Molt-dependent transcriptomic analysis of cement proteins in the barnacle Amphibalanus amphitrite.
BACKGROUND
A complete understanding of barnacle adhesion remains elusive as the process occurs within and beneath the confines of a rigid calcified shell. Barnacle cement is mainly proteinaceous and several individual proteins have been identified in the hardened cement at the barnacle-substrate interface. Little is known about the molt- and tissue-specific expression of cement protein genes but could offer valuable insight into the complex multi-step processes of barnacle growth and adhesion.


METHODS
The main body and sub-mantle tissue of the barnacle Amphibalanus amphitrite (basionym Balanus amphitrite) were collected in pre- and post-molt stages. RNA-seq technology was used to analyze the transcriptome for differential gene expression at these two stages and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) was used to analyze the protein content of barnacle secretions.


RESULTS
We report on the transcriptomic analysis of barnacle cement gland tissue in pre- and post-molt growth stages and proteomic investigation of barnacle secretions. While no significant difference was found in the expression of cement proteins genes at pre- and post-molting stages, expression levels were highly elevated in the sub-mantle tissue (where the cement glands are located) compared to the main barnacle body. We report the discovery of a novel 114kD cement protein, which is identified in material secreted onto various surfaces by adult barnacles and with the encoding gene highly expressed in the sub-mantle tissue. Further differential gene expression analysis of the sub-mantle tissue samples reveals a limited number of genes highly expressed in pre-molt samples with a range of functions including cuticular development, biominerialization, and proteolytic activity.


CONCLUSIONS
The expression of cement protein genes appears to remain constant through the molt cycle and is largely confined to the sub-mantle tissue. Our results reveal a novel and potentially prominent protein to the mix of cement-related components in A. amphitrite. Despite the lack of a complete genome, sample collection allowed for extended transcriptomic analysis of pre- and post-molt barnacle samples and identified a number of highly-expressed genes. Our results highlight the complexities of this sessile marine organism as it grows via molt cycles and increases the area over which it exhibits robust adhesion to its substrate.
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, 20375, USA. zheng.wang@nrl.navy.mil.
Zheng
Z
Wang
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, 20375, USA. dasha.leary@nrl.navy.mil.
Dagmar H
DH
Leary
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, 20375, USA. jinny.liu@nrl.navy.mil.
Jinny
J
Liu
Virginia Bioinformatics Institute, 1015 Life Science Circle, Blacksburg, VA, 24061, USA. rsettlage@vbi.vt.edu.
Robert E
RE
Settlage
Chemistry Division, Naval Research Laboratory, Washington, DC, 20375, USA. kenan.fears@nrl.navy.mil.
Kenan P
KP
Fears
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, 20375, USA. stella.north@nrl.navy.mil.
Stella H
SH
North
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, 20375, USA. mostagas@evms.edu.|Present address: Eastern Virginia Medical School, 700 West Olney Road, Norfolk, VA, 23507, USA. mostagas@evms.edu.
Anahita
A
Mostaghim
Chemistry Division, Naval Research Laboratory, Washington, DC, 20375, USA. tara.essock-burns@duke.edu.|Present address: Duke University Marine Laboratory, 135 Duke Marine Lab Rd. Beaufort, North Carolina, 28516, USA. tara.essock-burns@duke.edu.
Tara
T
Essock-Burns
Chemistry Division, Naval Research Laboratory, Washington, DC, 20375, USA. hayse@umich.edu.|Present address: Department of Chemistry, University of Michigan, 930 N. University Avenue, Ann Arbor, MI, 48109, USA. hayse@umich.edu.
Sarah E
SE
Haynes
Chemistry Division, Naval Research Laboratory, Washington, DC, 20375, USA. kathryn.wahl@nrl.navy.mil.
Kathryn J
KJ
Wahl
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, 20375, USA. christopher.spillmann@nrl.navy.mil.
Christopher M
CM
Spillmann
D000818:Animals; D019295:Computational Biology / Q000379:methods; D015870:Gene Expression; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D059014:High-Throughput Nucleotide Sequencing; D058977:Molecular Sequence Annotation; D018978:Molting / Q000235:genetics*; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D001468:Thoracica / Q000235:genetics* / Q000378:metabolism*; D059467:Transcriptome
D011506:Proteins