BACKGROUND
in insects, cholesterol is required as a structural component of cellular membranes and as a precursor for the insect molting hormone .
however, insects lack several enzymes in the cholesterol biosynthesis pathway  <cit> , which makes them auxotrophic for exogenous  cholesterol for satisfactory growth, development and reproduction  <cit> .
therefore, cholesterol uptake and transportation are critical for their survival.
uptake of cholesterol involves intracellular transfer and intercellular transportation.
intracellular cholesterol transfer enables the redistribution of cholesterol within a cell, and intercellular transport is needed for the distribution of cholesterol between cells.
it is very well documented that lipophorin  and lipid transport particles  are responsible for the intercellular transportation of cholesterol and lipids in insects  <cit> .
cholesterol is highly hydrophobic with solubility of ~ <dig> nm in water at room temperature  <cit> , can be easily inserted into the lipid bilayer and has a fast flip-flop rate  within the lipid bilayer  <cit> .
diffusion of cholesterol into the lipid bilayer can happen readily, and an aqueous diffusion model has been proposed to account for some of the cholesterol uptake by cells  <cit> .
however, the desorption of membrane-bound cholesterol is slow without the aid of a carrier protein  <cit>  to enhance the desorption of membrane-bound cholesterol through the collision with membrane-bound cholesterol  <cit> .
in vertebrates, sterol carrier protein- <dig> increases the rate of cholesterol transfer between membrane structures from t1/ <dig> =  <dig> days to t1/ <dig> = 7- <dig> minutes in vitro  <cit> , demonstrating the potential of lipid carrier proteins to enhance cellular lipid uptake.
insect cells do not transfer cholesterol between the midgut and the fat body via receptor mediated endocytosis of lp.
the diffusion of cholesterol from lp to target cell membranes is thought to be the main mechanism for the transport of cholesterol to cells  <cit> .
moreover, lp is the only apolipoprotein found in insects and lp is not synthesized in the midgut  <cit> , which excludes the possibility of efflux of absorbed lipids from the midgut into the hemolymph via exocytosis of apolipoprotein/lipid vehicles  <cit> .
therefore, nonvesicular transport of intracellular cholesterol may be important in insect cellular cholesterol uptake via intracellular sterol carrier protein to aid the desorption of membrane-bound cholesterol and the delivery of the cholesterol within the cytosol.
little is known about the protein involved in the intracellular transfer of cholesterol in insects.
several insect intracellular sterol carrier protein genes have been identified such as the sterol carrier protein-x  <cit> , the sterol carrier protein- <dig>  <cit> , and the steroid acute regulator protein -related lipid transfer protein  <cit> .
in most cases, the function of those insect intracellular sterol carrier proteins is deduced from the temporal and spatial expression profiles and from the known function of their vertebrate homologs.
genomic sequence data show that insects may have multiple copies of sterol carrier protein- <dig> -like proteins  <cit> , in contrast to the single copy of the scp-x/scp- <dig> gene in vertebrates  <cit>  and in the silkworm, b. mori  <cit> .
scp-x belongs to the scp- <dig> gene family, which includes scp- <dig>  scp-x, 17β-hydroxysteroid dehydrogenase iv, unc- <dig>  metallo-β-lactomase and stomatin  <cit> .
in vertebrates, the scp-x/scp- <dig> gene has two initiation sites for coding proteins.
one site codes for scp-x which is post-translationally cleaved into 2/3-oxoacyl-coa thiolase and scp- <dig>  a second site codes for scp- <dig>  both scp-x and scp- <dig> share a common scp- <dig> c-terminus.
increasingly, data suggest that 3-oxoacyl-coa thiolase is involved in the oxidation of branched chain fatty acids and scp- <dig> is involved in the transfer of lipids  <cit> .
whether insect scp- <dig> and scp-2-like proteins have similar or divergent functions is unknown.
some insect scp- <dig> and scp-2-like proteins lack the peroxisome targeting sequence at the c-termini, suggesting that they are cytosolic proteins  <cit> .
aescp- <dig>  an aedes aegypti scp- <dig> protein which is not produced from the scp-x gene, binds to both cholesterol  <cit>  and fatty acid  <cit> , which is consistent with the broad spectrum of lipid ligands of the vertebrate scp- <dig>  over-expression or knockdown of aescp- <dig> gene expression affects cholesterol uptake in vivo  <cit> .
chemical inhibitors of aescp- <dig> suppress cholesterol uptake in cultured cells from aedes aegypti and manduca sexta, indicating that m. sexta has a protein homologous to aescp- <dig>  <cit> .
the tobacco hornworm, manduca sexta, has been one of the primary experimental insect model systems for sterol metabolism studies  <cit> .
studies using m. sexta have contributed to a broad picture of lipid metabolism and have revealed that cholesterol is transported from the midgut to the fat body via lipophorin  in the hemolymph in the free form, and absorbed cholesterol is stored as cholesterol este
here, we report molecular cloning and characterization of cdna encoding scp-x and scp- <dig> from the tobacco hornworm , and the effect of sterol carrier protein- <dig> inhibitors  on cholesterol and fatty acid uptake in m. sexta larvae.
the results suggest that msscp-x/scp- <dig> is involved in cholesterol absorption and transportation in insects.
RESULTS
molecular cloning and nucleotide sequence analysis of msscp-x/scp-2
a pcr-based cloning strategy was used to clone the msscp-x/scp- <dig> gene, using cdnas from the larval midgut and fat body as templates.
degenerate primers  allowed us to obtain a cdna fragment highly homologous to the scp- <dig> domain of bmscp-x and slscp- <dig> .
the degenerated primer xnf and gene specific primer cr <dig> produced a cdna fragment of about  <dig> bp, which matches the 2/3-oxoacyl-coa thiolase domain of insect scp-x.
the 3'-race using msscp-cf <dig> primer  produced independent cdna clones of about  <dig> bp containing a putative translation stop site .
additional gene specific primers  derived from partial msscp-x cdna sequence were synthesized and used for the 5'-race to obtain the 5'-ends of the cdna.
the 5'-race using cr <dig> primer produced cdna clones of  <dig> bp containing a putative translation start site at position  <dig> .
all results taken together indicate that a putative full-length msscp-x/scp- <dig> gene spans a total of  <dig> bps and encodes a protein of  <dig> amino acids .
interestingly,  <dig> independent cdna clones from the cr <dig> primer 5'-race had  <dig> bp sequences at the 5'-end divergent from the msscp-x , suggesting that the msscp- <dig> transcript  may be made from alternative splicing or an alternative initiation site.
the deduced amino acid sequence of the coding region showed high similarity to members of the scp- <dig> gene family in other insect species .
the scp- <dig> domain of manduca scp-x has amino acid sequence identities of 97%, 93%, 79%, 69%, and 69% with bombyx mori, spodoptera littoralis, aedes aegypti, apis melifera, and drosophila melanogaster, respectively .
the msscp-x/scp- <dig> gene encodes the scp-x protein with much higher homology to bmscp-x and slscp-x than the dipteran scp-x such as aescp-x and dmscp-x .
tissue and stage expression profiles of msscp-x/scp-2
the transcription profiles of msscp- <dig> in different tissues and stages were determined by semi-quantitative rt-pcr.
the msscp- <dig> mrna level was significantly higher in the midgut and fat body in feeding larvae .
msscp- <dig> was strongly expressed throughout the examined larval stages and decreased slightly in the wandering and pupal stages .
in the fat body, there was a transient increase in msscp- <dig> transcripts at the end and beginning of each instar .
low levels of msscp- <dig> mrna were detected in the hemolymph, hindgut, epidermis and muscle .
however, primers corresponding to the 2/3-oxoacyl-coa thiolase domain detected msscp-x mrna at moderate and high levels in all tissues except the midgut .
data from rt-pcrs showed that the transcription of scp-x and scp- <dig> mrnas from the msscp-x/scp- <dig> gene is regulated differently in different tissues.
msscp- <dig> mrna was the predominant transcript from this gene in the midgut during the feeding stage .
in the midgut, the levels of msscp- <dig> transcript increased during the 5th larval instar without any increase in the level of msscp-x transcript .
this result implies that the msscp-x/scp- <dig> gene might produce two transcripts: scp-x mrna and scp- <dig> mrna, which is similar to the human scp-x/scp- <dig> gene and other lepidopteran scp-x/scp- <dig>  bmscp-x/bmscp- <dig>  <cit> .
western blotting analysis was performed to investigate the existence of an scp- <dig> homolog in tissues from day  <dig> 4th instars using affinity-purified anti-scp- <dig> domain polyclonal antibodies of mosquito scp-x, aescp-x  <cit> .
figure  <dig> shows that a protein similar to the molecular weight of  <dig> kda  was detected by anti-aescp-x antibodies, suggesting that m. sexta may have a scp- <dig> analog.
tissues expressing msscp- <dig> in m. sexta at high levels were the midgut and fat body, which was consistent with the results of rt-pcr .
trace amounts of scp- <dig> were also detected in the hindgut and epidermis .
a band of about  <dig> kda protein in the hemolymph was detected by the anti-scp- <dig> domain antibody, which was presumably the full-length msscp-x.
in both the hindgut and epidermis, the predominant transcript from the msscp-x/scp- <dig> gene was the scp-x mrna , whereas the msscp- <dig> protein was the only detectable product of the msscp-x/ <dig> gene .
the results indicate that the full length msscp-x protein was proteolytically cleaved to produce the scp- <dig> domain protein in those tissues.
the msscp-x protein was not processed to produce msscp- <dig> in the hemocytes due to the prominent scp-x band and the lack of the msscp- <dig> band .
ligand and scpis binding in msscp-2
the binding affinity  of recombinant msscp- <dig> to the cholesterol analog was determined using an nbd-cholesterol binding assay.
as shown in figure  <dig>  increasing the concentrations of msscp- <dig>  while maintaining the concentration of nbd-cholesterol constant  resulted in increased fluorescence emission of nbd-cholesterol.
analysis of the binding curve using the one site binding non-linear regression model in graphpad prism software  yielded high binding affinity for both msscp- <dig> and aescp- <dig> .
aedes aegypti scp- <dig>  is not transcribed from the scp-x gene  <cit>  but belongs to the scp- <dig> gene family, and has been shown to have high affinity to cholesterol  <cit> .
the binding affinity  was  <dig>  × 10- <dig> m  for msscp- <dig> and  <dig>  × 10- <dig> m  for aescp- <dig>  respectively.
however, the gst  showed little binding to nbd-cholesterol .
although scatchard plots are useful for visualizing data, they are not the most accurate estimation.
in this experiment, we used the one site binding non-linear regression model, since the linear transformation of scatchard plots distorts the experimental error.
the inhibitory effect of scpis on cholesterol binding to msscp- <dig> was measured using nbd-cholesterol competition assays.
scpi- <dig> and scpi- <dig> reduced nbd-cholesterol binding to msscp- <dig> in a concentration-dependent fashion .
aescp- <dig> and gst proteins were used as positive and negative controls, respectively.
scpis inhibited the binding of nbd-cholesterol to both msscp- <dig> and aescp- <dig>  however, scpis showed no effect on the binding of nbd-cholesterol and gst, which is a non-binding protein to lipid species .
the 50% maximal effective concentration  of scpi- <dig> and scpi- <dig> to block nbd-cholesterol-msscp- <dig> binding was determined using the one site competition binding, non-linear regression model in graphpad prism® software version  <dig>  .
the ec50s of scpi- <dig> were  <dig>  μm  and  <dig>  μm  for msscp- <dig> and aescp- <dig>  respectively .
the ec50s of scpi- <dig> were  <dig>  μm  and  <dig>  μm  for msscp- <dig> and aescp- <dig>  respectively .
the result suggests that scpis have high binding affinities to insect scp-2s and are able to compete with nbd-cholesterol for binding to msscp- <dig> and aescp- <dig>  the result also indicates that scpi- <dig> may be more effective in inhibiting msscp-2's function than that of scpi- <dig> , which is consistent with the observation in the mosquito aescp- <dig>  <cit> .
whether the differences in the ec <dig> values of scpi- <dig> and scpi- <dig> in inhibiting cholesterol binding to msscp- <dig> result in different biological effects will be described later.
effects of scpis on lipid uptake in m. sexta
to determine whether msscp- <dig> is involved in cholesterol uptake in vivo, we investigated the effects of scpis on cholesterol absorption in gate ii day  <dig> 4th instar larvae when msscp- <dig> expression was high in both the midgut and fat body .
larvae were fasted for  <dig> hours, then were fed  <dig>  g of diet containing -cholesterol and scpi- <dig> and scpi- <dig> at ld <dig> dosages  <cit> .
tracking the ingested -cholesterol in schistocerca gregaria showed that insects appear not to alter the 3h label on the -cholesterol in vivo, therefore, measuring the amount of -cholesterol can be used directly as an indication of cholesterol uptake  <cit> .
moreover, manduca sexta larvae absorb free -cholesterol readily with little modification of the absorbed -cholesterol  <cit> .
the lipophorin in the hemolymph uploads absorbed dietary lipids at the basal side of the midgut and transports the cholesterol to the fat body for storage  <cit> .
it was hypothesized that cytosolic lipid transport proteins in the midgut transfer absorbed dietary lipids toward the basal side of the epithelium where lipophorin picks up the lipids  <cit> .
we speculated that msscp- <dig> could be one of the midgut sterol carrier proteins that aid the uptake of cholesterol because msscp- <dig> was expressed at high levels in the midgut and fat body  and msscp- <dig> bound to a cholesterol analog .
to determine whether the inhibition of msscp- <dig> function would affect cholesterol uptake from the diet into the midgut,  -cholesterol in midgut tissue was measured at different time points after feeding.
in the midgut tissue, -cholesterol content decreased significantly in the controls between  <dig> and  <dig> hours post feeding , indicating the clearance of absorbed -cholesterol over time from the midgut tissue .
the result is consistent with the previous report of a slow but steady clearance of -cholesterol from the midgut within  <dig> hours post feeding  <cit> .
in scpi- <dig> treated larvae at the  <dig> hour time point, the total -cholesterol in the midgut tissue was slightly lower than that of the control group, whereas, the scpi-2-treated group had significantly much less  labeled cholesterol .
at  <dig> hours post feeding, there was a significant decrease of -cholesterol in the midgut in both the control and scpi-1-treated groups , indicating that absorbed cholesterol was likely transported out of the midgut tissues .
there was no significant change in the levels of labeled cholesterol  <dig> to  <dig> hours post feeding in scpi-2-treated groups , which suggests that there was little transfer of absorbed cholesterol.
over the observed time period, the total amount of -cholesterol in the midgut tissue was significantly lower in scpi- <dig> and scpi-2-treated larvae than in the controls .
the amount of -cholesterol in the midgut  <dig> to  <dig> hours post feeding was  <dig> ,  <dig> , and  <dig>  dmp/mg protein for the control, scpi-1-, and scpi-2-treated groups, respectively .
the results imply that the uptake of -cholesterol into the midgut tissue in scpi-treated larvae was less efficient than in the controls.
scpi- <dig> was more effective in inhibiting cholesterol binding to msscp- <dig> than that of scpi- <dig> .
scpi- <dig> had severer effects on msscp-2's function in vivo resulting in a 46% greater reduction in -cholesterol uptake/transport than the scpi-1-treatment  in the midgut tissue .
to determine whether the inhibition of msscp- <dig> function would affect cholesterol transfer from the midgut to the hemolymph, -cholesterol in the hemolymph was measured at different time points after the feeding.
in the hemolymph, constant high levels of -cholesterol were observed over the time period in the control group , which is consistent with the observation that hemolymph -cholesterol levels in the label/chase experiments remains steady 4- <dig> hours post feeding  <cit> .
however, the hemolymph -cholesterol levels were significantly lower  in the scpi-treated groups at all post feeding time points .
the amount of hemolymph -cholesterol at each time point was significantly higher in control larvae than that of scpi-1-treated larvae  whereas there were insignificant differences at the same time point in the amount of -cholesterol in the midgut tissue between the controls and scpi-1-treated larvae .
the results indicate that the efflux of absorbed cholesterol from the midgut into the hemolymph was affected in scpi-treated larvae, suggesting that there was an impairment of -cholesterol transport from the midgut into the hemolymph.
on the other hand, the amount of labeled cholesterol in the hemolymph  <dig> to  <dig> hours post feeding was  <dig>  and  <dig>  dpm/mg protein for scpi- <dig> and scpi- <dig> treated larvae, respectively , showing that the efflux of cholesterol from the midgut into the hemolymph was similar between scpi- <dig> and scpi- <dig> treated larvae even though scpi- <dig> treated larvae had significantly lower levels of labeled cholesterol in the midgut .
therefore, scpi- <dig> and scpi- <dig> had a similar effect on the efflux of cholesterol despite the differences in their efficiency in inhibiting cholesterol binding to msscp <dig> .
the insect fat body is the site of cholesterol storage during the feeding stage  <cit> .
the amount of -cholesterol increased 3-fold from  <dig> hours to  <dig> hours post feeding in the fat body of the control larvae, indicating an uptake of -cholesterol over time .
the results from the controls are consistent with other studies in which ingested -cholesterol is rapidly accumulated in the fat body in manduca larvae  <cit> .
in contrast, in scpi- <dig> or scpi- <dig> treated larvae, the accumulation of -cholesterol in the fat body within  <dig> hours post feeding was significantly lower  than that of in the controls .
this is consistent with the observation that the total amount of -cholesterol is significantly lower in the hemolymph of scpi-treated larvae .
the  <dig> hour-post feeding time represented the accumulation of labeled cholesterol stored in the fat body for the observed period.
scpi- <dig> and scpi- <dig> treatment suppressed cholesterol storage in m. sexta larvae within  <dig> hours of treatment by 37% and 69%, respectively .
scpi-2-treated larvae had significantly less stored -cholesterol than the scpi- <dig> treated larvae  although the amount of labeled cholesterol in the hemolymph in both treatments were similar .
the result implies that scpi- <dig> was more effective than scpi- <dig> in blocking the uptake of cholesterol from the hemolymph into the fat body.
the results of this study provide the evidence for the first time that scpis inhibit cholesterol uptake into the midgut, reduce the efflux of absorbed cholesterol into the hemolymph, and block the uptake of cholesterol in the fat body in vivo in m. sexta larvae.
vertebrate scp- <dig> binds to fatty acid and cholesterol  <cit> .
to investigate whether msscp- <dig> is also involved in fatty acid transport in the midgut, we fed labeled -palmatic acid in a label/chase experiment as described above.
in the midgut tissue of the control group, there was a significant rapid decrease  in the levels of -palmatic acid from  <dig> to  <dig> hours post feeding , indicating a very quick clearance of absorbed labeled fatty acid in the midgut.
this is consistent with the previous reports that showed rapid uptake and clearance of free fatty acid from the midgut into the hemolymph within  <dig> hours post feeding  <cit> .
in the scpi-treated larvae, there was a significant reduction of -palmatic acid levels  in the midgut tissue except at the  <dig> hour time point in the scpi- <dig> treated group , suggesting that there was a generalized suppression in the uptake of free fatty acid from the midgut lumen into the midgut tissue in scpi-treated larvae.
however, there were no significant differences between the controls and scpi-treated larvae in the quantity of -palmatic acid in the hemolymph  or in the fat body , implying that the clearance of absorbed fatty acid from the midgut and the storage of absorbed fatty acid in the fat body were not affected by scpi-treatment.
discussion
there are multiple steps in the cellular uptake of lipids.
this process may be important for insects since receptor or lipoprotein mediated endocytosis and/or exocytosis is not involved in cellular lipid transfer  <cit> .
lipids can easily diffuse into the outer layer of the cytoplasm membrane where they flip-flop across the membrane bilayer.
cytosolic lipid carriers such as cytosolic fatty acid binding protein and scp- <dig> desorbs the free lipids from the cytoplasm membrane and deliver them to the storage site or funnel them into a metabolic pathway  <cit> .
the mechanisms underlying msscp-2-mediated cellular cholesterol uptake are unclear.
aescp- <dig> is not a membrane bound protein  <cit> , and msscp-2-mediated lipid absorption is unlike membrane transporters, such as niemann-pick c1-like  <dig>   <cit> .
msscp- <dig> may enhance the desorption of membrane-bound ligand through the collision with membrane-bound cholesterol  <cit> , which, in turn, may lead to a msscp-2-mediated cholesterol diffusion into the cytosol.
the scp-2/membrane collision model is the proposed mode for fatty acid transport by the scp- <dig> of yarrowia lipolytica  <cit> .
in an earlier study, sterol carrier protein- <dig> inhibitors  were identified via high throughput screening using recombinant aescp- <dig>  <cit> .
aescp- <dig> is not a product of the aescp-x gene  <cit> .
however, both aescp-x and aescp- <dig> belong to the scp- <dig> gene family  <cit> .
the scpis inhibit ligand binding in both aescp- <dig> and the scp- <dig> domain protein of aescp-x, although the ec50s of the same scpi are higher for the scp- <dig> domain protein of aescp-x than for aescp- <dig>  <cit> .
scpis are lethal to both mosquito and manduca sexta larvae  <cit> .
it was speculated that the manduca sexta has a protein homologous to that of mosquito aescp- <dig>  which was inhibited by scpis to cause the biological effects in manduca larvae  <cit> .
to further investigate the possible existence of manduca sexta scp- <dig> , we identified msscp-x/scp- <dig> via reverse-transcriptase pcr based cloning strategy using degenerate primers designed on the basis of a consensus cdna sequence within insect scp-x/scp- <dig> genes .
in vertebrates, the scp-x/scp- <dig> gene codes two proteins that share a common  <dig> kda scp- <dig> domain:  <dig> kda scp-x and  <dig> kda pro-scp- <dig>  which is post-translationally cleaved to  <dig> kda scp- <dig>  <cit> .
msscp-x has the same domain architecture as vertebrate scp-x.
the full-length of the msscp-z/scp- <dig> cdna encodes a protein of  <dig> amino acids .
the amino acid sequence similarities between msscp- <dig> and the scp- <dig> domain of scp-x in other insect species were high .
using two pairs of primers that detect the n-terminal 2/3-oxoacyl-coa thiolase domain and the c-terminal scp- <dig> domain, it was discovered that the transcripts for msscp-x and msscp- <dig> were differentially regulated based on the abundance of each in the same samples .
the differential production of the transcripts was most evident in the midgut and fat body, at which time the midgut had predominantly msscp- <dig> mrna, whereas the fat body had a much higher level of msscp-x transcript .
in the cotton leafworm , two transcripts from a single copy of the scp-x/scp- <dig> gene were detected  <cit> .
however, in the silkworm , alternative splicing seemed to generate two transcripts from a single copy of the scp-x/scp- <dig> gene  <cit> .
the first  <dig> bps of the 5'end utr sequence of msscp-x is divergent from the msscp-x 5' utr or the sequence corresponding to the scp- <dig> domain , whereas the identical sequence in the 5' utr of msscp- <dig> to the scp- <dig> domain of the msscp-x transcript started at the same position as the splicing site in the b. mori scp-x/scp- <dig> gene .
whether the msscp-x/scp- <dig> gene may produce two transcripts via alternative splicing needs further investigation.
the spatial expression protein files of msscp-x are different from those described in s. littoralis and b. mori, in which the scp-x transcript level is higher in the midgut and malpighian tubules.
in contrast, the msscp-x mrna levels were much lower in the midgut than in other tissues in general .
the spatial transcription profiles of msscp-x and msscp- <dig> are similar to the mrna levels of the vertebrate scp- <dig> gene.
the vertebrate scp-x is detected essentially in all tissues, whereas scp- <dig> was in highest abundance in the liver and intestine  <cit> .
the msscp- <dig> transcription levels were higher in the midgut and fat body during the feeding stage ; the metabolic function of the fat body is similar to that of the vertebrate liver and adipose tissue.
however, the insect scp-x protein appears to be processed post-translationally to produce two proteins: the 2/3-oxoacyl-coa thiolase and the scp- <dig>  <cit> , which would render higher protein levels of scp- <dig> in tissues transcribing scp-x at higher levels.
the anti-scp- <dig> domain of aescp-x antibodies recognized the purified recombinant msscp- <dig>  and detected a similar molecular weight immune-reactive protein in the midgut, hindgut, and fat body .
this result coincides with the fact that the insect midgut is the main site for cholesterol and fatty acid uptake  <cit> .
this also showed the expression pattern of msscp-x/scp- <dig> is similar to other mosquito scp-x and scp- <dig> genes.
in aedes aegypti, aescp- <dig> and aescp-x were strongly expressed in the foregut and mostly in the midgut during the larval feeding stages  <cit> .
in aedes aegypti, spodoptera littoralis and bombyx mori, scp-xs had high levels of transcription in the midgut during the last larval feeding stages  <cit> , however, the scp- <dig> domain protein is the prominent product due to post-translational processing of the scp-x protein in the midgut  <cit> .
therefore, regardless of which transcript of the scp-x/scp- <dig> gene is predominant in the midgut, high levels of scp- <dig> domain protein may be the end result.
there was 56% less  stored -cholesterol  <dig> hours post feeding in the fat body in scpi-2-treated larvae than for the scpi-1-treatment .
in manduca sexta gv <dig> cells, scpi- <dig> and scpi- <dig> at  <dig> μm concentrations suppressed cholesterol uptake by 15% and 22%, respectively  <cit> .
the relatively higher binding affinities of scpi- <dig> than that of scpi- <dig> to insect scp- <dig>  <cit>   may result in the differences in biological effects between scpi- <dig> and scpi- <dig> treatments in which scpi- <dig> constantly showed higher efficacy in inhibiting cholesterol trafficking in vivo .
the results were consistent with the earlier observation that scpi- <dig> is more lethal than scpi- <dig> to manduca larvae  <cit> .
both scpi- <dig> and scpi- <dig> effectively inhibited cholesterol uptake .
there was little labeled cholesterol detected in the feces  <dig> hours post feeding and a significant increase in -cholesterol in the feces over the observed time period in the control group , although the excretion of labeled fatty acid during the same period was constant .
the underlying mechanism for the slower excretion of cholesterol than that of fatty acid in feces is unknown.
however, -cholesterol levels in the feces within the  <dig> hour period were  <dig> %  and  <dig> %  higher in scpi-1- and scpi- <dig> treated larvae, respectively, than in the controls .
the results indicate that unabsorbed dietary cholesterol was excreted via feces.
studies using the vertebrate model suggest that scp- <dig> mediates cholesterol trafficking and metabolism and is involved in fatty acid uptake  <cit> .
however, it is unclear whether insect scp- <dig> has similar functions in cholesterol and fatty acid trafficking and metabolism.
we investigated the function of msscp- <dig> in vivo using sterol carrier protein- <dig> inhibitors  to examine the biological effect of msscp- <dig> impairment on cholesterol and fatty acid uptake in m. sexta larvae.
inhibition of msscp-2's function by scpis resulted in arrested growth and development in surviving larvae, indicated by lower weight gain over time and delayed pupation .
when manduca larvae were fed lc <dig> concentrations of scpi- <dig> and scpi- <dig> for a short time , there was a significant reduction in the uptake of labeled cholesterol in the midgut and fat body in scpi-treated larvae compared to the controls .
similar results were obtained when nbd-cholesterol, a fluorescent cholesterol analog, was used as the tracer for cholesterol uptake in vivo .
however, the effect of scpi-treatment on palmatic acid was limited to the suppression of uptake into the midgut .
in insects, the mechanism for the uptake of lipids from the diet into the midgut is unclear.
it is hypothesized that lipids  may be transported into the midgut epithelium via specific membrane transporters, and then trafficked intracellularly by carrier proteins toward the basal side of the midgut for uploading onto the lp  <cit> .
the efficacy of a scpi in suppressing cholesterol absorption  seemed to correlate with its efficiency in inhibiting ligand-binding in scp- <dig>   <cit> , suggesting that intracellular transport plays a critical role in cellular uptake of cholesterol.
the fact that inhibition of msscp-2's function via scpis did not completely abolish cholesterol uptake points to the possibility that there may be more than one class of intracellular carrier proteins for cholesterol in insects.
another candidate as an intracellular cholesterol carrier is the steroid acute regulator protein -related lipid transfer protein  <cit> .
the mode of larvicidal activity of scpis against some insect species, including aedes aegypti, anopheles gambiae, culex pipiens and m. sexta is unclear  <cit> .
the relatively high binding affinities of scpi- <dig> and scpi- <dig> to insect scp-2s indicate that scpis may inhibit the biological functions of scp- <dig> by competing with cholesterol for binding to scp- <dig>  <cit> .
scpi- <dig> and scpi- <dig> suppressed -cholesterol efflux from the midgut to the hemolymph in m. sexta larvae within  <dig> hours of treatment , resulting in the reduction of stored -cholesterol by 37% and 69%, respectively .
although, scpi- <dig> and scpi- <dig> also suppressed -palmatic acid uptake in the midgut , there was no evidence that scpi-treatment impaired the efflux of absorbed -palmatic acid from the midgut into the hemolymph .
one plausible explanation for the lack of scpi's effect on the efflux of fatty acid into the hemolymph and ultimately storage in the fat body may be the redundant function of fatty acid binding proteins in the midgut and fat body  <cit> .
the results of this study provide evidence that scpis inhibit cholesterol uptake/storage in insects in vivo.
the effect of scpi-treatment on fatty acid uptake in larvae was limited to the midgut .
since insects can synthesize fatty acid de novo from acetate  <cit> , one possible mode of action of scpis' larvicidal activities is reduction in cholesterol uptake over time resulting in a deficiency in cholesterol that might lead to a high mortality rate in scpi-treated manduca larvae  <cit> .
the likely mechanism of scpis in inhibiting cholesterol absorption is the decrease in clearance of absorbed cholesterol from the midgut tissue , suggesting that msscp- <dig> may be involved in the cellular trafficking of cholesterol in the midgut.
CONCLUSIONS
in this study we tested the hypothesis that msscp- <dig> may be involved in cholesterol uptake and intracellular transportation.
our results show that manduca sexta sterol carrier protein-x and sterol carrier protein- <dig>  have a high degree of homology in the scp- <dig> domain to other insects' scp- <dig> and recombinant msscp- <dig> bound to nbd-cholesterol with high binding affinity.
transcripts of msscp- <dig> were highly expressed in the larval midgut and fat body.
the result suggests that msscp- <dig> may be involved in cholesterol uptake in the midgut, the main site for cholesterol absorption, and intracellular transportation in the fat body, the main site for cholesterol storage.
msscp- <dig> may function as a lipid carrier protein in vivo.
we have also shown that sterol carrier protein inhibitors  effectively reduced cholesterol uptake and cholesterol accumulation in the midgut and in fat body.
due to the total dependence of insects on exogenous cholesterol, targeting insect scp- <dig> may be a viable approach for the development of new insecticides.

 in insects, cholesterol is required as a structural component of cellular membranes and as a precursor for the insect molting hormone. however, insects lack several enzymes in the cholesterol biosynthesis pathway, which makes them auxotrophic for exogenous cholesterol for satisfactory growth, development and reproduction. therefore, cholesterol uptake and transportation are critical for their survival. uptake of cholesterol involves intracellular transfer and intercellular transportation. intracellular cholesterol transfer enables the redistribution of cholesterol within a cell, and intercellular transport is needed for the distribution of cholesterol between cells. it is very well documented that lipophorin and lipid transport particles are responsible for the intercellular transportation of cholesterol and lipids in insects. cholesterol is highly hydrophobic with solubility of ~ nm in water at room temperature, can be easily inserted into the lipid bilayer and has a fast flip-flop rate within the lipid bilayer. diffusion of cholesterol into the lipid bilayer can happen readily, and an aqueous diffusion model has been proposed to account for some of the cholesterol uptake by cells. however, the desorption of membrane-bound cholesterol is slow without the aid of a carrier protein to enhance the desorption of membrane-bound cholesterol through the collision with membrane-bound cholesterol. in vertebrates, sterol carrier protein- increases the rate of cholesterol transfer between membrane structures from t1/ = days to t1/ = 7- minutes in vitro, demonstrating the potential of lipid carrier proteins to enhance cellular lipid uptake. insect cells do not transfer cholesterol between the midgut and the fat body via receptor mediated endocytosis of lp. the diffusion of cholesterol from lp to target cell membranes is thought to be the main mechanism for the transport of cholesterol to cells. moreover, lp is the only apolipoprotein found in insects and lp is not synthesized in the midgut, which excludes the possibility of efflux of absorbed lipids from the midgut into the hemolymph via exocytosis of apolipoprotein/lipid vehicles. therefore, nonvesicular transport of intracellular cholesterol may be important in insect cellular cholesterol uptake via intracellular sterol carrier protein to aid the desorption of membrane-bound cholesterol and the delivery of the cholesterol within the cytosol. little is known about the protein involved in the intracellular transfer of cholesterol in insects. several insect intracellular sterol carrier protein genes have been identified such as the sterol carrier protein-x, the sterol carrier protein-, and the steroid acute regulator protein -related lipid transfer protein. in most cases, the function of those insect intracellular sterol carrier proteins is deduced from the temporal and spatial expression profiles and from the known function of their vertebrate homologs. genomic sequence data show that insects may have multiple copies of sterol carrier protein- -like proteins, in contrast to the single copy of the scp-x/scp- gene in vertebrates and in the silkworm, b. mori. scp-x belongs to the scp- gene family, which includes scp- scp-x, 17β-hydroxysteroid dehydrogenase iv, unc- metallo-β-lactomase and stomatin. in vertebrates, the scp-x/scp- gene has two initiation sites for coding proteins. one site codes for scp-x which is post-translationally cleaved into 2/3-oxoacyl-coa thiolase and scp- a second site codes for scp- both scp-x and scp- share a common scp- c-terminus. increasingly, data suggest that 3-oxoacyl-coa thiolase is involved in the oxidation of branched chain fatty acids and scp- is involved in the transfer of lipids. whether insect scp- and scp-2-like proteins have similar or divergent functions is unknown. some insect scp- and scp-2-like proteins lack the peroxisome targeting sequence at the c-termini, suggesting that they are cytosolic proteins. aescp- an aedes aegypti scp- protein which is not produced from the scp-x gene, binds to both cholesterol and fatty acid, which is consistent with the broad spectrum of lipid ligands of the vertebrate scp- over-expression or knockdown of aescp- gene expression affects cholesterol uptake in vivo. chemical inhibitors of aescp- suppress cholesterol uptake in cultured cells from aedes aegypti and manduca sexta, indicating that m. sexta has a protein homologous to aescp-. the tobacco hornworm, manduca sexta, has been one of the primary experimental insect model systems for sterol metabolism studies. studies using m. sexta have contributed to a broad picture of lipid metabolism and have revealed that cholesterol is transported from the midgut to the fat body via lipophorin in the hemolymph in the free form, and absorbed cholesterol is stored as cholesterol este here, we report molecular cloning and characterization of cdna encoding scp-x and scp- from the tobacco hornworm, and the effect of sterol carrier protein- inhibitors on cholesterol and fatty acid uptake in m. sexta larvae. the results suggest that msscp-x/scp- is involved in cholesterol absorption and transportation in insects.  molecular cloning and nucleotide sequence analysis of msscp-x/scp-2 a pcr-based cloning strategy was used to clone the msscp-x/scp- gene, using cdnas from the larval midgut and fat body as templates. degenerate primers allowed us to obtain a cdna fragment highly homologous to the scp- domain of bmscp-x and slscp-. the degenerated primer xnf and gene specific primer cr produced a cdna fragment of about bp, which matches the 2/3-oxoacyl-coa thiolase domain of insect scp-x. the 3'-race using msscp-cf primer produced independent cdna clones of about bp containing a putative translation stop site. additional gene specific primers derived from partial msscp-x cdna sequence were synthesized and used for the 5'-race to obtain the 5'-ends of the cdna. the 5'-race using cr primer produced cdna clones of bp containing a putative translation start site at position. all results taken together indicate that a putative full-length msscp-x/scp- gene spans a total of bps and encodes a protein of amino acids. interestingly, independent cdna clones from the cr primer 5'-race had bp sequences at the 5'-end divergent from the msscp-x, suggesting that the msscp- transcript may be made from alternative splicing or an alternative initiation site. the deduced amino acid sequence of the coding region showed high similarity to members of the scp- gene family in other insect species. the scp- domain of manduca scp-x has amino acid sequence identities of 97%, 93%, 79%, 69%, and 69% with bombyx mori, spodoptera littoralis, aedes aegypti, apis melifera, and drosophila melanogaster, respectively. the msscp-x/scp- gene encodes the scp-x protein with much higher homology to bmscp-x and slscp-x than the dipteran scp-x such as aescp-x and dmscp-x. tissue and stage expression profiles of msscp-x/scp-2 the transcription profiles of msscp- in different tissues and stages were determined by semi-quantitative rt-pcr. the msscp- mrna level was significantly higher in the midgut and fat body in feeding larvae. msscp- was strongly expressed throughout the examined larval stages and decreased slightly in the wandering and pupal stages. in the fat body, there was a transient increase in msscp- transcripts at the end and beginning of each instar. low levels of msscp- mrna were detected in the hemolymph, hindgut, epidermis and muscle. however, primers corresponding to the 2/3-oxoacyl-coa thiolase domain detected msscp-x mrna at moderate and high levels in all tissues except the midgut. data from rt-pcrs showed that the transcription of scp-x and scp- mrnas from the msscp-x/scp- gene is regulated differently in different tissues. msscp- mrna was the predominant transcript from this gene in the midgut during the feeding stage. in the midgut, the levels of msscp- transcript increased during the 5th larval instar without any increase in the level of msscp-x transcript. this result implies that the msscp-x/scp- gene might produce two transcripts: scp-x mrna and scp- mrna, which is similar to the human scp-x/scp- gene and other lepidopteran scp-x/scp- bmscp-x/bmscp-. western blotting analysis was performed to investigate the existence of an scp- homolog in tissues from day 4th instars using affinity-purified anti-scp- domain polyclonal antibodies of mosquito scp-x, aescp-x. figure shows that a protein similar to the molecular weight of kda was detected by anti-aescp-x antibodies, suggesting that m. sexta may have a scp- analog. tissues expressing msscp- in m. sexta at high levels were the midgut and fat body, which was consistent with the results of rt-pcr. trace amounts of scp- were also detected in the hindgut and epidermis. a band of about kda protein in the hemolymph was detected by the anti-scp- domain antibody, which was presumably the full-length msscp-x. in both the hindgut and epidermis, the predominant transcript from the msscp-x/scp- gene was the scp-x mrna, whereas the msscp- protein was the only detectable product of the msscp-x/ gene. the results indicate that the full length msscp-x protein was proteolytically cleaved to produce the scp- domain protein in those tissues. the msscp-x protein was not processed to produce msscp- in the hemocytes due to the prominent scp-x band and the lack of the msscp- band. ligand and scpis binding in msscp-2 the binding affinity of recombinant msscp- to the cholesterol analog was determined using an nbd-cholesterol binding assay. as shown in figure increasing the concentrations of msscp- while maintaining the concentration of nbd-cholesterol constant resulted in increased fluorescence emission of nbd-cholesterol. analysis of the binding curve using the one site binding non-linear regression model in graphpad prism software yielded high binding affinity for both msscp- and aescp-. aedes aegypti scp- is not transcribed from the scp-x gene but belongs to the scp- gene family, and has been shown to have high affinity to cholesterol. the binding affinity was × 10- m for msscp- and × 10- m for aescp- respectively. however, the gst showed little binding to nbd-cholesterol. although scatchard plots are useful for visualizing data, they are not the most accurate estimation. in this experiment, we used the one site binding non-linear regression model, since the linear transformation of scatchard plots distorts the experimental error. the inhibitory effect of scpis on cholesterol binding to msscp- was measured using nbd-cholesterol competition assays. scpi- and scpi- reduced nbd-cholesterol binding to msscp- in a concentration-dependent fashion. aescp- and gst proteins were used as positive and negative controls, respectively. scpis inhibited the binding of nbd-cholesterol to both msscp- and aescp- however, scpis showed no effect on the binding of nbd-cholesterol and gst, which is a non-binding protein to lipid species. the 50% maximal effective concentration of scpi- and scpi- to block nbd-cholesterol-msscp- binding was determined using the one site competition binding, non-linear regression model in graphpad prism® software version. the ec50s of scpi- were μm and μm for msscp- and aescp- respectively. the ec50s of scpi- were μm and μm for msscp- and aescp- respectively. the result suggests that scpis have high binding affinities to insect scp-2s and are able to compete with nbd-cholesterol for binding to msscp- and aescp- the result also indicates that scpi- may be more effective in inhibiting msscp-2's function than that of scpi-, which is consistent with the observation in the mosquito aescp-. whether the differences in the ec values of scpi- and scpi- in inhibiting cholesterol binding to msscp- result in different biological effects will be described later. effects of scpis on lipid uptake in m. sexta to determine whether msscp- is involved in cholesterol uptake in vivo, we investigated the effects of scpis on cholesterol absorption in gate ii day 4th instar larvae when msscp- expression was high in both the midgut and fat body. larvae were fasted for hours, then were fed g of diet containing -cholesterol and scpi- and scpi- at ld dosages. tracking the ingested -cholesterol in schistocerca gregaria showed that insects appear not to alter the 3h label on the -cholesterol in vivo, therefore, measuring the amount of -cholesterol can be used directly as an indication of cholesterol uptake. moreover, manduca sexta larvae absorb free -cholesterol readily with little modification of the absorbed -cholesterol. the lipophorin in the hemolymph uploads absorbed dietary lipids at the basal side of the midgut and transports the cholesterol to the fat body for storage. it was hypothesized that cytosolic lipid transport proteins in the midgut transfer absorbed dietary lipids toward the basal side of the epithelium where lipophorin picks up the lipids. we speculated that msscp- could be one of the midgut sterol carrier proteins that aid the uptake of cholesterol because msscp- was expressed at high levels in the midgut and fat body and msscp- bound to a cholesterol analog. to determine whether the inhibition of msscp- function would affect cholesterol uptake from the diet into the midgut, -cholesterol in midgut tissue was measured at different time points after feeding. in the midgut tissue, -cholesterol content decreased significantly in the controls between and hours post feeding, indicating the clearance of absorbed -cholesterol over time from the midgut tissue. the result is consistent with the previous report of a slow but steady clearance of -cholesterol from the midgut within hours post feeding. in scpi- treated larvae at the hour time point, the total -cholesterol in the midgut tissue was slightly lower than that of the control group, whereas, the scpi-2-treated group had significantly much less labeled cholesterol. at hours post feeding, there was a significant decrease of -cholesterol in the midgut in both the control and scpi-1-treated groups, indicating that absorbed cholesterol was likely transported out of the midgut tissues. there was no significant change in the levels of labeled cholesterol to hours post feeding in scpi-2-treated groups, which suggests that there was little transfer of absorbed cholesterol. over the observed time period, the total amount of -cholesterol in the midgut tissue was significantly lower in scpi- and scpi-2-treated larvae than in the controls. the amount of -cholesterol in the midgut to hours post feeding was,, and dmp/mg protein for the control, scpi-1-, and scpi-2-treated groups, respectively. the results imply that the uptake of -cholesterol into the midgut tissue in scpi-treated larvae was less efficient than in the controls. scpi- was more effective in inhibiting cholesterol binding to msscp- than that of scpi-. scpi- had severer effects on msscp-2's function in vivo resulting in a 46% greater reduction in -cholesterol uptake/transport than the scpi-1-treatment in the midgut tissue. to determine whether the inhibition of msscp- function would affect cholesterol transfer from the midgut to the hemolymph, -cholesterol in the hemolymph was measured at different time points after the feeding. in the hemolymph, constant high levels of -cholesterol were observed over the time period in the control group, which is consistent with the observation that hemolymph -cholesterol levels in the label/chase experiments remains steady 4- hours post feeding. however, the hemolymph -cholesterol levels were significantly lower in the scpi-treated groups at all post feeding time points. the amount of hemolymph -cholesterol at each time point was significantly higher in control larvae than that of scpi-1-treated larvae whereas there were insignificant differences at the same time point in the amount of -cholesterol in the midgut tissue between the controls and scpi-1-treated larvae. the results indicate that the efflux of absorbed cholesterol from the midgut into the hemolymph was affected in scpi-treated larvae, suggesting that there was an impairment of -cholesterol transport from the midgut into the hemolymph. on the other hand, the amount of labeled cholesterol in the hemolymph to hours post feeding was and dpm/mg protein for scpi- and scpi- treated larvae, respectively, showing that the efflux of cholesterol from the midgut into the hemolymph was similar between scpi- and scpi- treated larvae even though scpi- treated larvae had significantly lower levels of labeled cholesterol in the midgut. therefore, scpi- and scpi- had a similar effect on the efflux of cholesterol despite the differences in their efficiency in inhibiting cholesterol binding to msscp. the insect fat body is the site of cholesterol storage during the feeding stage. the amount of -cholesterol increased 3-fold from hours to hours post feeding in the fat body of the control larvae, indicating an uptake of -cholesterol over time. the results from the controls are consistent with other studies in which ingested -cholesterol is rapidly accumulated in the fat body in manduca larvae. in contrast, in scpi- or scpi- treated larvae, the accumulation of -cholesterol in the fat body within hours post feeding was significantly lower than that of in the controls. this is consistent with the observation that the total amount of -cholesterol is significantly lower in the hemolymph of scpi-treated larvae. the hour-post feeding time represented the accumulation of labeled cholesterol stored in the fat body for the observed period. scpi- and scpi- treatment suppressed cholesterol storage in m. sexta larvae within hours of treatment by 37% and 69%, respectively. scpi-2-treated larvae had significantly less stored -cholesterol than the scpi- treated larvae although the amount of labeled cholesterol in the hemolymph in both treatments were similar. the result implies that scpi- was more effective than scpi- in blocking the uptake of cholesterol from the hemolymph into the fat body. the results of this study provide the evidence for the first time that scpis inhibit cholesterol uptake into the midgut, reduce the efflux of absorbed cholesterol into the hemolymph, and block the uptake of cholesterol in the fat body in vivo in m. sexta larvae. vertebrate scp- binds to fatty acid and cholesterol. to investigate whether msscp- is also involved in fatty acid transport in the midgut, we fed labeled -palmatic acid in a label/chase experiment as described above. in the midgut tissue of the control group, there was a significant rapid decrease in the levels of -palmatic acid from to hours post feeding, indicating a very quick clearance of absorbed labeled fatty acid in the midgut. this is consistent with the previous reports that showed rapid uptake and clearance of free fatty acid from the midgut into the hemolymph within hours post feeding. in the scpi-treated larvae, there was a significant reduction of -palmatic acid levels in the midgut tissue except at the hour time point in the scpi- treated group, suggesting that there was a generalized suppression in the uptake of free fatty acid from the midgut lumen into the midgut tissue in scpi-treated larvae. however, there were no significant differences between the controls and scpi-treated larvae in the quantity of -palmatic acid in the hemolymph or in the fat body, implying that the clearance of absorbed fatty acid from the midgut and the storage of absorbed fatty acid in the fat body were not affected by scpi-treatment. discussion there are multiple steps in the cellular uptake of lipids. this process may be important for insects since receptor or lipoprotein mediated endocytosis and/or exocytosis is not involved in cellular lipid transfer. lipids can easily diffuse into the outer layer of the cytoplasm membrane where they flip-flop across the membrane bilayer. cytosolic lipid carriers such as cytosolic fatty acid binding protein and scp- desorbs the free lipids from the cytoplasm membrane and deliver them to the storage site or funnel them into a metabolic pathway. the mechanisms underlying msscp-2-mediated cellular cholesterol uptake are unclear. aescp- is not a membrane bound protein, and msscp-2-mediated lipid absorption is unlike membrane transporters, such as niemann-pick c1-like. msscp- may enhance the desorption of membrane-bound ligand through the collision with membrane-bound cholesterol, which, in turn, may lead to a msscp-2-mediated cholesterol diffusion into the cytosol. the scp-2/membrane collision model is the proposed mode for fatty acid transport by the scp- of yarrowia lipolytica. in an earlier study, sterol carrier protein- inhibitors were identified via high throughput screening using recombinant aescp-. aescp- is not a product of the aescp-x gene. however, both aescp-x and aescp- belong to the scp- gene family. the scpis inhibit ligand binding in both aescp- and the scp- domain protein of aescp-x, although the ec50s of the same scpi are higher for the scp- domain protein of aescp-x than for aescp-. scpis are lethal to both mosquito and manduca sexta larvae. it was speculated that the manduca sexta has a protein homologous to that of mosquito aescp- which was inhibited by scpis to cause the biological effects in manduca larvae. to further investigate the possible existence of manduca sexta scp-, we identified msscp-x/scp- via reverse-transcriptase pcr based cloning strategy using degenerate primers designed on the basis of a consensus cdna sequence within insect scp-x/scp- genes. in vertebrates, the scp-x/scp- gene codes two proteins that share a common kda scp- domain: kda scp-x and kda pro-scp- which is post-translationally cleaved to kda scp-. msscp-x has the same domain architecture as vertebrate scp-x. the full-length of the msscp-z/scp- cdna encodes a protein of amino acids. the amino acid sequence similarities between msscp- and the scp- domain of scp-x in other insect species were high. using two pairs of primers that detect the n-terminal 2/3-oxoacyl-coa thiolase domain and the c-terminal scp- domain, it was discovered that the transcripts for msscp-x and msscp- were differentially regulated based on the abundance of each in the same samples. the differential production of the transcripts was most evident in the midgut and fat body, at which time the midgut had predominantly msscp- mrna, whereas the fat body had a much higher level of msscp-x transcript. in the cotton leafworm, two transcripts from a single copy of the scp-x/scp- gene were detected. however, in the silkworm, alternative splicing seemed to generate two transcripts from a single copy of the scp-x/scp- gene. the first bps of the 5'end utr sequence of msscp-x is divergent from the msscp-x 5' utr or the sequence corresponding to the scp- domain, whereas the identical sequence in the 5' utr of msscp- to the scp- domain of the msscp-x transcript started at the same position as the splicing site in the b. mori scp-x/scp- gene. whether the msscp-x/scp- gene may produce two transcripts via alternative splicing needs further investigation. the spatial expression protein files of msscp-x are different from those described in s. littoralis and b. mori, in which the scp-x transcript level is higher in the midgut and malpighian tubules. in contrast, the msscp-x mrna levels were much lower in the midgut than in other tissues in general. the spatial transcription profiles of msscp-x and msscp- are similar to the mrna levels of the vertebrate scp- gene. the vertebrate scp-x is detected essentially in all tissues, whereas scp- was in highest abundance in the liver and intestine. the msscp- transcription levels were higher in the midgut and fat body during the feeding stage; the metabolic function of the fat body is similar to that of the vertebrate liver and adipose tissue. however, the insect scp-x protein appears to be processed post-translationally to produce two proteins: the 2/3-oxoacyl-coa thiolase and the scp-, which would render higher protein levels of scp- in tissues transcribing scp-x at higher levels. the anti-scp- domain of aescp-x antibodies recognized the purified recombinant msscp- and detected a similar molecular weight immune-reactive protein in the midgut, hindgut, and fat body. this result coincides with the fact that the insect midgut is the main site for cholesterol and fatty acid uptake. this also showed the expression pattern of msscp-x/scp- is similar to other mosquito scp-x and scp- genes. in aedes aegypti, aescp- and aescp-x were strongly expressed in the foregut and mostly in the midgut during the larval feeding stages. in aedes aegypti, spodoptera littoralis and bombyx mori, scp-xs had high levels of transcription in the midgut during the last larval feeding stages, however, the scp- domain protein is the prominent product due to post-translational processing of the scp-x protein in the midgut. therefore, regardless of which transcript of the scp-x/scp- gene is predominant in the midgut, high levels of scp- domain protein may be the end result. there was 56% less stored -cholesterol hours post feeding in the fat body in scpi-2-treated larvae than for the scpi-1-treatment. in manduca sexta gv cells, scpi- and scpi- at μm concentrations suppressed cholesterol uptake by 15% and 22%, respectively. the relatively higher binding affinities of scpi- than that of scpi- to insect scp- may result in the differences in biological effects between scpi- and scpi- treatments in which scpi- constantly showed higher efficacy in inhibiting cholesterol trafficking in vivo. the results were consistent with the earlier observation that scpi- is more lethal than scpi- to manduca larvae. both scpi- and scpi- effectively inhibited cholesterol uptake. there was little labeled cholesterol detected in the feces hours post feeding and a significant increase in -cholesterol in the feces over the observed time period in the control group, although the excretion of labeled fatty acid during the same period was constant. the underlying mechanism for the slower excretion of cholesterol than that of fatty acid in feces is unknown. however, -cholesterol levels in the feces within the hour period were % and % higher in scpi-1- and scpi- treated larvae, respectively, than in the controls. the results indicate that unabsorbed dietary cholesterol was excreted via feces. studies using the vertebrate model suggest that scp- mediates cholesterol trafficking and metabolism and is involved in fatty acid uptake. however, it is unclear whether insect scp- has similar functions in cholesterol and fatty acid trafficking and metabolism. we investigated the function of msscp- in vivo using sterol carrier protein- inhibitors to examine the biological effect of msscp- impairment on cholesterol and fatty acid uptake in m. sexta larvae. inhibition of msscp-2's function by scpis resulted in arrested growth and development in surviving larvae, indicated by lower weight gain over time and delayed pupation. when manduca larvae were fed lc concentrations of scpi- and scpi- for a short time, there was a significant reduction in the uptake of labeled cholesterol in the midgut and fat body in scpi-treated larvae compared to the controls. similar results were obtained when nbd-cholesterol, a fluorescent cholesterol analog, was used as the tracer for cholesterol uptake in vivo. however, the effect of scpi-treatment on palmatic acid was limited to the suppression of uptake into the midgut. in insects, the mechanism for the uptake of lipids from the diet into the midgut is unclear. it is hypothesized that lipids may be transported into the midgut epithelium via specific membrane transporters, and then trafficked intracellularly by carrier proteins toward the basal side of the midgut for uploading onto the lp. the efficacy of a scpi in suppressing cholesterol absorption seemed to correlate with its efficiency in inhibiting ligand-binding in scp-, suggesting that intracellular transport plays a critical role in cellular uptake of cholesterol. the fact that inhibition of msscp-2's function via scpis did not completely abolish cholesterol uptake points to the possibility that there may be more than one class of intracellular carrier proteins for cholesterol in insects. another candidate as an intracellular cholesterol carrier is the steroid acute regulator protein -related lipid transfer protein. the mode of larvicidal activity of scpis against some insect species, including aedes aegypti, anopheles gambiae, culex pipiens and m. sexta is unclear. the relatively high binding affinities of scpi- and scpi- to insect scp-2s indicate that scpis may inhibit the biological functions of scp- by competing with cholesterol for binding to scp-. scpi- and scpi- suppressed -cholesterol efflux from the midgut to the hemolymph in m. sexta larvae within hours of treatment, resulting in the reduction of stored -cholesterol by 37% and 69%, respectively. although, scpi- and scpi- also suppressed -palmatic acid uptake in the midgut, there was no evidence that scpi-treatment impaired the efflux of absorbed -palmatic acid from the midgut into the hemolymph. one plausible explanation for the lack of scpi's effect on the efflux of fatty acid into the hemolymph and ultimately storage in the fat body may be the redundant function of fatty acid binding proteins in the midgut and fat body. the results of this study provide evidence that scpis inhibit cholesterol uptake/storage in insects in vivo. the effect of scpi-treatment on fatty acid uptake in larvae was limited to the midgut. since insects can synthesize fatty acid de novo from acetate, one possible mode of action of scpis' larvicidal activities is reduction in cholesterol uptake over time resulting in a deficiency in cholesterol that might lead to a high mortality rate in scpi-treated manduca larvae. the likely mechanism of scpis in inhibiting cholesterol absorption is the decrease in clearance of absorbed cholesterol from the midgut tissue, suggesting that msscp- may be involved in the cellular trafficking of cholesterol in the midgut.  in this study we tested the hypothesis that msscp- may be involved in cholesterol uptake and intracellular transportation. our results show that manduca sexta sterol carrier protein-x and sterol carrier protein- have a high degree of homology in the scp- domain to other insects' scp- and recombinant msscp- bound to nbd-cholesterol with high binding affinity. transcripts of msscp- were highly expressed in the larval midgut and fat body. the result suggests that msscp- may be involved in cholesterol uptake in the midgut, the main site for cholesterol absorption, and intracellular transportation in the fat body, the main site for cholesterol storage. msscp- may function as a lipid carrier protein in vivo. we have also shown that sterol carrier protein inhibitors effectively reduced cholesterol uptake and cholesterol accumulation in the midgut and in fat body. due to the total dependence of insects on exogenous cholesterol, targeting insect scp- may be a viable approach for the development of new insecticides. 
we identified the manduca sexta sterol carrier protein-x and the sterol carrier protein- gene from the larval fat body and the midgut cdnas. the results suggest that msscp- may function as a lipid carrier protein in vivo, and targeting insect scp- may be a viable approach for the development of new insecticides. the msscp-x/scp- protein has a high degree of homology in the scp- domain to other insects' scp- transcripts of msscp- were detected at high levels in the midgut and the fat body of m. sexta during the larval stages. the midgut is the main site for cholesterol uptake in many insects. recombinant msscp- bound to nbd-cholesterol with high affinity, which was suppressed by sterol carrier protein- inhibitors. sterol carrier proteins, non-specific lipid transfer proteins, have been speculated to be involved in intracellular cholesterol transfer and metabolism in vertebrates. based on the high degree of homology in the conserved sterol transfer domain to rat and human scp- it is supposed that insect scp- has a parallel function to vertebrate scp- cholesterol uptake and transportation during the feeding larval stages are critical processes in insects because they are auxotrophic for exogenous cholesterol. however, the molecular mechanism by which dietary cholesterol is digested and absorbed within the midgut and then released into the hemolymph for transportation to utilization or storage sites is poorly understood.
 cholesterol uptake and transportation during the feeding larval stages are critical processes in insects because they are auxotrophic for exogenous cholesterol. the midgut is the main site for cholesterol uptake in many insects. however, the molecular mechanism by which dietary cholesterol is digested and absorbed within the midgut and then released into the hemolymph for transportation to utilization or storage sites is poorly understood. sterol carrier proteins, non-specific lipid transfer proteins, have been speculated to be involved in intracellular cholesterol transfer and metabolism in vertebrates. based on the high degree of homology in the conserved sterol transfer domain to rat and human scp- it is supposed that insect scp- has a parallel function to vertebrate scp-  we identified the manduca sexta sterol carrier protein-x and the sterol carrier protein- gene from the larval fat body and the midgut cdnas. the msscp-x/scp- protein has a high degree of homology in the scp- domain to other insects' scp- transcripts of msscp- were detected at high levels in the midgut and the fat body of m. sexta during the larval stages. recombinant msscp- bound to nbd-cholesterol with high affinity, which was suppressed by sterol carrier protein- inhibitors.  the results suggest that msscp- may function as a lipid carrier protein in vivo, and targeting insect scp- may be a viable approach for the development of new insecticides. 