Small RNAs and extracellular vesicles in filarial nematodes: From nematode development to diagnostics

Parasitic nematodes have evolved sophisticated mechanisms to communicate with their hosts in order to survive and successfully establish an infection. The transfer of RNA within extracellular vesicles (EVs) has recently been described as a mechanism that could contribute to this communication in filarial nematodes. It has been shown that these EVs are loaded with several types of RNAs, including microRNAs, leading to the hypothesis that parasites could actively use these molecules to manipulate host gene expression and to the exciting prospect that these pathways could result in new diagnostic and therapeutic strategies. Here, we review the literature on the diverse RNAi pathways that operate in nematodes and more specifically our current knowledge of extracellular RNA (exRNA) and EVs derived from filarial nematodes in vitro and within their hosts. We further detail some of the issues and questions related to the capacity of RNA‐mediated communication to function in parasite–host interactions and the ability of exRNA to enable us to distinguish and detect different nematode parasites in their hosts.

nematodes, making use of comparisons with studies in C. elegans in which many mechanistic aspects of RNAi were discovered. 9,10 In the last 8 years, it has been shown that the small RNAs involved in RNAi within cells are also found extracellularly. Their association with extracellular vesicles (EVs) in parasite infections may implicate them as novel players in the transmission of information between the parasites and their hosts. 11 We will describe recent evidence of extracellular RNAs derived from filarial nematodes, their potential use for diagnostics and current challenges and outstanding questions in the field.

| RNAI PATHWAYS IN FILARIAL NEMATODES
Three primary RNAi pathways have been characterized in animals: the microRNA (miRNA) pathway, the endo/exo-small interfering RNA (endo/ exo-siRNA) pathway and the P-element-induced wimpy testis (PIWI)interacting RNA (piRNA) pathway. 9,12 These pathways are distinguished by the origin and identity of the small RNA guide and target, as well as the properties of the Argonaute (AGO) protein to which they bind. In general, AGOs have two main functions: (1) recognizing and binding small RNA and (2) mediating the interaction with other proteins required for small RNA loading, association with targeted RNAs, gene silencing activity and/or subcellular localization. 8,13 From a structural standpoint, they are generally ~90-100-kDa monomeric proteins containing several domains a PAZ domain involved in 3′-end recognition and binding of the small RNA, a MID domain that binds the 5' end of the small RNA and a PIWI domain, that in some cases includes an RNaseH-like activity that can carry out endonucleolytic cleavage ("slicing") of the targets. 13,14 The ancestral AGOs that bind to miRNAs are called ALG1 and ALG2 (AGOlike gene). The piRNA pathway is thought primarily to operate in genome defence through targeting transposable elements, mediated by the PIWI clade of AGOs. Homologs to these proteins are not present in clade III nematodes; the phylogenetic classification proposed by Blaxter et al. 15 is used throughout this manuscript. Rather, it is thought instead that other AGOs and small RNA classes could be involved in genome defence in this clade. 16 Indeed, a remarkable feature of nematodes is their extended AGOs (27 identified in C. elegans 8,13,17 ), reflecting the diversity of RNAi pathways that can operate in these animals. The majority of the AGOs in C. elegans belong to the WAGO (worm-specific AGO) clade, and many members of this clade are expected to be found in filarial nematodes. 8,13 From studies in C. elegans, the WAGOs are thought to bind to a class of secondary siRNAs that can act through a range of mechanisms including chromosome segregation and epigenetic modifications [18][19][20] and can mediate transgenerational inheritance. 21 A more extensive description of different structural, functional and mechanistic aspects of AGO proteins is provided in recent reviews. 8,13,22

| BIOGENESIS OF MIRNAS
The microRNA pathway is one of the best characterized RNAi pathways in nematodes. 23 These molecules, first described in C. elegans over two decades ago, are encoded within the genome as stem-loop structures that undergo a series of maturation events to produce the short RNA guide. In nematodes, as in other animals, miRNAs can either derive from within intragenic sequences (generally within the introns) or from independent, intergenic transcriptional units. 24 These transcripts, termed the primary miRNAs (pri-miRNAs), are mostly derived from the activity of RNA polymerase II ( Figure 1). Some miRNAs are clustered together in discrete genomic regions suggesting coordinated expression. 10 Once transcribed, miRNA biogenesis involves a series of maturation events starting with cleavage by the microprocessor complex in the nucleus. 10,25 The microprocessor is composed of the RNase III endonuclease DROSHA and DCRG8, among other scaffold proteins, and cleaves the pri-miRNA to produce a shorter hairpin (pre-miRNA) with a 5′ monophosphate and a ~2-nt overhang at the 3′ end ( Figure 1).
The pre-miRNA is then actively transported to the cytoplasm by Ran-GTP protein and members of the exportin family (predominantly EXP-5). Once in the cytoplasm, the pre-miRNA is recognized by a second RNase III endonuclease called DICER that catalyses cleavage of the hairpin to produce a double-stranded duplex approximately 22 nt in length, where both 3′ ends display a ~2-nt overhang. 10,25 One strand of this miRNA duplex is then incorporated into the RNA-induced silencing complex (RISC) through association with the AGO protein ( Figure 1). The miRNA then guides RISC to target messenger RNAs to elicit inhibition of translation, accelerated mRNA de-adenylation and/ or endonucleolytic cleavage of the mRNA, depending on the degree of complementarity between the miRNA and its target. 10,25 In animals, miRNAs generally are not perfectly complementary to their targets and recognition is dominated by the "seed" site defined as nucleotides 2-7 in the 5′ end of the miRNA.

| MIRNA DISCOVERY AND EVOLUTION IN FILARIAL NEMATODES
A number of studies have now documented miRNAs in filarial nematodes as well as the related clade III nematodes Ascaris suum and Ascaris lumbricoides [26][27][28] (Table 1) were not conserved in the other. Some of the newly evolved miRNAs were highly abundant and/or showed stage-specific expression.
Beyond the studies with Brugia spp., miRNAs have also been identified in the dog heartworm parasite Dirofilaria immitis. 31 Here, a total of 1063 miRNA candidates were identified by sequence alignment of mixed adult libraries against the miRBase repository, 32 corresponding to 808 miRNA families. 31 While the large number of miRNAs reported here could reflect an expanded miRNA repertoire in this parasite, it also highlights the fact that different studies use different criteria for assigning a small RNA sequence as a miRNA. In the study by Winter et al. 30 , for example, the authors used both miReap and miRDeep prediction programs, but then further filtered the results manually with the requirement that both arms of the hairpin must be present in their data sets. All of these factors, along with the depth of sequencing that is carried out, will affect the number and identity of miRNAs identified in different nematode species, in addition to the quality of the genomes available. This becomes an issue when trying to examine acquisitions and losses, as well as species specificity of miRNAs for use in diagnostic applications (detailed further below).
While it is tempting to speculate that the evolution of miRNAs in filarial nematodes relates to parasitism, it should also be noted that a study comparing miRNAs in the free-living nematode Pristionchus pacificus to the Caenorhabditis spp. (clade V nematodes) also showed that the majority of miRNAs were not conserved. 33 Likewise, another study examining miRNAs in nematodes spanning clades I-V showed that at least 20% of C. elegans miRNAs were conserved. This work also demonstrated that homology inversely correlated with phylogenetic distance for both free-living and parasitic nematodes. 16 Consequently, it seems likely that different miRNAs follow diverse evolutionary trajectories linked to various aspects of nematode biology in both free-living and parasitic organisms. It is still challenging to pinpoint correlations between specific behavioural and physiological adaptations and the fluidity at which miRNA families are lost or gained. Gene duplication and "arm switching" (a process that leads to a switch in the arm from which the functional mature miRNA is derived) have been proposed as common mechanisms for the evolution of miRNAs and expansion of some miRNA family members. 30,34

| FUNCTIONAL IMPLICATIONS OF STAGE-SPECIFIC EXPRESSION OF MIRNAS
A number of observations suggest that discrete miRNA subsets might be important regulators of processes in a particular life stage of filarial F I G U R E 1 Simplified schematic of biogenesis and potential export pathways of microRNAs. miRNAs are generally produced from primary miRNA transcripts that are processed by the microprocessor in the nucleus and exported to the cytoplasm where they are further processed by Dicer to produce a 22-nt duplex RNA. One strand of the duplex (the mature miRNA) is loaded onto an Argonaute (AGO) protein and guides the RISC complex to mediate control of gene expression by translational repression or accelerated miRNA decay (A). The microRNA can also be exported out of the cell, either in association with AGO or in another form (the uncertainty is depicted with a question mark), through directly fusing with components of the plasma membrane into extracellular vesicles (EVs) termed microvesicles (B), or through incorporation into the exosomal biogenesis pathway into multivesicular bodies (MVBs) (C) It has also been reported that miR-71 was one of the most abundant miRNAs in small RNA libraries prepared from total RNA from mixed adult worms in D. immitis. 31 A later study in B. malayi showed that miR-71 represents ~27% of the total miRNA reads identified in Mf data sets and is 3-5X more enriched in Mf than adult worms. 36 It is possible that some of miR-71 detected in the study with D. immitis could potentially originate from Mf found in gravid female worms and not from adult worms per se, although this is still unclear. A recent report demonstrated the functional activity of miR-71 in developmentally competent B. malayi embryos using a luciferase reporter assay, concluding that miR-71 can act as a post-transcriptional repressor of mRNA targets in this life stage. 37 In C. elegans, miR-71 regulates longevity and life span, 38 where it is upregulated in L1 diapause and dauer larvae but not particularly in other life stages. 39 It has been shown that filarial nematodes adjust their developmental schedule and fecundity in response to host-derived immunological factors. 40 This is indicative of different developmental trajectories depending upon environmental signals, a phenomenon referred to as phenotypic plasticity. [41][42][43][44] miRNAs, as well as other RNAi pathways, have been shown to control developmental choices and life-history traits in post-dauer C. elegans, which shares behavioural and physiological traits with infective L3 larvae in parasitic nematodes. 41,43,[45][46][47] Therefore, it is likely that the same mechanisms operate in filarial nematodes to control development and fertility in response to immunological cues from the host. A comparative analysis evaluating the RNAi landscape throughout filarial development in different environmental contexts will help to clarify whether such molecular "switches" (discrete small RNA populations) could be the drivers or modulators of such morphological and developmental choices.

| ENDOGENOUS SMALL INTERFERING RNA (ENDO-SIRNA) PATHWAYS IN FILARIAL NEMATODES
Most commonly, RNAi pathways in parasitic nematodes are discussed in relation to the ability to trigger an RNAi-mediated gene silencing response upon stimulation with exogenous (or environmental) double-stranded RNAs (exo-dsRNAs). This requires uptake of double-stranded RNA (dsRNA), processing this into primary siRNAs, amplification involving an RNA-dependent RNA polymerase to produce the secondary siRNAs and ability to spread the signal, (reviewed in [48][49][50][51] ). In many nematodes, the absence of the dsRNA import protein SID-1 is thought to explain the lack of efficient RNAi carried out experimentally. 52 However, endogenous pathways are expected to exist in these organisms where siRNAs are generated by a variety of mechanisms and these can have a variety of functions. 50,53 In C. elegans, In Ascaris, it was shown that 26G-RNAs as well as 22G-RNAs were predominantly detected in the germline through to 128-cell embryos. 28 The majority of these endo-siRNAs mapped to a broad spectrum of coding genes in an antisense fashion. 28 On the other hand, a total of 40 repeat-associated siRNAs were identified in adult stages of B. malayi. 29 Similarly, several sense and antisense siRNAs were detected in the small RNA data from iL3s and mixed adult stages in B. pahangi, with at least eight sequences derived from repetitive elements. 30 A closer examination revealed that these sequences were mainly associated with retrotransposons and mapped to nonannotated repeats. Interestingly, a phylumwide survey suggested that in clade III nematodes, the 22G-RNAs preferentially target antisense to predicted repetitive elements and have been proposed as a mechanism to control transposon activity in the absence of piRNAs. 16

| EXTRACELLULAR VESICLES AND EXTRACELLULAR RNA IN FILARIAL NEMATODES
It is now recognized that RNA molecules can also operate beyond the limits of the cell. although it is not known whether these were associated with rRNA fragments that were also found. It is unclear whether or how different RNA processing pathways converge with EV biogenesis and secretion. In some systems, components of the RISC complex have been detected in EVs or shown to comigrate with endosomal MVB fractions in density gradients. 73 Interestingly, one AGO protein was also identified in both vesicle and vesicle-depleted fractions from H. polygyrus in vitro, 68 although the mechanistic aspects associated with secretion of AGO proteins in nematodes or others parasites are unknown.

| REGULATION AND PLASTICITY OF EV SECRETION?
The population of EVs detected in ES products seems to be variable between life stages, with reduced content observed in B. malayi gravid adult females compared to iL3s. 69 Interestingly, the EV release rate

| EXTRACELLULAR SMALL RNAS AS BIOMARKERS FOR FILARIASIS-TOWARDS DIAGNOSTIC APPLICATIONS
One potential application of these parasite-derived exRNAs is in the area of biomarkers for helminthiases. This is based on a key observation that parasite-derived exRNAs can be detected in biofluids from their hosts as demonstrated by small RNA sequencing and qRT-PCR.
This was first documented in schistosomiasis, [75][76][77] but has also been examined by multiple groups in the context of filarial infections [78][79][80] ( Table 2). In an initial report, Tritten et al. 78 documented a total of 245 miRNA candidates of potential nematode origin in the plasma of dogs infected with the heartworm D. immitis based on sequencing. In a subsequent report, they documented a total of 22 unique sequences derived from L. loa in human serum and 10 sequences derived from Onchocerca ochengi in infected cattle serum. 79 We have also identi- nematode-derived small RNAs in host fluids is also challenging given the dominance of host-derived sequences in these samples, and it may be appropriate to remove all sequences that could derive from the host prior to assignment of these as parasitic in origin.
While it might be expected that all nematode parasites can or do release exRNA and EVs, there are a number of factors that will influence the ability to detect these molecules in different host fluids.
It is logical that the localization of the parasite within the host dictates the presence of parasite-derived exRNAs in different biofluids.
In line with this, nematode miRNAs could be identified in the serum of mice infected with L. sigmodontis, but not in serum from mice infected with H. polygyrus (which resides in the small intestine) in a side-byside comparison. 68 The close contact between some filarial nema- suggest that, for instance, miR-71 can be used as a biomarker for filarial infection. [78][79][80] However, it is expected that several technological approaches will be considered to improve not only the platforms currently available for exRNA detection (reviewed in 82,83 ) but also the way in which these technologies can be transferred in a field-friendly manner. Advancing inexpensive technologies and streamlined purification protocols will certainly increase the likelihood of adopting small RNA-based biomarkers in the field.

| FINAL CONSIDERATIONS & OUTSTANDING QUESTIONS
The field of EVs and small RNAs in parasitic nematodes is in its infancy and rapidly growing alongside efforts to exploit these in ther- If this is the case, several aspects need to be considered. First, if the parasites effectively use EVs and small RNAs as a mechanism for invasion, colonization and immune evasion, then one possibility is that these functions are specifically compartmentalized within the parasites. We therefore should expect that certain tissues or organs be directly involved in their production and secretion/excretion, for example those with glandular functions such as the pharynx or cells producing ES products. Building from this idea, we could also propose that the profile and exRNA content of EVs, as well as the diversity  91,92 ) to engineer specific EVs cargos. These "tailored" EVs could be used as vehicles to further our understanding on how multiple organisms use these extracellular systems to transfer information and to maintain a dialogue with their surroundings. Towards this goal, further work will be required to improve the genetic manipulation toolkit currently available for filarial nematodes and to advance the basic research on EV and exRNA secretion and function in these parasites.

ACKNOWLEDGEMENTS
We thank our collaborators on filarial nematode projects for many