphy, 2005).Cyclic adenosine-30-50-monophosphate (cAMP) and

the enzymes adenylate cyclase and cAMP-dependentphosphodiesterase occur in insect haemocytes (Comp-ton and Mills, 1982; Vandenberg and Mills, 1975).Protein kinase A affects haemocyte activities, whichimplies that cAMP and the two aforementionedenzymes may participate in haemocyte non-self re-sponses. The involvement of cAMP and adenylatecyclase with haemocyte activities has seldom beenconsidered. G-protein mediated production of cAMPactivates both antibacterial cecropin gene induction(Choi et al., 1995) and protein kinase A (Taniai et al.,1996) in Bombyx mori haemocytes. The biogenic amines,5-hydroxytryptamine and octopamine, increase cAMPlevels in Periplaneta americana which, depending on thecAMP concentrations, inhibits or stimulates phagocy-tosis (Baines and Downer, 1992; Baines et al., 1992) withmediation by inositol triphosphate (Baines and Downer,1994). Cytosolic cAMP enhances nodulation (Baines etal., 1992). Octopamine also increases cAMP levels inLymantria disstria haemocytes (Yadwad and Downer,1993) and is linked to accelerated removal of bacteriafrom G. mellonella haemolymph in vivo by directlyactivating the haemocytes (Dunphy and Downer, 1994).We herein report that high concentrations of cAMP

impair larval G. mellonella haemocyte adhesion to glass

and bacterial-haemocyte contact in vitro. In vivoelevated cAMP diminished the removal of the twobacterial species Bacillus subtilis (a non-pathogen) andXenorhabdus nematophila (a virulent pathogen). Low-ering cAMP levels in haemocytes in vivo producedchanges in haemocyte types and induced nodulationsimilar to the insect’s responses to B. subtilis. Uniquelyreported is the contribution of phosphodiesterase type 4to haemocyte responses.

2. Material and methods

2.1. Insects and bacteria

Galleria mellonella were reared on a multigrain dietsupplemented with glycerol and vitamins at 30 1C(Dutky et al., 1962) under constant light. Fifth instarlarvae weighing 20075mg were used.Gram-negative entomopathogenic X. nematophila (F.

Enterobacteriaceae ATCC strain 19601) in the phaseone form [the form vectored into the insect haemocoelby the entomogenous nematode, Steinernema carpocap-

sae (Akhurst, 1982)] and non-pathogenic B. subtilis (F.Bacillaceae; Boreal Biological Co., St. Catharines,Canada) were cultured on Luria agar supplementedwith triphenyltetrazolium chloride (4mg/ml) andbromthymol blue (2.5mg/ml) and non-supplementedLuria agar, respectively, at 25 1C. Bacteria weresubcultured every fortnight and kept in darkness.Experimental bacteria were grown in 10ml of Luria

broth in 25ml scintillation vials at 30 1C on a horizontalgyrotary shaker (250 rpm) until the cells reached anoptical density reading of 0.6 at 660 nm, i.e., midloggrowth phase. The bacteria were washed three times bycentrifugation (12,000g, 2min, 20 1C) and resuspensionof the pellet in phosphate-buffered saline (pH 6.5,Dunphy and Halwani, 1997). Bacteria were killed byultraviolet irradiation for 2 h, stored at 5 1C overnight,and centrifuge-washed in 1ml of phosphate-bufferedsaline. The latter removed formyl peptides which affecthaemocyte activity (Alavo and Dunphy, 2004). Deathwas confirmed by the absence of change in opticaldensity of 10ml of Luria broth after 72 h incubationpost-inoculation with the bacteria.

2.2. Haemocyte adhesion to glass slides

Determining the effects of signal transduction modi-fiers on haemocyte adhesion to glass surfaces requiredthe formation of haemocyte monolayers using haemo-cyte suspensions.Haemocyte suspensions were made by adding 60 ml of

haemolymph (collected in chilled micro pipette tips)from six chilled larvae (5 1C, 10min) into 1ml of ice-coldCa2+ (10mM) supplemented phosphate-buffered saline

ARTICLE IN PRESS

Table 1

Inhibitors and activators of signal transduction enzymes

Compound (abbreviation,

supplier)

Solventa Mode of action Reference

Forskolin (Sigma Chem. Co.) DMSO (5% v/v)-PBS Direct, reversible adenylate

cyclase activation

Seamon et al. (1981)

9-(Tetrahydro-20-fury1) adenine

(SQ22536, Calbiochem)

PBS Inhibition of adenylate cyclase

by binding to P-site on catalytic

unit

Reid et al. (1990)

N-(cis-2-phenylcyclopentyl)

azacylotridecan-2-imine

hydrochloride (MDL,

Calbiochem)

PBS Irreversibly inhibits catalytic

site of adenyl cyclase

Guellaen et al. (1977)

3-Isobutyl-1-methylxanthine

(IBMX, Calbiochem)

DMSO (5% v/v)-PBS Non-specific inhibitor of cAMP

phosphodiesterases and agonist

of adenosine receptors

Beavo and Reifsnyder (1990);

Chen and Bayne (1995)

1-Ethyl-4-[(1-methylethyidene)

hydrazino-1H-pyrazolo [3,4-b]

pyridine-5-carboxylic acid ethyl

ester, HCl (etazolate,

Calbiochem)

PBS cAMP-specific

phosphodiesterase type 4

inhibitor

Wang et al. (1997)

aDMSO—dimethylsulfoxide (5% v/v for in vitro studies, 50% v/v for injection series), PBS—phosphate-buffered saline, pH 6.5.

D. Marin et al. / Journal of Insect Physiology 51 (2005) 575–586 577

(Zakarian et al., 2003). Ten ml of suspension were addedto a 95mm2 area on endotoxin-free slides previouslyinoculated with 10 ml of test solution. The test solutioncontained various concentrations of compounds knownto either increase or decrease cAMP levels in insects byaffecting adenylate cyclase or phosphodiesterases, ara-chidonic acid or prostaglandin E2 or the appropriatecontrol buffer (Table 1). The two lipids were chosenbecause they affect insect haemocyte activity (Mandatoet al., 1997; Stanley, 2000) and SQ22556 (an adenylatecyclase inhibitor (Reid et al., 1990)) inhibits ecosanoidmetabolism (Fedyk and Phipps, 1996). Slides were madeendotoxin-free prior to use by heating at 350 1C for 72 h.The studies were done in approximately 3.5% (v/v)

insect plasma since humoral factors from lepidopteranspecies affect haemocyte reactions (Wago, 1980; Koizu-mi et al., 1999; Lanz-Mendozoa et al., 1996; Zakarian etal., 2003). Haemocyte mixes were used to reflectadditionally the in vivo situation and preclude haemo-cyte isolation artefacts described by Yokoo et al. (1995).After incubating at 25 1C for 30min [the optimumcontrol reaction time (Zakarian et al., 2003)] the slideswere rinsed three times with 2ml of phosphate-bufferedsaline. Attached haemocytes were fixed for 30min informaldehyde-glutaraldehyde vapour, rinsed in phos-phate-buffered saline and mounted in 20% (v/v)glycerol-phosphate-buffered saline. The total numberof haemocytes and the number of adhering granularcells and plasmatocytes in each of three randomlyselected fields of view in five samples per each of fivereplicates were determined using phase contrast micro-scopy and expressed as cells per mm2. Haemocytes wereidentified according to Price and Ratcliffe (1974).

2.3. cAMP assay

To establish a linkage between haemocyte non-selfactivities and cAMP concentrations plasma free haemo-cytes were obtained as follows.Chilled larvae were injected with 60 ml of cold antic-

oagulant (Mandato et al., 1997) and incubated for 5minon ice to allow the anticoagulant to disperse. Six larvaewere bled from a prothoracic leg lesion into 1ml ofanticoagulant (4 1C). Haemocytes were washed free ofplasma and anticoagulant three times, each time bycentrifugation (2000g, 2min), removing the supernatantand resuspending the haemocyte pellet in 1ml of 4 1Cphosphate-buffered saline. The final suspension wasused in the following three protocols. Two protocolsexamined the effect of antigen-haemocyte contact onintracellular cAMP levels and the third experimentdetermined the effect of adenylate cyclase and phos-phodiesterases on cAMP levels. Protocol one examiningcAMP levels in attached and non-attached haemocytesinvolved adding 99 ml of haemocyte suspension to 10wells in a microtitre 96 well plate containing glass disks(5mm diam.) and 1 ml of buffer. Immediately the non-attached haemocytes in 5 wells were lysed by theaddition of 1 ml of 12N HCl and vigorous pipetting.The haemocytes in the remaining wells adhered to theglass during 30min of stationary incubation. The cellswere lysed by adding 1 ml of 12N HCl and pipetting.Immediately after lysis the solutions were frozen at�80 1C until assayed for cAMP. Protocol two deter-mined the effect of bacterial adhesion to haemocytes onintracellular cAMP concentrations. The protocol con-sisted of adding 1 ml of phosphate-buffered saline

ARTICLE IN PRESSD. Marin et al. / Journal of Insect Physiology 51 (2005) 575–586578

without (haemocyte control) and with B. subtilis

(1.0� 109 bacteria) to 98 ml of phosphate-buffered salinecontaining the haemocytes. To serve as the bacterialcontrol, bacteria were added to 98 ml of phosphate-buffered saline. Incubating the plates at 25 1C on ahorizontal gyrotary shaker (250 rpm) for 30min limitedhaemocyte adhesion to the surfaces of the wells (datanot shown) while facilitating bacterial contact with thehaemocytes. HCl (12N, 1 ml) was added to all the wellsand the lysed suspension frozen at �80 1C.The third protocol determined the effect of enzyme

modulators on haemocyte cAMP levels. One hundredmicroliters of haemocyte suspension were added to100 ml of adenylate cyclase or phosphodiesterase mod-ulators in 200 ml microcentrifuge tubes. The concentra-tions selected were based on modulator levels producingsignificant changes in haemocyte adhesion to slidesdescribed in the previous section. After 30min incuba-tion at 25 1C the haemocytes were twice pelleted bycentrifugation (2000g, 2min) and resuspended in 200 mlof phosphate-buffered saline. Upon a third centrifuga-tion, the haemocytes were lysed by resuspending in100 ml of 0.1N HCl. The lysate was frozen at �80 1C.The five replicates containing 5 samples from each of

the three protocols were analysed for cAMP levels usingthe acetylated version of a cAMP immunoassay kit(Cedarlane Labs, Hornby, Ontario).

2.4. Enzyme modulators in vivo

To establish the link between the effects of the enzymemodulators on haemocyte non-self responses in vivolarvae were injected at the base of a prothoracic leg with10 ml of increasing amounts of the test compounds orappropriate buffer (Table 1). The larvae were incubatedat 30 1C for 30min (unless stated otherwise) and 10 ml ofhaemolymph were added to 90 ml chilled phosphate-buffered saline. Aliquots were placed on a haemocyt-ometer for the total haemocyte counts. Also, 10ml wereadded to 10ml of phosphate-buffered saline on glass slidesand the haemocyte types and quantity determined for thedifferential haemocyte counts. In the case of forskolinand IBMX few haemocytes adhered to glass; however,these were identified (Price and Ratcliffe, 1974). The non-attached haemocytes were difficult to identify, cells in onegroup being phase bright, granulated and circular(possibly granular cells) and the other group the cellswere elliptical-fibroblastoid and phase dark (possiblyplasmatocytes). Changes in the haemogram induced byinjecting various amounts of B. subtilis in 10ml of buffercompared to control larvae with buffer were determinedas described for the test compounds.The effects of the modulators on the removal of both

bacterial species from the haemolymph was initiallyassessed by sequentially injecting 10 larvae with a testcompound in 10 ml of buffer (control larvae received

buffer only), incubating the insects for 5min at 25 1Cand injecting 10 ml of bacterial suspension (1.5� 108

bacteria). Thirty min later the larvae were bled (10 ml)into 90 ml of buffer and the number of bacteria notattached to haemocytes determined on a haemocyt-ometer by phase contrast microscopy. Co-injections oftest solutions with bacteria were also used to determinemodulator effects on bacterial levels 30min post-injection. Experiments were repeated six times.

2.5. Statistical analysis

All data were analysed using the 95% confidencelimits over-lap protocol (Sokal and Rohlf, 1969).Percentage data were analysed after aresin

ffiffiffi

pp-trans-

formed data. Graphic and tabular data are presented asthe mean7standard error of the mean. N contained atleast 5 replicates with a minimum of 5 samples perreplicate. Control values were pooled when the data fitthe assumptions of the analysis of variance (Sokal andRohlf, 1969).

3. Results

3.1. In vitro study of the effect of enzyme modulators and

signal transduction components on haemocyte adhesion

and cAMP levels

Forskolin, an adenyl cyclase activator, maximallydiminished the adhesion of granular cells and plasma-tocytes to slides by approximately 45% and 80%,respectively, by 22.5 mM (Fig. 1). The adenylate cyclaseinhibitor, SQ22536, increased granular cell adhesion by70% at 475 mM but did not affect the plasmatocytes(Fig. 2A). Arachidonic acid alone significantly increasedgranular cell adhesion without affecting the plasmato-cytes (Fig. 2B). However, the fatty acid lowered bothgranular cell and plasmatocyte adhesion in a concentra-tion-dependent manner when incubated with 250 mMSQ22536 (the lowest concentration inducing an increasein granular cell adhesion). Prostaglandin E2 when usedalone elevated plasmatocyte and granular cell adhesion,the effect being more pronounced for the granular cells(data not shown) and was similar in pattern but lesser inextent of effect to arachidonic acid. Incubation ofincreasing amounts of prostaglandin E2 with 250 mMSQ22536 substantially lowered granular cell adhesion(0 mM prosta glandin E2:491777.2 haemocytes/mm

2,1500 mM prostaglandin E2: 187.5712.6 haemocytes/mm2, Po0:05, slope ¼ 0.35) but not the plasmatocytes(0 mM prostaglandin E2: 201.1712.7 haemocytes/mm

2,2500 mM prostaglandin E2: 183.277.1 haemocytes/mm2, P40:05, slope ¼ 0.01). Results with the adenylatecyclase inhibitor MDL were less defined; both thegranular cells and plasmatocyte adhesion levels maxi-

ARTICLE IN PRESSD. Marin et al. / Journal of Insect Physiology 51 (2005) 575–586 579

mally declined at 100 mM and 275 mM, respectively (Fig.3). Thereafter granular cell adhesion increased whereasthe plasmatocyte levels remained constant. Because theresults with MDL and SQ22536 and the two lipidsdiffered the possible effects of the adenylate cyclase

Fig. 1. Effect of the adenylate cyclase activator forskolin added prior

to the addition of haemocytes of larval Galleria mellonella to glass

slides.

Fig. 2. The prior addition of (A) adenylate cyclase inhibitor, SQ22536, enh

slides. (B) Arachidonic acid alone elevated haemocyte adhesion whereas wit

inhibitors and eicosanoids on cAMP levels weredetermined.The cAMP levels compared to the control group

increased in haemocytes incubated with forskolin, butnot with arachidonic acid or prostaglandin E2 anddeclined in haemocytes with SQ22536 (Table 2) andMDL levels in excess of 100 mM diminished cAMPwhereas 100 mM unexpectedly increased cAMP levelsSQ22536 was a more effective adenylate cyclaseinhibitor than MDL. Low cAMP levels were correlatedwith an increase in total haemocyte adhesion(r ¼ 0:87;Po0:05) and high cAMP levels negativelycorrelated with total cell adhesion (r ¼ 0:92;Po0:05).IMBX, a general phosphodiesterase inhibitor, maxi-

mally lowered granular cell (90%) and plasmatocyte(75%) adhesion by 100 and 250 nM, respectively (Fig.4). Thereafter the decline rates were lower and compar-able for both haemocyte types. IBMX increased totalhaemocyte cAMP (Table 2) which was negativelycorrelated with total haemocyte adhesion(r ¼ 0:77;Po0:05). Etazolate, an inhibitor of phospho-diesterase type 4, up to a concentration of 100 nM,marginally increased granular cell adhesion (Fig. 5)without discernibly affecting cAMP levels (Table 2).At higher etazolate levels haemocyte counts declined(Fig. 5) as cAMP levels increased (Table 2). There wasno significant change in adhering plasmatocyte over theetazolate concentrations used (P40:05). Because

anced the adhesion of the granular cells but not the plasmatocytes to

h 250 mM of SQ22536, the unsaturated fatty acid impaired adhesion.

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Table 2

Effect of signal transduction modulators on the concentration of

cAMP in larval Galleria mellonella haemocytes

Modulator Concentration

(mM)cAMP

(pmol/ml)a

Forskolin 2.5 5.2170.07c

25.0 12.7670.09d

MDL 100.0 6.7170.03d

250.0 2.2570.05c

500.0 1.2670.04e

SQ22536 250.0 0.3570.09d

500.0 0.1070.11e

Etazolate 0.1 2.0070.02c

0.5 6.3170.06d

1.0 8.7070.07e

10.0 11.9270.08f

Forskolin (2.5 nM)+Etazolate 0.1 7.3270.030.5 9.7470.11*

IBMX 0.025 4.1170.08c

0.250 8.7270.06d

Prostaglandin E2 25.000 2.9170.13c

175.000 3.2170.12c

Arachidonic acid 0.250 3.9770.17c

0.500 4.1170.19c

1.000 3.8870.12c

Pooled controlb ___ 3.8270.08c

aMean7standard error of the mean, n ¼ 5 replicates each of which

had 3 samples. Total haemocyte used ¼ 5.0� 106/ml. Means with

different superscripts were significantly different from the pooled

control group, Po0:05. Means within a treatment with differentsuperscripts were significantly different from each other, Po0:05).bMeans of data with the same statistical value, homosecadasicity

and kurtosis from the different buffer regimes fitting the assumptions

of the analysis of variance were pooled.

Fig. 3. MDL, an adenylate cyclase inhibitor, impaired granular cell

and plasmatocyte adhesion at low concentrations and stimulated

adhesion of granular cells at higher concentrations.

D. Marin et al. / Journal of Insect Physiology 51 (2005) 575–586580

100 nM etazolate did not produce discernible changes incAMP (within the assay limitations) it is possible cAMPdid not achieve sufficient levels to inhibit haemocyteadhesion. This was tested by adding a concentration offorskolin that did not affect haemocyte adhesion tosolutions of increasing etazolate levels and determiningthe number of adhering haemocyte types. Incubatinghaemocytes with 2.5 nM forskolin did not alter haemo-cyte adhesion (total haemocytes ¼ 275.17 15.3 haemo-cytes/mm2, plasmatocytes ¼ 97.273.8 haemocytes/mm2; granular cells ¼ 233.7717.2 haemocytes/mm2)compared with the control buffer (see Fig. 1, P40:05).However, co-incubating haemocytes with 2.5 nM for-skolin and increasing amounts of etazolate significantlydecreased haemocyte counts (100 nM etazolate: totalhaemocytes ¼ 185.7712.1 haemocytes/mm2; plas-matocytes ¼ 72.576.1 haemocytes/mm2 granularcells ¼ 132.9711.2 haemocytes/mm2; 500 nM etazolate:total haemocytes ¼ 71.075.3 haemocytes/mm2, plas-matocytes ¼ 29.277.1 haemocytes/mm2 granularcells ¼ 32.1710.2 haemocytes/mm2, Po0:05) and in-creased cAMP levels (Table 2). The decrease inhaemocyte attachment was greater and occurred soonerthan for haemocytes with etazolate only.Collectively, the pharmacological data implied that

lowered intracellular cAMP concentrations were con-ducive to haemocyte adhesion. This was biochemicallydetermined by comparing cAMP levels of antigen-bound and free-floating haemocytes. Haemocytes at-tached to glass disks contained less cAMP (0.6570.11

pmol/ml) than non-attached haemocytes (3.2170.32 pmol/ml, Po0:05). Also haemocytes with adheringB. subtilis possessed less cAMP (0.7270.15 pmol/ml)than haemocytes without bacteria (3.9770.21 pmol/ml,Po0:05). There was no discernible cAMP in the deadbacteria.

3.2. In vivo study on the effects of the enzyme modulators

on the haemograms and bacterial removal from the

haemolymph

Injections of forskolin (Fig. 6A), and IBMX (Fig. 6B)into the larvae elevated the total haemocyte countswhereas SQ22536 (Fig. 6C), MDL (data not shown) andetazolate (without forskolin (Fig. 8) decreased the totalhaemocyte counts. Compared with SQ22536, the MDLeffect on total haemocyte counts was approximately30% of the former’s effect.Both adenylate cyclase inhibitors and etazolate as

opposed to forskolin and IBMX, triggered nodule

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Fig. 5. Effect of the phosphodiesterase type 4 inhibitor etazolate (in

vitro on haemocyte adhesion).

Fig. 4. IBMX, a general phosphodiesterase inhibitor, impaired

granular cell and plasmatocyte adhesion to slides.

Fig. 6. Effect of injected (A) forskolin and (B) IBMX on the

haemocyte totals and types in vivo. Haemocyte types were identified

by removing the haemolymph, placing it on glass slides and using

identifying parameters described in the text. (C) SQ22536 diminished

the total and types of haemocytes in the haemolymph in vivo.

D. Marin et al. / Journal of Insect Physiology 51 (2005) 575–586 581

formation at the injection site causing a correspond-ing decrease in the number of granular cells andplasmatocytes available for adhering to slides, the effectwith SQ22536 being three fold greater than for MDL(data not shown). Haemocytes from forskolin and

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Fig. 7. Changes in the total haemocytes and haemocyte types in larvae

injected with etazolate.Fig. 8. The effects of injected Bacillus subtilis on the haemograms of G.

mellonella.

Table 3

Effect of sequential signal transduction modulators on the removal of

bacteria from the haemolymph of larval Galleria mellonella 30min

post-bacterial-injectiona

Bacterial level (� 107/ml)b

Compound Concentration

(mM)Bacillus subtilis Xenorhabdus

nematophila

Forskolin 2.5 1.270.1a 2.470.2b

25 2.470.1b 12.670.5c

MDL 250 2.970.2c 3.370.1d

500 4.870.2d 7.270.2e

SQ22536 250 2.670.1c 2.470.1b

500 3.770.2d 4.270.1f

Etazolate 0.025 1.170.1a 1.670.1a

0.250 2.770.1a 3.070.1b

IBMX 0.025 0.970.3a 0.170.2a

0.250 2.770.2e 1.170.1h

Pooled controlc — 1.470.1a 1.770.1a

aLarvae were injected with 10 ml of buffer without and with selectedmodulator concentrations. After incubating for 5min insects were

injected with 10 ml of bacteria (1� 109 bacteria), incubated for 30minand the bacterial level determined.bMean7standard error of the mean, nX10 larvae, means within a

column with different superscripts were significantly different Pp0:05.

D. Marin et al. / Journal of Insect Physiology 51 (2005) 575–586582

IBMX-treated larvae consisted of non-adhering phase-bright granulated cells (possibly granular cells) andphase-dark elliptical cells (possibly plasmatocytes),which were frequently observed rolling over the slides.Such haemocytes were as viable (range 87–95%) asthose from control larvae (range 82–93%, P40:05).Larvae with SQ22536 (Fig. 6C) and MDL (data not

shown) contained fewer spherulocytes (Po0:05) thandid the controls; oenocytoid levels were not affected(P40:05). Forskolin and IBMX elevated both spher-ulocyte and oenocytoids levels (Fig. 6A and B; Po0:05).The granular cells, plasmatocytes and spherulocytesdiminished with low doses of etazolate (Fig. 7; Po0:05).Increasing amounts of injected B. subtilis alonedecreased the total haemocyte counts, and the granularcells, plasmatocytes and spherulocyte levels (Fig. 8,Po0:05).Injection of a given enzyme modulator followed

30min later with test bacteria produced higher bacteriallevels (i.e. less bacterial removal) with increasing drugconcentrations compared with the control insects (Table3). The effect was more pronounced for X. nematophila

than B. subtilis. Co-injections of forskolin or IBMXwith bacteria increased bacterial levels with increasingdrug concentration whereas, etazolate, MDL andSQ22536 diminished bacterial levels (Table 4). Theoverall responses to X. nematophila were more pro-nounced than for B. subtilis. Attempts to offset theetazolate effect by increasing cAMP levels using aforskolin concentration that did not alter the haemo-gram failed (data not shown).

4. Discussion

The secondary messenger, cAMP, exists in all tissuesexamined in numerous insect species from different orders(Vandenberg and Mills, 1975; Compton and Mills, 1982).However, studies on the involvement of cAMP in

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Table 4

Coinjections of signal transduction modulators with bacteria affect

bacterial removal from Galleria mellonella haemolymph 30min post-

injection.

Bacterial level (� 106/ml)a

Compound Concentration

(mM)Bacillus subtilis Xenorhabdus

nematophila

Forskolin 2.5 37.270.8d 52.771.3p

25.0 59.171.9e 67.270.7q

MDL 100.0 26.171.1f 32.271.2r

250.0 12.470.7g 9.271.1s

500.0 1.370.8h 0.771.5t

SQ22536 250.0 7.972.1i 5.270.6u

500.0 8.170.2j 0.370.1v

Etazolate 0.025 15.771.2c 13.271.9o

0.250 9.271.1l 6.472.3w

IBMX 0.025 46.571.7m 51.772.2x

0.250 62.172.5n 68.172.1y

Pooled

controlsb— 16.170.2e 21.170.2o

aMean7standard error of the mean, nX10 replicates each contain-

ing 5 larvae. Means within a column with different superscripts

differed significantly Pp0:05.bMeans from larvae injected with different buffer regimes which

were not significantly different (P40:05) and which fit the assumptionsof the analysis of variance were pooled.cMeans of values from larvae injected with different buffer regimes

which were not significantly different (P40:05) and which fit theassumptions of the analysis of variance were pooled (Sokal and Rohlf,

1969).

D. Marin et al. / Journal of Insect Physiology 51 (2005) 575–586 583

haemocytic non-self responses are limited to cecropin-gene activation in B. mori (Shimabukuro et al., 1996) and,depending on cAMP levels, enhanced or diminishedbacterial phagocytosis by P. americana haemocytes(Baines et al., 1992). Possibly cAMP participates in non-self responses of Malacosoma disstria haemocytes sincecAMP is elevated by octopamine in the haemocytes invivo and in the insects’ established haemocyte cell line,Md66 (Gole et al., 1982). Octopamine participates in theantimicrobial responses of G. mellonella haemocytes(Dunphy and Downer, 1994). Herein cAMP affected thein vitro adhesion of larval G. mellonella haemocytes toslides and nodulation and the removal of two bacterialspecies from the haemolymph in vivo.

4.1. In vitro experiments

The decline induced by the adenylate cyclase activator(forskolin) in haemocyte adhesion on slides and theincrease in haemocyte adhesion with the enzymeinhibitors (SQ22536 and high levels of MDL) corre-sponded with an increase and decrease in haemocytecAMP levels, respectively. This implies that adenylate

cyclase and cAMP are part of the non-self response. Thegranular cells were more sensitive to the drugs than werethe plasmatocytes. This is expected in view of the firstcontact importance of granular cells to nodulation andencapsulation. However, the plasmatocyte responseswere of a lesser magnitude possibly reflecting alterationsin proposed granular cell-plasmatocyte cross-talk (An-ggraeni and Ratcliffe, 1991) or differential sensitivity tothe test agents such as arachidonic acid and SQ22536.Additional support for the involvement of cAMPincluded higher cAMP levels in non-attached haemo-cytes than in haemocytes attached to glass disks and thedecline in cAMP in haemocytes with adhering bacteria.Hence a basal level of cAMP is required to maintain thehaemocytes in a quiescent state. This is similar to thereport by Baines et al. (1992) in which different levelscAMP in P. americana differentially affect bacterialphagocytosis. In contrast, low cytosolic cAMP inhibitsmolluscan haemocyte activities including phagocytosisin the bivalves Biomphalaria glabarata (Bezerra et al.,1999) Crassostrea gigas (Lacoste et al., 2001a) andsubstratum adhesion of Mytilus californicus haemocytes(Chen and Bayne, 1995).Arachidonic acid and prostaglandin E2 induce bacter-

ial nodulation in several insect orders including theLepidoptera (Stanley, 2000) and the unsaturated fattyacid enhances phagocytosis by and spreading of G.

mellonella haemocytes (Mandato et al., 1997). This andthe effects of SQ22536 on mouse b-lymphocyte eicosa-noid metabolism (Fedyk and Phipps, 1996) warrantedanalysis of the interaction of the lipids and SQ22536with insect haemocytes. That both arachidonic acid andprostaglandin E2 alone increased haemocyte adhesionwithout affecting cytoplasmic cAMP levels suggests thelipids may have stimulated an alternative cAMP-independent adhesion pathway. Although this is thefirst report of the lipids not influencing cAMP in insects,prostaglandin E2 does not affect adenylate cyclase in thesalivary glands of the female tick, Amblyomma amer-

icanum (Qian et al., 1997). A similar result occurs withprostaglandin E1 inhibiting human blood plateletaggregation induced by adenosine diphosphate or 1-epinephrine in which there was no change in plateletcAMP (Sinha and Colman, 1978). Herein eicosanoidstimulation may represent an alteration or formation ofstimulatory plasma-macromolecules, which regulateshaemocyte activity independent of cAMP. A fixedconcentration of SQ22536 with increasing eicosanoidconcentration inexplicably suppressed haemocyte adhe-sion.The IBMX results imply that inactive phosphodies-

terases by elevating cAMP limits the contact of G.

mellonella haemocytes to slides. Haemocytes in thequiescent state may lack active phosphodiesterases,cAMP levels being maintained by adenylate cyclase.This would be similar to P. americana haemocytes in

ARTICLE IN PRESSD. Marin et al. / Journal of Insect Physiology 51 (2005) 575–586584

which bursicon activation of adenylate cyclase occurswithout phosphodiesterase activity (Compton and Mills,1982). Although elevated cAMP limited haemocyteadhesion and degeneration herein, in D. melanogaster

bursicon induces cAMP elevation and initiates epider-mal wing cell degeneration (Kimura et al., 2004). Thesedifferences may be cell-type specific since cAMP elicitsapoptosis in mammalian thymocytes (McConkey et al.,1990) and avoids cell death of neutrophils (Parvathenaniet al., 1998). Herein the phosphodiesterase results areopposite those of molluscs in which the active enzymeenhances haemocyte adhesion to substrata (Chen andBayne, 1995) and phagocytosis (Lacoste et al., 2001a).Phosphodiesterase type 4 participates in the antimi-

crobial responses of mammals (Essayan, 2001) andmolluscs (Lacoste et al., 2001a, b). We used etazolate, aphosphodiesterase type 4 inhibitor (Wang et al., 1997) todetermine the contribution of this enzyme to the insecthaemocytes. Low etazolate levels did not elevate cAMPand marginally increased haemocyte adhesion. Etazo-late fails to inhibit all mammalian eosinophil functionsknown to be controlled by phosphodiesterase type 4unless additional cAMP is produced by adenylatecyclase (Ezeamuzie, 2001). This may apply to thepresent study because (1) 2.5 nM forskolin did not alterhaemocyte adhesion, (2) etazolate (0.1 and 0.5 mM)combined with 2.5 nM forskolin increased haemocyticcAMP concentrations and (3) together as opposed toseparately, the drugs lowered haemocyte adhesion. Thata decline in haemocyte adhesion occurred at highetazolate concentrations may indicate differential sensi-tivity of possible subtypes of the type 4 enzyme, higherconcentrations being required to elicit inhibitory cAMPlevels as proposed for human phosphodiesterase type 4subtype D expressed in the insect cell line Sf9 (Wang etal., 1997).Injections of inhibitors of adenylate cyclase and

phosphodiesterase including etazolate alone producedchanges in haemocyte totals and types similar tochanges caused by B. subtilis including nodulation.Forskolin and IBMX precluded these changes. Thus, inagreement with the in vitro results, lowering cAMP ispart of the initiation of haemocytic non-self responses.This was further confirmed by injecting SQ22536, MDL,etazolate, IBMX or forskolin followed by bacterialchallenge; bacterial removal diminished with increasingdrug concentration. The effect with SQ22536, MDL,and etazolate was likely due to a decline in floatinggranular cells and plasmatocytes induced by nodulation,which limited the number of available haemocytes thatreact with bacteria. Forskolin and IBMX impairedbacterial removal in a concentration dependent manner;impairment was not due to altered haemocyte viability.However, the absence of nodulation and diminishedadhesion to slides of haemocytes from larvae with thesedrugs suggest the haemocytes had been rendered

quiescent by elevating cAMP. Co-injections ofSQ22536, MDL, or etazolate with bacteria enhancedbacterial removal from the haemolymph in parallel witha decrease in haemocyte counts, induced nodulation andincreased adhesion to slides (in the in vitro study).Forskolin and IBMX did not lower bacterial concentra-tion possibly due to the haemocytes being shut-down byelevated cAMP levels. Attempts to modify the etazolateeffect by raising cAMP levels in vivo without alteringhaemograms failed for unknown reasons.The magnitude of the antibacterial responses in vivo

varied with the bacterial species. Possibly the antigenicdifferences may affect non-self generating pathwaysother than those affecting cAMP. In D. melanogaster

responses to different bacterial antigens represent theeffects of antigens activating common and differentantibacterial protein signaling pathways (Kurata, 2004).In summary, larval G. mellonella haemocytes are in

the quiescent, non-reactive state when the cytoplasmiccAMP levels are high due to active adenylate cyclaseand/or inhibited phosphodiesterases, possibly type 4.Haemocyte contact with foreign materials lowerscAMP. In vivo adenylate cyclase inhibitors loweredboth the total and types of haemocytes producingnodules much like those induced by B. subtilis andinfluenced bacterial removal from the haemolymph.Thus cAMP may contribute to haemocyte adhesion toforeign materials.

Acknowledgements

The research was supported by a grant to G.B.D. bythe Natural Sciences and Engineering Research Councilof Canada. C.A.M. is a Canada Research Chair.

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Further reading

Kurata, R., Takayangi, I., Hisayama, T., 1993. Eicosanoid-induced

Ca2+-release and sustained contraction in Ca2+-free media are

mediated by different signal transduction pathway in rat aorta.

British Journal of Pharmacology 110, 875–881.