Integrating temperature and nutrition – Environmental impacts on an insect immune system

Journal of Insect Physiology 64 (2014) 14–20

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Integrating temperature and nutrition – Environmental impacts on an insect immune system Stephanie S. Bauerfeind a,⇑, Klaus Fischer b a b

Department of Immunology, University Medicine of Greifswald, 17489 Greifswald, Germany Zoological Institute & Museum, University of Greifswald, Johann-Sebastian-Bach Str. 11/12, 17489 Greifswald, Germany

a r t i c l e

i n f o

Article history: Received 23 September 2013 Received in revised form 25 January 2014 Accepted 7 March 2014 Available online 15 March 2014 Keywords: Diet Food quality Haemocytes Lysozyme Phenoloxidase Pieris napi

a b s t r a c t Globally increasing temperatures may strongly affect insect herbivore performance. In contrast to direct effects of temperature on herbivores, indirect effects mediated via thermal effects on host-plant quality are only poorly understood, despite having the potential to substantially impact the herbivores’ performance. Part of this performance is the organisms’ immune system which may be of pivotal importance for local survival. We here use a full-factorial design to explore the direct (larvae were reared at 17 °C or 25 °C) and indirect effects (host plants were reared at 17 °C or 25 °C) of temperature on immune function of the temperate-zone butterfly Pieris napi. At the higher rearing temperature haemocyte numbers and prophenoloxidase activity were reduced. Plant temperature, in contrast, did not affect immune competence despite clear effects on insect growth patterns. Overall, thermal and dietary impacts on the insects’ immune responses were weak and trait-specific. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Global temperatures have increased at a rate of approximately 0.2 °C per decade in the last 30 years (Hansen et al., 2006) and will continue to rise in coming decades by 1–4 °C until 2100, depending on the emission scenario used (IPCC, 2007). Well documented responses of species to the environmental change already at hand include shifts in temporal and geographic niches (Altermatt, 2011; Hegland et al., 2009; Visser, 2008; Walther et al., 2002; Walther, 2010). Across taxa, the capacities of species to respond to climatic changes through plastic and genetic changes have been less widely studied, but are now widely acknowledged as the basis for in situ adaptation and thus the local persistence of species (Chown et al., 2010; Hofmann and Todgham, 2010; Mitchell et al., 2010; Nyamukondiwa et al., 2011). Environmental temperatures are known to have strong and pervasive effects on ectotherm life including e.g. developmental and life history traits (e.g. reviewed in Angilletta (2009), Bauerfeind and Fischer (2013a), Fischer et al. (2011)), behavioural traits (Berwaerts and Van Dyck, 2004; Geister and Fischer, 2007), reproductive traits (Fischer et al., 2003), stress responses and immune function (e.g. Hoffmann et al., 2003; Karl et al., 2011).

⇑ Corresponding author. Tel.: +49 (0)3834/865518; fax: +49 (0)3834/865545. E-mail addresses: [email protected] (S.S. Bauerfeind), klaus.fischer@ uni-greifswald.de (K. Fischer). http://dx.doi.org/10.1016/j.jinsphys.2014.03.003 0022-1910/Ó 2014 Elsevier Ltd. All rights reserved.

Insects have a highly developed innate immune system involving humoral and cellular defence mechanisms (Lavine and Strand, 2002). Humoral defences include the production of antimicrobial peptides (including lysozymes that are produced in the insect fat body and play a key role in the defence against gram+ bacteria; c.f. Franke and Fischer, 2013), reactive oxygen and nitrogen species, and enzymatic cascades that regulate coagulation or melanisation of the haemolymph (Lavine and Strand, 2002). A key enzyme involved in melanogenesis is phenoloxidase PO (for a review on PO function see González-Santoyo and Córdoba-Aguilar (2011)). If PO is needed for immune defence, its inactive zymogens (proPO) are converted into active PO (in order to prevent autoimmunological damage; González-Santoyo and Córdoba-Aguilar, 2011). As PO is active for only a short time after an immune challenge, measuring levels of inactive proPO may provide a more accurate measure of immunity (Cerenius et al., 2008). PO is a costly trait whose production and maintenance appear to have fitness costs and can be viewed as an indicator of an individual’s condition even more than being an indicator of resistance per se (GonzálezSantoyo and Córdoba-Aguilar, 2011). Cellular defences include haemocyte-mediated phagocytosis, nodulation, and encapsulation as well as clot formation in response to wounding (Lavine and Strand, 2002). The humoral and cellular defence systems considerably overlap and are well coordinated with one another (Lavine and Strand, 2002), e.g. after encapsulation, one of the agents killing the foreign organism is the production of toxic quinones and

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hydroquinones via the proPO cascade (see Lavine and Strand (2002) and references therein). Recently, environmental impacts on immune function have been acknowledged to be of potentially pivotal importance for local survival in changing environments (Martin et al., 2010; Polley and Thompson, 2009; Rohr et al., 2011). Environmental temperatures strongly affect overall resistance to a wide variety of parasites and impact on latency periods and the recovery from an infection (see Catalán et al. (2012), Murdock et al. (2012a,b)). Although immunological parameters have been shown to readily respond to thermal variation, the few studies available so far suggest that responses to environmental change are complex and general patterns seem difficult to formulate, yet (for a review see Murdock et al. (2012a, 2013)). Generally, warmer temperatures seem to increase insect immune performance through increases of haemocyte numbers and the enzymatic activity of phenoloxidase (PO) and lysozymes (Catalán et al., 2012; González-Santoyo and Córdoba-Aguilar, 2011; Lavine and Strand, 2002; Murdock et al., 2012b). Also, infected animals have been shown to prefer warmer microclimates (behavioural fever; Kluger et al., 1998). Furthermore, increases to stressful temperatures reduce insect immune performance which is likely due to a trade-off with molecular stress mechanisms (Karl et al., 2011). In nature, shifts in environmental temperatures as predicted in climate change scenarios will not only exert direct effects on ectotherms, but will simultaneously affect the availability and quality of dietary resources such as host plants of herbivorous species. Thermal conditions during plant growth impact on multiple processes including photosynthesis, respiration, evapo-transpiration, nitrogen mineralisation and availability to the plant, and the production and composition of plant biomass (e.g. Way and Oren, 2010). A recent meta-analysis by Zvereva and Kozlov (2006) revealed that increasing temperatures may only weakly affect plant nitrogen content, C/N ratio or leaf mechanical traits, while other characteristics may be more profoundly affected (e.g. leaf water content, carbohydrates, phenols, terpenoids; Bohinc and Trdan, 2012; Kuokkanen et al., 2001; Veteli et al., 2002). While the effects of thermal conditions on plant growth and metabolism are well known, the consequences thereof for herbivores are less well documented even though the interactions between temperature and nutrition are of a potentially high ecological importance (Bauerfeind and Fischer, 2013a,b; Jankovic´-Tomanic´ and Lazarevic´, 2012; Verdú et al., 2010; Wojewodzic et al., 2011). For instance, a high temperature during plant growth resulted in host plants that impeded development and diminished body mass of the herbivorous caterpillars of a Lepidopteran species (Bauerfeind and Fischer, 2013a). Thus, potential negative effects of global warming may be exaggerated by a combination of direct effects on the focal species itself and indirect effects mediated via changes in host plant quality (Cornelissen, 2011; Huey et al., 2012; Kingsolver et al., 2011; Netherer and Schopf, 2010). Such indirect thermal effects may also affect immune responses in herbivorous species, as both the constitutive and induced immune defence system depend on sufficient and adequate nutrition (Cotter et al., 2011; Ponton et al., 2013; Schmid-Hempel, 2005; Siva-Jothy and Thompson, 2002; Vogelweith et al., 2011). Different immune pathways have been shown to respond differently to dietary quality and are involved in mutual trade-offs (Cotter et al., 2011; Lee et al., 2008; Schmid-Hempel, 2005; Vogelweith et al., 2011). For instance, in caterpillars, lysozyme and phenoloxidase activity were maximised for different dietary protein and carbohydrate intake (Cotter et al., 2011). Therefore, it is important to consider various components of the immune system at a time. Given these profound effects of environmental conditions on the immune system, the last decade has seen the rise of a discipline

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that has been coined ‘ecological immunology’ and that focusses on the impact of ecological parameters on the complexities and withinand across-species variation of the immune system (e.g. Boughton et al., 2011; Rolff and Siva-Jothy, 2003; Ponton et al., 2011). Within this discipline, sub-fields such as ‘nutritional immunology’ have emerged (Ponton et al., 2013) that focus on single extrinsic factors. Our study contributes to this field by integrating two environmental factors (temperature and nutrition) and as such crossing sub-field boundaries (see also Triggs and Knell (2012)). We here use the temperate-zone pierid butterfly Pieris napi (Linnaeus, 1758) to investigate interactive effects between larval rearing and plant growth temperature on adult butterfly immune parameters, in order to assess detrimental effects on performance. Our design enables us to simultaneously assess the direct and indirect (mediated via host plant quality) effects of temperature on ectotherm performance (Bauerfeind and Fischer, 2013a). The measured parameters include the activity of phenol- and prophenoloxidase, lysozyme-like activity of the haemolymph and haemocyte numbers. Specifically, we predict (1) direct thermal effects: increasing mean temperatures should result in an increased immune reactivity (Angilletta, 2009; Murdock et al., 2012a, 2012b; Thomas and Blanford, 2003; Triggs and Knell, 2012) and (2) indirect thermal effects: growing plants at a higher temperature is expected to reduce plant quality, negatively affecting herbivore performance (Bauerfeind and Fischer, 2013a; Cornelissen, 2011) and thus reducing immune reactivity. 2. Materials and methods 2.1. Study organism P. napi is a temperate-zone butterfly that is widely distributed across northern Eurasia (Ebert and Rennwald, 1993) forming a complex of different subspecies (Espeland et al., 2007). The species is bi- to trivoltine in most parts of its range with the first butterflies appearing in April/May and the last ones in September/October in Northern Europe (Ebert and Rennwald, 1993), although populations with only one generation per year occur. The morphology and life history of P. napi varies depending on altitude and latitude with more strongly melanised phenotypes occurring at high elevations and latitudes (Tuomaala et al., 2012). The sexes show a pronounced dimorphism with the females being smaller than the males in this highly polyandrous species (Wiklund and Kaitala, 1995). P. napi hibernates as pupa. The principal larval host plants are several species of Brassicaceae (such as Cardamine pratensis and Alliaria petiolata); the species is of limited importance as a pest species in contrast to the closely related Pieris brassicae (Ebert and Rennwald, 1993). For this study 19 P. napi females were collected near Greifswald, north-eastern Germany. 2.2. Experimental design Field-caught females were transferred to climate cells at Greifswald University, and were provided with water, sucrose solution (ca. 30 vol.%) and leaves of A. petiolata and Sinapis alba for egg deposition (25 °C, 60% relative humidity, L:D 20:4). On day 5 after hatching, resulting larvae were individually transferred to translucent plastic pots (250 ml, lined with moist tissue) and randomly divided among two rearing temperatures (17 °C and 25 °C). 17 °C approximates the average mean temperature in June/July in the part of Germany where the experimental population was derived from (German weather service, www.dwd.de), while 25 °C was chosen to cover a thermal range sufficiently high to elicit physiological responses of both plants and butterflies. Thus, it was not intended to mimic predicted climate change here, but to include

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temperatures commonly experienced by the butterflies, especially in the more southern ranges of its distribution, in nature. Until day 7 of larval development, larvae were fed a mixture of A. petiolata and S. alba. Starting on day 7 after hatching, larvae of both temperature groups were randomly distributed to two host-plant qualities. Larvae were provided with S. alba leaves from plants grown at either 17 °C or 25 °C, thus resulting in four treatment groups: (1) larval rearing temperature (LT) = 17 °C, plant growth temperature (PT) = 17 °C; (2) LT = 17 °C, PT = 25 °C; (3) LT = 25 °C, PT = 17 °C and (4) LT = 25 °C, PT = 25 °C. Sample sizes ranged between 67 and 78 individuals per treatment group (Ntotal = 290). All food plants used here were reared in climate cabinets at either 17 °C or 25 °C under identical light conditions and in standard pots (ca. 10–15 plants per pot). The same soil was used for all plants, which were randomly divided among thermal treatment groups after potting. Plants were watered every other day and harvested for food when they had reached the 8-leaf stage, i.e. before the onset of flowering buds. All leaves used for feeding insects were replaced daily. Resulting pupae were placed in individual translucent plastic pots (250 ml, lined with moist tissue). Upon eclosion, butterflies were marked individually, weighed (on the day of eclosion, but not before the wings had dried and the meconium had been excreted). They were provided with water, a sucrose solution and flowers as found in the field (amongst others Bellis perennis, Trifolium pratense, Taraxacum officinale, Achillea millefolium) in a communal cylindrical gauze cage. Two days after eclosion, butterflies were frozen at 80 °C for further analysis. All immune parameters measured (see below) thus represent the constitutive amount present in the organism without hurting or infecting the butterflies. 2.3. Immune parameters The assessment of the butterflies’ immune function included measurements of phenoloxidase (PO) and prophenoloxidase (ProPO) activity, haemocyte numbers, and lysozyme-like activity of the haemolymph. The protocols followed Bauerfeind and Fischer (2013b) and Karl et al. (2011) with slight modifications. Prior to analyses, wings, head and legs were removed, and thorax and abdomen were separated. Thoraces were weighed and used for the determination of PO and ProPO activity and the number of haemocytes (cf. Bauerfeind and Fischer, 2013c; Rolff et al., 2004; Stoks et al., 2006). Haemolymph extract was obtained by perfusing the thorax of each animal with 0.35 ml cacodylate buffer (0.01 mol/L Na-Coc, 0.005 mol/L CaCl2, syringe: single-use fine dosage syringe (Injekt-F 1 ml; Braun) with disposable hypodermic needle, Sterican, Braun). To obtain haemocyte numbers, 10 ll of the haemolymph extract was transferred into one well of a multiwell slide. To each well 2.5 ll of Gel Red™ (Nucleic Acid Gel Stain, 10,000 in water, catalogue number 41,003, Biotium; applied dilution: 1:25 in PBS) were added as a fluorescent stain. After the dyeing of the cells, individual haemocytes can easily be counted under a UV light microscope as they usually do not form clots and appear not to lose their cellular integrity during the freezing. Haemocyte counts were obtained using a digital camera (Nikon DS-U2-Ri1, Nikon Corporation, Tokyo, Japan) connected to a Nikon Eclipse 50I fluorescence microscope (magnification 40) and the program NIS Elements. Each picture was subdivided with a grid of 1.25  1.25 mm and the number of haemocytes was counted in three replicate sample areas from the central part of each picture (covering ca. 30–40% of the whole picture). All statistical analyses were done on the mean of the three replicates. The quantification of PO activity closely followed the protocols of Rolff et al. (2004) and Stoks et al. (2006). The perfused thorax was transferred back to the haemolymph extract and disrupted

in a bead mill (Tissuelyser II, Qiagen, disruption of tissue for 2  20 s at 20 Hz). Coarse particles (such as cell walls) were removed via centrifugation (4 °C, 14,000 g, 10 min). Thereafter, 80 ll of the supernatant or cacodylate buffer (for blank measurements, two per microwell plate) were added to 120 ll L-DOPA (dihydrophenyl-L-alanine; 10 mM in cacodylate buffer). For the measurement of PO 5 ll milli-Q water, and for the measurement of the total of ProPO and PO 5 ll of chymotrypsin were added. Readings were taken every 30 s on a spectrophotometric plate reader (BioTek EL 808) at 30 °C and 490 nm for 45 min. Enzyme activity was measured as the slope during the linear phase of the reaction during which the enzyme catalyses the transition from L-DOPA to dopachrome. PO activity and the activity of the total PO and ProPO were each assayed twice per individual. ProPO activity was calculated as the difference between the total of PO and ProPO and PO activity. The mean of both readings (controlled by measures of the blank readings) was used in subsequent statistical analyses. The total protein content of each thorax was quantified using the Bio-Rad (Hercules, CA, USA) protein assay based on the Bradford method (Bradford, 1976). 1 ll of the supernatant (see above) was diluted in 160 ll milli-Q water and was combined with 40 ll of the dye reagent. Four replicates were used per sample. The absorbance was read at 593 nm after 10 min of incubation at 30 °C on a microplate reader (BioTek EL 808). A bovine gamma globulin standard (Calbiochem, catalogue number 126593) was used to construct standard curves. For the measurement of lysozyme-like activity of the haemolymph, the abdomen was used and weighed before the analysis. 50 ll of PBS (phosphate buffered saline, pH = 7.4) were added and the abdomen was homogenized in a bead mill (Tissuelyser II, Qiagen, disruption of tissue for 2  20 s at 20 Hz). After removing coarse particles via centrifugation (4 °C, 14,000g, 10 min), 20 ll of the supernatant and 80 ll of a suspension of Micrococcus lysodeikticus (MP Biomedicals, catalogue number 159972, 3 mg/ml in PBS) were loaded per well of a 96-well plate; each sample was measured in two replicate measures. In addition, two readings using PBS buffer instead of sample volume were taken per microwell plate as blanks. The optical density was measured every 30 s on a spectrophotometric plate reader (BioTek EL 808) at 30 °C and 490 nm for 5 h. Enzyme activity was measured as the difference between the measured optical densities at the start and the end of the 5 h period. The mean of the blank values was subtracted from the resulting optical densities of each plate. 2.4. Statistical analyses All statistical tests were performed using Statistica 8.0 (StatSoft, 2007). If not otherwise indicated, general linear (mixed) models (GLMs) with rearing temperature, plant temperature and sex as fixed factors, and masses (thorax mass for the analysis of protein content; abdomen mass for the analysis of lysozyme-like activity) or protein content (for the analysis of PO/ProPO activity) were used as covariates. Minimum adequate models were built by sequentially removing non-significant interaction terms. PO activity and haemocyte numbers were log-transformed prior to analyses to meet GLM assumptions. Correlations between continuous variables were estimated using Pearson moment product correlations. Throughout, mean values are given ± 1 SE. 3. Results Larval rearing temperature did not significantly affect thorax protein content, PO activity or lysozyme-like activity of the haemolymph (Table 1). However, a high larval rearing temperature

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significantly reduced haemocyte numbers (17 °C vs. 25 °C: 70.6 ± 4.1 vs. 43.3 ± 2.7) and tended to reduce ProPO activity (10.2 ± 0.7 vs. 8.5 ± 0.7, P = 0.092). Thermal effects on haemocyte numbers depended partly on plant temperature and sex. For both larval rearing temperatures highest haemocyte numbers were found when the larval temperature matched the plant temperature (significant larval by plant temperature interaction; Fig. 1A). Males responded much more strongly to rearing temperatures than females, showing a 54.3% reduction in haemocyte numbers compared with 17.3% in females (significant larval rearing temperature by sex interaction, Fig. 1B). Plant temperature, in contrast, did not significantly affect any of the traits measured, but a high plant temperature tended to reduce PO and to increase ProPO activity (PO: 20.3 ± 1.6 vs. 16.9 ± 1.3, P = 0.093; ProPO: 8.4 ± 0.8 vs. 10.3 ± 0.7, P = 0.065). Sex only affected lysozyme-like activity of the haemolymph, which was higher in males than in females (0.551 ± 0.015 DOD vs. 0.425 ± 0.019 DOD; Table 1). Thorax protein content was slightly lower in males than in females at the lower plant temperature, but slightly higher at the higher plant temperature (significant plant temperature by sex interaction). Note that differences in protein content, even though statistically significant, were exceedingly small and thus likely not biologically meaningful (Fig. 1C). Protein content and haemocyte numbers were significantly positively correlated with thorax mass (protein content: R = 0.16, t = 2.6, P = 0.010, N = 272; haemocyte numbers: R = 0.37, t = 6.6, P < 0.001, N = 271).

4. Discussion Larval rearing temperature impacted the adult butterfly immune system surprisingly little, even though the invertebrate

(a)

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immune system responds readily to changes in ambient temperatures (Fischer et al., 2011; Karl et al., 2011; Murdock et al., 2012a,b; Triggs and Knell, 2012). Generally, higher temperatures tend to increase immune reactivity in ectotherms (Adamo, 2012; Triggs and Knell, 2012) though the quality and extent of the thermal impact seems to strongly depend on the focal immune trait and concomitant environmental factors (Murdock et al., 2013). In contrast, in our study haemocyte numbers and to a lesser extent also proPO activity were reduced at the higher temperature, which may indicate that 25 °C already imposed stress on the butterflies (cf. Fischer et al., 2011; Karl et al., 2011). The fact that haemocyte numbers showed the strongest response to environmental temperature is in agreement with other studies, whereas other components of the insect immune system seem less reactive (Pandey and Tiwari, 2012). Plant temperature had no effect on P. napi immune function, although it has been shown that higher temperatures substantially reduce plant quality to the herbivorous larvae of P. napi. This resulted in reduced body mass, longer development time, and partial compensatory feeding with the effects of host plant quality being more pronounced at the higher temperature (Bauerfeind and Fischer, 2013a). Only one interaction between temperature and host plant was significant here, showing higher haemocyte numbers if the larval rearing temperature matched the plant temperature (Fig. 1A). Several reasons may account for the overall weak responses of the P. napi immune system to the changes in environmental conditions: (1) thermal and host plant treatments may not have been sufficiently different to elicit clear responses. Note though that earlier results documented clear effects on life history and growth parameters using the same species and conditions (Bauerfeind and Fischer, 2013a); (2) the immune system of P. napi may be relatively well buffered against changes in environmental conditions,

(b)

(c)

Fig. 1. Effects of larval rearing temperature, plant temperature and/or sex on haemocyte numbers (A, B) and thorax protein content (C) in Pieris napi. PT: plant temperature. Sample sizes range from 32 to 38/group.

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Table 1 General linear models for the effects of larval rearing temperature, plant growth temperature, and sex on thorax protein content and immune parameters in Pieris napi. Protein content, thorax or abdomen mass was added as covariate where appropriate. Minimum adequate models were constructed by sequentially removing non-significant interaction terms. PO: phenoloxidase, ProPO: prophenoloxidase; P < 0.05 in bold. Trait

Source

MS

Protein content R2 = 0.05

Rearing temperature Plant temperature Sex Plant temp.  sex Thorax mass Error

0.03  10 0.09  10 0.13  10 0.27  10 0.55  10 0.06  10

DF

F

P

1 1 1 1 1 266

0.4 1.4 2.1 4.3 8.8

0.514 0.238 0.153 0.040 0.003

PO activity R2 = 0.02

Rearing temperature Plant temperature Sex Protein content Error

0.39 0.73 <0.01 0.08 0.26

1 1 1 1 267

1.5 2.8 <0.1 0.3

0.218 0.093 0.968 0.580

ProPO activity R2 = 0.03

Rearing temperature Plant temperature Sex Protein content Error

202.3 243.6 178.8 1.8 70.7

1 1 1 1 267

2.9 3.4 2.5 <0.1

0.092 0.065 0.113 0.872

Lysozyme-like activity R2 = 0.10

Rearing temperature Plant temperature Sex Abdomen mass Error

0.19  10 3 5.0  10 3 806.9  10 3 82.1  10 3 38.8  10 3

1 1 1 1 264

<0.1 0.1 20.8 2.1

0.944 0.720 <0.001 0.147

Haemocyte No. R2 = 0.26

Rearing temperature Plant temperature Sex Rear. temp.  plant temp. Rear. temp.  sex Thorax mass Error

1.7 <0.1 <0.1 0.3 1.0 1.3 <0.1

1 1 1 1 1 1 264

23.5 0.2 0.4 4.2 14.2 18.6

<0.001 0.660 0.511 0.041 <0.001 <0.001

e.g. in order to prevent autoimmunological damage (Sadd and Siva-Jothy, 2006); (3) environmental effects on the insect immune system are known to be transient, such that measuring effects of the larval environment in adults is evidently difficult and may depend on the age of the adult butterfly (Piesk et al., 2013; Siva-Jothy and Thompson, 2002) and (4) a lack of statistical power with sample sizes being too low to detect an effect. Note in the latter context the tendencies found for plant rearing temperature in the case of PO and proPO that were affected by plant rearing temperature with P < 0.1). However, the sample sizes used here were substantial (between 67 and 78 individuals, pooled across sexes, per treatment group). While further increasing sample size may indeed result in some significant results, this is unlikely to change the biological meaning of the data obtained due to the obviously very low effect size. Sex differences were only apparent for lysozyme-like activity of the haemolymph and to a lesser degree for haemocyte numbers (interaction term): males had a much higher level of lysozyme-like activity and responded more strongly to temperatures (haemocytes) than females. In contrast to our results, females often tend to invest more into immune function than males as a result of differences in reproductive effort and energy-allocation strategies (Fanson et al., 2013; Nunn et al., 2009; Schmid-Hempel, 2005). Given the high reproductive investment of P. napi males (large spermatophores and concomitantly larger body mass than females), P. napi apparently does not readily fit such general patterns, but well the notion of a link to reproductive effort (Bergström and Wiklund, 2002; Wiklund and Kaitala, 1995). While for most immune traits consistent sex differences seem to be absent, the lack of a difference in PO activity is surprising as a recent meta-analysis found that PO activity is consistently higher in females (Fanson et al., 2013; Nunn et al., 2009). However, Prasai and Karlsson (2012) also found little sexual differences in the P. napi encapsulation response.

5 5 5 5 5 5

5. Conclusion Effects of environmental variation were overall weak and traitspecific. The higher rearing temperature reduced haemocyte numbers and tended to reduce prophenoloxidase activity, even across the metamorphic boundary. This corroborates the notion that higher temperatures do not necessarily increase insect immune competence. In contrast, variation in plant temperature did not affect immune competence despite clear effects on growth patterns (Bauerfeind and Fischer, 2013a). Interpreting these results needs to take into consideration that any response or the lack thereof in baseline immune parameters does not necessarily predict the insects’ actual resistance to pathogens (Adamo, 2004a,b). Investigating environmental effects on immune function by applying variation in the same stage in which the target traits are measured (i.e. both in the adult stage) and/or by measuring induced rather than constitutive levels of immune parameters may lead to different insights. However, we here used constitutive levels in the first place to assess an organism’s condition. Our study underlines the complex interactions between food quality and temperature which might affect immune function.

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