Density-dependent Prophylaxis Research Articles

Immunological regulation by Toll-1 and Spätzle-4 in larval density-dependent prophylaxis of the oriental armyworm, Mythimna separata

High population density has been shown to alter insect prophylactic immunity. Toll-Spätzle pathway performs a key function in insect innate immune response. To determine the role of Toll and Spätzle, two main components of Toll-Spätzle pathway, in the density-dependent prophylaxis of Mythimna separata. We identified full-length cDNA encoding the Toll-1 and Spätzle-4 genes in M. separata (designed MsToll-1 and Ms Spätzle-4). Both MsToll-1 and MsSpätzle-4 were expressed throughout all developmental stages. MsToll-1 expression was highly in fat body and brain and MsSpätzle-4 was highly expressed in brain and Malpighian tubule. With increased larval density, MsToll-1 expression was markedly up-regulated. MsSpätzle-4 expression was found to be raised in larvae that were fed in high density (5 and 10 larvae per jar). Co-immunoprecipitation assays demonstrated that MsToll-1 interacted with MsSpätzle-4. Immune-related genes transcriptions were considerably reduced in high-density larvae MsToll-1 (or MsSpätzle-4) was silenced by dsRNA injection. Meanwhile, a discernible reduction in the survival rate of the larvae exposed to Bacillus thuringiensis infection with silence of MsToll-1 (or MsSpätzle-4) was observed. This study implies that prophylactic immunity was influenced by crowded larvae via modulating the Toll-Spätzle pathway in M. separata and allow for a new understanding of into density-dependent prophylaxis in insects.

Effect of rearing density on female investment in reproduction and melanotic encapsulation response in the sand cricket (Gryllus firmus) (Orthoptera: Gryllidae)

Abstract The density-dependent prophylaxis hypothesis posits that because pathogens are expected to be transmitted among conspecifics in crowded conditions, individuals living in such conditions should minimize their chance of infection and maximize survival by elevating their baseline immunity. Further, if individuals developing in crowded conditions indeed allocate more resources to immunity then resource allocation to other life-history traits, such as reproduction, should decline. We tested these hypotheses by rearing sand crickets (Gryllus firmus) at either low or high density in the laboratory and quantified immune function (melanotic encapsulation response) and reproductive investment (ovary mass, egg size) at adulthood. The results did not support the density-dependent prophylaxis hypothesis, but we did find that rearing density significantly affected investment in two reproductive traits, with average ovary mass and egg size both being larger in low-density crickets. We discuss possible explanations for the lack of a trade-off between immunity and reproduction in our study.

High larval densities and high temperatures lead to a stronger immune response in the black soldier fly

Organisms are expected to invest more in their immune function when the risk of disease infection is high. However, induction of a robust immune response is costly and may not be achievable in suboptimal environments. High conspecific density could simultaneously imply high infection risk and a suboptimal environment for many insect species. We focus on the economically important dipteran species (black soldier fly, BSF) that represents the insect order that has been ignored in previous research on density effects on immunity. The experimental part of the study was carried out to evaluate the effect of larval density (three density treatments: 1, 5 and 10 larvae/cm2) and temperature (three thermal treatments: 23, 27 and 30 °C) on the immune function of BSF larvae. The larvae that were reared at high compared to low larval densities and at higher than lower temperatures had significantly higher activity of phenoloxidase, an enzyme that plays an essential role in insect immune function. Sex did not have a significant effect on phenoloxidase activity and prepupal mass, pupal mass and adult mass were not affected by the levels of phenoloxidase activity of fifth instar larvae. In addition, we give an overview of larval density effects on insect immunity and show that density-dependent prophylaxis (stronger immune response in high larval density environments) is indeed common in the results of published case studies. However, cases with no correlation between density and immunity traits were as frequent. Moreover, in more than half of the studies, qualitatively different within-species patterns in different immunity traits were observed. We conclude that BSF larvae exhibit density-dependent prophylaxis, and larvae invest more into their immune system at high larval densities and temperatures than they do at low larval densities and temperatures.

Increasing adult density compromises survival following bacterial infections in Drosophila melanogaster

The density-dependent prophylaxis hypothesis predicts that risk of pathogen transmission increases with increase in population density, and in response to this, organisms mount a prophylactic immune response when exposed to high density. This prophylactic response is expected to help organisms improve their chances of survival when exposed to pathogens. Alternatively, organisms living at high densities can exhibit compromised defense against pathogens due to lack of resources and density associated physiological stress; the crowding stress hypothesis. We housed adult Drosophila melanogaster flies at different densities and measured the effect this has on their post-infection survival and resistance to starvation. We find that flies housed at higher densities show greater mortality after being infected with bacterial pathogens, while also exhibiting increased resistance to starvation. Our results are more in line with the crowding stress hypothesis that postulates a compromised immune system when hosts are subjected to high densities.

Density-Dependent Prophylaxis in Freshwater Snails Driven by Oxylipin Chemical Cues.

While animal aggregations can benefit the fitness of group members, the behaviour may also lead to higher risks of parasite infection as group density increases. Some animals are known to moderate their investment in immunity relative to the risk of infection. These animals exhibit density-dependent prophylaxis (DDP) by increasing their immune investment as group density increases. Despite being documented in many taxa, the mechanisms of DDP remain largely unexplored. Snails are known to aggregate and experience large fluctuations in density and serve as required hosts for many parasites. Further, they are known to use chemical cues to aggregate. To test whether freshwater snails exhibit DDP and investigate the role that chemical signaling compounds may play in triggering this phenomenon, we performed four experiments on the freshwater snail Stagnicola elodes, which is a common host for many trematode parasite species. First, we tested if DDP occurred in snails in laboratory-controlled conditions (control vs snail-conditioned water) and whether differences in exposure to chemical cues affected immune function. Second, we used gas chromatography to characterize fatty acids expressed in snail-conditioned water to determine if precursors for particular signaling molecules, such as oxylipins, were being produced by snails. Third, we characterized the oxylipins released by infected and uninfected field-collected snails, to better understand how differences in oxylipin cocktails may play a role in inducing DDP. Finally, we tested the immune response of snails exposed to four oxylipins to test the ability of specific oxylipins to affect DDP. We found that snails exposed to water with higher densities of snails and raised in snail-conditioned water had higher counts of haemocytes. Additionally, lipid analysis demonstrated that fatty acid molecules that are also precursors for oxylipins were present in snail-conditioned water. Trematode-infected snails emitted 50 oxylipins in higher amounts, with 24 of these oxylipins only detected in this group. Finally, oxylipins that were higher in infected snails induced naïve snails to increase their immune responses compared to sham-exposed snails. Our results provide evidence that snails exhibit DDP, and the changes in oxylipins emitted by infected hosts may be one of the molecular mechanisms driving this phenomenon.

Realism in Immune Ecology Studies: Artificial Diet Enhances a Caterpillar's Immune Defense but Does Not Mask the Effects of a Plastic Immune Strategy.

The immune system is considered a functional trait in life-history theory and its modulation is predicted to be costly and highly dependent on the host's nutrition. Therefore, the nutritional status of an individual has a great impact on an animal's immune ecology. Herbivorous insects are commonly used as model organisms in eco-immunology studies and the use of an artificial diet is the predominant rearing procedure to test them. However, this diet differs from what herbivores experience in nature and it is unclear to what degree this distinction might impact on the relevance of these studies for the real world. Here, we compared plant-based vs. artificial diet in a set of three experiments to investigate the interaction of both diets with a plastic immune strategy known as Density-Dependent Prophylaxis (DDP). We used as a model organism the velvetbean caterpillar Anticarsia gemmatalis, which is known to adjust its immune defense in line with the DDP hypothesis. Our main results showed that larvae fed with artificial diet had 20.5% more hemocytes circulating in the hemolymph and died 20% more slowly when infected with an obligate (viral) pathogen. Crucially, however, we did not find any indication of fitness costs related to DDP. The use of artificial diet did not interact with that of DDP except in the case of host survival after infection, where the DDP effect was only observable in this diet. Our findings suggest the use of an artificial diet does not mask resource allocation conflicts between immune investment and fitness related traits, but to some extent it might lead to an overestimation of immune parameters and host survival time after infection. We believe that this is the first study to compare an artificial diet and a host plant covering all these aspects: immune parameters, life-history traits, and host survival after infection. Here we provide evidence that, besides the quantitative effects in immune parameters and host survival time, the use of artificial diet interacts only marginally with a density-dependent immune response. This provides support for the use of artificial diets in eco-immunology studies with insects.

Effects of Larval Density on Plutella xylostella Resistance to Granulosis Virus.

Simple SummaryGenerally, the transmission of pathogens is positively density-dependent; therefore, the risk of insects becoming infected by diseases increases with increasing population density. It has been reported that some phase-polyphenic insects from high-density conditions are more resistant (or susceptible or identical) to pathogens than those from low-density conditions. This phenomenon is termed “density-dependent prophylaxis” (DDP). The diamondback moth (DBM), Plutella xylostella, one of the most destructive insect pests affecting cruciferous crops, is non phase-polyphenic. Biological control, especially by pathogens, plays an important role in the integrated pest management program of DBM. However, whether the P. xylostella larval population exhibits DDP has not been elucidated. The resistance of DBM larvae to P. xylostella granulosis virus (Plxy GV) and their immune response to the virus under different density treatments were investigated under laboratory conditions. Our results demonstrated that P. xylostella larvae exhibited DDP within a certain limited density. This study may help to elucidate the biocontrol effect of different density populations of P. xylostella by granulosis virus and guide improvements in future management strategy.It has been reported that some phase-polyphenic insects from high-density conditions are more resistant to pathogens than those from low-density conditions. This phenomenon is termed “density-dependent prophylaxis” (DDP). However, whether non phase-polyphenic insects exhibit DDP has rarely been elucidated. The diamondback moth (DBM), Plutella xylostella, one of the most destructive insect pests affecting cruciferous crops, is non phase-polyphenic. In this study, the resistance of DBM larvae to P. xylostella granulosis virus (Plxy GV) and their immune response to the virus when reared at densities of 1, 2, 5, 10, 15, and 20 larvae per Petri dish were investigated under laboratory conditions. Compared with larvae reared at lower densities, larvae reared at moderate density showed a significantly higher survival rate, but the survival rate significantly decreased with further increases in rearing density. Furthermore, the phenoloxidase, lysozyme and antibacterial activity and total hemocyte count in the hemolymph of the larvae, regardless of whether they were challenged with the virus, from different larval densities corresponded to the observed differences in resistance to Plxy GV. These results demonstrated that P. xylostella larvae exhibited DDP within a certain limited density. This study may help to elucidate the biocontrol effect of different density populations of P. xylostella by granulosis virus and guide improvements in future management strategy.

Immunological regulation by a β-adrenergic-like octopamine receptor gene in crowded larvae of the oriental Armyworm, Mythmina separata

Recent reports demonstrate that octopamine plays an important immunological role in crowded larvae of the Oriental Armyworm, Mythmina separata. We identified an octopamine receptor, the β-adrenergic-like gene (designated MsOctβ2R), with a 1191 bp open reading frame that encodes 396 amino acids and contains seven conserved hydrophobic transmembrane domains. Multiple sequence alignments and a phylogenetic analysis indicated that MsOctβ2R was orthologous to Octβ2R that is present in other lepidopterans. MsOctβ2R was expressed throughout all developmental stages with higher relative expression during the fourth instar and adult stages. MsOctβ2R was highly expressed in the ventral nerve cord and the fat body relative to other examined tissues. Elevated MsOctβ2R expression was observed in larvae that were under higher-density conditions (7 and 10 larvae per jar). Silencing MsOctβ2R expression via dsRNA injections in larvae from higher-density conditions significantly decreased phenoloxidase (PO) and lysozyme activity, total haemocyte counts, and survival rates against Beauveria bassiana infections (54.06%, 9.91%, 36.22%, and 23.53%, respectively) when compared with control larvae. These results suggest that high-density conditions might alter prophylactic immunity in larvae by regulating the MsOctβ2R gene in M. separara and provide new insights into density-dependent prophylaxis in insects.

The Role of Tyramine β-Hydroxylase in Density Dependent Immunityof Oriental Armyworm (Mythmina separata) Larva.

High population density alters insect prophylactic immunity, with density-dependent prophylaxis (DDP) being reported in many polyphonic insects. However, the molecular mechanism for DDP remains unclear. In current study, the role of tyramine β-hydroxylase (Tβh) in the immune response of M. separata larvae that were subject to different rearing densities conditions was investigated. The tyramine β-hydroxylase activity of larvae from high density treatments (10 and 30 larvae per jar) was significantly higher than that of the larvae from low density treatments (one, two, and five larvae/jar). A tyramine β-hydroxylase (designated MsTβh) containing a 1779 bp open reading frame was identified. Multiple sequence alignment and phylogenetic analysis indicated that MsTβh was orthologous to the Tβh that was found in other lepidopterans. Elevated MsTβh expression was observed in larvae under high density (10 larvae per jar). Silencing MsTβh expression by the injection of dsRNA in larvae from the high density treatment produced a 25.1% reduction in octopamine levels, while at the same time, there was a significant decrease in phenoloxidase (PO) and lysozyme activity, total haemocyte counts, and survival against Beauveria infection 56.6%, 88.5%, 82.0%, and 55.8%, respectively, when compared to control larvae. Our findings provide the first insights into how MsTβh mediates the octopamine level, which in turn modulates the immune response of larvae under different population densities.

Transcriptomic insight into antimicrobial peptide factors involved in the prophylactic immunity of crowded Mythimna separata larvae

Similar to pathogenic infection, a high population density alters insect prophylactic immunity. Antimicrobial peptides (AMPs) are known to play critical roles in an insect's humoral immune response to microbial infection. We applied RNA sequencing to investigate differential gene expression levels in fat body and hemocyte samples from larvae reared in high- (10 larvae per jar) and low-density (1 larva per jar) conditions; the samples exhibited density-dependent prophylaxis. A number of AMP molecule-related proteins were annotated for the first time from 145,439 assembled unigenes from M. separata larvae. The transcript levels of AMP molecules such as gloverin-, defensin-, cecropin-, lebocin- and attacin-related unigenes were increased with the prophylactic immunity of high-density larvae. The pattern recognition receptor peptidoglycan recognition protein (PGRP), a key protein in the synthesis of AMPs in IMD- and Toll pathway-related unigenes, was also upregulated in the larvae from the high-density group. The resultant transcriptomic database was validated by the transcript levels of four selected AMP genes quantified from the high- and low-density larval groups with quantitative real-time PCR. The antimicrobial activity against gram-positive Staphylococcus aureus and Bacillus subtilis and gram-negative Edwardsiella ictaluri and Vibrio anguillarum in the hemolymph of larvae from the high-density group was significantly higher than that of larvae from the low-density group. Our findings provide the first insight into the role of AMP genes in the mechanisms of density-dependent prophylaxis in M. separata and provide new insight into the control of M. separata with biopesticides.

Altered immunity in crowded Mythimna separata is mediated by octopamine and dopamine

Similar to pathogenic infection, high population density alters insects’ prophylactic immunity. Density-dependent prophylaxis has been reported in many polyphenic insects, but the regulatory mechanism underlying this phenomenon remains unclear. The biogenic monoamines are known to play critical roles in mediating insect immune responses. In the current study, the immune capacity and the levels of three biogenic monoamines were investigated in the polyphenic larvae of Mythimna separata, reared at the densities of 1, 2, 5, 10, and 30 larvae per 650-mL jar. Concomitant with the increased phenoloxidase (PO) activity and total haemocyte count in the larvae at high densities (5, 10, 30 larvae/jar), the octopamine level was also increased. In contrast, the dopamine level was decreased, and the 5-hydroxytryptamine level was not significantly affected. Injection of octopamine induced significant increases in the total haemocyte count and PO activity. Conversely, epinastine, a specific antagonist of octopamine, decreased the total haemocyte count and PO activity. Another octopamine antagonist, phentolamine, inhibited the activity of PO and lysozymes. In addition, injection of dopamine induced a significant increase in PO activity and decreased the total haemocyte count and lysozyme activity. These results suggested that both octopamine and dopamine mediate the increases in total haemocyte count and PO activity in the crowded larvae.

Molecular Characterization of a Lysozyme Gene and Its Altered Expression Profile in Crowded Beet Webworm (Loxostege sticticalis).

There is growing evidence that insects living in high-density populations exhibit an increase in immune function to counter a higher risk of disease. This phenomenon, known as density-dependent prophylaxis, has been experimentally tested in a number of insect species. Although density-dependent prophylaxis is especially prevalent in insects exhibiting density-dependent phase polyphenism, the molecular mechanism remains unclear. Our previous study demonstrated that the antibacterial activity of lysozyme is important for this process in the beet webworm Loxostege sticticalis. In this study, a lysozyme cDNA from L. sticticalis was cloned and characterized. The full-length cDNA is 1078 bp long and contains an open reading frame of 426 bp that encodes 142 amino acids. The deduced protein possesses structural characteristics of a typical c-type lysozyme and clusters with c-type lysozymes from other Lepidoptera. LsLysozyme was found to be expressed throughout all developmental stages, showing the highest level in pupae. LsLysozyme was also highly expressed in the midgut and fat body. Elevated LsLysozyme expression was observed in L. sticticalis larvae infected by Beauveria bassiana and in larvae reared under crowding conditions. In addition, the expression level of LsLysozyme in infected larvae reared at a density of 10 larvae per jar was significantly higher compared to those reared at a density of l or 30 larvae per jar. These results suggest that larval crowding affects the gene expression profile of this lysozyme. This study provides additional insight into the expression of an immune-associated lysozyme gene and helps us to better understand the immune response of L. sticticalis under crowding conditions.

Survival after pathogen exposure in group-living insects: don't forget the stress of social isolation!

A major cost of group-living is its inherent risk of pathogen infection. To limit this risk, many group-living animals have developed the capability to prophylactically boost their immune system in the presence of group members and/or to mount collective defences against pathogens. These two phenomena, called density-dependent prophylaxis and social immunity, respectively, are often used to explain why, in group-living species, individuals survive better in groups than in isolation. However, this survival difference may also reflect an alternative and often overlooked process: a cost of social isolation on individuals' capability to fight against infections. Here, we disentangled the effects of density-dependent prophylaxis, social immunity and stress of social isolation on the survival after pathogen exposure in group-living adults of the European earwig Forficula auricularia. By manipulating the presence of group members both before and after pathogen exposure, we demonstrated that the cost of being isolated after infection, but not the benefits of social immunity or density-dependent prophylaxis, explained the survival of females. Specifically, females kept constantly in groups or constantly isolated had higher survival rates than females that were first in groups and then isolated after infection. Our results also showed that this cost of social isolation was absent in males and that social isolation did not reduce the survival of noninfected individuals. Overall, this study gives a new perspective on the role of pathogens in social evolution, as it suggests that an apparently nonadaptive, personal immune process may promote the maintenance of group-living under pathogenic environments.

Transgenerational effects modulate density-dependent prophylactic resistance to viral infection in a lepidopteran pest.

There is an increasing appreciation of the importance of transgenerational effects on offspring fitness, including in relation to immune function and disease resistance. Here, we assess the impact of parental rearing density on offspring resistance to viral challenge in an insect species expressing density-dependent prophylaxis (DDP); i.e. the adaptive increase in resistance or tolerance to pathogen infection in response to crowding. We quantified survival rates in larvae of the cotton leafworm (Spodoptera littoralis) from either gregarious- or solitary-reared parents following challenge with the baculovirus S. littoralis nucleopolyhedrovirus. Larvae from both the parental and offspring generations exhibited DDP, with gregarious-reared larvae having higher survival rates post-challenge than solitary-reared larvae. Within each of these categories, however, survival following infection was lower in those larvae from gregarious-reared parents than those from solitary-reared, consistent with a transgenerational cost of DDP immune upregulation. This observation demonstrates that crowding influences lepidopteran disease resistance over multiple generations, with potential implications for the dynamics of host-pathogen interactions.

Stability Switches in a Host–Pathogen Model as the Length of a Time Delay Increases

The destabilising effects of a time delay in mathematical models are well known. However, delays are not necessarily destabilising. In this paper, we explore an example of a biological system where a time delay can be both stabilising and destabilising. This example is a host–pathogen model, incorporating density-dependent prophylaxis (DDP). DDP describes when individual hosts invest more in immunity when population densities are high, due to the increased risk of infection in crowded conditions. In this system, as the delay length increases, there are a finite number of switches between stable and unstable behaviour. These stability switches are demonstrated and characterised using a combination of numerical methods and analysis.

Density-dependent prophylaxis in crowded Beet Webworm, Loxostege sticticalis (Lepidoptera: Pyralidae) larvae to a parasitoid and a fungal pathogen

Transmission of parasites and pathogens is generally positively density-dependent: as an insect population's density increases, the risk of an individual becoming attacked or infected also increases. In some insect species, individuals experiencing crowded conditions are more resistant to natural enemies than those experiencing low density conditions, and they are predicted to divert resources to increase resistance. This phenomenon is called density-dependent prophylaxis. Here, possible expression of prophylaxis in fifth-instar larvae of Beet Webworm, Loxostege sticticalis, to biocontrol agents was investigated under rearing densities of 1, 10, and 30 larvae per jar (650 mL). Larvae reared at the moderate density and those reared in isolation displayed the greatest and lowest resistance, respectively, to an entomopathogenic fungus and a parasitoid. Moreover, larvae from the moderate density treatment exhibited elevated phenoloxidase, total haemocyte count and antibacterial activity in the haemolymph, whereas phenoloxidase levels in the midgut were not affected. The results suggest that larval rearing density significantly affects the immune system, and they provide evidence for density-dependent prophylaxis of larval L. sticticalis against its biocontrol agents. These results have implications for understanding the population dynamics and biocontrol of beet webworm.

Two's a crowd: phenotypic adjustments and prophylaxis in Anticarsia gemmatalis larvae are triggered by the presence of conspecifics.

Defence from parasites and pathogens involves a cost. Thus, it is expected that organisms use this only at high population densities, where the risk of pathogen transmission may be high, as proposed by the "density-dependent prophylaxis" (DDP) hypothesis. These predictions have been tested in a wide range of insects, both in comparative and experimental studies. We think it pertinent to consider a continuum between solitarious and gregarious living insects, wherein: (1) solitarious insects are those that are constitutively solitary and do not express any phenotypic plasticity, (2) the middle of the continuum is represented by insects that are subject to fluctuations in local density and show a range of facultative and plastic changes; and (3) constitutively gregarious forms live gregariously and show the gregarious phenotype even in the absence of crowding stimuli. We aimed to chart some of the intermediary continuum with an insect that presents solitarious aspects, but that is subject to fluctuations in density. Thus, Anticarsia gemmatalis (Lepidoptera: Noctuidae) larvae reared at higher densities showed changes in coloration, a greater degree of encapsulation, had higher hemocyte densities and were more resistant to Baculovirus anticarsia, but not to Bacillus thuringiensis. Meanwhile, with increased rearing density there was reduced capsule melanization. Hemocyte density was the only variable that did not vary according to larval phenotype. The observed responses were not a continuous function of larval density, but an all-or-nothing response to the presence of a conspecific. As A. gemmatalis is not known for gregarious living, yet shows these density-dependent changes, it thus seems that this plastic phenotypic adjustment may be a broader phenomenon than previously thought.

Density-dependent prophylaxis in the coral-eating crown-of-thorns sea star, Acanthaster planci

The density-dependent prophylaxis hypothesis predicts that individuals at high density will invest more resources into immune defence than individuals at lower densities as a counter-measure to density-dependent pathogen transmission rates. Evidence has been found for this hypothesis in insects, but not in a non-arthropod taxon. To investigate this hypothesis in the coral-eating crown-of-thorns sea star, Acanthaster planci, density treatments were set up over 21 days, and pathogen infection was simulated with bacterial injection. Five immune responses: amoebocyte count, amoebocyte viability, lysosomal membrane integrity, respiratory burst and peroxidase activity were all upregulated at high density. These results demonstrate that immune investment shows phenotypic plasticity with adult population density in agreement with the density-dependent prophylaxis hypothesis. Here I show that the density-dependent prophylaxis hypothesis is neither dependent on larval density nor restricted to insects, and hence may potentially have important consequences on disease dynamics in any species with widely fluctuating population densities. This is the first demonstration of the density-dependent prophylaxis hypothesis outside arthropods.

Interactions between environmental variables determine immunity in the Indian meal moth Plodia interpunctella

1. Animals raised in good environmental conditions are expected to have more resources to invest in immunity than those raised in poor conditions. Variation in immune activity and parasite resistance in response to changes in environmental temperature, population density and food quality have been shown in many invertebrate species. 2. Almost all studies to date have examined the effects of individual variables in isolation. The aim of this study was to address whether environmental factors interact to produce synergistic effects on phenoloxidase (PO) activity and haemocyte count, both indicators of immune system activity. Temperature, food quality and density were varied in a fully factorial design for a total of eight treatment combinations. 3. Strong interactions between the three environmental variables led to the magnitude and in some cases the direction of the effect of most variables changing as the other environmental factors were altered. Overall, food quality had the most important and consistent influence, larvae raised on a good-quality diet having substantially higher PO activity in every case and substantially higher haemocyte counts in all treatments except unheated/low density. 4. When food quality was good, the larvae showed 'density-dependent prophylaxis': raising their investment in immunity when population density is high. When food quality was poor and the temperature low, however, those larvae raised at high densities invested less in immunity. 5. Increased temperature is often thought to lead to increased immune reactivity in ectotherms, but we found that the effect of temperature was strongly dependent on the values of other environmental variables. PO activity increased with temperature when larvae were raised on good food or when density was high, but when food was poor and density low, a higher temperature led to reduced PO activity. A higher temperature led to higher haemocyte counts when density was high and food quality was poor, but in all other cases, the effect of increased temperature was either close to zero or somewhat negative. 6. Although PO activity and haemocyte count were weakly correlated across the whole data set, there were a number of treatments where the two measures responded in different ways to environmental change. Overall, effect sizes for PO activity were substantially higher than those for haemocyte count, indicating that the different components of the immune system vary in their sensitivity to environmental change. 7. Predictions of the effect of environmental or population change on immunity and disease dynamics based on laboratory experiments that only investigate the effects of single variable are likely to be inaccurate or even entirely wrong.

The population dynamical consequences of density-dependent prophylaxis

When infectious disease transmission is density-dependent, the risk of infection will tend to increase with host population density. Since host defence mechanisms can be costly, individual hosts may benefit from increasing their investment in immunity in response to increasing population density. Such “density-dependent prophylaxis” (DDP) has now indeed been demonstrated experimentally in several species. However, it remains unclear how DDP will affect the population dynamics of the host–pathogen interaction, with previous theoretical work making conflicting predictions. We develop a general host–pathogen model and assess the role of DDP on the population dynamics. The ability of DDP to drive population cycles is critically dependent on the time delay between the change in density and the subsequent phenotypic change in the level of resistance. When the delay is absent or short, DDP destabilises the system. As the delay increases, its destabilising effect first diminishes and then DDP becomes increasingly stabilising. Our work highlights the significance of the time delay and suggests that it must be estimated experimentally or varied in theoretical investigations in order to understand the implications of DDP for the population dynamics of particular systems.