BACKGROUND
in insects, hemocyanin superfamily proteins accumulate apparently to serve as sources of amino acids during metamorphosis, reproduction and periods when food is unavailable and the demand for amino acids is high  <cit> .
before the initiation of insect metamorphosis and reproduction, these proteins are progressively stored in larval hemolymph.
hexamerins are mainly synthesized by the fat body during larval development, stored in the hemolymph, and sequestered in the fat body, where they serve as sources of nitrogen and amino acids for pupae and adults during metamorphosis and reproduction  <cit> .
the insect tracheal system has many respiratory proteins that transport oxygen in the hemolymph and, in preparing for metamorphosis, insect larvae store a huge amount of protein in hemolymph  <cit> .
these specialized oxygen-transport proteins evolved from the copper-containing hemocyanins  <cit> .
in most hexapoda, gas exchange is mediated by the tracheal system, a network of tubules that open to the atmosphere on the cuticle and radiate to all parts of the body.
oxygen is delivered through the trachea and tracheoles in the gaseous phase  <cit> ..
insect hexamerins share sequence similarities with other proteins of divergent functions  <cit>  and belong to a protein superfamily that also comprises arthropod phenoloxidases, crustacean pseudohemocyanins, insect storage hexamerins and dipteran hexamerin receptors  <cit> .
the most abundant and widely distributed storage proteins that accumulate in the hemolymph or fat body are composed of six identical or similar subunits of ~ <dig> kda, and thus are also called hexamerins  <cit> .
storage hexamerins include the hexamerins, juvenile hormone-related protein, riboflavin-binding hexamerin precursor, methionine-rich storage protein , very-high-density lipoprotein, tyrosine-rich proteins and hemocyanin-related proteins  <cit> .
genes for storage hexamerins have been cloned from plants, bacteria and fungi.
gordadze et al.
cloned two hexamerin cdnas from the mosquito aedes aegypti  <cit> .
so far, sps and hexamerins have been cloned and studied in musca domestica  <cit> , apriona germari  <cit> , apis mellifera  <cit> , drosophila melanogaster  <cit>  and reticulitermes flavipes  <cit> .
in lepidoptera, sps and hexamerins have been cloned and studied in spodoptera litura  <cit> , plodia interpunctella  <cit> , corcyra cephalonica  <cit> , manduca sexta  <cit> , amsacta albistriga  <cit> , plutella xylostella  <cit> , helicoverpa zea  <cit> , omphisa fuscidentalis  <cit> , and sesamia nonagrioides  <cit> .
spodoptera exigua, commonly called the beet armyworm, is a worldwide agricultural pest that has developed resistance to many chemical pesticides.
in the present study, we cloned cdna for two hexamerin genes from s. exigua .
we observed expression of these two genes not only in fat body, but also in malpighian tubules and other tissues.
furthermore, the genes exhibited differential expression patterns in fat body and they play a role in starvation.
we used rna interference  to investigate the functions of the genes.
RESULTS
sehex and sesp <dig> cdna sequence analysis
sehex had an open reading frame of  <dig> bp, encoding a protein of  <dig> amino acids  with a predicted molecular weight  of approximately  <dig>  kda and an isoelectric point  of  <dig> .
sp <dig> had an open reading frame of  <dig> bp, encoding a protein of  <dig> amino acids  with a predicted mw of approximately  <dig>  kda and pi of  <dig> .
sehex cdna  has 43-74% identity to other known hex genes .
sehex is most similar to hex from the lepidopteran h. armigera .
it is also similar to hex genes from trichoplusia ni, hyalophora cecropia, galleria mellonella and o. fuscidentalis .
sesp <dig> cdna  has 28-96% identity to other known sp <dig> genes .
sesp <dig> is most similar to sp <dig> from the lepidopteran s. litura .
it is also similar to sp <dig> genes from t. ni, s. nonagrioides, h. cecropia, hyphantria cunea, m. sexta, samia cynthia, heliconius erato, bombyx mori, chilo suppressalis, p. interpunctella, a. aegypti, culex quinquefasciatus, anopheles gambiae, periplaneta americana, perla marginata, thermobia domestica, reticulitermes speratus, a. germari, sinella curviseta, tribolium castaneum, tenebrio molitor and r. flavipes .
protein sequence analysis
in figure  <dig>  underlined amino acid residues 1- <dig> and 1- <dig> represent the signal peptides for sehex and sesp <dig>  respectively.
the deduced amino acid sequences for sehex and sesp <dig> contain not only four signature motifs of the hemocyanin family, such as erl , rdp , rlnh  and gfp , but also other conserved motifs.
sequence alignment for lepidopteran hex proteins revealed three highly conserved regions .
alignment of lepidopteran sp <dig> proteins revealed ten highly conserved motifs .
four potential n-glycosylation sites  were found in sehex, but only one potential site  in sesp <dig> .
results for two hexamerin genes from the termite r. flavipes  revealed conserved hexamerin signature motifs   <cit> .
the protein sequence encoded by sehex had only two signature motifs  in common with rfhex.
sequence alignment revealed four conserved signature motifs  for insect hexamerins and storage proteins.
three conserved motifs  were observed for lepidopteran hex genes .
moreover, lepidopteran sp <dig> genes had ten highly conserved regions  .
tissue distribution of hex and sp <dig> in s. exigua
northern blotting revealed strong sehex bands for fat body and midgut and weak bands for cuticle and malpighian tubules, but no transcripts were detected for brain, spermary and trachea samples from s. exigua larvae .
the results suggest that sehex is specifically expressed in fat body, midgut, cuticle and malpighian tubules.
northern blotting results for sesp <dig> transcripts revealed expression in fat body, malpighian tubules and tracheae, but not in brain, cuticle, midgut and spermary .
developmental expression of sehex and sesp1
sehex and sesp <dig> mrnas were expressed in fat body at different levels from fifth instar larvae to pupae.
sehex transcripts were highly expressed in fat body in wandering larvae , as well as in day- <dig> and day- <dig> pupae.
transcripts were present at lower levels in fat body of day- <dig> and day- <dig> fifth instar larvae and the day- <dig> and day- <dig> pupae.
sehex transcripts were very low in fat body of day- <dig> and day- <dig> pupae .
moreover, sehex expression steadily decreased from wandering larvae to 6-day-old pupae.
the results suggest that sehex mrna is constitutively expressed at a rather high level in fat body from the second day of larva stages to the fourth day of pupa stages.
sesp <dig> transcripts were highly expressed in fat body in wandering larvae , as well as in day- <dig> fifth instar larvae and day- <dig>  day- <dig> and day- <dig> pupae.
it was present at lower levels on day- <dig> fifth instar larvae and day- <dig>  day- <dig> and day- <dig> pupae .
comparative rt-pcr results showed that sehex and sesp <dig> mrnas were expressed at very low levels in fat body from first instar larvae to day- <dig> fifth instar larvae.
sehex and sesp <dig> mrnas expression was higher in day- <dig> fifth instar larvae .
sehex and sesp <dig> proteins' function identified on the condition of starvation
rt-pcr analysis showed that sehex and sesp <dig> transcripts were mainly lowly expressed when the insects were been on the condition of starvation.
and sehex and sesp <dig> transcripts expressed in the same mode or tread in the different starvation treatment .
in the group of starvation with  <dig> h, once stress after finals, sehex and sesp <dig> mrna expressional levels lower than control, but higher than the group of starvation with  <dig> h at  <dig> h,  <dig> h and  <dig> h .
in the group of starvation with  <dig> h, once stress was finished, sehex and sesp <dig> mrna expressed decreased and keep the lower expressional levels.
survival rate and sehex and sesp <dig> transcript analysis after double-stranded rna injection
double-stranded rna for sehex and sesp <dig>  was injected into day- <dig> fifth instar larvae.
the survival rate of insects injected with dssehex was  <dig> %,  <dig> %,  <dig> % and  <dig> % at  <dig>   <dig>   <dig> and  <dig> h after injection, respectively, which is significantly lower than the survival of insects in the two control groups .
a sharp decrease in survival rate was observed between  <dig> and  <dig> h after dssehex injection.
the survival rate of insects injected with dssesp <dig> was  <dig> %,  <dig> %,  <dig> % and  <dig> % at  <dig>   <dig>   <dig> and  <dig> h after injection, respectively, which is significantly lower than the survival of insects in the two control groups .
two sharp decreases in survival rate were observed from  <dig> to  <dig> h and from  <dig> to  <dig> h after dssesp <dig> injection.
prior to death, these larvae usually became less vital and smaller in size.
to test the rnai efficiency, semi-quantitative rt-pcr was performed to detect sehex and sesp <dig> transcripts.
sehex and sesp <dig> mrna levels substantially decreased at  <dig>   <dig> and  <dig> h after injection compared to the negative controls  .
however, transcription of both genes recovered to some extent  <dig> h after injection.
discussion
many different storage proteins are found in larvae and pupae at different stages, but not all species have all the storage proteins.
ryan et al.
were the first to identify a 74-kda hexamerin subunit in a. mellifera larval hemolymph  <cit> .
two storage proteins have also been investigated in the honeybee a. mellifera  <cit> , the ant camponotus festinatus  <cit> , and several other ant species  <cit> .
in lepidoptera, two different types of methionine-rich hexamerins can be classified according to their common amino acid composition  <cit> , and three or four sps are found in some species.
storage proteins first reported in t. ni in final-stage larvae  <cit>  and two sps in m. sexta have been separated and cloned  <cit> .
six storage proteins of four different types have been found in caplodes ethlius  <cit> .
in the present study, two storage proteins were cloned from s. exigua .
the results in figure  <dig> show that three storage proteins  are found in the hymenoptera a. aegypti and c. quinquefasciatus.
moreover, no more than three different storage proteins have been found in lepidoptera .
storage proteins serve as a source of amino acids for tissue metamorphosis during pupal development and have been shown to be a component of the sclerotizing system of the cuticle  <cit> .
they also serve as an ecdysteroid carrier in the hemolymph and function in nutrient uptake and storage; some are also capable of binding the insect morphogenetic hormone juvenile hormone  <cit> .
storage proteins are mainly synthesized in fat body during larval development and stored in the hemolymph  <cit> .
corcyra cephalonica hexamerin protein  <dig> was expressed in fat body and carcass tissue, but not in salivary gland, midgut or malpighian tubules  <cit> .
kim et al.
reported that a. germari hexamerin was expressed not only in fat body, but also in midgut, in agreement with our results for sehex <dig>  <cit> .
we found that sehex was expressed not only in fat body and midgut, but also in cuticle and malpighian tubules .
sp <dig> in amsacta albistriga was detected in fat body and pupal ovary  <cit> .
however, the results for sesp <dig> reveal that it is expressed in fat body, malpighian tubules and tracheae .
storage proteins do not bind copper or transport oxygen to the hemolymph  <cit> , and lower expression levels were detected in malpighian tubules and tracheae .
this is consistent with the theory of hemocyanin superfamily evolution.
storage proteins accumulate in large quantities in hemolymph during final instar larvae, are taken up by fat body cells and serve as a reservoir for subsequent development  <cit> .
cchex <dig> mrna was present at all stages of larval development of c. cephalonica and reached a maximum in fat body of final instar larvae  <cit> .
aalsp <dig> expression gradually increased from day- <dig> to day- <dig> final instar larvae  <cit> .
it has been reported that expression levels of insect storage proteins  reach a maximum in the last stage of final instar larvae  <cit> .
sehex and sesp <dig> mrna expression also reached a maximum in the final stage  of last instar larvae .
however, sehex and sesp <dig> mrna expression was much lower before the first day of fifth instar larvae, so it was not detected in the  <dig> μg of total rna used for northern blotting.
in order to get the exact expression patterns of sehex and sesp <dig> mrna in the larvae developmental stages, the more sensitive way of rt-pcr is used.
and rt-pcr results confirmed that expression of these storage proteins is high in final instar larvae .
this result showed that storage protein can be accumulated on the good nutritional status, just as the last larvae in s. exigua.
the function of storage proteins is clear.
in m. sexta, starvation is effective in reducing mrna levels of both hexamerin genes  <cit> .
in the group of starvation with  <dig> h, sehex and sesp <dig> mrna expression was much lower than control , that showed storage proteins are key in the stress of starvation.
hakim and colleagues reported that a monomeric α-arylphorin storage protein can stimulate stem cell proliferation in the lepidopterans m. sexta and spodoptera littoralis and in the beetle leptinotarsa decemlineata.
in addition, feeding an artificial diet containing arylphorin increased the growth rate of several insect species  <cit> .
it is known that storage proteins are crucial for insect development and disruption has a negative effect on the natural development and movement of an insect, which may eventually die.
rnai was used to investigate the function of sehex and sesp <dig> in the present study and resulted in adult survival rates of  <dig> % and  <dig> %, respectively .
moreover, sehex and sesp <dig> mrna was substantially lower at  <dig> h after injection compared to the negative control .
and the expression levels of some control larvae are weird may result in the side effect of inject.
the results confirm that sehex and sesp <dig> mrna expression is crucial for insect life.
abnormal morphological development was not observed after rnai treatment.
it is also found that sehex and sesp <dig> mrna are lower on the condition of starvation.
hence, a lack of nutritional reserves or developmental failure may be key reasons for the lethality.
nevertheless, the sehex and sesp <dig> rnai results suggest that a potential method for control of this pests is to disrupt the regulation of storage proteins.
CONCLUSIONS
we demonstrated that genes for two storage hexamerins exist in s. exigua.
sehex transcripts were expressed not only in fat body, but also in cuticle, midgut and malpighian tubules.
sesp <dig> mrna was expressed in fat body, malpighian tubules and tracheae.
furthermore, sehex and sesp <dig> have differential expression patterns in fat body.
the results of starvation treatment suggest that sehex and sesp <dig> have important functions in s. exigua.
rnai results showed that these proteins are critical for s. exigua metamorphosis, indicating that a potential method for control of this pest is to disrupt the regulation of storage proteins.

 in insects, hemocyanin superfamily proteins accumulate apparently to serve as sources of amino acids during metamorphosis, reproduction and periods when food is unavailable and the demand for amino acids is high. before the initiation of insect metamorphosis and reproduction, these proteins are progressively stored in larval hemolymph. hexamerins are mainly synthesized by the fat body during larval development, stored in the hemolymph, and sequestered in the fat body, where they serve as sources of nitrogen and amino acids for pupae and adults during metamorphosis and reproduction. the insect tracheal system has many respiratory proteins that transport oxygen in the hemolymph and, in preparing for metamorphosis, insect larvae store a huge amount of protein in hemolymph. these specialized oxygen-transport proteins evolved from the copper-containing hemocyanins. in most hexapoda, gas exchange is mediated by the tracheal system, a network of tubules that open to the atmosphere on the cuticle and radiate to all parts of the body. oxygen is delivered through the trachea and tracheoles in the gaseous phase.. insect hexamerins share sequence similarities with other proteins of divergent functions and belong to a protein superfamily that also comprises arthropod phenoloxidases, crustacean pseudohemocyanins, insect storage hexamerins and dipteran hexamerin receptors. the most abundant and widely distributed storage proteins that accumulate in the hemolymph or fat body are composed of six identical or similar subunits of ~ kda, and thus are also called hexamerins. storage hexamerins include the hexamerins, juvenile hormone-related protein, riboflavin-binding hexamerin precursor, methionine-rich storage protein, very-high-density lipoprotein, tyrosine-rich proteins and hemocyanin-related proteins. genes for storage hexamerins have been cloned from plants, bacteria and fungi. gordadze et al. cloned two hexamerin cdnas from the mosquito aedes aegypti. so far, sps and hexamerins have been cloned and studied in musca domestica, apriona germari, apis mellifera, drosophila melanogaster and reticulitermes flavipes. in lepidoptera, sps and hexamerins have been cloned and studied in spodoptera litura, plodia interpunctella, corcyra cephalonica, manduca sexta, amsacta albistriga, plutella xylostella, helicoverpa zea, omphisa fuscidentalis, and sesamia nonagrioides. spodoptera exigua, commonly called the beet armyworm, is a worldwide agricultural pest that has developed resistance to many chemical pesticides. in the present study, we cloned cdna for two hexamerin genes from s. exigua. we observed expression of these two genes not only in fat body, but also in malpighian tubules and other tissues. furthermore, the genes exhibited differential expression patterns in fat body and they play a role in starvation. we used rna interference to investigate the functions of the genes.  sehex and sesp cdna sequence analysis sehex had an open reading frame of bp, encoding a protein of amino acids with a predicted molecular weight of approximately kda and an isoelectric point of. sp had an open reading frame of bp, encoding a protein of amino acids with a predicted mw of approximately kda and pi of. sehex cdna has 43-74% identity to other known hex genes. sehex is most similar to hex from the lepidopteran h. armigera. it is also similar to hex genes from trichoplusia ni, hyalophora cecropia, galleria mellonella and o. fuscidentalis. sesp cdna has 28-96% identity to other known sp genes. sesp is most similar to sp from the lepidopteran s. litura. it is also similar to sp genes from t. ni, s. nonagrioides, h. cecropia, hyphantria cunea, m. sexta, samia cynthia, heliconius erato, bombyx mori, chilo suppressalis, p. interpunctella, a. aegypti, culex quinquefasciatus, anopheles gambiae, periplaneta americana, perla marginata, thermobia domestica, reticulitermes speratus, a. germari, sinella curviseta, tribolium castaneum, tenebrio molitor and r. flavipes. protein sequence analysis in figure underlined amino acid residues 1- and 1- represent the signal peptides for sehex and sesp respectively. the deduced amino acid sequences for sehex and sesp contain not only four signature motifs of the hemocyanin family, such as erl, rdp, rlnh and gfp, but also other conserved motifs. sequence alignment for lepidopteran hex proteins revealed three highly conserved regions. alignment of lepidopteran sp proteins revealed ten highly conserved motifs. four potential n-glycosylation sites were found in sehex, but only one potential site in sesp. results for two hexamerin genes from the termite r. flavipes revealed conserved hexamerin signature motifs. the protein sequence encoded by sehex had only two signature motifs in common with rfhex. sequence alignment revealed four conserved signature motifs for insect hexamerins and storage proteins. three conserved motifs were observed for lepidopteran hex genes. moreover, lepidopteran sp genes had ten highly conserved regions. tissue distribution of hex and sp in s. exigua northern blotting revealed strong sehex bands for fat body and midgut and weak bands for cuticle and malpighian tubules, but no transcripts were detected for brain, spermary and trachea samples from s. exigua larvae. the results suggest that sehex is specifically expressed in fat body, midgut, cuticle and malpighian tubules. northern blotting results for sesp transcripts revealed expression in fat body, malpighian tubules and tracheae, but not in brain, cuticle, midgut and spermary. developmental expression of sehex and sesp1 sehex and sesp mrnas were expressed in fat body at different levels from fifth instar larvae to pupae. sehex transcripts were highly expressed in fat body in wandering larvae, as well as in day- and day- pupae. transcripts were present at lower levels in fat body of day- and day- fifth instar larvae and the day- and day- pupae. sehex transcripts were very low in fat body of day- and day- pupae. moreover, sehex expression steadily decreased from wandering larvae to 6-day-old pupae. the results suggest that sehex mrna is constitutively expressed at a rather high level in fat body from the second day of larva stages to the fourth day of pupa stages. sesp transcripts were highly expressed in fat body in wandering larvae, as well as in day- fifth instar larvae and day- day- and day- pupae. it was present at lower levels on day- fifth instar larvae and day- day- and day- pupae. comparative rt-pcr results showed that sehex and sesp mrnas were expressed at very low levels in fat body from first instar larvae to day- fifth instar larvae. sehex and sesp mrnas expression was higher in day- fifth instar larvae. sehex and sesp proteins' function identified on the condition of starvation rt-pcr analysis showed that sehex and sesp transcripts were mainly lowly expressed when the insects were been on the condition of starvation. and sehex and sesp transcripts expressed in the same mode or tread in the different starvation treatment. in the group of starvation with h, once stress after finals, sehex and sesp mrna expressional levels lower than control, but higher than the group of starvation with h at h, h and h. in the group of starvation with h, once stress was finished, sehex and sesp mrna expressed decreased and keep the lower expressional levels. survival rate and sehex and sesp transcript analysis after double-stranded rna injection double-stranded rna for sehex and sesp was injected into day- fifth instar larvae. the survival rate of insects injected with dssehex was %, %, % and % at and h after injection, respectively, which is significantly lower than the survival of insects in the two control groups. a sharp decrease in survival rate was observed between and h after dssehex injection. the survival rate of insects injected with dssesp was %, %, % and % at and h after injection, respectively, which is significantly lower than the survival of insects in the two control groups. two sharp decreases in survival rate were observed from to h and from to h after dssesp injection. prior to death, these larvae usually became less vital and smaller in size. to test the rnai efficiency, semi-quantitative rt-pcr was performed to detect sehex and sesp transcripts. sehex and sesp mrna levels substantially decreased at and h after injection compared to the negative controls. however, transcription of both genes recovered to some extent h after injection. discussion many different storage proteins are found in larvae and pupae at different stages, but not all species have all the storage proteins. ryan et al. were the first to identify a 74-kda hexamerin subunit in a. mellifera larval hemolymph. two storage proteins have also been investigated in the honeybee a. mellifera, the ant camponotus festinatus, and several other ant species. in lepidoptera, two different types of methionine-rich hexamerins can be classified according to their common amino acid composition, and three or four sps are found in some species. storage proteins first reported in t. ni in final-stage larvae and two sps in m. sexta have been separated and cloned. six storage proteins of four different types have been found in caplodes ethlius. in the present study, two storage proteins were cloned from s. exigua. the results in figure show that three storage proteins are found in the hymenoptera a. aegypti and c. quinquefasciatus. moreover, no more than three different storage proteins have been found in lepidoptera. storage proteins serve as a source of amino acids for tissue metamorphosis during pupal development and have been shown to be a component of the sclerotizing system of the cuticle. they also serve as an ecdysteroid carrier in the hemolymph and function in nutrient uptake and storage; some are also capable of binding the insect morphogenetic hormone juvenile hormone. storage proteins are mainly synthesized in fat body during larval development and stored in the hemolymph. corcyra cephalonica hexamerin protein was expressed in fat body and carcass tissue, but not in salivary gland, midgut or malpighian tubules. kim et al. reported that a. germari hexamerin was expressed not only in fat body, but also in midgut, in agreement with our results for sehex. we found that sehex was expressed not only in fat body and midgut, but also in cuticle and malpighian tubules. sp in amsacta albistriga was detected in fat body and pupal ovary. however, the results for sesp reveal that it is expressed in fat body, malpighian tubules and tracheae. storage proteins do not bind copper or transport oxygen to the hemolymph, and lower expression levels were detected in malpighian tubules and tracheae. this is consistent with the theory of hemocyanin superfamily evolution. storage proteins accumulate in large quantities in hemolymph during final instar larvae, are taken up by fat body cells and serve as a reservoir for subsequent development. cchex mrna was present at all stages of larval development of c. cephalonica and reached a maximum in fat body of final instar larvae. aalsp expression gradually increased from day- to day- final instar larvae. it has been reported that expression levels of insect storage proteins reach a maximum in the last stage of final instar larvae. sehex and sesp mrna expression also reached a maximum in the final stage of last instar larvae. however, sehex and sesp mrna expression was much lower before the first day of fifth instar larvae, so it was not detected in the μg of total rna used for northern blotting. in order to get the exact expression patterns of sehex and sesp mrna in the larvae developmental stages, the more sensitive way of rt-pcr is used. and rt-pcr results confirmed that expression of these storage proteins is high in final instar larvae. this result showed that storage protein can be accumulated on the good nutritional status, just as the last larvae in s. exigua. the function of storage proteins is clear. in m. sexta, starvation is effective in reducing mrna levels of both hexamerin genes. in the group of starvation with h, sehex and sesp mrna expression was much lower than control, that showed storage proteins are key in the stress of starvation. hakim and colleagues reported that a monomeric α-arylphorin storage protein can stimulate stem cell proliferation in the lepidopterans m. sexta and spodoptera littoralis and in the beetle leptinotarsa decemlineata. in addition, feeding an artificial diet containing arylphorin increased the growth rate of several insect species. it is known that storage proteins are crucial for insect development and disruption has a negative effect on the natural development and movement of an insect, which may eventually die. rnai was used to investigate the function of sehex and sesp in the present study and resulted in adult survival rates of % and %, respectively. moreover, sehex and sesp mrna was substantially lower at h after injection compared to the negative control. and the expression levels of some control larvae are weird may result in the side effect of inject. the results confirm that sehex and sesp mrna expression is crucial for insect life. abnormal morphological development was not observed after rnai treatment. it is also found that sehex and sesp mrna are lower on the condition of starvation. hence, a lack of nutritional reserves or developmental failure may be key reasons for the lethality. nevertheless, the sehex and sesp rnai results suggest that a potential method for control of this pests is to disrupt the regulation of storage proteins.  we demonstrated that genes for two storage hexamerins exist in s. exigua. sehex transcripts were expressed not only in fat body, but also in cuticle, midgut and malpighian tubules. sesp mrna was expressed in fat body, malpighian tubules and tracheae. furthermore, sehex and sesp have differential expression patterns in fat body. the results of starvation treatment suggest that sehex and sesp have important functions in s. exigua. rnai results showed that these proteins are critical for s. exigua metamorphosis, indicating that a potential method for control of this pest is to disrupt the regulation of storage proteins. 
these findings provide new data on the tissue distribution, expression patterns and the function in starvation of storage proteins. although insects possess storage hexamerins, very little is known about the character and specific functions of hexamerin and storage protein in insect development. rna interference results revealed that storage protein genes are key in metamorphosis, reproduction and insect development. northern blotting analyses revealed that sehex mrna is expressed in the fat body, cuticle, midgut and malpighian tubules and sesp in fat body, malpighian tubules and tracheae. sehex and sesp mrnas were expressed in fat body at different levels from first instar larvae to pupae, with expression was much lower from first instar larvae to first-day fifth instar larvae. sesp transcript expression was high in fat body of wandering larvae, 2-day-old fifth instar larvae and 2-, 4- and 7-day-old pupae. storage hexamerins are important members of the hemocyanin superfamily. the results for sehex and sesp interference reveal that a potential method to control this pest is to disrupt the regulation of storage proteins. in insects, hemocyanin superfamily proteins accumulate apparently to serve as sources of amino acids during metamorphosis, reproduction and development. s. exigua hexamerin cdna contained an open reading frame of nucleotides encoding a protein of amino acids with a predicted molecular weight of kda. s. exigua storage protein cdna contained an open reading frame of bp encoding a protein of amino acids with a predicted molecular weight of ~ kda. to gain insight into the function of storage proteins in insects, cdnas for two storage proteins were cloned from the fat body of spodoptera exigua. sehex transcript expression was high in fat body of wandering larvae and steadily decreased to the seventh pupal day.
 in insects, hemocyanin superfamily proteins accumulate apparently to serve as sources of amino acids during metamorphosis, reproduction and development. storage hexamerins are important members of the hemocyanin superfamily. although insects possess storage hexamerins, very little is known about the character and specific functions of hexamerin and storage protein in insect development.  to gain insight into the function of storage proteins in insects, cdnas for two storage proteins were cloned from the fat body of spodoptera exigua. s. exigua hexamerin cdna contained an open reading frame of nucleotides encoding a protein of amino acids with a predicted molecular weight of kda. s. exigua storage protein cdna contained an open reading frame of bp encoding a protein of amino acids with a predicted molecular weight of ~ kda. northern blotting analyses revealed that sehex mrna is expressed in the fat body, cuticle, midgut and malpighian tubules and sesp in fat body, malpighian tubules and tracheae. sehex and sesp mrnas were expressed in fat body at different levels from first instar larvae to pupae, with expression was much lower from first instar larvae to first-day fifth instar larvae. sehex transcript expression was high in fat body of wandering larvae and steadily decreased to the seventh pupal day. sesp transcript expression was high in fat body of wandering larvae, 2-day-old fifth instar larvae and 2-, 4- and 7-day-old pupae. sehex and sesp mrnas levels were expressed lower than control on the condition of starvation at h. of insects injected with sehex and sesp dsrna, % and % survived to h after treatment, respectively. this was significantly lower than in the controls groups.  these findings provide new data on the tissue distribution, expression patterns and the function in starvation of storage proteins. rna interference results revealed that storage protein genes are key in metamorphosis, reproduction and insect development. the results for sehex and sesp interference reveal that a potential method to control this pest is to disrupt the regulation of storage proteins. 