Number of KO terms assigned to differentially represented transcripts in the <i>M. rosenbergii</i> developmental transcriptomic database.

<p>Number of KO terms assigned to differentially represented transcripts in the <i>M. rosenbergii</i> developmental transcriptomic database.</p

Post-Embryonic Transcriptomes of the Prawn <em>Macrobrachium rosenbergii:</em> Multigenic Succession through Metamorphosis

<div><p>Like many metazoans, the freshwater prawn <em>Macrobrachium rosenbergii</em> begins its post-embryonic life with a set of morphologically distinct planktonic larval stages, followed by a benthic post-larval stage during which the maturing organism differs from the larvae both ecologically and physiologically. Understanding of the molecular basis underlying morphogenesis in crustaceans is limited to the observation that methyl farnesoate, the non-epoxidated form of the insect juvenile hormone, acts as the active crustacean juvenoid. Molt steroids were also linked to morphogenesis and several other molecular pathways, such as Hedgehog and Wnt, are known to underlie morphogenesis in all metazoans examined and, as such, are thought to do the same in crustaceans. Using next generation sequencing, we deep-sequenced the transcriptomes of several larval and post-larval stages. <em>De novo</em> assembly, followed by bioinformatics analysis, revealed that many novel transcripts are over-expressed in either larvae- or post-larvae-stage prawn, shedding light on the molecular basis underlying <em>M. rosenbergii</em> metamorphosis. Fast larval molting rates and periodic morphological changes were reflected in over-expression of transcripts annotated to the cell cycle, DNA replication and morphogenic pathways (i.e., Hedgehog and Wnt). Further characterization of transcripts assigned to morphogenic pathways by real-time RT-PCR reconfirmed their over-expression in larvae, albeit with a more complex expression pattern when examined in the individual developmental stages. The expression level of an orthologue of cytochrome P450, 15A1, known to epoxidize methyl farnesoate in insects, was increased in the late larval and early post-larval stages, in accordance with the role of methyl farnesoate in crustacean metamorphosis. This study exemplifies the applicability of a high-throughput sequencing approach for studying complex traits, including metamorphosis, providing new insight into this unexplored area of crustacean research.</p> </div

Number of transcripts differentially represented in the <i>M. rosenbergii</i> developmental transcriptomic database.

<p>Number of transcripts differentially represented in the <i>M. rosenbergii</i> developmental transcriptomic database.</p

Most prominent GO terms assigned to the <i>M. rosebergii</i> developmental transcriptome.

<p>The most prominent GO terms assigned to level 2 of biological processes (top) and levels 2 and 3 of molecular functions (middle) and cellular components (bottom) are listed, as calculated by Blast2GO software suite. A small fraction of 161 GO terms (∼1.4%) are assigned to developmental processes.</p

Volcano plot of transcript expression differences between larval and post-larval prawns.

<p>The magnitude of the difference in gene expression between larval and post-larval <i>M. rosenbergii</i> is shown on the X-axis on a log₂ scale (post-larval/larval) of intensity (with minimum 2-fold change). The significance of the difference is given as the -log₁₀ (<i>P</i>-value) obtained from a t test (with a minimum <i>P</i>-value  = 0.05). The mass of transcripts that are slightly over-represented in larvae (2–8-fold) are circled. Dashed lines represent the border of 8-fold change in larvae (-8 and left) and post-larvae (8 and right).</p

Comparison of whole ovaries obtained from dsRNA and DDW-injected intersex crayfish.

<p>The whole ovary from a dsRNA-injected intersex animal (A) contained yellowish oocytes, as in a mature female ovary (B), with an average diameter of approximately 900 µm. The ovary of a DDW-injected intersex animal (C) contained whitish oocytes, approximately 300 µm in diameter (bar  = 2 mm).</p

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

<p>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 <i>Macrobrachium rosenbergii</i> by knocking down the transcript level of an insulin-like androgenic gland hormone (<i>Mr-IAG</i>) through injections of dsRNA of the entire <i>Mr-IAG</i> ORF sequence (ds<i>Mr-IAG</i> – 518bp). Interestingly, <i>in-vivo</i> knockdown success and ds<i>Mr-IAG</i> 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 <i>M. rosenbergii</i>. We discovered the <i>Mr-Dicer</i> and <i>Mr-Argonaute</i> gene families, associated with the major knockdown pathways, in our <i>M. rosenbergii</i> transcriptomic library. In response to ds<i>Mr-IAG</i> administration, only post-transcriptional pathway-related gene transcript levels were upregulated. In addition, a passive dsRNA channel (a <i>SID1</i> gene ortholog) that allows external dsRNA to enter cells was found. Its function was validated by observing <i>Mr-SID1</i> 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 <i>in-vitro</i>, the case presented here demonstrates length dependency <i>in-vivo</i>, suggesting further complexity in the context of the entire organism.</p

Levels of <i>Cq-IAG</i> transcripts following <i>in vivo</i> dsRNA injections in male crayfish.

<p>Relative <i>Cq-IAG</i> transcript levels were quantified in crayfish males by real-time RT-PCR following short-term silencing. Three different groups were injected with either ds<i>Cq-IAG</i> (n = 6), ds<i>Mr-IAG</i> (n = 6) or DDW (n = 6). The groups were found to be statistically different (Kruskal-Wallis statist: H (df = 2, N = 18) = 7.450, p = 0.0241). Followed by a multiple pair-wise comparison ds<i>Cq-IAG</i> group was found significantly different from the DDW (P = 0.0386) and showed a difference that turned out to be non-significant (P = 0.0798) from the ds<i>Mr-IAG</i> group. Different letters represent significant difference and error bars represent SEM.</p

Effects of ds<i>Cq-IAG</i> injection on the reproductive system of intersex individuals.

<p>Hematoxylin- and eosin-stained cross-sections used for structure description. Components of the reproductive system of <i>Cq-IAG</i> dsRNA-injected intersex animals showed an empty sperm duct (black arrowhead, A) and inactive testicular lobules (black arrowheads, B), along with an activated ovary containing enlarged yolk-accumulating oocytes (C). A filled sperm duct (E), spermatogenic testis (F) and an arrested ovary (G) were observed in the control intersex animal. Enlarged areas within the ovaries of both groups are shown in the right hand side (D and H). Bar  = 500 µm in ovarian sections, 50 µm in sperm duct, testis and ovary, high magnification sections.</p

Levels of vitellogenin and expression of its encoding gene following <i>Cq-IAG</i> silencing in intersex crayfish.

<p>(A) <i>Cq-Vg</i> transcription in the hepatopancreas was demonstrated by RT-PCR. A single band was observed in the ds<i>Cq-IAG</i>-injected intersex animal sample but not in the control intersex sample. Samples of a vitellogenic female and a male served as controls. (B) Detection of the vitellogenin protein in the hemolymph was conducted by ELISA using anti-Cq-Vg antibodies. Hemolymph samples were collected from DDW-injected intersex (INX) animals (n = 7), ds<i>Cq-IAG</i>-injected INX (n = 3), vitellogenic females (n = 6) and mature males (n = 6). Egg high density lipoprotein (HDL) equivalent levels in dsRNA-injected INX were similar to those of vitellogenic females (Kruskal-Wallis statist: H (df = 3, N = 22) = 16.137, p = 0.001 followed by multiple pair-wise comparison). Negligible levels of egg HDL equivalents were detected in control intersex and mature males. Different letters represent significant differences (p<0.05±SEM).</p