Hydractinia symbiolongicarpus is an emerging model organism in which cutting-edge genomic tools and resources are being developed for use in a growing number of research fields. One limitation of this model system is the lack of long-term storage for genetic resources. The goal of this study was to establish a generalizable cryopreservation approach for Hydractinia that would support future repository development for other cnidarian species. Specific objectives were to: (1) characterize basic parameters related to sperm quality; (2) develop a generalizable approach for sperm collection; (3) assess the feasibility of in vitro fertilization (IVF) with sperm after refrigerated storage; (4) assess the feasibility of IVF with sperm cryopreserved with various sperm concentrations; (5) evaluate feasibility of cryopreservation with various freezing conditions, and (6) explore the feasibility of cryopreservation by use of a 3-D printed open-hardware (CryoKit) device. Animal husbandry and sperm collection were facilitated by use of 3-D printed open hardware. Hydractinia sperm at a concentration of 2 × 10⁷ cells/mL stored at 4 °C for 6 d were able to achieve 50% fertilization rate. It appeared that relatively higher sperm concentration (>5 × 10⁷ cells/mL) for cryopreservation could promote fertilization. A fertilization rate of 41–69% was observed using sperm equilibrated with 5, 10, or 15% (v/v) cryoprotectant (dimethyl sulfoxide or methanol) for 20 min, cooled at a rate of 5, 10, or 20 °C/min from 4 °C to −80 °C, at a cell concentration of 10⁸/mL, in 0.25 mL French straws. Samples cryopreserved with the CryoKit produced a fertilization rate of 72–82%. Establishing repository capabilities for the Hydractinia research community will be essential for future development, maintenance, protection, and distribution of genetic resources. More broadly, these generalizable approaches can be used as a model to develop germplasm repositories for other cnidarian species.

Aquatic germplasm repositories can play a pivotal role in securing the genetic diversity of natural populations and agriculturally important aquatic species. However, existing technologies for repository development and operation face challenges in terms of accuracy, precision, efficiency, and cost-effectiveness, especially for microdevices used in gamete quality evaluation. Quality management is critical throughout genetic resource protection processes from sample collection to final usage. In this study, we examined the potential of using three-dimensional (3-D) stereolithography resin printing to address these challenges and evaluated the overall capabilities and limitations of a representative industrial 3-D resin printer with a price of US$18,000, a consumer-level printer with a price <US$700, and soft lithography, a conventional microfabrication method. A standardized test object, the Integrated Geometry Sampler (IGS), and a device with application in repository quality management, the Single-piece Sperm Counting Chamber (SSCC), were printed to determine capabilities and evaluate differences in targeted versus printed depths and heights. The IGS design had an array of negative and positive features with dimensions ranging from 1 mm to 0.02 mm in width and depth. The SSCC consisted of grid and wall features to facilitate cell counting. The SSCC was evaluated with polydimethylsiloxane (PDMS) devices cast from a typical photoresist and silicon mold. Fabrication quality was evaluated by optical profilometry for parameters such as dimensional accuracy, precision, and visual morphology. Fabrication time and cost were also evaluated. The precision, reliability, and surface quality of industrial-grade 3-D resin printing were satisfactory for operations requiring depths or heights larger than 0.1 mm due to a low discrepancy between targeted and measured dimensions across a range of 1 mm to 0.1 mm. Meanwhile, consumer-grade printers were suitable for microdevices with depths or heights larger than 0.2 mm. While the performance of either of these printers could be further optimized, their current capabilities, broad availability, low cost of operation, high throughput, and simplicity offer great promise for rapid development and widespread use of standardized microdevices for numerous applications, including gamete quality evaluation and "laboratory-on-a-chip" applications in support of aquatic germplasm repositories.

Carbonic anhydrases (CA; EC 4.2.1.1.) play a vital role in dissolved inorganic carbon (DIC) transport to photosynthetic microalgae residing in symbiotic cnidarians. The temperate sea anemone Anthopleura elegantissima can occur in three symbiotic states: hosting Breviolum muscatinei (brown), Elliptochloris marina (green) or without algal symbionts (aposymbiotic). This provides a basis for A. elegantissima to be a model for detailed studies of the role of CA in DIC transport. This study investigated the effects of symbiosis, body size, and light on CA activity and expression, and suggests that A. elegantissima has a heterotrophy-dominated trophic strategy. We identified putative A. elegantissima CA genes and performed phylogenetic analyses to infer subcellular localization in anemones. We performed experiments on field-collected anemones to compare (1) CA activity and expression from anemones in different symbiotic states, (2) CA activity in brown anemones as a function of size, and (3) CA activity in anemones of different symbiotic states that were exposed to different light intensities. Carbonic anhydrase activity in brown anemones was highest, whereas activity in green and aposymbiotic anemones was low. Several CAs had expression patterns that mirrored activity while another had expression that was inversely correlated to activity suggesting that symbionts may induce different DIC transport pathways. Finally, CA activity was inversely correlated with anemone size. Our results suggest that the observed CA activity and expression patterns are not only affected by symbiosis but also by other factors in the host physiology including trophic strategy as it relates to body size and cellular pH homeostasis.

The California sea hare (Aplysia californica) provides a powerful biomedical model system for studying aspects of neurological development and damage, behavior, aging, and hypoxia. Aplysia encapsulate their zygotes within strands that result in tangled egg masses that greatly complicate culture and experimentation. The historical and current importance of Aplysia for biomedical research and the mounting climate crisis necessitates protection of Aplysia genetic resources. The goal of this work was to prototype open-hardware sizing, processing, and packaging devices for A. californica early life stages suitable for integration into a cryopreservation pathway. The Strand Centi-Sizer was a low-cost, fused filament fabrication 3-D printable device that increased experiment preparation efficiency and standardized the cutting of egg strands customizable to user needs. A downstream system of 3-D printed devices was also prototyped to address inefficiencies in handling of egg strand sections for processing and packaging into existing cryopreservation straw platforms. Time studies were conducted comparing manual methods (i.e., no specialized equipment) with open hardware to demonstrate utility of the devices and to encourage community members to design and prototype new devices to address recurrent and novel problems in other aquatic animals that produce egg strands. Improvements in design could further increase efficiency, standardization, and reproducibility, and extend the application of these devices to other research communities, such as shrimp or salamander spermatophores, sea anemone body part (e.g., pedal lacerate) cryopreservation, or study areas such as vitrification.

Precise control of microscale object rotation is essential for numerous biomedical and microelectromechanical applications. For example, somatic cell nuclear transfer for aquatic biomedical models such as zebrafish faces significant technical challenges, particularly in egg trapping and alignment of an injection needle with the micropyle. In this study, we developed a 3D resin-printed microdevice to achieve frequency-selective electrorotation of dielectric microspheres using a quadrupole electrode configuration driven by phase-shifted alternating current (AC). Theoretical analysis based on the Clausius-Mossotti factor, which governs the polarization of a particle concerning its surrounding environment, highlights the critical role of its imaginary component in the induced dipole moment from the AC field that generates torque. Simulations conducted in COMSOL Multiphysics confirmed the formation of symmetric torque-driven rotation without significant micro-scale object translation. The frequency response of angular velocity exhibited a unimodal profile, with a peak near 4 MHz corresponding to maximum torque efficiency. Experimental validation using 700-µm polystyrene microspheres in Dulbecco's Phosphate Buffered Saline demonstrated consistent clockwise rotation, with a peak angular velocity of 8.1 Degrees/s observed at 900 kHz and 16 Vp-p. Although the experimental peak angular velocity occurred at a lower frequency than the theoretical maximum, the rotational trend followed the polarization relaxation behavior captured by Im[K cm ]. Parameter studies further revealed that increasing microscale object permittivity amplified torque generation, while higher medium permittivity reduced it, underscoring the tunability of electrorotation via dielectric properties. This work demonstrates a robust and scalable platform for manipulating large microscale objects. It lays the foundation for future applications involving biologically relevant objects, such as eggs of biomedical research models.

Carbonic anhydrase (CA) is an enzyme which catalyzes the interconversion of bicarbonate ions (HCO3-) and carbon dioxide (CO2). The activity levels of CA can be measured by taking advantage of the chemical reaction that it catalyzes. When CO2 is added to a tissue homogenate, the CA present converts the CO2 into HCO3- and a hydrogen ion (H+), which results in a decrease in pH. The rate at which the pH drops can be measured and converted into a CA activity mesaure. If there is no CA activity, the pH level decreases slowly and if there is a high CA activity, the pH decreases quickly.