BACKGROUND
the ixodid ticks rhipicephalus appendiculatus and rhipicephalus  microplus are important pests of livestock.
besides causing direct production losses and leather damage due to their blood-feeding habit, both ticks are able to transmit a wide variety of pathogens.
both tick species overlap in their distribution, but r. microplus is more widespread and occurs in subtropical and tropical areas of the world whereas the distribution of r. appendiculatus, also known as the brown ear tick, is limited to areas with a humid climate from southern sudan to the southeastern coast of south africa.
their life cycle differs quite dramatically too: r. microplus is a one-host tick species with all life stages feeding on the same, usually bovine, host whereas r. appendiculatus is a three-host tick species with each life stage requiring a new host to feed on.
as a consequence of this, r. microplus can complete its life cycle in less than  <dig> months, whereas r. appendiculatus takes about  <dig> months to complete its life cycle under the most favorable conditions  <cit> .
control of ticks worldwide relies principally on the use of acaricides, but two vaccines targeting r. microplus were commercialized in the 1990s: tickgard plus® in australia and gavac® in cuba.
both are based on the same recombinant antigen named bm <dig>  a glycoprotein of unknown function which is located predominantly on the surface of midgut digest cells  <cit> .
although bm86-based vaccines showed cross-protection against various other tick species, e.g.
rhipicephalus  annulatus  <cit> , rhipicephalus  decoloratus, hyalomma anatolicum and hyalomma dromedarii  <cit> , they were not effective against amblyomma variegatum and r. appendiculatus  <cit> .
due to the veterinary and economical importance of r. microplus and r. appendiculatus in subtropical and tropical areas of the world, expressed sequence tag  datasets for these tick species have been established  <cit> .
the availability of these data greatly facilitates research in tick biology and tick-host-pathogen interactions.
microarrays and quantitative rt-pcr are two important techniques measuring gene expression which may help in unraveling such interactions and provide insight into the complex regulatory networks behind biological processes.
output data require normalization to control for variables such as the intrinsic variability of rna, impurities during rna extraction, reverse transcription and pcr efficiencies  <cit> .
a frequently used method for the accurate normalization of quantitative rt-pcr data involves the measurement of internal reference genes .
such genes should ideally have a stable expression independent of cell or tissue type, or experimental condition.
a survey of  <dig> papers using quantitative rt-pcr in tick research published between  <dig> and  <dig> shows β-actin as the most popular reference gene used for normalization in  <dig> publications, with the one remaining article employing the 18s rrna gene.
however, the presumed expression stability of these genes in ticks has never been examined and the use of a single reference gene may lead to erroneous normalization  <cit> .
in this study, the mrna transcript levels of nine commonly used reference genes from different functional classes: β-actin , β-tubulin , elongation factor 1α , glyceraldehyde 3-phosphate dehydrogenase , glutathione s-transferase , h <dig> histone family 3a , cyclophilin , ribosomal protein l <dig>  and tata box binding protein  were measured by quantitative rt-pcr in all life stages of whole r. microplus and r. appendiculatus ticks.
the results were evaluated using genorm  <cit>  and normfinder  <cit> .
although both programs have the same aim of identifying the most stably expressed reference genes, they make use of different strategies.
genorm is a software application which determines the expression stability of reference genes by calculating a gene-stability measure  for each gene.
this measure relies on the principle that the expression ratio of two ideal reference genes is identical in all samples, regardless of the experimental condition or cell type.
pairwise variation for each combination of reference genes is determined and assigned a value for m, and genes with the highest m value  are progressively eliminated until the two most stably expressed genes remain.
it thus ranks the reference genes according to the similarity in expression profiles across the samples  <cit> .
incorporation of co-regulated reference genes will affect the outcome of this approach and care must therefore be taken in selecting candidate reference genes from different functional classes.
normfinder is an application on a model-based approach which ranks the reference genes according to the estimated intra- and intergroup expression variation.
normalization with the six most stable expressed reference genes of the bm <dig> mrna transcript levels in all life stages of r. microplus and r. appendiculatus was carried out with the aim to elucidate the role of antigen abundance in bm <dig> vaccine susceptibility.
RESULTS
quantitative rt-pcr
the efficiencies of the quantitative rt-pcr's were uniformly high and ranged from 91% to 103%, making all assays suitable for quantitative analysis .
all pcr's generated a single band and the absence of primer dimer formation was confirmed by a dissociation assay performed with each assay .
none of the primer combinations amplified cdna synthesized from bovine blood rna, which excludes interference with the pcr results caused by the possible presence of host rna in fed ticks.
raw ct values ranged from  <dig>   to  <dig>   in r. microplus and from  <dig>   to  <dig>   in r. appendiculatus .
gapdh, gst and tbp were expressed at low levels in both tick species with median ct values above  <dig> cycles.
the smallest ct variation between all samples of r. microplus was exhibited by tbp  and by gapdh  in r. appendiculatus.
gst showed the most variable expression between all samples for both tick species;  <dig>  in r. microplus and  <dig>  in r. appendiculatus.
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arm, r. microplus; ra, r. appendiculatus
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arm, r. microplus; ra, r. appendiculatus
genorm and normfinder analysis
the gene expression stability of nine candidate reference genes over the life cycle of r. microplus and r. appendiculatus was analyzed using the genorm and normfinder software applications .
the genorm approach identified elf1a and rpl <dig> as the best pair of reference genes over the life cycle of both r. microplus and r. appendiculatus, as well as in a combined analysis of all samples from both species.
normfinder ranked these genes as second and third best in r. microplus and r. appendiculatus with tbp and gapdh being indicated as the best reference gene, respectively.
a combined analysis of both species by normfinder ranked elf1a as best reference gene followed by gapdh, tbp, ppia, rpl <dig>  h3f3a, btub, actb and gst.
gst and actb were identified as the least stable genes in all groups by both methods.
r. microplus
r. appendiculatus
the candidates are listed with decreasing expression stability from top to bottom.
average expression stability values are shown between parentheses.
to determine the minimum number of reference genes necessary for accurate normalization, calculation of the pairwise variation  was performed by genorm.
the lowest v values were found to be  <dig>  for v5/ <dig> in r. microplus and  <dig>  for v7/ <dig> in r. appendiculatus.
combined analysis of both tick species yielded a lowest v value of  <dig>  at v6/ <dig>
bm <dig> and ra <dig> sequence analysis
one sequence for bm <dig>  and two sequences from the bm <dig> homologue of r. appendiculatus, ra86- <dig> and ra86- <dig>  were obtained by 3'race with degenerate primer ra86-f, which is located one amino acid downstream of the signal peptide of bm <dig>  the open reading frame  of the bm <dig>  nucleotide sequence is  <dig> bp, coding for a protein of  <dig> amino acids which is  <dig> % identical to the bm <dig> yeerongpilly reference strain  with similar structural properties.
the  <dig> amino acid gap reported previously in a second bm <dig>  sequence  was not detected in any of the five sequenced clones.
both r. appendiculatus sequences contain a  <dig> bp-long orf which encodes for  <dig> amino acids.
the alleles differ by  <dig> nucleotides of which  <dig> are silent mutations and  <dig> result in a change in the deduced amino acid sequence of the protein .
the identity of the amino acid sequence of ra86- <dig> and ra86- <dig> are  <dig> % and  <dig> % with the bm <dig> proteins of r. microplus  and  <dig> % and  <dig> % with r. microplus  respectively.
a comparison between the amino acid sequence of ra <dig> and bm <dig> shows that ra <dig> contains the same epidermal growth factor -like domains as bm <dig>  <cit> .
these domains are also present in ba <dig>  bd <dig> and haa <dig>  the bm <dig> homologues of r. annulatus, r. decoloratus and hy.
a. anatolicum respectively .
the ra <dig> sequences contain  <dig> potential sites for n-linked glycosylation  and ra86- <dig> has  <dig> potential o-glycosylation site .
both ra86- <dig> and ra86- <dig> are predicted to contain a glycosylphosphatidylinositol  modification site at position  <dig> , which provides linkage of the molecule to the cell membrane.
the presence of a gpi anchor is a common feature found in all ixodid tick bm <dig> homologues characterized thus far.
western blot analysis showed that ovine bm <dig> antiserum recognized bands of the expected ra <dig> protein size in the isolated midguts from partially fed r. appendiculatus females whereas serum from a sheep vaccinated with adjuvant only did not .
bm86/ra <dig> expression analysis
the expression profile of bm86/ra <dig> mrna  in both r. microplus and r. appendiculatus was obtained by normalizing its expression with six reference genes that ranked highest in the genorm and normfinder analysis of the combined r. microplus and r. appendiculatus samples: elf1a, gapdh, h3f3a, ppia, rpl <dig> and tbp .
in eggs of r. microplus, bm <dig> expression was detected at low levels in eggs  <dig> and  <dig> days after initiation of the oviposition  and increased by three-fold in eggs collected  <dig> days p.o.
this formed the start of a rapid increase in the expression of bm <dig> in the third trimester of the embryogenesis to levels similar to that found in unfed larvae.
bm <dig> expression decreased with feeding and molting in the immature life stages, with the lowest expression found in the pharate life stages.
the decrease of bm <dig> expression levels following feeding of immatures was significantly more pronounced in the larvae and nymphs of r. appendiculatus compared to r. microplus where a more continuous expression pattern was observed during the life cycle with less dramatic variation.
the expression level of bm <dig> in adults did not differ significantly between males and females of both species.
discussion
to minimize rt-pcr specific errors and correct for sample-to-sample variation in order to make a comparison of the bm <dig> expression profiles from r. microplus and r. appendiculatus possible, appropriate normalization is required.
the use of reference genes is most frequently applied to normalize the mrna fraction, but validation of the expression stability of such genes in ticks has not been reported until now.
actb is the most commonly used reference gene in tick research, but recent findings in mammals revealed that this gene and other commonly used 'classical' reference genes such as gapdh may be inappropriate for use as a reference gene because of their variability under experimental conditions  <cit> .
the ideal reference gene should be expressed at a constant level in the tissue of interest at all stages of development and be unaffected by the specific experimental treatment being examined.
however, no such universal reference gene has yet been identified and probably does not exist  <cit> .
normalization with multiple selected reference genes has been proposed as an alternative to overcome this problem and several tools to evaluate the expression stability of candidate reference genes have been developed.
in this study, two of these tools, the genorm  <cit>  and normfinder  <cit>  programs, were employed to evaluate the expression stability of nine selected candidate reference genes.
besides the two 'classical' reference genes actb and gapdh, other candidate reference genes evaluated in this study were from different functional classes and were selected based on their reported expression stability in other organisms and their presence in the est libraries of r. microplus and r. appendiculatus  <cit> .
rpl <dig> for instance was among the thirteen ribosomal proteins which were ranked in the top  <dig> of most stable expressed reference genes in a meta-analysis performed on a large dataset of human gene arrays  <cit> .
other ribosomal proteins were not included in this study to prevent bias in the ranking of the reference genes due to correlated expression of proteins belonging to the same functional class.
the outcome of the gene stability evaluation differed between the programs used, which is not surprising in light of the different algorithms they employ.
only elf1a was consistently ranked first or second by both programs and is suitable for use as a reference gene under the conditions described here.
rpl <dig> is consistently ranked as the most stable expressed gene together with elf1a by genorm but not by normfinder.
since both elf1a and rpl <dig> play a role in protein translation, co-regulation cannot be ruled out and this may have affected the outcome of the genorm analysis.
normfinder is less sensitive to the incorporation of co-regulated genes since it focuses on the intra- and intergroup variation in selecting the most stable expressed genes  <cit> .
this may altogether explain the discordance in ranking of rpl <dig> between the genorm and normfinder programs.
gst turned out to be the least stable gene in all conducted analyses .
gst is known to be differentially expressed under different conditions  <cit>  and so could be expected to perform poorly as a reference gene.
of the 'traditional' reference genes, actb was ranked among the least stable genes by both methods whereas gapdh was ranked first in the normfinder analysis of the r. appendiculatus life stages.
this is a direct result of the small ct variation observed in the gapdh expression in this species, which also explains the high ranking of tbp in the normfinder analysis of the r. microplus life stages where this gene showed the least ct variation over all samples.
analysis of the pairwise variation  of the samples by genorm returned values slightly lower or higher than the arbitrarily chosen threshold of  <dig>   <cit> , reflecting the heterogeneous nature of the analyzed whole tick samples which varied from egg to feeding adults.
a direct consequence is the need of using a larger number of reference genes for optimal normalization.
to be able to compare the bm <dig> mrna expression between all life stages of r. microplus and r. appendiculatus, normalization with six reference genes: elf1a, rpl <dig>  tbp, h3f3a, ppia and gapdh was conducted.
these reference genes were evaluated as being the most stable by both genorm and normfinder in a combined analysis of all samples from both tick species and returned the lowest pairwise variation value in the genorm analysis .
the protein sequence of the bm <dig> gene from the mozambique r. microplus strain was highly identical to previously reported bm <dig> sequences from australia and south america and shared a maximum identity of  <dig> % with the bm <dig> sequence from a r. microplus strain from central brazil .
a second bm <dig> sequence isolated from the viscera of partially fed females originating from the same mozambique tick colony  contains a  <dig> amino acid gap which was not present in the bm <dig> sequence identified in this study  <cit> .
this sequence was not found in any of the five sequenced clones and may be less abundant, have been the result of alternative splicing or represent an allele which was lost in the r. microplus tick population since the source material was collected several generations earlier.
the presence of bm <dig> alleles within the same tick population has been previously reported  <cit> .
two alleles, ra86- <dig> and ra86- <dig>  were also found to be transcribed in the midgut of r. appendiculatus females.
the orfs were similar in size but the alleles differed by  <dig> amino acids.
these differences do not appear to have a striking effect on the main properties of these proteins since both ra86- <dig> and ra86- <dig> are predicted to contain a gpi-anchor and contain egf-like domains similar to those found in bm <dig> , but ra86- <dig> does have a single potential o-glycosylation site which is absent from ra86- <dig>  since boophilus species were recently synonymized with the rhipicephalus genus  <cit> , it is not surprising that the ra <dig> protein shows a higher amino acid sequence identity with bm <dig>  compared to the haa <dig> protein, the bm <dig> homologue from the two-host tick hy.
anatolicum .
however, feeding of hy.
anatolicum on cattle vaccinated with a recombinant bm <dig> vaccine does result in a deleterious effect against this tick species which is not seen in r. appendiculatus  <cit> .
since western blot analysis showed that ovine bm <dig> antisera does indeed recognize r. appendiculatus proteins , other biological factors such as conformational epitopes, amount of blood/antibodies ingested, or antigen abundance may play a role in the biology of bm <dig> vaccine susceptibility.
to investigate the latter hypothesis, the bm <dig> and ra <dig> transcript levels were measured throughout the life cycle of both tick species feeding on the same host by quantitative rt-pcr using a single primer pair which amplifies all known alleles of bm <dig> and ra <dig>
the normalized bm <dig> mrna expression levels were monitored in various stages of embryonic development in r. microplus and were found to increase exponentially during the last  <dig> days prior to hatching, simultaneous with the development of the midgut in embryos which takes place in the third trimester of embryogenesis in ixodid ticks  <cit>  .
at day  <dig> p.o.
expression levels of bm <dig> were  <dig>  times higher in r. appendiculatus eggs compared to r. microplus eggs, a difference that might in part be explained by a more advanced egg development in r. appendiculatus eggs as they were noted to hatch one day earlier than eggs from r. microplus.
the same large difference in bm <dig> expression level was also observed in unfed larvae and nymphs where bm <dig> expression levels were approximately tenfold higher in r. appendiculatus immatures in anticipation of a blood meal compared to unfed r. microplus larvae and nymphs.
it should be noted that all samples were collected at single well defined points from each life stage and fluctuations possibly occurring during these life stages could therefore have been missed.
the bm <dig> expression decreased significantly during feeding and particularly during molting in r. appendiculatus, a decrease which was present in r. microplus nymphs as well but to a far lesser extent.
bm <dig> expression levels of adults from both species were similar and so the total amount of bm <dig> expressed during blood feeding and exposure to the host immune system may be comparable between adults of the two species, assuming that the expression profile of bm <dig> mrna is indicative for the amount of expressed bm <dig> protein.
if so, differences in bm <dig> vaccination susceptibility could perhaps be sought in the prolonged exposure to imbibed blood and the host immune system of r. microplus which is adapted for continuous development on one host compared to the three-host tick r. appendiculatus.
the latter has a longer 'recovery' period during molting at which time no or very little bm <dig> is expressed.
hence little reaction between ingested antibodies and the bm <dig> protein would be expected to occur.
however, effects of vaccination with bm <dig> are predominantly seen in adults of r. microplus  <cit> .
this is corroborated by the fact that if r. microplus are raised to the stage of unfed adults on non-vaccinated cattle, then transferred to either vaccinated sheep  <cit>  or to an in-vitro feeding system using blood from vaccinated cattle  <cit> , strong vaccine effects are seen.
as mentioned earlier, this effect is not seen in r. appendiculatus adults feeding on cows vaccinated with bm <dig>  <cit> , although both r. microplus and r. appendiculatus have comparable bm <dig> expression levels in both unfed and fed adults.
although the nomenclature used to distinguish the various cell types present in the midgut of ticks is not unanimous, the midgut is thought to consist of the following epithelial cell types: stem cells, also referred to as undifferentiated reserve cell  <cit>  or replacement cell  <cit> , various stages of digest cells, secretory cells and albeit controversial, a basophilic cell type  <cit> .
the digest cells are thought to derive from the stem cells and transform from a prodigest cell type to a sessile digest cell following the absorption of blood meal haemoglobin.
sessile cells may detach from the basal lamina into the gut lumen, thus becoming detached or motile digest cells.
upon release of their hematin granules and other indigestible products into the lumen they are termed spent or degenerating digest cells  <cit> .
exhausted digest cells are replaced by consecutive cycles of growth and differentiation from undifferentiated cells so multiple generations of a digest cell type may be present at the same time, which has led to some confusion in the interpretation of these events  <cit> .
while it has been reported that the bm <dig> protein is located predominantly on the microvilli surface of digest cells, the exact cell type could not be determined for technical reasons  <cit> .
the high bm <dig> expression levels found in eggs in the third trimester of embryogenesis and unfed larvae suggest that stem cells and/or prodigest cells are expressing bm <dig> protein as well.
this would be in concordance with the hypothesized function of bm <dig> in the regulation of cell growth based on its sequence and structural homology to epidermal growth factor precursors  <cit>  and the greater abundance of bm <dig> towards the apical tips of gut digest cells associating it with a regulatory role in the apical growth  <cit> .
since antibodies from vaccination sera will bind to tick gut cells and inhibit their endocytotic function, involvement of bm <dig> in endocytosis of the blood meal seemed possible.
however, the inhibition of endocytosis was suggested to be an indirect effect of bm <dig> antibodies binding to the bm <dig> protein  <cit> .
the low levels of bm <dig> expression in feeding and pharate immature ticks and comparable expression levels between unfed and feeding adults make a role for bm <dig> in endocytosis more unlikely since the expression of proteins involved in endocytosis is expected to increase during the uptake of a bloodmeal.
CONCLUSIONS
nine candidate reference genes from different functional classes were identified in the est databases of r. microplus and r. appendiculatus and their expression stability throughout the life cycle of these two tick species was evaluated.
elf1a was found to be the most stable expressed gene in both tick species following analysis by both the genorm and normfinder software applications, gst showed the least stability.
the six most stable expressed genes were used for normalization of the expression profile of the tick-protective antigen bm <dig> for both r. microplus and r. appendiculatus.
this expression profile revealed a more continuous bm <dig> antigen abundance in r. microplus throughout its one-host life cycle compared to the three-host tick r. appendiculatus where large variations were observed between the different life stages.
the observed differences in bm <dig> expression profile between the two species alone can not adequately explain the lack of a bm <dig> vaccination effect in r. appendiculatus.

 the ixodid ticks rhipicephalus appendiculatus and rhipicephalus microplus are important pests of livestock. besides causing direct production losses and leather damage due to their blood-feeding habit, both ticks are able to transmit a wide variety of pathogens. both tick species overlap in their distribution, but r. microplus is more widespread and occurs in subtropical and tropical areas of the world whereas the distribution of r. appendiculatus, also known as the brown ear tick, is limited to areas with a humid climate from southern sudan to the southeastern coast of south africa. their life cycle differs quite dramatically too: r. microplus is a one-host tick species with all life stages feeding on the same, usually bovine, host whereas r. appendiculatus is a three-host tick species with each life stage requiring a new host to feed on. as a consequence of this, r. microplus can complete its life cycle in less than months, whereas r. appendiculatus takes about months to complete its life cycle under the most favorable conditions. control of ticks worldwide relies principally on the use of acaricides, but two vaccines targeting r. microplus were commercialized in the 1990s: tickgard plus® in australia and gavac® in cuba. both are based on the same recombinant antigen named bm a glycoprotein of unknown function which is located predominantly on the surface of midgut digest cells. although bm86-based vaccines showed cross-protection against various other tick species, e.g. rhipicephalus annulatus, rhipicephalus decoloratus, hyalomma anatolicum and hyalomma dromedarii, they were not effective against amblyomma variegatum and r. appendiculatus. due to the veterinary and economical importance of r. microplus and r. appendiculatus in subtropical and tropical areas of the world, expressed sequence tag datasets for these tick species have been established. the availability of these data greatly facilitates research in tick biology and tick-host-pathogen interactions. microarrays and quantitative rt-pcr are two important techniques measuring gene expression which may help in unraveling such interactions and provide insight into the complex regulatory networks behind biological processes. output data require normalization to control for variables such as the intrinsic variability of rna, impurities during rna extraction, reverse transcription and pcr efficiencies. a frequently used method for the accurate normalization of quantitative rt-pcr data involves the measurement of internal reference genes. such genes should ideally have a stable expression independent of cell or tissue type, or experimental condition. a survey of papers using quantitative rt-pcr in tick research published between and shows β-actin as the most popular reference gene used for normalization in publications, with the one remaining article employing the 18s rrna gene. however, the presumed expression stability of these genes in ticks has never been examined and the use of a single reference gene may lead to erroneous normalization. in this study, the mrna transcript levels of nine commonly used reference genes from different functional classes: β-actin, β-tubulin, elongation factor 1α, glyceraldehyde 3-phosphate dehydrogenase, glutathione s-transferase, h histone family 3a, cyclophilin, ribosomal protein l and tata box binding protein were measured by quantitative rt-pcr in all life stages of whole r. microplus and r. appendiculatus ticks. the results were evaluated using genorm and normfinder. although both programs have the same aim of identifying the most stably expressed reference genes, they make use of different strategies. genorm is a software application which determines the expression stability of reference genes by calculating a gene-stability measure for each gene. this measure relies on the principle that the expression ratio of two ideal reference genes is identical in all samples, regardless of the experimental condition or cell type. pairwise variation for each combination of reference genes is determined and assigned a value for m, and genes with the highest m value are progressively eliminated until the two most stably expressed genes remain. it thus ranks the reference genes according to the similarity in expression profiles across the samples. incorporation of co-regulated reference genes will affect the outcome of this approach and care must therefore be taken in selecting candidate reference genes from different functional classes. normfinder is an application on a model-based approach which ranks the reference genes according to the estimated intra- and intergroup expression variation. normalization with the six most stable expressed reference genes of the bm mrna transcript levels in all life stages of r. microplus and r. appendiculatus was carried out with the aim to elucidate the role of antigen abundance in bm vaccine susceptibility.  quantitative rt-pcr the efficiencies of the quantitative rt-pcr's were uniformly high and ranged from 91% to 103%, making all assays suitable for quantitative analysis. all pcr's generated a single band and the absence of primer dimer formation was confirmed by a dissociation assay performed with each assay. none of the primer combinations amplified cdna synthesized from bovine blood rna, which excludes interference with the pcr results caused by the possible presence of host rna in fed ticks. raw ct values ranged from to in r. microplus and from to in r. appendiculatus. gapdh, gst and tbp were expressed at low levels in both tick species with median ct values above cycles. the smallest ct variation between all samples of r. microplus was exhibited by tbp and by gapdh in r. appendiculatus. gst showed the most variable expression between all samples for both tick species; in r. microplus and in r. appendiculatus. rm a ra a rm a ra a arm, r. microplus; ra, r. appendiculatus rm a ra a rm a ra a rm a ra a rm a ra a arm, r. microplus; ra, r. appendiculatus genorm and normfinder analysis the gene expression stability of nine candidate reference genes over the life cycle of r. microplus and r. appendiculatus was analyzed using the genorm and normfinder software applications. the genorm approach identified elf1a and rpl as the best pair of reference genes over the life cycle of both r. microplus and r. appendiculatus, as well as in a combined analysis of all samples from both species. normfinder ranked these genes as second and third best in r. microplus and r. appendiculatus with tbp and gapdh being indicated as the best reference gene, respectively. a combined analysis of both species by normfinder ranked elf1a as best reference gene followed by gapdh, tbp, ppia, rpl h3f3a, btub, actb and gst. gst and actb were identified as the least stable genes in all groups by both methods. r. microplus r. appendiculatus the candidates are listed with decreasing expression stability from top to bottom. average expression stability values are shown between parentheses. to determine the minimum number of reference genes necessary for accurate normalization, calculation of the pairwise variation was performed by genorm. the lowest v values were found to be for v5/ in r. microplus and for v7/ in r. appendiculatus. combined analysis of both tick species yielded a lowest v value of at v6/ bm and ra sequence analysis one sequence for bm and two sequences from the bm homologue of r. appendiculatus, ra86- and ra86- were obtained by 3'race with degenerate primer ra86-f, which is located one amino acid downstream of the signal peptide of bm the open reading frame of the bm nucleotide sequence is bp, coding for a protein of amino acids which is % identical to the bm yeerongpilly reference strain with similar structural properties. the amino acid gap reported previously in a second bm sequence was not detected in any of the five sequenced clones. both r. appendiculatus sequences contain a bp-long orf which encodes for amino acids. the alleles differ by nucleotides of which are silent mutations and result in a change in the deduced amino acid sequence of the protein. the identity of the amino acid sequence of ra86- and ra86- are % and % with the bm proteins of r. microplus and % and % with r. microplus respectively. a comparison between the amino acid sequence of ra and bm shows that ra contains the same epidermal growth factor -like domains as bm. these domains are also present in ba bd and haa the bm homologues of r. annulatus, r. decoloratus and hy. a. anatolicum respectively. the ra sequences contain potential sites for n-linked glycosylation and ra86- has potential o-glycosylation site. both ra86- and ra86- are predicted to contain a glycosylphosphatidylinositol modification site at position, which provides linkage of the molecule to the cell membrane. the presence of a gpi anchor is a common feature found in all ixodid tick bm homologues characterized thus far. western blot analysis showed that ovine bm antiserum recognized bands of the expected ra protein size in the isolated midguts from partially fed r. appendiculatus females whereas serum from a sheep vaccinated with adjuvant only did not. bm86/ra expression analysis the expression profile of bm86/ra mrna in both r. microplus and r. appendiculatus was obtained by normalizing its expression with six reference genes that ranked highest in the genorm and normfinder analysis of the combined r. microplus and r. appendiculatus samples: elf1a, gapdh, h3f3a, ppia, rpl and tbp. in eggs of r. microplus, bm expression was detected at low levels in eggs and days after initiation of the oviposition and increased by three-fold in eggs collected days p.o. this formed the start of a rapid increase in the expression of bm in the third trimester of the embryogenesis to levels similar to that found in unfed larvae. bm expression decreased with feeding and molting in the immature life stages, with the lowest expression found in the pharate life stages. the decrease of bm expression levels following feeding of immatures was significantly more pronounced in the larvae and nymphs of r. appendiculatus compared to r. microplus where a more continuous expression pattern was observed during the life cycle with less dramatic variation. the expression level of bm in adults did not differ significantly between males and females of both species. discussion to minimize rt-pcr specific errors and correct for sample-to-sample variation in order to make a comparison of the bm expression profiles from r. microplus and r. appendiculatus possible, appropriate normalization is required. the use of reference genes is most frequently applied to normalize the mrna fraction, but validation of the expression stability of such genes in ticks has not been reported until now. actb is the most commonly used reference gene in tick research, but recent findings in mammals revealed that this gene and other commonly used'classical' reference genes such as gapdh may be inappropriate for use as a reference gene because of their variability under experimental conditions. the ideal reference gene should be expressed at a constant level in the tissue of interest at all stages of development and be unaffected by the specific experimental treatment being examined. however, no such universal reference gene has yet been identified and probably does not exist. normalization with multiple selected reference genes has been proposed as an alternative to overcome this problem and several tools to evaluate the expression stability of candidate reference genes have been developed. in this study, two of these tools, the genorm and normfinder programs, were employed to evaluate the expression stability of nine selected candidate reference genes. besides the two'classical' reference genes actb and gapdh, other candidate reference genes evaluated in this study were from different functional classes and were selected based on their reported expression stability in other organisms and their presence in the est libraries of r. microplus and r. appendiculatus. rpl for instance was among the thirteen ribosomal proteins which were ranked in the top of most stable expressed reference genes in a meta-analysis performed on a large dataset of human gene arrays. other ribosomal proteins were not included in this study to prevent bias in the ranking of the reference genes due to correlated expression of proteins belonging to the same functional class. the outcome of the gene stability evaluation differed between the programs used, which is not surprising in light of the different algorithms they employ. only elf1a was consistently ranked first or second by both programs and is suitable for use as a reference gene under the conditions described here. rpl is consistently ranked as the most stable expressed gene together with elf1a by genorm but not by normfinder. since both elf1a and rpl play a role in protein translation, co-regulation cannot be ruled out and this may have affected the outcome of the genorm analysis. normfinder is less sensitive to the incorporation of co-regulated genes since it focuses on the intra- and intergroup variation in selecting the most stable expressed genes. this may altogether explain the discordance in ranking of rpl between the genorm and normfinder programs. gst turned out to be the least stable gene in all conducted analyses. gst is known to be differentially expressed under different conditions and so could be expected to perform poorly as a reference gene. of the'traditional' reference genes, actb was ranked among the least stable genes by both methods whereas gapdh was ranked first in the normfinder analysis of the r. appendiculatus life stages. this is a direct result of the small ct variation observed in the gapdh expression in this species, which also explains the high ranking of tbp in the normfinder analysis of the r. microplus life stages where this gene showed the least ct variation over all samples. analysis of the pairwise variation of the samples by genorm returned values slightly lower or higher than the arbitrarily chosen threshold of, reflecting the heterogeneous nature of the analyzed whole tick samples which varied from egg to feeding adults. a direct consequence is the need of using a larger number of reference genes for optimal normalization. to be able to compare the bm mrna expression between all life stages of r. microplus and r. appendiculatus, normalization with six reference genes: elf1a, rpl tbp, h3f3a, ppia and gapdh was conducted. these reference genes were evaluated as being the most stable by both genorm and normfinder in a combined analysis of all samples from both tick species and returned the lowest pairwise variation value in the genorm analysis. the protein sequence of the bm gene from the mozambique r. microplus strain was highly identical to previously reported bm sequences from australia and south america and shared a maximum identity of % with the bm sequence from a r. microplus strain from central brazil. a second bm sequence isolated from the viscera of partially fed females originating from the same mozambique tick colony contains a amino acid gap which was not present in the bm sequence identified in this study. this sequence was not found in any of the five sequenced clones and may be less abundant, have been the result of alternative splicing or represent an allele which was lost in the r. microplus tick population since the source material was collected several generations earlier. the presence of bm alleles within the same tick population has been previously reported. two alleles, ra86- and ra86- were also found to be transcribed in the midgut of r. appendiculatus females. the orfs were similar in size but the alleles differed by amino acids. these differences do not appear to have a striking effect on the main properties of these proteins since both ra86- and ra86- are predicted to contain a gpi-anchor and contain egf-like domains similar to those found in bm, but ra86- does have a single potential o-glycosylation site which is absent from ra86- since boophilus species were recently synonymized with the rhipicephalus genus, it is not surprising that the ra protein shows a higher amino acid sequence identity with bm compared to the haa protein, the bm homologue from the two-host tick hy. anatolicum. however, feeding of hy. anatolicum on cattle vaccinated with a recombinant bm vaccine does result in a deleterious effect against this tick species which is not seen in r. appendiculatus. since western blot analysis showed that ovine bm antisera does indeed recognize r. appendiculatus proteins, other biological factors such as conformational epitopes, amount of blood/antibodies ingested, or antigen abundance may play a role in the biology of bm vaccine susceptibility. to investigate the latter hypothesis, the bm and ra transcript levels were measured throughout the life cycle of both tick species feeding on the same host by quantitative rt-pcr using a single primer pair which amplifies all known alleles of bm and ra the normalized bm mrna expression levels were monitored in various stages of embryonic development in r. microplus and were found to increase exponentially during the last days prior to hatching, simultaneous with the development of the midgut in embryos which takes place in the third trimester of embryogenesis in ixodid ticks. at day p.o. expression levels of bm were times higher in r. appendiculatus eggs compared to r. microplus eggs, a difference that might in part be explained by a more advanced egg development in r. appendiculatus eggs as they were noted to hatch one day earlier than eggs from r. microplus. the same large difference in bm expression level was also observed in unfed larvae and nymphs where bm expression levels were approximately tenfold higher in r. appendiculatus immatures in anticipation of a blood meal compared to unfed r. microplus larvae and nymphs. it should be noted that all samples were collected at single well defined points from each life stage and fluctuations possibly occurring during these life stages could therefore have been missed. the bm expression decreased significantly during feeding and particularly during molting in r. appendiculatus, a decrease which was present in r. microplus nymphs as well but to a far lesser extent. bm expression levels of adults from both species were similar and so the total amount of bm expressed during blood feeding and exposure to the host immune system may be comparable between adults of the two species, assuming that the expression profile of bm mrna is indicative for the amount of expressed bm protein. if so, differences in bm vaccination susceptibility could perhaps be sought in the prolonged exposure to imbibed blood and the host immune system of r. microplus which is adapted for continuous development on one host compared to the three-host tick r. appendiculatus. the latter has a longer'recovery' period during molting at which time no or very little bm is expressed. hence little reaction between ingested antibodies and the bm protein would be expected to occur. however, effects of vaccination with bm are predominantly seen in adults of r. microplus. this is corroborated by the fact that if r. microplus are raised to the stage of unfed adults on non-vaccinated cattle, then transferred to either vaccinated sheep or to an in-vitro feeding system using blood from vaccinated cattle, strong vaccine effects are seen. as mentioned earlier, this effect is not seen in r. appendiculatus adults feeding on cows vaccinated with bm, although both r. microplus and r. appendiculatus have comparable bm expression levels in both unfed and fed adults. although the nomenclature used to distinguish the various cell types present in the midgut of ticks is not unanimous, the midgut is thought to consist of the following epithelial cell types: stem cells, also referred to as undifferentiated reserve cell or replacement cell, various stages of digest cells, secretory cells and albeit controversial, a basophilic cell type. the digest cells are thought to derive from the stem cells and transform from a prodigest cell type to a sessile digest cell following the absorption of blood meal haemoglobin. sessile cells may detach from the basal lamina into the gut lumen, thus becoming detached or motile digest cells. upon release of their hematin granules and other indigestible products into the lumen they are termed spent or degenerating digest cells. exhausted digest cells are replaced by consecutive cycles of growth and differentiation from undifferentiated cells so multiple generations of a digest cell type may be present at the same time, which has led to some confusion in the interpretation of these events. while it has been reported that the bm protein is located predominantly on the microvilli surface of digest cells, the exact cell type could not be determined for technical reasons. the high bm expression levels found in eggs in the third trimester of embryogenesis and unfed larvae suggest that stem cells and/or prodigest cells are expressing bm protein as well. this would be in concordance with the hypothesized function of bm in the regulation of cell growth based on its sequence and structural homology to epidermal growth factor precursors and the greater abundance of bm towards the apical tips of gut digest cells associating it with a regulatory role in the apical growth. since antibodies from vaccination sera will bind to tick gut cells and inhibit their endocytotic function, involvement of bm in endocytosis of the blood meal seemed possible. however, the inhibition of endocytosis was suggested to be an indirect effect of bm antibodies binding to the bm protein. the low levels of bm expression in feeding and pharate immature ticks and comparable expression levels between unfed and feeding adults make a role for bm in endocytosis more unlikely since the expression of proteins involved in endocytosis is expected to increase during the uptake of a bloodmeal.  nine candidate reference genes from different functional classes were identified in the est databases of r. microplus and r. appendiculatus and their expression stability throughout the life cycle of these two tick species was evaluated. elf1a was found to be the most stable expressed gene in both tick species following analysis by both the genorm and normfinder software applications, gst showed the least stability. the six most stable expressed genes were used for normalization of the expression profile of the tick-protective antigen bm for both r. microplus and r. appendiculatus. this expression profile revealed a more continuous bm antigen abundance in r. microplus throughout its one-host life cycle compared to the three-host tick r. appendiculatus where large variations were observed between the different life stages. the observed differences in bm expression profile between the two species alone can not adequately explain the lack of a bm vaccination effect in r. appendiculatus. 
the expression profile of bm in r. appendiculatus and r. microplus revealed a more continuous bm antigen abundance in r. microplus throughout its one-host life cycle compared to the three-host tick r. appendiculatus where large variations were observed between different life stages. to demonstrate the usefulness of these results, an unresolved issue in tick vaccine development was examined. based on these results, elf1a can be proposed as an initial reference gene for normalization of quantitative rt-pcr data in whole r. microplus and r. appendiculatus ticks. in most studies on ticks where quantitative rt-pcr is employed, normalization occurs with a single reference gene, usually β-actin, without validation of its presumed expression stability. the transcription levels of nine potential reference genes: β-actin, β-tubulin, elongation factor 1α, glyceraldehyde 3-phosphate dehydrogenase, glutathione s-transferase, h histone family 3a, cyclophilin, ribosomal protein l and tata box binding protein were measured in all life stages of r. microplus and r. appendiculatus. in fact, bm86-based vaccines give better protection against some tick species with lower bm sequence homology. the most stable reference genes were therefore used for normalization of the bm expression profile in all life stages of both species to examine whether antigen abundance plays a role in bm vaccine susceptibility. commercial vaccines against r. microplus were developed based on the recombinant antigen bm but despite a high degree of sequence homology, these vaccines are not effective against r. appendiculatus. one possible explanation is the variation in bm expression levels between r. microplus and r. appendiculatus. for accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is essential. the first goal of this study was to evaluate the expression stability of commonly used reference genes in rhipicephalus appendiculatus and rhipicephalus microplus ticks. the observed differences in bm expression profile between the two species alone can not adequately explain the lack of a bm vaccination effect in r. appendiculatus. elf1a was found to be the most stable expressed gene in both species following analysis by both genorm and normfinder software applications, gst showed the least stability.
 for accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is essential. in most studies on ticks where quantitative rt-pcr is employed, normalization occurs with a single reference gene, usually β-actin, without validation of its presumed expression stability. the first goal of this study was to evaluate the expression stability of commonly used reference genes in rhipicephalus appendiculatus and rhipicephalus microplus ticks. to demonstrate the usefulness of these results, an unresolved issue in tick vaccine development was examined. commercial vaccines against r. microplus were developed based on the recombinant antigen bm but despite a high degree of sequence homology, these vaccines are not effective against r. appendiculatus. in fact, bm86-based vaccines give better protection against some tick species with lower bm sequence homology. one possible explanation is the variation in bm expression levels between r. microplus and r. appendiculatus. the most stable reference genes were therefore used for normalization of the bm expression profile in all life stages of both species to examine whether antigen abundance plays a role in bm vaccine susceptibility.  the transcription levels of nine potential reference genes: β-actin, β-tubulin, elongation factor 1α, glyceraldehyde 3-phosphate dehydrogenase, glutathione s-transferase, h histone family 3a, cyclophilin, ribosomal protein l and tata box binding protein were measured in all life stages of r. microplus and r. appendiculatus. elf1a was found to be the most stable expressed gene in both species following analysis by both genorm and normfinder software applications, gst showed the least stability. the expression profile of bm in r. appendiculatus and r. microplus revealed a more continuous bm antigen abundance in r. microplus throughout its one-host life cycle compared to the three-host tick r. appendiculatus where large variations were observed between different life stages.  based on these results, elf1a can be proposed as an initial reference gene for normalization of quantitative rt-pcr data in whole r. microplus and r. appendiculatus ticks. the observed differences in bm expression profile between the two species alone can not adequately explain the lack of a bm vaccination effect in r. appendiculatus. 