BACKGROUND
an increasing set of data is shedding light on the role of microorganisms that have co-evolved with their hosts, including humans  <cit> .
they illustrate the high diversity of endosymbiotic forms among living organisms.
moreover the evidence of gene transfer between bacterial cells or viruses and eukaryotic cells supports the theory of symbiotic relationships as a major force driving evolution  <cit>  and as a source of phenotypic complexity  <cit> .
multiple new symbionts are regularly discovered in the same host, which can compete or cooperate  <cit> .
normally, they play a role in host nutrition; defence against pathogens remains an underappreciated benefit of such associations, both in invertebrates and vertebrates  <cit> .
social insects are particularly concerned as they are highly susceptible to infectious diseases, due to their lifestyle, and have evolved several associations with microorganisms  <cit> .
endosymbionts are very common among insects, especially in those sucking plant sap, feeding on vertebrate blood for their entire life span, and those that eat wood and keratin.
as they are all strict specialists in nourishment, it is assumed that endosymbionts play a role in providing complementary elements absent from these restricted diets.
camponotus genus, carpenter ants, have established an association with intracellular endosymbionts blochmannia, a taxon of γ-proteobacteria, found in all camponotus species studied hitherto  <cit> .
the bacteria live within specialized cells, the bacteriocytes.
the function of the endosymbionts is not fully elucidated but their role as dietary complement suppliers has been pointed out after the genome sequence analysis of two blochmannia species.
the bacteria is probably able to supply nitrogen and sulphur compounds to the host  <cit> .
moreover, bacteria elimination using antibiotic treatment is deleterious and chemically defined diets can complement bacteria suppression  <cit>  demonstrating the necessary nutritional role of bacteria.
however, the presence of blochmannia in omnivorous camponotus species suggests that bacteria may also have other functions beneficial to the ants.
some studies have suggested that blochmannia may play a more important role during the colony founding phase and growth rather than in adult worker maintenance  <cit>  or may play a role in pheromone production  <cit> .
microbes that forms chronic infections in a host lineage may evolve to promote host survival or benefits to its host, as this will help to maintain its immediate ecological resource  <cit> .
in this context, secondary endosymbionts can provide hosts with defences against parasites, beyond nutritional advantages  <cit> .
so far, no similar example with primary endosymbionts has been reported.
externally located bacteria are also capable of conferring protection to insect hosts against parasite infections  <cit> .
here, we tested the hypotheses that blochmannia provide faster colony development in the initial stages  as previously stated  <cit>  and/or improve the host immune system of the host.
we used the encapsulation rate as an index of the immune response and analysed whether it was correlated or not with the number of bacteria.
the use of incipient colonies, obtained from founding queens, is a suitable choice since it allows the study of animals of similar ages and reduces the effects of natural selection operating on colonies throughout their development.
RESULTS
endosymbiont identification
the 16s rdna endosymbiont sequence was deposited in the genbank database under accession number ef <dig>  according to the ribosomal database project  <cit> , the 16s rdna sequence of camponotus fellah endosymbiont correspond to an unclassified γ-proteobacteria closely related to 16rdna sequences from blochmannia endosymbionts bacteria of various camponotus ant species.
this sequence has g+c content of 47% which is near to that of other blochmannia symbionts.
when compared with the nucleic sequences of other blochmannia , maximum identity ranged from 91–93%.
however, other blochmannia species present in genbank exhibit up to 98% of identity to each other.
phylogenetic comparisons showed the existence of a monophyletic group containing classified and unclassified endosymbionts from camponotus ant species, closer to other insect endosymbionts and distinct from other outgroup bacteria .
the use of fish with primers specific for eubacteria and blochmannia endosymbionts showed that bacteriocytes of midgut preparations were full of bacteria.
in these preparations it was possible to see the individual bacterium and its rod form.
the bacteriocytes were also detected in the oocytes by fish as well.
effectiveness of antibiotic treatment
the quantity of blochmannia in midgut bacteriocytes was estimated after rifampin treatment using two complementary methods: real-time quantitative pcr and fluorescent in situ hybridization .
the two methods showed a reduction of blochmannia numbers in midgut bacteriocytes after 12-weeks of antibiotic treatment.
within this period, fish did not detect the presence of blochmania in the bacteriocytes .
however quantitative real-time pcr indicated that the bacteria were not completely eliminated as a low quantity of 16s rdna bacteria molecules can be detected in the midgut.
treated and control groups differed significantly in their content of blochmannia measured as 16s rdna molecules  .
the treatment reduced the quantity of bacteria by 75%.
moreover, the individual variation in bacteria amount was more constant in antibiotic treated colonies than in control colonies.
evaluation of colony development
each colony was composed of at least one larva, pupa or worker and queen.
colonies composed only with the queen or colonies with a dying queen during the experiment were excluded.
after seven months, seven control colonies and nine treated colonies were kept for further analysis.
workers, larvae and pupae numbers were not significantly different during the first three months after the beginning of the experiments.
after this time, untreated colonies displayed more accentuated larvae production and had a higher number of adult workers .
pupae number varied significantly throughout the time of the experiment but no difference between treated and control colonies was observed .
the variation in workers numbers was significatively different between treated and control colonies with untreated colonies having more workers .
mean number of larvae, pupae and workers analysed by anova.
significance levels are *p <  <dig> , **p <  <dig>  and ***p ≤  <dig> .
amount of blochmannia endosymbiont versus encapsulation response
when expressing encapsulation rate versus 16s rdna molecules amount , control and treated colonies displayed different patterns of immune response.
we found a significant positive correlation between encapsulation rate and bacteria amount in the ants from control colonies: the bacteria did facilitate the encapsulation response .
on the contrary, ants from treated colonies did not display a correlation between the amount of bacteria in the midgut and the encapsulation response .
thus, it seems that antibiotic treatment eliminated the bacterial effects on the immune encapsulation response.
an ancova analysis with the encapsulation rate as independent variable showed that treated workers present a significant increase in encapsulation rate .
the regression inclinations of treated and control groups differed significantly .
discussion and
CONCLUSIONS
in this study, we confirmed that blochmannia plays an important role for camponotus ants by improving the colony growth.
we also demonstrated for the first time that blochmannia interacts with the ant immune defence.
antibiotic treatment with rifampin considerably reduced the endosymbiont number in the midgut, although they were never totally eliminated and there was a great variability between workers.
this may be due to different access to the antibiotic and some ants may not drink the antibiotic solution or, as observed by feldhaar et al.
, may be explained by the fact that dna of the endosymbiont may still be detectable by qrt-pcr when bacteria are not alive or active.
additionally, it was confirmed that bacterial sequences were not integrated in the genome of the ant by a pcr test performed on ant dna from legs using blochmannia 16s rdna and ant 18s rdna primers .
the treatment had a remarkable impact on colony development by reducing larvae production and worker numbers, corroborating previous worker  <cit> .
carrying out the studies in entire incipient colonies, we can demonstrate the importance of endosymbionts in this phase of colony development.
according feldhaar et al.
, essential amino acids provided by endosymbionts improve workers ability to raise pupae.
here, we have verified that control colonies exhibited a bigger population in the first seven months of colony development.
since the establishment phase is critical for new colonies, harbouring more bacteria may have major ecological consequences in a context of inter and intraspecific competition: more workers confers a special advantage to maintain a young colony, occupy and monopolize food resources.
indeed, animal protein food resources are more unpredictable in the time-space scale.
blochmannia presence could signify a possible adaptation for ants to fluctuations in protein availability, permitting the colony growth even in absence of preys.
we do not know the mechanisms allowing an increase in brood production, beyond the direct nutritional effects on treated queen, but several mechanisms are plausible, including a direct oogenesis control.
for example, it has been demonstrated that wolbachia bacteria are necessary for the host oogenesis in a particular strain of the parasitic wasp asobara tabida  <cit> .
furthermore, it was evidenced that apoptosis prevention of nurse cells by wolbachia can regulate the host oogenesis  <cit> .
we have demonstrated that blochmannia play another important function by improving camponotus host immune system.
the encapsulation rate measured in rifampin treated workers was significantly higher when compared with control colonies.
although no evident toxic effect  was observed, it is expected that antibiotic treatment has stressing effects on workers and that the increase of the encapsulation rate might correspond to an adaptive response to stress.
furthermore, antibiotic treatment seemed to mask the effects of endosymbiont number on encapsulation response observed in control colonies, where the bacteria favoured the encapsulation response.
positive effects of symbionts on host immune system have been described in the last years.
for example, the facultative symbionts of acyrthosiphon pisum  confer it resistance to parasitoid attacks  <cit> .
recently, it has been demonstrated that wolbachia confer vigorous antiviral protection to drosophila  <cit> .
the mechanisms by which the resistance is expressed is still unknown, but in another example it was showed that symbiotic bacteria could compete directly for space and resources and thus prevent host colonization by pathogens  <cit> .
encapsulation is the principal physiological response against parasitoids suggesting an important role of the stimulation induced by blochmannia in the protection against parasites.
this strong interaction between symbiotic bacteria and ants may explain the persistence and broad occurrence of symbiotic bacteria in the camponotus genus.
ants from camponotus genus are abundant almost everywhere in the world where ants are found, comprising more than  <dig> described species within an estimated number greater than  <dig>  species  <cit> .
its large distribution, the diversity of forms and food behaviour and the occurrence on diverse environments make the system camponotus/blochmannia an interesting model to study how ecological forces determine symbiont characteristics and how bacteria determine the ant traits.
for example, it is interesting to determine how genetic differences found among different species of blochmannia could be related to host ecological characteristics.
the social habits of the ants make them particularly vulnerable to several parasites and parasitoids.
phoridae flies are frequently found around camponotus nests and their influence is fundamental in regulating the ant communities  <cit> .
so, it can be expected that camponotus species more exposed to phoridae attack should harbour more bacteria.
the physiological mechanism linking bacterial amount and encapsulation response remains unknown.
although the better workers "quality" due to extra nutrients furnished by bacteria is the more probable explanation, direct production of biomolecules in stress situation should not be excluded.
an efficient immune system is a major trait allowing the existence of social insect colonies with thousand of individuals, genetically related  <cit> , living close together, constantly exposed to parasitic disease risks.
competition in the first stages of colony growth constitutes also a great challenge to reach the reproductive stage.
thus, blochmannia endosymbionts appear to be a fundamental partner, responsible for the ecological success of camponotus ants.
as more than 10% of insect species depend on obligate bacterial mutualists for their viability and reproduction  <cit> , the research on symbiosis between bacteria and animals appears to be a new and promising field, particularly in social insects.

 an increasing set of data is shedding light on the role of microorganisms that have co-evolved with their hosts, including humans. they illustrate the high diversity of endosymbiotic forms among living organisms. moreover the evidence of gene transfer between bacterial cells or viruses and eukaryotic cells supports the theory of symbiotic relationships as a major force driving evolution and as a source of phenotypic complexity. multiple new symbionts are regularly discovered in the same host, which can compete or cooperate. normally, they play a role in host nutrition; defence against pathogens remains an underappreciated benefit of such associations, both in invertebrates and vertebrates. social insects are particularly concerned as they are highly susceptible to infectious diseases, due to their lifestyle, and have evolved several associations with microorganisms. endosymbionts are very common among insects, especially in those sucking plant sap, feeding on vertebrate blood for their entire life span, and those that eat wood and keratin. as they are all strict specialists in nourishment, it is assumed that endosymbionts play a role in providing complementary elements absent from these restricted diets. camponotus genus, carpenter ants, have established an association with intracellular endosymbionts blochmannia, a taxon of γ-proteobacteria, found in all camponotus species studied hitherto. the bacteria live within specialized cells, the bacteriocytes. the function of the endosymbionts is not fully elucidated but their role as dietary complement suppliers has been pointed out after the genome sequence analysis of two blochmannia species. the bacteria is probably able to supply nitrogen and sulphur compounds to the host. moreover, bacteria elimination using antibiotic treatment is deleterious and chemically defined diets can complement bacteria suppression demonstrating the necessary nutritional role of bacteria. however, the presence of blochmannia in omnivorous camponotus species suggests that bacteria may also have other functions beneficial to the ants. some studies have suggested that blochmannia may play a more important role during the colony founding phase and growth rather than in adult worker maintenance or may play a role in pheromone production. microbes that forms chronic infections in a host lineage may evolve to promote host survival or benefits to its host, as this will help to maintain its immediate ecological resource. in this context, secondary endosymbionts can provide hosts with defences against parasites, beyond nutritional advantages. so far, no similar example with primary endosymbionts has been reported. externally located bacteria are also capable of conferring protection to insect hosts against parasite infections. here, we tested the hypotheses that blochmannia provide faster colony development in the initial stages as previously stated and/or improve the host immune system of the host. we used the encapsulation rate as an index of the immune response and analysed whether it was correlated or not with the number of bacteria. the use of incipient colonies, obtained from founding queens, is a suitable choice since it allows the study of animals of similar ages and reduces the effects of natural selection operating on colonies throughout their development.  endosymbiont identification the 16s rdna endosymbiont sequence was deposited in the genbank database under accession number ef according to the ribosomal database project, the 16s rdna sequence of camponotus fellah endosymbiont correspond to an unclassified γ-proteobacteria closely related to 16rdna sequences from blochmannia endosymbionts bacteria of various camponotus ant species. this sequence has g+c content of 47% which is near to that of other blochmannia symbionts. when compared with the nucleic sequences of other blochmannia, maximum identity ranged from 91–93%. however, other blochmannia species present in genbank exhibit up to 98% of identity to each other. phylogenetic comparisons showed the existence of a monophyletic group containing classified and unclassified endosymbionts from camponotus ant species, closer to other insect endosymbionts and distinct from other outgroup bacteria. the use of fish with primers specific for eubacteria and blochmannia endosymbionts showed that bacteriocytes of midgut preparations were full of bacteria. in these preparations it was possible to see the individual bacterium and its rod form. the bacteriocytes were also detected in the oocytes by fish as well. effectiveness of antibiotic treatment the quantity of blochmannia in midgut bacteriocytes was estimated after rifampin treatment using two complementary methods: real-time quantitative pcr and fluorescent in situ hybridization. the two methods showed a reduction of blochmannia numbers in midgut bacteriocytes after 12-weeks of antibiotic treatment. within this period, fish did not detect the presence of blochmania in the bacteriocytes. however quantitative real-time pcr indicated that the bacteria were not completely eliminated as a low quantity of 16s rdna bacteria molecules can be detected in the midgut. treated and control groups differed significantly in their content of blochmannia measured as 16s rdna molecules. the treatment reduced the quantity of bacteria by 75%. moreover, the individual variation in bacteria amount was more constant in antibiotic treated colonies than in control colonies. evaluation of colony development each colony was composed of at least one larva, pupa or worker and queen. colonies composed only with the queen or colonies with a dying queen during the experiment were excluded. after seven months, seven control colonies and nine treated colonies were kept for further analysis. workers, larvae and pupae numbers were not significantly different during the first three months after the beginning of the experiments. after this time, untreated colonies displayed more accentuated larvae production and had a higher number of adult workers. pupae number varied significantly throughout the time of the experiment but no difference between treated and control colonies was observed. the variation in workers numbers was significatively different between treated and control colonies with untreated colonies having more workers. mean number of larvae, pupae and workers analysed by anova. significance levels are *p <, **p < and ***p ≤. amount of blochmannia endosymbiont versus encapsulation response when expressing encapsulation rate versus 16s rdna molecules amount, control and treated colonies displayed different patterns of immune response. we found a significant positive correlation between encapsulation rate and bacteria amount in the ants from control colonies: the bacteria did facilitate the encapsulation response. on the contrary, ants from treated colonies did not display a correlation between the amount of bacteria in the midgut and the encapsulation response. thus, it seems that antibiotic treatment eliminated the bacterial effects on the immune encapsulation response. an ancova analysis with the encapsulation rate as independent variable showed that treated workers present a significant increase in encapsulation rate. the regression inclinations of treated and control groups differed significantly. discussion and  in this study, we confirmed that blochmannia plays an important role for camponotus ants by improving the colony growth. we also demonstrated for the first time that blochmannia interacts with the ant immune defence. antibiotic treatment with rifampin considerably reduced the endosymbiont number in the midgut, although they were never totally eliminated and there was a great variability between workers. this may be due to different access to the antibiotic and some ants may not drink the antibiotic solution or, as observed by feldhaar et al., may be explained by the fact that dna of the endosymbiont may still be detectable by qrt-pcr when bacteria are not alive or active. additionally, it was confirmed that bacterial sequences were not integrated in the genome of the ant by a pcr test performed on ant dna from legs using blochmannia 16s rdna and ant 18s rdna primers. the treatment had a remarkable impact on colony development by reducing larvae production and worker numbers, corroborating previous worker. carrying out the studies in entire incipient colonies, we can demonstrate the importance of endosymbionts in this phase of colony development. according feldhaar et al., essential amino acids provided by endosymbionts improve workers ability to raise pupae. here, we have verified that control colonies exhibited a bigger population in the first seven months of colony development. since the establishment phase is critical for new colonies, harbouring more bacteria may have major ecological consequences in a context of inter and intraspecific competition: more workers confers a special advantage to maintain a young colony, occupy and monopolize food resources. indeed, animal protein food resources are more unpredictable in the time-space scale. blochmannia presence could signify a possible adaptation for ants to fluctuations in protein availability, permitting the colony growth even in absence of preys. we do not know the mechanisms allowing an increase in brood production, beyond the direct nutritional effects on treated queen, but several mechanisms are plausible, including a direct oogenesis control. for example, it has been demonstrated that wolbachia bacteria are necessary for the host oogenesis in a particular strain of the parasitic wasp asobara tabida. furthermore, it was evidenced that apoptosis prevention of nurse cells by wolbachia can regulate the host oogenesis. we have demonstrated that blochmannia play another important function by improving camponotus host immune system. the encapsulation rate measured in rifampin treated workers was significantly higher when compared with control colonies. although no evident toxic effect was observed, it is expected that antibiotic treatment has stressing effects on workers and that the increase of the encapsulation rate might correspond to an adaptive response to stress. furthermore, antibiotic treatment seemed to mask the effects of endosymbiont number on encapsulation response observed in control colonies, where the bacteria favoured the encapsulation response. positive effects of symbionts on host immune system have been described in the last years. for example, the facultative symbionts of acyrthosiphon pisum confer it resistance to parasitoid attacks. recently, it has been demonstrated that wolbachia confer vigorous antiviral protection to drosophila. the mechanisms by which the resistance is expressed is still unknown, but in another example it was showed that symbiotic bacteria could compete directly for space and resources and thus prevent host colonization by pathogens. encapsulation is the principal physiological response against parasitoids suggesting an important role of the stimulation induced by blochmannia in the protection against parasites. this strong interaction between symbiotic bacteria and ants may explain the persistence and broad occurrence of symbiotic bacteria in the camponotus genus. ants from camponotus genus are abundant almost everywhere in the world where ants are found, comprising more than described species within an estimated number greater than species. its large distribution, the diversity of forms and food behaviour and the occurrence on diverse environments make the system camponotus/blochmannia an interesting model to study how ecological forces determine symbiont characteristics and how bacteria determine the ant traits. for example, it is interesting to determine how genetic differences found among different species of blochmannia could be related to host ecological characteristics. the social habits of the ants make them particularly vulnerable to several parasites and parasitoids. phoridae flies are frequently found around camponotus nests and their influence is fundamental in regulating the ant communities. so, it can be expected that camponotus species more exposed to phoridae attack should harbour more bacteria. the physiological mechanism linking bacterial amount and encapsulation response remains unknown. although the better workers "quality" due to extra nutrients furnished by bacteria is the more probable explanation, direct production of biomolecules in stress situation should not be excluded. an efficient immune system is a major trait allowing the existence of social insect colonies with thousand of individuals, genetically related, living close together, constantly exposed to parasitic disease risks. competition in the first stages of colony growth constitutes also a great challenge to reach the reproductive stage. thus, blochmannia endosymbionts appear to be a fundamental partner, responsible for the ecological success of camponotus ants. as more than 10% of insect species depend on obligate bacterial mutualists for their viability and reproduction, the research on symbiosis between bacteria and animals appears to be a new and promising field, particularly in social insects. 
in this study, we establish links between blochmannia, growth of starting new colonies and the host immune response. the immune response of ants from control and treated colonies was estimated by inserting nylon filaments in the gaster and removing it after h. in the control colonies, the encapsulation response was positively correlated to the bacterial amount, while no correlation was observed in treated colonies. indeed, antibiotic treatment increased the encapsulation response of the workers, probably due to stress conditions. furthermore, the bacteria improve the workers encapsulation response. the increased growth rate observed in non-treated colonies confirms the importance of blochmannia in this phase of colony development. this would provide an important selective advantage during colony founding, where the colonies are faced with severe inter and intraspecific competition. incipient rifampin treated colonies had significantly lower numbers of brood and adult workers than control colonies. thus, these ants are likely to be less susceptible to various pathogen attacks, such as the phoridae fly parasitoids, normally found in the vicinity of camponotus nests. all carpenter ants studied hitherto harbour primary endosymbiotic bacteria of the blochmannia genus. efficiency of the treatment was estimated by quantitative polymerase chain reaction and fluorescent in situ hybridization, using blochmannia specific primers and two fluorescent probes.
 microorganisms are a large and diverse form of life. many of them live in association with large multicellular organisms, developing symbiotic relations with the host and some have even evolved to form obligate endosymbiosis. all carpenter ants studied hitherto harbour primary endosymbiotic bacteria of the blochmannia genus. the role of these bacteria in ant nutrition has been demonstrated but the omnivorous diet of these ants lead us to hypothesize that the bacteria might provide additional advantages to their host. in this study, we establish links between blochmannia, growth of starting new colonies and the host immune response.  we manipulated the number of bacterial endosymbionts in incipient laboratory-reared colonies of camponotus fellah by administrating doses of an antibiotic mixed in honey-solution. efficiency of the treatment was estimated by quantitative polymerase chain reaction and fluorescent in situ hybridization, using blochmannia specific primers and two fluorescent probes. very few or no bacteria could be detected in treated ants. incipient rifampin treated colonies had significantly lower numbers of brood and adult workers than control colonies. the immune response of ants from control and treated colonies was estimated by inserting nylon filaments in the gaster and removing it after h. in the control colonies, the encapsulation response was positively correlated to the bacterial amount, while no correlation was observed in treated colonies. indeed, antibiotic treatment increased the encapsulation response of the workers, probably due to stress conditions.  the increased growth rate observed in non-treated colonies confirms the importance of blochmannia in this phase of colony development. this would provide an important selective advantage during colony founding, where the colonies are faced with severe inter and intraspecific competition. furthermore, the bacteria improve the workers encapsulation response. thus, these ants are likely to be less susceptible to various pathogen attacks, such as the phoridae fly parasitoids, normally found in the vicinity of camponotus nests. these advantages might explain the remarkable ecological success of this ant genus, comprising more than species. 