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The effects of immune challenge on phenoloxidase activity in locust salivary glands in vitro

The effects of immune challenge on phenoloxidase activity in locust salivary glands in vitro Volume 1 † Number 2 † June 2008 10.1093/biohorizons/hzn015 ......................................................................................................................................................................................................................................... Research article The effects of immune challenge on phenoloxidase activity in locust salivary glands in vitro Kirsty L. Wells* Birkbeck College, University of London, Malet Street, London WC1E 7HX, UK. * Corresponding author: Birkbeck College, London WC1E 7HX, UK. Email: kirsty.wells@kcl.ac.uk Supervisor: Professor Graham J. Goldsworthy, Birkbeck, University of London, London, UK. ........................................................................................................................................................................................................................................ Homogenates of salivary glands from Locusta migratoria possess phenoloxidase (PO) activity. This study investigates the activation of prophenoloxidase (PPO) in these glands in vitro. When freshly dissected salivary glands from L. migratoria are incubated with the immu- nogen laminarin, and then homogenized, a 4-fold increase in PO activity (expressed as a percentage of the total PO) can be measured within 20 min. Addition of laminarin to the incubation medium is best made prior to addition of salivary gland tissue, because when laminarin is added 10 min after the addition of tissue, the response to laminarin is reduced by 50%. When salivary glands are incu- 2þ bated in Ca -free Ringer, the response to laminarin cannot be demonstrated. Addition of a calcium ionophore to the incubation in normal Ringer does not initiate a response on its own, but does augment the response to laminarin. Addition of phorbol ester to an incubation containing normal Ringer has no effect on PO activity, and does not augment the response to laminarin. In contrast, addition of okadaic acid to normal Ringer has no effect on its own, but does augment the response to laminarin. Activation of PPO in response to 2þ laminarin is therefore calcium-dependant, possibly involving an influx of extracellular Ca , and modulated by protein phosphatases. Future work should aim to clarify the function of salivary glands in the immune defence of the locust and to investigate the exact source of the PO associated with the glands. Key words: Locusta migratoria, phenoloxidase, salivary glands, laminarin. ........................................................................................................................................................................................................................................ Introduction non-specific responses to infection and can be said to com- prise three types of defence: physical, cellular and humoral. Insects possess a highly efficient immune system. An under- The cuticle is an insect’s first line of defence against invad- standing of this system of immune defence is important not ing microorganisms and it presents an impenetrable physical only in terms of potential applications to the biocontrol of 4, 5 barrier to many potential pathogens. However, if a patho- pest insects, but also because insects are useful models for gen is able to overcome cuticular defences, or cross the gut investigating immune systems. Therefore insect immunity is wall, coordinated responses of immune cells in the haemo- increasingly well-studied, especially in Drosophila, which lymph are initiated in response to pathogen detection. has become a particularly valuable model for studying This involves detection by pattern recognition proteins innate immunity over the past 10 years due to the power of (PRPs) present both in the haemolymph and on the surfaces molecular genetics. Locusts are also commonly used as a of haemocytes in the haemolymph. Haemocytes are cells model in studies of insect immunity due to their prevalence involved in haemolymph clotting and defence. They are as a pest in parts of the world and their relatively large size. able to bind pathogens, initiating signalling pathways Invertebrates do not show adaptive immunity and do not leading to the encapsulation or phagocytosis of the produce antibodies in response to infection. Instead, they rely foreign body by the haemocyte. Haemocytes can respond on a system of innate immunity to protect them from to infection in a coordinated manner, trapping foreign pathogens. The insect immune system consists of rapid, ......................................................................................................................................................................................................................................... 2008 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 122 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... bodies within aggregates of many haemocytes called polysaccharides found in fungal cell walls) into the haemo- nodules. Nodules are often visible to the naked eye as lymph: the acini of the salivary glands in dissected locusts blackened spots along the internal body wall of an infected became intensely black. It was thought that this phenomenon insect that has undergone dissection. did not occur in adult locusts, however, it has been shown Insects also synthesize a variety of extracellular humoral subsequently that the salivary glands of adult locusts, while compounds in response to immune challenge. Some of the showing a less pronounced melanization response to injected various signal transduction pathways initiated as a result of laminarin, do exhibit a strong response in terms of increased pathogen detection culminate in an alteration of gene PO activity in the glands (G.J. Goldsworthy, personal expression in specialist cells, primarily in the fat body, communication). causing these cells to produce and secrete infection-fighting This previously unreported phenomenon raises a number substances into the haemolymph. For example, lysozymes of questions. For example, what are the characteristics of are synthesized in the fat body of lepidopterans in response the PO response in locust salivary glands? What are the sig- to bacterial injection; broad-spectrum antibacterial peptides nalling pathways mediating the activation of PPO in locust (cecropins) are synthesized in the fat body, cuticle and salivary glands, and how is the immunogen being detected endothelium of lepidopterans and dipterans in response to by the salivary glands? This study of PO activity in salivary bacterial challenge; and the antifungal drosomycin from glands of adult locusts addresses these questions. Drosophila melanogaster also has antibacterial activity. These examples represent a small fraction of the many anti- Materials and methods bacterial and antifungal compounds that have been identified and sequenced from many different insect species. Insects One of the most important and ubiquitous compounds L. migratoria migratorioides were reared under crowded released by insects in response to immune challenge, and of conditions at 30ºC in a light:dark 12:12 h photocycle, and particular relevance to this study, is the enzyme phenoloxi- fed daily with fresh grass and wheat seedlings supplemented dase (PO). PO is activated in the cuticle or the haemolymph with bran. Adult male locusts between 15 and 25 days after of many invertebrates in response to immune challenge or the final moult were used in all experiments. wounding and is activated via the prophenoloxidase (PPO) cascade, PPO being the inactive zymogen of PO. PPO is Materials present in the haemolymph of Locusta migratoria and is Locust Ringer (168 mM NaCl, 6.4 mM KCl, activated in response to immune challenge. 3.6 mM MgCl 6 mM NaH PO 2H O, 2.1 mM NaHCO 2, 2 4 2 3, Detection of a pathogen or immunogen by a PRP leads to 2þ 20 mM Hepes, 2.1 mM CaCl 4% Sucrose, pH 7.0) was 2, aCa -dependant cascade of serine proteases, the final com- prepared, autoclaved and stored at 58C until used as incu- ponent of which, the PPO activating enzyme, is thought to be 2þ bation medium. A Ca -free Ringer was used in some exper- a clip domain serine protease that cleaves PPO to PO. PO, iments (no CaCl , 1 mM EGTA added). an oxidoreductase, catalyses the oxidation of phenols All chemicals were purchased from Sigma Chemical present in the haemolymph to cytotoxic quinones. These Company. Laminarin was dissolved in locust Ringer to give quinones polymerize non-enzymatically to melanin. Both a10 mg mL solution. A protease inhibitor, phenylmetha- quinones and melanin are toxic to microorganisms. The nesulphonylfluoride (PMSF), was dissolved in propanol to deposition of melanin causes parasites to become blackened give a 0.5% solution. Immediately prior to use, dopamine in the haemolymph, and in nodules due to melanization of was dissolved in 0.01 M phosphate buffer (pH 5.9) to give haemocytes encapsulating foreign bodies. a 3 mg mL solution. Absolute methanol was used to acti- Although the cuticle is an effective barrier to most patho- vate PPO. Calcimycin, a calcium ionophore (A23187) was gens, many isolates of entomopathogenic fungi are able to dissolved in DMSO to give a 10 mM stock solution. penetrate the insect cuticle. As a result, over the past two Phorbol 12-myristate 13-acetate (PMA) and okadaic acid decades there has been considerable interest in these fungi were each dissolved in DMSO to give separate 100 nM as biocontrol agents. After a short delay, topically applied stock solutions. conidia of Metarhizium, for example, send out apressoria that can penetrate the cuticle. To circumvent the delay and Dissection produce synchronized infection, Mullen and Goldsworthy studied PO activity in haemolymph of L. migratoria after The head of the insect was removed using sharp scissors and injection of Metarhizium blastospores. During the course a mid-dorsal cut made along the full length of the body. The of these experiments, Mullen and Goldsworthy observed body was pinned ‘dry’ onto a corkboard with the inner that salivary glands of 5th instar L. migratoria nymphs mel- ventral surface of the body wall uppermost. The salivary anized in response to injection of blastospores or high doses glands were viewed under a binocular microscope and of laminarin (a b1–3 glucan that is representative of removed using two pairs of fine forceps. Each insect ......................................................................................................................................................................................................................................... 123 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... usually yielded sufficient salivary gland tissue for two incubations. Measurement of PO activity Salivary gland tissue was incubated in 1 ml plastic centrifuge tubes containing 150 ml of locust Ringer (and other incu- bation constituents as appropriate) at room temperature for 60 min unless otherwise stated. The room temperature did not vary appreciably between different experiments, but control incubations were always included to allow for poss- ible differences due to any variations in room temperature. Incubations were stopped by the addition of 1 ml of PMSF to prevent further serine protease activity. After homogeniz- Figure 1. Percentage of total PO activated in salivary gland tissue incu- ation by sonication (Cole-Parmer for 10 s) and centrifugation bated with 4 ml laminarin (added to the incubation prior to tissue) for (13 000 rpm (10 000g) for 2 min), two 50 ml samples of various time periods. A control group of salivary glands was incubated for 60 min in a mixture to which 1 ml PMSF had been added before supernatant were taken from each tube and pipetted into tissue (0 min incubation time). Data points and vertical lines represent separate wells of a microtitre plate. Total PPO was activated mean % total PO+ SE, respectively. Data points with different letters are in one of the wells by adding 50 ml of methanol, mixing and significantly different from each other taking the level of significance as discarding 50 ml of the mixture to leave 50 ml in the well. P  0.05. Numbers in parentheses refer to the number of observations After addition of 150 ml of dopamine solution to both per group. wells, PO activity was assessed by determining the initial linear increase in absorbance at 492 nm. Absorbance incubations that did not have any addition of laminarin, values were read every minute for 15 min in a Labsystems there was no statistically significance increase in PO activity Multiskan Bichromatic plate reader. Appropriate allowance when laminarin was added to the incubation 10 min after the was made for the fact that the total PO activity related to tissue (Fig. 2). half the amount of salivary gland homogenate than was present in the sample not reacted with methanol. Calcium dependency When salivary gland tissue samples were incubated with Data analysis 2þ laminarin in Ca -free Ringer, PO activity was reduced by Data were initially evaluated using Microsoft Excel to calcu- 80% in comparison with incubations in normal Ringer late the slope of a line of best fit for the absorbance measure- 2þ and was similar to that in either Ca -free or standard ments taken from each microtitre-plate well between 0 and Ringer in the absence of laminarin (Fig. 3A). 15 min. The PO activity in each well that had not been acti- vated with methanol was expressed as a percentage of the PO Calcium ionophore activity in its corresponding well in which total PPO had When salivary glands were incubated in normal Ringer been activated with methanol. Data were analysed statisti- with calcimycin only (no laminarin) PO activity did not cally using a one-way ANOVA followed by Fisher’s one-way multiple comparison tests. All statistical tests were undertaken using Minitab. Results PO activity in response to addition of laminarin and the effect of time of incubation When laminarin was present in the incubation medium at the start of the incubations, PO activity in the homogenates of the glands did not increase significantly within the first Figure 2. Percentage of total PO activated in salivary gland tissue incu- 10 min of incubation, but subsequently it increased to bated with 4 ml laminarin which had been added to the incubation before tissue (open bar, time ¼ 0) and 10 min after tissue (shaded bar). reach a maximal level by about 20 min that persisted for A control group of salivary glands was incubated without laminarin up to 60 min (Fig. 1). Delaying the addition of laminarin (open bar, No Lam). Bars and vertical lines represent mean % total PO+ to the incubations of salivary glands by 10 min reduced PO SE, respectively. Bars with different letters are significantly different from activity by  50% in comparison with the levels when lami- each other taking the level of significance as P  0.05. Numbers in parenth- narin was added before the glands. In comparison with eses refer to the number of observations per group. ......................................................................................................................................................................................................................................... 124 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... absence of laminarin, there was no indication that the PMA had enhanced the increase in PO activity due to the laminarin (Fig. 3C). Okadaic acid Addition of okadaic acid on its own did not change the PO activity significantly. When salivary glands were incubated with okadaic acid and laminarin together, PO activity increased  6-fold in comparison with incubations in which laminarin was absent, which represented a statistically significant  2-fold enhancement of the increase in PO activity in comparison with incubations with laminarin on its own (Fig. 3D). Discussion Locust salivary glands comprise secretory acini bundled together in a grape-like arrangement. Associated with each acinus of L. migratoria there are three or four nephro- cytes. These are pericardial cells and, like haemocytes, originate from the mesoderm, but unlike haemocytes are static cells associated with tissues. They are involved in clearance of substances from the haemolymph and are able to take up material by endocytosis and release it by exocyto- sis. In L. migratoria, nephrocytes are involved in immuno- gen uptake leading to haemocyte recruitment and nodule 12, 17 formation. It seems likely that salivary gland melanization in locusts Figure 3. Percentage of total PO activated in salivary gland tissue in 12 in vivo as described by Mullen and Goldsworthy is a two- various incubation mixtures. Control groups of salivary glands (open phase process, whereby nephrocytes recognize foreign bars) were incubated with 4 ml laminarin added to the incubation prior material and then themselves become a focus for ‘attack’ to tissue (the first bar on the left of each panel), and without laminarin (the third bar from the left of each panel). Bars and vertical lines represent by haemocytes. This leads to a very focused formation of mean % total PO+ SE, respectively. Bars with different letters are signifi- melanized nodules associated with the acini of the salivary cantly different from each other taking the level of significance as P glands. The present study concerns only the first phase of 0.05. Numbers in parentheses refer to the number of observations per this response because dissected salivary gland tissue incu- group. (A) Salivary glands incubated in calcium-free ringer with and bated in vitro will contain few, if any, of the haemocytes without laminarin. (B) Salivary glands incubated in normal Ringer with and without laminarin, with 3 ml of calcimycin added to the incubation that circulate normally in the haemolymph. Therefore it is before salivary gland tissue. (C) Salivary glands incubated in normal doubtful that the PO activity observed in salivary glands Ringer with and without laminarin, with 3 ml of PMA added to the incu- in vitro originates from haemocytes, especially because an bation before salivary gland tissue. (D) Salivary glands incubated in increase in PO activity has been observed in locust salivary normal Ringer with and without laminarin, with 15 ml of okadaic acid gland tissue that has been rinsed in Ringer before being incu- added to the incubation before salivary gland tissue. bated with laminarin in vitro (G.J. Goldsworthy, personal communication). Therefore it is thought that the nephrocytes change significantly. When salivary glands were incubated associated with the acini are the source of the PO activity with calcimycin and laminarin there was a statistically measured in this study. Since haemocytes and nephrocytes significant greater increase in PO activity in comparison share the same embryological origin, this hypothesis seems with incubations of laminarin alone (Fig. 3B). logical, but remains to be demonstrated. Phorbol ester Effect of incubation time When salivary glands were incubated with PMA only (no The laminarin-induced increase in PO activity in salivary laminarin), there was no statistically significant increase in gland tissue in vitro occurs within 20 min. In terms of the PO. However, when salivary gland tissue was incubated speed of the response, this is consistent with findings by with PMA and laminarin together, although the PO activity Goldsworthy et al. who measured changes in PO activity increased 4-fold in comparison with incubations in the in vivo in samples of haemolymph taken from adult locusts ......................................................................................................................................................................................................................................... 125 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... at various times after injection with laminarin, but the the incubation with calcimycin with the same effect on PO response in the haemolymph is more prolonged. In the activity. However, calcimycin alone does not bring about present study, incubations need only have been left for PPO activation in the salivary glands but, interestingly, 20 min, but were in fact allowed to run for 60 min because there is significant augmentation of the rise of PO activity this allowed time for completion of all dissections before in response to laminarin when salivary glands are incubated the addition of protease inhibitor. Note that PO activity with calcimycin and laminarin together. The reasons for this increases from minimal to maximal levels between 10 and are unclear, but the results suggest that an influx of extra- 20 min, suggesting that signalling activity leading to PPO cellular calcium is involved in the PO response, but that activation takes place during the first 10 min after tissue other cellular events triggered by laminarin need to take comes into contact with immunogen. In terms of magnitude place for PPO to become activated. of response, the percentage activation of the total PPO in the Other second messengers salivary glands is greater (at around 20%) than that observed in the haemolymph (around 10%) under the most favourable The possible role of cAMP as a second messenger in PPO conditions by Goldsworthy et al. activation in the salivary glands was not examined in this Interestingly, the PO response to laminarin in salivary study because the cAMP activator forskolin is ineffective glands is lost when the addition of immunogen to the incu- when added to incubations of salivary glands in vitro bation mixture is delayed for 10 min after the addition of (G.J. Goldsworthy, personal communication). However, the salivary gland tissue. The reason for this is unclear, but the results of this study suggest that protein kinase C it could be that the cells responsible for PPO activation (PKC) is also not involved in the activation cascade for become refractory soon after being extracted from the PPO induced by laminarin. Phorbol esters such as PMA acti- animal and therefore need to come into contact with immu- vate PKC, and many (although not all) kinases in this family nogen directly after dissection for a response to take place. are calcium-dependent and it could therefore have been This loss of sensitivity to laminarin in vitro may explain possible that the calcium-dependency of the PO response is partly why the response to laminarin increases for only due to the presence of calcium-dependent kinases in the 20 min in the salivary gland in vitro (this study), and for PPO activation cascade. However, addition of PMA, either up to 60 min in the haemolymph in vivo. in the presence or absence of laminarin, does not influence PO activity in the salivary glands in vitro. Calcium dependency Okadaic acid is an inhibitor of phosphatases that remove 2þ When salivary glands are incubated in Ca -free Ringer phosphate groups from proteins, often returning proteins to (with EGTA), the increase in PO activity in response to lami- an ‘inactive’ state. Their presence in the PPO cascade would narin is lost. The loss in PO activation by laminarin in the modulate PPO activation. Thus, if the laminarin-sensitive absence of calcium is not caused by toxicity of the EGTA pathway for PPO activation is constitutively active, incubating 2þ because salivary glands incubated in a Ca -free Ringer salivary glands with a phosphatase inhibitor could mimic the without EGTA do not show an increase in PO activity in PPO activating effect of laminarin. Incubation with okadaic response to laminarin. This suggests strongly that activation acid alone showed that this is not the case, but the response of the PPO cascade in locust salivary glands is calcium- to laminarin is augmented markedly when salivary glands dependent. This is not surprising because PPO in insect are incubated with okadaic acid and laminarin together, haemolymph and cuticle is activated by a calcium-dependant suggesting that the PO response to laminarin in the salivary serine protease cascade. Further, the addition of PMSF glands is modulated by protein phosphatases. However, (a non-competitive inhibitor of serine proteases) to the incu- other cellular events triggered by laminarin are required to bation prior to salivary gland tissue prevents any increase in initiate PPO activation. The final concentration of okadaic PO activity in response to laminarin. Therefore, at least in acid in the incubation in these studies was 10 nM. this respect the evidence suggests that PPO in the salivary According to Scho¨nthal, a minimum okadaic acid concen- glands is activated by a mechanism similar to that operating tration of 20 nM is required for 50% inhibition of PP1, and elsewhere in the animal. a maximum 1 nM concentration for 50% inhibition of A rise in intracellular calcium concentration is a ubiqui- PP2A. It may therefore be informative to test different concen- tous signal in biological systems and can be achieved either trations of okadaic acid in incubations of salivary glands. by the release of calcium from intracellular stores, or the Physiological role of PO activity associated influx of extracellular calcium due to the opening of 2þ with insect salivary glands Ca -specific ion channels in the plasma membrane. Calcimycin is a carrier of calcium ions, allowing calcium The present study is not the first in which PO activity has to cross the cell membrane. Therefore if the action of lami- been detected in the salivary glands of insects. Miles narin is only to trigger the opening of plasma membrane stained the salivary glands of aphids with dopamine and pro- 2þ Ca channels, in theory, laminarin could be replaced in vided evidence of the presence of PO. More recently, Hattori ......................................................................................................................................................................................................................................... 126 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... et al. used SDS–PAGE to reveal the presence of two types References of PO (a laccase and a PO) in salivary gland homogenates 1. Gillespie JP, Kanost MR, Trenczek T (1997) Biological mediators of insect from the green rice leafhopper. This raises a question: what immunity. Annu Rev Entomol 42: 611–643. is the physiological role of PO activity in insect salivary 2. Hoffmann JA (2003) The immune response of Drosophila. Nature 426: 33–38. glands? One hypothesis is that PO is secreted into the 3. Lavine MD (2002) Strand MR Insect hemocytes and their role in immunity. saliva with the purpose of attacking pathogens or oxidizing Insect Biochem Mol Biol 32: 1295–1309. toxins in the mouth or gut. For example, Hattori et al. 4. Brey PT, Lee WJ, Yamakawa M et al. (1993) Role of the integument suggested that a possible function of the leafhopper salivary in insect immunity: epicuticular abrasion and induction of cecropin synthesis in cuticular epithelial cells. Proc Natl Acad Sci USA 90: laccase may be to rapidly oxidize toxins while feeding on 6275–6279. plants. It seems possible that PO may be secreted in locust 5. Ashida M, Brey PT (1995) Role of the integument in insect defense: pro- saliva for the purpose of oxidizing toxins in the food, or it phenol oxidase cascade in the cuticular matrix. Proc Natl Acad Sci USA 92: may even be transferred to the cuticle via grooming behaviour 10698–10702. where it could have a protective effect against infection. 6. Leonard C, Ratcliffe NA, Rowley AF (1985) The role of prophenoloxidase acti- However, there is as yet no evidence that the PO detected in vation in non-self recognition and phagocytosis by insect blood-cells. locust salivary glands in vitro is actually secreted into the J Insect Physiol 31: 789–799. saliva in vivo, although the presence of PO in the saliva has 7. Cherqui A, Duvic B, Brehelin M (1996) Purification and characterization of prophenoloxidase from the haemolymph of Locusta migratoria. Arch Insect been demonstrated in other insects. For example, an immuno- Biochem Physiol 32: 225–235. histochemical study on the salivary proteins of aphids by 8. Goldsworthy GJ, Opoku-Ware K, Mullen L (2002) Adipokinetic hormone Cherqui and Tjallingii confirmed the presence of PO in enhances laminarin and bacterial lipopolysaccharide-induced activation of the saliva. Hattori et al. analysed leafhopper saliva that the prophenoloxidase cascade in the African migratory locust, Locusta had been deposited on the food while feeding and showed migratoria. J Insect Physiol 48: 601–608. that the laccase found in the salivary gland homogenates of 9. Ashida M (1990) The prophenoloxidase cascade in insect immunity. Res the leafhoppers is secreted in the watery saliva. Immunol 141: 908–910. 10. Soderhall K, Cerenius L (1998) Role of the prophenoloxidase-activating system in invertebrate immunity. Curr Opin Immunol 10: 23–28. Conclusions 11. Hajek AE, St Leger RJ (1994) Interactions between fungal pathogens and insect hosts. Annu Rev Entomol 39: 293–322. This study has shown that adult locust salivary glands 12. Mullen LM, Goldsworthy GJ (2006) Immune responses of locusts to chal- possess PPO activity and that this can be activated in lenge with the pathogenic fungus Metarhizium or high doses of laminarin. response to laminarin in vitro. The response is calcium- J Insect Physiol 52: 389–398. dependent, possibly involving an influx of extracellular 13. Keating C, Orchard I (2001) Dopamine induces hyperpolarization of locust 2þ Ca , and is modulated by protein phosphatases. Future salivary gland acinar cells via D-1-like receptors. J Insect Physiol 47: 667–673. research should aim to analyse locust saliva to establish 14. Albrecht FO (1953) The Anatomy of the Migratory Locust. University of whether PO is present, and if so whether the levels change London: Athlone Press. during immune challenge. It is hoped that such work 15. Wigglesworth VB (1972) The Principles of Insect Physiology. London: Chapman & Hall. would help to clarify the function of salivary glands in the 16. Crossley AC (1985) Nephrocytes and pericardial cells. In Kerkut GA, Gilbert immune defence of the locust and to shed further light on LIeds, Comprehensive Insect Physiology, Biochemistry and Pharmacology. this intriguing aspect of insect immunity. Oxford: Pergamon Press Ltd, Vol. 3, pp. 487–515. 17. Goldsworthy GJ, Chandrakant S, Opoku-Ware K (2003) Adipokinetic hormone enhances nodule formation and phenoloxidase activation in Acknowledgements adult locusts injected with bacterial lipopolysaccharide. J Insect Physiol 49: I am grateful to Professor Graham Goldsworthy, Birkbeck 795–803. College, University of London, for supervision and encour- 18. Scho¨ntal AH (1998) Role of PP2A in intracellular signal transduction path- ways. Front Biosci 3: 1262–1273. agement, and Mary Lightfoot, Birkbeck College, University 19. Miles PW (1964) Studies on the salivary physiology of plant-bugs: the sali- of London, for general technical guidance and for providing vary secretions of aphids. J Insect Physiol 11: 1261–1262. live insects for my research. 20. Hattori M, Konishi H, Tamura Y et al. (2005) Laccase-type phenoloxidase in salivary glands and watery saliva of the green rice leafhopper, Nephotettix cincticeps. J Insect Physiol 51: 1359–1365. Funding 21. Cherqui A, Tjallingii WF (2000) Salivary proteins of aphids, a pilot study on This work was funded by the School of Biological and identification, separation and immunolocalisation. J Insect Physiol 46: Chemical Sciences, Birkbeck College, University of London. 1177–1186. ........................................................................................................................................................................................................................................ Submitted on 26 September 2007; accepted on 17 December 2007; advance access publication 17 April 2008 ......................................................................................................................................................................................................................................... http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

The effects of immune challenge on phenoloxidase activity in locust salivary glands in vitro

Bioscience Horizons , Volume 1 (2) – Jun 17, 2008

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Volume 1 † Number 2 † June 2008 10.1093/biohorizons/hzn015 ......................................................................................................................................................................................................................................... Research article The effects of immune challenge on phenoloxidase activity in locust salivary glands in vitro Kirsty L. Wells* Birkbeck College, University of London, Malet Street, London WC1E 7HX, UK. * Corresponding author: Birkbeck College, London WC1E 7HX, UK. Email: kirsty.wells@kcl.ac.uk Supervisor: Professor Graham J. Goldsworthy, Birkbeck, University of London, London, UK. ........................................................................................................................................................................................................................................ Homogenates of salivary glands from Locusta migratoria possess phenoloxidase (PO) activity. This study investigates the activation of prophenoloxidase (PPO) in these glands in vitro. When freshly dissected salivary glands from L. migratoria are incubated with the immu- nogen laminarin, and then homogenized, a 4-fold increase in PO activity (expressed as a percentage of the total PO) can be measured within 20 min. Addition of laminarin to the incubation medium is best made prior to addition of salivary gland tissue, because when laminarin is added 10 min after the addition of tissue, the response to laminarin is reduced by 50%. When salivary glands are incu- 2þ bated in Ca -free Ringer, the response to laminarin cannot be demonstrated. Addition of a calcium ionophore to the incubation in normal Ringer does not initiate a response on its own, but does augment the response to laminarin. Addition of phorbol ester to an incubation containing normal Ringer has no effect on PO activity, and does not augment the response to laminarin. In contrast, addition of okadaic acid to normal Ringer has no effect on its own, but does augment the response to laminarin. Activation of PPO in response to 2þ laminarin is therefore calcium-dependant, possibly involving an influx of extracellular Ca , and modulated by protein phosphatases. Future work should aim to clarify the function of salivary glands in the immune defence of the locust and to investigate the exact source of the PO associated with the glands. Key words: Locusta migratoria, phenoloxidase, salivary glands, laminarin. ........................................................................................................................................................................................................................................ Introduction non-specific responses to infection and can be said to com- prise three types of defence: physical, cellular and humoral. Insects possess a highly efficient immune system. An under- The cuticle is an insect’s first line of defence against invad- standing of this system of immune defence is important not ing microorganisms and it presents an impenetrable physical only in terms of potential applications to the biocontrol of 4, 5 barrier to many potential pathogens. However, if a patho- pest insects, but also because insects are useful models for gen is able to overcome cuticular defences, or cross the gut investigating immune systems. Therefore insect immunity is wall, coordinated responses of immune cells in the haemo- increasingly well-studied, especially in Drosophila, which lymph are initiated in response to pathogen detection. has become a particularly valuable model for studying This involves detection by pattern recognition proteins innate immunity over the past 10 years due to the power of (PRPs) present both in the haemolymph and on the surfaces molecular genetics. Locusts are also commonly used as a of haemocytes in the haemolymph. Haemocytes are cells model in studies of insect immunity due to their prevalence involved in haemolymph clotting and defence. They are as a pest in parts of the world and their relatively large size. able to bind pathogens, initiating signalling pathways Invertebrates do not show adaptive immunity and do not leading to the encapsulation or phagocytosis of the produce antibodies in response to infection. Instead, they rely foreign body by the haemocyte. Haemocytes can respond on a system of innate immunity to protect them from to infection in a coordinated manner, trapping foreign pathogens. The insect immune system consists of rapid, ......................................................................................................................................................................................................................................... 2008 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 122 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... bodies within aggregates of many haemocytes called polysaccharides found in fungal cell walls) into the haemo- nodules. Nodules are often visible to the naked eye as lymph: the acini of the salivary glands in dissected locusts blackened spots along the internal body wall of an infected became intensely black. It was thought that this phenomenon insect that has undergone dissection. did not occur in adult locusts, however, it has been shown Insects also synthesize a variety of extracellular humoral subsequently that the salivary glands of adult locusts, while compounds in response to immune challenge. Some of the showing a less pronounced melanization response to injected various signal transduction pathways initiated as a result of laminarin, do exhibit a strong response in terms of increased pathogen detection culminate in an alteration of gene PO activity in the glands (G.J. Goldsworthy, personal expression in specialist cells, primarily in the fat body, communication). causing these cells to produce and secrete infection-fighting This previously unreported phenomenon raises a number substances into the haemolymph. For example, lysozymes of questions. For example, what are the characteristics of are synthesized in the fat body of lepidopterans in response the PO response in locust salivary glands? What are the sig- to bacterial injection; broad-spectrum antibacterial peptides nalling pathways mediating the activation of PPO in locust (cecropins) are synthesized in the fat body, cuticle and salivary glands, and how is the immunogen being detected endothelium of lepidopterans and dipterans in response to by the salivary glands? This study of PO activity in salivary bacterial challenge; and the antifungal drosomycin from glands of adult locusts addresses these questions. Drosophila melanogaster also has antibacterial activity. These examples represent a small fraction of the many anti- Materials and methods bacterial and antifungal compounds that have been identified and sequenced from many different insect species. Insects One of the most important and ubiquitous compounds L. migratoria migratorioides were reared under crowded released by insects in response to immune challenge, and of conditions at 30ºC in a light:dark 12:12 h photocycle, and particular relevance to this study, is the enzyme phenoloxi- fed daily with fresh grass and wheat seedlings supplemented dase (PO). PO is activated in the cuticle or the haemolymph with bran. Adult male locusts between 15 and 25 days after of many invertebrates in response to immune challenge or the final moult were used in all experiments. wounding and is activated via the prophenoloxidase (PPO) cascade, PPO being the inactive zymogen of PO. PPO is Materials present in the haemolymph of Locusta migratoria and is Locust Ringer (168 mM NaCl, 6.4 mM KCl, activated in response to immune challenge. 3.6 mM MgCl 6 mM NaH PO 2H O, 2.1 mM NaHCO 2, 2 4 2 3, Detection of a pathogen or immunogen by a PRP leads to 2þ 20 mM Hepes, 2.1 mM CaCl 4% Sucrose, pH 7.0) was 2, aCa -dependant cascade of serine proteases, the final com- prepared, autoclaved and stored at 58C until used as incu- ponent of which, the PPO activating enzyme, is thought to be 2þ bation medium. A Ca -free Ringer was used in some exper- a clip domain serine protease that cleaves PPO to PO. PO, iments (no CaCl , 1 mM EGTA added). an oxidoreductase, catalyses the oxidation of phenols All chemicals were purchased from Sigma Chemical present in the haemolymph to cytotoxic quinones. These Company. Laminarin was dissolved in locust Ringer to give quinones polymerize non-enzymatically to melanin. Both a10 mg mL solution. A protease inhibitor, phenylmetha- quinones and melanin are toxic to microorganisms. The nesulphonylfluoride (PMSF), was dissolved in propanol to deposition of melanin causes parasites to become blackened give a 0.5% solution. Immediately prior to use, dopamine in the haemolymph, and in nodules due to melanization of was dissolved in 0.01 M phosphate buffer (pH 5.9) to give haemocytes encapsulating foreign bodies. a 3 mg mL solution. Absolute methanol was used to acti- Although the cuticle is an effective barrier to most patho- vate PPO. Calcimycin, a calcium ionophore (A23187) was gens, many isolates of entomopathogenic fungi are able to dissolved in DMSO to give a 10 mM stock solution. penetrate the insect cuticle. As a result, over the past two Phorbol 12-myristate 13-acetate (PMA) and okadaic acid decades there has been considerable interest in these fungi were each dissolved in DMSO to give separate 100 nM as biocontrol agents. After a short delay, topically applied stock solutions. conidia of Metarhizium, for example, send out apressoria that can penetrate the cuticle. To circumvent the delay and Dissection produce synchronized infection, Mullen and Goldsworthy studied PO activity in haemolymph of L. migratoria after The head of the insect was removed using sharp scissors and injection of Metarhizium blastospores. During the course a mid-dorsal cut made along the full length of the body. The of these experiments, Mullen and Goldsworthy observed body was pinned ‘dry’ onto a corkboard with the inner that salivary glands of 5th instar L. migratoria nymphs mel- ventral surface of the body wall uppermost. The salivary anized in response to injection of blastospores or high doses glands were viewed under a binocular microscope and of laminarin (a b1–3 glucan that is representative of removed using two pairs of fine forceps. Each insect ......................................................................................................................................................................................................................................... 123 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... usually yielded sufficient salivary gland tissue for two incubations. Measurement of PO activity Salivary gland tissue was incubated in 1 ml plastic centrifuge tubes containing 150 ml of locust Ringer (and other incu- bation constituents as appropriate) at room temperature for 60 min unless otherwise stated. The room temperature did not vary appreciably between different experiments, but control incubations were always included to allow for poss- ible differences due to any variations in room temperature. Incubations were stopped by the addition of 1 ml of PMSF to prevent further serine protease activity. After homogeniz- Figure 1. Percentage of total PO activated in salivary gland tissue incu- ation by sonication (Cole-Parmer for 10 s) and centrifugation bated with 4 ml laminarin (added to the incubation prior to tissue) for (13 000 rpm (10 000g) for 2 min), two 50 ml samples of various time periods. A control group of salivary glands was incubated for 60 min in a mixture to which 1 ml PMSF had been added before supernatant were taken from each tube and pipetted into tissue (0 min incubation time). Data points and vertical lines represent separate wells of a microtitre plate. Total PPO was activated mean % total PO+ SE, respectively. Data points with different letters are in one of the wells by adding 50 ml of methanol, mixing and significantly different from each other taking the level of significance as discarding 50 ml of the mixture to leave 50 ml in the well. P  0.05. Numbers in parentheses refer to the number of observations After addition of 150 ml of dopamine solution to both per group. wells, PO activity was assessed by determining the initial linear increase in absorbance at 492 nm. Absorbance incubations that did not have any addition of laminarin, values were read every minute for 15 min in a Labsystems there was no statistically significance increase in PO activity Multiskan Bichromatic plate reader. Appropriate allowance when laminarin was added to the incubation 10 min after the was made for the fact that the total PO activity related to tissue (Fig. 2). half the amount of salivary gland homogenate than was present in the sample not reacted with methanol. Calcium dependency When salivary gland tissue samples were incubated with Data analysis 2þ laminarin in Ca -free Ringer, PO activity was reduced by Data were initially evaluated using Microsoft Excel to calcu- 80% in comparison with incubations in normal Ringer late the slope of a line of best fit for the absorbance measure- 2þ and was similar to that in either Ca -free or standard ments taken from each microtitre-plate well between 0 and Ringer in the absence of laminarin (Fig. 3A). 15 min. The PO activity in each well that had not been acti- vated with methanol was expressed as a percentage of the PO Calcium ionophore activity in its corresponding well in which total PPO had When salivary glands were incubated in normal Ringer been activated with methanol. Data were analysed statisti- with calcimycin only (no laminarin) PO activity did not cally using a one-way ANOVA followed by Fisher’s one-way multiple comparison tests. All statistical tests were undertaken using Minitab. Results PO activity in response to addition of laminarin and the effect of time of incubation When laminarin was present in the incubation medium at the start of the incubations, PO activity in the homogenates of the glands did not increase significantly within the first Figure 2. Percentage of total PO activated in salivary gland tissue incu- 10 min of incubation, but subsequently it increased to bated with 4 ml laminarin which had been added to the incubation before tissue (open bar, time ¼ 0) and 10 min after tissue (shaded bar). reach a maximal level by about 20 min that persisted for A control group of salivary glands was incubated without laminarin up to 60 min (Fig. 1). Delaying the addition of laminarin (open bar, No Lam). Bars and vertical lines represent mean % total PO+ to the incubations of salivary glands by 10 min reduced PO SE, respectively. Bars with different letters are significantly different from activity by  50% in comparison with the levels when lami- each other taking the level of significance as P  0.05. Numbers in parenth- narin was added before the glands. In comparison with eses refer to the number of observations per group. ......................................................................................................................................................................................................................................... 124 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... absence of laminarin, there was no indication that the PMA had enhanced the increase in PO activity due to the laminarin (Fig. 3C). Okadaic acid Addition of okadaic acid on its own did not change the PO activity significantly. When salivary glands were incubated with okadaic acid and laminarin together, PO activity increased  6-fold in comparison with incubations in which laminarin was absent, which represented a statistically significant  2-fold enhancement of the increase in PO activity in comparison with incubations with laminarin on its own (Fig. 3D). Discussion Locust salivary glands comprise secretory acini bundled together in a grape-like arrangement. Associated with each acinus of L. migratoria there are three or four nephro- cytes. These are pericardial cells and, like haemocytes, originate from the mesoderm, but unlike haemocytes are static cells associated with tissues. They are involved in clearance of substances from the haemolymph and are able to take up material by endocytosis and release it by exocyto- sis. In L. migratoria, nephrocytes are involved in immuno- gen uptake leading to haemocyte recruitment and nodule 12, 17 formation. It seems likely that salivary gland melanization in locusts Figure 3. Percentage of total PO activated in salivary gland tissue in 12 in vivo as described by Mullen and Goldsworthy is a two- various incubation mixtures. Control groups of salivary glands (open phase process, whereby nephrocytes recognize foreign bars) were incubated with 4 ml laminarin added to the incubation prior material and then themselves become a focus for ‘attack’ to tissue (the first bar on the left of each panel), and without laminarin (the third bar from the left of each panel). Bars and vertical lines represent by haemocytes. This leads to a very focused formation of mean % total PO+ SE, respectively. Bars with different letters are signifi- melanized nodules associated with the acini of the salivary cantly different from each other taking the level of significance as P glands. The present study concerns only the first phase of 0.05. Numbers in parentheses refer to the number of observations per this response because dissected salivary gland tissue incu- group. (A) Salivary glands incubated in calcium-free ringer with and bated in vitro will contain few, if any, of the haemocytes without laminarin. (B) Salivary glands incubated in normal Ringer with and without laminarin, with 3 ml of calcimycin added to the incubation that circulate normally in the haemolymph. Therefore it is before salivary gland tissue. (C) Salivary glands incubated in normal doubtful that the PO activity observed in salivary glands Ringer with and without laminarin, with 3 ml of PMA added to the incu- in vitro originates from haemocytes, especially because an bation before salivary gland tissue. (D) Salivary glands incubated in increase in PO activity has been observed in locust salivary normal Ringer with and without laminarin, with 15 ml of okadaic acid gland tissue that has been rinsed in Ringer before being incu- added to the incubation before salivary gland tissue. bated with laminarin in vitro (G.J. Goldsworthy, personal communication). Therefore it is thought that the nephrocytes change significantly. When salivary glands were incubated associated with the acini are the source of the PO activity with calcimycin and laminarin there was a statistically measured in this study. Since haemocytes and nephrocytes significant greater increase in PO activity in comparison share the same embryological origin, this hypothesis seems with incubations of laminarin alone (Fig. 3B). logical, but remains to be demonstrated. Phorbol ester Effect of incubation time When salivary glands were incubated with PMA only (no The laminarin-induced increase in PO activity in salivary laminarin), there was no statistically significant increase in gland tissue in vitro occurs within 20 min. In terms of the PO. However, when salivary gland tissue was incubated speed of the response, this is consistent with findings by with PMA and laminarin together, although the PO activity Goldsworthy et al. who measured changes in PO activity increased 4-fold in comparison with incubations in the in vivo in samples of haemolymph taken from adult locusts ......................................................................................................................................................................................................................................... 125 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... at various times after injection with laminarin, but the the incubation with calcimycin with the same effect on PO response in the haemolymph is more prolonged. In the activity. However, calcimycin alone does not bring about present study, incubations need only have been left for PPO activation in the salivary glands but, interestingly, 20 min, but were in fact allowed to run for 60 min because there is significant augmentation of the rise of PO activity this allowed time for completion of all dissections before in response to laminarin when salivary glands are incubated the addition of protease inhibitor. Note that PO activity with calcimycin and laminarin together. The reasons for this increases from minimal to maximal levels between 10 and are unclear, but the results suggest that an influx of extra- 20 min, suggesting that signalling activity leading to PPO cellular calcium is involved in the PO response, but that activation takes place during the first 10 min after tissue other cellular events triggered by laminarin need to take comes into contact with immunogen. In terms of magnitude place for PPO to become activated. of response, the percentage activation of the total PPO in the Other second messengers salivary glands is greater (at around 20%) than that observed in the haemolymph (around 10%) under the most favourable The possible role of cAMP as a second messenger in PPO conditions by Goldsworthy et al. activation in the salivary glands was not examined in this Interestingly, the PO response to laminarin in salivary study because the cAMP activator forskolin is ineffective glands is lost when the addition of immunogen to the incu- when added to incubations of salivary glands in vitro bation mixture is delayed for 10 min after the addition of (G.J. Goldsworthy, personal communication). However, the salivary gland tissue. The reason for this is unclear, but the results of this study suggest that protein kinase C it could be that the cells responsible for PPO activation (PKC) is also not involved in the activation cascade for become refractory soon after being extracted from the PPO induced by laminarin. Phorbol esters such as PMA acti- animal and therefore need to come into contact with immu- vate PKC, and many (although not all) kinases in this family nogen directly after dissection for a response to take place. are calcium-dependent and it could therefore have been This loss of sensitivity to laminarin in vitro may explain possible that the calcium-dependency of the PO response is partly why the response to laminarin increases for only due to the presence of calcium-dependent kinases in the 20 min in the salivary gland in vitro (this study), and for PPO activation cascade. However, addition of PMA, either up to 60 min in the haemolymph in vivo. in the presence or absence of laminarin, does not influence PO activity in the salivary glands in vitro. Calcium dependency Okadaic acid is an inhibitor of phosphatases that remove 2þ When salivary glands are incubated in Ca -free Ringer phosphate groups from proteins, often returning proteins to (with EGTA), the increase in PO activity in response to lami- an ‘inactive’ state. Their presence in the PPO cascade would narin is lost. The loss in PO activation by laminarin in the modulate PPO activation. Thus, if the laminarin-sensitive absence of calcium is not caused by toxicity of the EGTA pathway for PPO activation is constitutively active, incubating 2þ because salivary glands incubated in a Ca -free Ringer salivary glands with a phosphatase inhibitor could mimic the without EGTA do not show an increase in PO activity in PPO activating effect of laminarin. Incubation with okadaic response to laminarin. This suggests strongly that activation acid alone showed that this is not the case, but the response of the PPO cascade in locust salivary glands is calcium- to laminarin is augmented markedly when salivary glands dependent. This is not surprising because PPO in insect are incubated with okadaic acid and laminarin together, haemolymph and cuticle is activated by a calcium-dependant suggesting that the PO response to laminarin in the salivary serine protease cascade. Further, the addition of PMSF glands is modulated by protein phosphatases. However, (a non-competitive inhibitor of serine proteases) to the incu- other cellular events triggered by laminarin are required to bation prior to salivary gland tissue prevents any increase in initiate PPO activation. The final concentration of okadaic PO activity in response to laminarin. Therefore, at least in acid in the incubation in these studies was 10 nM. this respect the evidence suggests that PPO in the salivary According to Scho¨nthal, a minimum okadaic acid concen- glands is activated by a mechanism similar to that operating tration of 20 nM is required for 50% inhibition of PP1, and elsewhere in the animal. a maximum 1 nM concentration for 50% inhibition of A rise in intracellular calcium concentration is a ubiqui- PP2A. It may therefore be informative to test different concen- tous signal in biological systems and can be achieved either trations of okadaic acid in incubations of salivary glands. by the release of calcium from intracellular stores, or the Physiological role of PO activity associated influx of extracellular calcium due to the opening of 2þ with insect salivary glands Ca -specific ion channels in the plasma membrane. Calcimycin is a carrier of calcium ions, allowing calcium The present study is not the first in which PO activity has to cross the cell membrane. Therefore if the action of lami- been detected in the salivary glands of insects. Miles narin is only to trigger the opening of plasma membrane stained the salivary glands of aphids with dopamine and pro- 2þ Ca channels, in theory, laminarin could be replaced in vided evidence of the presence of PO. More recently, Hattori ......................................................................................................................................................................................................................................... 126 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... et al. used SDS–PAGE to reveal the presence of two types References of PO (a laccase and a PO) in salivary gland homogenates 1. Gillespie JP, Kanost MR, Trenczek T (1997) Biological mediators of insect from the green rice leafhopper. This raises a question: what immunity. Annu Rev Entomol 42: 611–643. is the physiological role of PO activity in insect salivary 2. Hoffmann JA (2003) The immune response of Drosophila. Nature 426: 33–38. glands? One hypothesis is that PO is secreted into the 3. Lavine MD (2002) Strand MR Insect hemocytes and their role in immunity. saliva with the purpose of attacking pathogens or oxidizing Insect Biochem Mol Biol 32: 1295–1309. toxins in the mouth or gut. For example, Hattori et al. 4. Brey PT, Lee WJ, Yamakawa M et al. (1993) Role of the integument suggested that a possible function of the leafhopper salivary in insect immunity: epicuticular abrasion and induction of cecropin synthesis in cuticular epithelial cells. Proc Natl Acad Sci USA 90: laccase may be to rapidly oxidize toxins while feeding on 6275–6279. plants. It seems possible that PO may be secreted in locust 5. Ashida M, Brey PT (1995) Role of the integument in insect defense: pro- saliva for the purpose of oxidizing toxins in the food, or it phenol oxidase cascade in the cuticular matrix. Proc Natl Acad Sci USA 92: may even be transferred to the cuticle via grooming behaviour 10698–10702. where it could have a protective effect against infection. 6. Leonard C, Ratcliffe NA, Rowley AF (1985) The role of prophenoloxidase acti- However, there is as yet no evidence that the PO detected in vation in non-self recognition and phagocytosis by insect blood-cells. locust salivary glands in vitro is actually secreted into the J Insect Physiol 31: 789–799. saliva in vivo, although the presence of PO in the saliva has 7. Cherqui A, Duvic B, Brehelin M (1996) Purification and characterization of prophenoloxidase from the haemolymph of Locusta migratoria. Arch Insect been demonstrated in other insects. For example, an immuno- Biochem Physiol 32: 225–235. histochemical study on the salivary proteins of aphids by 8. Goldsworthy GJ, Opoku-Ware K, Mullen L (2002) Adipokinetic hormone Cherqui and Tjallingii confirmed the presence of PO in enhances laminarin and bacterial lipopolysaccharide-induced activation of the saliva. Hattori et al. analysed leafhopper saliva that the prophenoloxidase cascade in the African migratory locust, Locusta had been deposited on the food while feeding and showed migratoria. J Insect Physiol 48: 601–608. that the laccase found in the salivary gland homogenates of 9. Ashida M (1990) The prophenoloxidase cascade in insect immunity. Res the leafhoppers is secreted in the watery saliva. Immunol 141: 908–910. 10. Soderhall K, Cerenius L (1998) Role of the prophenoloxidase-activating system in invertebrate immunity. Curr Opin Immunol 10: 23–28. Conclusions 11. Hajek AE, St Leger RJ (1994) Interactions between fungal pathogens and insect hosts. Annu Rev Entomol 39: 293–322. This study has shown that adult locust salivary glands 12. Mullen LM, Goldsworthy GJ (2006) Immune responses of locusts to chal- possess PPO activity and that this can be activated in lenge with the pathogenic fungus Metarhizium or high doses of laminarin. response to laminarin in vitro. The response is calcium- J Insect Physiol 52: 389–398. dependent, possibly involving an influx of extracellular 13. Keating C, Orchard I (2001) Dopamine induces hyperpolarization of locust 2þ Ca , and is modulated by protein phosphatases. Future salivary gland acinar cells via D-1-like receptors. J Insect Physiol 47: 667–673. research should aim to analyse locust saliva to establish 14. Albrecht FO (1953) The Anatomy of the Migratory Locust. University of whether PO is present, and if so whether the levels change London: Athlone Press. during immune challenge. It is hoped that such work 15. Wigglesworth VB (1972) The Principles of Insect Physiology. London: Chapman & Hall. would help to clarify the function of salivary glands in the 16. Crossley AC (1985) Nephrocytes and pericardial cells. In Kerkut GA, Gilbert immune defence of the locust and to shed further light on LIeds, Comprehensive Insect Physiology, Biochemistry and Pharmacology. this intriguing aspect of insect immunity. Oxford: Pergamon Press Ltd, Vol. 3, pp. 487–515. 17. Goldsworthy GJ, Chandrakant S, Opoku-Ware K (2003) Adipokinetic hormone enhances nodule formation and phenoloxidase activation in Acknowledgements adult locusts injected with bacterial lipopolysaccharide. J Insect Physiol 49: I am grateful to Professor Graham Goldsworthy, Birkbeck 795–803. College, University of London, for supervision and encour- 18. Scho¨ntal AH (1998) Role of PP2A in intracellular signal transduction path- ways. Front Biosci 3: 1262–1273. agement, and Mary Lightfoot, Birkbeck College, University 19. Miles PW (1964) Studies on the salivary physiology of plant-bugs: the sali- of London, for general technical guidance and for providing vary secretions of aphids. J Insect Physiol 11: 1261–1262. live insects for my research. 20. Hattori M, Konishi H, Tamura Y et al. (2005) Laccase-type phenoloxidase in salivary glands and watery saliva of the green rice leafhopper, Nephotettix cincticeps. J Insect Physiol 51: 1359–1365. Funding 21. Cherqui A, Tjallingii WF (2000) Salivary proteins of aphids, a pilot study on This work was funded by the School of Biological and identification, separation and immunolocalisation. J Insect Physiol 46: Chemical Sciences, Birkbeck College, University of London. 1177–1186. ........................................................................................................................................................................................................................................ Submitted on 26 September 2007; accepted on 17 December 2007; advance access publication 17 April 2008 .........................................................................................................................................................................................................................................

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Bioscience HorizonsOxford University Press

Published: Jun 17, 2008

Keywords: Key words Locusta migratoria phenoloxidase salivary glands laminarin

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