BACKGROUND
avian malaria  is a globally widespread disease but considerable variation exists in both prevalence and parasitaemia at small spatial scales  <cit> .
this variation can be generated by several ecological, demographical and immunological mechanisms, including variation in vector and host densities and host susceptibility.
for example, temperature, altitude and proximity to water bodies are small-scale ecological factors known to influence vector  and/or parasite distribution and abundance  <cit> .
this in turn increases the probability of transmission from vector to host.
furthermore, if an area with malaria transmission is of poor quality , this may reduce the bird’s condition and ability to resist a parasitic infection  <cit> , further compounding the rate of infection.
similarly, although transmission rate of vector borne diseases are predicted to be independent of host density  <cit> , high densities of birds may reduce the availability of food or other resources, thereby further impairing resistance.
other demographic parameters that may influence infection rates include sex and age structures of the population arising from differences in behaviour, reproductive effort and immune capacities  <cit> .
the impact of avian malaria on wild birds has been difficult to estimate, especially in areas where avian malaria is common and the host-parasites have co-evolved.
however, recent long-term and experimental studies have shown that there are effects on both survival and reproductive success  <cit> .
on naïve hosts such as island species and captive birds in zoo the impacts have been devastating  <cit> .
for example on the hawaiian islands there was an accidental introduction of plasmodium relictum and one of its vectors which caused direct die-offs during the acute infection state which continues to play a significant role in species distribution and is a serious threat for endangered species  <cit> .
in the early stages of infection, the first line of defense is the host’s innate, non-specific immune defence.
immune cells generate and release reactive oxygen species  that attack the parasite  <cit> .
the advantage of oxidative burst is that it can act within minutes after transmission, in contrast to the acquired immune defence, which needs longer to develop the target-specific antibodies.
however, the non-specificity and general cytotoxicity of ros make it impossible for the host to distinguish between self and parasite tissue, causing oxidative damage to all types of adjacent molecules, which if not repaired, can lead to haemolysis and cellular dysfunction .
if the parasite resists the host’s ros attack and starts to multiply and grow, it also starts to generate ros as a by-product of haemoglobin digestion  <cit> .
consequently, the detrimental and beneficial effects of ros need to be fine-tuned by the host’s antioxidant defences during different stages of infection  <cit> , which can result in a quadratic relationship between oxidative stress and malaria severity.
this association can depend on other host- and environment-specific characters, including age and nutrition which influences the oxidative status  <cit> .
here we assess the relative importance of ecological , demographic , life-history  and physiological  factors for natural variation in plasmodium infection and parasitaemia  in a population of great tits, parus major.
the sampling took place in the spring, which suggests that most infections are relapse infections from previous autumn, since most vectors have not yet emerged  <cit> .
assessment of the relative importance of a broad range of factors, from ecology to physiology, to explain variation in patterns of avian malaria infection in the wild has rarely been done  <cit> , but is important for understanding the significance of effects found under controlled laboratory situations.
in addition, the present population allows test of experimentally increased host density on patterns of prevalence.
RESULTS
in the present population of great tits,  <dig> of the  <dig> screened birds were infected with plasmodium ;  <dig> with p. relictum  and  <dig> with p. ciricumflexum  morphospecies; only two birds had mixed infection .
the infection for first year breeders was 17% for both morphospecies, and the corresponding figures for older birds were 26% and 14%, for, p.relictum and p. ciricumflexum, respectively .
infection of plasmodium relictum
in table  <dig>  the top ten models are presented along with the averaged model estimates for the parameters included .
for p. relictum infection, there were only small differences in the aicc between the top ten models , so no model could be confidently singled out as the best.
however, five out of the ten parameters were important for explaining variation in p. relictum infection; namely age, sex, clutch size, spring date and rom.
the experimental manipulation of density was not important in any of the top ten models for p. relictum .
the raw data from the top three parameters revealed a 12% increase in infection from first year to older breeders and males were less infected  than females .
in terms of clutch size, uninfected birds had laid a larger clutch size  compared to the infected birds .
regarding spring date, it should be mentioned that it was highly correlated to gsh:gssg ratio  i.e., the earlier in the spring the more reduced to oxidized glutathione.
the top ten models are presented along with relative parameter importance .
cs clutch size, d breeding density, gssg oxidized glutathione, q habitat quality, rom reactive oxygen metabolites, tgsh total glutathione, rel.
imp relative parameters importance, ci confidence interval , ω model weight.
when interactions were included the top model retained tgsh, age×tgsh and sex×tgsh apart from the parameters in model  <dig> .
although not significant, the interactions suggest that young and male great tits have higher tgsh when infected compared to when uninfected; and vice versa for older and female birds.
infection of plasmodium circumflexum
in table  <dig>  the top ten models are presented along with the averaged model estimates for the parameters included.
for p. circumflexum, the manipulation of bird density was the best explanatory variable, followed by gssg .
in line with our prediction, great tits breeding in high density areas showed a twofold increase in prevalence  compared to birds breeding in the low density areas .
moreover, p. circumflexum infected birds had a lower gssg than uninfected birds .
when interactions were added, the best fitting model included density, gssg, age and age×density .
closer examination revealed that the density pattern was found to be stronger in older great tits, with 3% p.circumflexum infection in low density compared to 19% in the high density habitats.
for first year breeders the p. circumflexum infection was 18% in both densities.
the top ten models are presented along with relative parameter importance .
cs clutch size, d breeding density, gssg oxidized glutathione, q habitat quality, rom reactive oxygen metabolites, tgsh total glutathione, rel.
imp relative parameters importance, ci confidence interval , ω model weight.
in addition, parasite density of both plasmodium species was quantified.
however, the species-specific models did not explain any significant variation in parasitaemia, so will not be presented.
there were no significant associations between species-specific parasitaemia and any of the physiological variables .
although we are aware of the difference in virulence of the two different plasmodium species  <cit> , the digestion of haemoglobin by both species releases iron  which can generate rom  <cit>  .
a correlational analysis were performed and it revealed the predicted positive association between rom and parasitaemia , but not for tgsh , gssg  and the gssg:tgsh ratio .
discussion
in the present study, we combined experimental manipulation of host density across two contrasting environments with assessment of host physiology and life-history in a natural population of great tits to understand causes of variation in plasmodium infection.
the key and novel findings of this study were, firstly, that manipulation of host density increases p. circumflexum infection, but not infection with p. relictum; and secondly; that host antioxidants and rom physiology show species-specific patterns in relation to infection, and thirdly, that rom increases with plasmodium parasitaemia.
avian malaria, ecology and demography
host density was found to be the only ecological and demographic parameter of importance for p. circumflexum infection.
this link was absent for infection with p.relictum.
this suggests that population density reduces the bird’s ability to resist infection by p. circumflexum, the more virulent of the two plasmodium species  <cit> .
possibly, this is via more intense competition over resources or it could be mediated via higher levels of testosterone or stress hormones in high density areas which have been shown to supress the immune system  <cit> .
alternatively, the vectors transmitting p. circumflexum either aggregate where hosts are abundant or that vectors carrying the parasite are able to infect several birds, whereas the vector transmitting p.relictum may be more widely distributed or only feed on one host at one point in time  <cit>  but see  <cit> .
in wytham woods; a woodland in the proximity of bagley, recent work on great tits and the closely related sympatric host species, blue tits , have shown that the two plasmodium species have pronounced differences in their spatial distributions and impacts on both hosts species  <cit> .
while p. circumflexum infections exhibit pronounced spatial structuring that is stable over years in both host species; p. relictum infections are effectively randomly distributed in space .
likewise, p. circumflexum infections are linked with reduced survival, particularly during the acute stage of infection  <cit> ; whereas p. relictum infections are associated with reproductive costs  <cit> .
hence the species-specific relationships between ecological factors and parasite prevalence detected in the present study are not unexpected.
moreover, in wytham woods it was shown that territory size was linked to host density i.e., the larger territory the lower density.
this estimate of host density did not explain patterns of plasmodium infection in the conspecific blue tits, cyanistes caeruleus <cit> .
similar to the present study, however, a positive association was found between large-scale breeding density  and leucocytozoons prevalence and parasitaemia in nestling eagle owls   <cit> .
in temperate regions plasmodium species show a bimodal peak in infection, one in autumn and one in spring, and the spring peak  is most often a relapse from the autumn infection gained in the previous year  <cit> .
since juvenile great tits disperse during late summer-early autumn, the impact of the current environment on first-year breeders may be small compared to the older great tits which have strong site fidelity.
indeed, the association of host density manipulation was stronger among the older great tits for infection with p. circumflexum.
in contrast to our prediction, habitat quality and spring date did not explain much  variation in infection patterns in either of the two morphospecies.
avian malaria, life-history and oxidative stress
there was no overall important life-history or oxidative stress parameter that explained variation in infection of both morphospecies.
for p. relictum infection, age was one of the best predictors and in accordance to our prediction; older great tits were to a greater extent infected compared to younger great tits.
however, there was no sign of an age effect on p. circumflexum infection, which is possibly due to the strong influence of host density .
naturally, the probability of getting bitten by an infected vector increases with time regardless of species  <cit> .
despite that, older individuals may have an acquired immune defence that is familiar to plasmodium parasites, so an overall decline in cellular mechanisms and resistance with age can be an explanation for the influence of age  <cit> .
indeed, since most birds are likely to have a relapse infection, the low tgsh in old p. relictum infected birds can be a result of a more rapid decline of the antioxidant defences  compared to uninfected ageing birds.
for first year breeders, tgsh was higher when infected compared to those that were not infected, which is what we predicted during a chronic malaria infection.
alternatively, young and old individuals have different hormetic responses to parasites  <cit>  or selective disappearance of the old individuals with a high tgsh in response to infection.
the cause for this age difference in tgsh in response to infection warrants further investigation.
given the difference between the sexes in behaviour and physiology during the breeding season, it was not surprising that sex explained some variation in p.relictum infection  <cit> .
generally, in both mammals and birds, males are to a greater extent infected by malaria, but in the present population females have slightly higher presence of infection, but not significantly.
similarly, in a nearby forest, wytham woods, female blue tits showed a significantly higher presence of infection of p. relictum <cit> .
possibly, the direction the association is a result of the timing of sampling i.e., breeding season .
furthermore, there was an interaction between sex and tgsh, revealing that infected males have a higher tgsh compared to uninfected males, whereas infected females have lower  tgsh compared to uninfected females.
interestingly, sex shows a similar pattern to age with regards to the interaction .
the group with relatively lower presence of infection  had higher tgsh when infected compared to the uninfected birds for the same group, whereas the group with relatively higher presence of infection  had lower tgsh when infected.
this sex- and age-specific response of tgsh to p. relictum parasites may play a role in susceptibility to this infection.
alternatively, and independently of the similar age pattern, the investment into reproduction by infected females is associated with a greater physiological costs compared to uninfected females and compared to males, thus, females may not be able to up-regulate their antioxidant system  <cit> .
recently, a study of great tits revealed that increasing brood size results in an increase in malaria parasitaemia in males, but that both sexes show a decrease in resistance to oxidative stress i.e., red blood cell resistance to an external free radical attack  <cit> .
however, a direct link between oxidative stress and parasitaemia was not found.
here clutch size was an important predictor of p. relictum infection, with uninfected birds having larger clutch size than infected birds, supporting the previously found fitness cost  <cit> .
rom was slightly higher in the uninfected birds, thus the production of hydroperoxyl radicals when exposed to oxidizing agent is less likely in p. relictum prevalent birds.
this seem to be in contrast to overall parasitaemia, where rom increases with parasite density , when analysed separately per species only a trend was found .
as mentioned above, the overall effect of parasitaemia may be independent of parasite species virulence but rather a result of parasite metabolism  <cit> .
when a parasite digests haemoglobin it releases haem with iron in its ferrous state .
this state is highly reactive and can easily oxidize to fe3+ and thereby generate rom  <cit> .
thus, increased parasite metabolism and abundance may be followed by increased oxidative damage  unless the antioxidant defence is adequate.
in a recent study of seychelles warbler , malaria infection was linked to a higher rom, but parasitaemia was not measured  <cit> .
an alternative explanation for the patterns in both seychelles warblers and great tits is that high generation of hydroperoxyl radicals may be linked to other unmeasured abiotic or biotic factors that increase the susceptibility to generate them, resulting in a positive association between avian malaria and rom.
in contrast to p. relictum, gssg  rather than the tgsh explained variation in p. circumflexum prevalence.
generally, a high gssg indicates that the cell experiences an oxidative challenge, and that the gsh antioxidant system is in action  <cit> .
however, in an infected cell the antioxidant efficiency of gsh has been shown to be suppressed by a down-regulation of glutathione peroxidase and a low gssg can be detected even though the oxidative challenge is high  <cit> , see also  <cit> .
this is perhaps the most likely interpretation of the lower gssg in p. circumflexum prevalent birds.
however, it should be noted that in the present study all erythrocytes were measured i.e.
not only the infected ones.
thus, the redox environment for the parasite may be different to the average host cell gsh/gssg redox homeostasis.
possibly, the individuals with low gssg are more susceptible to p. cirumflexum infection or there has been selective disappearance of individuals with high intracellular gssg independent of infection.
in captivity, healthy partridges gssg show a quadratic relationship with age  <cit> .
unfortunately, the present study is not able to disentangle the effect of age and infection on gssg and gsh.
the species difference can be a result of different manipulations of the host gsh system, parasite metabolism or virulence  <cit> .
CONCLUSIONS
the present study is the first experimental test showing that bird breeding density increases infection of an avian malaria species - p. circumflexum.
patterns of p. relictum infection, on the other hand, were better explained by host factors: sex, age and clutch size.
as in human malaria, gsh redox system seems to form a part of the physiological response to avian malaria, but in a species-specific pattern and depending on the bird’s age and sex.
however, independent of plasmodium species and host factors, plasmodium abundance showed a positive association with generation of rom.
this could form part of a mechanistic explanation for the recently documented negative impact of infection on fitness in passerines  <cit>  and suggest that oxidative stress may be an important cost of infection in wild populations.
future experimental studies will be important for determining the causal relationship between the different avian malaria species and parameters of oxidative stress.

 avian malaria is a globally widespread disease but considerable variation exists in both prevalence and parasitaemia at small spatial scales. this variation can be generated by several ecological, demographical and immunological mechanisms, including variation in vector and host densities and host susceptibility. for example, temperature, altitude and proximity to water bodies are small-scale ecological factors known to influence vector and/or parasite distribution and abundance. this in turn increases the probability of transmission from vector to host. furthermore, if an area with malaria transmission is of poor quality, this may reduce the bird’s condition and ability to resist a parasitic infection, further compounding the rate of infection. similarly, although transmission rate of vector borne diseases are predicted to be independent of host density, high densities of birds may reduce the availability of food or other resources, thereby further impairing resistance. other demographic parameters that may influence infection rates include sex and age structures of the population arising from differences in behaviour, reproductive effort and immune capacities. the impact of avian malaria on wild birds has been difficult to estimate, especially in areas where avian malaria is common and the host-parasites have co-evolved. however, recent long-term and experimental studies have shown that there are effects on both survival and reproductive success. on naïve hosts such as island species and captive birds in zoo the impacts have been devastating. for example on the hawaiian islands there was an accidental introduction of plasmodium relictum and one of its vectors which caused direct die-offs during the acute infection state which continues to play a significant role in species distribution and is a serious threat for endangered species. in the early stages of infection, the first line of defense is the host’s innate, non-specific immune defence. immune cells generate and release reactive oxygen species that attack the parasite. the advantage of oxidative burst is that it can act within minutes after transmission, in contrast to the acquired immune defence, which needs longer to develop the target-specific antibodies. however, the non-specificity and general cytotoxicity of ros make it impossible for the host to distinguish between self and parasite tissue, causing oxidative damage to all types of adjacent molecules, which if not repaired, can lead to haemolysis and cellular dysfunction. if the parasite resists the host’s ros attack and starts to multiply and grow, it also starts to generate ros as a by-product of haemoglobin digestion. consequently, the detrimental and beneficial effects of ros need to be fine-tuned by the host’s antioxidant defences during different stages of infection, which can result in a quadratic relationship between oxidative stress and malaria severity. this association can depend on other host- and environment-specific characters, including age and nutrition which influences the oxidative status. here we assess the relative importance of ecological, demographic, life-history and physiological factors for natural variation in plasmodium infection and parasitaemia in a population of great tits, parus major. the sampling took place in the spring, which suggests that most infections are relapse infections from previous autumn, since most vectors have not yet emerged. assessment of the relative importance of a broad range of factors, from ecology to physiology, to explain variation in patterns of avian malaria infection in the wild has rarely been done, but is important for understanding the significance of effects found under controlled laboratory situations. in addition, the present population allows test of experimentally increased host density on patterns of prevalence.  in the present population of great tits, of the screened birds were infected with plasmodium; with p. relictum and with p. ciricumflexum morphospecies; only two birds had mixed infection. the infection for first year breeders was 17% for both morphospecies, and the corresponding figures for older birds were 26% and 14%, for, p.relictum and p. ciricumflexum, respectively. infection of plasmodium relictum in table the top ten models are presented along with the averaged model estimates for the parameters included. for p. relictum infection, there were only small differences in the aicc between the top ten models, so no model could be confidently singled out as the best. however, five out of the ten parameters were important for explaining variation in p. relictum infection; namely age, sex, clutch size, spring date and rom. the experimental manipulation of density was not important in any of the top ten models for p. relictum. the raw data from the top three parameters revealed a 12% increase in infection from first year to older breeders and males were less infected than females. in terms of clutch size, uninfected birds had laid a larger clutch size compared to the infected birds. regarding spring date, it should be mentioned that it was highly correlated to gsh:gssg ratio i.e., the earlier in the spring the more reduced to oxidized glutathione. the top ten models are presented along with relative parameter importance. cs clutch size, d breeding density, gssg oxidized glutathione, q habitat quality, rom reactive oxygen metabolites, tgsh total glutathione, rel. imp relative parameters importance, ci confidence interval, ω model weight. when interactions were included the top model retained tgsh, age×tgsh and sex×tgsh apart from the parameters in model. although not significant, the interactions suggest that young and male great tits have higher tgsh when infected compared to when uninfected; and vice versa for older and female birds. infection of plasmodium circumflexum in table the top ten models are presented along with the averaged model estimates for the parameters included. for p. circumflexum, the manipulation of bird density was the best explanatory variable, followed by gssg. in line with our prediction, great tits breeding in high density areas showed a twofold increase in prevalence compared to birds breeding in the low density areas. moreover, p. circumflexum infected birds had a lower gssg than uninfected birds. when interactions were added, the best fitting model included density, gssg, age and age×density. closer examination revealed that the density pattern was found to be stronger in older great tits, with 3% p.circumflexum infection in low density compared to 19% in the high density habitats. for first year breeders the p. circumflexum infection was 18% in both densities. the top ten models are presented along with relative parameter importance. cs clutch size, d breeding density, gssg oxidized glutathione, q habitat quality, rom reactive oxygen metabolites, tgsh total glutathione, rel. imp relative parameters importance, ci confidence interval, ω model weight. in addition, parasite density of both plasmodium species was quantified. however, the species-specific models did not explain any significant variation in parasitaemia, so will not be presented. there were no significant associations between species-specific parasitaemia and any of the physiological variables. although we are aware of the difference in virulence of the two different plasmodium species, the digestion of haemoglobin by both species releases iron which can generate rom. a correlational analysis were performed and it revealed the predicted positive association between rom and parasitaemia, but not for tgsh, gssg and the gssg:tgsh ratio. discussion in the present study, we combined experimental manipulation of host density across two contrasting environments with assessment of host physiology and life-history in a natural population of great tits to understand causes of variation in plasmodium infection. the key and novel findings of this study were, firstly, that manipulation of host density increases p. circumflexum infection, but not infection with p. relictum; and secondly; that host antioxidants and rom physiology show species-specific patterns in relation to infection, and thirdly, that rom increases with plasmodium parasitaemia. avian malaria, ecology and demography host density was found to be the only ecological and demographic parameter of importance for p. circumflexum infection. this link was absent for infection with p.relictum. this suggests that population density reduces the bird’s ability to resist infection by p. circumflexum, the more virulent of the two plasmodium species. possibly, this is via more intense competition over resources or it could be mediated via higher levels of testosterone or stress hormones in high density areas which have been shown to supress the immune system. alternatively, the vectors transmitting p. circumflexum either aggregate where hosts are abundant or that vectors carrying the parasite are able to infect several birds, whereas the vector transmitting p.relictum may be more widely distributed or only feed on one host at one point in time but see. in wytham woods; a woodland in the proximity of bagley, recent work on great tits and the closely related sympatric host species, blue tits, have shown that the two plasmodium species have pronounced differences in their spatial distributions and impacts on both hosts species. while p. circumflexum infections exhibit pronounced spatial structuring that is stable over years in both host species; p. relictum infections are effectively randomly distributed in space. likewise, p. circumflexum infections are linked with reduced survival, particularly during the acute stage of infection; whereas p. relictum infections are associated with reproductive costs. hence the species-specific relationships between ecological factors and parasite prevalence detected in the present study are not unexpected. moreover, in wytham woods it was shown that territory size was linked to host density i.e., the larger territory the lower density. this estimate of host density did not explain patterns of plasmodium infection in the conspecific blue tits, cyanistes caeruleus. similar to the present study, however, a positive association was found between large-scale breeding density and leucocytozoons prevalence and parasitaemia in nestling eagle owls. in temperate regions plasmodium species show a bimodal peak in infection, one in autumn and one in spring, and the spring peak is most often a relapse from the autumn infection gained in the previous year. since juvenile great tits disperse during late summer-early autumn, the impact of the current environment on first-year breeders may be small compared to the older great tits which have strong site fidelity. indeed, the association of host density manipulation was stronger among the older great tits for infection with p. circumflexum. in contrast to our prediction, habitat quality and spring date did not explain much variation in infection patterns in either of the two morphospecies. avian malaria, life-history and oxidative stress there was no overall important life-history or oxidative stress parameter that explained variation in infection of both morphospecies. for p. relictum infection, age was one of the best predictors and in accordance to our prediction; older great tits were to a greater extent infected compared to younger great tits. however, there was no sign of an age effect on p. circumflexum infection, which is possibly due to the strong influence of host density. naturally, the probability of getting bitten by an infected vector increases with time regardless of species. despite that, older individuals may have an acquired immune defence that is familiar to plasmodium parasites, so an overall decline in cellular mechanisms and resistance with age can be an explanation for the influence of age. indeed, since most birds are likely to have a relapse infection, the low tgsh in old p. relictum infected birds can be a result of a more rapid decline of the antioxidant defences compared to uninfected ageing birds. for first year breeders, tgsh was higher when infected compared to those that were not infected, which is what we predicted during a chronic malaria infection. alternatively, young and old individuals have different hormetic responses to parasites or selective disappearance of the old individuals with a high tgsh in response to infection. the cause for this age difference in tgsh in response to infection warrants further investigation. given the difference between the sexes in behaviour and physiology during the breeding season, it was not surprising that sex explained some variation in p.relictum infection. generally, in both mammals and birds, males are to a greater extent infected by malaria, but in the present population females have slightly higher presence of infection, but not significantly. similarly, in a nearby forest, wytham woods, female blue tits showed a significantly higher presence of infection of p. relictum. possibly, the direction the association is a result of the timing of sampling i.e., breeding season. furthermore, there was an interaction between sex and tgsh, revealing that infected males have a higher tgsh compared to uninfected males, whereas infected females have lower tgsh compared to uninfected females. interestingly, sex shows a similar pattern to age with regards to the interaction. the group with relatively lower presence of infection had higher tgsh when infected compared to the uninfected birds for the same group, whereas the group with relatively higher presence of infection had lower tgsh when infected. this sex- and age-specific response of tgsh to p. relictum parasites may play a role in susceptibility to this infection. alternatively, and independently of the similar age pattern, the investment into reproduction by infected females is associated with a greater physiological costs compared to uninfected females and compared to males, thus, females may not be able to up-regulate their antioxidant system. recently, a study of great tits revealed that increasing brood size results in an increase in malaria parasitaemia in males, but that both sexes show a decrease in resistance to oxidative stress i.e., red blood cell resistance to an external free radical attack. however, a direct link between oxidative stress and parasitaemia was not found. here clutch size was an important predictor of p. relictum infection, with uninfected birds having larger clutch size than infected birds, supporting the previously found fitness cost. rom was slightly higher in the uninfected birds, thus the production of hydroperoxyl radicals when exposed to oxidizing agent is less likely in p. relictum prevalent birds. this seem to be in contrast to overall parasitaemia, where rom increases with parasite density, when analysed separately per species only a trend was found. as mentioned above, the overall effect of parasitaemia may be independent of parasite species virulence but rather a result of parasite metabolism. when a parasite digests haemoglobin it releases haem with iron in its ferrous state. this state is highly reactive and can easily oxidize to fe3+ and thereby generate rom. thus, increased parasite metabolism and abundance may be followed by increased oxidative damage unless the antioxidant defence is adequate. in a recent study of seychelles warbler, malaria infection was linked to a higher rom, but parasitaemia was not measured. an alternative explanation for the patterns in both seychelles warblers and great tits is that high generation of hydroperoxyl radicals may be linked to other unmeasured abiotic or biotic factors that increase the susceptibility to generate them, resulting in a positive association between avian malaria and rom. in contrast to p. relictum, gssg rather than the tgsh explained variation in p. circumflexum prevalence. generally, a high gssg indicates that the cell experiences an oxidative challenge, and that the gsh antioxidant system is in action. however, in an infected cell the antioxidant efficiency of gsh has been shown to be suppressed by a down-regulation of glutathione peroxidase and a low gssg can be detected even though the oxidative challenge is high, see also. this is perhaps the most likely interpretation of the lower gssg in p. circumflexum prevalent birds. however, it should be noted that in the present study all erythrocytes were measured i.e. not only the infected ones. thus, the redox environment for the parasite may be different to the average host cell gsh/gssg redox homeostasis. possibly, the individuals with low gssg are more susceptible to p. cirumflexum infection or there has been selective disappearance of individuals with high intracellular gssg independent of infection. in captivity, healthy partridges gssg show a quadratic relationship with age. unfortunately, the present study is not able to disentangle the effect of age and infection on gssg and gsh. the species difference can be a result of different manipulations of the host gsh system, parasite metabolism or virulence.  the present study is the first experimental test showing that bird breeding density increases infection of an avian malaria species - p. circumflexum. patterns of p. relictum infection, on the other hand, were better explained by host factors: sex, age and clutch size. as in human malaria, gsh redox system seems to form a part of the physiological response to avian malaria, but in a species-specific pattern and depending on the bird’s age and sex. however, independent of plasmodium species and host factors, plasmodium abundance showed a positive association with generation of rom. this could form part of a mechanistic explanation for the recently documented negative impact of infection on fitness in passerines and suggest that oxidative stress may be an important cost of infection in wild populations. future experimental studies will be important for determining the causal relationship between the different avian malaria species and parameters of oxidative stress. 
instead rom significantly increased with plasmodium parasitaemia. first, the infection of p. circumflexum was significantly higher in areas with experimental increased host density, whereas variation in p. relictum infection was mainly attributed to age, sex and reproduction. second, great tit antioxidant responses – total and oxidizied glutathione - showed age-, sex- and plasmodium species-specific patterns between infected and uninfected individuals, but reactive oxygen metabolites showed only a weak explanatory power for patterns of p. relictum infection. these results identify some key factors that influence plasmodium infection in wild birds, and provide a potential explanation for the underlying physiological basis of recently documented negative effects of chronic avian malaria on survival and reproductive success. avian malaria is globally widespread, but considerable variation exists in infection patterns at small spatial scales. here we use experimental and correlational approaches to assess the relative importance of host density, habitat composition, individual level variation and redox physiology for plasmodium infection in a wild population of great tits, parus major.
 avian malaria is globally widespread, but considerable variation exists in infection patterns at small spatial scales. this variation can be driven by variation in ecology, demography, and phenotypic characters, in particular those that influence the host’s resistance. generation of reactive oxygen species is one of the host’s initial immune responses to combat parasitic invasion. however, long-term ros exposure can harm the host and the redox response therefore needs to be adjusted according to infection stage and host phenotype. here we use experimental and correlational approaches to assess the relative importance of host density, habitat composition, individual level variation and redox physiology for plasmodium infection in a wild population of great tits, parus major.  we found that 36% of the great tit population was infected with plasmodium and that patterns of infection were plasmodium species-specific. first, the infection of p. circumflexum was significantly higher in areas with experimental increased host density, whereas variation in p. relictum infection was mainly attributed to age, sex and reproduction. second, great tit antioxidant responses – total and oxidizied glutathione - showed age-, sex- and plasmodium species-specific patterns between infected and uninfected individuals, but reactive oxygen metabolites showed only a weak explanatory power for patterns of p. relictum infection. instead rom significantly increased with plasmodium parasitaemia.  these results identify some key factors that influence plasmodium infection in wild birds, and provide a potential explanation for the underlying physiological basis of recently documented negative effects of chronic avian malaria on survival and reproductive success. antioxidantsglutathionehost densityhydroperoxidesoxidative stressparus majorplasmodium 