Abstract
Like many metazoans, the freshwater prawn Macrobrachium rosenbergii 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. De novo 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 M. rosenbergii 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.
Highlights
Metamorphosis refers to the set of drastic post-embryonic anatomical and physiological changes that occur mostly in arthropods and amphibians when an immature individual transforms into an adult, and is usually accompanied by a change of habitat and/or behavior [1]
Transcriptome Profiling One hundred bp-paired end sequencing in a single lane by an Illumina Genome Analyzer (HighSeq 2000, Illumina, San Diego, CA) generated,242 million reads from the cDNA of 200 M. rosenbergii larvae and PL2–28 (2– 28 day post-metamorphosis, 4 pooled samples)
As compared with the recently reported M. rosenbergii adult tissues transcriptome [23], our transcriptome is much more comprehensive, with 24.2 Gbp sequenced in total, yielding more transcripts (66,152, as compared with 8,411) of higher average length (921, as compared with 845, see Fig. S1 for length and coverage distribution). 11,528 Gene onthology (GO) terms were assigned to 7,076 transcripts, of which 4,011 GO terms were assigned biological processes, 5,172 were assigned molecular functions and 2,345 were assigned cellular components (Fig. S2)
Summary
Metamorphosis refers to the set of drastic post-embryonic anatomical and physiological changes that occur mostly in arthropods and amphibians when an immature individual transforms into an adult, and is usually accompanied by a change of habitat and/or behavior [1]. A common feature of metamorphosis in the animal kingdom is a biphasic life cycle where pelagic larvae metamorphose into benthic adults. This pelago-benthic transition, that occurs in crustaceans such as the study organism, Macrobrachium rosenbergii, is orchestrated by numerous factors. Apart from the fundamental understanding that 20hydroxy ecdysone is the key molting regulator and that methyl farnesoate (MF), the non-epoxidated form of the insect juvenile hormone (JH III), governs the metamorphic transition [2,3], little is known. MF is epoxidated by CYP15A1 in the corpora allata, yielding JH III [4], the active juvenile hormone in insects [5]. MF was previously identified in M. rosenbergii [6], with its administration prolonging late larval stages [7], further strengthening the notion of MF being the crustacean juvenile hormone
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