BACKGROUND
innate immunity is of great importance to insects and crustaceans because they lack antibodies  <cit> .
innate immunity involves phagocytosis, encapsulation, hemocyte coagulation and activation of the prophenoloxidase  or melanization cascade  <cit> .
activation of propo generates phenoloxidase , which catalyzes the oxygenation of monophenols to o-diphenols and the oxidation of o-diphenols to the corresponding o-quinones  <cit> .
these are reactive intermediates for melanin synthesis and other physiological processes such as cuticle sclerotization, wound healing and pathogen sequestration  <cit> .
in most cases, the propo cascade is triggered by a small amount of microbe-derived molecules such as lipopolysaccharides , β- <dig>  3-glucans, or peptidoglycan.
pattern-recognition proteins bind these molecules and initiate the propo system through a quick proteolytic cascade, and many proteins involved in the proteolytic cascade are serine proteinases  <cit> .
the final serine proteinase that converts the inactive propo into its active form is called prophenoloxidase-activating enzyme   <cit> .
the propo genes of insects and crustaceans have been intensively studied  <cit> , and models for the serine proteinase cascades regulating propo activation have been studied in detail in some insects such as manduca sexta and tenebrio molitor  <cit> .
studies on propo-activating cascades are limited in crustaceans.
hitherto, few ppaes in crustaceans have been reported, except for in crayfish, pacifastacus leniusculus and shrimp, penaeus monodon  <cit> .
in this study, the ppae <dig> of white shrimp litopenaeus vannamei was identified and its expression feature was reported.
the new findings of the study will contribute to our understanding of po activating system of crustaceans.
RESULTS
isolation and lvppae <dig> sequence
the full-length lvppae <dig> cdna was  <dig> bp, with an orf of  <dig>  a 5'-utr of  <dig> bp, and a 3'-utp of  <dig> bp.
the theoretical pi and mw were  <dig>  and  <dig>  kda.
the proteolytic activation site was found between arg <dig> and ile <dig>  the pi and mw of activated lvppae <dig> were  <dig>  and  <dig>  kda.
the lvppae <dig> protein showed 94% identity to pmppae <dig> and 61% to plppae.
three amino acid residues  corresponded to the catalytic triad of the sp domain and another three amino acid residues  to the substrate binding sites.
multiple alignments between the sequences of lvppae <dig>  plppae, and pmppae <dig> showed that this gene is relatively well conserved in crustaceans .
lvppae <dig> exhibited a typical clip-domain structure, composed of six cysteines at the n-terminus.
similar to other crustacean ppaes, a glycine-rich domain was found between residues  <dig> and  <dig>  with  <dig> % glycines , and a proline-rich domain was found between residues  <dig> and  <dig>  with  <dig> % prolines .
lvppae phylogenetic analysis
a phylogenetic tree was constructed using the neighbor-joining method  by comparing the deduced amino acid sequence of the conserved sp domain to the  <dig> clip-domain serine proteinases showing the most similarity or any reported ppaes.
the  <dig> proteinases were separated into two main branches: one mainly composed of insect prophenoloxidase-activating proteins and the other composed of insect pro-clotting enzymes.
the ppaes of crustaceans were classified into the second main branch and they shared the same and a unique sub-branch.
except for seven pro-clotting enzymes and five trypsins, the biological roles of the clip-domain serine proteinases from the second main branch are still unclear.
tissue-specific gene expression in healthy shrimp and bacteria-challenged shrimp
lvppae <dig> gene expression was analyzed by qrt-pcr.
lvppae <dig> in spf shrimps was mainly expressed in hemocytes and gill, whilst low level was detected in the other tissues.
the relative expression in fhs was about  <dig> -fold in gill.
forty eight hours after vibrio challenge, the relative expression level of lvppae <dig> in gill was as same as that in fhs, which indicated an increase in the gill .
gill samples from the challenged shrimps were checked using microscope with phase contrast mode.
intensive hemocyte aggregation was found in gill canals .
temporal expression of lvppae <dig> corresponding to bacterial challenge
hemocytes and gill were sampled at each time point after the challenge.
qrt-pcr showed that the lvppae <dig> mrna expression level began to decline in fhs in a short time post challenge.
this suppression became more obvious after  <dig> hpc ; the expression level decreased by 60% compared to the control .
after  <dig> hpc, the expression recovered slightly but then declined again and remained suppressed after  <dig> hpc.
multiple comparison between challenged and saline injected groups at each time point showed the suppression of lvppae <dig> were significant at  <dig>   <dig>   <dig> and  <dig> hpc.
temporal expression in saline injected shrimp was not changed significantly.
the two results, normalized to  <dig> s and β-actin separately, were well matched .
however, the relative change in expression in gills was different.
at  <dig> hpc, lvppae <dig> expression in gill was significantly enhanced to about  <dig> -fold than that of the control .
after a relative decline at  <dig> hpc, the expression level increased to 3-fold compared to the control and the high expression was sustained for  <dig> hpc .
gene expression analysis in shrimp reared in different bacterial environments
vibrio in the biofloc and filtered seawater was calculated following colony forming unit method after a 24-h culture.
the vibrio number in biofloc seawater was  <dig>  ×  <dig> cfu/ml, whereas  <dig> cfu/ml were found in filtered seawater .
the relative lvppae <dig> mrna expression level in fhs of shrimp reared in different bacterial environments was analyzed by qrt-pcr.
five samples were taken as replicates for expression analysis.
the relative lvppae <dig> mrna expression level in shrimps exposed to a higher bacteria environment was  <dig> -fold than that of those exposed to a lower level of bacteria .
rna interference of lvppae1
qrt-pcr was used to detect the dsrna interference effect  <dig> and  <dig> h after the first dsrna injection and  <dig> h after the second injection.
total rna extracted from the hemocytes of gene-silenced shrimp was analyzed.
rnai strongly inhibited lvppae <dig> expression.
the amount of mrna decreased to less than 5% of the control  <dig> h after the second lvppae <dig> dsrna injection .
susceptibility of shrimp to vibrio harveyi after gene silencing
susceptibility of shrimps to v. harveyi was tested after gene silence.
about  <dig> h after the challenge, shrimps in the gene-silenced group began to show symptoms of slowed mobility and imbalance, as well as mortality after  <dig> h. saline-injected shrimp showed symptoms after at least  <dig> h, and mortality began after  <dig> h. mortality in the saline pre-injected group reached about 50% after  <dig> h, and all gene-silenced shrimp died within  <dig> h. no mortality was found in the saline-only injected group and occasional mortality was found in the lvppae <dig> dsrna-only injected group .
po activity assay
po activity was analyzed in shrimp injected with gene-specific dsrna, gfp dsrna, or saline solution.
po-induced activity in the gene-silenced shrimp decreased by approximately 50% compared to the saline-injected control group.
no such decrease was found in the gfp dsrna-injected shrimp .
discussion
we identified and characterized a clip-domain serine proteinase from the white shrimp, l. vannamei.
the deduced amino acid of this gene showed high similarity to that of plppae  and particularly high similarity to pmppae <dig> , which is the terminal enzyme in the prophenoloxidase-activating cascade  <cit> .
thus, we named this gene lvppae <dig>  multiple alignment results indicated that this gene is rather well conserved in crustaceans.
interestingly, phylogenetic analyses revealed that all ppaes in crustaceans branched off into the cluster of insect pro-clotting proteinases rather than insect ppaes.
besides with molecular structure deviation, ppaes in crustaceans can proteolyses propo dependently  <cit> , unlike most of insect ppaes that need cofactors to active propos  <cit> .
another major difference between ppaes in crustaceans and insects is the different regulation type to pathogen invasion.
three prophenoloxidase activating proteinase of the horn worm manduca sexta were reported to be stimulated or up-regulated by injection of micrococcus lysodeikticus or escherichia coli  <cit> .
five putative ppaes of the fly drosophila were induced to higher levels after e. coli and micrococcus luteus infection  <cit> .
two potential ppaes of the mosquito anopheles gambiae were up-regulated by bacteria e. coli or m. luteus and parasite plasmodium berghei  <cit> .
a prophenoloxidase activation proteinase of the silk worm samia cynthia ricini was inducible by cell wall components of gram-positive bacteria  <cit> .
contrary to those insect ppaes, the crayfish ppae transcript level was reported to be unaffected upon aeromonas hydrophila challenge  <cit> .
the pmppae <dig> transcript level in p. monodon was down regulated 3- <dig> h after vibrio injection  <cit> .
in the present study, the lvppae <dig> transcript abundance was reduced within  <dig> h after vibrio challenge.
all reported crustacean ppaes as well as our study showed their transcript levels were either not affected or down-regulated within  <dig> h, contrary to ppaes in insects that were up-regulated or stimulated within  <dig> h after challenge.
dissimilarity in proteinase structures and functional features imply certain degree of difference exists between crustaceans and insects for the prophenoloxidase-activating mechanism, particularly in the down stream.
it has been reported that the ppae <dig> mrna of crustaceans were found specially in hemocyte and free from other tissues  <cit> .
our tissue distribution analysis by qrt-pcr also revealed that lvppae <dig> transcript level was very low in muscle, antenna gland, nerve cord and digesting system, and this minimum expression also could be caused by a few amount of hemocytes penetrated into the tissues  <cit> .
the most convincing proof was that the lvppae <dig> transcript level in those tissues didn't change after bacteria challenge, suggesting that these tissues were in fact free of lvppae <dig> transcripts.
this conclusion is in accordance with the previous report  <cit> .
however, it is notable that during bacteria clearance process in shrimps, bacteria or foreign materials would be encapsulated by hemocytes to form hemocyte-foreign material aggregation and this kind of aggregation happens mainly in gill, which is the main bacteria or alien materials clearance site  <cit> .
this phenomenon was also confirmed by our microscope observation .
in another word, gill is closely related with hemocytic immunity in host defense of shrimps.
by sensitive qrt-pcr method, we successfully found out the inevitable repercussion of this close relation: the lvppae <dig> mrna transcripts represented a considerable level and even higher after vibrio challenge.
our rnai experiment showed silence of lvppae <dig> significantly reduced the po activity and increased the susceptibility to v. harveyi.
qrt-pcr revealed shrimps exposed in a bacteria rich environment for a long-term presented a higher lvppae <dig> transcript level.
those results suggested the importance of lvppae <dig> in the host defense of l. vannamei.
it is thus odd to find the expression of lvppae <dig> was depressed when shrimps tried to fight against the injected bacteria.
same phenomenon was found in another shrimp species p. monodon, yet the author didn't give an explanation.
our findings in gill gave a clue to answer the doubtful question.
although the transcript expression of lvppae <dig> in gill was in fact due to the fixed hemocytes , the transcript level of ghs showed different regulation type than in fhs.
contrary to the continuous dropping trend in fhs, the transcript level of lvppae <dig> in gill was stimulated quickly and arrived to a peak at  <dig> h when the depression in fhs was at the upmost degree.
higher lvppae <dig> mrna transcript level was maintained in gill within  <dig> h when the lvppae <dig> was continuously depressed in fhs.
by now, a new conclusion was drawn that the lvppae <dig> was not simply down-regulated by vibrio challenge but also up-regulated in bacteria clearance site.
by the current data, it can not be asserted that the overall lvppae <dig> transcript level was depressed or enhanced shortly after the vibrio challenge; we thus call the expression feature as the "selective enhanced expression".
because the selective enhanced expression in gill happens simultaneously with the suppression in fluid hemocytes, the endogenous regulation mechanism for the short time enhanced expression in bacteria clearance site is probably different from the enhanced expression in fhs under a long-term, bacteria-rich environment stress.
our current study on lvppae <dig> indicated that the endogenous regulation mechanism for shrimp immunity is far from clarified.
CONCLUSIONS
a prophenoloxidase activating enzyme  of the white shrimp l. vannamei was cloned and sequenced.
in vivo gene silence of lvppae <dig> significantly deduced the po activity and increased the susceptibility of shrimps to gram negative bacteria v. harveyi.
the overall tissue distribution analysis showed the lvppae <dig> was mainly expressed in fluid hemocytes  and gill due to the aggregated hemocytes in canal .
the lvppae <dig> expression was significantly enhanced in gill within  <dig> h after v. harveyi challenge.
in the case of fh, lvppae <dig> expression was suppressed within  <dig> h after a single challenge, whereas higher expression was found when shrimp was exposed to a high bacterial environment over the long term.
the different gene expression responses to bacterial challenge in fh and gill suggest enhanced lvppae <dig> expression at the bacteria-clearance site.
in conclusion, the lvppae <dig> is involved in melanization cascade and plays an important role in shrimp host defense against bacteria.
the expression in fh was suppressed in a short term after v. harveyi challenge but was up regulated under bacterial press for a long term.
the lvppae <dig> represents a feature of selective enhanced expression in bacteria clearance site after challenge.
this is a novel characteristic for crustacean ppaes expression.

 innate immunity is of great importance to insects and crustaceans because they lack antibodies. innate immunity involves phagocytosis, encapsulation, hemocyte coagulation and activation of the prophenoloxidase or melanization cascade. activation of propo generates phenoloxidase, which catalyzes the oxygenation of monophenols to o-diphenols and the oxidation of o-diphenols to the corresponding o-quinones. these are reactive intermediates for melanin synthesis and other physiological processes such as cuticle sclerotization, wound healing and pathogen sequestration. in most cases, the propo cascade is triggered by a small amount of microbe-derived molecules such as lipopolysaccharides, β- 3-glucans, or peptidoglycan. pattern-recognition proteins bind these molecules and initiate the propo system through a quick proteolytic cascade, and many proteins involved in the proteolytic cascade are serine proteinases. the final serine proteinase that converts the inactive propo into its active form is called prophenoloxidase-activating enzyme. the propo genes of insects and crustaceans have been intensively studied, and models for the serine proteinase cascades regulating propo activation have been studied in detail in some insects such as manduca sexta and tenebrio molitor. studies on propo-activating cascades are limited in crustaceans. hitherto, few ppaes in crustaceans have been reported, except for in crayfish, pacifastacus leniusculus and shrimp, penaeus monodon. in this study, the ppae of white shrimp litopenaeus vannamei was identified and its expression feature was reported. the new findings of the study will contribute to our understanding of po activating system of crustaceans.  isolation and lvppae sequence the full-length lvppae cdna was bp, with an orf of a 5'-utr of bp, and a 3'-utp of bp. the theoretical pi and mw were and kda. the proteolytic activation site was found between arg and ile the pi and mw of activated lvppae were and kda. the lvppae protein showed 94% identity to pmppae and 61% to plppae. three amino acid residues corresponded to the catalytic triad of the sp domain and another three amino acid residues to the substrate binding sites. multiple alignments between the sequences of lvppae plppae, and pmppae showed that this gene is relatively well conserved in crustaceans. lvppae exhibited a typical clip-domain structure, composed of six cysteines at the n-terminus. similar to other crustacean ppaes, a glycine-rich domain was found between residues and with % glycines, and a proline-rich domain was found between residues and with % prolines. lvppae phylogenetic analysis a phylogenetic tree was constructed using the neighbor-joining method by comparing the deduced amino acid sequence of the conserved sp domain to the clip-domain serine proteinases showing the most similarity or any reported ppaes. the proteinases were separated into two main branches: one mainly composed of insect prophenoloxidase-activating proteins and the other composed of insect pro-clotting enzymes. the ppaes of crustaceans were classified into the second main branch and they shared the same and a unique sub-branch. except for seven pro-clotting enzymes and five trypsins, the biological roles of the clip-domain serine proteinases from the second main branch are still unclear. tissue-specific gene expression in healthy shrimp and bacteria-challenged shrimp lvppae gene expression was analyzed by qrt-pcr. lvppae in spf shrimps was mainly expressed in hemocytes and gill, whilst low level was detected in the other tissues. the relative expression in fhs was about -fold in gill. forty eight hours after vibrio challenge, the relative expression level of lvppae in gill was as same as that in fhs, which indicated an increase in the gill. gill samples from the challenged shrimps were checked using microscope with phase contrast mode. intensive hemocyte aggregation was found in gill canals. temporal expression of lvppae corresponding to bacterial challenge hemocytes and gill were sampled at each time point after the challenge. qrt-pcr showed that the lvppae mrna expression level began to decline in fhs in a short time post challenge. this suppression became more obvious after hpc; the expression level decreased by 60% compared to the control. after hpc, the expression recovered slightly but then declined again and remained suppressed after hpc. multiple comparison between challenged and saline injected groups at each time point showed the suppression of lvppae were significant at and hpc. temporal expression in saline injected shrimp was not changed significantly. the two results, normalized to s and β-actin separately, were well matched. however, the relative change in expression in gills was different. at hpc, lvppae expression in gill was significantly enhanced to about -fold than that of the control. after a relative decline at hpc, the expression level increased to 3-fold compared to the control and the high expression was sustained for hpc. gene expression analysis in shrimp reared in different bacterial environments vibrio in the biofloc and filtered seawater was calculated following colony forming unit method after a 24-h culture. the vibrio number in biofloc seawater was × cfu/ml, whereas cfu/ml were found in filtered seawater. the relative lvppae mrna expression level in fhs of shrimp reared in different bacterial environments was analyzed by qrt-pcr. five samples were taken as replicates for expression analysis. the relative lvppae mrna expression level in shrimps exposed to a higher bacteria environment was -fold than that of those exposed to a lower level of bacteria. rna interference of lvppae1 qrt-pcr was used to detect the dsrna interference effect and h after the first dsrna injection and h after the second injection. total rna extracted from the hemocytes of gene-silenced shrimp was analyzed. rnai strongly inhibited lvppae expression. the amount of mrna decreased to less than 5% of the control h after the second lvppae dsrna injection. susceptibility of shrimp to vibrio harveyi after gene silencing susceptibility of shrimps to v. harveyi was tested after gene silence. about h after the challenge, shrimps in the gene-silenced group began to show symptoms of slowed mobility and imbalance, as well as mortality after h. saline-injected shrimp showed symptoms after at least h, and mortality began after h. mortality in the saline pre-injected group reached about 50% after h, and all gene-silenced shrimp died within h. no mortality was found in the saline-only injected group and occasional mortality was found in the lvppae dsrna-only injected group. po activity assay po activity was analyzed in shrimp injected with gene-specific dsrna, gfp dsrna, or saline solution. po-induced activity in the gene-silenced shrimp decreased by approximately 50% compared to the saline-injected control group. no such decrease was found in the gfp dsrna-injected shrimp. discussion we identified and characterized a clip-domain serine proteinase from the white shrimp, l. vannamei. the deduced amino acid of this gene showed high similarity to that of plppae and particularly high similarity to pmppae, which is the terminal enzyme in the prophenoloxidase-activating cascade. thus, we named this gene lvppae multiple alignment results indicated that this gene is rather well conserved in crustaceans. interestingly, phylogenetic analyses revealed that all ppaes in crustaceans branched off into the cluster of insect pro-clotting proteinases rather than insect ppaes. besides with molecular structure deviation, ppaes in crustaceans can proteolyses propo dependently, unlike most of insect ppaes that need cofactors to active propos. another major difference between ppaes in crustaceans and insects is the different regulation type to pathogen invasion. three prophenoloxidase activating proteinase of the horn worm manduca sexta were reported to be stimulated or up-regulated by injection of micrococcus lysodeikticus or escherichia coli. five putative ppaes of the fly drosophila were induced to higher levels after e. coli and micrococcus luteus infection. two potential ppaes of the mosquito anopheles gambiae were up-regulated by bacteria e. coli or m. luteus and parasite plasmodium berghei. a prophenoloxidase activation proteinase of the silk worm samia cynthia ricini was inducible by cell wall components of gram-positive bacteria. contrary to those insect ppaes, the crayfish ppae transcript level was reported to be unaffected upon aeromonas hydrophila challenge. the pmppae transcript level in p. monodon was down regulated 3- h after vibrio injection. in the present study, the lvppae transcript abundance was reduced within h after vibrio challenge. all reported crustacean ppaes as well as our study showed their transcript levels were either not affected or down-regulated within h, contrary to ppaes in insects that were up-regulated or stimulated within h after challenge. dissimilarity in proteinase structures and functional features imply certain degree of difference exists between crustaceans and insects for the prophenoloxidase-activating mechanism, particularly in the down stream. it has been reported that the ppae mrna of crustaceans were found specially in hemocyte and free from other tissues. our tissue distribution analysis by qrt-pcr also revealed that lvppae transcript level was very low in muscle, antenna gland, nerve cord and digesting system, and this minimum expression also could be caused by a few amount of hemocytes penetrated into the tissues. the most convincing proof was that the lvppae transcript level in those tissues didn't change after bacteria challenge, suggesting that these tissues were in fact free of lvppae transcripts. this conclusion is in accordance with the previous report. however, it is notable that during bacteria clearance process in shrimps, bacteria or foreign materials would be encapsulated by hemocytes to form hemocyte-foreign material aggregation and this kind of aggregation happens mainly in gill, which is the main bacteria or alien materials clearance site. this phenomenon was also confirmed by our microscope observation. in another word, gill is closely related with hemocytic immunity in host defense of shrimps. by sensitive qrt-pcr method, we successfully found out the inevitable repercussion of this close relation: the lvppae mrna transcripts represented a considerable level and even higher after vibrio challenge. our rnai experiment showed silence of lvppae significantly reduced the po activity and increased the susceptibility to v. harveyi. qrt-pcr revealed shrimps exposed in a bacteria rich environment for a long-term presented a higher lvppae transcript level. those results suggested the importance of lvppae in the host defense of l. vannamei. it is thus odd to find the expression of lvppae was depressed when shrimps tried to fight against the injected bacteria. same phenomenon was found in another shrimp species p. monodon, yet the author didn't give an explanation. our findings in gill gave a clue to answer the doubtful question. although the transcript expression of lvppae in gill was in fact due to the fixed hemocytes, the transcript level of ghs showed different regulation type than in fhs. contrary to the continuous dropping trend in fhs, the transcript level of lvppae in gill was stimulated quickly and arrived to a peak at h when the depression in fhs was at the upmost degree. higher lvppae mrna transcript level was maintained in gill within h when the lvppae was continuously depressed in fhs. by now, a new conclusion was drawn that the lvppae was not simply down-regulated by vibrio challenge but also up-regulated in bacteria clearance site. by the current data, it can not be asserted that the overall lvppae transcript level was depressed or enhanced shortly after the vibrio challenge; we thus call the expression feature as the "selective enhanced expression". because the selective enhanced expression in gill happens simultaneously with the suppression in fluid hemocytes, the endogenous regulation mechanism for the short time enhanced expression in bacteria clearance site is probably different from the enhanced expression in fhs under a long-term, bacteria-rich environment stress. our current study on lvppae indicated that the endogenous regulation mechanism for shrimp immunity is far from clarified.  a prophenoloxidase activating enzyme of the white shrimp l. vannamei was cloned and sequenced. in vivo gene silence of lvppae significantly deduced the po activity and increased the susceptibility of shrimps to gram negative bacteria v. harveyi. the overall tissue distribution analysis showed the lvppae was mainly expressed in fluid hemocytes and gill due to the aggregated hemocytes in canal. the lvppae expression was significantly enhanced in gill within h after v. harveyi challenge. in the case of fh, lvppae expression was suppressed within h after a single challenge, whereas higher expression was found when shrimp was exposed to a high bacterial environment over the long term. the different gene expression responses to bacterial challenge in fh and gill suggest enhanced lvppae expression at the bacteria-clearance site. in conclusion, the lvppae is involved in melanization cascade and plays an important role in shrimp host defense against bacteria. the expression in fh was suppressed in a short term after v. harveyi challenge but was up regulated under bacterial press for a long term. the lvppae represents a feature of selective enhanced expression in bacteria clearance site after challenge. this is a novel characteristic for crustacean ppaes expression. 
the prophenoloxidase-activating system plays an important role in the crustacean innate immunity, particularly in wound healing and pathogen defense. here we report on a ppae of white shrimp, litopenaeus vannamei, which showed 94% similarity to ppae of penaeus monodon. although lvppae was down-regulated in fluid hemocytes by vibrio challenge, its expression increased significantly in gill after bacteria injection, which is the primary bacteria-clearance tissue. we found that lvppae in fluid hemocytes was down regulated after challenge by vibrio harveyi but was enhanced when shrimps were exposed to a bacteria-rich environment for long-term. in vivo gene silence of lvppae by rnai can significantly reduce the phenoloxidase activity and increase the susceptibility of shrimps to v. harveyi. suppressed expression in fluid hemocytes and enhanced expression in gill indicates selectively enhanced expression at the bacterial clearance site. the results will contribute to our understanding of the po activating system in crustaceans.
 the prophenoloxidase-activating system plays an important role in the crustacean innate immunity, particularly in wound healing and pathogen defense. a key member of this system is prophenoloxidase-activating enzyme, which is the direct activator of prophenoloxidase. despite their importance in crustacean po activating system, the studies on them remain limited.  here we report on a ppae of white shrimp, litopenaeus vannamei, which showed 94% similarity to ppae of penaeus monodon. we found that lvppae in fluid hemocytes was down regulated after challenge by vibrio harveyi but was enhanced when shrimps were exposed to a bacteria-rich environment for long-term. in vivo gene silence of lvppae by rnai can significantly reduce the phenoloxidase activity and increase the susceptibility of shrimps to v. harveyi. although lvppae was down-regulated in fluid hemocytes by vibrio challenge, its expression increased significantly in gill after bacteria injection, which is the primary bacteria-clearance tissue.  suppressed expression in fluid hemocytes and enhanced expression in gill indicates selectively enhanced expression at the bacterial clearance site. this is a novel feature for ppae expression. the results will contribute to our understanding of the po activating system in crustaceans. 