The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z

Additional File 1:

Video clip of spawning female. Demonstration of spawning female and eggs prior to fertilization.

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Additional File 2:

Video clip of spawning male. Demonstration of spawning male and sperm prior to fertilization.

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Video clip of fertilization procedure. Demonstration of fertilization procedure and collection of fertilized eggs.

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Additional File 4:

Video clip of large scale larvae culture (10 l). Demonstration of the culture conditions of larvae in artificial seawater at 10 l-scale.

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Additional File 5:

Video microscopy of 2-day larvae. Video microscopic (32× objective) demonstration of the behaviour of 2-day larvae. Note that the larvae are already in the early veliger stage and have a D-shaped shell.

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Additional File 6:

Video microscopy of 5-day larvae. Video microscopic (32× objective) demonstration of the behaviour of 5-day larvae. The size of the velum and the motility of the larvae increased. Note that the hinge of the larval shell became straight.

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Additional File 7:

Video microscopy of 8-day larvae. Video microscopic (32× objective) demonstration of the behaviour of 8-day larvae. The motility of the larvae is similar to 5-day larvae. The size of the larval shells slightly increased. Most of the shells are still in D-shape.

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Additional File 8:

Video microscopy of 12-day larvae. Video microscopic (20× objective) demonstration of the behaviour of 12-day larvae. Note that the size of the shell increased, and the larvae switched from the D-shape stage to the umbo stage of shell formation.

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Additional File 9:

Video microscopy of 31-day larvae. Video microscopic (20× objective) demonstration of the behaviour of 31-day larvae. Larvae are in different developmental stages, such as veliger larvae and pediveliger larvae. The latter are indicated by the developing foot and a degenerate, vanishing velum that is retracted into the shell.

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Additional File 10:

Video microscopy of 36-day larvae. Video microscopic (20× objective) demonstration of the behaviour of 36-day larvae. At this age, the velum disappeared, and metamorphosis into the adult was completed as indicated by the functional foot.

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Additional File 11:

Table: Viability and malformation rate estimated in Mytilus galloprovincialis larvae populations in the presence of NikkomycinZ. Data source (estimated values) of survival rate and malformation rate of mollusc larvae populations reared in the presence of 0 μM, 5 μM, and 10 μM NikkomycinZ as presented in figure 2 and figure 3.

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Additional File 12:

Video microscopy of 8-day larvae after 6 days of NikkomycinZ treatment. Video microscopic (32× objective) demonstration of the behaviour and phenotype of 8-day larvae after 6 days of treatment with the chitin synthase inhibitor NikkomycinZ. The overall motility of the larvae decreased as compared to untreated control cultures. Note that NikkomycinZ appears to have a dramatic effect on shell development. The size of the shells is comparable to 2-day larvae. The hinge is not a straight line, and shell edges appear irregular or undulated.

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Additional File 13:

Video microscopy of 12-day larvae after 7 days of NikkomycinZ treatment. Video microscopic (32× objective) demonstration of the behaviour and phenotype of 12-day larvae after 7 days of treatment with the chitin synthase inhibitor NikkomycinZ. The overall motility of the larvae decreased as compared to untreated control cultures. Note that NikkomycinZ appears to have a dramatic effect on shell development. The formation of the umbo was prevented (see additional file 8). The size of the shells is comparable to 5-day larvae. The most prominent features were curved hinges and malformed shell edges. Shells of some individuals were too small to host the organism completely.

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Additional File 14:

Video microscopy of 12-day larvae after 4 days of NikkomycinZ treatment. Video microscopic (32× objective) demonstration of the behaviour and phenotype of 12-day larvae treated from the 8^th day on with the chitin synthase inhibitor NikkomycinZ. Even four days of treatment with NikkomycinZ have similar effects on shell development of individuals as described in additional file 13. Note that less affected individuals were highly motile and therefore out of focus in this data set.

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Additional File 15:

Video microscopy of 15-day larvae after 7 days of NikkomycinZ treatment. Video microscopic (32× objective) demonstration of the behaviour and phenotype of 15-day larvae treated from the 8^th day on with the chitin synthase inhibitor NikkomycinZ. Even in later developmental stages, NikkomycinZ induced characteristic effects on the shell development of living individuals. Note that the previously straight hinge (see additional files 7 &8) appears now curved. This indicates that NikkomycinZ interferes with the remodelling of the hinge region. The fact that shells are much smaller than in the untreated control cultures (additional file 8) suggests that either the solubility of the newly formed shell is increased, or lateral shell growth is blocked by the chitin synthase inhibitor drug. Note that also shell remnants of larvae that died at undefined age are present in this data set.

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