BACKGROUND
the life rhythmicities in caenorhabditis elegans and drosophila melanogaster result from programmed mediation of a series of heterchronic genes  <cit> .
two small noncoding rnas, lin- <dig> and let- <dig>  are essential components of the heterochronic pathway dictating temporal decisions of cell fate from one stage to the next  <cit> .lin- <dig> has long been thought to regulate the transition from the first to the second larval stages in c. elegans  <cit> , and let- <dig> was also confirmed to be undetectable until the last larval stage and functions as a temporal switch promoting the transition from larval to adult stages  <cit> .
however, the latest studies showed that lin- <dig> and one of its target genes, lin- <dig>  also regulate life span in the adult of c. elegans  <cit> , and that let- <dig> can even be found in the 3rd instar larvae of c.elegans  <cit> , and may be a master temporal regulator of late larval development in c. elegans  <cit> .
in drosophila,let- <dig> rna first appears at the end of the third larval instar, a few hours before puparium formation, and reaches high levels during pupal development stage  <cit> .
both the temporal rnas are functional in a mechanism through post-transcriptionally or translationally repressing their target genes.
lin- <dig> inhibits the translations of lin- <dig> and lin- <dig> by base-pairing to partially complementary sites in the 3'-utrs of their mrnas  <cit> , and let- <dig> has been confirmed to inhibit lin-41's expression in a similar fashion through binding to complementary sites in its 3'utr  <cit> .
let- <dig> rna is consistently detectable in samples from diverse animal phyla, including chordates, hemichordates, echinoderms, mollusks, annelids, and arthropods, but cannot be found in basal metazoans, such as cnidarians and poriferans, as well as in plants and unicellular organisms  <cit> .
the diverse expression patterns of let- <dig> family in various species may be responsible for its important role in developmental regulation  <cit> .
the expression level of temporal micrornas was confirmed to respond to the pulse of steroid hormone 20-hydroxyecdysone  and/or juvenile hormone   <cit> .
ecd and jh control the development of drosophila from embryogensis to adulthood  <cit> .
they exert the opposite effects on the expression of temporal mirna genes, such as mir- <dig>  mir- <dig>  mir- <dig> and let- <dig>  <cit> .
furthermore, the opposite effects of jh and ecd signals could be mediated by broad-complex   <cit> .
the major developmental transitions in the life cycle of drosophila are directed by the pulses of ecdysone  <cit> , and ecdysone signals the stage-specific programmed cell death  of the larval salivary glands during drosophila metamorphosis  <cit> , and also triggers the programmed cell death in anterior silk glands  of bombyx mori  <cit> .
early ecd-inducible genes,e <dig>  e <dig> and br-c, coordinate with the temporal and spatial activation of downstream genes, initiating a genetic cascade that leads to distinct metamorphic processes such as pupation, pcd of larval tissues, remodeling of the central nervous system, and proliferation and morphogenesis of imaginal discs  <cit> .
ecdysone and the ecd-inducible gene br-c are required in the upregulation of some micrornas including let- <dig> as well as the downregulation of other micrornas  <cit> .
although a bevy of let- <dig> family members exist in various organisms, their dos-a-dos in b. mori has not been described experimentally  <cit> .
b. mori is a typical member of the family bombycidae with about  <dig> moth species in the order lepidoptera.
it has recently become a model for studying the harmful lepidopteran insects and the commercial production of useful biological substances called interleukins  <cit> , especially after the completion of draft sequence of domesticated silkworm genome  <cit> .
the overt roles of lin- <dig> and let- <dig> in switching the transition of metamorphosis in other organisms raise the question of whether similar regulatory rnas are involved in the control of metamorphosis development of b. mori and how the temporal identity conferred by the heterochronic genes is related to the major developmental landmarks that define the silkworm life cycle.
as a step towards a better understanding of roles of let- <dig>  we seek to explore whether they exist in silkworm genome and how they are expressed during the life cycle of this insect.
RESULTS
one homology of let- <dig> in the silkworm genome
by using of blastn based on sequence homology, we identified one orthologue of let- <dig> in the silkworm genome, which was named bmo-let- <dig> according to a common criterion   <cit> .
the same site on the genome sequence of aadk <dig> was further confirmed by using the mirscan program combined with rnafold.
the mature sequence and its precursor are at 10858– <dig> and 10846– <dig>  respectively, or at 10857– <dig> and 10854– <dig>  respectively.
the mature sequence, either bmo-let- <dig> or bmo-let-7*, is on the 5' arm of the foldback precursor .
the potential alternative forms of this homology were submitted to northern blotting analysis later in order to see if stable results could be obtained.
using the same prediction methods, however, we did not find cel-lin- <dig> homologue in the silkworm genome.
the extending let- <dig> family shares common "mirna seed" and represents restricted species diversities
there have been over  <dig> members so far in let- <dig> family that contains about  <dig> different mature sequences, ranging in length from  <dig> nt to  <dig> nt.
all members share the two common fragments, gagguag and uug , and they might be the common "mirna seed" which could be used to search for target genes according to the complementarity to sequences in the 3' utr of all expressed genes  <cit> .
seven of ten shared positions are located at the 5' end of the mature sequence, and the other three at the 3' end, consistent with the proposal that the 5' end of the functional small rna is crucial for the stability and proper loading of let- <dig> into the mirisc complex though both ends are required for downregulation of a target gene  <cit> .
twelve positions exhibit exchangeable if the mature seqence of cel-let- <dig>  5'-ugagguaguagguuguauaguu-3' , is used as the reference, u at 5' end being position  <dig>  for example, it is a at position  <dig> in five let-7d members, g at position  <dig> in three let-7e members, and u at position  <dig> in three other let-7d members.
at position  <dig>  g is replaced by a in three let-7h and eleven mir- <dig> members, or by u in both fru-let-7j and tni-let-7j.
position  <dig> is shared by three nucleotides, g, u and a in the mature sequences of all members.
four nucleotides near the 3' end, namely, positions  <dig> to  <dig> are also replaceable.
compared with the reference sequence , bmo-let- <dig> substitutes the nucleotide u with a at 3' end, as an exception to uridine preference at both ends.
in addition, deletion of nucleotides can take place at the 3'end.
for example, ssc-let- <dig> is three nucleotides shorter than the control sequence and twenty-two members are one nucleotide shorter than the norm sequence .
taken together, the divergent sequences and replaceable nucleotides may imply their function diversities among species to some extent.
the extending let- <dig> family might have evolved from a common ancient precursor
c. briggsae, c. elegans, b. mori, d. melanogaster, d. pseudoobscura and a. gambiae are ecdysozoa, protostomia, and all let- <dig> precursors of them are clustered in the same clade .
b. mori belongs to insecta, arthropoda, and bmo-let- <dig> precursor is clustered with those of other insects, accordingly.
homo sapiens, fugu rubripes, danio rerio, xenopus tropicalis, bos taurus belong to chordata deuterostomia, and let- <dig> precursors of them, except for has-let- <dig> and ssc-let- <dig>  are clustered in one clade.
the conserved nucleotides of the precursors are those that tend to form the mature sequences.
every small rna locates closely to the 5' end of its precursor.
however, the loop regions are significantly more divergent than their mature sequences between mammalians and insects .
the conserved fragments on both arms of the precursors and the variations in loop regions support the hypothesis that an ancient precursor of the let- <dig> genes may have been common to the earliest animal lineages  <cit> .
it is detectable in the silkworm from the early first molt to the adult, suggesting that bmo-let- <dig> might even function in early larval stages of silkworm
let- <dig> is notably induced at the last larval stage in c. elegans, promoting the transformation from the larva to the adult  <cit> , and highly expressed in the late third instar larvae of d. melanogaster when a pulse of the ecdysone triggers puparium formation and onset of metamorphosis  <cit> .
in the case of bmo-let- <dig>  we observed a wide expression profile by using of fifteen stage-specific samples across the lifespan of the silkworm .
the expression of bmo-let- <dig> started at the early first molt, remained at low levels until the early third instar, and rapidly reached high level at the early third molt, still increased gradually to the maximum at pupa and imago.
the whole larval duration from the first to the fifth instars could be divided into two parts with the boundary at the third instar on the basis of expression level of bmo-let- <dig> , in accordance with the division determined by the pulse of ecdysone in the first four instars  <cit> .
approximately, the general dynamic changes in bmo-let- <dig> levels is also close to the pulse of broad-complext  transcriptional factor of silkworm, which is hardly detectable before the fourth instar, lowly expressed in the fifth instar, and largely induced during cocooning and pupation when the ecdysone titer increases  <cit> .
the coordinated changes imply that the developmental transitions of silkworm are more likely to be triggered by the cooperation of ecdysone, br-c, bmo-let- <dig> and many other downstream genes.
the wide-range emergence implies that bmo-let- <dig> not only controls the metamorphosis from late laval stage to the pupa, as do the other members in let- <dig> family confirmed in c. elegans  <cit>  and d. melanogaster  <cit> , but might even function in the early larval stages of silkworm.
the antisense probes of bmo-let- <dig> and bmo-let-7* brought out the same results in our sequential and parallel blotting experiments.
the two sense probes, however, gave no signals in all samples, confirming the single strand of this small functional rna.
cel-lin- <dig> was beyond detection in the silkworm by northern blotting with the antisense and sense probes, consistent with the prediction results.
detailed expression profiles from unfertilized egg  to the third molt and from the early fourth instar to the late fifth instar
after studying the general expression profile of bmo-let- <dig> across the whole lifespan  with fifteen samples , we further investigated its expression patterns at the turning point of each stage.
as shown in the expression profiles from unfertilized egg  to the adult and from ova to the third molt , as well as from the early fourth instar to day- <dig> in the fifth instar , the signal intensity at the beginning of each molt was stronger than that at the latest former early instar.
for example, the signal intensities at the early second and third molts almost doubled those at the early second and third instars, respectively ; and the signal intensity at the early fourth molt was also higher than that at the early fourth instar .
high level of ecdysone is required to induce some heterochronic genes before molt, and let- <dig> was also confirmed to be induced by pulse of ecdysone in d. melanogaster  <cit> .
both let- <dig> and ecdysone may be crucial for the transformation of larval stages  <cit> .
during the development of silkworm, ecdysone peak comes several hours before the beginning of each molt, and a fall occurs at ecdysis  <cit> , slightly ahead of scheduel of the expression profile peak of bmo-let- <dig> in our tests, well supporting the proposal that bmo-let- <dig> may be induced by the pulse of ecdysone.
it has been demonstrated that not conjugated but free ecdysone plays an important role in the control of cuticle formation in the embryo  <cit> .
the unfertilized egg  before oviposition removed from the female abdomen gave a weak signal of this microrna , probably due to the slight induction by maternity genes or/and by large amount of free ecdysone in the adult organ, oviduct, which overly expressed bmo-let- <dig> .
bmo-let- <dig> was undetectable in diapause eggs  because of very low free ecdysteroid fraction  <cit> .
for the same reason, no signal was found in the embryonic development stages at day- <dig>  day- <dig> and day- <dig> after oviposition at 25°c , and in the first instar larval stages, even though the free ecdysteroid fraction is much larger than the conjugated ecdysteroid fraction in early developing eggs from the second to the fifth day <cit> .
probably, it is not the conjugated ecdysteroid but the free ecdysone that functions in inducing the expression of bmo-let- <dig> and only when the amount of free ecdysone is over a threshold can function properly.
the signal intensity at the beginning of molt was slightly stronger than that at the end of molt.
for example, the expression levels of bmo-let- <dig> were higher at the early first and fourth molts than those at the late first and fourth molts, respectively .
the instar stages, however, showed different expression profiles.
the signal intensity at the beginning of the second and fourth instars was almost identical to that at the end of the second and fourth instars, respectively.
a big jump in the expression profile was observed relative to at the beginning of third instar, further confirming the watershed between the third instar and fourth instar where comes the maximum peak of ecdysone  <cit> .
the duration of the fifth instar is usually  <dig> days, and the expression pattern during this period was relatively broad.
bmo-let- <dig> was highly expressed at day  <dig> and day  <dig>  relatively lowly expressed at day  <dig> and day  <dig>  then again highly expressed at the end of the fifth instar.
moreover, there was no expression difference between female and male during this stage .
taken together, each upsurge occurs at the turning point between developmental stages before maturing, suggesting that bmo-let- <dig> is required for the transformation of larval stages.
a falling in the high level of expression profile is shared by female and male pupa metamorphosis at pupal-moth ecdysis
a strong signal was observed in the new female pupa and adult .
northern blotting in this assay, however, revealed an extraordinarily high expression profile across the whole process of pupation and eclosion of females and males.
a strong signal of the expression was observed at day- <dig> of the fifth instar when the silkworm is ripe for mounting or spinning cocoons .
after spinning, the fifth instar larvae enter a prepupation stage.
there was no expression difference between the beginning and the end of spinning stage, implying that bmo-let- <dig> is unlikely related to the process of spinning.
the silkworm prepupa undergoes a molt before pupation, which requires higher titer of ecdysone  <cit> , as confirmed in the process of transition from prepupa to pupa in d. melanogaster  <cit> .
after pupation, histolysis and histogenesis are occurring intensively and simultaneously, much more of this functional rna should be required for the double missions, accordingly.
the expression profile of bmo-let- <dig> fell at day  <dig> pupa in female group, and at day  <dig> in male group, when the ecdysone is also lowly produced  <cit> , then resumed to the maximum just before adult emergence corresponding to the rising ecdysone  <cit> .
the expression falling during pupal-adult metamorphosis of both female and male should have occurred synchronously if they had been incubated under the same temperature and humidity.
jh and ecd function oppositely on the developmental metamorphosis of the silkworm.
jh can induce the expression of some genes and repress the expression of other genes activated by ecd  <cit> .
jh is highly secreted by corpus allatum at early period of each larva stage, and ecd is highly secreted by prothoracic gland at late period of each larva stage  <cit> .
insect metamorphosis is triggered by ecd in the absence of jh, and is carried out by self-destructive mechanisms of programmed cell death   <cit> .
the let- <dig> temporal expression alterations are in synchronization with the pulse of ecdysone  <cit> , as was further confirmed by our northern blotting results .
large amount of ecd is secreted in the late fifth instar or in just ripe silkworm in favor of spinning cocoons and molting in pupation  <cit> .
jh is required at late pupa stage to promote the development of ovary and accumulation of yelk  and to maintain the functions of testicles.
a previous study revealed that in b.mori, the prothoracic gland hormone secreted during the first day after larval-pupal ecdysis might be responsible for the development of the ovaries during the pupal period, especially essential for the initiation of ovarian development  <cit> .
after pupation, high level ecdysone is required to drive the tissue differentiation and organ formation of adult moth  <cit> .
therefore, the ecd-induced microrna, bmo-let- <dig>  is highly expressed in pupae.
however, the mechanism of how bmo-let- <dig> responds to ecdysone and broad-complex pathways in the silkworm is worthy of further study.
bmo-let- <dig> is widely expressed in tissues/organs of female and male individuals of the fifth-instar day- <dig> larvae
bmo-let- <dig> was moderately expressed in female and male individuals at the fifth instar day- <dig> .
to correlate expression domains with the functions, we determined the tissue specificity of bmo-let- <dig> expression in various tissues of larvae in day- <dig> of the fifth instar.
reproducible and consistent results were confirmed by independently parallel northern blotting experiments with the two antisense probes .
the let- <dig> small rna of  <dig> nt or  <dig> nt was present in all tissues studied .
moderate signals were observed in body wall, midgut, gonad and malpighian tubule, whereas a weak positive signal appeared in anterior silk gland and posterior silk gland, and strong signal was observed in the heads.
sex differences in the expression level were prominent in fat body and hemocyte, however.
the female fat body showed weak signal but the male fat body exhibited very strong one.
on the contrary, bmo-let- <dig> was highly expressed in the female hemocyte but lowly in male hemocyte.
the distinct differences in the expression of bmo-let- <dig> imply that some unknown mechanisms responsible for its transcription might be functioning differently in fat body and hemocyte of female and male silkworm.
tissue-specific or spatial expression patterns have also been demonstrated in other organisms.
as shown in d. melanogaster, let- <dig> is widely expressed in a variety of tissues from prepupae, such as brains, imaginal discs, fat bodies, salivary glands, and malphigian tubules, with relatively higher levels in fat bodies and imaginal discs, and it was also detected in adult ovaraies and carcasses, suggesting that it could regulate diverse metamorphic processes, such as the terminal differentiation of imaginal discs and apoptosis of salivary glands and fat bodies  <cit> .
furthermore, various tissues from human also express let- <dig> rna, including brain, heart, kidney, liver, lung, trachea, bone marrow, colon, small intestine, spleen, stomach, and thymus, and the lowest level of human let- <dig> is observed in bone marrow likely due to a large proportion of immature cell in it  <cit> .
different expression profiles in various tissues from the silkworm indicate that bmo-let- <dig> might not only function in triggering transitions of temporal stages, but more broadly in a variety of metabolisms because each microrna can control hundreds of target genes  <cit> .
expression profile in tissues from just mounting to day- <dig> pupae suggests its potential roles in histolysis and histogenesis
tissues from different stages here presented profiles breadthwise and lengthwise .
the prospective fates of organs and tissues such as head, body wall, silk gland, midgut, fat body and gonad during metamorphic transformation are different.
silk gland and midgut will be completely histolyzed during the pupal stage  <cit> , and fat body undergoes reorganization as well as histolysis during larval-pupal transformation  <cit>  and predetermined imaginal tissues differentiate and grow into organs and external structures  <cit> .
the histolysis of larval silk gland, fat body and midgut is typical of programmed cell death  <cit> .
thus, the expression profile of bmo-let- <dig> in tissues from just mounting to day- <dig> pupae may reflect its association with histolysis and apoptosis.
the individuals at just mounting expressed large amount of bmo-let- <dig> small rna , but the body wall at this period only showed very weak signal .
silk gland and fat body at this time shared a moderate amount.
midgut, however, showed very high level, suggesting that midgut histolysis was triggered once the silkworm stopped eating or just before mounting.
when silkworms have finished spinning cocoons three days after mounting, the silk gland accomplishes its mission and undergoes histolysis.
at this time, high level expression of let- <dig> was detected in silk gland and testis; moderate level was observed in midgut and fat body, and a weak signal appeared in head and body wall, suggesting that let- <dig> also promotes the apoptosis of silk gland and the maturation of testisis, as demonstrated during the metamorphosis in d. melanogaster  <cit> .
just after pupation , silk gland enters the late stage of histolysis, body wall and fat body are newly reorganized.
let- <dig> was very highly expressed in body wall and fat body, but very lowly expressed in silk gland of the newborn pupa, suggesting that let- <dig> functions in the histogenesis of body wall and fat body on the basis of the conclusion that lin- <dig> and let- <dig> control the timing of cell differentiation and proliferation  <cit> .
at day- <dig> pupae, weak expression signals were observed in midgut and silk gland because these two tissues are almost completely histolyzed, but intensive signals appeared in body wall and fat body which are still undergoing reorganization as well as histolysis.
taken together, during the metamorphosis from just mounting to day- <dig> pupa, the signal intensity in body wall and fat body was rising to the utmost at newborn pupa, then fell at day - <dig> pupa.
the high level expression of bmo-let- <dig> in fat body and other tissues is very likely to be resulted from the ecdysone induction, considering the evidence that the programmed autophagy in the drosophila fat body is induced by ecdysone  <cit> .
silk gland showed the highest expression level just after spinning , and exhibited a falling signal during the undergoing of apoptosis.
in midgut, however, the expression level of bmo-let- <dig> reached the maximum at just mounting, and then decreased.
although bmo-let- <dig> expression is generally response to ecdysone pulse in various tissues as well as in developmental stages, more precise time points and tissues will be investigated to further determine if the expression of bmo-let- <dig> is directly induced by ecdysone in the silkworm.
effect of ecdysone change on the expression of bmo-let- <dig> in cultured cells
the expression of bmo-let- <dig> has been systematically investigated in the whole body and tissues or organs during the development of silkworm.
the results suggested that bmo-let- <dig> generally responds to the pulse of ecdysone.
cell line culture experiments were then carried out in order to test whether ecdysone is required to sustain the expression of bmo-let- <dig> and how the concentrations of ecdysone are influencing its expression.
this small rna expression trends differed even if it was detectable in all cell samples, which have been incubated in the presence of different levels of ecdysone for different periods .
for the control cells , bmo-let- <dig> rna level increased straightly during the whole culture time, with a gentle inflexion at  <dig> h .
when the cells were treated with  <dig> μm ecdysone, bmo-let- <dig> was initially up-regulated, then reached a plateau shared by the control at  <dig> h. lower level of ecdysone, such as  <dig> μm, seems to exert diverse influence on bmo-let- <dig>  down-regulating it slightly before  <dig> h, up-regulating it distinctly from  <dig> h to  <dig> h, then down-regulating it again .
to avoid the confusion resulted from possible experiment discrepancy, we especially investigated the effects of different levels of ecdysone at  <dig> h after incubation .
bmo-let- <dig> was slightly upregulated in the presence of  <dig> μm ecdysone and most effectively induced by  <dig> μm ecdysone.
however, after treated with  <dig> μmecdysone, bmo-let- <dig> rna level was almost always stable during the whole examined span of  <dig> h, and dramatically decreased in the presence of  <dig> μm.
this suggests that the expression of bmo-let- <dig> might be induced by low-level ecdysone but repressed by high-level ecdysone.
the silkworm ovary itself has been confirmed to express bmo-let- <dig> , and ovarian cell line bmn-swu <dig> was also validated to produce this small rna even if no ecdysone was added into the culture medium .
during the culture periods, the untreated cells are very likely to produce ecdysone, so we cannot rule out the possibility that the endogenetic and adscititious ecdysones are functioning together in inducing or repressing the expression of bmo-let- <dig> rna.
since bmo-let- <dig> is undetectable during the development of silkworm's embryo , the embryo cell line is also unlikely to produce detectable bmo-let- <dig> rna.
it would be interesting to look at whether the expression of bmo-let- <dig> rna could be observed when the embryo cell line would have been incubated for certain length of time in the presence of ecdysone.
the tissue-specificity and the timing of let- <dig> expression are conserved in invertebrates, suggesting that it could exert widely in mediating diverse metamorphic processes  <cit> .
the programmed ecdysis behaviour in larval, pupal and adult stages occurs in all of the holometabolous insects, and depends on coordinated expression of a series of genes related to peptide signaling  <cit> .
very low-level expression of let- <dig> can even be observed in the npr <dig> mutants of drosophila, which are unresponsive to the ecdysone pulse at the end of the last larval stage  due to the lack of four br-c isoforms  <cit> .
it could be speculated that ecdysone might be only one of the factors regulating bmo-let- <dig>  and some higher upstream components of the heterochronic pathway could also contribute to its expression.
CONCLUSIONS
a new candidate member  of let- <dig> family in the silkworm  was identified by computational approach and its expression was profiled by northern blotting.
lines of evidence confirmed that bmo-let- <dig> is temporally and spatially expressed in the silkworm and the expression is in response to the pulse of ecdysone, and might be related to a number of biological processes such as cell proliferation, histogenesis, histolysis, organogenesis and apoptosis.
it is shown from the cell culture experiment that induction or repression effects of ecdysone on the expression of bmo-let- <dig> are concentration-dependent.

 the life rhythmicities in caenorhabditis elegans and drosophila melanogaster result from programmed mediation of a series of heterchronic genes. two small noncoding rnas, lin- and let- are essential components of the heterochronic pathway dictating temporal decisions of cell fate from one stage to the next.lin- has long been thought to regulate the transition from the first to the second larval stages in c. elegans, and let- was also confirmed to be undetectable until the last larval stage and functions as a temporal switch promoting the transition from larval to adult stages. however, the latest studies showed that lin- and one of its target genes, lin- also regulate life span in the adult of c. elegans, and that let- can even be found in the 3rd instar larvae of c.elegans, and may be a master temporal regulator of late larval development in c. elegans. in drosophila,let- rna first appears at the end of the third larval instar, a few hours before puparium formation, and reaches high levels during pupal development stage. both the temporal rnas are functional in a mechanism through post-transcriptionally or translationally repressing their target genes. lin- inhibits the translations of lin- and lin- by base-pairing to partially complementary sites in the 3'-utrs of their mrnas, and let- has been confirmed to inhibit lin-41's expression in a similar fashion through binding to complementary sites in its 3'utr. let- rna is consistently detectable in samples from diverse animal phyla, including chordates, hemichordates, echinoderms, mollusks, annelids, and arthropods, but cannot be found in basal metazoans, such as cnidarians and poriferans, as well as in plants and unicellular organisms. the diverse expression patterns of let- family in various species may be responsible for its important role in developmental regulation. the expression level of temporal micrornas was confirmed to respond to the pulse of steroid hormone 20-hydroxyecdysone and/or juvenile hormone. ecd and jh control the development of drosophila from embryogensis to adulthood. they exert the opposite effects on the expression of temporal mirna genes, such as mir- mir- mir- and let-. furthermore, the opposite effects of jh and ecd signals could be mediated by broad-complex. the major developmental transitions in the life cycle of drosophila are directed by the pulses of ecdysone, and ecdysone signals the stage-specific programmed cell death of the larval salivary glands during drosophila metamorphosis, and also triggers the programmed cell death in anterior silk glands of bombyx mori. early ecd-inducible genes,e e and br-c, coordinate with the temporal and spatial activation of downstream genes, initiating a genetic cascade that leads to distinct metamorphic processes such as pupation, pcd of larval tissues, remodeling of the central nervous system, and proliferation and morphogenesis of imaginal discs. ecdysone and the ecd-inducible gene br-c are required in the upregulation of some micrornas including let- as well as the downregulation of other micrornas. although a bevy of let- family members exist in various organisms, their dos-a-dos in b. mori has not been described experimentally. b. mori is a typical member of the family bombycidae with about moth species in the order lepidoptera. it has recently become a model for studying the harmful lepidopteran insects and the commercial production of useful biological substances called interleukins, especially after the completion of draft sequence of domesticated silkworm genome. the overt roles of lin- and let- in switching the transition of metamorphosis in other organisms raise the question of whether similar regulatory rnas are involved in the control of metamorphosis development of b. mori and how the temporal identity conferred by the heterochronic genes is related to the major developmental landmarks that define the silkworm life cycle. as a step towards a better understanding of roles of let- we seek to explore whether they exist in silkworm genome and how they are expressed during the life cycle of this insect.  one homology of let- in the silkworm genome by using of blastn based on sequence homology, we identified one orthologue of let- in the silkworm genome, which was named bmo-let- according to a common criterion. the same site on the genome sequence of aadk was further confirmed by using the mirscan program combined with rnafold. the mature sequence and its precursor are at 10858– and 10846– respectively, or at 10857– and 10854– respectively. the mature sequence, either bmo-let- or bmo-let-7*, is on the 5' arm of the foldback precursor. the potential alternative forms of this homology were submitted to northern blotting analysis later in order to see if stable results could be obtained. using the same prediction methods, however, we did not find cel-lin- homologue in the silkworm genome. the extending let- family shares common "mirna seed" and represents restricted species diversities there have been over members so far in let- family that contains about different mature sequences, ranging in length from nt to nt. all members share the two common fragments, gagguag and uug, and they might be the common "mirna seed" which could be used to search for target genes according to the complementarity to sequences in the 3' utr of all expressed genes. seven of ten shared positions are located at the 5' end of the mature sequence, and the other three at the 3' end, consistent with the proposal that the 5' end of the functional small rna is crucial for the stability and proper loading of let- into the mirisc complex though both ends are required for downregulation of a target gene. twelve positions exhibit exchangeable if the mature seqence of cel-let- 5'-ugagguaguagguuguauaguu-3', is used as the reference, u at 5' end being position for example, it is a at position in five let-7d members, g at position in three let-7e members, and u at position in three other let-7d members. at position g is replaced by a in three let-7h and eleven mir- members, or by u in both fru-let-7j and tni-let-7j. position is shared by three nucleotides, g, u and a in the mature sequences of all members. four nucleotides near the 3' end, namely, positions to are also replaceable. compared with the reference sequence, bmo-let- substitutes the nucleotide u with a at 3' end, as an exception to uridine preference at both ends. in addition, deletion of nucleotides can take place at the 3'end. for example, ssc-let- is three nucleotides shorter than the control sequence and twenty-two members are one nucleotide shorter than the norm sequence. taken together, the divergent sequences and replaceable nucleotides may imply their function diversities among species to some extent. the extending let- family might have evolved from a common ancient precursor c. briggsae, c. elegans, b. mori, d. melanogaster, d. pseudoobscura and a. gambiae are ecdysozoa, protostomia, and all let- precursors of them are clustered in the same clade. b. mori belongs to insecta, arthropoda, and bmo-let- precursor is clustered with those of other insects, accordingly. homo sapiens, fugu rubripes, danio rerio, xenopus tropicalis, bos taurus belong to chordata deuterostomia, and let- precursors of them, except for has-let- and ssc-let- are clustered in one clade. the conserved nucleotides of the precursors are those that tend to form the mature sequences. every small rna locates closely to the 5' end of its precursor. however, the loop regions are significantly more divergent than their mature sequences between mammalians and insects. the conserved fragments on both arms of the precursors and the variations in loop regions support the hypothesis that an ancient precursor of the let- genes may have been common to the earliest animal lineages. it is detectable in the silkworm from the early first molt to the adult, suggesting that bmo-let- might even function in early larval stages of silkworm let- is notably induced at the last larval stage in c. elegans, promoting the transformation from the larva to the adult, and highly expressed in the late third instar larvae of d. melanogaster when a pulse of the ecdysone triggers puparium formation and onset of metamorphosis. in the case of bmo-let- we observed a wide expression profile by using of fifteen stage-specific samples across the lifespan of the silkworm. the expression of bmo-let- started at the early first molt, remained at low levels until the early third instar, and rapidly reached high level at the early third molt, still increased gradually to the maximum at pupa and imago. the whole larval duration from the first to the fifth instars could be divided into two parts with the boundary at the third instar on the basis of expression level of bmo-let-, in accordance with the division determined by the pulse of ecdysone in the first four instars. approximately, the general dynamic changes in bmo-let- levels is also close to the pulse of broad-complext transcriptional factor of silkworm, which is hardly detectable before the fourth instar, lowly expressed in the fifth instar, and largely induced during cocooning and pupation when the ecdysone titer increases. the coordinated changes imply that the developmental transitions of silkworm are more likely to be triggered by the cooperation of ecdysone, br-c, bmo-let- and many other downstream genes. the wide-range emergence implies that bmo-let- not only controls the metamorphosis from late laval stage to the pupa, as do the other members in let- family confirmed in c. elegans and d. melanogaster, but might even function in the early larval stages of silkworm. the antisense probes of bmo-let- and bmo-let-7* brought out the same results in our sequential and parallel blotting experiments. the two sense probes, however, gave no signals in all samples, confirming the single strand of this small functional rna. cel-lin- was beyond detection in the silkworm by northern blotting with the antisense and sense probes, consistent with the prediction results. detailed expression profiles from unfertilized egg to the third molt and from the early fourth instar to the late fifth instar after studying the general expression profile of bmo-let- across the whole lifespan with fifteen samples, we further investigated its expression patterns at the turning point of each stage. as shown in the expression profiles from unfertilized egg to the adult and from ova to the third molt, as well as from the early fourth instar to day- in the fifth instar, the signal intensity at the beginning of each molt was stronger than that at the latest former early instar. for example, the signal intensities at the early second and third molts almost doubled those at the early second and third instars, respectively; and the signal intensity at the early fourth molt was also higher than that at the early fourth instar. high level of ecdysone is required to induce some heterochronic genes before molt, and let- was also confirmed to be induced by pulse of ecdysone in d. melanogaster. both let- and ecdysone may be crucial for the transformation of larval stages. during the development of silkworm, ecdysone peak comes several hours before the beginning of each molt, and a fall occurs at ecdysis, slightly ahead of scheduel of the expression profile peak of bmo-let- in our tests, well supporting the proposal that bmo-let- may be induced by the pulse of ecdysone. it has been demonstrated that not conjugated but free ecdysone plays an important role in the control of cuticle formation in the embryo. the unfertilized egg before oviposition removed from the female abdomen gave a weak signal of this microrna, probably due to the slight induction by maternity genes or/and by large amount of free ecdysone in the adult organ, oviduct, which overly expressed bmo-let-. bmo-let- was undetectable in diapause eggs because of very low free ecdysteroid fraction. for the same reason, no signal was found in the embryonic development stages at day- day- and day- after oviposition at 25°c, and in the first instar larval stages, even though the free ecdysteroid fraction is much larger than the conjugated ecdysteroid fraction in early developing eggs from the second to the fifth day. probably, it is not the conjugated ecdysteroid but the free ecdysone that functions in inducing the expression of bmo-let- and only when the amount of free ecdysone is over a threshold can function properly. the signal intensity at the beginning of molt was slightly stronger than that at the end of molt. for example, the expression levels of bmo-let- were higher at the early first and fourth molts than those at the late first and fourth molts, respectively. the instar stages, however, showed different expression profiles. the signal intensity at the beginning of the second and fourth instars was almost identical to that at the end of the second and fourth instars, respectively. a big jump in the expression profile was observed relative to at the beginning of third instar, further confirming the watershed between the third instar and fourth instar where comes the maximum peak of ecdysone. the duration of the fifth instar is usually days, and the expression pattern during this period was relatively broad. bmo-let- was highly expressed at day and day relatively lowly expressed at day and day then again highly expressed at the end of the fifth instar. moreover, there was no expression difference between female and male during this stage. taken together, each upsurge occurs at the turning point between developmental stages before maturing, suggesting that bmo-let- is required for the transformation of larval stages. a falling in the high level of expression profile is shared by female and male pupa metamorphosis at pupal-moth ecdysis a strong signal was observed in the new female pupa and adult. northern blotting in this assay, however, revealed an extraordinarily high expression profile across the whole process of pupation and eclosion of females and males. a strong signal of the expression was observed at day- of the fifth instar when the silkworm is ripe for mounting or spinning cocoons. after spinning, the fifth instar larvae enter a prepupation stage. there was no expression difference between the beginning and the end of spinning stage, implying that bmo-let- is unlikely related to the process of spinning. the silkworm prepupa undergoes a molt before pupation, which requires higher titer of ecdysone, as confirmed in the process of transition from prepupa to pupa in d. melanogaster. after pupation, histolysis and histogenesis are occurring intensively and simultaneously, much more of this functional rna should be required for the double missions, accordingly. the expression profile of bmo-let- fell at day pupa in female group, and at day in male group, when the ecdysone is also lowly produced, then resumed to the maximum just before adult emergence corresponding to the rising ecdysone. the expression falling during pupal-adult metamorphosis of both female and male should have occurred synchronously if they had been incubated under the same temperature and humidity. jh and ecd function oppositely on the developmental metamorphosis of the silkworm. jh can induce the expression of some genes and repress the expression of other genes activated by ecd. jh is highly secreted by corpus allatum at early period of each larva stage, and ecd is highly secreted by prothoracic gland at late period of each larva stage. insect metamorphosis is triggered by ecd in the absence of jh, and is carried out by self-destructive mechanisms of programmed cell death. the let- temporal expression alterations are in synchronization with the pulse of ecdysone, as was further confirmed by our northern blotting results. large amount of ecd is secreted in the late fifth instar or in just ripe silkworm in favor of spinning cocoons and molting in pupation. jh is required at late pupa stage to promote the development of ovary and accumulation of yelk and to maintain the functions of testicles. a previous study revealed that in b.mori, the prothoracic gland hormone secreted during the first day after larval-pupal ecdysis might be responsible for the development of the ovaries during the pupal period, especially essential for the initiation of ovarian development. after pupation, high level ecdysone is required to drive the tissue differentiation and organ formation of adult moth. therefore, the ecd-induced microrna, bmo-let- is highly expressed in pupae. however, the mechanism of how bmo-let- responds to ecdysone and broad-complex pathways in the silkworm is worthy of further study. bmo-let- is widely expressed in tissues/organs of female and male individuals of the fifth-instar day- larvae bmo-let- was moderately expressed in female and male individuals at the fifth instar day-. to correlate expression domains with the functions, we determined the tissue specificity of bmo-let- expression in various tissues of larvae in day- of the fifth instar. reproducible and consistent results were confirmed by independently parallel northern blotting experiments with the two antisense probes. the let- small rna of nt or nt was present in all tissues studied. moderate signals were observed in body wall, midgut, gonad and malpighian tubule, whereas a weak positive signal appeared in anterior silk gland and posterior silk gland, and strong signal was observed in the heads. sex differences in the expression level were prominent in fat body and hemocyte, however. the female fat body showed weak signal but the male fat body exhibited very strong one. on the contrary, bmo-let- was highly expressed in the female hemocyte but lowly in male hemocyte. the distinct differences in the expression of bmo-let- imply that some unknown mechanisms responsible for its transcription might be functioning differently in fat body and hemocyte of female and male silkworm. tissue-specific or spatial expression patterns have also been demonstrated in other organisms. as shown in d. melanogaster, let- is widely expressed in a variety of tissues from prepupae, such as brains, imaginal discs, fat bodies, salivary glands, and malphigian tubules, with relatively higher levels in fat bodies and imaginal discs, and it was also detected in adult ovaraies and carcasses, suggesting that it could regulate diverse metamorphic processes, such as the terminal differentiation of imaginal discs and apoptosis of salivary glands and fat bodies. furthermore, various tissues from human also express let- rna, including brain, heart, kidney, liver, lung, trachea, bone marrow, colon, small intestine, spleen, stomach, and thymus, and the lowest level of human let- is observed in bone marrow likely due to a large proportion of immature cell in it. different expression profiles in various tissues from the silkworm indicate that bmo-let- might not only function in triggering transitions of temporal stages, but more broadly in a variety of metabolisms because each microrna can control hundreds of target genes. expression profile in tissues from just mounting to day- pupae suggests its potential roles in histolysis and histogenesis tissues from different stages here presented profiles breadthwise and lengthwise. the prospective fates of organs and tissues such as head, body wall, silk gland, midgut, fat body and gonad during metamorphic transformation are different. silk gland and midgut will be completely histolyzed during the pupal stage, and fat body undergoes reorganization as well as histolysis during larval-pupal transformation and predetermined imaginal tissues differentiate and grow into organs and external structures. the histolysis of larval silk gland, fat body and midgut is typical of programmed cell death. thus, the expression profile of bmo-let- in tissues from just mounting to day- pupae may reflect its association with histolysis and apoptosis. the individuals at just mounting expressed large amount of bmo-let- small rna, but the body wall at this period only showed very weak signal. silk gland and fat body at this time shared a moderate amount. midgut, however, showed very high level, suggesting that midgut histolysis was triggered once the silkworm stopped eating or just before mounting. when silkworms have finished spinning cocoons three days after mounting, the silk gland accomplishes its mission and undergoes histolysis. at this time, high level expression of let- was detected in silk gland and testis; moderate level was observed in midgut and fat body, and a weak signal appeared in head and body wall, suggesting that let- also promotes the apoptosis of silk gland and the maturation of testisis, as demonstrated during the metamorphosis in d. melanogaster. just after pupation, silk gland enters the late stage of histolysis, body wall and fat body are newly reorganized. let- was very highly expressed in body wall and fat body, but very lowly expressed in silk gland of the newborn pupa, suggesting that let- functions in the histogenesis of body wall and fat body on the basis of the conclusion that lin- and let- control the timing of cell differentiation and proliferation. at day- pupae, weak expression signals were observed in midgut and silk gland because these two tissues are almost completely histolyzed, but intensive signals appeared in body wall and fat body which are still undergoing reorganization as well as histolysis. taken together, during the metamorphosis from just mounting to day- pupa, the signal intensity in body wall and fat body was rising to the utmost at newborn pupa, then fell at day - pupa. the high level expression of bmo-let- in fat body and other tissues is very likely to be resulted from the ecdysone induction, considering the evidence that the programmed autophagy in the drosophila fat body is induced by ecdysone. silk gland showed the highest expression level just after spinning, and exhibited a falling signal during the undergoing of apoptosis. in midgut, however, the expression level of bmo-let- reached the maximum at just mounting, and then decreased. although bmo-let- expression is generally response to ecdysone pulse in various tissues as well as in developmental stages, more precise time points and tissues will be investigated to further determine if the expression of bmo-let- is directly induced by ecdysone in the silkworm. effect of ecdysone change on the expression of bmo-let- in cultured cells the expression of bmo-let- has been systematically investigated in the whole body and tissues or organs during the development of silkworm. the results suggested that bmo-let- generally responds to the pulse of ecdysone. cell line culture experiments were then carried out in order to test whether ecdysone is required to sustain the expression of bmo-let- and how the concentrations of ecdysone are influencing its expression. this small rna expression trends differed even if it was detectable in all cell samples, which have been incubated in the presence of different levels of ecdysone for different periods. for the control cells, bmo-let- rna level increased straightly during the whole culture time, with a gentle inflexion at h. when the cells were treated with μm ecdysone, bmo-let- was initially up-regulated, then reached a plateau shared by the control at h. lower level of ecdysone, such as μm, seems to exert diverse influence on bmo-let- down-regulating it slightly before h, up-regulating it distinctly from h to h, then down-regulating it again. to avoid the confusion resulted from possible experiment discrepancy, we especially investigated the effects of different levels of ecdysone at h after incubation. bmo-let- was slightly upregulated in the presence of μm ecdysone and most effectively induced by μm ecdysone. however, after treated with μmecdysone, bmo-let- rna level was almost always stable during the whole examined span of h, and dramatically decreased in the presence of μm. this suggests that the expression of bmo-let- might be induced by low-level ecdysone but repressed by high-level ecdysone. the silkworm ovary itself has been confirmed to express bmo-let-, and ovarian cell line bmn-swu was also validated to produce this small rna even if no ecdysone was added into the culture medium. during the culture periods, the untreated cells are very likely to produce ecdysone, so we cannot rule out the possibility that the endogenetic and adscititious ecdysones are functioning together in inducing or repressing the expression of bmo-let- rna. since bmo-let- is undetectable during the development of silkworm's embryo, the embryo cell line is also unlikely to produce detectable bmo-let- rna. it would be interesting to look at whether the expression of bmo-let- rna could be observed when the embryo cell line would have been incubated for certain length of time in the presence of ecdysone. the tissue-specificity and the timing of let- expression are conserved in invertebrates, suggesting that it could exert widely in mediating diverse metamorphic processes. the programmed ecdysis behaviour in larval, pupal and adult stages occurs in all of the holometabolous insects, and depends on coordinated expression of a series of genes related to peptide signaling. very low-level expression of let- can even be observed in the npr mutants of drosophila, which are unresponsive to the ecdysone pulse at the end of the last larval stage due to the lack of four br-c isoforms. it could be speculated that ecdysone might be only one of the factors regulating bmo-let- and some higher upstream components of the heterochronic pathway could also contribute to its expression.  a new candidate member of let- family in the silkworm was identified by computational approach and its expression was profiled by northern blotting. lines of evidence confirmed that bmo-let- is temporally and spatially expressed in the silkworm and the expression is in response to the pulse of ecdysone, and might be related to a number of biological processes such as cell proliferation, histogenesis, histolysis, organogenesis and apoptosis. it is shown from the cell culture experiment that induction or repression effects of ecdysone on the expression of bmo-let- are concentration-dependent. 
the expression was also detected in various tissues of day of the fifth instar and of from day of the fifth to pupa, suggesting a wide distributing pattern with various signal intensities. lin- and let- the two founding members of heterochronic microrna genes, are firstly confirmed in caenorhabditis elegans to control the proper timing of developmental programs in a heterochronic pathway. the detailed expression profiles in the whole life cycle and cultured cell line of silkworm showed a clear association with ecdysone pulse and a variety of biological processes. the expression levels were higher at the end of each instar and at the beginning of each molt than at other periods, coinciding with the pulse of ecdysone and br-c as a whole. all known members of this family share the same nucleotides at ten positions within the mature sequences.
 lin- and let- the two founding members of heterochronic microrna genes, are firstly confirmed in caenorhabditis elegans to control the proper timing of developmental programs in a heterochronic pathway. let- has been thought to trigger the onset of adulthood across animal phyla. ecdysone and broad-complex are required for the temporal expression of let- in drosophila melanogaster. for a better understanding of the conservation and functions of let- we seek to explore how it is expressed in the silkworm.  one member of let- family has been identified in silkworm computationally and experimentally. all known members of this family share the same nucleotides at ten positions within the mature sequences. sequence logo and phylogenetic tree show that they are not only conserved but diversify to some extent among some species. the bmo-let- was very lowly expressed in ova harvested from newborn unmated female adult and in individuals from the first molt to the early third instar, highly expressed after the third molt, and the most abundant expression was observed after mounting, particularly after pupation. the expression levels were higher at the end of each instar and at the beginning of each molt than at other periods, coinciding with the pulse of ecdysone and br-c as a whole. using cultured ovary cell line, bmn-swu we examined the effect of altered ecdysone levels on bmo-let- expression. the expression was also detected in various tissues of day of the fifth instar and of from day of the fifth to pupa, suggesting a wide distributing pattern with various signal intensities.  bmo-let- is stage- and tissue-specifically expressed in the silkworm. although no signals were detected during embryonic development and first larval instar stages, the expression of bmo-let- was observed from the first molt, suggesting that it might also function at early larval stage of the silkworm. the detailed expression profiles in the whole life cycle and cultured cell line of silkworm showed a clear association with ecdysone pulse and a variety of biological processes. 