BACKGROUND
cryptosporidium parvum is a globally distributed zoonotic pathogen belonging to the phylum apicomplexa that contains many important human and animal pathogens, such as plasmodium, toxoplasma, eimeria, babesia and theileria.
cryptosporidium can cause severe watery diarrhea in humans and animals, and is an important opportunistic pathogen in immunocompromised individuals such as aids patients
<cit> .
it is also listed as one of the category b agents in the national institutes of health  biodefense program.
despite its importance to both human and animal heaths, research on cryptosporidium is limited not only by the difficulties in manipulating the parasite, but also by the lack of tools for genetic study such as robust culture systems, transfection methods and parasite-specific microarrays.
cryptosporidium is transmitted between humans and/or animals by the infectious oocysts that can survive in the natural environments for a substantial length of time.
each c. parvum oocyst contains four sporozoites enclosed within an oocyst wall structure that is highly resistant to disinfectants, including commonly used chlorine treatments
<cit> .
molecular and cellular studies on the oocyst stage have been largely devoted to understanding the composition of oocyst wall, such as the family of oocyst wall proteins
<cit> , and little is known on the biochemical events of this parasite stage.
understanding the global gene expression profiles in oocysts would not only deepen our understanding on the biology and metabolic features of this opportunistic pathogen, but also provide new insight into how the oocysts survive without any nutrient supply in the field and withstand various stresses in the natural environments.
for example, c. parvum lacks a citrate cycle and a cytochrome-based respiratory chain, thus relying solely on glycolysis and fermentation as an energy and carbon source
<cit> .
it is therefore important to understand how the oocysts function in the oxygen-rich external environment, with metabolic machinery that is seemingly specialized for the anaerobic conditions of the host gut, as well as streamlined for nutrient scavenging.
oocysts need to survive in the external environment for months or years, where they might face a range of hostile environmental stresses.
determining the gene expression profile changes in oocysts in response ultraviolent  irradiation will help to understand how the parasite responds to stress, with practical relevance to the uv exposure encountered in the external environment, as well as uv irradiation commonly used by humans to treat drinking and surface waters
<cit> .
here we report the development of an agilent platform-based, c. parvum-specific microarray using 60-base oligonucleotides that represent all predicted protein-encoding genes in the parasite.
assays of transcript levels using this microarray permitted us for the first time to systematically study gene transcript profiles for a global view of unique metabolic features of parasite oocysts, as well response to a common stress uv irradiation.
RESULTS
quality of the cparray15k chips and data
the newly developed cparray15k is an agilent microarray that contains  <dig>  spots, spanning  <dig>  unique probes, covering all predicted protein coding genes in the c. parvum genome.
the array was used to profile global gene transcript levels in oocysts, with or without uv irradiation, as described in the materials and methods.
medians of signal and background intensities were acquired for subsequent analysis.
correlation plots of normalized signals from all  <dig> hybridized arrays suggested no or minimal bias between cy <dig> and cy5-labeled samples or between two biological replicates .
we calculated the means and standard deviations  of all individual probes among the  <dig> arrays to evaluate the inter-array variations, as well as those for all individual genes from multiple probes to evaluate the inter-probe variations .
although a few of the inter-probe variations were relatively large, there were no contradictory results among different probes and arrays, confirming that both inter-array and inter-probe variations were minimal.
we also validated the microarray data by real-time rt-pcr analysis on a panel of selected parasite genes, and observed a general agreement between microarray and qrt-pcr data, both in the relative level of expression in oocysts  and in the fold changes in expressions and directions in response to uv-treatment .
collectively, these observations confirm the overall quality of microarray design and the acquired signal data.
metabolic features in the c. parvum oocysts
the experimental design included determination of the gene expression profiles in untreated oocyst samples, as a means to understand metabolic features in the oocyst stage.
to minimize the effect of temperature change on gene expression, parasite oocysts were removed from 4°c storage and placed overnight at room temperature.
during this process they appeared to maintain their morphological integrity .
since all oocysts had matured to contain  <dig> sporozoites, the gene expression levels in oocysts, as well as their fold changes in response to uv irradiation as described in later sections, represent metabolism of sporozoites within the oocysts.
from the total of  <dig> untreated samples, and based on the signal to noise ratio  > <dig>  and signal to background ratio  > <dig>  criterions
<cit> , we have identified a total of  <dig>  genes that were positively expressed in oocysts .
this number represents 51% of the total  <dig>  genes in the c. parvum genome.
among the remaining unexpressed genes,  <dig>  genes had snr ≤ <dig>  and sbr ≤ <dig>  in all probes and all arrays  that were considered as unexpressed or extremely lowly expressed in the oocysts.
the remaining  <dig> genes  with positive signals in some, but not all, probes or arrays could be judged as lowly expressed, rather than unexpressed.
based on the expression levels, we ranked the group i genes by quartiles, and categorized them into major functional groups in comparison with unexpressed genes in group ii.
among the  <dig>  group i genes, only  <dig> genes  were annotated and could be assigned into functional categories .
the remaining  <dig>  genes  were hypothetical, including those with undefined functions, containing certain conserved domains, or possible membrane proteins.
a complete list of all categorized genes in groups i and ii were provided as part of the supplementary materials .
additionally, we compiled a list of the top 10%  highest expressed genes with more detailed subcategories .
* a list of genes with detailed information on the normalized expression levels, their ranks, and categories is provided in additional file
<dig>  table s <dig>
approximately 41% of the annotated group i genes were associated with gene expression functions, including those involved in core functions in expression ; ribosome biogenesis ; and rna metabolism or modification  .
among these genes, those involved in ribosome biogenesis, with a single exception, were expressed at top three quartiles and the majority of them  were ranked at top quartile .
another group of related genes were those associated with protein degradation, as indicated by the active expression of a large number of genes encoding proteosome subunits and ubiquitin pathway , suggesting that proteins in the oocysts were actively recycled and suggesting a finite lifespan.
genes encoding enzymes, excluding a few mitochondrial genes that were separately listed, constituted the second major group of expressed genes.
among them, 25%  were expressed at varied levels, relatively evenly across the  <dig> quartiles .
a slightly less number of enzyme genes  were either lowly or unexpressed.
nearly half of the expressed enzymes were involved in post-translational modification of proteins , including protein phosphatases, protein kinases, peptidases and protein folding elements.
in contrast, dna metabolism was much less active than the gene expression-associated pathways .
among them, relatively more genes associated with the dna repair than those involved in dna replication were expressed, although none of them had the top quartile signals.
these observations indicate that c. parvum devotes significant resources to the gene expression and the synthesis, modification and degradation of proteins in the oocysts.
considering the inability of cryptosporidium to synthesize any nutrients de novo, including amino acids
<cit> , we speculate that the parasite relies heavily on protein degradation pathways to recycle amino acids.
in this manner a protein synthetic capacity is maintained in environmental oocysts that have no access to the host cells to scavenge nutrients.
in addition to protein modifications, various core metabolic pathways were active in oocysts.
as expected, many enzymes involved in energy metabolism, such as glycolysis and fermentation, were expressed at relatively high levels .
oocysts appear to be able to use both amylopectin and hexoses as an energy and carbon source.
remarkably and highly unique, the lactate dehydrogenase  gene  was expressed at the highest level among all genes .
because c. parvum lacks a citrate cycle and cytochrome-based respiratory chain, the parasite relies solely on the glycolysis as an energy and carbon source.
to maintain the carbon flow it is capable of producing lactate, alcohol and acetate as organic end products.
however, the expressions of other key enzymes for producing acetate  and alcohol  were much lower than that of ldh .
these observations prompted us to investigate the roles of the three end products in other parasite life cycle stages.
our qrt-pcr analysis of ldh, adh-e, adh <dig> and acecl <dig> genes showed that the ldh gene was expressed at >100-fold higher levels in the oocysts and free sporozoites than in the intracellular developmental stages .
in contrast, the expression levels of genes encoding the acetate- and alcohol-producing enzymes were low in the oocysts and free sporozoites, but elevated in the intracellular stages , suggesting that these genes played differentiated roles during the complex parasite life cycle.
recently, mauzy and colleagues have reported a comprehensive transcriptome analysis of all annotated genes in c. parvum during intracellular development by qrt-pcr, albeit the lack of data on the oocysts and free sprozoites
<cit> .
data extracted from their study on the expressions of ldh, adh-e, adh <dig> and acecl genes during intracellular stages followed similar patterns as observed in this study; that is, peak expression at  <dig> h post-infection for ldh, but at 48– <dig> h for the other three genes .
cryptosporidium ldh gene is unique among apicomplexan orthologs, as it is known to have evolved from the malate dehydrogenase  gene following gene duplication after the genus diverged from other apicomplexans
<cit> .
our data indicate that this unique gene plays a more important role in the energy metabolism in the parasite oocysts than in the intracellular stages.
as expected, oocysts were active in maintaining intracellular ion homeostasis and organellar ph, as well as subcellular transport of nutrients.
specifically, 9%  of the group i genes encoded transporters that were expressed at varied levels; including atp-binding cassette  family proteins, vacuolar  atpases, various p-type atpases responsible for ion transports and a subset of nutrient transporters  .
structure protein genes represented 4% of the expressed genes, which included mainly cytoskeletal proteins  and five oocyst wall proteins, although most of them were expressed at low quartile levels.
among cytoskeletal proteins, actin expression was the highest, along with many other microfilamental proteins .
in contrast, tubulins and other microtubule-associated proteins were mostly expressed at low to near background levels, which implies that the parasite oocysts are probably more active in maintaining intracellular trafficking than maintaining its structure.
the membrane protein group included mucins, trap and other thrombospondin domain-containing proteins
<cit> , and proteins involved in membrane or vesicular sorting or trafficking.
other hypothetical proteins and enzymes could also be membrane-associated.
around 5% of the expressed genes belonged to this group .
a subset of genes involved in membrane/vesicular sorting/transporting was expressed, indicating that the oocysts maintain certain level of membrane trafficking.
the highest observed expression levels were among mucin-like proteins, particularly cpmuc <dig>  that has been described as a marker in c. parvum infection
<cit> .
the data suggest that mucin-like proteins are also important in the oocysts, possibly in maintaining and stabilizing the membrane structure under the hostile environment, or localization on the surface membrane of sporozoites in preparation to encounter the midgut environment and interaction with host cells.
stress-related genes consists of various chaperonins/heat shock proteins  and proteins for maintaining redox homeostasis.
among the  <dig> chaperon proteins of various classes,  <dig> were expressed.
there were  <dig> major classes of proteins involved in cellular redox homeostasis .
among them, all three glutaredoxin-associated genes and  <dig>  thioredoxin-associated genes were expressed at various levels.
however, signals for superoxide dismutases  and ferredoxin/ferredoxin reductase  were at or near background level, implying that in oocysts the sod and the mitochondrial fdx/fdxr pair were less important than the glutaredoxin and thioredoxin-associated pathways.
among proteins predicted to reside in the relict mitochondria in c. parvum, a subset of genes encoding mitochondrial carrier proteins and translocases were active.
like the fdx/fdxr pair, alternative oxidase  was not or lowly expressed.
in contrast, the ion-sulfur  cluster assembly pathway is active.
it is known that c. parvum does not possess a complete f-atpase, but only has two headpieces
<cit> , for which their functional role remains to be defined.
these two subunits were highly expressed in the oocysts , suggesting that the standalone headpiece complex may play a role in the environmental oocyst stage.
a total of  <dig>  hypothetical genes with undefined functions  were actively expressed at different levels in the oocysts ; as well as proteins containing apicomplexan-specific domains , or predicted membrane proteins  .
investigations on these genes may help to elucidate their functional roles in the survival and stress-resistance of parasite oocysts in the environment.
the environmental oocyst stage is also present in other intestinal and cyst-forming coccidia, such as eimeria tenella and toxoplasma gondii; although global gene expression profile data in the sporulated oocyst stage is at present only available for t. gondii <cit> .
although the report mainly summarized the highly regulated t. gondii genes between tachyzoites, bradyzoites, and oocysts sporulated for various days
<cit> , we were able to extract the expression data on individual developmental stages from toxodb  for a snapshot comparison with those in c. parvum.
among the top  <dig> highly expressed genes in fully sporulated t. gondii oocysts , the majority  were annotated as hypothetical.
among the remaining  <dig> genes, the largest group belonged to the secretory antigens/proteins , including micronemal proteins , surface antigen 1-related sequences  and dense granule proteins , followed by enzymes  , which differed from the expression profiles in c. parvum oocysts that was characterized by the high number of genes associated with ribosomal biogenesis, enzymes, and gene expression .
we also analyzed ldh gene expression profiles in t. gondii; namely, two ldh genes that are known to be differentially expressed in tachyzoites and bradyzoites
<cit> .
the microarray data by fritz and colleagues confirmed this finding, and also showed that the two ldh genes are differentially expressed in t. gondii oocysts.
specifically, the ldh <dig> gene  was expressed at much higher levels than the ldh <dig> gene  in oocysts with various sporulation times .
however, unlike the ldh gene in c. parvum, neither ldh <dig> nor ldh <dig> genes in t. gondii displayed extremely higher levels of expression in oocysts in comparison with either their expressions in other developmental stages or the expression of other genes in the oocysts .
thus the difference in gene expression profiles in sporulated oocysts between c. parvum and t. gondii might reflect different metabolic features and adaptation of life cycle styles between these two apicomplexans.
gene expression in the oocysts in response to the uv irradiation
to identify an appropriate dose of uv treatment to study the global gene expression responses, we first determined the survival curve of c. parvum oocysts against increasing uv <dig> doses, as determined by a qrt-pcr-based assay of their ability to infect hct- <dig> cells in vitro .
we then selected the id <dig> dose  that could introduce sufficient damages, but also allow the recovery of the majority of the irradiated oocysts.
both control and uv-treated oocysts were allowed to recover for  <dig>  h and  <dig> h to represent relatively short and long post-irradiation recovery times.
our microarray analysis identified  <dig>  and  <dig>  probes in the  <dig>  h and  <dig> h groups, respectively, that were statistically significantly changed in expressions , which gave a total number of  <dig>  probes  that were statistically significantly changed in one or both groups .
the means of signal intensity of individual genes were determined from multiple probes and arrays, and used to calculate p-values by student t-test and the false discovery rate  q-values by the storey and tibshirani method
<cit> .
using q ≤  <dig>  as the cutoff value, we identified  <dig>  and  <dig> genes in the  <dig>  h and  <dig> h groups, respectively, that were significantly changed in expression.
our qrt-pcr analysis of selected genes with different fold changes in the  <dig>  h group also confirmed that the microarray data were generally reliable, although the exact fold changes might slightly vary between the two sets of data .
among these regulated genes, we observed changes ranging from  <dig> -fold  to  <dig> -fold  in the  <dig>  h group, and from  <dig>   to  <dig>   in the  <dig> h group .
to determine the patterns of gene expression changes in oocysts, we selected ± <dig> -fold  change as the empirical cutoff value for “highly regulated” expressions, and genes were grouped into  <dig> clusters: > <dig>  ; between  <dig>  and  <dig>  ; and < <dig>  .
this categorization allowed us to address the dynamics in the gene expressions after short  and long  post-irradiation recovery times with  <dig> distinguished clusters ; cluster  <dig> genes were changed from up-regulated to down-regulated, and so forth) .
a complete list of genes grouped by major functions under the  <dig> patterns is provided in additional file
6: table s <dig>  and summarized in table
<dig>
note: up = up-regulated , mid = less significantly regulated “middle” group , down = down-regulated .
based on the ± <dig> -fold cutoff,  <dig>   were considered as “less regulated ” in both  <dig>  h and  <dig> h groups  .
most of the other  <dig> genes fell into the following  <dig> clusters:  <dig>  or  <dig>  genes were first up- or down-regulated in  <dig>  h and then returned to the middle range in  <dig> h, respectively;  <dig>  and  <dig>  genes were first in the mid-range  and then became up- or down-regulated, respectively.
we were able to categorize  <dig> genes  into major functional groups, in which the largest group was enzymes, followed by those involved in rna metabolism, core gene expression and ribosome biogenesis, among others .
these observations indicated that gene expressions in the oocysts were dynamically and comprehensively regulated during the cause of recovery after uv-irradiation.
at the early  <dig>  h recovery time, in a few groups the proportion of up-regulated members appeared to exceed the down-regulated ones, which included those involved in cell cycle control , dna metabolism , chromatin associated protein, cytoskeletal proteins and various transporters .
the data agreed with our general assumptions that oocysts need to activate pathways to repair dna, other macromolecules, and cellular components damaged by uv-irradiation.
the cytoskeletal rearrangement became more active, which was probably associated with the repair of damaged cytoskeletal components and intracellular trafficking based on the elevated expressions of motor molecules  and a number of membrane-trafficking associated proteins .
a large subset of transporters was regulated.
each group of transporters possessed both up- and down-regulated members, with the top up-regulated genes were predominantly abc-type, ion, nutrient and nuclear transporters that were mostly resumed to near normal levels in  <dig> h. the highly down-regulated genes were predominantly vacuolar atpase subunits that remained down-regulated in  <dig> h. the increased expressions of several oocyst wall proteins; articulin proteins that are known to be important in maintaining cell shapes in euglena and ciliates
<cit> , and mucin-like proteins suggestive of active repairing on some cell/oocyst surface molecules.
among stress-related genes, predominant up-regulated members included chaperonins and redox homeostasis, three putative t-complex protein  <dig>  subunits and several thioredoxin-associated genes; whereas various hsp/dnaj family members, as well as sod and glutaredoxin-related genes were either much less regulated or down-regulated.
it has been reported that a hsp <dig> gene  exhibits >10-fold increased expression in response to heat-shock at 45°c for  <dig> min
<cit> .
these data suggest that different proteins within this functional group are responsible to handle different stresses.
the chaperonin containing tcp- <dig>  is known to be required for the production/folding of actin and tubulin among the others
<cit> .
the up-regulated expression of tcp- <dig> subunits correlates well with those of actin and several other cytoskeletal genes.
in the  <dig> h group, the majority of the highly up- or down-regulated genes resumed, or displayed a clear trend toward the middle-range levels of expression .
only  <dig> or  <dig> genes stayed either up- or down-regulated expressions, respectively, in both  <dig>  h and  <dig> h groups , in which only a small number of genes could be functionally categorized.
among them, genes encoding a dna replication licensing factor  and dna polymerase epsilon subunit  remained up-regulated in both  <dig>  h and  <dig> h groups, which suggested that dna repair machinery in the oocysts was activated soon after uv-irradiation, and lasting for > <dig> h. the data were generally in agreement with our previous observations on a select panel of dna repair-associated genes in the parasite in response to uv-irradiation
<cit> .
additionally, the long-lasting up-regulated expressions of γ-tubulin and a microtubule-associated protein indicated that the cytoskeletal elements underwent substantial rearrangements in the parasite, probably as needed for transporting and recycling the damaged macromolecules and cellular components.
several genes involved in the ribosome biogenesis and rna metabolism remained down-regulated in both groups, although they were expressed at relatively high levels in normal oocysts.
few genes had reversed their expression patterns .
however, a substantial number of genes went up or down from the middle range , in which mostly were enzymes among the annotatable genes.
the proteasome and ubiquitin associated genes appeared to be the group after the enzymes with more genes went up in  <dig> h group from mid-range expressions in  <dig>  h group .
in this group, there were also three and one lowered expressed genes bounced back to mid-range or higher levels of expression, separately , but none went further down, indicating that the activities associated with protein degradation and amino acid recycling remained relatively high for a substantial long time after uv-irradiation.
more regulated genes could not be annotated into functional groups, which included those encoding hypothetical or membrane proteins .
further functional analyses of these genes may not only assist in their annotations, but also shed new light into their functions and roles in response to environmental stresses.
CONCLUSIONS
we have developed an agilent microarray for c. parvum  that covers all predicted orfs in the parasite genome.
using cparray15k coupled with real-time qrt-pcr of selected genes, we have observed that the parasite oocysts are highly active in protein synthesis based on the high levels of expressions of genes associated with ribosome biogenesis, transcription and translation.
proteasome and ubiquitin associated components are also highly active, implying that this environmental stage of parasite may employ protein degradation pathways to recycle amino acids in order to overcome an inability to synthesize amino acids de novo.
the energy metabolism in oocysts is highly active, as expected, and featured by the highest level of expression of the ldh gene in the oocysts.
cryptosporidium is able to produce lactate, alcohol and acetate as organic end products.
our follow-up qrt-pcr analysis confirms that the oocysts rely on lactate production, whereas the intracellular parasite developmental stages likely depend more on alcohol and acetate end products to maintain essential carbon flow.
in response to uv-irradiation, the parasite oocysts undergo complex and dynamic regulations in gene expression.
the majority of the top regulated genes changed their expression levels by around + <dig>  and − <dig>  folds, respectively, with only a few genes showing ≥2-fold changes.
increased activations in dna repair and intracellular trafficking were also observed.
among the stress-related genes, tcp- <dig> family members and some thioredoxin-associated genes appear to play important roles in the recovery of uv-induced damages in the oocysts.
our observations also implied increased activities in cytoskeletal rearrangement and intracellular membrane trafficking in the parasite oocysts upon uv irradiation.
cparray15k is the first microarray chip developed for c. parvum, which provides the cryptosporidium research community a needed tool to study parasite transcriptome and functional genomics.
although next generation of sequencing -based rna-seq has been used increasingly in transcriptome analysis, microarray remains a powerful complementary technology with advantages on low costs, short turn-round time and ease of data generation
<cit> .
cparray15k has been successfully used in profiling the gene expressions in the parasite oocysts as well as their responses to uv-irradiation.
the current version may be further expanded to include additional probes, such as the non-coding regions and snps, by taking advantage of the small parasite genome similar to the multi-functional array developed for toxoplasma gondii <cit> .

 cryptosporidium parvum is a globally distributed zoonotic pathogen belonging to the phylum apicomplexa that contains many important human and animal pathogens, such as plasmodium, toxoplasma, eimeria, babesia and theileria. cryptosporidium can cause severe watery diarrhea in humans and animals, and is an important opportunistic pathogen in immunocompromised individuals such as aids patients. it is also listed as one of the category b agents in the national institutes of health biodefense program. despite its importance to both human and animal heaths, research on cryptosporidium is limited not only by the difficulties in manipulating the parasite, but also by the lack of tools for genetic study such as robust culture systems, transfection methods and parasite-specific microarrays. cryptosporidium is transmitted between humans and/or animals by the infectious oocysts that can survive in the natural environments for a substantial length of time. each c. parvum oocyst contains four sporozoites enclosed within an oocyst wall structure that is highly resistant to disinfectants, including commonly used chlorine treatments. molecular and cellular studies on the oocyst stage have been largely devoted to understanding the composition of oocyst wall, such as the family of oocyst wall proteins, and little is known on the biochemical events of this parasite stage. understanding the global gene expression profiles in oocysts would not only deepen our understanding on the biology and metabolic features of this opportunistic pathogen, but also provide new insight into how the oocysts survive without any nutrient supply in the field and withstand various stresses in the natural environments. for example, c. parvum lacks a citrate cycle and a cytochrome-based respiratory chain, thus relying solely on glycolysis and fermentation as an energy and carbon source. it is therefore important to understand how the oocysts function in the oxygen-rich external environment, with metabolic machinery that is seemingly specialized for the anaerobic conditions of the host gut, as well as streamlined for nutrient scavenging. oocysts need to survive in the external environment for months or years, where they might face a range of hostile environmental stresses. determining the gene expression profile changes in oocysts in response ultraviolent irradiation will help to understand how the parasite responds to stress, with practical relevance to the uv exposure encountered in the external environment, as well as uv irradiation commonly used by humans to treat drinking and surface waters. here we report the development of an agilent platform-based, c. parvum-specific microarray using 60-base oligonucleotides that represent all predicted protein-encoding genes in the parasite. assays of transcript levels using this microarray permitted us for the first time to systematically study gene transcript profiles for a global view of unique metabolic features of parasite oocysts, as well response to a common stress uv irradiation.  quality of the cparray15k chips and data the newly developed cparray15k is an agilent microarray that contains spots, spanning unique probes, covering all predicted protein coding genes in the c. parvum genome. the array was used to profile global gene transcript levels in oocysts, with or without uv irradiation, as described in the materials and methods. medians of signal and background intensities were acquired for subsequent analysis. correlation plots of normalized signals from all hybridized arrays suggested no or minimal bias between cy and cy5-labeled samples or between two biological replicates. we calculated the means and standard deviations of all individual probes among the arrays to evaluate the inter-array variations, as well as those for all individual genes from multiple probes to evaluate the inter-probe variations. although a few of the inter-probe variations were relatively large, there were no contradictory results among different probes and arrays, confirming that both inter-array and inter-probe variations were minimal. we also validated the microarray data by real-time rt-pcr analysis on a panel of selected parasite genes, and observed a general agreement between microarray and qrt-pcr data, both in the relative level of expression in oocysts and in the fold changes in expressions and directions in response to uv-treatment. collectively, these observations confirm the overall quality of microarray design and the acquired signal data. metabolic features in the c. parvum oocysts the experimental design included determination of the gene expression profiles in untreated oocyst samples, as a means to understand metabolic features in the oocyst stage. to minimize the effect of temperature change on gene expression, parasite oocysts were removed from 4°c storage and placed overnight at room temperature. during this process they appeared to maintain their morphological integrity. since all oocysts had matured to contain sporozoites, the gene expression levels in oocysts, as well as their fold changes in response to uv irradiation as described in later sections, represent metabolism of sporozoites within the oocysts. from the total of untreated samples, and based on the signal to noise ratio > and signal to background ratio > criterions, we have identified a total of genes that were positively expressed in oocysts. this number represents 51% of the total genes in the c. parvum genome. among the remaining unexpressed genes, genes had snr ≤ and sbr ≤ in all probes and all arrays that were considered as unexpressed or extremely lowly expressed in the oocysts. the remaining genes with positive signals in some, but not all, probes or arrays could be judged as lowly expressed, rather than unexpressed. based on the expression levels, we ranked the group i genes by quartiles, and categorized them into major functional groups in comparison with unexpressed genes in group ii. among the group i genes, only genes were annotated and could be assigned into functional categories. the remaining genes were hypothetical, including those with undefined functions, containing certain conserved domains, or possible membrane proteins. a complete list of all categorized genes in groups i and ii were provided as part of the supplementary materials. additionally, we compiled a list of the top 10% highest expressed genes with more detailed subcategories. * a list of genes with detailed information on the normalized expression levels, their ranks, and categories is provided in additional file table s approximately 41% of the annotated group i genes were associated with gene expression functions, including those involved in core functions in expression; ribosome biogenesis; and rna metabolism or modification. among these genes, those involved in ribosome biogenesis, with a single exception, were expressed at top three quartiles and the majority of them were ranked at top quartile. another group of related genes were those associated with protein degradation, as indicated by the active expression of a large number of genes encoding proteosome subunits and ubiquitin pathway, suggesting that proteins in the oocysts were actively recycled and suggesting a finite lifespan. genes encoding enzymes, excluding a few mitochondrial genes that were separately listed, constituted the second major group of expressed genes. among them, 25% were expressed at varied levels, relatively evenly across the quartiles. a slightly less number of enzyme genes were either lowly or unexpressed. nearly half of the expressed enzymes were involved in post-translational modification of proteins, including protein phosphatases, protein kinases, peptidases and protein folding elements. in contrast, dna metabolism was much less active than the gene expression-associated pathways. among them, relatively more genes associated with the dna repair than those involved in dna replication were expressed, although none of them had the top quartile signals. these observations indicate that c. parvum devotes significant resources to the gene expression and the synthesis, modification and degradation of proteins in the oocysts. considering the inability of cryptosporidium to synthesize any nutrients de novo, including amino acids, we speculate that the parasite relies heavily on protein degradation pathways to recycle amino acids. in this manner a protein synthetic capacity is maintained in environmental oocysts that have no access to the host cells to scavenge nutrients. in addition to protein modifications, various core metabolic pathways were active in oocysts. as expected, many enzymes involved in energy metabolism, such as glycolysis and fermentation, were expressed at relatively high levels. oocysts appear to be able to use both amylopectin and hexoses as an energy and carbon source. remarkably and highly unique, the lactate dehydrogenase gene was expressed at the highest level among all genes. because c. parvum lacks a citrate cycle and cytochrome-based respiratory chain, the parasite relies solely on the glycolysis as an energy and carbon source. to maintain the carbon flow it is capable of producing lactate, alcohol and acetate as organic end products. however, the expressions of other key enzymes for producing acetate and alcohol were much lower than that of ldh. these observations prompted us to investigate the roles of the three end products in other parasite life cycle stages. our qrt-pcr analysis of ldh, adh-e, adh and acecl genes showed that the ldh gene was expressed at >100-fold higher levels in the oocysts and free sporozoites than in the intracellular developmental stages. in contrast, the expression levels of genes encoding the acetate- and alcohol-producing enzymes were low in the oocysts and free sporozoites, but elevated in the intracellular stages, suggesting that these genes played differentiated roles during the complex parasite life cycle. recently, mauzy and colleagues have reported a comprehensive transcriptome analysis of all annotated genes in c. parvum during intracellular development by qrt-pcr, albeit the lack of data on the oocysts and free sprozoites. data extracted from their study on the expressions of ldh, adh-e, adh and acecl genes during intracellular stages followed similar patterns as observed in this study; that is, peak expression at h post-infection for ldh, but at 48– h for the other three genes. cryptosporidium ldh gene is unique among apicomplexan orthologs, as it is known to have evolved from the malate dehydrogenase gene following gene duplication after the genus diverged from other apicomplexans. our data indicate that this unique gene plays a more important role in the energy metabolism in the parasite oocysts than in the intracellular stages. as expected, oocysts were active in maintaining intracellular ion homeostasis and organellar ph, as well as subcellular transport of nutrients. specifically, 9% of the group i genes encoded transporters that were expressed at varied levels; including atp-binding cassette family proteins, vacuolar atpases, various p-type atpases responsible for ion transports and a subset of nutrient transporters. structure protein genes represented 4% of the expressed genes, which included mainly cytoskeletal proteins and five oocyst wall proteins, although most of them were expressed at low quartile levels. among cytoskeletal proteins, actin expression was the highest, along with many other microfilamental proteins. in contrast, tubulins and other microtubule-associated proteins were mostly expressed at low to near background levels, which implies that the parasite oocysts are probably more active in maintaining intracellular trafficking than maintaining its structure. the membrane protein group included mucins, trap and other thrombospondin domain-containing proteins, and proteins involved in membrane or vesicular sorting or trafficking. other hypothetical proteins and enzymes could also be membrane-associated. around 5% of the expressed genes belonged to this group. a subset of genes involved in membrane/vesicular sorting/transporting was expressed, indicating that the oocysts maintain certain level of membrane trafficking. the highest observed expression levels were among mucin-like proteins, particularly cpmuc that has been described as a marker in c. parvum infection. the data suggest that mucin-like proteins are also important in the oocysts, possibly in maintaining and stabilizing the membrane structure under the hostile environment, or localization on the surface membrane of sporozoites in preparation to encounter the midgut environment and interaction with host cells. stress-related genes consists of various chaperonins/heat shock proteins and proteins for maintaining redox homeostasis. among the chaperon proteins of various classes, were expressed. there were major classes of proteins involved in cellular redox homeostasis. among them, all three glutaredoxin-associated genes and thioredoxin-associated genes were expressed at various levels. however, signals for superoxide dismutases and ferredoxin/ferredoxin reductase were at or near background level, implying that in oocysts the sod and the mitochondrial fdx/fdxr pair were less important than the glutaredoxin and thioredoxin-associated pathways. among proteins predicted to reside in the relict mitochondria in c. parvum, a subset of genes encoding mitochondrial carrier proteins and translocases were active. like the fdx/fdxr pair, alternative oxidase was not or lowly expressed. in contrast, the ion-sulfur cluster assembly pathway is active. it is known that c. parvum does not possess a complete f-atpase, but only has two headpieces, for which their functional role remains to be defined. these two subunits were highly expressed in the oocysts, suggesting that the standalone headpiece complex may play a role in the environmental oocyst stage. a total of hypothetical genes with undefined functions were actively expressed at different levels in the oocysts; as well as proteins containing apicomplexan-specific domains, or predicted membrane proteins. investigations on these genes may help to elucidate their functional roles in the survival and stress-resistance of parasite oocysts in the environment. the environmental oocyst stage is also present in other intestinal and cyst-forming coccidia, such as eimeria tenella and toxoplasma gondii; although global gene expression profile data in the sporulated oocyst stage is at present only available for t. gondii. although the report mainly summarized the highly regulated t. gondii genes between tachyzoites, bradyzoites, and oocysts sporulated for various days, we were able to extract the expression data on individual developmental stages from toxodb for a snapshot comparison with those in c. parvum. among the top highly expressed genes in fully sporulated t. gondii oocysts, the majority were annotated as hypothetical. among the remaining genes, the largest group belonged to the secretory antigens/proteins, including micronemal proteins, surface antigen 1-related sequences and dense granule proteins, followed by enzymes, which differed from the expression profiles in c. parvum oocysts that was characterized by the high number of genes associated with ribosomal biogenesis, enzymes, and gene expression. we also analyzed ldh gene expression profiles in t. gondii; namely, two ldh genes that are known to be differentially expressed in tachyzoites and bradyzoites. the microarray data by fritz and colleagues confirmed this finding, and also showed that the two ldh genes are differentially expressed in t. gondii oocysts. specifically, the ldh gene was expressed at much higher levels than the ldh gene in oocysts with various sporulation times. however, unlike the ldh gene in c. parvum, neither ldh nor ldh genes in t. gondii displayed extremely higher levels of expression in oocysts in comparison with either their expressions in other developmental stages or the expression of other genes in the oocysts. thus the difference in gene expression profiles in sporulated oocysts between c. parvum and t. gondii might reflect different metabolic features and adaptation of life cycle styles between these two apicomplexans. gene expression in the oocysts in response to the uv irradiation to identify an appropriate dose of uv treatment to study the global gene expression responses, we first determined the survival curve of c. parvum oocysts against increasing uv doses, as determined by a qrt-pcr-based assay of their ability to infect hct- cells in vitro. we then selected the id dose that could introduce sufficient damages, but also allow the recovery of the majority of the irradiated oocysts. both control and uv-treated oocysts were allowed to recover for h and h to represent relatively short and long post-irradiation recovery times. our microarray analysis identified and probes in the h and h groups, respectively, that were statistically significantly changed in expressions, which gave a total number of probes that were statistically significantly changed in one or both groups. the means of signal intensity of individual genes were determined from multiple probes and arrays, and used to calculate p-values by student t-test and the false discovery rate q-values by the storey and tibshirani method. using q ≤ as the cutoff value, we identified and genes in the h and h groups, respectively, that were significantly changed in expression. our qrt-pcr analysis of selected genes with different fold changes in the h group also confirmed that the microarray data were generally reliable, although the exact fold changes might slightly vary between the two sets of data. among these regulated genes, we observed changes ranging from -fold to -fold in the h group, and from to in the h group. to determine the patterns of gene expression changes in oocysts, we selected ± -fold change as the empirical cutoff value for “highly regulated” expressions, and genes were grouped into clusters: >; between and; and <. this categorization allowed us to address the dynamics in the gene expressions after short and long post-irradiation recovery times with distinguished clusters; cluster genes were changed from up-regulated to down-regulated, and so forth). a complete list of genes grouped by major functions under the patterns is provided in additional file 6: table s and summarized in table note: up = up-regulated, mid = less significantly regulated “middle” group, down = down-regulated. based on the ± -fold cutoff, were considered as “less regulated ” in both h and h groups. most of the other genes fell into the following clusters: or genes were first up- or down-regulated in h and then returned to the middle range in h, respectively; and genes were first in the mid-range and then became up- or down-regulated, respectively. we were able to categorize genes into major functional groups, in which the largest group was enzymes, followed by those involved in rna metabolism, core gene expression and ribosome biogenesis, among others. these observations indicated that gene expressions in the oocysts were dynamically and comprehensively regulated during the cause of recovery after uv-irradiation. at the early h recovery time, in a few groups the proportion of up-regulated members appeared to exceed the down-regulated ones, which included those involved in cell cycle control, dna metabolism, chromatin associated protein, cytoskeletal proteins and various transporters. the data agreed with our general assumptions that oocysts need to activate pathways to repair dna, other macromolecules, and cellular components damaged by uv-irradiation. the cytoskeletal rearrangement became more active, which was probably associated with the repair of damaged cytoskeletal components and intracellular trafficking based on the elevated expressions of motor molecules and a number of membrane-trafficking associated proteins. a large subset of transporters was regulated. each group of transporters possessed both up- and down-regulated members, with the top up-regulated genes were predominantly abc-type, ion, nutrient and nuclear transporters that were mostly resumed to near normal levels in h. the highly down-regulated genes were predominantly vacuolar atpase subunits that remained down-regulated in h. the increased expressions of several oocyst wall proteins; articulin proteins that are known to be important in maintaining cell shapes in euglena and ciliates, and mucin-like proteins suggestive of active repairing on some cell/oocyst surface molecules. among stress-related genes, predominant up-regulated members included chaperonins and redox homeostasis, three putative t-complex protein subunits and several thioredoxin-associated genes; whereas various hsp/dnaj family members, as well as sod and glutaredoxin-related genes were either much less regulated or down-regulated. it has been reported that a hsp gene exhibits >10-fold increased expression in response to heat-shock at 45°c for min. these data suggest that different proteins within this functional group are responsible to handle different stresses. the chaperonin containing tcp- is known to be required for the production/folding of actin and tubulin among the others. the up-regulated expression of tcp- subunits correlates well with those of actin and several other cytoskeletal genes. in the h group, the majority of the highly up- or down-regulated genes resumed, or displayed a clear trend toward the middle-range levels of expression. only or genes stayed either up- or down-regulated expressions, respectively, in both h and h groups, in which only a small number of genes could be functionally categorized. among them, genes encoding a dna replication licensing factor and dna polymerase epsilon subunit remained up-regulated in both h and h groups, which suggested that dna repair machinery in the oocysts was activated soon after uv-irradiation, and lasting for > h. the data were generally in agreement with our previous observations on a select panel of dna repair-associated genes in the parasite in response to uv-irradiation. additionally, the long-lasting up-regulated expressions of γ-tubulin and a microtubule-associated protein indicated that the cytoskeletal elements underwent substantial rearrangements in the parasite, probably as needed for transporting and recycling the damaged macromolecules and cellular components. several genes involved in the ribosome biogenesis and rna metabolism remained down-regulated in both groups, although they were expressed at relatively high levels in normal oocysts. few genes had reversed their expression patterns. however, a substantial number of genes went up or down from the middle range, in which mostly were enzymes among the annotatable genes. the proteasome and ubiquitin associated genes appeared to be the group after the enzymes with more genes went up in h group from mid-range expressions in h group. in this group, there were also three and one lowered expressed genes bounced back to mid-range or higher levels of expression, separately, but none went further down, indicating that the activities associated with protein degradation and amino acid recycling remained relatively high for a substantial long time after uv-irradiation. more regulated genes could not be annotated into functional groups, which included those encoding hypothetical or membrane proteins. further functional analyses of these genes may not only assist in their annotations, but also shed new light into their functions and roles in response to environmental stresses.  we have developed an agilent microarray for c. parvum that covers all predicted orfs in the parasite genome. using cparray15k coupled with real-time qrt-pcr of selected genes, we have observed that the parasite oocysts are highly active in protein synthesis based on the high levels of expressions of genes associated with ribosome biogenesis, transcription and translation. proteasome and ubiquitin associated components are also highly active, implying that this environmental stage of parasite may employ protein degradation pathways to recycle amino acids in order to overcome an inability to synthesize amino acids de novo. the energy metabolism in oocysts is highly active, as expected, and featured by the highest level of expression of the ldh gene in the oocysts. cryptosporidium is able to produce lactate, alcohol and acetate as organic end products. our follow-up qrt-pcr analysis confirms that the oocysts rely on lactate production, whereas the intracellular parasite developmental stages likely depend more on alcohol and acetate end products to maintain essential carbon flow. in response to uv-irradiation, the parasite oocysts undergo complex and dynamic regulations in gene expression. the majority of the top regulated genes changed their expression levels by around + and − folds, respectively, with only a few genes showing ≥2-fold changes. increased activations in dna repair and intracellular trafficking were also observed. among the stress-related genes, tcp- family members and some thioredoxin-associated genes appear to play important roles in the recovery of uv-induced damages in the oocysts. our observations also implied increased activities in cytoskeletal rearrangement and intracellular membrane trafficking in the parasite oocysts upon uv irradiation. cparray15k is the first microarray chip developed for c. parvum, which provides the cryptosporidium research community a needed tool to study parasite transcriptome and functional genomics. although next generation of sequencing -based rna-seq has been used increasingly in transcriptome analysis, microarray remains a powerful complementary technology with advantages on low costs, short turn-round time and ease of data generation. cparray15k has been successfully used in profiling the gene expressions in the parasite oocysts as well as their responses to uv-irradiation. the current version may be further expanded to include additional probes, such as the non-coding regions and snps, by taking advantage of the small parasite genome similar to the multi-functional array developed for toxoplasma gondii. 
the proteasome and ubiquitin associated components were also highly active, implying that oocysts might employ protein degradation pathways to recycle amino acids in order to overcome the inability to synthesize amino acids de novo. oocyst stage parasites were found to be highly active in protein synthesis, based on the high transcript levels of genes associated with ribosome biogenesis, transcription and translation. notable changes included increased transcript levels of genes involved in dna repair and intracellular trafficking. cparray15k is the first microarray chip developed for c. parvum, which provides the cryptosporidium research community a needed tool to study the parasite transcriptome and functional genomics. our observations also suggest that uv irradiation of oocysts results in increased activities in cytoskeletal rearrangement and intracellular membrane trafficking. global transcriptome analysis using cparray15k coupled with real-time qrt-pcr uncovered a number of unique metabolic features in oocysts, the infectious and environmental stage of the parasite. these observations shed light on how the parasite oocysts might adapt and respond to the hostile external environment and associated stress such as uv irradiation. energy metabolism in oocysts was featured by the highest level of expression of lactate dehydrogenase gene. here we report the development of the first agilent microarray for c. parvum that covers all predicted orfs in the parasite genome. we also studied parasite responses to uv-irradiation, and observed complex and dynamic regulations of gene expression. cparray15k has been successfully used in profiling the gene expressions in the parasite oocysts as well as their responses to uv-irradiation. cryptosporidium parvum is a globally distributed zoonotic parasite and an important opportunistic pathogen in immunocompromised patients. among the stress-related genes, tcp- family members and some thioredoxin-associated genes appear to play more important roles in the recovery of uv-induced damages in the oocysts.
 cryptosporidium parvum is a globally distributed zoonotic parasite and an important opportunistic pathogen in immunocompromised patients. little is known on the metabolic dynamics of the parasite, and study is hampered by the lack of molecular and genetic tools. here we report the development of the first agilent microarray for c. parvum that covers all predicted orfs in the parasite genome. global transcriptome analysis using cparray15k coupled with real-time qrt-pcr uncovered a number of unique metabolic features in oocysts, the infectious and environmental stage of the parasite.  oocyst stage parasites were found to be highly active in protein synthesis, based on the high transcript levels of genes associated with ribosome biogenesis, transcription and translation. the proteasome and ubiquitin associated components were also highly active, implying that oocysts might employ protein degradation pathways to recycle amino acids in order to overcome the inability to synthesize amino acids de novo. energy metabolism in oocysts was featured by the highest level of expression of lactate dehydrogenase gene. we also studied parasite responses to uv-irradiation, and observed complex and dynamic regulations of gene expression. notable changes included increased transcript levels of genes involved in dna repair and intracellular trafficking. among the stress-related genes, tcp- family members and some thioredoxin-associated genes appear to play more important roles in the recovery of uv-induced damages in the oocysts. our observations also suggest that uv irradiation of oocysts results in increased activities in cytoskeletal rearrangement and intracellular membrane trafficking.  cparray15k is the first microarray chip developed for c. parvum, which provides the cryptosporidium research community a needed tool to study the parasite transcriptome and functional genomics. cparray15k has been successfully used in profiling the gene expressions in the parasite oocysts as well as their responses to uv-irradiation. these observations shed light on how the parasite oocysts might adapt and respond to the hostile external environment and associated stress such as uv irradiation. apicomplexancryptosporidium parvumoocystsagilent microarraytranscriptomeultraviolent irradiation 