BACKGROUND
the mosquito anopheles gambiae is the main malaria vector in sub-saharan africa.
resistance to anti-malaria drugs and insecticides together with the lack of vaccines highlight the need for novel strategies in malaria control.
such a strategy could be the interruption of the transmission cycle within the mosquito.
the mosquito becomes infected with the malaria parasite by taking a blood meal.
the blood meal itself brings metabolic changes and induces a state of oxidative stress  <cit> .
this is further increased by the presence of plasmodium parasites in the blood meal  <cit> .
during mosquito response to infection, active nitrogen and oxygen radicals are produced to contain plasmodium infection  <cit> .
these products may represent potential oxidative stress that can be ameliorated or eliminated by detoxification enzymes.
for example several glutathione s-transferases  have peroxidase activity and some can also metabolise reactive α,β-aldehydes  <cit> .
gst expression can also be induced by reactive oxygen species   <cit> .
while gsts help to eliminate ros, cytochrome p <dig> monooxygenases  may actually contribute towards its generation  <cit> .
although transcription alteration of detoxification genes in response to bacteria and plasmodium  <cit>  has been described, the nature of this response hasn't been fully discussed.
in this study we describe the impact of p. berghei infection at two time points  on the expression of detoxification genes in the midgut and fat body.
we identified several genes, previously implicated in the detoxification of xenobiotics, which are differentially expressed in relation to parasite infection in the midgut and fat body.
the possible role of detoxification enzymes in modulating malaria transmission is discussed.
RESULTS
microarray
tissues for microarray analyses were collected at two critical time points of the plasmodium cycle in the mosquito host:  <dig> day following the blood meal, during which parasites invade the midgut epithelium, and  <dig> days after the blood meal when sporozoites are starting to be released to the hemolymph, as demonstrated by detection of parasite's dna in the hemolymph .
the mosquitoes were fed on mice that were either infected with the parasite or uninfected.
the success of infection was indirectly confirmed by randomly selecting up to 19- <dig> mosquitoes that were screened for the presence of oocysts .
most of the mosquitoes were found to be positive  and hence it can be assumed that the tissues used in the gene expression studies were infected too.
the microarray experiment was developed to answer the following questions, regarding midgut and fat body tissues:
<dig>  which genes respond to plasmodium midgut epithelium invasion
<dig>  which genes respond to the release of sporozoites into the hemolymph , and
<dig>  which genes respond differently between the two events .
in the microarray analysis  <dig> loci were differentially expressed in at least one of the comparisons made.
the results for all comparisons are given in table s <dig> .
the microarray results were validated by comparing the mean values for the expression data  for genes from three independent replicates obtained by microarray analysis with the corresponding mean expression values obtained with the multiplex quantitative rt-pcr.
the pearson correlation coefficient  demonstrates a high degree of correlation between the two methods .
genes differentially expressed in infected versus uninfected mosquitoes at day  <dig> post blood meal
at day  <dig> post blood meal more changes were observed in the midgut as compared to the fat body.
while in the midgut  <dig> genes were differentially expressed, only  <dig> were different in the fat body .
in the midgut,  <dig> cyps were differentially expressed with the majority  being down-regulated.
in the fat body, five out of the six cyps differentially expressed in response to plasmodium infection were up-regulated.
the vast majority of these differentially expressed cyps belong to families primarily associated with detoxification roles  rather than families implicated in hormone biosynthetic pathways  <cit> .
similarly in the gst family the two classes primarily associated with xenobiotic detoxification, delta and epsilon  <cit> , were generally repressed in response to parasite infection with the notable exception of gstd <dig> which was strongly up-regulated  in infected vs. uninfected midguts.
in both a. gambiae and a. stephensi, plasmodium parasite invasion induces an increase of nitric oxide synthase  expression and in turn an increase in nitric oxide  and no metabolites  <cit> .
no has been shown to down regulate cyp gene expression in other organisms  <cit> .
we hypothesize therefore that the observed down-regulation of cyps in the midgut may also be linked to increased levels of no.
of the up-regulated cyps, cyp6m <dig> showed the greatest response to infection .
this gene has already been reported to be over-expressed in response to p. berghei infection  <cit>  and implicated in resistance to pyrethroid insecticides  <cit> .
one possible explanation for this up-regulation is a response to an endogenous mediator increased upon the infection process.
as an example, prostaglandins have been shown to induce expression of cyps in human liver cells  <cit> .
superoxide dismutases constitute part of the first line of defence against ros and reactive nitrogen oxide species   <cit> .
however, sod <dig> was down-regulated  <dig> day post infection suggesting that down-regulation of oxidative stress response genes could be part of the defence response triggered by parasite invasion.
a similar mechanism has been described for other oxidative stress response genes such as catalase in response to plasmodium invasion  <cit> .
cytoskeleton reorganization and up-regulation of genes related to folding and movement of microtubules suggest that cytoskeleton dynamics and remodelling function as key elements of plasmodium invasion of the anopheles midgut  <cit> .
this epithelium rearrangement is a robust molecular response to ookinetes penetration.
in a whole genome microarray study seven tubulins were differentially up-regulated during the invasion period  <cit> .
here too, three cytoskeletal genes represented on the detox array, tubulin b, tubulin a and actin, were up-regulated at day  <dig> post infection .
in mammals, microtubule disruption leads to down-regulation of several cyps  <cit>  and perhaps similar responses also lead to down-regulation of cyps during parasite invasion.
genes differentially expressed in infected versus uninfected mosquitoes  <dig> days post blood meal
at this time point, when sporozoites are released from oocysts to the hemocoel a less pronounced effect on the transcript levels of detoxification genes was observed as compared to midgut invasion .
two of the genes up-regulated on day  <dig>  cyp4h <dig> and cyp4h <dig>  were down-regulated at day  <dig> , suggesting that their suppression is linked to the invasion of the midgut epithelium by the parasite, while their up-regulation on day  <dig> may be associated with subtle changes in midgut structure as sporozoites are leaving oocysts.
gst <dig> was up-regulated at both time points  which indicates that this enzyme is directly involved in the response to parasites at both stages.
although at this stage the fat body would have had direct contact with parasites or at least molecules released by parasites during midgut egress, the transcriptional response in the fat body was more pronounced at day  <dig> than on day  <dig> post infection.
cyp6m <dig> was down-regulated on day  <dig> but was up-regulated at day  <dig> , indicating that this cyp responds to particular events of the parasites life cycle.
genes that show a different response between plasmodium midgut epithelium invasion and release of sporozoites into the hemolymph
the interaction term between the two time points was investigated to compare responses to plasmodium invasion of the midgut epithelium  and to the release of sporozoites into the hemolymph .
heat diagrams with the genes that presented significant positive  and negative interaction  in midgut and fat body are shown in figure  <dig>  the number of genes under positive interaction was higher in the midgut while the opposite was seen in the fat body, reflecting the active site of infection.
abc transporters from family c showed a strong negative interaction in the midgut and to a lesser extent in the fat body, implying that these cytoplasmic membrane transporters are important for infection control probably by transporting glutathione conjugates or lipid-derived eicosanoids that are known to be involved in insect response to infection  <cit> .
the interaction analysis confirmed that there is a considerable difference between the gene expression levels between day  <dig> and day  <dig> in response to plasmodium infection.
there were a high number of genes that had different levels of expression in response to the ookinetes invasion of the midgut and in response to the release of sporozoites in the hemolymph, showing that these genes have the ability of changing their expression levels according with the time of infection.
in the midgut, the majority  of differentially expressed genes between day  <dig> and day  <dig> were the same both in uninfected and infected mosquitoes, as was the direction of change, indicating that these genes were responding mainly to the blood meal, as it represents a strong oxidative insult.
however, this total concordance was not observed in the fat body where only 26% of genes were regulated in the same direction between infected and uninfected while 38% were regulated in opposite directions .
the trend of expression of both tissues suggests that differences observed are due to fat body response to parasite released from the oocysts into the hemocoel.
the mosquito response to sporozoites in the hemolymph triggers effector mechanisms like melanization  <cit> , and a burst of expression of genes encoding constituents of the immune system including the production of free radicals  <cit>  that needs a counter detoxification reaction.
after excluding genes similarly regulated in both infected and uninfected groups, fat body cyp genes were down-regulated, at day  <dig>  as observed for the midgut at day  <dig>  sod <dig> was down-regulated and seems to be determinant for parasite control.
tpx <dig> was up-regulated confirming its role on infection detoxification mediated by the fat body.
the fat body has an important role in the detoxification and in the immune response of the mosquito on day  <dig> of infection when compared with day  <dig> post infection, which is not observed when we compare infected and uninfected mosquitoes on day  <dig>
CONCLUSIONS
this study determined transcription profiles of detoxification enzymes during plasmodium infection in a. gambiae, showing important changes in the expression of several detoxification enzymes, as well as membrane associated abc transporters.
interestingly, genes coding for detoxification enzymes revealed a variable response, being differentially induced or repressed depending on the tissue and stage of infection.
although the mechanism underlying these changes is presently unclear, this differential regulation of detoxification genes observed during plasmodium infection may be due to 1) the increasing oxidative stress caused by the presence of the parasite; 2) the epithelium rearrangement involving alterations in cytoskeleton genes caused by the ookinetes invasion and the oocysts burst; or 3) a combination of both.
a hypothetical scenario for the inter-relationship between infection and detoxifying molecules is depicted in figure  <dig>
in contrast to the majority of differentially expressed p450s which were down-regulated in response to midgut invasion, cyp6m <dig> expression was induced in response to p. berghei infection.
this cyp is involved in resistance to pyrethroid insecticides  <cit> .
this observation together with regulation of other genes, such as membrane abc transporters involved in xenobiotic elimination, lead us to speculate that there might be an association between the response to plasmodium infection and insecticide resistance, enhancing the importance of further studying their interaction.

 the mosquito anopheles gambiae is the main malaria vector in sub-saharan africa. resistance to anti-malaria drugs and insecticides together with the lack of vaccines highlight the need for novel strategies in malaria control. such a strategy could be the interruption of the transmission cycle within the mosquito. the mosquito becomes infected with the malaria parasite by taking a blood meal. the blood meal itself brings metabolic changes and induces a state of oxidative stress. this is further increased by the presence of plasmodium parasites in the blood meal. during mosquito response to infection, active nitrogen and oxygen radicals are produced to contain plasmodium infection. these products may represent potential oxidative stress that can be ameliorated or eliminated by detoxification enzymes. for example several glutathione s-transferases have peroxidase activity and some can also metabolise reactive α,β-aldehydes. gst expression can also be induced by reactive oxygen species. while gsts help to eliminate ros, cytochrome p monooxygenases may actually contribute towards its generation. although transcription alteration of detoxification genes in response to bacteria and plasmodium has been described, the nature of this response hasn't been fully discussed. in this study we describe the impact of p. berghei infection at two time points on the expression of detoxification genes in the midgut and fat body. we identified several genes, previously implicated in the detoxification of xenobiotics, which are differentially expressed in relation to parasite infection in the midgut and fat body. the possible role of detoxification enzymes in modulating malaria transmission is discussed.  microarray tissues for microarray analyses were collected at two critical time points of the plasmodium cycle in the mosquito host: day following the blood meal, during which parasites invade the midgut epithelium, and days after the blood meal when sporozoites are starting to be released to the hemolymph, as demonstrated by detection of parasite's dna in the hemolymph. the mosquitoes were fed on mice that were either infected with the parasite or uninfected. the success of infection was indirectly confirmed by randomly selecting up to 19- mosquitoes that were screened for the presence of oocysts. most of the mosquitoes were found to be positive and hence it can be assumed that the tissues used in the gene expression studies were infected too. the microarray experiment was developed to answer the following questions, regarding midgut and fat body tissues: which genes respond to plasmodium midgut epithelium invasion which genes respond to the release of sporozoites into the hemolymph, and which genes respond differently between the two events. in the microarray analysis loci were differentially expressed in at least one of the comparisons made. the results for all comparisons are given in table s. the microarray results were validated by comparing the mean values for the expression data for genes from three independent replicates obtained by microarray analysis with the corresponding mean expression values obtained with the multiplex quantitative rt-pcr. the pearson correlation coefficient demonstrates a high degree of correlation between the two methods. genes differentially expressed in infected versus uninfected mosquitoes at day post blood meal at day post blood meal more changes were observed in the midgut as compared to the fat body. while in the midgut genes were differentially expressed, only were different in the fat body. in the midgut, cyps were differentially expressed with the majority being down-regulated. in the fat body, five out of the six cyps differentially expressed in response to plasmodium infection were up-regulated. the vast majority of these differentially expressed cyps belong to families primarily associated with detoxification roles rather than families implicated in hormone biosynthetic pathways. similarly in the gst family the two classes primarily associated with xenobiotic detoxification, delta and epsilon, were generally repressed in response to parasite infection with the notable exception of gstd which was strongly up-regulated in infected vs. uninfected midguts. in both a. gambiae and a. stephensi, plasmodium parasite invasion induces an increase of nitric oxide synthase expression and in turn an increase in nitric oxide and no metabolites. no has been shown to down regulate cyp gene expression in other organisms. we hypothesize therefore that the observed down-regulation of cyps in the midgut may also be linked to increased levels of no. of the up-regulated cyps, cyp6m showed the greatest response to infection. this gene has already been reported to be over-expressed in response to p. berghei infection and implicated in resistance to pyrethroid insecticides. one possible explanation for this up-regulation is a response to an endogenous mediator increased upon the infection process. as an example, prostaglandins have been shown to induce expression of cyps in human liver cells. superoxide dismutases constitute part of the first line of defence against ros and reactive nitrogen oxide species. however, sod was down-regulated day post infection suggesting that down-regulation of oxidative stress response genes could be part of the defence response triggered by parasite invasion. a similar mechanism has been described for other oxidative stress response genes such as catalase in response to plasmodium invasion. cytoskeleton reorganization and up-regulation of genes related to folding and movement of microtubules suggest that cytoskeleton dynamics and remodelling function as key elements of plasmodium invasion of the anopheles midgut. this epithelium rearrangement is a robust molecular response to ookinetes penetration. in a whole genome microarray study seven tubulins were differentially up-regulated during the invasion period. here too, three cytoskeletal genes represented on the detox array, tubulin b, tubulin a and actin, were up-regulated at day post infection. in mammals, microtubule disruption leads to down-regulation of several cyps and perhaps similar responses also lead to down-regulation of cyps during parasite invasion. genes differentially expressed in infected versus uninfected mosquitoes days post blood meal at this time point, when sporozoites are released from oocysts to the hemocoel a less pronounced effect on the transcript levels of detoxification genes was observed as compared to midgut invasion. two of the genes up-regulated on day cyp4h and cyp4h were down-regulated at day, suggesting that their suppression is linked to the invasion of the midgut epithelium by the parasite, while their up-regulation on day may be associated with subtle changes in midgut structure as sporozoites are leaving oocysts. gst was up-regulated at both time points which indicates that this enzyme is directly involved in the response to parasites at both stages. although at this stage the fat body would have had direct contact with parasites or at least molecules released by parasites during midgut egress, the transcriptional response in the fat body was more pronounced at day than on day post infection. cyp6m was down-regulated on day but was up-regulated at day, indicating that this cyp responds to particular events of the parasites life cycle. genes that show a different response between plasmodium midgut epithelium invasion and release of sporozoites into the hemolymph the interaction term between the two time points was investigated to compare responses to plasmodium invasion of the midgut epithelium and to the release of sporozoites into the hemolymph. heat diagrams with the genes that presented significant positive and negative interaction in midgut and fat body are shown in figure the number of genes under positive interaction was higher in the midgut while the opposite was seen in the fat body, reflecting the active site of infection. abc transporters from family c showed a strong negative interaction in the midgut and to a lesser extent in the fat body, implying that these cytoplasmic membrane transporters are important for infection control probably by transporting glutathione conjugates or lipid-derived eicosanoids that are known to be involved in insect response to infection. the interaction analysis confirmed that there is a considerable difference between the gene expression levels between day and day in response to plasmodium infection. there were a high number of genes that had different levels of expression in response to the ookinetes invasion of the midgut and in response to the release of sporozoites in the hemolymph, showing that these genes have the ability of changing their expression levels according with the time of infection. in the midgut, the majority of differentially expressed genes between day and day were the same both in uninfected and infected mosquitoes, as was the direction of change, indicating that these genes were responding mainly to the blood meal, as it represents a strong oxidative insult. however, this total concordance was not observed in the fat body where only 26% of genes were regulated in the same direction between infected and uninfected while 38% were regulated in opposite directions. the trend of expression of both tissues suggests that differences observed are due to fat body response to parasite released from the oocysts into the hemocoel. the mosquito response to sporozoites in the hemolymph triggers effector mechanisms like melanization, and a burst of expression of genes encoding constituents of the immune system including the production of free radicals that needs a counter detoxification reaction. after excluding genes similarly regulated in both infected and uninfected groups, fat body cyp genes were down-regulated, at day as observed for the midgut at day sod was down-regulated and seems to be determinant for parasite control. tpx was up-regulated confirming its role on infection detoxification mediated by the fat body. the fat body has an important role in the detoxification and in the immune response of the mosquito on day of infection when compared with day post infection, which is not observed when we compare infected and uninfected mosquitoes on day  this study determined transcription profiles of detoxification enzymes during plasmodium infection in a. gambiae, showing important changes in the expression of several detoxification enzymes, as well as membrane associated abc transporters. interestingly, genes coding for detoxification enzymes revealed a variable response, being differentially induced or repressed depending on the tissue and stage of infection. although the mechanism underlying these changes is presently unclear, this differential regulation of detoxification genes observed during plasmodium infection may be due to 1) the increasing oxidative stress caused by the presence of the parasite; 2) the epithelium rearrangement involving alterations in cytoskeleton genes caused by the ookinetes invasion and the oocysts burst; or 3) a combination of both. a hypothetical scenario for the inter-relationship between infection and detoxifying molecules is depicted in figure in contrast to the majority of differentially expressed p450s which were down-regulated in response to midgut invasion, cyp6m expression was induced in response to p. berghei infection. this cyp is involved in resistance to pyrethroid insecticides. this observation together with regulation of other genes, such as membrane abc transporters involved in xenobiotic elimination, lead us to speculate that there might be an association between the response to plasmodium infection and insecticide resistance, enhancing the importance of further studying their interaction. 
most toxicity-related reactions occur in the midgut shortly after the ingestion of an infected blood meal. using a custom-made dna microarray, transcript levels of loci primarily belonging to three major detoxification enzyme families were compared in infected and uninfected mosquitoes both during ookinete invasion and the release of sporozoites into the hemocoel. while many alterations are directly related to the mosquito's innate immune response, parasite invasion is also expected to generate toxic by-products such as free radicals. the current study aimed at identifying which loci coding for detoxification enzymes are differentially expressed as a function of plasmodium berghei infection in midgut and fat body tissues. interestingly, many detoxification genes including a large number of p450s were down-regulated at this stage. anopheles gambiae has been shown to change its global gene expression patterns upon plasmodium infection. while most gene expression changes were tissue-related, cyp6m a cyp previously associated with insecticide resistance, was over-expressed both in the midgut and fat body during ookinete invasion. strong up-regulation of cyp6m in the midgut and the fat body as well as its previous association with insecticide resistance shows its broad role in metabolic detoxification. in the fat body, while less dramatic, gene expression alterations were also observed and occurred during the ookinete invasion and during the release of sporozoites into the hemocoel.
 anopheles gambiae has been shown to change its global gene expression patterns upon plasmodium infection. while many alterations are directly related to the mosquito's innate immune response, parasite invasion is also expected to generate toxic by-products such as free radicals. the current study aimed at identifying which loci coding for detoxification enzymes are differentially expressed as a function of plasmodium berghei infection in midgut and fat body tissues.  using a custom-made dna microarray, transcript levels of loci primarily belonging to three major detoxification enzyme families were compared in infected and uninfected mosquitoes both during ookinete invasion and the release of sporozoites into the hemocoel. the greatest changes in gene expression were observed in the midgut in response to ookinete invasion. interestingly, many detoxification genes including a large number of p450s were down-regulated at this stage. in the fat body, while less dramatic, gene expression alterations were also observed and occurred during the ookinete invasion and during the release of sporozoites into the hemocoel. while most gene expression changes were tissue-related, cyp6m a cyp previously associated with insecticide resistance, was over-expressed both in the midgut and fat body during ookinete invasion.  most toxicity-related reactions occur in the midgut shortly after the ingestion of an infected blood meal. strong up-regulation of cyp6m in the midgut and the fat body as well as its previous association with insecticide resistance shows its broad role in metabolic detoxification. 