BACKGROUND
the molecular mechanisms by which eukaryotic pathogens infect their hosts remain key questions in infection biology, as improved understanding of these events could lead to new methods of control.
this is particularly so for vector-borne parasites, where the ability to survive and differentiate within the new environment of a mammalian host is critical for successful transmission.
in this paper we explore the molecular events underlying the transmission of filarial nematodes from the mosquito to the mammalian host.
we focus on micrornas , which are small  non-coding rnas that regulate gene expression in animals, plants and viruses.
mirnas bind to complementary sequences, often in the 3′ untranslated region  of mrnas, leading to translational repression and mrna destabilisation  <cit> .
mirnas are essential during development in the free-living nematode caenorhabditis elegans, with functions in developmental timing  <cit> , lifespan and stress responses  <cit> , and embryogenesis  <cit> .
c. elegans lin- <dig>  the first mirna to be identified  <cit> , regulates developmental switching during the early larval stages  by repressing the heterochronic proteins lin- <dig>  <cit>  and lin- <dig>  <cit> , allowing epidermal cell fate transitions.
c. elegans let- <dig> was the second mirna to be identified and acts from the l <dig> stage onwards to regulate the transition from l <dig> to adult by down-regulating a number of targets, including lin- <dig>  <cit> .
let- <dig> is the founding member of a family of c. elegans mirnas that share identity in the seed sequence .
this family includes a further six mirnas; mir- <dig>  mir- <dig>  mir- <dig>  mir- <dig>  mir- <dig>  and mir- <dig>  <cit> .
while let- <dig> is involved in specifying larval-adult cell fate, mir- <dig>  mir- <dig> and mir- <dig> are co-activated at an earlier time point where they co-ordinate the l2-l <dig> transition  <cit> .
although information continues to accrue on mirnas in the model nematode , much less is known in parasitic nematodes.
we recently identified  <dig> mirnas from the filarial nematode brugia pahangi  <cit> , the sister species to the human pathogen b. malayi.
in our study the mirna identification process required that all sequences matched perfectly to the b. malayi genome and we therefore consider these mirnas common to both species, with a recent study confirming this  <cit> .
b. malayi and the related wuchereria bancrofti are the causative agents of lymphatic filariasis.
collectively, filarial worms infect around  <dig> million individuals in  <dig> countries worldwide  <cit> .
these parasites cause a wide spectrum of pathology in the infected individual, including the debilitating conditions of elephantiasis and hydrocele.
lymphatic filariae have complex life cycles with no free-living forms; the parasites develop from first-stage larvae  to infective l <dig> within the mosquito intermediate host, and then from l <dig> to adults within the mammalian definitive host.
following transmission from an infected mosquito, the l <dig> migrate to the lymphatics where they develop through two moults to adults, which have a reproductive life-span of approximately 8 years  <cit> .
mated females release millions of microfilariae into the bloodstream where they are available for ingestion by a mosquito taking a blood meal.
lymphatic filariae therefore have two periods of developmental arrest, as l <dig> in the mosquito and as microfilariae in the blood of the mammalian host.
while parasite development is dependent upon transmission between hosts, little is known of the molecular mechanisms that control arrest and development within the different hosts.
in this paper we show that a novel member of the let- <dig> mirna family, bpa-mir- <dig>  is highly up-regulated to coincide with the transition of the l <dig> from mosquito to mammalian host.
the role of bpa-mir- <dig> in the infection event was investigated by identifying potential mrna targets using bioinformatic predictions, comparative genomics, and transcriptomic analysis, with selected targets verified using a mammalian cell transfection system.
we propose that by regulating specific mrnas, bpa-mir- <dig> plays a key role in the transmission of the l <dig> stage from mosquito to mammalian host.
this is the first example of a parasitic nematode mirna for which function has been investigated and its mrna targets identified and confirmed experimentally.
RESULTS
expression profiling identifies developmentally regulated b. pahangi mirnas
the complete lifecycle of b. pahangi is maintained in our laboratory allowing access to specific developmental stages.
therefore, from the mirnas found in our previous genome-wide discovery study  <cit> , we aimed to identify those with important developmental functions by detailed expression profiling at six key developmental time-points.
the following life cycle stages were analysed: mosquito-derived l <dig>  l <dig> isolated from the mammalian host at day  <dig> and day  <dig> post-infection , l <dig> isolated at day  <dig> p.i., and sexually mature adult males and females isolated approximately 3 months p.i.
the mosquito-derived l <dig> and day  <dig> p.i.
l <dig> time-points were selected in order to identify changes occurring coincident with infection, day  <dig> p.i.
l <dig> and day  <dig> p.i.
l <dig> to identify changes prior to and after a moult, and males and females to define mirna profiles specific to the adult stages.
rna was prepared from worms recovered at each time-point using six biological replicates, with three replicates used for microarray and three for qrt-pcr.
a custom array  contained probes against brugia spp.
<cit>  and c. elegans  mirna sequences.
this paper focuses on mirnas differentially expressed in the infective stage as it transits from the mosquito to the mammalian host; our analysis of mirnas functioning specifically in adult parasites will be presented elsewhere.
the microarray data for the larval stages is provided in additional file  <dig>
significant changes in mirna profiles are linked to invasion of the host
to identify high confidence sets of mirnas that were differentially expressed during larval development, the processed microarray results were filtered to retain only those with t-test p-values of <  <dig>  and log <dig> fold changes of >  <dig> between two larval time-points .
these criteria identified a set of  <dig> probes which were then further refined to five differentially expressed mirnas .
importantly, this set included the let- <dig> mirna family members bpa-let- <dig> and bpa-mir- <dig>  suggesting that other b. pahangi mirnas with important developmental functions could potentially be identified by this analysis.figure  <dig>
brugia mirnas are developmentally regulated.
volcano plots of pairwise comparison for larval stage microarray results.
probes in red represent those with t-test p-values of < <dig>   and log <dig> fold changes of > <dig>  a) mosquito-derived l <dig> versus day  <dig> p.i.
l <dig>  b) mosquito-derived l <dig> versus day  <dig> p.i.
l <dig>  c) mosquito-derived l <dig> versus day  <dig> p.i.
l <dig>  d) day  <dig> p.i.
l <dig> versus day  <dig> p.i.
l <dig>  e) day  <dig> p.i.
l <dig> versus day  <dig> p.i.
l <dig>  f) day  <dig> p.i.
l <dig> vs day  <dig> p.i.
l <dig>
mean
bpa-mir-5364
microarray signal in five
b. pahangi
life cycle stages
reporter name
mosquito-derived l3
day 1
day 5
day 10
adult male
adult female
bpa-let-7
bpa-mir-84-5p
bpa-mir-5364
cel-mir-789
bpa-mir-5853
eight probes shown in additional file  <dig> are not included here for the following reasons: cel-let- <dig>  as its profile was essentially identical to bpa-let-7; bpa-mir-84-3p*, as it sequence is identical to bpa-mir-84-5p; bpa <dig>  as its sequence corresponds to a trna  <cit> ; bpa-mir-5853*, cel-mir- <dig> and bpa-mir- <dig>  due to low expression ; cel-mir- <dig> and cel-mir- <dig>  as the corresponding b. pahangi sequences could not be identified by pcr amplification of candidate sequences found from re-analysis of our previous small rna deep sequence data  <cit> .
in addition to bpa-let- <dig> and bpa-mir- <dig>  the three other mirnas that met the criteria were bpa-mir- <dig>  cel-mir- <dig> and bpa-mir- <dig>  we chose to focus on bpa-mir- <dig> for the following reasons: its expression profile is particularly striking as it is up-regulated ~ <dig> fold  within 24 hours of infection of the mammalian host and doubles again over the period to day  <dig> p.i.
; bpa-mir- <dig> shares sequence similarity with the let- <dig> mirna family and appears to be specific to clade iii parasitic nematodes ; it is one of the most abundant mirnas with only bpa-lin- <dig> and bpa-mir- <dig> found at higher levels at day  <dig> p.i.
.
abundant mirna are often evolutionarily ancient  <cit>  suggesting important function.
bpa-mir- <dig> is a novel member of the let- <dig> mirna family
in the free-living nematode c. elegans, the let- <dig> mirna family controls developmental transitions and consists of seven members of which only two  are found in brugia spp.
<cit> .
although bpa-mir- <dig> is clearly a novel sequence, it can be considered a member of the let- <dig> family due to conservation around the mirna seed sequence .
this led us to hypothesise that bpa-mir- <dig> may also influence a developmental transition, in this case from the mosquito vector to the mammalian host.
comparison of the overall microarray expression profiles of bpa-mir- <dig> with the b. pahangi let- <dig> family mirnas reveals a related but distinct pattern: while the expression profiles of all three mirnas mirror each other, bpa-mir- <dig> is found at a much higher level than bpa-let- <dig> and bpa-mir- <dig> and is up-regulated a minimum of 24 hours earlier .figure  <dig>
mir- <dig> is a novel member of the let- <dig> mirna family.
a. clustal alignment comparing mirna sequences from the c. elegans let- <dig> family with bpa-mir- <dig>  bpa-let- <dig> and bpa-mir- <dig>  shaded nucleotides are identical.
note the level of identity particularly over the seed sequence .
b. abundance of bpa-mir- <dig>  bpa-let- <dig> and bpa-mir- <dig> increase in parallel from mosquito-derived  l <dig> to adult as determined by microarray analysis .
the mean of three biological replicates is shown +/− standard deviation.
expression of bpa-mir- <dig> is induced in culture
the bpa-mir- <dig> developmental expression profile defined by microarray was confirmed by qrt-pcr, with the striking increase in abundance of the mature mirna clearly evident within 24 hours post-infection of the mammalian host .
to test if this rapid increase in bpa-mir- <dig> level could be replicated in vitro, mosquito-derived l <dig> were cultured for 24 hours at 37°c in medium without serum, or with the addition of 5% or 10% serum.
qrt-pcr demonstrated that bpa-mir- <dig> abundance increased after 24 hours in culture to a level similar to that found in 24 hour p.i.
l <dig> analysed directly ex vivo, with the addition of serum causing no significant difference  .figure  <dig> expression of bpa-mir- <dig> is upregulated following host invasion and in cultured larvae.
a. qrt-pcr confirms bpa-mir- <dig> is up-regulated between mosquito-derived l <dig> and 24 h p.i.
l <dig>  levels of bpa-mir- <dig> were assessed relative to the constitutively expressed bpa-mir-100c and are expressed as log2fold change relative to mosquito-derived  l <dig>  the mean of three biological replicates is shown +/− standard deviation; replicates were independent from those used in the microarray analysis.
b. bpa-mir- <dig> expression determined by qrt-pcr in b. pahangi l <dig> cultured at 37°c in worm culture medium that was either fcs-free, or contained 5% or 10% fcs.
levels of bpa-mir- <dig> were normalised to bpa-mir-100c and are expressed as log2fold change relative to mosquito-derived  l <dig>  the mean of three technical replicates from a single biological replicate is shown +/− standard deviation.
to examine potential mechanisms regulating expression, the genomic sequences 5′ and 3′ of bpa-mir- <dig> were analysed.
brugia spp.
mir- <dig> is an intergenic mirna with the closest protein coding region residing  <dig>  kb upstream and is therefore likely to be regulated by its own promoter.
however, analysis of this region revealed no significant transcription factor binding sites .
in c. elegans, let- <dig> is autoregulated via a let- <dig> binding site situated at the 3′ end of the primary transcript approximately 500 bp downstream of the sequence encoding the mature mirna  <cit> .
autoregulation of c. elegans lin- <dig> also occurs, in this case by activation of lin- <dig> transcription via a lin- <dig> binding site in its promoter  <cit> .
the b. malayi mir- <dig> genomic sequence was analysed using the probability of interaction by target accessibility  algorithm  <cit> .
interestingly, predicted binding sites for mir- <dig> were found  <dig> bases downstream  and  <dig> bases upstream  of the mir- <dig> mature region.
this suggests that similar mechanisms of autoregulation to those described for let- <dig> and lin- <dig> in c. elegans might exist for mir- <dig> in the parasite.
mir- <dig> is specific to clade iii parasitic nematodes
mirbase   shows that, to date, mir- <dig> has been identified only in the parasitic nematodes b. pahangi  <cit>  and ascaris suum  <cit> , both of which are members of nematode clade iii  <cit> .
mir- <dig> is not present in the deep-sequencing-defined mirna sets from the animal parasitic nematodes haemonchus contortus   <cit>  or strongyloides ratti   <cit> , nor in the free-living nematode species c. elegans, c. briggsae, c. brenneri, c. remanei, pristionchus pacificus  or panagrellus redivivus   <cit> , suggesting that mir- <dig> mirna is specific to clade iii parasitic nematodes.
to investigate this further, we examined a number of other nematode genomes to determine the extent of mir- <dig> conservation.
using the bpa-mir- <dig> precursor sequence as a query for blastn analysis at  <dig> nematode genomes , mir- <dig> was found in all clade iii parasitic nematode genomes for which sequence was available.
regions of ≥ 84% identity were identified in the filarial nematodes dirofilaria immitis, loa loa, wuchereria bancrofti, litomosoides sigmodontis and onchocerca ochengi  with complete conservation of the mature mirna sequence in all five species .
when folded using mfold  <cit>   the regions of precursor homology identified from each species formed mirna-like hairpins  which also passed the mirna hairpin classifier algorithm mipred   <cit>  .
in contrast, our bioinformatic analyses showed no evidence for mir- <dig> orthologs in the genomic data at  <dig> nematode genomes of the non-clade iii nematodes bursaphelenchus xylophilus, heligosomoides polygyrus, howardula aoronymphium or oscheius tipulae using either the precursor or mature sequences as blast queries.
although it is possible that divergent sequences might not have been detected using blastn, mir- <dig> was not found in h. polygyrus by deep sequencing of small rnas  <cit> .
therefore, as mir- <dig> was also recently experimentally confirmed in b. malayi  <cit> , this mirna is highly conserved in eight clade iii parasitic nematodes but has not been identified in any other species to date.
computational and phylogenetic analysis predicts target mrnas
as mirnas bind to partially complementary sequences in their target mrnas, computational analysis can be used to predict targets  <cit> .
genome-wide target predictions for mir- <dig> were performed utilising b. malayi and d. immitis genomic data  <cit> .
although mirna target sites have been defined throughout genes  <cit> , we focussed on the 3′utr, as many well-characterised target sites reside in this region  <cit> .
also, rather than rely on annotated 3′utrs, which are often dependent on developmental stage  <cit>  or cell type, and for which the current b. malayi and d. immitis data are incomplete, we used 1 kb of genomic sequence downstream of the annotated end of all genes.
the mirna target prediction algorithms pita  <cit>  and miranda  <cit>  were used and only predictions found using both programs were retained.
the genome-wide mir- <dig> target predictions for both parasites are provided in additional file  <dig>
to identify high confidence mir- <dig> targets, the large number of predictions were refined by first considering only those with pita ΔΔg energies of < − <dig>  this resulted in  <dig> target sites in  <dig> b. malayi genes and  <dig> targets in  <dig> d. immitis genes.
we then defined the genome-wide set of b. malayi and d. immitis orthologs using inparanoid  <cit>   and retained only those genes where a mir- <dig> binding site was found in the downstream region of orthologous genes.
this resulted in  <dig> predicted mir- <dig> binding sites in  <dig> b. malayi genes, and  <dig> sites in  <dig> d. immitis genes .
target-site positioning was then considered and predictions retained only if the site occurred with similar positioning  in the two species.
this process produced a final set of  <dig> orthologous genes that were high confidence predicted targets of mir- <dig> .
three of these genes were chosen for further study; bm1_ <dig> , bm1_ <dig>  and bm1_ <dig> .
for bm1_ <dig> and bm1_ <dig> the mir- <dig> target sites were < <dig> nucleotides downstream of the stop codon, an arrangement that can result in potent silencing  <cit> , and the target site positioning was within one nucleotide and  <dig> nucleotides, respectively, in the two species .
the third predicted target, bm1_ <dig> was included as proteins of this type are known dna-binding proteins  <cit> .
further support that our predictions represented bona fide mir- <dig> targets was provided by searching for sites in the orthologs of bm1_ <dig>  bm1_ <dig> and bm1_ <dig> in the clade iii parasitic nematodes l. loa, w. bancrofti and l. sigmodontis .
3′ race was performed for the three proposed target genes, using b. pahangi material, which confirmed the annotated stop and the presence of the mir- <dig> target sequence in the 3′utrs.table  <dig>
predicted mrna targets of
bpa-mir-5364
b. malayi g
ene annotation
b. malayi
ortholog
d. immitis
ortholog
c. elegans
ortholog
reciprocal best blast matches with b. malayi in all cases except y48b6a.14/bm1_ <dig>
the expression profile of predicted target mrnas correlates inversely with that of bpa-mir-5364
if the mrnas identified are indeed targeted by bpa-mir- <dig> a decrease in abundance would be expected as mirna abundance increases.
to examine this, qrt-pcr was performed on b. pahangi mosquito-derived l <dig> and on day  <dig> p.i.
l <dig>  the time point at which maximal expression of bpa-mir- <dig> was observed, using b. pahangi β-tubulin as a normalising gene  <cit> .
the abundance of all three selected target genes decreased significantly in day  <dig> p.i.
l <dig> compared to mosquito-derived l <dig> .
the inverse relationship between mirna and mrna abundance supports the bioinformatics prediction that these three transcripts are targeted by bpa-mir- <dig>  the expression profiles of each of these three predicted targets of bpa-mir- <dig> were also analysed from our transcriptomic data covering the l <dig> and later larval stages.
an inverse relationship between mirna and mrna abundance was also observed here for the three target genes .
additional analysis of the transcriptome data demonstrated that a further three mrnas included in the final set of  <dig> bpa-mir- <dig> targets showed a pronounced decrease in abundance between mosquito-derived l <dig> and day  <dig> p.i.
larvae .figure  <dig> expression profiles of three predicted mrna targets are inversely correlated with bpa-mir- <dig>  graph shows levels of expression of bm1_ <dig> , bm1_ <dig>  and bm1_ <dig>  in day  <dig> post-infective l <dig> compared to mosquito-derived l <dig>  as b. pahangi material was used throughout, protein-coding genes with identifiers of the form “bm1_00001” refer, here, to the b. pahangi orthologs of the b. malayi genes.
the mean of three biological replicates is shown +/− standard deviation.
results were normalised to β tubulin and are expressed as log <dig> fold change relative to mosquito-derived  l <dig>
bpa-mir- <dig> interacts directly with predicted mrna targets
for brugia spp., and related parasites, there are currently no suitable transgenic approaches or parasite-derived cell lines that could be used to directly investigate mirna/mrna interactions.
the predicted bpa-mir-5364/mrna interactions were therefore assessed using a mammalian cell system.
3′utrs from the b. pahangi orthologs of bm1_ <dig>  bm1_ <dig> and bm1_ <dig> were cloned downstream of firefly luciferase and transiently transfected into hek <dig> cells along with a plasmid containing bpa-mir- <dig>  successful expression of bpa-mir- <dig> in this cell line was confirmed by qrt-pcr .
levels of luciferase in the presence or absence of bpa-mir- <dig> were compared.
for all three 3′utrs tested, a significant reduction in expression was observed in the presence of bpa-mir- <dig>  a synthetic gene containing five copies of a perfect bpa-mir- <dig> binding site was included as a positive control in these experiments and gave a reduction in signal of 42% in the presence of bpa-mir- <dig> versus plasmid with the mirna insert in the reverse orientation  in the representative experiment shown in figure  <dig>  for the proposed target 3′utrs, a 57% reduction in expression was observed in the presence of bpa-mir- <dig> for bm1_ <dig>  a 31% reduction for bm1_ <dig>  and a 45% reduction for bm1_ <dig> versus mirna insert in reverse orientation .figure  <dig>
bpa-mir- <dig> interacts with predicted target gene 3′utrs.
graph shows the mean ratio +/− standard deviation of 5– <dig> replicate wells comparing firefly to renilla luciferase signal in the presence of mir- <dig> and a synthetic gene containing five repeats of a perfect mir- <dig> binding site or the 3′utrs of predicted target mrnas, bm1_ <dig>  bm1_ <dig> and bm1_ <dig>  ratios using mir- <dig> cloned in the forward orientation  and in reverse orientation  are indicated.
percentage reduction in signal was calculated by comparison of the two.
figures show data from one of three representative experiments, except for plasmid bm1_ <dig> which was repeated twice with comparable results.
attempts at bpa-mir- <dig> inhibition using an antisense oligonucleotide
to examine the effect of bpa-mir- <dig> inhibition in cultured parasites, conditions for effective uptake of labelled small rnas by b. pahangi larvae were first determined.
mosquito-derived l <dig> or day  <dig> p.i.
l <dig> maintained in culture show uptake of cy3-labeled sirna or cy3-labeled anti-mir  after 24 hours and to a greater level after 48 hours culture .
having verified uptake, we attempted to inhibit bpa-mir- <dig> using l <dig> recovered from the mammalian host at 4– <dig> days p.i.
larvae were maintained in culture for 48 hours in the presence of an antisense rna oligonucleotide  designed to inhibit bpa-mir- <dig> or a sequence-scrambled control oligo and levels of bm1_ <dig>  bm1_ <dig> and bm1_ <dig> mrna assessed by qrt-pcr.
however, no de-repression of any target mrna, nor any phenotypic difference, was observed between aso treated larvae and controls.figure  <dig> uptake of fluorescently labelled oligonucleotides from culture medium by b. pahangi.
uptake of small rnas from media was assessed after 24– <dig> hours of culture using  <dig>  μm cy3-labelled sirna.
panels a-d show mosquito-derived l <dig> after 48 hours .
panels e & f show uptake in day  <dig> p.i.
l <dig> after 24 hours .
no fluorescence was observed in controls cultured in the absence of cy3-labelled sirna .
we also tested the effect of rnai of the single predicted mirna-specific argonaute gene bm1_ <dig>  <cit>  using endoribonuclease-prepared pools of complex sirna   <cit> , together with the bpa-mir- <dig> aso.
this approach is similar to that used in c. elegans where mirna mutants were analysed in the background of global reduction in mirna levels due to alg- <dig> mutation  <cit> .
this combined approach did not produce any phenotype or de-repression of target mrnas in b. pahangi .
co-ordinated regulation of mrna targets by multiple mirnas
as individual mrnas are often regulated by multiple mirnas, this most likely explains the lack of de-repression with the mir- <dig> aso.
therefore predictions of additional mirnas that might function alongside bpa-mir- <dig> to target bm1_ <dig>  bm1_ <dig> and bm1_ <dig>  and their d. immitis orthologs, were made using pita and a set of mirna which included all  <dig> mature brugia spp.
mirna identified previously  <cit> .
predicted mirna target sites for the three target genes in both parasite species are shown in additional file  <dig>  interestingly, conserved sites for let- <dig> and mir- <dig> are found for bm1_ <dig> with almost identical positioning in each species.
given the sequence similarity between mir- <dig> and the let- <dig> family  this might be expected; however, the binding sites for mir- <dig> and let-7/mir- <dig> are not identically positioned .
in addition, the c. elegans ortholog of bm1_ <dig>  c33a <dig>   also known as tag- <dig>  has predicted binding sites for let- <dig> family mirnas using pita  and targetscan  <cit>  , indicating that regulation of bm1_ <dig> and its orthologs by this important mirna family may be a feature conserved throughout the nematode phylum.
similarly, using pita the c. elegans ortholog of bm1_ <dig>  has a site for the let- <dig> family member mir- <dig> .
predicted binding sites for the orthologous mirnas bantam and mir- <dig>  <cit>  are found for bm1_ <dig>  and in the targetscan predictions for the c. elegans ortholog , suggesting conservation of mirna-mediated regulation across species.
further work will reveal if these interactions are functional and if these mirnas act in conjunction with mir- <dig> to regulate its targets.
discussion
infection of the mammalian host by the l <dig> stage of filarial worms is a key event in the life cycle: how the parasite survives and differentiates within its new environment is poorly understood.
here we show that bpa-mir- <dig>  which is specific to clade iii parasitic nematodes and related to the let- <dig> family, is highly up-regulated to coincide with transmission to the mammalian host and may be involved in the infection event by targeting specific mrnas.
the l <dig> of many parasitic nematodes show parallels with the developmentally arrested dauer larva of c. elegans.
in a recent study, four c. elegans mirnas were found to be up-regulated in samples of mixed stages compared to dauers, two of which are the let- <dig> family members mir- <dig> and mir- <dig>  <cit> .
similarly, in a detailed analysis of mirna expression following dauer or continuous development, let- <dig> family members showed increased expression post-dauer  <cit> .
brugia spp.
possess three let- <dig> family members, let- <dig>  mir- <dig> and mir- <dig>  all of which are up-regulated post-infection, with expression of mir- <dig> increasing first, within 24 hours, and rising to a significantly greater level than either let- <dig> or mir- <dig>  suggesting that in brugia spp.
mir- <dig> has a dominant role.
we therefore aimed to identify the possible functions of this mirna by identifying its target genes.
identifying mrna targets is complex because most bioinformatics tools search primarily for complementarity between the short mirna seed sequence  and the mrna, and then take other factors into account, including the free energy of the mirna-mrna duplex and position of binding within the 3′utr.
thus, bioinformatics tools potentially identify hundreds of possible targets, many of which may not be verifiable.
in an attempt to overcome this problem, the availability of other filarial genomes was exploited in a comparative genomics approach.
we identified orthologous mrnas in different clade iii nematodes that have conserved binding sites for mir- <dig>  with the further requirement that the site be similarly positioned.
this approach, which has been used previously in vertebrates and nematodes  <cit> , significantly reduced the number of possible target mrnas from over  <dig> to  <dig>  three of these were selected for in-depth study: bm1_ <dig> encoding an ets domain transcription factor, bm1_ <dig> encoding an n-recognin-type zinc finger protein, and bm1_ <dig> which encodes a putative high mobility group protein.
all three genes were found to have a reciprocal expression profile to bpa-mir- <dig>  both from analysis of transcriptomic data and by qrt-pcr, consistent with mirna-mediated negative regulation.
to demonstrate a direct interaction between mirna and target we used transient transfection of mammalian hek <dig> cells and a dual luciferase assay system and found that the presence of bpa-mir- <dig> resulted in the down-regulation of the luciferase reporter gene for all three 3′utrs, providing further evidence that these genes are true targets of this mirna.
all three verified targets of bpa-mir- <dig> could potentially function to alter gene and/or protein levels and a decrease in the levels of these post-infection may be important for larval activation and/or developmental progression in the mammalian host.
the human ortholog of bm1_ <dig>  belongs to the ets domain transcription factor family, which are characterised by epithelial-specific expression  and by tight tissue-specific and temporal regulation.
for example, in prostate cancer this transcription factor controls the balance between cell proliferation and differentiation  <cit> .
the c. elegans homolog of bm1_ <dig>  is upregulated in mutants of the mirna-specific argonaute gene alg- <dig>  <cit>  and is enriched in ain- <dig>  pull-downs  <cit> , consistent with mirna regulation.
bm1_ <dig> contains an n-recognin zinc finger motif conserved in ubiquitin-ligases, which target proteins for degradation.
in c. elegans, an important function of let- <dig> is the negative regulation of lin- <dig>  <cit> , which contains a ubiquitin ligase domain.
in turn, lin- <dig> negatively regulates levels of the transcription factor lin- <dig>  which is required for adult cell fates  <cit> .
it is possible that in brugia spp.
l <dig>  bm1_ <dig> may perform a similar function to lin- <dig> in c. elegans, as proteins with ubiquitin ligase activity could potentially target a subset of proteins for degradation.
the final mir- <dig> target studied, bm1_ <dig>  is predicted to encode an hmg protein.
these bind to chromatin and can act in the regulation of transcription during development, dna repair, apoptosis or cell senescence  <cit> .
in mammalian cells, hmga <dig> is abundant during early development and levels decrease during later stages of development and differentiation, via let- <dig> regulation  <cit> .
a reduction in hmg bm1_ <dig> following transmission to the mammalian host may allow larval development and/or exit from an arrested state.
it is important to note that whatever the function of bpa-mir- <dig>  it is not likely to be restricted to the l <dig> stage, as in the life cycle stages tested, expression peaked in day  <dig> p.i.
l <dig>  an actively growing stage and continued into adulthood.
whether the target mrnas are the same between the p.i.
l <dig> and subsequent developmental stages of brugia spp.
is currently unknown.
even in c. elegans where mirnas are comparatively well defined, little is known of target conservation between different life cycle stages.
in an attempt to define the possible function of the mrna targets in b. pahangi, we tested the effects of inhibiting bpa-mir- <dig> in the larval stages using a specific aso.
asos bind with sequence complementarity to the mature mirna to disrupt binding to mrna targets.
while we found efficient uptake of labelled rna by b. pahangi l <dig> and p.i.
l3s in the culture conditions tested, no de-repression of any of the three verified target genes was found using the aso.
as loss of a single mirna is often not sufficient to reveal function, we also tested the effect of the mir- <dig> aso along with sirnas targeting the brugia spp.
mirna-specific argonaute, but found no observable effect.
therefore, it may be necessary to include specific inhibitors of additional mirnas to reveal the effect of bpa-mir- <dig> disruption.
however, the conditions required for inhibition of mirna function in parasitic nematodes are completely unknown, with this being the first study to attempt to inhibit mirnas.
factors such as the concentration of aso required, efficiency of uptake in different cells and tissues, and the ability of exogenously applied inhibitors to enter the risc and block target binding will all influence the ability to inhibit mirna function.
as expression of bpa-mir- <dig> increased 12-fold between vector derived l <dig> and 24 h p.i.l <dig>  it was of interest to determine what factors might induce the expression of this mirna.
previous studies on the control of gene expression in vector-derived l <dig> versus 24 hour p.i.
l <dig> highlighted a number of mrnas which appeared to be regulated largely by the temperature shift associated with infection  <cit> .
by simply moving l <dig> to 37°c in an atmosphere of 5% co <dig> in air, the expression of bpa-mir- <dig> was induced, with serum having minimal additional effect.
recent studies in arabidopsis have shown that increased temperature and co <dig> lead to significant changes in expression of several mirnas  <cit> , but whether temperature or co <dig> has any role in the induction of bpa-mir- <dig> requires further study.
in addition, autoregulation of let- <dig> has been identified in c. elegans, in which binding of let- <dig> to its own 3′utr acts to increase processing of the pri-mirna by argonaute, leading to higher levels of the mature mirna  <cit> .
similarly, binding of c. elegans lin- <dig> to a site in its own promoter activates lin- <dig> transcription  <cit> .
in this study, we identified potential bpa-mir- <dig> binding sites in genomic sequence upstream and downstream of the mature mirna region, suggesting that autoregulation could also be important for control of this parasite mirna.
mir- <dig> appears to be restricted to clade iii nematodes as, to date, related sequences have not been found in other parasitic or free-living nematode genomes.
in addition to mir- <dig> in brugia spp.
<cit> , we found this mirna in the genomes of five additional clade iii filarial parasites, with a recent study confirming expression in d. immitis  <cit> .
mir- <dig> is not restricted to filarial nematodes as it is also expressed in the clade iii gastrointestinal nematode a. suum  <cit>  suggesting its role is not specific to tissue-dwelling nematodes.
therefore mir- <dig> may have diverged from the let- <dig> family during the evolution of clade iii nematodes, an event that occurred 350– <dig> million years ago  <cit> .
these observations support the concept that small rna sequence and function have evolved and diversified between nematode clades, as suggested previously following the identification of 21u  rnas in clade v nematodes, but not, to date, in clade iii  <cit>  or clade iv  <cit>  nematodes.
CONCLUSIONS
in conclusion, we report a b. pahangi mirna that is highly up-regulated to coincide with the transition between hosts and identify some of its mrna targets.
bpa-mir- <dig> is a novel member of the let- <dig> family, consistent with the function of other members of that family in nematode development.
infection of the mammalian host is the key event in the filarial life cycle and our data indicate that post-transcriptional regulation of gene expression may be necessary for this event.

 the molecular mechanisms by which eukaryotic pathogens infect their hosts remain key questions in infection biology, as improved understanding of these events could lead to new methods of control. this is particularly so for vector-borne parasites, where the ability to survive and differentiate within the new environment of a mammalian host is critical for successful transmission. in this paper we explore the molecular events underlying the transmission of filarial nematodes from the mosquito to the mammalian host. we focus on micrornas, which are small non-coding rnas that regulate gene expression in animals, plants and viruses. mirnas bind to complementary sequences, often in the 3′ untranslated region of mrnas, leading to translational repression and mrna destabilisation. mirnas are essential during development in the free-living nematode caenorhabditis elegans, with functions in developmental timing, lifespan and stress responses, and embryogenesis. c. elegans lin- the first mirna to be identified, regulates developmental switching during the early larval stages by repressing the heterochronic proteins lin- and lin-, allowing epidermal cell fate transitions. c. elegans let- was the second mirna to be identified and acts from the l stage onwards to regulate the transition from l to adult by down-regulating a number of targets, including lin-. let- is the founding member of a family of c. elegans mirnas that share identity in the seed sequence. this family includes a further six mirnas; mir- mir- mir- mir- mir- and mir-. while let- is involved in specifying larval-adult cell fate, mir- mir- and mir- are co-activated at an earlier time point where they co-ordinate the l2-l transition. although information continues to accrue on mirnas in the model nematode, much less is known in parasitic nematodes. we recently identified mirnas from the filarial nematode brugia pahangi, the sister species to the human pathogen b. malayi. in our study the mirna identification process required that all sequences matched perfectly to the b. malayi genome and we therefore consider these mirnas common to both species, with a recent study confirming this. b. malayi and the related wuchereria bancrofti are the causative agents of lymphatic filariasis. collectively, filarial worms infect around million individuals in countries worldwide. these parasites cause a wide spectrum of pathology in the infected individual, including the debilitating conditions of elephantiasis and hydrocele. lymphatic filariae have complex life cycles with no free-living forms; the parasites develop from first-stage larvae to infective l within the mosquito intermediate host, and then from l to adults within the mammalian definitive host. following transmission from an infected mosquito, the l migrate to the lymphatics where they develop through two moults to adults, which have a reproductive life-span of approximately 8 years. mated females release millions of microfilariae into the bloodstream where they are available for ingestion by a mosquito taking a blood meal. lymphatic filariae therefore have two periods of developmental arrest, as l in the mosquito and as microfilariae in the blood of the mammalian host. while parasite development is dependent upon transmission between hosts, little is known of the molecular mechanisms that control arrest and development within the different hosts. in this paper we show that a novel member of the let- mirna family, bpa-mir- is highly up-regulated to coincide with the transition of the l from mosquito to mammalian host. the role of bpa-mir- in the infection event was investigated by identifying potential mrna targets using bioinformatic predictions, comparative genomics, and transcriptomic analysis, with selected targets verified using a mammalian cell transfection system. we propose that by regulating specific mrnas, bpa-mir- plays a key role in the transmission of the l stage from mosquito to mammalian host. this is the first example of a parasitic nematode mirna for which function has been investigated and its mrna targets identified and confirmed experimentally.  expression profiling identifies developmentally regulated b. pahangi mirnas the complete lifecycle of b. pahangi is maintained in our laboratory allowing access to specific developmental stages. therefore, from the mirnas found in our previous genome-wide discovery study, we aimed to identify those with important developmental functions by detailed expression profiling at six key developmental time-points. the following life cycle stages were analysed: mosquito-derived l l isolated from the mammalian host at day and day post-infection, l isolated at day p.i., and sexually mature adult males and females isolated approximately 3 months p.i. the mosquito-derived l and day p.i. l time-points were selected in order to identify changes occurring coincident with infection, day p.i. l and day p.i. l to identify changes prior to and after a moult, and males and females to define mirna profiles specific to the adult stages. rna was prepared from worms recovered at each time-point using six biological replicates, with three replicates used for microarray and three for qrt-pcr. a custom array contained probes against brugia spp. and c. elegans mirna sequences. this paper focuses on mirnas differentially expressed in the infective stage as it transits from the mosquito to the mammalian host; our analysis of mirnas functioning specifically in adult parasites will be presented elsewhere. the microarray data for the larval stages is provided in additional file significant changes in mirna profiles are linked to invasion of the host to identify high confidence sets of mirnas that were differentially expressed during larval development, the processed microarray results were filtered to retain only those with t-test p-values of <  and log fold changes of >  between two larval time-points. these criteria identified a set of probes which were then further refined to five differentially expressed mirnas. importantly, this set included the let- mirna family members bpa-let- and bpa-mir- suggesting that other b. pahangi mirnas with important developmental functions could potentially be identified by this analysis.figure brugia mirnas are developmentally regulated. volcano plots of pairwise comparison for larval stage microarray results. probes in red represent those with t-test p-values of < and log fold changes of > a) mosquito-derived l versus day p.i. l b) mosquito-derived l versus day p.i. l c) mosquito-derived l versus day p.i. l d) day p.i. l versus day p.i. l e) day p.i. l versus day p.i. l f) day p.i. l vs day p.i. l mean bpa-mir-5364 microarray signal in five b. pahangi life cycle stages reporter name mosquito-derived l3 day 1 day 5 day 10 adult male adult female bpa-let-7 bpa-mir-84-5p bpa-mir-5364 cel-mir-789 bpa-mir-5853 eight probes shown in additional file are not included here for the following reasons: cel-let- as its profile was essentially identical to bpa-let-7; bpa-mir-84-3p*, as it sequence is identical to bpa-mir-84-5p; bpa as its sequence corresponds to a trna; bpa-mir-5853*, cel-mir- and bpa-mir- due to low expression; cel-mir- and cel-mir- as the corresponding b. pahangi sequences could not be identified by pcr amplification of candidate sequences found from re-analysis of our previous small rna deep sequence data. in addition to bpa-let- and bpa-mir- the three other mirnas that met the criteria were bpa-mir- cel-mir- and bpa-mir- we chose to focus on bpa-mir- for the following reasons: its expression profile is particularly striking as it is up-regulated ~ fold within 24 hours of infection of the mammalian host and doubles again over the period to day p.i.; bpa-mir- shares sequence similarity with the let- mirna family and appears to be specific to clade iii parasitic nematodes; it is one of the most abundant mirnas with only bpa-lin- and bpa-mir- found at higher levels at day p.i.. abundant mirna are often evolutionarily ancient suggesting important function. bpa-mir- is a novel member of the let- mirna family in the free-living nematode c. elegans, the let- mirna family controls developmental transitions and consists of seven members of which only two are found in brugia spp.. although bpa-mir- is clearly a novel sequence, it can be considered a member of the let- family due to conservation around the mirna seed sequence. this led us to hypothesise that bpa-mir- may also influence a developmental transition, in this case from the mosquito vector to the mammalian host. comparison of the overall microarray expression profiles of bpa-mir- with the b. pahangi let- family mirnas reveals a related but distinct pattern: while the expression profiles of all three mirnas mirror each other, bpa-mir- is found at a much higher level than bpa-let- and bpa-mir- and is up-regulated a minimum of 24 hours earlier.figure mir- is a novel member of the let- mirna family. a. clustal alignment comparing mirna sequences from the c. elegans let- family with bpa-mir- bpa-let- and bpa-mir- shaded nucleotides are identical. note the level of identity particularly over the seed sequence. b. abundance of bpa-mir- bpa-let- and bpa-mir- increase in parallel from mosquito-derived l to adult as determined by microarray analysis. the mean of three biological replicates is shown +/− standard deviation. expression of bpa-mir- is induced in culture the bpa-mir- developmental expression profile defined by microarray was confirmed by qrt-pcr, with the striking increase in abundance of the mature mirna clearly evident within 24 hours post-infection of the mammalian host. to test if this rapid increase in bpa-mir- level could be replicated in vitro, mosquito-derived l were cultured for 24 hours at 37°c in medium without serum, or with the addition of 5% or 10% serum. qrt-pcr demonstrated that bpa-mir- abundance increased after 24 hours in culture to a level similar to that found in 24 hour p.i. l analysed directly ex vivo, with the addition of serum causing no significant difference.figure expression of bpa-mir- is upregulated following host invasion and in cultured larvae. a. qrt-pcr confirms bpa-mir- is up-regulated between mosquito-derived l and 24 h p.i. l levels of bpa-mir- were assessed relative to the constitutively expressed bpa-mir-100c and are expressed as log2fold change relative to mosquito-derived l the mean of three biological replicates is shown +/− standard deviation; replicates were independent from those used in the microarray analysis. b. bpa-mir- expression determined by qrt-pcr in b. pahangi l cultured at 37°c in worm culture medium that was either fcs-free, or contained 5% or 10% fcs. levels of bpa-mir- were normalised to bpa-mir-100c and are expressed as log2fold change relative to mosquito-derived l the mean of three technical replicates from a single biological replicate is shown +/− standard deviation. to examine potential mechanisms regulating expression, the genomic sequences 5′ and 3′ of bpa-mir- were analysed. brugia spp. mir- is an intergenic mirna with the closest protein coding region residing  kb upstream and is therefore likely to be regulated by its own promoter. however, analysis of this region revealed no significant transcription factor binding sites. in c. elegans, let- is autoregulated via a let- binding site situated at the 3′ end of the primary transcript approximately 500 bp downstream of the sequence encoding the mature mirna. autoregulation of c. elegans lin- also occurs, in this case by activation of lin- transcription via a lin- binding site in its promoter. the b. malayi mir- genomic sequence was analysed using the probability of interaction by target accessibility algorithm. interestingly, predicted binding sites for mir- were found bases downstream and bases upstream of the mir- mature region. this suggests that similar mechanisms of autoregulation to those described for let- and lin- in c. elegans might exist for mir- in the parasite. mir- is specific to clade iii parasitic nematodes mirbase shows that, to date, mir- has been identified only in the parasitic nematodes b. pahangi and ascaris suum, both of which are members of nematode clade iii. mir- is not present in the deep-sequencing-defined mirna sets from the animal parasitic nematodes haemonchus contortus or strongyloides ratti, nor in the free-living nematode species c. elegans, c. briggsae, c. brenneri, c. remanei, pristionchus pacificus or panagrellus redivivus, suggesting that mir- mirna is specific to clade iii parasitic nematodes. to investigate this further, we examined a number of other nematode genomes to determine the extent of mir- conservation. using the bpa-mir- precursor sequence as a query for blastn analysis at nematode genomes, mir- was found in all clade iii parasitic nematode genomes for which sequence was available. regions of ≥ 84% identity were identified in the filarial nematodes dirofilaria immitis, loa loa, wuchereria bancrofti, litomosoides sigmodontis and onchocerca ochengi with complete conservation of the mature mirna sequence in all five species. when folded using mfold the regions of precursor homology identified from each species formed mirna-like hairpins which also passed the mirna hairpin classifier algorithm mipred. in contrast, our bioinformatic analyses showed no evidence for mir- orthologs in the genomic data at nematode genomes of the non-clade iii nematodes bursaphelenchus xylophilus, heligosomoides polygyrus, howardula aoronymphium or oscheius tipulae using either the precursor or mature sequences as blast queries. although it is possible that divergent sequences might not have been detected using blastn, mir- was not found in h. polygyrus by deep sequencing of small rnas. therefore, as mir- was also recently experimentally confirmed in b. malayi, this mirna is highly conserved in eight clade iii parasitic nematodes but has not been identified in any other species to date. computational and phylogenetic analysis predicts target mrnas as mirnas bind to partially complementary sequences in their target mrnas, computational analysis can be used to predict targets. genome-wide target predictions for mir- were performed utilising b. malayi and d. immitis genomic data. although mirna target sites have been defined throughout genes, we focussed on the 3′utr, as many well-characterised target sites reside in this region. also, rather than rely on annotated 3′utrs, which are often dependent on developmental stage or cell type, and for which the current b. malayi and d. immitis data are incomplete, we used 1 kb of genomic sequence downstream of the annotated end of all genes. the mirna target prediction algorithms pita and miranda were used and only predictions found using both programs were retained. the genome-wide mir- target predictions for both parasites are provided in additional file to identify high confidence mir- targets, the large number of predictions were refined by first considering only those with pita ΔΔg energies of < − this resulted in target sites in b. malayi genes and targets in d. immitis genes. we then defined the genome-wide set of b. malayi and d. immitis orthologs using inparanoid and retained only those genes where a mir- binding site was found in the downstream region of orthologous genes. this resulted in predicted mir- binding sites in b. malayi genes, and sites in d. immitis genes. target-site positioning was then considered and predictions retained only if the site occurred with similar positioning in the two species. this process produced a final set of orthologous genes that were high confidence predicted targets of mir-. three of these genes were chosen for further study; bm1_, bm1_ and bm1_. for bm1_ and bm1_ the mir- target sites were < nucleotides downstream of the stop codon, an arrangement that can result in potent silencing, and the target site positioning was within one nucleotide and nucleotides, respectively, in the two species. the third predicted target, bm1_ was included as proteins of this type are known dna-binding proteins. further support that our predictions represented bona fide mir- targets was provided by searching for sites in the orthologs of bm1_ bm1_ and bm1_ in the clade iii parasitic nematodes l. loa, w. bancrofti and l. sigmodontis. 3′ race was performed for the three proposed target genes, using b. pahangi material, which confirmed the annotated stop and the presence of the mir- target sequence in the 3′utrs.table predicted mrna targets of bpa-mir-5364 b. malayi g ene annotation b. malayi ortholog d. immitis ortholog c. elegans ortholog reciprocal best blast matches with b. malayi in all cases except y48b6a.14/bm1_ the expression profile of predicted target mrnas correlates inversely with that of bpa-mir-5364 if the mrnas identified are indeed targeted by bpa-mir- a decrease in abundance would be expected as mirna abundance increases. to examine this, qrt-pcr was performed on b. pahangi mosquito-derived l and on day p.i. l the time point at which maximal expression of bpa-mir- was observed, using b. pahangi β-tubulin as a normalising gene. the abundance of all three selected target genes decreased significantly in day p.i. l compared to mosquito-derived l. the inverse relationship between mirna and mrna abundance supports the bioinformatics prediction that these three transcripts are targeted by bpa-mir- the expression profiles of each of these three predicted targets of bpa-mir- were also analysed from our transcriptomic data covering the l and later larval stages. an inverse relationship between mirna and mrna abundance was also observed here for the three target genes. additional analysis of the transcriptome data demonstrated that a further three mrnas included in the final set of bpa-mir- targets showed a pronounced decrease in abundance between mosquito-derived l and day p.i. larvae.figure expression profiles of three predicted mrna targets are inversely correlated with bpa-mir- graph shows levels of expression of bm1_, bm1_ and bm1_ in day post-infective l compared to mosquito-derived l as b. pahangi material was used throughout, protein-coding genes with identifiers of the form “bm1_00001” refer, here, to the b. pahangi orthologs of the b. malayi genes. the mean of three biological replicates is shown +/− standard deviation. results were normalised to β tubulin and are expressed as log fold change relative to mosquito-derived l bpa-mir- interacts directly with predicted mrna targets for brugia spp., and related parasites, there are currently no suitable transgenic approaches or parasite-derived cell lines that could be used to directly investigate mirna/mrna interactions. the predicted bpa-mir-5364/mrna interactions were therefore assessed using a mammalian cell system. 3′utrs from the b. pahangi orthologs of bm1_ bm1_ and bm1_ were cloned downstream of firefly luciferase and transiently transfected into hek cells along with a plasmid containing bpa-mir- successful expression of bpa-mir- in this cell line was confirmed by qrt-pcr. levels of luciferase in the presence or absence of bpa-mir- were compared. for all three 3′utrs tested, a significant reduction in expression was observed in the presence of bpa-mir- a synthetic gene containing five copies of a perfect bpa-mir- binding site was included as a positive control in these experiments and gave a reduction in signal of 42% in the presence of bpa-mir- versus plasmid with the mirna insert in the reverse orientation in the representative experiment shown in figure  for the proposed target 3′utrs, a 57% reduction in expression was observed in the presence of bpa-mir- for bm1_ a 31% reduction for bm1_ and a 45% reduction for bm1_ versus mirna insert in reverse orientation.figure bpa-mir- interacts with predicted target gene 3′utrs. graph shows the mean ratio +/− standard deviation of 5– replicate wells comparing firefly to renilla luciferase signal in the presence of mir- and a synthetic gene containing five repeats of a perfect mir- binding site or the 3′utrs of predicted target mrnas, bm1_ bm1_ and bm1_ ratios using mir- cloned in the forward orientation and in reverse orientation are indicated. percentage reduction in signal was calculated by comparison of the two. figures show data from one of three representative experiments, except for plasmid bm1_ which was repeated twice with comparable results. attempts at bpa-mir- inhibition using an antisense oligonucleotide to examine the effect of bpa-mir- inhibition in cultured parasites, conditions for effective uptake of labelled small rnas by b. pahangi larvae were first determined. mosquito-derived l or day p.i. l maintained in culture show uptake of cy3-labeled sirna or cy3-labeled anti-mir after 24 hours and to a greater level after 48 hours culture. having verified uptake, we attempted to inhibit bpa-mir- using l recovered from the mammalian host at 4– days p.i. larvae were maintained in culture for 48 hours in the presence of an antisense rna oligonucleotide designed to inhibit bpa-mir- or a sequence-scrambled control oligo and levels of bm1_ bm1_ and bm1_ mrna assessed by qrt-pcr. however, no de-repression of any target mrna, nor any phenotypic difference, was observed between aso treated larvae and controls.figure uptake of fluorescently labelled oligonucleotides from culture medium by b. pahangi. uptake of small rnas from media was assessed after 24– hours of culture using  μm cy3-labelled sirna. panels a-d show mosquito-derived l after 48 hours. panels e & f show uptake in day p.i. l after 24 hours. no fluorescence was observed in controls cultured in the absence of cy3-labelled sirna. we also tested the effect of rnai of the single predicted mirna-specific argonaute gene bm1_ using endoribonuclease-prepared pools of complex sirna, together with the bpa-mir- aso. this approach is similar to that used in c. elegans where mirna mutants were analysed in the background of global reduction in mirna levels due to alg- mutation. this combined approach did not produce any phenotype or de-repression of target mrnas in b. pahangi. co-ordinated regulation of mrna targets by multiple mirnas as individual mrnas are often regulated by multiple mirnas, this most likely explains the lack of de-repression with the mir- aso. therefore predictions of additional mirnas that might function alongside bpa-mir- to target bm1_ bm1_ and bm1_ and their d. immitis orthologs, were made using pita and a set of mirna which included all mature brugia spp. mirna identified previously. predicted mirna target sites for the three target genes in both parasite species are shown in additional file interestingly, conserved sites for let- and mir- are found for bm1_ with almost identical positioning in each species. given the sequence similarity between mir- and the let- family this might be expected; however, the binding sites for mir- and let-7/mir- are not identically positioned. in addition, the c. elegans ortholog of bm1_ c33a also known as tag- has predicted binding sites for let- family mirnas using pita and targetscan, indicating that regulation of bm1_ and its orthologs by this important mirna family may be a feature conserved throughout the nematode phylum. similarly, using pita the c. elegans ortholog of bm1_ has a site for the let- family member mir-. predicted binding sites for the orthologous mirnas bantam and mir- are found for bm1_ and in the targetscan predictions for the c. elegans ortholog, suggesting conservation of mirna-mediated regulation across species. further work will reveal if these interactions are functional and if these mirnas act in conjunction with mir- to regulate its targets. discussion infection of the mammalian host by the l stage of filarial worms is a key event in the life cycle: how the parasite survives and differentiates within its new environment is poorly understood. here we show that bpa-mir- which is specific to clade iii parasitic nematodes and related to the let- family, is highly up-regulated to coincide with transmission to the mammalian host and may be involved in the infection event by targeting specific mrnas. the l of many parasitic nematodes show parallels with the developmentally arrested dauer larva of c. elegans. in a recent study, four c. elegans mirnas were found to be up-regulated in samples of mixed stages compared to dauers, two of which are the let- family members mir- and mir-. similarly, in a detailed analysis of mirna expression following dauer or continuous development, let- family members showed increased expression post-dauer. brugia spp. possess three let- family members, let- mir- and mir- all of which are up-regulated post-infection, with expression of mir- increasing first, within 24 hours, and rising to a significantly greater level than either let- or mir- suggesting that in brugia spp. mir- has a dominant role. we therefore aimed to identify the possible functions of this mirna by identifying its target genes. identifying mrna targets is complex because most bioinformatics tools search primarily for complementarity between the short mirna seed sequence and the mrna, and then take other factors into account, including the free energy of the mirna-mrna duplex and position of binding within the 3′utr. thus, bioinformatics tools potentially identify hundreds of possible targets, many of which may not be verifiable. in an attempt to overcome this problem, the availability of other filarial genomes was exploited in a comparative genomics approach. we identified orthologous mrnas in different clade iii nematodes that have conserved binding sites for mir- with the further requirement that the site be similarly positioned. this approach, which has been used previously in vertebrates and nematodes, significantly reduced the number of possible target mrnas from over to three of these were selected for in-depth study: bm1_ encoding an ets domain transcription factor, bm1_ encoding an n-recognin-type zinc finger protein, and bm1_ which encodes a putative high mobility group protein. all three genes were found to have a reciprocal expression profile to bpa-mir- both from analysis of transcriptomic data and by qrt-pcr, consistent with mirna-mediated negative regulation. to demonstrate a direct interaction between mirna and target we used transient transfection of mammalian hek cells and a dual luciferase assay system and found that the presence of bpa-mir- resulted in the down-regulation of the luciferase reporter gene for all three 3′utrs, providing further evidence that these genes are true targets of this mirna. all three verified targets of bpa-mir- could potentially function to alter gene and/or protein levels and a decrease in the levels of these post-infection may be important for larval activation and/or developmental progression in the mammalian host. the human ortholog of bm1_ belongs to the ets domain transcription factor family, which are characterised by epithelial-specific expression and by tight tissue-specific and temporal regulation. for example, in prostate cancer this transcription factor controls the balance between cell proliferation and differentiation. the c. elegans homolog of bm1_ is upregulated in mutants of the mirna-specific argonaute gene alg- and is enriched in ain- pull-downs, consistent with mirna regulation. bm1_ contains an n-recognin zinc finger motif conserved in ubiquitin-ligases, which target proteins for degradation. in c. elegans, an important function of let- is the negative regulation of lin-, which contains a ubiquitin ligase domain. in turn, lin- negatively regulates levels of the transcription factor lin- which is required for adult cell fates. it is possible that in brugia spp. l bm1_ may perform a similar function to lin- in c. elegans, as proteins with ubiquitin ligase activity could potentially target a subset of proteins for degradation. the final mir- target studied, bm1_ is predicted to encode an hmg protein. these bind to chromatin and can act in the regulation of transcription during development, dna repair, apoptosis or cell senescence. in mammalian cells, hmga is abundant during early development and levels decrease during later stages of development and differentiation, via let- regulation. a reduction in hmg bm1_ following transmission to the mammalian host may allow larval development and/or exit from an arrested state. it is important to note that whatever the function of bpa-mir- it is not likely to be restricted to the l stage, as in the life cycle stages tested, expression peaked in day p.i. l an actively growing stage and continued into adulthood. whether the target mrnas are the same between the p.i. l and subsequent developmental stages of brugia spp. is currently unknown. even in c. elegans where mirnas are comparatively well defined, little is known of target conservation between different life cycle stages. in an attempt to define the possible function of the mrna targets in b. pahangi, we tested the effects of inhibiting bpa-mir- in the larval stages using a specific aso. asos bind with sequence complementarity to the mature mirna to disrupt binding to mrna targets. while we found efficient uptake of labelled rna by b. pahangi l and p.i. l3s in the culture conditions tested, no de-repression of any of the three verified target genes was found using the aso. as loss of a single mirna is often not sufficient to reveal function, we also tested the effect of the mir- aso along with sirnas targeting the brugia spp. mirna-specific argonaute, but found no observable effect. therefore, it may be necessary to include specific inhibitors of additional mirnas to reveal the effect of bpa-mir- disruption. however, the conditions required for inhibition of mirna function in parasitic nematodes are completely unknown, with this being the first study to attempt to inhibit mirnas. factors such as the concentration of aso required, efficiency of uptake in different cells and tissues, and the ability of exogenously applied inhibitors to enter the risc and block target binding will all influence the ability to inhibit mirna function. as expression of bpa-mir- increased 12-fold between vector derived l and 24 h p.i.l it was of interest to determine what factors might induce the expression of this mirna. previous studies on the control of gene expression in vector-derived l versus 24 hour p.i. l highlighted a number of mrnas which appeared to be regulated largely by the temperature shift associated with infection. by simply moving l to 37°c in an atmosphere of 5% co in air, the expression of bpa-mir- was induced, with serum having minimal additional effect. recent studies in arabidopsis have shown that increased temperature and co lead to significant changes in expression of several mirnas, but whether temperature or co has any role in the induction of bpa-mir- requires further study. in addition, autoregulation of let- has been identified in c. elegans, in which binding of let- to its own 3′utr acts to increase processing of the pri-mirna by argonaute, leading to higher levels of the mature mirna. similarly, binding of c. elegans lin- to a site in its own promoter activates lin- transcription. in this study, we identified potential bpa-mir- binding sites in genomic sequence upstream and downstream of the mature mirna region, suggesting that autoregulation could also be important for control of this parasite mirna. mir- appears to be restricted to clade iii nematodes as, to date, related sequences have not been found in other parasitic or free-living nematode genomes. in addition to mir- in brugia spp., we found this mirna in the genomes of five additional clade iii filarial parasites, with a recent study confirming expression in d. immitis. mir- is not restricted to filarial nematodes as it is also expressed in the clade iii gastrointestinal nematode a. suum suggesting its role is not specific to tissue-dwelling nematodes. therefore mir- may have diverged from the let- family during the evolution of clade iii nematodes, an event that occurred 350– million years ago. these observations support the concept that small rna sequence and function have evolved and diversified between nematode clades, as suggested previously following the identification of 21u rnas in clade v nematodes, but not, to date, in clade iii or clade iv nematodes.  in conclusion, we report a b. pahangi mirna that is highly up-regulated to coincide with the transition between hosts and identify some of its mrna targets. bpa-mir- is a novel member of the let- family, consistent with the function of other members of that family in nematode development. infection of the mammalian host is the key event in the filarial life cycle and our data indicate that post-transcriptional regulation of gene expression may be necessary for this event. 
micrornas are small non-coding rna molecules that regulate target mrnas and we set out to investigate whether they play a role in the infection event. one of these, bpa-mir- was selected for further study as it is upregulated ~12-fold at 24 hours post-infection, is specific to clade iii nematodes, and is a novel member of the let- family, which are known to have key developmental functions in the free-living nematode caenorhabditis elegans. this study is the first to identify parasitic nematode mrnas that are verified targets of specific micrornas and demonstrates that post-transcriptional control of gene expression via stage-specific expression of micrornas may be important in the success of filarial infection. the molecular mechanisms underpinning survival and differentiation of these parasites following transmission are poorly understood. these data provide new insight into the molecular mechanisms underpinning the transmission of third stage larvae of filarial nematodes from vector to mammal. filarial nematodes are important pathogens in the tropics transmitted to humans via the bite of blood sucking arthropod vectors. predicted mrna targets of bpa-mir- were identified using bioinformatics and comparative genomics approaches that relied on the conservation of mir- binding sites in the orthologous mrnas of other filarial nematodes. micrornas differentially expressed during the early post-infective stages of brugia pahangi l were identified by microarray analysis.
 filarial nematodes are important pathogens in the tropics transmitted to humans via the bite of blood sucking arthropod vectors. the molecular mechanisms underpinning survival and differentiation of these parasites following transmission are poorly understood. micrornas are small non-coding rna molecules that regulate target mrnas and we set out to investigate whether they play a role in the infection event.  micrornas differentially expressed during the early post-infective stages of brugia pahangi l were identified by microarray analysis. one of these, bpa-mir- was selected for further study as it is upregulated ~12-fold at 24 hours post-infection, is specific to clade iii nematodes, and is a novel member of the let- family, which are known to have key developmental functions in the free-living nematode caenorhabditis elegans. predicted mrna targets of bpa-mir- were identified using bioinformatics and comparative genomics approaches that relied on the conservation of mir- binding sites in the orthologous mrnas of other filarial nematodes. finally, we confirmed the interaction between bpa-mir- and three of its predicted targets using a dual luciferase assay.  these data provide new insight into the molecular mechanisms underpinning the transmission of third stage larvae of filarial nematodes from vector to mammal. this study is the first to identify parasitic nematode mrnas that are verified targets of specific micrornas and demonstrates that post-transcriptional control of gene expression via stage-specific expression of micrornas may be important in the success of filarial infection. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords brugialymphatic filariasisnematodesmicrornasissue-copyright-statement© the author 2015 