Elsevier

Theriogenology

Volume 90, 1 March 2017, Pages 191-196
Theriogenology

Embryonic development of the grass pufferfish (Takifugu niphobles): From egg to larvae

https://doi.org/10.1016/j.theriogenology.2016.12.005Get rights and content

Abstract

Tetraodontidae (pufferfish) family members carry the smallest genomes among vertebrates, and these pocket-sized genomes have directly contributed to our understanding of the structure and evolution of higher animals. The grass pufferfish (Takifugu niphobles) could be considered a potential new model organism for comparative genomics and development due to the potential access to embryos, and availability of sequence data for two similar genomes: that of spotted green pufferfish (Tetraodon nigroviridis) and Fugu (Takifugu rubripes). In this study, we provide the first description of the normal embryonic development of T. niphobles, by drawing comparisons with the closely related species cited above. Embryos were obtained by in vitro fertilization of eggs, and subsequent development was monitored at a constant temperature consistent with natural conditions. T. niphobles development was divided into seven periods of embryogenesis: the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods; and stages subdividing these periods are defined based on morphological characteristics. The developmental stage series described in this study aims to provide the utilization of T. niphobles as an experimental model organism for comparative developmental studies.

Introduction

The grass pufferfish (T. niphobles) is a teleost fish with a wide distribution in the Northwest Pacific Ocean. This species is one of around twenty four pufferfish species in the genus Takifugu, and there are some reasons that justify its study: i) it is placed on the IUCN Red List due to the reduced knowledge about the stage of its current populations, making it a possible endangered species [1]; ii) the closely related species (like Takifugu rubripes) is widely-kept by scientists as a model organism for genomics [2], [3]; and iii) some species of this genus are considered a popular food in Japan.

The genome of the congeneric species T. rubripes (Fugu) has been sequenced and assembled recently, the second vertebrate genome to be sequenced and the shortest known genome of any vertebrate species [4]. In this respect, the pocket-sized genome of Fugu should help to resolve contentious estimates of human gene number, where the genome of Fugu has directly contributed to the annotation of protein-coding genes on 11 human chromosomes and has also helped unearth nearly 1000 new human genes [5], [6]. In this regard, closely related species such as T. niphobles could be similarly applied in this purpose due to its small and similar genome. One advantage of T. niphobles over the other pufferfish species currently used for genomic studies is the potential for the study of essential steps in development: staging series based on morphological traits will provide in-depth knowledge of the developmental processes governing teleost fish [7], [8].

Staging by morphological criteria is an useful tool for generating developmental comparisons between different species and, in this sense, to determine the underlying mechanisms of evolutionary changes among them [9]. For Fugu (T. rubripes), a developmental stage series has been published [10], but a standard and cost-effective laboratory breeding protocol is not available. In contrast, T. niphobles, with a high fertility rate during a wide spawning period (offering the availability of thousands of eggs [11]), can be kept and maturated in laboratory conditions [12]. As a result, both species have remained virtual models, mostly confined to genome sequence analyses. In this study, we have set out to promote T. niphobles as a laboratory model for functional and comparative genomic and developmental projects. We report the embryonic development of T. niphobles, raised under laboratory conditions, describing the series of embryonic stages and provide fundamental data to facilitate its use for future developmental studies.

Section snippets

Fish handling

Takifugu niphobles shows a singular spawning behavior at Arai Beach near Misaki Marine Biological Station (MMBS, Japan). Large schools of fish (200–1000 [13]) arrive to the beach around the new or full moon at spring tide during the spawning season, which occurs between May and July. Spawning takes place repeatedly from 2 h before the sunset and includes a beach-spawning behavior, where the fish are routinely found out of the water on the beach until the next wave. During this time, males and

Zygote period

The zygote period started from in vitro fertilization until the onset of cleavage period, when the embryonic polar cell mass transitioned from the 1-cell stage to the 2-cell stage (Fig. 1A–B). Zygote period spanned 0–1.7 hpf for reaching the cleavage.

Cleavage period

During the cleavage period of Takifugu niphobles embryonic development, a single cell (1st blastomere), formed at the animal pole by separation of cytoplasm from the yolk, was divided (cleaved) into an increasing number of smaller cells, decreasing

Discussion

In this study, we report the developmental stages of T. niphobles based on morphological characteristics. This information is anticipated to allow the use of pufferfish as a model for developmental studies [19], uncovering the morphological diversification of this group of highly derived teleost fishes. Regarding the different embryonic stages during the egg development, cell division cycle from the 2-cell to the 1k-cell stage lasted approximately 12 h in T. niphobles. These intervals were

Acknowledgements

Funded by the Spanish Ministry of Economy and Competitiveness (MINECO; AGL2010-16009). VG had a predoctoral grant (MINECO; BES-2009-020310) and had been granted by a fellowship (EEBB-I-12-05858) of the MINECO's Spanish Personnel Research Training Programme to carry out this research in the Misaki Marine Biological Station (Miura, Japan). This research was also supported by the Great Britain Sasakawa Foundation (grant to GJF). The authors would like to thank to L. Clement for her help in the

References (30)

  • V. Gallego et al.

    Sperm motility parameters and spermatozoa morphometric characterization in marine species: a study of swimmer and sessile species

    Theriogenology

    (2014)
  • K. Shao et al.

    Takifugu niphobles

  • Y. Yamanoue et al.

    Explosive speciation of Takifugu: another use of fugu as a model system for evolutionary biology

    Mol Biol Evol

    (2009)
  • S. Aparicio et al.

    Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes

    Science

    (2002)
  • S. Brenner et al.

    Characterization of the pufferfish (Fugu) genome as a compact model vertebrate genome

    Nature

    (1993)
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