BACKGROUND
substances produced by the male accessory glands  of insects and transferred into the female genital tract during mating are known to reduce pathogen transmission, to form mating plugs or spermatophores, and to be important in sperm competition.
in addition, they trigger fundamental changes in female physiology, behavior, and reproduction  <cit> .
the major biologically active components of mag secretions are carbohydrates, lipids, and in particular accessory gland proteins   <cit> .
in drosophila melanogaster, more than  <dig> acps have been identified  <cit> .
they play roles in the regulation of female receptivity  <cit> , defense against bacteria  <cit> , the activation of the female immune system  <cit> , the efficiency of sperm storage  <cit> , and the stimulation of ovulation and egg maturation  <cit> .
they are also decisive for the fertilization of eggs, as shown in experiments in which the activity of sperm of infertile mutant males lacking mags could be restored by subsequently re-mating females with males that provided acps but no sperm  <cit> .
in addition to stimulating immediate female fertility, mag products appear to negatively affect female lifespan  <cit> .
this reflects inter-sexual conflict  <cit> : the reproductive interests of males and females concerning the consequences of a mating act may differ, in particular when females later re-mate with other partners.
males prefer the female to maximize short-term investment into the offspring resulting from the present copulation, while females may benefit more from saving resources for later reproduction with other males.
sexual conflict may result in sexually antagonistic co-evolution and acps indeed appear to evolve very rapidly, as predicted from the ensuing arms race  <cit> .
compared to the wealth of information on accessory gland products of drosophilids, studies on other insect species are seriously lagging behind  <cit> .
this is particularly the case for eusocial hymenoptera, despite the special implications that their life style and evolutionary history have on sexual conflict.
in contrast to drosophila and other non-social insects, eusocial hymenoptera are characterized by life-long pair-bonding, even though the males frequently die after mating  <cit> .
investigations have shown that monogamy is the ancestral state of all truly eusocial organisms  <cit> , and lifetime monogamy seems to be a universal precondition for the evolution of obligate eusociality  <cit> .
the queens of ants, eusocial bees, and wasps indeed mate only once or a few times early in their adult lives without ever re-mating again.
sperm received during these matings is stored for months, years, or even decades, and utilized to fertilize eggs throughout the queens' lives.
furthermore, queens first rear large numbers of sterile workers from fertilized eggs before switching to the production of female sexuals.
hence, in contrast to males of non-social insects, eusocial insect males are expected to avoid harming the female, as this would reduce the total amount of female sexuals produced and, consequently, male reproductive success.
instead, males should have evolved mechanisms that promote female performance.
even where multiple mating has evolved after the monogamy window has been passed, competition among ejaculates  <cit>  is not expected to negatively affect female life span and future fecundity.
inter-sexual conflict might thus be replaced by "inter-sexual cooperation", though intra-sexual conflict might still evolve.
indeed, ant males appear to positively affect queen life span: regardless of being mated with a fertile or sterilized male, mated queens of cardiocondyla obscurior lived considerably longer than virgin queens  <cit> .
like in other insects, mag products of eusocial hymenoptera are involved in the formation of mating plugs  <cit>  and ejaculate  competition  <cit> .
in addition, they presumably elicit rapid changes in the physiology and behavior of freshly mated queens, e.g., the shedding of wings, reluctance of mating again, and changes in cuticular hydrocarbons  <cit> .
it is likely that mag products, and in particular acps, are also involved in the life-prolonging effects of mating  <cit> .
nevertheless, very little is known about acps in eusocial hymenoptera other than the honey bee, apis mellifera  <cit> .
we therefore examined, which genes are differentially expressed in the male accessory glands of the ant leptothorax gredleri.
virgin queens of l. gredleri attract males through "female calling", i.e., they climb up grass stems or similar, elevated positions near their maternal nests and release droplets of a sex pheromone from their poison glands  <cit> .
during mating, sperm is transferred within a gelatinous substance, functionally similar to a spermatophore.
the latter stays inside the female's genital tract for several hours, while the sperm is slowly transferred into the spermatheca  <cit> .
females have been observed to copulate with multiple males but it is unclear whether ejaculate is transferred during all these matings.
genetic analyses document single paternity of their offspring  <cit> .
in order to obtain information on gene products specifically produced by the accessory glands of l. gredleri males we generated a suppression subtractive library for transcripts using a representational difference analysis  protocol.
such an approach enriches tissue-specific transcripts  by removing from the library sequences that are shared with control tissue .
RESULTS
rda library characteristics and bioinformatic analyses
to detect differentially expressed genes in l. gredleri mags, we sequenced  <dig> clones from the subtracted gland library, resulting in  <dig> sequencing reads, of which  <dig> reads representing  <dig> different clones were of appropriate quality .
after quality analysis and following cap <dig> assembly we obtained  <dig> contigs and  <dig> singlets as unique sequences .
the sequences were submitted to genbank .
blastx analyses of the uss  revealed significant matches  for  <dig> uss.
the remaining  <dig> uss showed no significant matches to genes of other species.
shown are the predicted gene, the number of ests assembled to each us, us size in bp, the respective organism, followed by the respective similarity index  and corresponding gene ontology terms obtained from flybase http://flybase.bio.indiana.edu/.
most of the ests  clustered to a sequence corresponding to an apis mellifera predicted gene similar to cg1486-pa. based on domain predictions, the fly ortholog of this gene is annotated in flybase as being involved in carboxylic acid metabolism.
another us represented by  <dig> ests showed similarity to a heat shock factor binding protein 1-like from apis mellifera, with the corresponding cg5446-pa in drosophila melanogaster.
further uss were also formed by clustering considerable numbers of ests , such as us <dig> consisting of  <dig> ests, and us <dig> composed of  <dig> ests.
none of these transcripts, however, presented matches when submitted to blastx analysis.
in functional terms, the searches for orthologs revealed that, seven of the l. gredleri uss corresponded to functionally defined genes , with best hits to predicted a. mellifera proteins.
by judging from gene ontology attributes, two are expected to be involved in energy-dependent transport processes, such as the atp synthase gamma chain and nervana  <dig>  a sodium/potassium-transporting atpase subunit.
the leukocyte-antigen-related-like protein may function as a protein tyrosine phosphatase, while the kokopelli gene has a cyclin-dependent protein kinase regulator activity and is involved in male germ-line stem cell division.
the final two sequences with predicted molecular functions are cg6910-pa, with inositol oxygenase activity, and an apis mellifera ribosomal protein lp <dig>  given as being a structural constituent of ribosomes.
still unknown are biological processes or molecular functions for the five remaining sequences with blastx matches.
sjchgc <dig> and loc <dig> are both predicted proteins of the parasitic wasp nasonia vitripennis, and ciao- <dig> and cg32432-pa and heat shock factor binding protein 1-like are predicted proteins for a. mellifera.
neither of these had matches with drosophila genes.
quantitative rt-pcr analysis of rda library genes
the rda method is applicable to systems where genomic information is either completely lacking or still in an incipient stage.
rdas are able to detect both known and so far unknown transcripts, including regulatory rnas.
however, being a high throughput approach, transcripts indicated by rda as differentially expressed should be further analyzed by specific gene-expression analyses, such as quantitative rt-pcr .
thus, we designed gene-specific primers for  <dig> of the  <dig> rda products.
one product contained repeats, and so it was not considered suitable for primer design.
for six of the  <dig> products we obtained primers, shown in table  <dig>  that reliably amplified specific products and, thus, were suitable for qrt-pcr.
these were primers for the genes leukocyte-antigen-related-like  and cg <dig>  representing two genes with blastx matches and predicted functions, for ciao- <dig> and cg <dig>  being genes with matches but of unknown function, and the final two being for us <dig> and us <dig>  representing no-match sequences.
for five of the six genes we could show that their mean transcript levels are at least two-times higher in the l. gredleri mags relative to body carcasses, this holding for all three of the analyzed male life cycle stages, except for ciao- <dig> in the males running outside the nest that were sexually mature  samples .
for the sixth gene, us <dig>  overexpression was not validated.
this may, thus, represent false positive rda identification, represented by a single sequencing read .
when testing for statistical significance of differential expression by randomization tests built into rest software we could show significant overexpression in two genes.
cg <dig>  a gene with predicted carboxy-lyase activity, was not only represented by the highest number of ests , but was also confirmed as significantly overexpressed in mags of dark-pigmented male pupae  and sexually mature males .
the other gene, cg <dig>  was shown to be significantly overexpressed in sexually mature males  .
for all of these five genes biological variation in expression levels was considerable, a problem frequently encountered in qrt-pcr analyses, even with highly controlled cell cultures  <cit> .
discussion and
CONCLUSIONS
using the rda method we successfully identified a set of  <dig> genes as being differentially expressed in mags of the ant l. gredleri.
unfortunately, a fully sequenced ant genome is not yet available, so an annotation against a genome was not feasible.
such an annotation would be needed to identify proteins that are secreted into the lumen of l. gredleri mags, as their rnas should have hydrophobic amino-terminal signal sequences  <cit> .
for example, swanson et al.
<cit>  found that 24% of the  <dig> identified genes in their study of drosophila male reproductive proteins have such a putative signal sequence.
all best hit matches were against a. mellifera or n. vitripennis predicted genes, emphasizing the importance of these hymenopteran reference genomes in future gene expression studies in eusocial insects.
nevertheless, most of our ests represented functionally undefined genes or ones without matches to known genes.
at first sight, this might indicate rapid evolution and species specificity in mag secretion products, similar to what has been shown for mags of drosophila  <cit> , where about 50% of the drosophila acps have no homologs outside the drosophilid species complex.
when comparing the genes predicted to be differentially expressed in mags of l. gredleri with those identified in a proteome analysis of honey bee seminal fluid  <cit>  we did not find any matching gene products for the set with geneontology functional attributes.
this finding would also be in favor of a rapid evolution hypothesis for mag products.
queens of leptothorax are predominantly singly-mated  <cit> , but occasional multiple mating might introduce intra-sexual conflict and thus increase the evolutionary tempo of seminal fluid proteins.
in eusocial species with multiple mating, the beneficial effect of seminal fluid on sperm survival is reduced when seminal fluid is mixed with alien sperm  <cit> .
however, this may actually serve the interests of the queen rather than inflicting damage.
furthermore, the queen appears to be in control of ejaculate competition, since the negative impact of seminal fluid on foreign sperm can be neutralized by secretions from the queen sperm-storage organ  <cit> .
in this case, intra-sexual conflict seems to be controlled by inter-sexual cooperation.
concerning our studies, nothing is as yet known on the molecular nature of mag secretions of other ant species, and with apis mellifera and nasonia vitripennis being the closest species for genomic comparisons, the percentage of functionally undefined genes and of no-match ests might simply reflect the large phylogenetic distance separating ants, bees, and parasitic wasps.
speculating about the speed of the evolution of l. gredleri acps would, at this point, be premature, especially since, 2d-electrophoreses of acps suggest that they are conserved in l. gredleri and related taxa relative to, e.g., muscle proteins .
furthermore, not all seminal fluid genes can be subject to rapid evolution, because successful reproduction must be assured.
in a cross-species comparison of drosophila mag protein encoding genes, mueller et al.
<cit>  found not only rapidly evolving but also conserved genes, and functionally similar proteins may be represented and relevant in the seminal fluid of other animals  <cit> .
members of protein classes conserved across organisms in seminal fluids may reflect their general importance in reproductive physiology  <cit>  and might therefore be beneficial for both males and females.
in this context, immune defense proteins are of special interest.
mags secrete proteins of antimicrobial activity  <cit>  and proteins, which regulate the expression of genes with antimicrobial activity in females after sperm transfer  <cit> .
such acps might successively protect sperm from microbial attack in the reproductive tracts of both sexes, as well as coat eggs when they are laid.
a l. gredleri est, whose ortholog cg <dig> is known to be upregulated in d. melanogaster females after mating  <cit> , might play such a role.
the drosophila gene is annotated as possibly showing oxidoreductase activity and being downregulated after bacterial infection.
for the elucidation of its function in ants, it should be of interest to see whether the gene similar to cg <dig> is also differentially expressed in virgin and mated l. gredleri queens.
females are not 'silent partners' when it comes to seminal fluids and it is always an interplay of male and female factors that coordinates the gametes for fertilization  <cit> .
the cg <dig> protein predicted in the honey bee genome as a drosophila ortholog with undefined function was found to be significantly overexpressed in sexually mature l. gredleri males, and one gene similar to cg1486-pa was found to be overexpressed in two male life cycle stages.
this latter protein is predicted to have a carboxy-lyase activity and might be involved in carboxylic acid metabolism, catalyzing the non-hydrolytic addition or removal of a carboxyl group to or from a compound.
carboxylic acids are widespread in nature, giving rise to fatty acids.
fatty acids, such as palmitic-, linoleic-, oleic-, and stearic-acids, have been described as components of the mating plug in the bumblebee bombus terrestris, which form a physical barrier  <cit>  and prevent re-mating  <cit> .
mating plugs were also found in fire ants  <cit> , queenless dinoponera ants  <cit> , attine ants  <cit> , stingless bees  <cit> , and in the honey bee.
in the latter, however, they do not play a function as a plug, but rather as a mating sign that may even promote multiple matings  <cit> .
in l. gredleri, the existence of a spermatophore that could be comparable to a mating plug was already shown  <cit> .
the overexpression of a gene with carboxy-lyase activity in the late pupal stage, when the accessory glands are already formed, and in sexually mature males found outside the nest supports the idea that accessory gland products are crucial for spermatophore formation in this species.
taken together, our current study represents a first step addressing at a molecular level the reproductive interplay between l. gredleri males and their mates.
mag products identified in this study may play different roles, either as conserved or as species-specific proteins.
understanding the molecular underpinnings of differences in reproductive strategies among non-social and eusocial species, and in the latter among monogamous  and polygamous ones  will still require sequencing of mag products in a range of species.
nevertheless, we believe that at this interface of "molecular communication" between the sexes, much can be gained towards comprehending the evolution of eusociality at the passage through the monogamy window  <cit> , with inter-sexual conflict being replaced by inter-sexual cooperation.

 substances produced by the male accessory glands of insects and transferred into the female genital tract during mating are known to reduce pathogen transmission, to form mating plugs or spermatophores, and to be important in sperm competition. in addition, they trigger fundamental changes in female physiology, behavior, and reproduction. the major biologically active components of mag secretions are carbohydrates, lipids, and in particular accessory gland proteins. in drosophila melanogaster, more than acps have been identified. they play roles in the regulation of female receptivity, defense against bacteria, the activation of the female immune system, the efficiency of sperm storage, and the stimulation of ovulation and egg maturation. they are also decisive for the fertilization of eggs, as shown in experiments in which the activity of sperm of infertile mutant males lacking mags could be restored by subsequently re-mating females with males that provided acps but no sperm. in addition to stimulating immediate female fertility, mag products appear to negatively affect female lifespan. this reflects inter-sexual conflict: the reproductive interests of males and females concerning the consequences of a mating act may differ, in particular when females later re-mate with other partners. males prefer the female to maximize short-term investment into the offspring resulting from the present copulation, while females may benefit more from saving resources for later reproduction with other males. sexual conflict may result in sexually antagonistic co-evolution and acps indeed appear to evolve very rapidly, as predicted from the ensuing arms race. compared to the wealth of information on accessory gland products of drosophilids, studies on other insect species are seriously lagging behind. this is particularly the case for eusocial hymenoptera, despite the special implications that their life style and evolutionary history have on sexual conflict. in contrast to drosophila and other non-social insects, eusocial hymenoptera are characterized by life-long pair-bonding, even though the males frequently die after mating. investigations have shown that monogamy is the ancestral state of all truly eusocial organisms, and lifetime monogamy seems to be a universal precondition for the evolution of obligate eusociality. the queens of ants, eusocial bees, and wasps indeed mate only once or a few times early in their adult lives without ever re-mating again. sperm received during these matings is stored for months, years, or even decades, and utilized to fertilize eggs throughout the queens' lives. furthermore, queens first rear large numbers of sterile workers from fertilized eggs before switching to the production of female sexuals. hence, in contrast to males of non-social insects, eusocial insect males are expected to avoid harming the female, as this would reduce the total amount of female sexuals produced and, consequently, male reproductive success. instead, males should have evolved mechanisms that promote female performance. even where multiple mating has evolved after the monogamy window has been passed, competition among ejaculates is not expected to negatively affect female life span and future fecundity. inter-sexual conflict might thus be replaced by "inter-sexual cooperation", though intra-sexual conflict might still evolve. indeed, ant males appear to positively affect queen life span: regardless of being mated with a fertile or sterilized male, mated queens of cardiocondyla obscurior lived considerably longer than virgin queens. like in other insects, mag products of eusocial hymenoptera are involved in the formation of mating plugs and ejaculate competition. in addition, they presumably elicit rapid changes in the physiology and behavior of freshly mated queens, e.g., the shedding of wings, reluctance of mating again, and changes in cuticular hydrocarbons. it is likely that mag products, and in particular acps, are also involved in the life-prolonging effects of mating. nevertheless, very little is known about acps in eusocial hymenoptera other than the honey bee, apis mellifera. we therefore examined, which genes are differentially expressed in the male accessory glands of the ant leptothorax gredleri. virgin queens of l. gredleri attract males through "female calling", i.e., they climb up grass stems or similar, elevated positions near their maternal nests and release droplets of a sex pheromone from their poison glands. during mating, sperm is transferred within a gelatinous substance, functionally similar to a spermatophore. the latter stays inside the female's genital tract for several hours, while the sperm is slowly transferred into the spermatheca. females have been observed to copulate with multiple males but it is unclear whether ejaculate is transferred during all these matings. genetic analyses document single paternity of their offspring. in order to obtain information on gene products specifically produced by the accessory glands of l. gredleri males we generated a suppression subtractive library for transcripts using a representational difference analysis protocol. such an approach enriches tissue-specific transcripts by removing from the library sequences that are shared with control tissue.  rda library characteristics and bioinformatic analyses to detect differentially expressed genes in l. gredleri mags, we sequenced clones from the subtracted gland library, resulting in sequencing reads, of which reads representing different clones were of appropriate quality. after quality analysis and following cap assembly we obtained contigs and singlets as unique sequences. the sequences were submitted to genbank. blastx analyses of the uss revealed significant matches for uss. the remaining uss showed no significant matches to genes of other species. shown are the predicted gene, the number of ests assembled to each us, us size in bp, the respective organism, followed by the respective similarity index and corresponding gene ontology terms obtained from flybase http://flybase.bio.indiana.edu/. most of the ests clustered to a sequence corresponding to an apis mellifera predicted gene similar to cg1486-pa. based on domain predictions, the fly ortholog of this gene is annotated in flybase as being involved in carboxylic acid metabolism. another us represented by ests showed similarity to a heat shock factor binding protein 1-like from apis mellifera, with the corresponding cg5446-pa in drosophila melanogaster. further uss were also formed by clustering considerable numbers of ests, such as us consisting of ests, and us composed of ests. none of these transcripts, however, presented matches when submitted to blastx analysis. in functional terms, the searches for orthologs revealed that, seven of the l. gredleri uss corresponded to functionally defined genes, with best hits to predicted a. mellifera proteins. by judging from gene ontology attributes, two are expected to be involved in energy-dependent transport processes, such as the atp synthase gamma chain and nervana a sodium/potassium-transporting atpase subunit. the leukocyte-antigen-related-like protein may function as a protein tyrosine phosphatase, while the kokopelli gene has a cyclin-dependent protein kinase regulator activity and is involved in male germ-line stem cell division. the final two sequences with predicted molecular functions are cg6910-pa, with inositol oxygenase activity, and an apis mellifera ribosomal protein lp given as being a structural constituent of ribosomes. still unknown are biological processes or molecular functions for the five remaining sequences with blastx matches. sjchgc and loc are both predicted proteins of the parasitic wasp nasonia vitripennis, and ciao- and cg32432-pa and heat shock factor binding protein 1-like are predicted proteins for a. mellifera. neither of these had matches with drosophila genes. quantitative rt-pcr analysis of rda library genes the rda method is applicable to systems where genomic information is either completely lacking or still in an incipient stage. rdas are able to detect both known and so far unknown transcripts, including regulatory rnas. however, being a high throughput approach, transcripts indicated by rda as differentially expressed should be further analyzed by specific gene-expression analyses, such as quantitative rt-pcr. thus, we designed gene-specific primers for of the rda products. one product contained repeats, and so it was not considered suitable for primer design. for six of the products we obtained primers, shown in table that reliably amplified specific products and, thus, were suitable for qrt-pcr. these were primers for the genes leukocyte-antigen-related-like and cg representing two genes with blastx matches and predicted functions, for ciao- and cg being genes with matches but of unknown function, and the final two being for us and us representing no-match sequences. for five of the six genes we could show that their mean transcript levels are at least two-times higher in the l. gredleri mags relative to body carcasses, this holding for all three of the analyzed male life cycle stages, except for ciao- in the males running outside the nest that were sexually mature samples. for the sixth gene, us overexpression was not validated. this may, thus, represent false positive rda identification, represented by a single sequencing read. when testing for statistical significance of differential expression by randomization tests built into rest software we could show significant overexpression in two genes. cg a gene with predicted carboxy-lyase activity, was not only represented by the highest number of ests, but was also confirmed as significantly overexpressed in mags of dark-pigmented male pupae and sexually mature males. the other gene, cg was shown to be significantly overexpressed in sexually mature males. for all of these five genes biological variation in expression levels was considerable, a problem frequently encountered in qrt-pcr analyses, even with highly controlled cell cultures. discussion and  using the rda method we successfully identified a set of genes as being differentially expressed in mags of the ant l. gredleri. unfortunately, a fully sequenced ant genome is not yet available, so an annotation against a genome was not feasible. such an annotation would be needed to identify proteins that are secreted into the lumen of l. gredleri mags, as their rnas should have hydrophobic amino-terminal signal sequences. for example, swanson et al. found that 24% of the identified genes in their study of drosophila male reproductive proteins have such a putative signal sequence. all best hit matches were against a. mellifera or n. vitripennis predicted genes, emphasizing the importance of these hymenopteran reference genomes in future gene expression studies in eusocial insects. nevertheless, most of our ests represented functionally undefined genes or ones without matches to known genes. at first sight, this might indicate rapid evolution and species specificity in mag secretion products, similar to what has been shown for mags of drosophila, where about 50% of the drosophila acps have no homologs outside the drosophilid species complex. when comparing the genes predicted to be differentially expressed in mags of l. gredleri with those identified in a proteome analysis of honey bee seminal fluid we did not find any matching gene products for the set with geneontology functional attributes. this finding would also be in favor of a rapid evolution hypothesis for mag products. queens of leptothorax are predominantly singly-mated, but occasional multiple mating might introduce intra-sexual conflict and thus increase the evolutionary tempo of seminal fluid proteins. in eusocial species with multiple mating, the beneficial effect of seminal fluid on sperm survival is reduced when seminal fluid is mixed with alien sperm. however, this may actually serve the interests of the queen rather than inflicting damage. furthermore, the queen appears to be in control of ejaculate competition, since the negative impact of seminal fluid on foreign sperm can be neutralized by secretions from the queen sperm-storage organ. in this case, intra-sexual conflict seems to be controlled by inter-sexual cooperation. concerning our studies, nothing is as yet known on the molecular nature of mag secretions of other ant species, and with apis mellifera and nasonia vitripennis being the closest species for genomic comparisons, the percentage of functionally undefined genes and of no-match ests might simply reflect the large phylogenetic distance separating ants, bees, and parasitic wasps. speculating about the speed of the evolution of l. gredleri acps would, at this point, be premature, especially since, 2d-electrophoreses of acps suggest that they are conserved in l. gredleri and related taxa relative to, e.g., muscle proteins. furthermore, not all seminal fluid genes can be subject to rapid evolution, because successful reproduction must be assured. in a cross-species comparison of drosophila mag protein encoding genes, mueller et al. found not only rapidly evolving but also conserved genes, and functionally similar proteins may be represented and relevant in the seminal fluid of other animals. members of protein classes conserved across organisms in seminal fluids may reflect their general importance in reproductive physiology and might therefore be beneficial for both males and females. in this context, immune defense proteins are of special interest. mags secrete proteins of antimicrobial activity and proteins, which regulate the expression of genes with antimicrobial activity in females after sperm transfer. such acps might successively protect sperm from microbial attack in the reproductive tracts of both sexes, as well as coat eggs when they are laid. a l. gredleri est, whose ortholog cg is known to be upregulated in d. melanogaster females after mating, might play such a role. the drosophila gene is annotated as possibly showing oxidoreductase activity and being downregulated after bacterial infection. for the elucidation of its function in ants, it should be of interest to see whether the gene similar to cg is also differentially expressed in virgin and mated l. gredleri queens. females are not'silent partners' when it comes to seminal fluids and it is always an interplay of male and female factors that coordinates the gametes for fertilization. the cg protein predicted in the honey bee genome as a drosophila ortholog with undefined function was found to be significantly overexpressed in sexually mature l. gredleri males, and one gene similar to cg1486-pa was found to be overexpressed in two male life cycle stages. this latter protein is predicted to have a carboxy-lyase activity and might be involved in carboxylic acid metabolism, catalyzing the non-hydrolytic addition or removal of a carboxyl group to or from a compound. carboxylic acids are widespread in nature, giving rise to fatty acids. fatty acids, such as palmitic-, linoleic-, oleic-, and stearic-acids, have been described as components of the mating plug in the bumblebee bombus terrestris, which form a physical barrier and prevent re-mating. mating plugs were also found in fire ants, queenless dinoponera ants, attine ants, stingless bees, and in the honey bee. in the latter, however, they do not play a function as a plug, but rather as a mating sign that may even promote multiple matings. in l. gredleri, the existence of a spermatophore that could be comparable to a mating plug was already shown. the overexpression of a gene with carboxy-lyase activity in the late pupal stage, when the accessory glands are already formed, and in sexually mature males found outside the nest supports the idea that accessory gland products are crucial for spermatophore formation in this species. taken together, our current study represents a first step addressing at a molecular level the reproductive interplay between l. gredleri males and their mates. mag products identified in this study may play different roles, either as conserved or as species-specific proteins. understanding the molecular underpinnings of differences in reproductive strategies among non-social and eusocial species, and in the latter among monogamous and polygamous ones will still require sequencing of mag products in a range of species. nevertheless, we believe that at this interface of "molecular communication" between the sexes, much can be gained towards comprehending the evolution of eusociality at the passage through the monogamy window, with inter-sexual conflict being replaced by inter-sexual cooperation. 
a gene with carboxy-lyase activity and one of unpredicted function were significantly overexpressed in accessory glands of sexually mature males. in this study, we investigated genes, which are preferentially expressed in male accessory glands of the ant leptothorax gredleri, to determine which proteins might be transferred in the seminal fluid. our findings indicate that male accessory glands of l. gredleri express a series of genes that are unique to this species, possibly representing novel genes, in addition to conserved ones for which functions can be predicted. identifying differentially expressed genes might help to better understand molecular mechanisms involved in reproductive processes in eusocial hymenoptera. twelve had mutual best matches with genes predicted for apis mellifera and nasonia vitripennis. during mating, insect males eject accessory gland proteins into the female genital tract. indeed, mating appears to increase female longevity in at least one ant species. while the novel genes could account for rapidly evolving ones driven by intra-sexual conflict between males, conserved genes imply that rather beneficial traits might get fixed by a process described as inter-sexual cooperation between males and females. by a suppression subtractive hybridization protocol we obtained unique sequences. six genes were further analyzed by quantitative rt-pcr in three life cycle stages of male ants. whereas sexual conflict abounds in non-social species, the peculiar life history of social insects with lifelong pair-bonding and no re-mating aligns the reproductive interests of the sexes.
 during mating, insect males eject accessory gland proteins into the female genital tract. these substances are known to affect female post-mating behavior and physiology. in addition, they may harm the female, e.g., in reducing its lifespan. this is interpreted as a consequence of sexual antagonistic co-evolution. whereas sexual conflict abounds in non-social species, the peculiar life history of social insects with lifelong pair-bonding and no re-mating aligns the reproductive interests of the sexes. harming the female during mating would negatively affect male fitness and sexual antagonism is therefore not expected. indeed, mating appears to increase female longevity in at least one ant species. acps are presumed to play a role in this phenomenon, but the underlying mechanisms are unknown. in this study, we investigated genes, which are preferentially expressed in male accessory glands of the ant leptothorax gredleri, to determine which proteins might be transferred in the seminal fluid.  by a suppression subtractive hybridization protocol we obtained unique sequences. twelve had mutual best matches with genes predicted for apis mellifera and nasonia vitripennis. functional information was available only for seven of these, including intracellular signaling, energy-dependent transport and metabolic enzyme activities. the remaining eight uss did not match sequences from other species. six genes were further analyzed by quantitative rt-pcr in three life cycle stages of male ants. a gene with carboxy-lyase activity and one of unpredicted function were significantly overexpressed in accessory glands of sexually mature males.  our study is the first one to investigate differential gene expression in ants in a context related to mating. our findings indicate that male accessory glands of l. gredleri express a series of genes that are unique to this species, possibly representing novel genes, in addition to conserved ones for which functions can be predicted. identifying differentially expressed genes might help to better understand molecular mechanisms involved in reproductive processes in eusocial hymenoptera. while the novel genes could account for rapidly evolving ones driven by intra-sexual conflict between males, conserved genes imply that rather beneficial traits might get fixed by a process described as inter-sexual cooperation between males and females. 