Injection Of dsRNA Research Articles

RNA interference in vivo in Schistosoma japonicum: Establishing and optimization of RNAi mediated suppression of gene expression by long dsRNA in the intra-mammalian life stages of worms

Schistosomes are parasitic platyhelminths that threaten over 600 million people globally. In recent years, RNA interference (RNAi) has been widely used as a molecular tool in research into the genomic function of parasites. We aim to develop effective protocols for application of RNAi technology in the intra-mammalian life stages of Schistosoma japonicum. In this work, the expression of the parasite gene encoding cathepsin B1 (SjCB1) was targeted by exposing the worms to 10 μg of long dsRNA dissolved in 0.1 ml of 0.7% NaCl injected into the tail vein of infected mice. This method was effective and specific for eliciting SjCB1 gene suppression in both male and female adult worms in vivo (>79.4% in male and >91.5% in female knockdown relative to control). In 60 cercaria infected mice, RNAi suppression of gene expression was best achieved by using 10 μg of target dsRNA for at least 4 days. The recommended procedure for interference producing long-term suppression was an injection of dsRNA on the first day of infection with booster injections administered every 4 days for up to 26 days. Long-term suppression of three published functional genes (peroxiredoxin-1, mago nashi, insulin receptor) in S. japonicum provided more information about the role of the expression of these genes in producing particular phenotypes. The protocols described here may be more convenient, economical and applicable, than currently available technology and have contributed to the observation of more phenotypes during worm development from schistosomula to adult. These approaches may promote and facilitate further studies into functional schistosome genomics.

Biochemical Comparison of dsRNA Degrading Nucleases in Four Different Insects.

Double stranded RNAs (dsRNA) degrading nuclease is responsible for the rapid degradation of dsRNA molecules, and thus accounts for variations in RNA interference (RNAi) efficacy among insect species. Here, the biochemical properties and tissue-specific activities of dsRNA degrading nucleases in four insects (Spodoptera litura, Locusta migratoria, Periplaneta americana, and Zophobas atratus) from different orders were characterized using a modified assay method. The results revealed that all insect dsRNA degrading nucleases tested showed high activity in alkaline environments at optimal Mg2+ concentrations and elevated temperatures. We also found that enzymes from different insects varied in terms of their optimal reaction conditions and kinetic parameters. Whole body enzyme activity differed dramatically between insect species, although enzymes with higher substrate affinities (lower Km) were usually balanced by a smaller Vmax to maintain a proper level of degradative capacity. Furthermore, enzyme activities varied significantly between the four tested tissues (whole body, gut, hemolymph, and carcass) of the insect species. All the insects tested showed several hundred-fold higher dsRNA degrading activity in their gut than in other tissues. Reaction environment analysis demonstrated that physiological conditions in the prepared gut fluid and serum of different insects were not necessarily optimal for dsRNA degrading nuclease activity. Our data describe the biochemical characteristics and tissue distributions of dsRNA degrading activities in various insects, not only explaining why oral delivery of dsRNA often produces lower RNAi effects than injection of dsRNA, but also suggesting that dsRNA-degrading activities are regulated by physiological conditions. These results allow for a better understanding of the properties of dsRNA degrading nucleases, and will aid in the development of successful RNAi strategies in insects.

Effects of cold acclimation and dsRNA injections on Gs1l gene splicing in Drosophila montana

Alternative splicing, in which one gene produce multiple transcripts, may influence how adaptive genes respond to specific environments. A newly produced transcriptome of Drosophila montana shows the Gs1-like (Gs1l) gene to express multiple splice variants and to be down regulated in cold acclimated flies with increased cold tolerance. Gs1l’s effect on cold tolerance was further tested by injecting cold acclimated and non-acclimated flies from two distantly located northern and southern fly populations with double stranded RNA (dsRNA) targeting Gs1l. While both populations had similar cold acclimation responses, dsRNA injections only effected the northern population. The nature of splicing expression was then investigated in the northern population by confirming which Gs1l variants are present, by comparing the expression of different gene regions and by predicting the protein structures of splices using homology modelling. We find different splices of Gs1l not only appear to have independent impacts on cold acclimation but also elicit different effects in populations originating from two very different environments. Also, at the protein level, Gs1l appears homologous to the human HDHD1A protein and some splices might produce functionally different proteins though this needs to be verified in future studies by measuring the particular protein levels. Taken together, Gs1l appears to be an interesting new candidate to test how splicing influences adaptations.

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches.

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

Role of ABC transporters White, Scarlet and Brown in brown planthopper eye pigmentation.

The brown planthopper ATP-binding cassette (ABC) proteins White (W), Scarlet (St) and Brown (Bw) belong to the ABC transporter superfamily and are responsible for the transportation of guanine and tryptophan precursors of eye pigments. In the present study, the brown planthopper White (NlW), S t(NlSt) and Bw (NlBw) genes were cloned, and subsequent phylogenetic analysis showed that these genes are clustered with their respective homologues, with a genetic relationship observed between NlW and its Bemisia tabaci homologue having the highest similarity. Sequence alignments showed that these three proteins have a highly conserved Walker A domain, an ABC "signature sequence" and a Walker B domain. QRT-PCR demonstrated that W, St and Bw are highly expressed in the head of long-winged males and are highly expressed in both egg and male. Adult eye colour was altered after the downregulation of NlW, NlSt and NlBw in the 1st to 3rd instar nymph. The eye colours of emerged adults became white, dark and red after injection of dsNlW, dsNlSt and dsNlBw, respectively. The eye pigment content assay revealed that xanthommatin and pteridine were significantly decreased after the injection of dsRNAs, and the range of variation was inversely correlated with nymph age. The present study provides a theoretical basis for understanding the function of ABC transporters at the molecular and biochemical levels.

A Systematic Study of RNAi Effects and dsRNA Stability in Tribolium castaneum and Acyrthosiphon pisum, Following Injection and Ingestion of Analogous dsRNAs.

RNA interference (RNAi) effects in insects are highly variable and may be largely dependent upon the stability of introduced double-stranded RNAs to digestion by nucleases. Here, we report a systematic comparison of RNAi effects in susceptible red flour beetle (Tribolium castaneum) and recalcitrant pea aphid (Acyrthosiphon pisum) following delivery of dsRNAs of identical length targeting expression of V-type ATPase subunit E (VTE) and inhibitor of apoptosis (IAP) genes. Injection and ingestion of VTE and IAP dsRNAs resulted in up to 100% mortality of T. castaneum larvae and sustained suppression (>80%) of transcript levels. In A. pisum, injection of VTE but not IAP dsRNA resulted in up to 65% mortality and transient suppression (ca. 40%) of VTE transcript levels. Feeding aphids on VTE dsRNA reduced growth and fecundity although no evidence for gene suppression was obtained. Rapid degradation of dsRNAs by aphid salivary, haemolymph and gut nucleases contrasted with stability in T. castaneum larvae where it appears that exo-nuclease activity is responsible for relatively slow digestion of dsRNAs. This is the first study to directly compare RNAi effects and dsRNA stability in receptive and refractory insect species and provides further evidence that dsRNA susceptibility to nucleases is a key factor in determining RNAi efficiency.

A Prokineticin (PK)-like cytokine from Chinese mitten crab Eriocheir sinensis promotes the production of hemocytes via reactive oxygen species

Astakine is a cytokine-like factor containing a prokineticin domain, which directly participates in hematopoiesis and blood cell differentiation. In the present study, a novel Astakine gene was identified from Chinese mitten crab Eriocheir sinensis (designated as EsAst). The full-length cDNA of EsAst was of 1163 bp, consisting of a 5′ untranslated region (UTR) of 120 bp, a 3′ UTR of 656 bp, and an open reading frame (ORF) of 387 bp encoding a polypeptide of 128 amino acids. There were a signal peptide and a prokineticin domain with nine conserved cysteine residues in the deduced amino acid sequence of EsAst. EsAst shared higher similarity with Astakines from Penaeus monodon and Pacifastacus leniusculus, and it was closely clustered with the Astakine from shrimp P. monodon in the phylogenetic tree. The EsAst mRNA transcript was higher expressed in hemocytes and hepatopancreas. The relative expression level of EsAst in hemocytes was continuously increased from 1.5 to 48 h after Vibro anguillarum challenge compared that in the untreated control group. After Pichia pastoris GS115 challenge, the relative expression level of EsAst in hemocytes was also up-regulated. After rEsAst injection, ROS levels in HPT cells were also increased at 12 and 24 h, and the total hemocyte counts were also significantly increased at 6, 9, 12, and 24 h post rEsAst injection. The interference of EsAst expression with dsRNA injection could delay the recovery of hemocytes production post A. hydrophila stimulation. When mitochondrial complexes I was knock down by dsRNA, ROS levels were decreased and THCs were also decreased. Recovery of hemocyte production inducing by A. hydrophila stimulation and rEsAst injection were delayed with dsEsbc1 injection. When ROS levels were increased after RNAi of Lon protease, THCs were also increased. The expression levels of five genes (EsJNK, EsSTAT, EsPI3K, EsAKT1, EsP70S6K) involved in SAPK-JNK and mTOR signaling pathways were up-regulated at 12 and 24 h in rEsAst group and EsLon dsRNA group compared with that in EGFP dsRNA group, and were similar to the trend of ROS levels. These results collectively suggested that EsAst should be a novel Astakine to promote the production of hemocytes in a ROS-dependent way in E. sinensis.

Transgenic soybean plants expressing Spb18S dsRNA exhibit enhanced resistance to the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Olethreutidae).

The soybean pod borer [SPB; Leguminivora glycinivorella (Mats.) Obraztsov] is a major soybean pest in northeastern Asia. A useful method for addressing this problem is the generation of transgenic plants producing double-stranded RNA (dsRNA) that target essential insect genes. In this study, we confirmed that 18S ribosomal RNA is critical for SPB development. Downregulated Spb18S expression induced by dsRNA injection increased larval mortality rates and resulted in early pupation. We also assessed whether Spb18S is silenced in SPB larvae fed on transgenic soybean expressing Spb18S dsRNA. Transgenic plants downregulated Spb18S expression levels and second-instar larval survival rates. Moreover, such plants were less damaged by SPB larvae than control plants under field conditions.

Improving RNAi in the Brown Marmorated Stink Bug: Identification of target genes and reference genes for RT-qPCR

The brown marmorated stink bug (BMSB) is native to Asia and recently invaded the USA. RNA interference (RNAi) is a gene silencing mechanism in which the introduction of double-stranded RNA (dsRNA) inhibits gene function by degrading target mRNA. In dsRNA stability assays, the dsRNases present in the hemolymph and salivary gland secretions of BMSB showed lower activity than those in the hemolymph of Heliothis virescens. We evaluated six housekeeping genes (18S rRNA, EF1-α, Actin, Ubiquitin, 60S RP and β-Tubulin) across dsRNA treatments (injection and feeding) in nymphs and adults of BMSB and identified 18S rRNA and 60S RP as the best genes to use as a reference in reverse-transcriptase quantitative PCR (RT-qPCR). Homologs of 13 genes that were shown to function as effective RNAi targets in other insects were identified and evaluated by injecting dsRNA targeting these homologs into BMSB adults. Five out of 13 dsRNAs tested caused more than 70% mortality by seven days after injection of dsRNA. Feeding dsRNA targeting five of these genes (IAP, ATPase, SNF7, GPCR, and PPI) to nymphs caused more than 70% mortality by three of the five dsRNAs tested. These data suggest that feeding dsRNA causes target gene knockdown and mortality in BMSB.

Expression of Neuropeptide F Gene and Its Regulation of Feeding Behavior in the Pea Aphid, Acyrthosiphon pisum.

Neuropeptide F (NPF) signaling systems are widespread and highly evolutionarily conserved from vertebrates to invertebrates. In fact, NPF has been identified in many insect species and plays regulatory roles in diverse physiological processes, such as feeding, learning, reproduction and stress responses. NPF operates by interacting with the NPF receptor (NPFR). Here, we characterized and determined the presumed role of NPF signaling in the wingless parthenogenetic pea aphid, Acyrthosiphon pisum. Quantitative real-time reverse transcription-PCR (qRT-PCR) revealed that the expression levels of both NPF and NPFR transcripts varied across developmental stages, which implies that the NPF signaling system might participate in the developmental regulation of aphid physiological processes or behaviors. The NPF transcript was mainly detected in the head but not in the gut, whereas the NPFR transcript was mainly detected in both the gut and head. In addition, the NPF transcript levels were markedly up-regulated in starved aphids compared with satiated aphids, and the transcript levels recovered after re-feeding. In contrast, the NPFR transcript levels remained stable in starved and re-fed aphids. Furthermore, RNAi knockdown by the injection of NPF dsRNA into wingless adult aphids significantly reduced their food intake. Further analysis of the modification of aphid feeding behavior on broad bean plants using electrical penetration graphs (EPGs) revealed that both the probing time and the total duration of phloem activity decreased significantly in the NPF treatment group. These results indicated a lower appetite for food after NPF knockdown, which could explain the reduction in aphid food intake. NPF silencing was also shown to reduce reproduction but not survival in aphids. Overall, the results of these experiments suggest that NPF plays an important role in regulation of feeding in A. pisum.

RNAi-Mediated Knockdown of vATPase Subunits Affects Survival and Reproduction of Bed Bugs (Hemiptera: Cimicidae).

The common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae) has resurged as one of the most troublesome household pests affecting people across the globe. Bed bug infestations have increased in recent years primarily due to the evolution of insecticide resistance and the insect's ability to hitchhike with travelers. vATPases are one of the most evolutionarily conserved holoenzymes in eukaryotes, which are mainly involved in proton transport across the plasma membranes and intracellular organelles. RNA interference (RNAi) has been developed as a promising tool for insect control. In this study, we used RNAi as an approach to knock down subunits A and E of the vATPase gene of bed bugs. Delivery of 0.2 µg/insect of dsRNA specific to vATPase-A and vATPase-E into female bed bugs dramatically impaired the laying and viability of eggs over time. Injection of the vATPase-E dsRNA decreased survival of the bed bugs over 30 d. Our results also showed that the knockdown of mRNA is highly effective and persistent up to 30 d post injection. This research demonstrated that silencing of the two vATPase subunits A and E offers a potential strategy to suppress bed bug populations.

Reduction in Musca domestica fecundity by dsRNA-mediated gene knockdown.

House flies (Musca domestica) are worldwide agricultural pests with estimated control costs at $375 million annually in the U.S. Non-target effects and widespread resistance challenge the efficacy of traditional chemical control. Double stranded RNA (dsRNA) has been suggested as a biopesticide for M. domestica but a phenotypic response due to the induction of the RNAi pathway has not been demonstrated in adults. In this study female house flies were injected with dsRNA targeting actin-5C or ribosomal protein (RP) transcripts RPL26 and RPS6. Ovaries showed highly reduced provisioning and clutch reductions of 94–99% in RP dsRNA treated flies but not in actin-5C or GFP treated flies. Gene expression levels were significantly and specifically reduced in dsRNA injected groups but remained unchanged in the control dsGFP treated group. Furthermore, injections with an Aedes aegypti conspecific dsRNA designed against RPS6 did not impact fecundity, demonstrating species specificity of the RNAi response. Analysis of M. domestica tissues following RPS6 dsRNA injection showed significant reduction of transcript levels in the head, thorax, and abdomen but increased expression in ovarian tissues. This study demonstrates that exogenous dsRNA is specifically effective and has potential efficacy as a highly specific biocontrol intervention in adult house flies. Further work is required to develop effective methods for delivery of dsRNA to adult flies.

Identification and characterization of opsin gene and its role in ovarian maturation in the oriental river prawn Macrobrachium nipponense.

Opsins are photoreceptors with important roles in reproductive regulation in birds and fishes. In the present study, we identified an opsin gene from the eyes of the oriental river prawn Macrobrachium nipponense using expressed sequence tag analysis and rapid amplification of cDNA ends. The full-length transcript contained 1382 base pairs, encoding 375 amino acids. It was classified into the long-wavelength opsin group by phylogenetic analysis, and designated Mn-LW. Mn-LW expression demonstrated significant seasonal variation in somatic tissues from both male and female prawns, with the highest expression in the eyes, and expression also shown in the ovary. The expression profiles of Mn-LW in eyes and ovary were positively related to ovarian development. In situ hybridization showed that Mn-LW was present in retinular cells in the eye and oocytes in the ovary. Injection of Mn-LW dsRNA in vivo effectively down-regulated Mn-LW expression levels compared with control levels. Mn-LW dsRNA injection also significantly reduced vitellogenin (Vg) expression, indicating a close relationship between Mn-LW and Vg in ovarian development. These results suggest that Mn-LW may play an important role in Vg synthesis and accumulation during ovarian maturation in M. nipponense.

Administration of co-expressed Penaeus stylirostris densovirus-like particles and dsRNA-YHV-Pro provide protection against yellow head virus in shrimp

The activation of the innate RNA interference pathway through double-stranded RNAs (dsRNAs) is one of the approaches to protecting shrimp from viruses. Previous studies have shown that injection of specific dsRNAs can successfully inhibit viral infection in shrimp. However, inhibition requires high levels of dsRNA and dsRNA stability in shrimp is limited. Virus-like particles (VLPs) have been applied to deliver nucleic acids into host cells because of the protection of dsRNAs from host endonucleases as well as the target specificity provided by VLPs. Therefore, this study aimed to develop Penaeus stylirostris densovirus (PstDNV) VLPs for dsRNA deliver to shrimp. The PstDNV capsid protein was expressed and can be self-assembled to form PstDNV VLPs. Co-expression of dsRNA-YHV-Pro and PstDNV capsid protein was achieved in the same bacterial cells, whose structure was displayed as the aggregation of VLPs by TEM. Tested for their inhibiting yellow head virus (YHV) from infecting shrimp, the dsRNA-YHV-Pro-PstDNV VLPs gave higher levels of YHV suppression and a greater reduction in shrimp mortality than the delivery of naked dsRNA-YHV-Pro. Therefore, PstDNV-VLPs are a promising vehicle for dsRNA delivery that maintains the anti-virus activity of dsRNA in shrimp over a longer period of time as compared to native dsRNAs.

RNA Interference in Planarians: Feeding and Injection of Synthetic dsRNA.

RNA interference (RNAi) is one of the simplest and easiest methods for specifically perturbing gene function in an organism. In planarian research, RNAi is one of the essential methods for defining gene functions not only during regeneration, but also during other life history stages. Since the first report of the efficacy of RNAi in planarians in 1999, several RNAi protocols have been reported. Here, we describe protocols to synthesize and deliver synthetic double-stranded RNA (dsRNA) to planarians, either by injection or by feeding. Both are easy, effective, and economical means of investigating gene functions in planarians.

Knockdown of the Chromatin Remodeling Gene Brahma by RNA Interference Reduces Reproductive Fitness and Lifespan in Common Bed Bug (Hemiptera: Cimicidae).

The common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae) is a nuisance household pest causing significant medical and economic impacts. RNA interference (RNAi) of genes that are involved in vital physiological processes can serve as potential RNAi targets for insect control. Brahma is an ATPase subunit of a chromatin-remodeling complex involved in transcription of several genes for cellular processes, most importantly the homeotic genes. In this study, we used a microinjection technique to deliver double stranded RNA into female bed bugs. Delivery of 0.05 and 0.5 µg/insect of brahma dsRNA directly into hemocele resulted substantial reduction in oviposition. Eggs laid by bed bugs receiving both doses of brahma dsRNA exhibited significantly lower hatching percentage as compared to controls. In addition, brahma RNAi in female bed bugs caused significant mortality. Our results disclosed the potential of brahma RNAi to suppress bed bug population through injection of specific dsRNA, suggesting a critical function of this gene in bed bugs' reproduction and survival. Based on our data, brahma can be a promising RNAi target for suppression of bed bug population.

The Single von Willebrand factor C-domain protein (SVC) coding gene is not involved in the hymenoptaecin upregulation after Israeli acute paralysis virus (IAPV) injection in the bumblebee Bombus terrestris

Within insects, inductions of antimicrobial peptides (AMPs) have been reported after different virus challenges. It is believed that this link is not directly induced by the virus itself, but rather indirectly induced by secondary effects of virus infection. Here we explored if direct sensing of the virus could trigger AMP expression. Recently, a cytokine-like molecule vago, a member of the Single von Willebrand factor C-domain (SVC) protein family, has been shown to be induced by virus infection in a Dicer-2 dependent manner. SVCs are also reported to be responsive in relation to multiple environmental challenges including bacterial infections and the nutritional status in the model species Drosophila melanogaster. Within the bumblebee Bombus terrestris only one SVC member has been identified and is proven to be involved in both the host antiviral defense and the basal expression of AMP genes, thereby it is a possible candidate linking virus infection and AMPs induction. Here we showed that the injection of Israeli acute paralysis virus (IAPV) resulted in a higher hymenoptaecin expression at 1dpi. This expression is IAPV specific as neither injection of slow bee paralysis virus (SBPV) nor random dsRNA results in a similar induction at 1dpi. We could not prove that hymenoptaecin expression after IAPV treatment was related to BtSVC, as a silencing experiment did not lower hymenoptaecin induction. This leaves indirect activation by secondary effects of IAPV infection as a mechanism of AMP genes induction, or that IAPV infection influences the AMP expression dynamics which is initially induced by non-virus related triggers.

Nuclear receptor HR3 controls locust molt by regulating chitin synthesis and degradation genes of Locusta migratoria

During growth and development of insects, the steroid hormone 20-Hydroxyecdysone (20E) regulates the molting process through activation of a series of genes including E74, E75 and HR3 by the 20E receptor EcR. Here, we analyzed the function of LmHR3 in the migratory locust Locusta migratoria. By sequence comparison, we first identified and characterized the putative nuclear receptor protein (LmHR3) based on L. migratoria transcriptome data. The full length cDNA is 2272 bp long encoding a protein of 455 amino acids that contains a DNA binding domain (zinc finger) and a ligand binding domain. Phylogenetic analyses showed that LmHR3 has a high homology with the ortholog from Blattaria. RT-qPCR results revealed that LmHR3 has a low level expression in the early days of 5th instar nymphs, and then increases and peaks at day 6, followed by a decrease to low levels before ecdysis. The LmHR3, hence, coincides with the profile of circulating 20E levels. Indeed, we show that transcription of LmHR3 is induced by 20E in vivo, and significantly suppressed by successfully knocking down expression of LmEcR. After injection of dsRNA for LmHR3 (dsLmHR3) at day 2 of earlier instar nymphs (3rd and 4th instar) and final instar nymphs (5th instar), none of the nymphs were able to molt normally, and eventually died. Chitin staining and ultra-structural analysis showed that both the synthesis of the new cuticle and the degradation of the old cuticle were blocked in the dsLmHR3 treated nymphs. Especially, chitin synthesis genes (LmUAP1 and LmCHS1) and chitinase genes (LmCHT5 and LmCHT10) were significantly down-regulated in the dsLmHR3 treatment group. Together, our results suggest that LmHR3 is involved in the control of chitin synthesis and degradation during L. migratoria molting.

Short versus long double-stranded RNA activation of a post-transcriptional gene knockdown pathway

ABSTRACTRNA interference (RNAi) utilizes a conserved cellular autoimmune defense mechanism involving the internalization of dsRNA into cells and the activation of a set of RNAi related genes. Using RNAi, complete sex reversal is achievable in males of the prawn Macrobrachium rosenbergii by knocking down the transcript level of an insulin-like androgenic gland hormone (Mr-IAG) through injections of dsRNA of the entire Mr-IAG ORF sequence (dsMr-IAG – 518bp). Interestingly, in-vivo knockdown success and dsMr-IAG lengths seemed to correlate, with long dsRNA being the most effective and short dsRNA fragments showing no effect. However, little is known about the RNAi machinery in M. rosenbergii. We discovered the Mr-Dicer and Mr-Argonaute gene families, associated with the major knockdown pathways, in our M. rosenbergii transcriptomic library. In response to dsMr-IAG administration, only post-transcriptional pathway-related gene transcript levels were upregulated. In addition, a passive dsRNA channel (a SID1 gene ortholog) that allows external dsRNA to enter cells was found. Its function was validated by observing Mr-SID1 specific upregulation dependent on dsRNA lengths, while attempted loss-of-function experiments were lethal. Our results, which suggest differential systemic responses to dsRNA lengths, provide evidence that the above RNAi-based manipulation occurs via the post-transcriptional pathway. The temporal nature of the latter pathway supports the safety of using such RNAi-based biotechnologies in aquaculture and environmental applications. Unlike reports of RNAi driven by the administration of small dsRNA fragments in-vitro, the case presented here demonstrates length dependency in-vivo, suggesting further complexity in the context of the entire organism.

Identification and function analysis of ras-related nuclear protein from Macrobrachium rosenbergii involved in Spiroplasma eriocheiris infection

A ras-related nuclear protein (Ran) protein was obtained from Macrobrachium rosenbergii, named MrRan. Phylogenetic analysis results showed that MrRan was clustered in one group together with other crustaceans. Tissue distribution analysis revealed that MrRan was expressed mainly in gill, intestine and stomach, and expressed weakly in muscle. The MrRan expression levels in gill and hemocyte of prawns were significantly up-regulated after challenged by Spiroplasma eriocheiris. The copy number of S. eriocheiris in MrRan dsRNA injection group was significantly less than control groups during infection. Meanwhile, silencing MrRan obviously increased the survival rate of prawns. The subcellular localization experiment suggested that recombinant MrRan was mainly located in the nucleus, and relatively weak in the cytoplasm. Finally, over-expression in Drosophila S2 cell indicated that MrRan could increase copies of S. eriocheiris and decrease of cell viability. The present study suggested that MrRan participated in regulating the phagocytosis of S. eriocheiris in M. rosenbergii.