Temporal patterns in immune responses to a range of microbial insults (Tenebrio molitor)
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ARTICLE IN PRESS Journal of Insect Physiology 54 (2008) 1090– 1097
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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Temporal patterns in immune responses to a range of microbial insults (Tenebrio molitor) Eleanor R. Haine a,, Laura C. Pollitt a,1, Yannick Moret b, Michael T. Siva-Jothy a, Jens Rolff a a b
Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK ´osciences, Universite´ de Bourgogne, 6 Boulevard Gabriel, 21000 Dijon, France Equipe Ecologie Evolutive, UMR CNRS 5561 Bioge
a r t i c l e in f o
a b s t r a c t
Article history: Received 28 January 2008 Received in revised form 9 April 2008 Accepted 15 April 2008
Much work has elucidated the pathways and mechanisms involved in the production of insect immune effector systems. However, the temporal nature of these responses with respect to different immune insults is less well understood. This study investigated the magnitude and temporal variation in phenoloxidase and antimicrobial activity in the mealworm beetle Tenebrio molitor in response to a number of different synthetic and real immune elicitors. We found that antimicrobial activity in haemolymph increased rapidly during the first 48 h after a challenge and was maintained at high levels for at least 14 days. There was no difference in the magnitude of responses to live or dead Escherichia coli or Bacillus subtilis. While peptidoglylcan also elicited a long-lasting antimicrobial response, the response to LPS was short lived. There was no long-lasting upregulation of phenoloxidase activity, suggesting that this immune effector system is not involved in the management of microbial infections over a long time scale. & 2008 Elsevier Ltd. All rights reserved.
Keywords: Insect immunity Antimicrobial peptides Haemolymph Zone of inhibition Long-lasting immunity Phenoloxidase Prophenoloxidase
1. Introduction The success of insects at avoiding and surviving infections may be largely attributable to the way they manage their suite of immune responses. Insect immune effector system upregulation and activity varies both temporally, and according to the type of pathogen challenge (reviewed by Lemaitre et al., 1997; Hultmark, 2003; Siva-Jothy et al., 2005). The production of antimicrobial peptides following an immune challenge is possibly the major induced component of insect immunity (reviewed in Dunn, 1986; Boman and Hultmark, 1987), and the biochemical pathways and molecular machinery involved in their upregulation have been well studied in some instances (reviewed by Bulet et al., 1999, 2004). In Drosophila, the temporal nature of the upregulation of genes involved in the production of antimicrobial peptides is relatively well documented (e.g. Lemaitre et al., 1997; Leulier et al., 2003). The expression of antimicrobial peptide genes begins 1–3 h after an immune challenge (Lemaitre et al., 1997), peaks at 3–12 h after a challenge, and may remain at high levels for relatively long periods, depending on the challenge and the antimicrobial peptide assayed Corresponding author. Tel.: +44 114 222 0105; fax: +44 114 222 0002.
E-mail address: e.haine@sheffield.ac.uk (E.R. Haine). Present address: Institutes of Evolution, Infection and Immunology Research, School of Biological Sciences, The King’s Buildings, University of Edinburgh, Edinburgh EH9 3JT, Scotland, UK 1
0022-1910/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2008.04.013
(Lemaitre et al., 1997). Drosomycin expression, for example, is still at high levels 72 h after D. melanogaster were challenged with Beauveria bassiana (Lemaitre et al., 1997). Antimicrobial peptide gene expression is also detectable in the haemolymph of the moth Pseudoplusia includens 2 h after an immune challenge and peaks at 8–24 h post-challenge (Lavine et al., 2005). In order to understand which factors have had important roles in the evolution of insect antimicrobial peptides, it is important to investigate how antimicrobial peptide expression varies temporally, and according to the type of pathogen challenge received in a number of different insects. The appearance of antimicrobial peptides in insect haemolymph varies. It is detectable in the bumblebee Bombus terrestris 2 h after a challenge (Korner and Schmid-Hempel, 2004); 4 h after a challenge in the locust Locusta migratoria (Hoffmann (1980); 10 h after challenge in the silkmoth Samia cynthia (Faye et al., 1975); 6–12 h after challenge in the beetle Zophobas atratus (Bulet et al., 1991); 24–48 h after challenge in Rhodnius prolixus prolixus (Azambuja et al., 1986). The duration of antimicrobial activity in insect haemolymph can be long: at least 5 days in the butterfly Pieris brassicae and the wax moth Galleria mellonella (Jarosz, 1993); up to 9 days in the silkmoth S. cynthia (Faye et al., 1975); 11 days in R. prolixus (Azambuja et al., 1986) and the locust L. migratoria (Hoffmann, 1980); at least 14 days in the bumblebee B. terrestris (Korner and Schmid-Hempel, 2004); and is still at high levels 28 days after challenge in the beetle Z. atratus (Bulet et al., 1991) and 44 days after the challenge in the dragonfly Aeschna cyanea (Bulet et al., 1992).
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In the mealworm beetle Tenebrio molitor, the antimicrobial response is maintained for at least 7 days after insult, a trait that is adaptive via its effect on resistance against subsequent pathogen insults (Moret and Siva-Jothy, 2003). However, while the fact that insect immune responses are long lasting has been known for a number of years (Boman and Hultmark, 1987), few (if any) studies have systematically sampled haemolymph antimicrobial activity in relation to both time and more than two types of immune challenge (e.g. variation according to challenge only: Kanost et al.,1988; temporal variation only: Dunn et al., 1985 (3 days); Postlethwait et al.,1988 (8 days)), i.e. little is known about how the type of challenge impacts on the temporal nature of the response at the physiological level. Another important humoral component of insect immune responses is the phenoloxidase (PO) cascade. Pathogen recognition elicits the production of the inactive zymogen prophenoloxidase and its conversion into the enzyme PO (Ashida and Brey, 1995). PO is an enzyme involved in the conversion of phenols to quinones and the subsequent production of melanin. The production of melanin is likely to cut off the pathogen’s supply of oxygen and nutrients, eventually killing it. PO is therefore thought to have a major role in insect defences against pathogens (Cerenius and So¨derhall, 2004). The PO cascade is upregulated within 1 h after a pathogen challenge, and may continue to be expressed for more than 24 h (Korner and Schmid-Hempel, 2004). Little is known about how the type of challenge impacts upon the temporal nature of PO responses, or whether PO activity trades-off with other immune defences (but see Korner and Schmid-Hempel, 2004). Mounting any long-lasting immune response is potentially costly in terms of the metabolic expense of producing the defensive compound(s) and the potential to cause self harm (e.g. Poulsen et al., 2002; Armitage et al., 2003; Sadd and Siva-Jothy, 2006; Meylaers et al., 2007). Despite these costs, insects upregulate a suite of cytotoxic and antimicrobial responses upon immune insult, and they do this over a long period of time (reviewed in Boman and Hultmark, 1987). This strongly suggests that there is an adaptive advantage to mounting long-lasting responses. The aim of this study was to measure the temporal variation in antimicrobial and PO activities in response to challenges by different groups of live and dead bacteria and their associated immune elicitors. We used the mealworm beetle T. molitor as a model insect for this study because (a) it is known to mount long-lasting immune responses (Moret and Siva-Jothy, 2003), but relatively little is understood about the shape of the response curve(s); (b) it incurs different types of costs directly as a result of mounting an immune response (Armitage et al., 2003; Sadd and Siva-Jothy, 2006); and (c) its relatively large size makes it easy to measure immune activity in the haemolymph of individuals. This study quantifies the temporal nature of the levels of immune activity in response to different challenges at the physiological level of individuals, rather than examining the qualitative nature of the response (i.e. differential gene expression in response to pathogen recognition). In addition, by comparing the production of one induced and one constitutive component of the insect immune system, we aim to generate data that may reveal any correlations between different immune-responsive pathways.
2. Materials and methods Before solution preparation, all glass and plasticware were rinsed in 1% (w/v) E-Toxa Clean solution (Sigma) several times before autoclaving, and all solutions were made up in endotoxin-
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free water. This preparation served to minimize any contaminating lipopolysaccharide or peptidoglycan remnants before addition of LPS, peptidoglycan or Ringer. All injections were performed through the pleural membrane between the second and third abdominal sternites using sterilized (see earlier) glass capillaries that had been pulled out to a fine point with an electrode puller (Narishige PC-10). All experiments were performed at ambient temperature (2071 1C) unless otherwise stated. The size of all animals was measured as wet mass.
2.1. Insect culturing Experimental T. molitor beetles from a stock culture maintained at the University of Sheffield were reared and maintained in an insectory at 2572 1C with a light/dark (LD) 12:12 h photo cycle, and supplied with an ad libitum diet of rat chow and water, supplemented by apple. Newly eclosed pupae were collected, sexed and weighed. Only individuals weighing between 140 and 170 mg were used for the experiments and were maintained individually in grid box containers. At adult eclosion, beetles were allocated to one of the eight treatment groups (‘naı¨ve’, ‘Ringer’, ‘LPS’, ‘Peptidoglycan’, ‘Dead Escherichia coli’, ‘Live E. coli’, ‘Dead Bacillus subtilis’ and ‘Live B. subtilis’. All treatments were performed 7 days post-adult eclosion, and all individuals were virgin. All experimental beetles were therefore controlled for differences in age, gender, reproductive status and size.
2.2. Immune challenges Our experimental protocol consisted of the following eight treatments. ‘Naı¨ve’ animals did not undergo any treatment. Individuals in the ‘Ringer’ treatment were injected with 5 ml of sterile insect Ringer solution (128 mM NaCl, 18 mM CaCl2, 1.3 mM KCl, 2.3 mM NaHCO3). Individuals in the ‘‘LPS’’ treatment were injected with 0.5 mg/ml purified LPS (Sigma L4524) in 5 ml of sterile Ringer solution. LPS is a non-pathogenic surface molecule derived from E. coli. It is a highly immunogenic component of Gram-negative bacteria and elicits the production of antimicrobial peptides (Lemaitre et al., 1997; Soderhall, 1982; Ratcliffe et al., 1985). Commercial LPS often contains contaminating peptidoglycan fragments so only LPS that had been purified by both phenol extraction and ion exchange chromatography was used for this study. The ‘peptidoglycan’ treatment involved injection of 0.5 mg/ ml peptidoglycan derived from B. subtilis (Fluka 69554) in 5 ml sterile Ringer solution; peptidoglycan is a highly immunogenic component of Gram-positive bacterial cell walls. The final four treatments involved injection of dead or live B. subtilis A1 or E. coli 54.8T. To kill bacteria, 1 ml of freshly grown overnight culture was heat-killed at 95 1C for 30 min, centrifuged for 10 min at 10,000 g, rinsed with sterile Ringer solution and finally resuspended in 1 ml of sterile Ringer. Five microlitres of this solution (approximately 106 bacteria per ml) was then used for dead bacteria challenges. For live bacteria treatments, the bacteria were grown as described earlier but were not subjected to the heat treatment. The bacterial dose used was non-lethal, to ensure that the challenged individuals survived at least 14 days so that we could measure haemolymph antimicrobial activity over time. It was also in the range used in other invertebrate immunology studies (e.g. Sadd and Schmid-Hempel, 2006). Five beetles of each gender were assigned to each treatment group at each of the nine time intervals; an additional five beetles for each gender were included in the ‘naı¨ve’ and ‘Ringer’ treatments to make up for samples lost during assay calibrations.
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2.3. Collection of haemolymph Haemolymph samples were collected from individual beetles at 1 h and 1, 2, 4, 6, 8, 10, 12 and 14 days after treatment. Individuals were chilled on ice before 2–10 ml of haemolymph was collected from a wound in the beetle’s neck. The haemolymph was stored in a microcentrifuge tube at 80 1C. 2.4. Measurement of antimicrobial activity The antimicrobial activity in the haemolymph samples was measured using a ‘zone of inhibition’ assay (Moret and SchmidHempel, 2000). Our purpose was to measure the temporal nature of antimicrobial activity in T. molitor haemolymph after different treatments, but not to test the specificity of the haemolymph activity against different bacteria. Previous attempts to use E. coli and B. subtilis for this assay failed as these bacteria grew too quickly, and we could only measure haemolymph antimicrobial activity repeatably using Arthrobacter globiformis (Y. Moret, personal observation). Briefly, therefore, an overnight culture of A. globiformis was used to seed 1% agar plates in which wells were made using a sterilised glass Pasteur pipette. One microlitre of each haemolymph sample was placed in each well and the plates were incubated at 30 1C for 48 h. After incubation, a clear zone could be seen around the wells where the antimicrobial activity of the haemolymph inhibited bacterial growth. There is a linear relationship between the diameter of the zone squared and the log of antimicrobial peptide concentration (Hultmark et al., 1982). Qualitatively, however, there is no difference between presenting the data as the diameter or the diameter squared, and to avoid compounding marginal measurement errors by multiplying them, the diameter of the clear zone was used as the measure of haemolymph antimicrobial activity. 2.5. Measurement of phenoloxidase and prophenoloxidase activity PO activity was monitored spectrophotometrically as the formation of dopachrome. Haemolymph samples from the same individuals used for the measures of haemolymph antimicrobial activity were defrosted in ice water and diluted to a 1:20 ratio of haemolymph: phosphate buffered saline (PBS): 149.6 mM NaCl,
10 mM Na2PO4, pH 6.5). Cell walls were removed by centrifugation (4 1C, 6500 rpm, 15 min, Sanyo MSE Hawk 15/05 centrifuge). Eight microlitres of the supernatant were added to 8 ml L-DOPA (4 mg/ml in H2O), 8 ml PBS and 56 ml distilled water. The reaction was allowed to proceed for 60 min at 30 1C. During the reaction, the enzyme catalyses the conversion of L-DOPA to dopachrome, dopachrome can then be measured spectrophotometrically. Readings were taken every 15 s on a spectrophotometer (Molecular Devices VERSAmax). Enzyme activity was measured as Vmax (the slope of the reaction curve during the linear phase) because previous studies established that the concentration of L-DOPA used satisfied Michaelis–Menten kinetics, and the slope of the reaction curve therefore directly correlates with the concentration of PO in the sample (Thompson, 2002). Prophenoloxidase (ProPO) activity was measured after activation using chymotrypsin. Reaction mixtures contained 8 ml haemolymph supernatant, 5 ml chymotrypsin (5 mg/ml in distilled water), 8 ml PBS and 51 ml distilled water. The mixture was incubated in a 96-well plate for 10 min at room temperature before addition of 8 ml L-DOPA (4 mg/ ml in H2O). Vmax was determined as for PO activity.
2.6. Statistical analyses All analyses were conducted using the statistical package R (R Development Core Team, 2004). To test whether there were significant differences in haemolymph antimicrobial activity over time between the different treatments, the data were analysed using non-linear regression (Crawley, 2002) because the relationship between antimicrobial activity and time could not be linearised by transformation. The function that best describes the data is a two-parameter Ricker curve (y ¼ ax ebx, Ricker, 1975). The first parameter (a) describes the slope of the curve up to a peak activity, and the second parameter (b) describes the decline in the slope with time—lower estimates of ‘b’ indicate a slower decline, and higher values of ‘b’ indicate a faster decline. Values and standard errors for parameters were estimated for each treatment and were then used to fit curves to the data. Significant differences between parameter estimates for each treatment were assessed on the basis of non-overlapping 95% confidence intervals (Crawley, 2002) (see Fig. 2). The effects of treatment on PO activity and ProPO activity were tested with time
Fig. 1. Graph showing the change in antimicrobial activity over time, as measured as the diameter (in mm) of a zone of bacterial growth inhibition. Each point is the mean of 10 individuals.
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Fig. 2. Bar charts to illustrate the differences in estimates of parameters ‘a’ and ‘b’ for the function y ¼ ax ebx. Lines above and below parameter estimates for each treatment represent 95% confidence intervals. Inset: graphs represent the effect of changing the values of ‘a’ and ‘b’ on the shape of the fitted non-linear regression curves.
and gender as covariates in separate ANCOVAs. Non-significant interactions between treatments and the covariates were dropped from final analyses (Crawley, 2002). We started with models including all higher order interactions between the factors and proceeded with stepwise simplification of the models removing the highest order interaction term that was not significant first (Crawley, 2002).
3. Results 3.1. Antimicrobial peptide activity There was an increase in haemolymph antimicrobial activity against A. globiformis in all treatment groups except for naı¨ve individuals during the first 48 h. This started falling between days 2 and 4 (Figs. 1 and 3). In order to determine whether there were any significant differences in the shape of the response over time, the relationship between antimicrobial activity and time for each
treatment was analysed using non-linear regression (Crawley, 2002). As stated in the methods, the first parameter (a) describes the slope of the curve up to a peak activity, and the second parameter (b) describes the decline in the slope with time—lower estimates of ‘b’ indicate a slower decline, and higher values of ‘b’ indicate a faster decline (Fig. 2, Table 1). There were significant differences between parameter ‘a’ estimates for naı¨ve, Ringer and LPS treatment individuals, and individuals in the remaining five treatment groups (Fig. 2, Table 1). There were also significant differences between parameter ‘b’ estimates for LPS individuals, and the other treatments (Fig. 2, Table 1). As expected, naı¨ve individuals showed no change in antimicrobial activity. Interestingly, Ringer individuals showed an increase in antimicrobial activity, although to significantly lower levels than other, nonnaı¨ve, treatments. The response to LPS treatment was a rapid increase in antimicrobial activity to similar levels as in the peptidoglycan treatment, and dead and live B. subtilis and E. coli treatments. However, compared to these latter five treatments, the antimicrobial activity rapidly decreased to levels similar to the
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Table 1 Parameter estimates and 95% confidence intervals for the curve y ¼ ax ebx fitted to the data for each treatment Treatment
Parameter ‘a’ estimate
95% CI (a)
Significance level (a)
Parameter ‘b’ estimate
95% CI (b)
Significance level (b)
Naı¨ve Ringer LPS Dead E. coli Live E. coli Peptidoglycan Dead B. subtilis Live B. subtilis
1.351 6.647 28.714 17.102 17.117 17.747 13.496 13.593
1.238 1.784 6.794 2.934 2.659 3.174 2.717 2.348
a b c d d d d d
0.148 0.255 0.572 0.279 0.264 0.329 0.285 0.252
0.103 0.092 0.033 0.028 0.037 0.029 0.028 0.044
a a b a, c a c a, c a
Parameter a is the slope of the curve and b is the shape of the decline from the peak antimicrobial activity with time. Significance levels were determined for estimates of a and b for each treatment where the confidence intervals were non-overlapping. For each parameter, treatments with a different letter are significantly different from each other.
Ringer individuals by day 4 (Fig. 3). The antimicrobial activities of the remaining treatments declined with time but at a lower rate than the LPS treatment. Interestingly, there was no significant difference between the shapes of the functions fitted to any of these remaining five treatments (Peptidoglycan, Dead B. subtilis, Live B. subtilis, Dead E. coli, Live E. coli) (Fig. 2, Table 1). There were no significant differences between antimicrobial activities of males and females except for dead B. subtilis treatment for parameter ‘a’, and Ringer for parameter ‘b’. The antimicrobial activity in response to dead B. subtilis was significantly lower at all time points in females than in males (data not shown). The antimicrobial activity of Ringer-treated individuals was higher in females between days 6 and 14 than in males (data not shown). 3.2. Phenoloxidase and prophenoloxidase activities Both PO (data not shown) and ProPO (Fig. 4) activity increased with time post-challenge. An ANCOVA testing for the effects of gender, time and treatment on PO activity revealed a significant interaction between treatment and time (F7,846 ¼ 2.340, P ¼ 0.0228) and significant main effects of treatment (F7,846 ¼ 5.984, Po0.0001), and time (F1,846 ¼ 67.424, Po0.0001). There was no clear pattern of differences in PO activity over time according to treatment. An ANCOVA testing for the effects of gender, time and treatment on ProPO activity revealed significant main effects of treatment (F7,784 ¼ 7.100, Po0.0001), and time (F1,784 ¼ 37.304, Po0.0001). While there are significant effects of treatment, there is no clear pattern (Fig. 4). In order to further examine the kinetics of the PO and ProPO activities, two groups of 10 females were challenged with either LPS or peptidoglycan, and the levels of PO and ProPO were measured, as descried earlier, at 2, 6, 12 and 18 h after injection (Table 2). As can be seen in Fig. 4 and Table 2, the relationships between PO and ProPO activities and the different treatments are complex over all time points.
4. Discussion This study demonstrates that the T. molitor antimicrobial response can be long lasting, irrespective of the type of pathogen challenge used. The expression of different antimicrobial peptide genes has previously been shown to vary temporally, or according to the type of pathogen challenge (e.g. Kanost et al., 1988; Dunn et al., 1985; Postlethwait et al., 1988; Lemaitre et al., 1997; Lavine et al., 2005). However, few studies have systematically sampled haemolymph antimicrobial activity in relation to both time and type of immune challenge. An early study followed the antimicrobial activity of silkmoth haemolymph over 6 days after injection of three different bacteria; however, it is unclear how many replicates were used and their data were not analysed
statistically (Faye et al., 1975). Our study is therefore valuable as one of the very few systematic studies that have explored the temporal nature of an insect’s antimicrobial response after different immune challenges, in particular in comparing live and dead bacteria, and components of bacterial cell walls. Our results suggest that the functional outcome of the expression of different antimicrobial genes is a long-lasting antimicrobial activity in T. molitor haemolymph that lasts at least 14 days. The first 24 h after a challenge are critical for the clearance of pathogens from insect haemolymph (Dunn and Drake, 1983; Vodovar et al., 2005). However, in our study, haemolymph antimicrobial activity did not reach maximal levels until 24–48 h after a challenge, which in the time course of an infection is relatively late. For example, in vivo, 99% of injected E. coli are cleared from T. molitor haemolymph in the first hour after injection, presumably as a result of cellular immune defences (Clare, Haine, Rolff, Siva-Jothy, unpublished data). Antimicrobial peptides reach maximum levels 24–48 h after an immune challenge, and this suggests that their role is to control the few surviving pathogens that escape the effector systems responsible for the bulk of attrition (Dunn, 1986). In other insects, the time at which haemolymph antimicrobial activity peaks after a challenge varies between 24 and 48 h (Bulet et al., 1991; Korner and SchmidHempel, 2004), and 4–6 days post-challenge (Faye et al., 1975; Hoffmann, 1980; Azambuja et al., 1986; Postlethwait et al., 1988; Jarosz, 1993). Further work in other insects and using other pathogens is required to determine whether the time postchallenge at which haemolymph antimicrobial activity peaks is insect taxon-specific, pathogen-type dependent, or determined by exogenous factors. There was no clear effect of treatment on PO and ProPO activities, even between naı¨ve animals and those that had received challenges, but levels of both increased over the 14 days. The relationships between treatments and activities of both PO and ProPO were complex up to 24 h and between 24 h and 14 days, and there was an overall increase in PO and ProPO activity over time. It is possible that PO and ProPO activity increase with age in T. molitor. On the day of challenge, each beetle was 7 days postadult eclosion and at day 14 they would therefore have been 21 days post-adult eclosion. The increase in investment in PO and ProPO activity over time may reflect an increase in immune investment with time, or reflect other age-related physiological effects (e.g. Rolff, 2001). There was no obvious correlation between the activities of PO/ProPO and antimicrobial activity over 14 days, and this result is supported by data from the bumblebee B. terrestris (Korner and Schmid-Hempel, 2004). Interestingly, a recent study suggested that ProPO activation is not required for survival to microbial infections in Drosophila (Leclerc et al., 2006), and total PO activity is not correlated with disease resistance in crickets (Adamo, 2004).
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Fig. 3. Plot of antimicrobial activity (diameter (in mm) of a zone of bacterial growth inhibition) against time (days) for each treatment. Open circles are the actual data, closed triangles are the means for all individuals (n ¼ 10) for each time point, curves are the plotted fit of the non-linear regression curve y ¼ ax ebx.
Our results show there were no differences between antimicrobial activity elicited after challenges by peptidoglycan and live/dead B. subtilis and E. coli treatments, but there were significant differences between these and the other treatments. As expected, naı¨ve animals had a low level of antimicrobial activity over 14 days. An injection of sterile Ringer, however, resulted in an increase in antimicrobial activity, confirming other studies that suggest that a wound alone can upregulate the synthesis of antimicrobial substances in insect haemolymph (e.g. Postlethwait et al., 1988). The most interesting result is that while LPS elicits antimicrobial activity in haemolymph to similar levels as peptidoglycan and live and dead bacteria, the response is much shorter lived than for those treatments. The long-term production of antimicrobial responses in T. molitor haemolymph must there-
fore depend on the recognition of peptidoglycan and other bacterial cell wall constituents rather than LPS. Indeed, LPS has been shown not to induce the expression of the antimicrobial peptide diptericin in Drosophila (Leulier et al., 2003) and only weakly in Drosophila cell lines (Werner et al., 2003). Our result contrasts with a study on bumblebees in which haemolymph antimicrobial activity remained high 14 days after injection of LPS (Korner and Schmid-Hempel, 2004); however, our study used purified LPS and the longer lasting response in the bumblebee study may have been elicited by peptidoglycan contamination in their LPS preparation. Further work could investigate whether the temporal nature of the antimicrobial response is the same when T. molitor is exposed to DAP-type or Lys-type peptidoglycans, which activate different immune pathways (Kurata et al., 2006).
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Fig. 4. Graph showing the change in prophenoloxidase activity over time, for (a) B. subtilis treatments, and (b) E. coli treatments, with controls shown in both. Each point is the mean of 10 individuals.
Table 2 Activities of PO and ProPO during the first 18 h after a challenge by either LPS or peptidoglycan Time (h)
2 6 12 18
PO activity (Vmax)
ProPO activity (Vmax)
Naı¨ve
LPS
Peptidoglycan
Naı¨ve
LPS
Peptidoglycan
10.8272.69
3.6772.47 5.5971.44 6.2471.92 4.9971.07
0.9970.49 5.1571.85 5.7572.53 3.9471.11
20.4175.50
11.8273.72 21.8176.06 14.3472.02 27.1378.96
5.0770.88 8.8272.03 10.4071.51 11.7473.19
Means are shown7S.E. of the mean.
The fact that there are no significant differences in activity in response to B. subtilis (Gram positive) and E. coli (Gram negative) treatments suggests that the temporal nature of the response to these two bacteria is the same. And while their study ended at 8 days post-challenge, Postlethwait et al. (1988) showed that the response of Medfly haemolymph antimicrobial activity to an
injection of Enterobacter cloacae is qualitatively the same as the response we find in this study, but further work could examine the temporal nature of the antimicrobial response against a wider selection of bacteria. Finally, the similarities in the temporal nature of antimicrobial responses between T. molitor and other insects (e.g. Postlethwait
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et al., 1988; Bulet et al., 1991; Bulet et al., 1992; Korner and Schmid-Hempel, 2004) suggest that the long-lasting nature of antimicrobial responses in insects is a general phenomenon. But, mounting immune responses is costly to insects (reviewed in Schmid-Hempel, 2003), generating the question ‘‘why has it evolved?’’ Long-lasting responses may have evolved as a prophylactic response where there is a high probability of secondary infection (Moret and Siva-Jothy, 2003). Alternatively, microbes that survive beyond 24 h in the host may have done so because they evade the host immune system by locating themselves in cells (Hurst et al., 2003), or because they survive phagocytosis. Therefore, it is possible that these microbes have selected insects to maintain a response long after the pathogen appears to have been eliminated.
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