On the safety of RNAi usage in aquaculture: The case of all-male prawn stocks generated through manipulation of the insulin-like androgenic gland hormone

Lezer, Yaara; Aflalo, Eliahu D.; Manor, Rivka; Sharabi, Omri; Abilevich, Lihie Katzir; Sagi, Amir

doi:10.1016/j.aquaculture.2014.09.040

Cited by 49 publications

(31 citation statements)

References 53 publications

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“…Moreover, our observations that the elapsed times until disappearance of the exogenous dsRNA from prawn tissues and the return of Mr-IAG transcript levels to normal during the post silencing period were comparable, suggesting that RNAi-based technology is temporal and indeed safe. 23,35 The current study, which examined the mechanism of the knockdown pathway, provided additional support for the temporal nature of the RNAi-based production of all-male cultures and rejects the possible involvement of permanent or epigenetic interference processes on the genome associated with the transcriptional silencing pathway.…”

Section: Discussionmentioning

confidence: 68%

“…3,33,34 The use of such a biotechnology was previously suggested to be safe based on the temporal nature of the intervention and its lack of any overtly apparent long-term consequences. 23,35 These studies presented evidence based on social structure, growth performance and masculine reproductive characters at the anatomic and molecular levels. Moreover, our observations that the elapsed times until disappearance of the exogenous dsRNA from prawn tissues and the return of Mr-IAG transcript levels to normal during the post silencing period were comparable, suggesting that RNAi-based technology is temporal and indeed safe.…”

Section: Discussionmentioning

confidence: 99%

“…Each experiment included a non-injected group as a negative control and an entire dsMr-IAG ORF injected group as a positive control. It is important to note that the determination of which animals would constitute the non-injected control groups was based on previous studies, which also used exogenous dsRNA or DDW injection as described in Lezer et al 35 and in Sharabi et al, 42 with similar results. Each experimental group included 6-8 small M. rosenbergii males (Mean body weight D 12.5 g).…”

Section: Synthesis Of Double-stranded Rnamentioning

confidence: 99%

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

et al. 2017

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RNA 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.

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Section: Discussionmentioning

confidence: 68%

Section: Discussionmentioning

confidence: 99%

Section: Synthesis Of Double-stranded Rnamentioning

confidence: 99%

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

et al. 2017

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“…It has been well known that IAG plays a vital role in masculinization and maintaining male characteristics. IAG mRNA silencing could cause male‐to‐female sex reversal during juvenile stage in the prawn Macrobrachium rosenbergii (Lezer et al, ; Rosen et al, ; Ventura et al, ). Besides its role in generating sex reversal of juvenile individuals, IAG silencing at a young stage could induce the reduction of sperm production, and result in extensive testicular degeneration in the male crayfish Cherax quadricarinatus (Rosen et al, ).…”

Section: Introductionmentioning

confidence: 99%

Identification of an insulin‐like androgenic gland hormone transcript highly expressed in male precociousness of the Chinese mitten crab Eriocheir sinensis

2018

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The androgenic gland (AG) hormone is well known as a key peptide to induce masculinization and maintains male characteristic in crustaceans. In this study, a fulllength cDNA encoding insulin-like androgenic gland hormone (EsIAG) was identified from the AG of the Chinese mitten crab Eriocheir sinensis. The full length of EsIAG cDNA contains 1,481 nucleotides, which encodes for a deduced protein of 151 amino acid residues. The predicted pre-peptide is composed of a signal peptide of 19 amino acids (aa), A chain of 44 aa, B chain of 31 aa, and C peptide of 57 aa. Moreover, six conserved cysteine residues and a putative N-linked glycosylated site were found in the deduced protein as reported in other crustaceans. Reverse transcription polymerase chain reaction and in situ hybridization analysis showed EsIAG was only expressed in the AG with especially high abundance during the mature stage of testis. Interestingly, a significantly higher expression level of EsIAG was also detected in precocious individuals than that in the normal grown ones, suggesting that the abnormally high expression of EsIAG might contribute to male precociousness in E. sinensis. K E Y W O R D SEriocheir sinensis, insulin-like androgenic gland hormone, precociousness

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“…Recent trialing of RNA-i technologies in a number of aquaculture species (Itsathitphaisarn et al, 2017;Lezer et al, 2015;Pindaro et al, 2013; have been successful and for GFP have generated preferred allmale monosex cultures that show improved yields. A very recent study by Shpak et al, 2017 demonstrated normal morphology and performance of all-male cultures across three generations in GFP.…”

Section: Other Directions For Improving Gfp Stock Quality and Productmentioning

confidence: 99%

Adult male morphotype variation in giant freshwater prawn (Macrobrachium rosenbergii): Genomic and experimental analysis of factors affecting the male social dominance hierarchy

Aziz¹

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On the safety of RNAi usage in aquaculture: The case of all-male prawn stocks generated through manipulation of the insulin-like androgenic gland hormone

Cited by 49 publications

References 53 publications

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

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

Identification of an insulin‐like androgenic gland hormone transcript highly expressed in male precociousness of the Chinese mitten crab Eriocheir sinensis

Adult male morphotype variation in giant freshwater prawn (Macrobrachium rosenbergii): Genomic and experimental analysis of factors affecting the male social dominance hierarchy

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