BACKGROUND
microorganisms constitute more than half of the total biomass of earth; however, we have just made the first steps in discovering and measuring the diversity, abundance and function of these microbial ecosystems  <cit> .
macroorganisms live, therefore, with a pool of microorganisms, being in close contact and continuously interacting with them.
some of the microorganisms are beneficial and mutualistic relationships have evolved with the host, while others can have detrimental effects on the host  <cit> .
to defend themselves against these potential parasites and maintain their health status, host organisms have evolved a complex system of behavioral, mechanical and chemical defenses  <cit> , the immune system being of the utmost importance.
in birds, some bacteria inhabiting the feathers are detrimental to the bird by degrading keratin  <cit> , thus altering thermoregulation, flight and ultimately fitness  <cit> .
to maintain feather condition, birds have evolved several anti-bacterial defenses  <cit> , including the deposition of preen secretions onto feathers .
preening may have antibacterial effects either by the direct activity of antibacterial compounds  <cit> , by antibacterial substances secreted by the uropygial gland-associated symbiotic bacteria  <cit>  or by forming a mechanical barrier between the bacteria and the feather surface  <cit> .
recently it has been shown that preen secretion production and preening frequency are adjusted to feather bacterial load, suggesting that they are energetically costly inducible antibacterial defenses  <cit> .
preen secretions are composed mainly of oily substances  <cit>  which probably require considerable energy in their anabolism.
in addition, preening is an energetically costly behavior  <cit>  that takes a substantial portion of a bird’s time budget  <cit> .
preening and preen secretion therefore probably compete with other costly life-history traits.
in particular, investment in preen gland associated traits  have been suggested to trade-off with investment in immune defense.
accordingly, immune-challenged or experimentally-infected tawny owls  and house sparrows  develop smaller preen glands  <cit> , while apapanes  infected with plasmodium preen less frequently  <cit> .
although feather bacteria load is essentially controlled by preen secretions, some feather bacteria can gain access into the tissue under specific conditions and become opportunistic pathogens, as demonstrated for some usually benign skin bacteria in humans  <cit> .
therefore, selection may favor maintaining an appropriate level of immune defense - the final line of defense against parasites - when facing high feather bacterial load.
how birds invest in the energetically competing defenses that are preen secretions and the immune system when facing varying degree of feather bacteria load is, however, unknown.
in a previous study, we have shown that preen secretion quantity and preening frequency increased with experimentally increased feather bacterial load in captive feral pigeons   <cit> .
therefore, to shed light on how birds balance the trade-off between immunity and preen secretions when facing high or low feather bacterial loads, we investigated the effect of feather bacteria load on immune defense in the same experimental pigeons.
given the potential trade-off between preen secretion and immunity, birds experiencing high bacterial load are expected to have decreased overall immune response.
however, they are expected also to maintain immune defenses against microparasites, against which the first line of defense is mediated by constitutive innate immunity, a mixture of humoral  and cellular components   <cit> .
providing a broad characterization of the immune system of vertebrates is a non-trivial challenge in immunoecological studies  <cit> , and simultaneous measurements of multiple immune parameters are necessary.
here we used five measures to assess the immunity of pigeons:  the response to phytohemagglutinin , a measure of induced pro-inflammatory capacity  <cit> ;  the hemagglutination and the  hemolysis capacity of plasma, a measure of the natural antibody and complement levels, respectively  <cit> ;  the bactericidal capacity of whole blood, a measure of the capacity to eliminate bacterial pathogens  <cit> ; and  the response against keyhole limpet hemocyanin , a measure of induced humoral immune response.
finally, because variation in immunity is usually associated with increased susceptibility to pathogens, we measured the intensity of blood parasite infection.
RESULTS
bact- birds had lower bacterial load on feathers than control  birds, while bact+ birds had higher bacterial load than co birds .
body condition did not differ among treatments .
pha response varied with treatment .
bact+ birds had lower pha response than bact- and co birds .
pha response tended to increase with wing length  but it did not depend upon sex  or morph .figure  <dig>
tukey boxplot of pha response  in bact- co and bact+ birds.
bact+ and bact- birds had higher bacterial killing ability  than co birds for all three bacteria strains tested .
however, differences in bka between treatments was significant only for the two strains of e. coli , while it was not significant for s. aureus .
bact- birds had significantly higher bka against e. coli than co birds , while bact+ birds had intermediate bka against e. coli that did not differ with co or bact- birds .
bka did not depend upon sex  or color morph .figure  <dig>
tukey boxplot of whole blood bacterial killing ability against
e. coli
atcc  <dig>
e. coli
atcc  <dig> and
s. aureus
in bact-, co and bact+ birds.
hemagglutination and hemolysis were not correlated with one another .
agglutination and lysis were unrelated to treatment .
agglutination was unrelated to sex , while lysis was higher in males than females .
agglutination and lysis did not vary with color morph .
primary and secondary responses to klh challenge were unrelated to treatment , sex  and color morph .figure  <dig>
anti-klh antibody levels in plasma of bact-, co and bact+ birds, over the days after klh first and second injection.
captivity tended to decrease blood parasite load .
the change in blood parasite load between day  <dig> and day  <dig> of treatment was not different between bact-, bact+ and co birds .
it did not vary with sex  and color morph .
discussion
in this study, we investigated how pigeons invest in the immune system when facing high or low feather bacterial load.
birds with increased bacterial load on plumage had lower response to pha skin swelling test compared to control birds and birds with decreased bacterial load.
pha injection generates acute inflammation  <cit> , a known energetically costly process  <cit> , which is traded-off with various life-history traits, such as molt, breeding or growth  <cit> .
in a previous study, we have found that pigeons with increased feather bacterial load invested more in behavioral and chemical defenses against feather bacteria than control birds  <cit> .
investment in these antibacterial defenses has been suggested to be energetically costly  <cit> , and birds with high bacteria load on their plumage, investing more in preen secretions and preening  <cit> , may not be able to sustain the costs of pha-induced inflammation.
our result is, therefore, consistent with a trade-off between immunity and preening in birds  <cit> .
it also suggests that birds facing high bacterial load preferentially invest in preen secretions – the first line of defense against feather bacteria – rather than in costly inflammatory processes.
the pha response is the only immune process that varied with increased bacterial load.
the bactericidal capacity of whole blood did not differ between birds with increased feather bacterial load and controls.
this assay estimates the capacity of the blood to rapidly thwart a potential bacterial pathogen.
this activity is a combination of several mechanisms of the innate immune system, including phagocytosis and the activities of humoral proteins such as natural antibodies, complement, antibacterial enzymes and peptides  <cit> .
thus, it is considered as the most general and integrative in vitro measurement of the constituent elements of the innate immune system  <cit> .
bactericidal activity of blood has been shown to be positively correlated with experimental or natural infection in several species  <cit> .
feather bacteria can gain entry into damaged skin and can potentially pass into the body through the gastrointestinal barrier once ingested during preening activities  <cit> .
birds facing high feather bacterial load may therefore be more at risk of infection by feather bacteria and may need to maintain investment in immune components against microparasites measured by the blood bactericidal activity test.
further studies are needed to determine whether pigeons with experimentally increased feather bacterial load do face higher bacterial ingestion and entry.
non-exclusively, pigeons facing high bacterial load may maintain blood bactericidal activity while decreasing pha-induced response, because the later is more costly and thus the first to be traded-off against preen secretion.
in contrast, birds with decreased bacterial load had higher bacterial killing ability against e. coli than control birds.
birds with decreased bacteria load on plumage may save energy from decreased preening frequency and/or investment in preen secretion  <cit>  and therefore may be able to invest more in these components of the innate immunity.
we did not find evidence for effects of feather bacterial load on acquired immunity in pigeons.
experimental pigeons injected with klh, a novel antigen, did not vary the primary and secondary antibody response .
we also did not find evidence from the hemagglutination-hemolysis assay for effects of feather bacterial load on the levels of natural antibodies  and complement.
nabs are part of the innate immune system and delay pathogen replication until the developing acquired humoral and cellular immune responses clear the infection  <cit> .
nabs are thought to be relatively insensitive to short-term changes in environmental conditions  <cit>  and have been shown to be unaffected by experimental infections or food limitation  <cit> .
our results are in line with these studies showing that non-specific inflammation  is more sensitive to changes in host condition and/or workload than are antibody responses.
for example, in pied flycatchers , females with experimentally-reduced clutch size have lower response to pha  <cit> , but similar antibody responses as controls  <cit> .
CONCLUSIONS
in conclusion, our study suggests that increased feather bacterial load in pigeons negatively affected the components of the induced innate immune response involved in pha-induced inflammation, while decreased feather bacterial load positively affected the components associated with the constitutive innate immune system that are measured by the bacterial killing ability test.
these results are consistent with a trade-off between investment in preen secretion and immunity.
no other components of the immune system were, however, found to be affected by manipulation of feather bacterial load.
whether these components were maintained to protect against feather bacteria that may enter the body or not sufficiently costly to be traded-off with preen secretion requires further study.
more generally, this study reinforces the idea that feathers microbiota may have a strong impact on ecology and evolution of the avian host.

 microorganisms constitute more than half of the total biomass of earth; however, we have just made the first steps in discovering and measuring the diversity, abundance and function of these microbial ecosystems. macroorganisms live, therefore, with a pool of microorganisms, being in close contact and continuously interacting with them. some of the microorganisms are beneficial and mutualistic relationships have evolved with the host, while others can have detrimental effects on the host. to defend themselves against these potential parasites and maintain their health status, host organisms have evolved a complex system of behavioral, mechanical and chemical defenses, the immune system being of the utmost importance. in birds, some bacteria inhabiting the feathers are detrimental to the bird by degrading keratin, thus altering thermoregulation, flight and ultimately fitness. to maintain feather condition, birds have evolved several anti-bacterial defenses, including the deposition of preen secretions onto feathers. preening may have antibacterial effects either by the direct activity of antibacterial compounds, by antibacterial substances secreted by the uropygial gland-associated symbiotic bacteria or by forming a mechanical barrier between the bacteria and the feather surface. recently it has been shown that preen secretion production and preening frequency are adjusted to feather bacterial load, suggesting that they are energetically costly inducible antibacterial defenses. preen secretions are composed mainly of oily substances which probably require considerable energy in their anabolism. in addition, preening is an energetically costly behavior that takes a substantial portion of a bird’s time budget. preening and preen secretion therefore probably compete with other costly life-history traits. in particular, investment in preen gland associated traits have been suggested to trade-off with investment in immune defense. accordingly, immune-challenged or experimentally-infected tawny owls and house sparrows develop smaller preen glands, while apapanes infected with plasmodium preen less frequently. although feather bacteria load is essentially controlled by preen secretions, some feather bacteria can gain access into the tissue under specific conditions and become opportunistic pathogens, as demonstrated for some usually benign skin bacteria in humans. therefore, selection may favor maintaining an appropriate level of immune defense - the final line of defense against parasites - when facing high feather bacterial load. how birds invest in the energetically competing defenses that are preen secretions and the immune system when facing varying degree of feather bacteria load is, however, unknown. in a previous study, we have shown that preen secretion quantity and preening frequency increased with experimentally increased feather bacterial load in captive feral pigeons. therefore, to shed light on how birds balance the trade-off between immunity and preen secretions when facing high or low feather bacterial loads, we investigated the effect of feather bacteria load on immune defense in the same experimental pigeons. given the potential trade-off between preen secretion and immunity, birds experiencing high bacterial load are expected to have decreased overall immune response. however, they are expected also to maintain immune defenses against microparasites, against which the first line of defense is mediated by constitutive innate immunity, a mixture of humoral and cellular components. providing a broad characterization of the immune system of vertebrates is a non-trivial challenge in immunoecological studies, and simultaneous measurements of multiple immune parameters are necessary. here we used five measures to assess the immunity of pigeons: the response to phytohemagglutinin, a measure of induced pro-inflammatory capacity; the hemagglutination and the hemolysis capacity of plasma, a measure of the natural antibody and complement levels, respectively; the bactericidal capacity of whole blood, a measure of the capacity to eliminate bacterial pathogens; and the response against keyhole limpet hemocyanin, a measure of induced humoral immune response. finally, because variation in immunity is usually associated with increased susceptibility to pathogens, we measured the intensity of blood parasite infection.  bact- birds had lower bacterial load on feathers than control birds, while bact+ birds had higher bacterial load than co birds. body condition did not differ among treatments. pha response varied with treatment. bact+ birds had lower pha response than bact- and co birds. pha response tended to increase with wing length but it did not depend upon sex or morph.figure tukey boxplot of pha response in bact- co and bact+ birds. bact+ and bact- birds had higher bacterial killing ability than co birds for all three bacteria strains tested. however, differences in bka between treatments was significant only for the two strains of e. coli, while it was not significant for s. aureus. bact- birds had significantly higher bka against e. coli than co birds, while bact+ birds had intermediate bka against e. coli that did not differ with co or bact- birds. bka did not depend upon sex or color morph.figure tukey boxplot of whole blood bacterial killing ability against e. coli atcc e. coli atcc and s. aureus in bact-, co and bact+ birds. hemagglutination and hemolysis were not correlated with one another. agglutination and lysis were unrelated to treatment. agglutination was unrelated to sex, while lysis was higher in males than females. agglutination and lysis did not vary with color morph. primary and secondary responses to klh challenge were unrelated to treatment, sex and color morph.figure anti-klh antibody levels in plasma of bact-, co and bact+ birds, over the days after klh first and second injection. captivity tended to decrease blood parasite load. the change in blood parasite load between day and day of treatment was not different between bact-, bact+ and co birds. it did not vary with sex and color morph. discussion in this study, we investigated how pigeons invest in the immune system when facing high or low feather bacterial load. birds with increased bacterial load on plumage had lower response to pha skin swelling test compared to control birds and birds with decreased bacterial load. pha injection generates acute inflammation, a known energetically costly process, which is traded-off with various life-history traits, such as molt, breeding or growth. in a previous study, we have found that pigeons with increased feather bacterial load invested more in behavioral and chemical defenses against feather bacteria than control birds. investment in these antibacterial defenses has been suggested to be energetically costly, and birds with high bacteria load on their plumage, investing more in preen secretions and preening, may not be able to sustain the costs of pha-induced inflammation. our result is, therefore, consistent with a trade-off between immunity and preening in birds. it also suggests that birds facing high bacterial load preferentially invest in preen secretions – the first line of defense against feather bacteria – rather than in costly inflammatory processes. the pha response is the only immune process that varied with increased bacterial load. the bactericidal capacity of whole blood did not differ between birds with increased feather bacterial load and controls. this assay estimates the capacity of the blood to rapidly thwart a potential bacterial pathogen. this activity is a combination of several mechanisms of the innate immune system, including phagocytosis and the activities of humoral proteins such as natural antibodies, complement, antibacterial enzymes and peptides. thus, it is considered as the most general and integrative in vitro measurement of the constituent elements of the innate immune system. bactericidal activity of blood has been shown to be positively correlated with experimental or natural infection in several species. feather bacteria can gain entry into damaged skin and can potentially pass into the body through the gastrointestinal barrier once ingested during preening activities. birds facing high feather bacterial load may therefore be more at risk of infection by feather bacteria and may need to maintain investment in immune components against microparasites measured by the blood bactericidal activity test. further studies are needed to determine whether pigeons with experimentally increased feather bacterial load do face higher bacterial ingestion and entry. non-exclusively, pigeons facing high bacterial load may maintain blood bactericidal activity while decreasing pha-induced response, because the later is more costly and thus the first to be traded-off against preen secretion. in contrast, birds with decreased bacterial load had higher bacterial killing ability against e. coli than control birds. birds with decreased bacteria load on plumage may save energy from decreased preening frequency and/or investment in preen secretion and therefore may be able to invest more in these components of the innate immunity. we did not find evidence for effects of feather bacterial load on acquired immunity in pigeons. experimental pigeons injected with klh, a novel antigen, did not vary the primary and secondary antibody response. we also did not find evidence from the hemagglutination-hemolysis assay for effects of feather bacterial load on the levels of natural antibodies and complement. nabs are part of the innate immune system and delay pathogen replication until the developing acquired humoral and cellular immune responses clear the infection. nabs are thought to be relatively insensitive to short-term changes in environmental conditions and have been shown to be unaffected by experimental infections or food limitation. our results are in line with these studies showing that non-specific inflammation is more sensitive to changes in host condition and/or workload than are antibody responses. for example, in pied flycatchers, females with experimentally-reduced clutch size have lower response to pha, but similar antibody responses as controls.  in conclusion, our study suggests that increased feather bacterial load in pigeons negatively affected the components of the induced innate immune response involved in pha-induced inflammation, while decreased feather bacterial load positively affected the components associated with the constitutive innate immune system that are measured by the bacterial killing ability test. these results are consistent with a trade-off between investment in preen secretion and immunity. no other components of the immune system were, however, found to be affected by manipulation of feather bacterial load. whether these components were maintained to protect against feather bacteria that may enter the body or not sufficiently costly to be traded-off with preen secretion requires further study. more generally, this study reinforces the idea that feathers microbiota may have a strong impact on ecology and evolution of the avian host. 
the decrease in the energetically costly inflammatory response of birds experiencing high bacterial load suggests a trade-off between investment in preen secretion and immunity and reinforces the idea that feather microbiota may have a strong impact on the ecology and evolution of the avian host. to shed light on how birds balance the trade-off between immunity and preen secretions when facing high or low feather bacterial load, we experimentally manipulated feather bacteria load of feral pigeons, and investigated the effects on immune defenses. birds facing high feather bacterial load had lower immune response to pha skin-swelling test than controls, while birds facing low feather bacterial load had higher blood bacterial killing ability than controls. no other components of the immune system were found to be affected by feather bacterial load. pigeons had previously been shown to adjust preening to feather bacterial load. the immune system is only triggered when bacteria gain access into the body. preening is, however, costly and may trade-off with investment in the immune system. microbiotaphabacterial killing abilityimmune systempigeonscolumba liviaissue-copyright-statement© the author 2015
 complex communities of bacteria inhabit the feathers of all birds. under normal conditions, individuals maintain a healthy state by defending themselves against these potential invaders by preening. the immune system is only triggered when bacteria gain access into the body. preening is, however, costly and may trade-off with investment in the immune system. to shed light on how birds balance the trade-off between immunity and preen secretions when facing high or low feather bacterial load, we experimentally manipulated feather bacteria load of feral pigeons, and investigated the effects on immune defenses.  birds facing high feather bacterial load had lower immune response to pha skin-swelling test than controls, while birds facing low feather bacterial load had higher blood bacterial killing ability than controls. no other components of the immune system were found to be affected by feather bacterial load.  pigeons had previously been shown to adjust preening to feather bacterial load. the decrease in the energetically costly inflammatory response of birds experiencing high bacterial load suggests a trade-off between investment in preen secretion and immunity and reinforces the idea that feather microbiota may have a strong impact on the ecology and evolution of the avian host. keywords microbiotaphabacterial killing abilityimmune systempigeonscolumba liviaissue-copyright-statement© the author 2015 