Molt-dependent transcriptomic analysis of cement proteins in the barnacle Amphibalanus amphitrite

Abstract 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.http://deepblue.lib.umich.edu/bitstream/2027.42/115487/1/12864_2015_Article_2076.pd

Exploring the Interface Between Macroorganisms and Microorganisms: Biochemical, Ecological, and Evolutionary Contexts

<p>The focus of this dissertation is the extension of the innate immune response in wound healing and non-wound healing contexts. I am interested in interactions at the interface between macroorganisms and microorganisms from marine/aqueous environments. This dissertation explored two aspects of the interactions: 1) the presence and function of macroorganism secretions and 2) the role of secretions in managing microfouling on macroorganism surfaces. Particularly of interest are how barriers are biochemically reinforced to mitigate microfouling and the potential consequences of a breach in those barriers. The innate immune response, an evolutionary conserved system in vertebrates and invertebrates, provides an evolutionary context for developing the hypotheses. </p><p>In this dissertation the biochemical composition and uses of crustacean secretions are explored for barnacles, fiddler crabs and blue crabs. Fluids of interest were secretions released during barnacle settlement and metamorphosis and those collected from living adult barnacles, fluids on fiddler crab sensory appendages including dactyl washings and buccal secretions, and fluids from blue crab egg masses. The biochemical composition was determined using a combination of fluorescent probes and confocal microscopy, proteomics, and enzyme-specific substrates with a spectrophotometer. </p><p>I demonstrated that self-wounding is inherent to the critical period of settlement and metamorphosis, in barnacles. Wounding occurs during cuticle expansion and organization and generates proteinaceous secretions, which function as a secondary mode of attachment that facilitates the transition to a sessile juvenile. I showed extensive proteomic evidence for components of all categories of the innate immune response, especially coagulation and pathogen defense during attachment and metamorphosis. This work provides insight into wound healing mechanisms that facilitate coagulation of proteinaceous material and expands the knowledge of potential glue curing mechanisms in barnacles. </p><p>In order to test macroorganism secretions in a non-wound healing context, I examined fluids sampled from body parts that macroorganisms must keep free of microorganisms. I showed that two types of decapod crustaceans can physically manage microorganisms on most parts of their body, but certain parts are particularly sensitive or difficult to clean mechanically. I examined sensory regions on the fiddler crab, including dactyls that are important for chemoreception and the buccal cavity that is used to remove microorganisms from sand particles, and blue crab egg mass fluids that protect egg masses from fouling through embryo development. </p><p>This dissertation explores organismal interactions across scales in size, space, and time. The findings from the barnacle work inform mechanisms of attachment and glue curing, both central to understanding bioadhesion. The work on fiddler crabs and blue crabs contributes to our understanding of chemoreception of feeding and reproductive behaviors.</p><p>The work presented here highlights how biological secretions from macroorganisms serve multifaceted roles. In cases of physical breaches of barriers, or wounding, secretions coagulate to obstruct loss of hemolymph and have antimicrobial capabilities to prevent infection by microorganisms. In non-wounding cases, secretions remove microorganisms from surfaces, whether that is on the body of the macroorganism or in the immediate environment.</p>Dissertatio

Phenotypic differences between interfertile Chlamydomonas species- high-resolution confocal z-stacks for visualizing organelle morphology

&lt;p&gt;This repository contains high-resolution confocal z-stacks of two interfertile &lt;i&gt;Chlamydomonas&lt;/i&gt; algal species. The protocol to generate this data is described in the associated publication, "Phenotypic differences between interfertile &lt;i&gt;Chlamydomonas&lt;/i&gt; species", and briefly summarized here. Cells were collected from agar plates with TAP medium and suspended in 500 µl of liquid TAP medium in a 1.5 ml eppendorf tube overnight. Cells were pelleted using a microcentrifuge at 2000 x g for 2 min and the supernatant removed. For staining mitochondria, PKMito orange was used at a 1:500 concentration and cells were moved to opaque black microcentrifuge tubes and placed on a tube rotator for 45 min. Cells were pelleted again and washed twice with fresh TAP medium. After the final wash and supernatant removal, cells were resuspended in 25 µl of 1.25% low gelling agar in TAP medium (kept at 45 C). Then 1 µl of the cell/agar mixture was mounted on a #1.5 coverslip with a small wax circle drawn to retain the droplet. Coverslips were flipped and placed on a slide and sealed with VALAP.&nbsp;&lt;/p&gt;&lt;p&gt;Images were collected on a Nikon CSU W-1 SoRA spinning disk confocal microscope equipped with an ORCA-Fusion BT digital scMOS camera. In order to apply deconvolution in the downstream processing, we needed to oversample (sample beyond Nyquist) in z resolution. To do this, we used a 100×/1.45 NA objective in 2.8× SoRa magnification mode, using ROIs of either 670 × 670 × 81 or 850 × 850 × 91. We imaged with a z-step size of 100 nm for sub-Nyquist sampling. We imaged bright-field first, then 640 nm excitation autofluorescence of chloroplasts, and then 561 nm excitation for PKmito orange dye, because the chloroplasts would bleach after 561 nm excitation. We set exposures to 300 ms with 30% and 50% laser power for 640 and 561, respectively.&lt;/p&gt;&lt;p&gt;We have included a set of demo data (10 images per species) that accompany the pub hosted on the Arcadia Science webpage (3Dmorpho_demo_data). In addition, we included all of the raw data we collected in this experiment (3Dmorpho_raw_data). Please use the point spread functions (PSF) from the zipped folders for each respective dataset (demo or raw).&nbsp;&lt;/p&gt

Natural glues and fouling management by interfering with glue curing

Multidisciplinary approaches and modern technology provide insights to glue curing that are stimulatingand controversial. Our team applies classic and modern theory and techniques to the study of barnacle glue. Techniques include physical measures, bacteriology, behavior, physiology, biochemistry, microscopy, spectroscopy, tomography, tandem mass spectrometry, molecular biology and proteomics. Theory is grounded in evolution and previous literature. Here, we use data from these techniques to support the hypothesis that barnacle glue curing is similar toblood clotting and propose a model for how glue cures. Similar to blood clotting, barnacle glue curing involves enzymatic activation of precursors and rearrangement of structural molecules to form a crosslinked material. Barnacle larval settlement, bacteriology and biochemical data show glue contains large amounts of small peptides. Their role in glue curing has been overlooked. The peptides comprise 15 to 30% of partially cured glue. Because they have little secondary structure, the peptides can associate with binding domains on the substrate and interface with the larger, well-described structural proteins known in barnacle glue. Enzymes participate in curing of barnacle glue. Siloxanes impact glue-curing enzymes. They potentiate trypsin activity and inhibit transglutaminase activity. Changing enzymeactivity impacts how glue cures. Disrupting the curing process of biological glues is central to effective cleaning strategies for fouling management. Thus silicones that interfere with enzyme activity have potential as additives in easy cleansurfaces. The environmental impacts of organosilicones that are generated by biological processes need to be addresse

Interactions of Symbiotic Partners Drive the Development of a Complex Biogeography in the Squid-Vibrio Symbiosis

The complexity, inaccessibility, and time scales of initial colonization of most animal microbiomes present challenges for the characterization of how the bacterial symbionts influence the form and function of tissues in the minutes to hours following the initial interaction of the partners. Here, we use the naturally occurring binary squid-vibrio association to explore this phenomenon. Imaging of the spatiotemporal landscape of this symbiosis during its onset provides a window into the impact of differences in both host-tissue maturation and symbiont strain phenotypes on the establishment of a dynamically stable symbiotic system. These data provide evidence that the symbionts shape the host-tissue landscape and that tissue maturation impacts the influence of strain-level differences on the daily rhythms of the symbiosis, the competitiveness for colonization, and antibiotic sensitivity.Microbes live in complex microniches within host tissues, but how symbiotic partners communicate to create such niches during development remains largely unexplored. Using confocal microscopy and symbiont genetics, we characterized the shaping of host microenvironments during light organ colonization of the squid Euprymna scolopes by the bacterium Vibrio fischeri. During embryogenesis, three pairs of invaginations form sequentially on the organ’s surface, producing pores that lead to interior compressed tubules at different stages of development. After hatching, these areas expand, allowing V. fischeri cells to enter and migrate ∼120 μm through three anatomically distinct regions before reaching blind-ended crypt spaces. A dynamic gatekeeper, or bottleneck, connects these crypts with the migration path. Once V. fischeri cells have entered the crypts, the bottlenecks narrow, and colonization by the symbiont population becomes spatially restricted. The actual timing of constriction and restriction varies with crypt maturity and with different V. fischeri strains. Subsequently, starting with the first dawn following colonization, the bottleneck controls a lifelong cycle of dawn-triggered expulsions of most of the symbionts into the environment and a subsequent regrowth in the crypts. Unlike other developmental phenotypes, bottleneck constriction is not induced by known microbe-associated molecular patterns (MAMPs) or by V. fischeri-produced bioluminescence, but it does require metabolically active symbionts. Further, while symbionts in the most mature crypts have a higher proportion of live cells and a greater likelihood of expulsion at dawn, they have a lower resistance to antibiotics. The overall dynamics of these distinct microenvironments reflect the complexity of the host-symbiont dialogue

The noncoding small RNA SsrA is released by Vibrio fischeri and modulates critical host responses.

The regulatory noncoding small RNAs (sRNAs) of bacteria are key elements influencing gene expression; however, there has been little evidence that beneficial bacteria use these molecules to communicate with their animal hosts. We report here that the bacterial sRNA SsrA plays an essential role in the light-organ symbiosis between Vibrio fischeri and the squid Euprymna scolopes. The symbionts load SsrA into outer membrane vesicles, which are transported specifically into the epithelial cells surrounding the symbiont population in the light organ. Although an SsrA-deletion mutant (ΔssrA) colonized the host to a normal level after 24 h, it produced only 2/10 the luminescence per bacterium, and its persistence began to decline by 48 h. The host's response to colonization by the ΔssrA strain was also abnormal: the epithelial cells underwent premature swelling, and host robustness was reduced. Most notably, when colonized by the ΔssrA strain, the light organ differentially up-regulated 10 genes, including several encoding heightened immune-function or antimicrobial activities. This study reveals the potential for a bacterial symbiont's sRNAs not only to control its own activities but also to trigger critical responses promoting homeostasis in its host. In the absence of this communication, there are dramatic fitness consequences for both partners

Maturation state of colonization sites promotes symbiotic resiliency in the Euprymna scolopes-Vibrio fischeri partnership

Abstract Background Many animals and plants acquire their coevolved symbiotic partners shortly post-embryonic development. Thus, during embryogenesis, cellular features must be developed that will promote both symbiont colonization of the appropriate tissues, as well as persistence at those sites. While variation in the degree of maturation occurs in newborn tissues, little is unknown about how this variation influences the establishment and persistence of host-microbe associations. Results The binary symbiosis model, the squid-vibrio (Euprymna scolopes-Vibrio fischeri) system, offers a way to study how an environmental gram-negative bacterium establishes a beneficial, persistent, extracellular colonization of an animal host. Here, we show that bacterial symbionts occupy six different colonization sites in the light-emitting organ of the host that have both distinct morphologies and responses to antibiotic treatment. Vibrio fischeri was most resilient to antibiotic disturbance when contained within the smallest and least mature colonization sites. We show that this variability in crypt development at the time of hatching allows the immature sites to act as a symbiont reservoir that has the potential to reseed the more mature sites in the host organ when they have been cleared by antibiotic treatment. This strategy may produce an ecologically significant resiliency to the association. Conclusions The data presented here provide evidence that the evolution of the squid-vibrio association has been selected for a nascent organ with a range of host tissue maturity at the onset of symbiosis. The resulting variation in physical and chemical environments results in a spectrum of host-symbiont interactions, notably, variation in susceptibility to environmental disturbance. This “insurance policy” provides resiliency to the symbiosis during the critical period of its early development. While differences in tissue maturity at birth have been documented in other animals, such as along the infant gut tract of mammals, the impact of this variation on host-microbiome interactions has not been studied. Because a wide variety of symbiosis characters are highly conserved over animal evolution, studies of the squid-vibrio association have the promise of providing insights into basic strategies that ensure successful bacterial passage between hosts in horizontally transmitted symbioses. Video Abstrac

Additional file 7: of Molt-dependent transcriptomic analysis of cement proteins in the barnacle Amphibalanus amphitrite

Detailed information about protein identifications from the various secreted samples. (XLSX 117 kb

Oxidase Activity of the Barnacle Adhesive Interface Involves Peroxide-Dependent Catechol Oxidase and Lysyl Oxidase Enzymes

Oxidases are found to play a growing role in providing functional chemistry to marine adhesives for the permanent attachment of macrofouling organisms. Here, we demonstrate active peroxidase and lysyl oxidase enzymes in the adhesive layer of adult Amphibalanus amphitrite barnacles through live staining, proteomic analysis, and competitive enzyme assays on isolated cement. A novel full-length peroxinectin (AaPxt-1) secreted by barnacles is largely responsible for oxidizing phenolic chemistries; AaPxt-1 is driven by native hydrogen peroxide in the adhesive and oxidizes phenolic substrates typically preferred by phenoloxidases (POX) such as laccase and tyrosinase. A major cement protein component AaCP43 is found to contain ketone/aldehyde modifications via 2,4-dinitrophenylhydrazine (DNPH) derivatization, also called Brady’s reagent, of cement proteins and immunoblotting with an anti-DNPH antibody. Our work outlines the landscape of molt-related oxidative pathways exposed to barnacle cement proteins, where ketone- and aldehyde-forming oxidases use peroxide intermediates to modify major cement components such as AaCP43