Chitin utilization by marine picocyanobacteria and the evolution of a planktonic lifestyle.

Marine picocyanobacteria Prochlorococcus and Synechococcus, the most abundant photosynthetic cells in the oceans, are generally thought to have a primarily single-celled and free-living lifestyle. However, while studying the ability of picocyanobacteria to supplement photosynthetic carbon fixation with the use of exogenous organic carbon, we found the widespread occurrence of genes for breaking down chitin, an abundant source of organic carbon that exists primarily as particles. We show that cells that encode a chitin degradation pathway display chitin degradation activity, attach to chitin particles, and show enhanced growth under low light conditions when exposed to chitosan, a partially deacetylated soluble form of chitin. Marine chitin is largely derived from arthropods, which underwent major diversifications 520 to 535 Mya, close to when marine picocyanobacteria are inferred to have appeared in the ocean. Phylogenetic analyses confirm that the chitin utilization trait was acquired at the root of marine picocyanobacteria. Together this leads us to postulate that attachment to chitin particles allowed benthic cyanobacteria to emulate their mat-based lifestyle in the water column, initiating their expansion into the open ocean, seeding the rise of modern marine ecosystems. Subsequently, transitioning to a constitutive planktonic life without chitin associations led to cellular and genomic streamlining along a major early branch within Prochlorococcus. Our work highlights how the emergence of associations between organisms from different trophic levels, and their coevolution, creates opportunities for colonizing new environments. In this view, the rise of ecological complexity and the expansion of the biosphere are deeply intertwined processes.

Shifts in predator behaviour following climate induced disturbance on coral reefs.

Coral reefs are increasingly ecologically destabilized across the globe due to climate change. Behavioural plasticity in corallivore behaviour and short-term trophic ecology in response to bleaching events may influence the extent and severity of coral bleaching and subsequent recovery potential, yet our understanding of these interactions in situ remains unclear. Here, we investigated interactions between corallivory and coral bleaching during a severe high thermal event (10.3-degree heating weeks) in Belize. We found that parrotfish changed their grazing behaviour in response to bleaching by selectively avoiding bleached Orbicella spp. colonies regardless of bleaching severity or coral size. For bleached corals, we hypothesize that this short-term respite from corallivory may temporarily buffer coral energy budgets by not redirecting energetic resources to wound healing, and may therefore enable compensatory nutrient acquisition. However, colonies that had previously been heavily grazed were also more susceptible to bleaching, which is likely to increase mortality risk. Thus, short-term respite from corallivory during bleaching may not be sufficient to functionally rescue corals during prolonged bleaching. Such pairwise interactions and behavioural shifts in response to disturbance may appear small scale and short term, but have the potential to fundamentally alter ecological outcomes, especially in already-degraded ecosystems that are vulnerable and sensitive to change.

Chitin degradation by Synechococcus WH7803.

Chitin is an abundant, carbon-rich polymer in the marine environment. Chitinase activity has been detected in spent media of Synechococcus WH7803 cultures-yet it was unclear which specific enzymes were involved. Here we delivered a CRISPR tool into the cells via electroporation to generate loss-of-function mutants of putative candidates and identified ChiA as the enzyme required for the activity detected in the wild type.

Filter Plating Method for Rendering Picocyanobacteria Cultures Free of Heterotrophic Bacterial Contaminants and Clonal.

Isolates of the marine picocyanobacteria, Prochlorococcus and Synechococcus, are often accompanied by diverse heterotrophic "contaminating" bacteria, which can act as confounding variables in otherwise controlled experiments. Traditional microbiological methods for eliminating contaminants, such as direct streak-plating, are often unsuccessful with this particular group of microorganisms. While they will grow in pour plates, colonies often remain contaminated with heterotrophic bacteria that can migrate through the soft agar. Additionally, axenic clones of picocyanobacteria can be recovered via dilution-to-extinction in liquid medium, but the efficiency of recovery is low, often requiring large numbers of 96-well plates. Here, we detail a simple and effective protocol for rendering cultures of Synechococcus and Prochlorococcus strains free of bacterial contaminants while at the same time yielding clonal isolates. We build on the fact that co-culture with specific heterotrophs-"helper heterotrophs"-is often necessary to grow colonies of picocyanobacteria from single cells in agar. Suspecting that direct physical contact between the helper and the picocyanobacterial cells was not necessary for the "helper effect," we developed a protocol in which the helper cells are embedded in soft agar pour plates, a filter overlaid on the surface, and a picocyanobacterial culture is diluted and then spotted on top of the filter. With this approach, motile contaminants cannot swim to the colonies, and it is possible to obtain the expected number of colonies from a given input (i.e., a Poisson distribution of colonies with an expected value equal to the input number of cells), thus ensuring clonal colonies. Using this protocol, we rendered three strains of Synechococcus, two strains of Prochlorococcus, and 19 new strains of Synechococcus from coastal seawater clonal and free of heterotrophic bacteria. The simplicity of this approach should expand the repertoire of axenic picocyanobacterial strains available for controlled physiological experiments. It will also enable the study of microdiversity in populations of picocyanobacteria by facilitating large-scale isolation of picocyanobacterial clones from a single source, including direct isolation from natural seawater.

High-Precision Stereolithography of Biomicrofluidic Devices.

Stereolithography (SL) is emerging as an attractive alternative to soft lithography for fabricating microfluidic devices due to its low cost and high design efficiency. Low molecular weight poly(ethylene glycol)diacrylate (MW = 258) (PEG-DA-258) has been used for SL 3D-printing of biocompatible microdevices at submillimeter resolution. However, 3D-printing resins that simultaneously feature high transparency, high biocompatibility, and high resolution are still lacking. It is found that photosensitizer isopropyl thioxanthone can, in a concentration-dependent manner, increase the absorbance of the resin (containing PEG-DA-258 and photoinitator Irgacure-819) by over an order of magnitude. This increase in absorbance allows for SL printing of microdevices at sub pixel resolution with commercially available desktop printers and without compromising transparency or biocompatibility. The assembly-free, rapid (<15 h) 3D-printing of a variety of complex 3D microfluidic devices such as a 3D-fluid router, a passive chaotic micro-mixer, an active micro-mixer with pneumatic microvalves, and high-aspect ratio (37:1) microchannels of single pixel width is demonstrated. These manufacturing capabilities are unavailable in conventional microfluidic rapid prototyping techniques. The low absorption of small hydrophobic molecules and microfluidic labeling of cultured mammalian cells in 3D-printed PEG-DA-258 microdevices is demonstrated, indicating the potential of PEG-DA-based fabrication of cell-based assays, drug discovery, and organ-on-chip platforms.

A Laser-Engraving Technique for Portable Micropneumatic Oscillators.

Microfluidic automation technology is at a stage where the complexity and cost of external hardware control often impose severe limitations on the size and functionality of microfluidic systems. Developments in autonomous microfluidics are intended to eliminate off-chip controls to enable scalable systems. Timing is a fundamental component of the digital logic required to manipulate fluidic flow. The authors present a self-driven pneumatic ring oscillator manufactured by assembling an elastomeric sheet of polydimethylsiloxane (PDMS) between two laser-engraved polymethylmethacrylate (PMMA) layers via surface activation through treatment with 3-aminopropyltriethoxysilane (APTES). The frequency of the fabricated oscillators is in the range of 3⁻7.5 Hz with a maximum of 14 min constant frequency syringe-powered operation. The control of a fluidic channel with the oscillator stages is demonstrated. The fabrication process represents an improvement in manufacturability compared to previous molding or etching approaches, and the resulting devices are inexpensive and portable, making the technology potentially applicable for wider use.

Digital Manufacturing of Selective Porous Barriers in Microchannels Using Multi-Material Stereolithography.

We have developed a sequential stereolithographic co-printing process using two different resins for fabricating porous barriers in microfluidic devices. We 3D-printed microfluidic channels with a resin made of poly(ethylene glycol) diacrylate (MW = 258) (PEG-DA-258), a UV photoinitiator, and a UV sensitizer. The porous barriers were created within the microchannels in a different resin made of either PEG-DA (MW = 575) (PEG-DA-575) or 40% (w/w in water) PEG-DA (MW = 700) (40% PEG-DA-700). We showed selective hydrogen ion diffusion across a 3D-printed PEG-DA-575 porous barrier in a cross-channel diffusion chip by observing color changes in phenol red, a pH indicator. We also demonstrated the diffusion of fluorescein across a 3D-printed 40% PEG-DA-700 porous barrier in a symmetric-channel diffusion chip by measuring fluorescence intensity changes across the porous barrier. Creating microfluidic chips with integrated porous barriers using a semi-automated 3D printing process shortens the design and processing time, avoids assembly and bonding complications, and reduces manufacturing costs compared to micromolding processes. We believe that our digital manufacturing method for fabricating selective porous barriers provides an inexpensive, simple, convenient and reproducible route to molecule delivery in the fields of molecular filtration and cell-based microdevices.