We are grateful to Garth Ilsley for his support in establishing the culture system. We acknowledge Ritsuko Suyama&#8217;s and Sylvain Guillot&#8217;s contributions to early sampling and species identification efforts. Special thanks are due to Hiroki Nishida, Takeshi Onuma, and Tatsuya Omotezako for their generous support and guidance throughout, including initial establishment of the local culturing system and sharing animals and microalgal culture. We also thank Daniel Chourrout, Jean-Marie Bouquet, Anne Aasjord, Cristian Ca&#241;estro, and Alfonso Ferr&#225;ndez-Rold&#225;n for sharing their expertise on sampling and culturing. Jai Denton, Charles Plessy, and Jeffrey Jolly provided invaluable feedback on the manuscript. Charlotte West formulated a generalized equation for algal calculation. Finally, we thank OIST for funding, Mary Collins and the OIST Fieldwork Safety Committee for advice on safe sampling procedures, the staff of OIST machine shop for the construction of culturing and sampling equipment, and Koichi Toda for delivering seawater.

The author has nothing to declare.

To facilitate flexibility in the establishment of O. dioica culture, it is important to understand the animals&#8217; natural habitat. Seasonal data provides information about the ranges of physical parameters, which can be used to guide laboratory culturing conditions. It also helps in understanding seasonal fluctuations in the abundance of animals. In Okinawa, O. dioica is most reliably found from June to October. However, in Tokyo bay, populations peak in February and October41. Although culturing of O. dioica is often reported at 20 &#176;C or lower27,28,29, Okinawan O. dioica shows better survival at temperatures above 20 &#176;C; this might be explained by the fact the minimum surface seawater temperature in Okinawa is ~20 &#176;C (Figure 6). The abundance of O. dioica might also be influenced by phytoplankton blooms42 and predator abundance43,44. Regardless of where O. dioica are collected, understanding the seasonality of local populations maximizes the chance of sampling and culturing success.
Given the appropriate season and location, net sampling is an effective way of collecting large numbers of Oikopleura with minimal effort. Plankton nets with smaller mesh size (60-70 &#181;m) may also be used to collect all stages of the animals. Fully mature animals are rarely found in the net, perhaps due to their fragility at the end of the lifecycle. Therefore, species identification followed by sampling is achieved by microscopic observation of subchordal cells. Mature individuals usually appear one or two days post-sampling as animals continue to grow in the laboratory. Although net sampling is efficient, alternative sampling methods might be necessary in different circumstances. For example, net sampling near urban areas can collect large numbers of phytoplankton, making it difficult to isolate Oikopleura. In such cases, simple bucket sampling to collect surface seawater or boat sampling from areas beyond the harbor are recommended. The results showed that the gradual change in salinity due to consecutive days of rain did not affect the abundance of O. dioica; however, shore sampling immediately after extreme weather events such as tropical cyclones should be avoided. These events cause sudden and drastic biogeochemical changes in a sheltered body of water45,46. The stormwater runoff may carry pollutants, sediments, and excess nutrients, which increase turbidity and lower water quality47. Filter-feeding plankton, such as Oikopleura, may be especially susceptible to these changes due to their mode of feeding and limited mobility. In such a circumstance, we recommend postponing sampling for a few days until the local conditions return to normal.
The introduction of a multi-step filter system is essential to maintain small, filter-feeding organisms such as O. dioica. Using poorly filtered seawater (for example, a 25 &#181;m mesh in the previous culture system), the culture was often unstable especially during summer, potentially due to the higher abundance of phytoplankton. Although some phytoplankton are beneficial to O. dioica growth, others produce biotoxins that can cause abnormal development of O. dioica embryos48. In addition, a high concentration of diatoms such as Chaetoceros spp. are potentially harmful to O. dioica growth as they can possess long setae which can clog the house and prevent efficient feeding49. We frequently observed houses of small animals being clogged by C. calcitrans setae; therefore, we now feed C. calcitrans only to animals at Day 2 and older (Table 3).
Although it was not a problem here, small scale long-term culturing of O. dioica can experience sudden drops in population size due to a genetic bottleneck; in such cases, Mart&#237;-Solans et al.29 recommend adding new wild individuals to the culture every 20 generations.
The Oikopleura culture system is flexible. A stable culture can be established within a week. Long-term culturing of O. dioica is possible on a modest budget with non-specialist equipment. The daily effort required for the maintenance of 5-10 beakers of Oikopleura is generally less than 2 hours with 2 people. O. dioica can also be maintained in artificial seawater, which is beneficial to those without access to natural seawater28. Long-term storage of algal food is possible using solid-culture and cryopreservation29. Moreover, O. dioica sperm can be cryopreserved, and remain viable for more than a year50. All of these factors mean that cultures can be easily re-established. Finally, past experience with accidental culturing of Pleurobrachia sp. may suggest that the culturing system developed for Oikopleura could potentially be extended to a broader community of fragile pelagic organisms.
O. dioica continues to provide powerful insights into various biological fields. An understanding of local seasonality, a meticulous culture system, and a few dedicated individuals allow effective culture to be established with little effort. Oikopleura culture system provides the baseline resources to investigate a wide range of biological fields relating to ecology, development, genomics, and evolution of this unique marine chordate.

Model organisms have been instrumental in addressing many biological questions including those relating to development, genetics, and physiology. Moreover, additional model organisms facilitate new discoveries and therefore are crucial to achieve a greater understanding of nature1,2. Marine zooplankton are diverse groups of organisms that play an important role in ocean ecosystems3,4,5,6. Despite their abundance and ecological importance, gelatinous organisms such as planktonic tunicates are often under-represented in plankton biodiversity studies because their transparency and fragility make field collection and identification challenging7,8. Adapted sampling techniques and laboratory culturing allow closer observation of the animals in vitro, which has furthered the knowledge in the biology of planktonic tunicates9,10,11,12.
Larvaceans (Appendicularians) are a class of free-swimming marine tunicates comprising around 70 described species worldwide8,13. As they are one of the most abundant groups within zooplankton communities14,15,16,17, larvaceans represent a primary food source for larger planktonic organisms such as fish larvae18,19. Unlike ascidians-the sessile tunicates-larvaceans retain a tadpole-like morphology and remain planktonic throughout their lives20. Each animal lives inside a self-built, intricate filter-feeding structure known as a house. They accumulate particulates in their houses by creating water currents through the undulating motion of their tails21. Clogged houses are discarded throughout the day, some of which form carbon aggregates and eventually sink to the seabed22; thus, larvaceans play a major role in global carbon flux23. Most species are reported to live in the pelagic zone within the upper 100 m of the water column13; however, the giant larvacean Bathochordaeus is known to inhabit the depths of 300 m24. A study on Bathochordaeus in Monterey Bay, California revealed that the animals also serve as a biological vector of microplastics, suggesting a potential importance in understanding the role of appendicularians in the vertical transport and distribution of microplastics in the oceans25.
Oikopleura dioica, a species of larvacean, has attracted attention in recent years as a model organism due to several remarkable characteristics. It is commonly reported throughout the world&#8217;s oceans. It is especially abundant in coastal waters26, which allows easy sampling from the shore. Long-term, stable culturing is possible with both natural and artificial seawater27,28,29. Temperature dependent generation times are as short as 4-9 days in laboratory conditions. It has high fecundity with each female capable of producing &#62;300 eggs throughout the year. As a tunicate, it occupies an important phylogenetic position for understanding chordate evolution30,31. At 70 Mb, O. dioica has the smallest identified genome amongst all chordates32. Among larvaceans, O. dioica is the only described non-hermaphroditic species thus far33.
The first successful O. dioica culture with laboratory grown microalgae was reported by Paffenho&#776;fer34. The original culture protocol using synchronous motors and paddles was developed by Fenaux and Gorsky35 and later adopted by multiple laboratories. More recently, Fujii et al.36 reported O. dioica culturing in artificial seawater, a robust culture system and field collection were described by Bouquet et al.27 and an optimized protocol for a simplified, affordable system was reported by Marti-Solans et al.29. Aside from the traditional Oikopleura culture system, a newly reported design with a double tube rearing tank also has the potential to culture Oikopleura sp.37.
We present a detailed protocol for initiating an O. dioica monoculture based on a combination of protocols developed by major Oikopleura research groups at the Sars International Centre for Marine Molecular Biology27, the University of Barcelona29, Osaka University28, and our own observations. In previously published culture protocols, detailed information regarding the composition of algal media, shore sampling techniques, and Oikopleura identification were only roughly described, leaving a lot of ambiguity. Here, with the aid of visual information in the video protocol, we have assembled all the critical information needed to set up an O. dioica culture from the ground up in a straightforward, step-by-step manner. We describe how to distinguish O. dioica from another commonly reported species, O. longicauda, which is one of the most challenging steps. Although the existing culture systems are applicable for the cultivation of O. dioica worldwide, we highlight the importance of protocol adjustment based on local environmental conditions. The presented information combines widely published data as well as knowledge gained through experience. The current protocol is ideally suited for researchers interested in establishing a culture from scratch.

<table><tbody><tr><td>Activated charcoal</td><td>Sigma</td><td>C2764-2.5KG</td><td></td></tr><tr><td>Alluminum pulley</td><td>Rainbow Products</td><td>10604-10607</td><td></td></tr><tr><td>Biotin</td><td>Sigma</td><td>B4501-100MG</td><td></td></tr><tr><td>Boric acid</td><td>Wako</td><td>021-02195</td><td></td></tr><tr><td>Cobalamin (B12)</td><td>Sigma</td><td>V2876-100MG</td><td></td></tr><tr><td>Cobalt(II) chloride hexahydrate</td><td>Wako</td><td>036-03682</td><td></td></tr><tr><td>Copper(II) sulfate pentahydrate</td><td>Wako</td><td>039-04412</td><td></td></tr><tr><td>Disodium edetate hydrate</td><td>Wako</td><td>044-29525</td><td></td></tr><tr><td>Hexaammonium heptamolybdate tetrahydrate</td><td>Wako</td><td>019-03212</td><td></td></tr><tr><td>Hexagon wrench</td><td>Anex</td><td>No.6600</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Wako</td><td>080-01066</td><td></td></tr><tr><td>Iron(III) chloride hexahydrate</td><td>Wako</td><td>091-00872</td><td></td></tr><tr><td>Jebao programmable auto dosing pump</td><td>Jebao</td><td>DP-4</td><td></td></tr><tr><td>Magnet pump</td><td>REI-SEA</td><td>RMD-201</td><td></td></tr><tr><td>Manganese(II) chloride tetrahydrate</td><td>Wako</td><td>134-15302</td><td></td></tr><tr><td>Polypropylene wound cartridge filter</td><td>Advantec</td><td>TCW-10N-PPS</td><td></td></tr><tr><td></td><td></td><td>TCW-5N-PPS</td><td></td></tr><tr><td></td><td></td><td>TCW-1N-PPS</td><td></td></tr><tr><td>Screwless terminal block</td><td>SATO PARTS</td><td>SL4500</td><td></td></tr><tr><td>Simple plankton net</td><td>RIGO, Japan</td><td>5512-C</td><td></td></tr><tr><td>Sodium metasilicate</td><td>Sigma</td><td>307815-1KG</td><td></td></tr><tr><td>Sodium nitrate</td><td>Wako</td><td>195-02545</td><td></td></tr><tr><td>Sodium phosphate monobasic anhydrous</td><td>MP Biomedicals</td><td>194740</td><td></td></tr><tr><td>Streptomycin sulfate salt</td><td>Sigma</td><td>S6501-25G</td><td></td></tr><tr><td>Synchronous electric motor</td><td>Servo</td><td>D5N6Z15M</td><td></td></tr><tr><td>Thiamin hydrochloride</td><td>Wako</td><td>201-00852</td><td></td></tr><tr><td>UV sterilizer</td><td>Iwaki</td><td>UVF-1000</td><td></td></tr><tr><td>Zinc chloride</td><td>MP Biomedicals</td><td>194858</td><td></td></tr></tbody></table>

streamlined sampling and cultivation of the pelagic cosmopolitan larvacean, oikopleura dioica

Oikopleura dioica is a planktonic chordate with exceptional filter-feeding ability, rapid generation time, conserved early development, and a compact genome. For these reasons, it is considered a useful model organism for marine ecological studies, evolutionary developmental biology, and genomics. As research often requires a steady supply of animal resources, it is useful to establish a reliable, low-maintenance culture system. Here we describe a step-by-step method for establishing an O. dioica culture. We describe how to select potential sampling sites, collection methods, target animal identification, and the set-up of the culturing system. We provide troubleshooting advice based on our own experiences. We also highlight critical factors that help sustain a robust culture system. Although the culture protocol provided here is optimized for O. dioica, we hope our sampling technique and culture setup will inspire new ideas for maintaining other fragile pelagic invertebrates.

Watch this Scientific Journal Video about Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica at JoVE.com

Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica

Oikopleura dioica is a tunicate model organism in various fields of biology. We describe sampling methods, species identification, culturing setup, and culturing protocols for the animals and algal feed. We highlight key factors that helped strengthen the culture system and discuss the possible problems and resolutions.

Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica

Oikopleura dioica is a planktonic chordate with exceptional filter-feeding ability, rapid generation time, conserved early development, and a compact ...

streamlined-sampling-cultivation-pelagic-cosmopolitan-larvacean

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

8.8K Views.  Okinawa Institute of Science and Technology Graduate University. Oikopleura dioica is a tunicate model organism in various fields of biology. We describe sampling methods, species identification, culturing setup, and culturing protocols for the animals and algal feed. We highlight key factors that helped strengthen the culture system and discuss the possible problems and resolutions.

Video: Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica

Integrated Field Lysimetry and Porewater Sampling for Evaluation of Chemical Mobility in Soils and Established Vegetation

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals is often not well understood. Here we demonstrate an integrated field lysimetry and porewater sampling method for evaluating the mobility of chemicals applied to soils and established vegetation. Lysimeters, open columns made of metal or plastic, are driven into bareground or vegetated soils. Porewater samplers, which are commercially available and use vacuum to collect percolating soil water, are installed at predetermined depths within the lysimeters. At prearranged times following chemical application to experimental plots, porewater is collected, and lysimeters, containing soil and vegetation, are exhumed. By analyzing chemical concentrations in the lysimeter soil, vegetation, and porewater, downward leaching rates, soil retention capacities, and plant uptake for the chemical of interest may be quantified. Because field lysimetry and porewater sampling are conducted under natural environmental conditions and with minimal soil disturbance, derived results project real-case scenarios and provide valuable information for chemical management. As chemicals are increasingly applied to land worldwide, the described techniques may be utilized to determine whether applied chemicals pose adverse effects to human health or the environment.

Field lysimetry and porewater sampling allow researchers to evaluate the fate of chemicals applied to soils and established vegetation. The goal of this protocol is to demonstrate how to install required instrumentation and collect samples for chemical analysis during integrated field lysimetry and porewater sampling experiments.

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals ...

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

An Ultra-clean Multilayer Apparatus for Collecting Size Fractionated Marine Plankton and Suspended Particles

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those of suspended/sinking particles. Here, we present an all plastic (Polypropylene and Polycarbonate), multi-layer filtration system for collection of suspended particulate matter (SPM) at sea. This ultra-clean sampling device has been designed and developed specifically for trace element studies. Meticulous selection of all non-metallic materials and utilization of an in-line flow-through procedure minimizes any possible metal contamination during sampling. This system has been successfully tested and tweaked for determining trace metals (e.g., Fe, Al, Mn, Cd, Cu, Ni) on particles of varying size in coastal and open ocean waters. Results from the South China Sea at the South East Asia Time-Series (SEATS) station indicate that diurnal variations and spatial distribution of plankton in the euphotic zone can be easily resolved and recognized. Chemical analysis of size-fractionated particles in surface waters of the Taiwan Strait suggests that the larger particles (&#62;153 &#181;m) were mostly biologically derived, while the smaller particles (10 - 63 &#181;m) were mostly composed of inorganic matter. Apart from Cd, the concentrations of metals (Fe, Al, Mn, Cu, Ni) decreased with increasing size.

Plankton and suspended particles play a major role in the biogeochemical cycles in the ocean. Here, we provide an ultra-clean, low stress method for the collection of various sizes of particles and plankton at sea with the capability of handling large volumes of seawater.

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those ...

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Immunology and Infection

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri (Uljanin 1884) for Experimental Studies

Gelatinous zooplanktons play a crucial role in ocean ecosystems. However, it is generally difficult to investigate their physiology, growth, fecundity, and trophic interactions primarily due to methodological challenges, including the ability to culture them. This is particularly true for the doliolid, Dolioletta gegenbauri. D. gegenbauri commonly occurs in productive subtropical continental shelf systems worldwide, often at bloom concentrations capable of consuming a large fraction of daily primary production. In this study, we describe cultivation approaches for collecting, rearing, and maintaining D. gegenbauri for the purpose of conducting laboratory-based studies. D. gegenbauri and other doliolid species can be captured live using obliquely towed conical 202 &micro;m mesh plankton nets from a drifting ship. Cultures are most reliably established when water temperatures are below 21 &deg;C and are started from immature gonozooids, maturing phorozooids, and large nurses. Cultures can be maintained in rounded culture vessels on a slowly rotating plankton wheel and sustained on a diet of cultured algae in natural seawater for many generations. In addition to the ability to establish laboratory cultures of D. gegenbauri, we demonstrate that the collection condition, algae concentration, temperature, and exposure to naturally conditioned seawater are all critical to the culture establishment, growth, survival, and reproduction of D. gegenbauri.

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri Uljanin 1884 for Experimental Studies

Doliolids, including the species Dolioletta gegenbauri, are small gelatinous marine zooplankton of ecological significance found on productive subcontinental shelf systems worldwide. The difficulty of culturing these delicate organisms limits their investigation. In this study, we describe cultivation approaches for collecting, rearing, and maintaining the doliolid Dolioletta gegenbauri.

Gelatinous zooplanktons play a crucial role in ocean ecosystems. However, it is generally difficult to investigate their physiology, growth, fecundity, ...

Field Collection and Laboratory Maintenance of Canopy-Forming Giant Kelp to Facilitate Restoration

Canopy-forming kelps are essential foundation species, supporting biodiversity and providing ecosystem services valued at more than USD$500 billion annually. The global decline of giant kelp forests due to climate-driven ecological stressors underscores the need for innovative restoration strategies. An emerging restoration technique known as &#39;green gravel&#39; aims to seed young kelps over large areas without extensive underwater labor and represents a promising restoration tool due to cost-effectiveness and scalability. This video article illustrates a protocol and tools for culturing giant kelp, Macrocystis pyrifera. It also provides a resource for further studies to address the successes and limitations of this method in field settings. We outline field and laboratory-based methods for collecting reproductive tissue, sporulating, inoculating, rearing, maintaining, and monitoring substrates seeded with early life stages using the &#39;green gravel&#39;&#160;technique. The protocol simplifies and centralizes current restoration practices in this field to support researchers, managers, and stakeholders in meeting kelp conservation objectives.

This protocol describes the field collection and regular laboratory maintenance of substrates seeded with canopy-forming giant kelp for use in restoration trials to address the success and limitations of the 'green gravel' technique in field settings.

Canopy-forming kelps are essential foundation species, supporting biodiversity and providing ecosystem services valued at more than USD$500 billion ...

Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter 1. These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms 2.
Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling 3 and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms 4, 2. Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web 5. Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism 6, 7. Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited 8, 4, 9, 10, 5. A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle.
We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity 4, 11, as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms.
RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs 12. In this study, we applied a radioisotope assay modified for filtered samples 13 to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.

Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a ...

Biology

Ex Situ Culture of Pocillopora acuta and Effective Feeding Techniques

Effective Techniques for the Feeding and Ex Situ Culture of a Brooding Scleractinian Coral, Pocillopora acuta

Climate change is affecting the survival, growth, and recruitment of corals globally, with large-scale shifts in abundance and community composition expected in reef ecosystems over the next several decades. Recognition of this reef degradation has prompted a range of novel research- and restoration-based active interventions. Ex situ aquaculture can play a supporting role through the establishment of robust coral culture protocols (e.g., to improve health and reproduction in long-term experiments) and through the provision of a consistent broodstock supply (e.g., for use in restoration projects). Here, simple techniques for the feeding and ex situ culture of brooding scleractinian corals are outlined using the common and well-studied coral, Pocillopora acuta, as an example. To demonstrate this approach, coral colonies were exposed to different temperatures (24 &#176;C vs. 28 &#176;C) and feeding treatments (fed vs. unfed) and the reproductive output and timing, as well as the feasibility of feeding Artemia nauplii to corals at both temperatures, was compared. Reproductive output showed high variation across colonies, with differing trends observed between the temperature treatments; at 24 &#176;C, fed colonies produced more larvae than unfed colonies, but the opposite was found in colonies cultured at 28 &#176;C. All colonies reproduced before the full moon, and differences in reproductive timing were only found between unfed colonies in the 28 &#176;C treatment and fed colonies in the 24 &#176;C treatment (mean lunar day of reproduction &#177; standard deviation: 6.5 &#177; 2.5 and 11.1 &#177; 2.6, respectively). The coral colonies fed efficiently on Artemia nauplii at both treatment temperatures. These proposed feeding and culture techniques focus on the reduction of coral stress and the promotion of reproductive longevity in a cost-effective and customizable manner, with versatile applicability in both flow-through and recirculating aquaculture systems.

Author Spotlight: Advancing Coral Research by Exploring Climate Change Resistance, Ex Situ Aquaculture, and Reproduction Strategies 

Climate change is impacting coral reef ecosystems globally. Corals sourced from ex situ aquaculture systems can help support restoration and research efforts. Herein, feeding and coral culture techniques that can be used to promote the long-term maintenance of brooding scleractinian corals ex situ are outlined.

Climate change is affecting the survival, growth, and recruitment of corals globally, with large-scale shifts in abundance and community composition ...

Culturing Larvae of Crepidula fornicata in Laboratory and Field Conditions

Laboratory and Field Culture of Larvae of The Slipper Limpet, Crepidula fornicata

The calyptraeid gastropod mollusk, Crepidula fornicata, has been widely used for studies of larval developmental biology, physiology, and ecology. Brooded veliger larvae of this species were collected by siphoning onto a sieve after natural release by adults, distributed into the culture at a density of 200/L, and fed with Isochrysis galbana (strain T-ISO) at 1 x 105 cells/mL. Shell growth and acquisition of competence for metamorphosis were documented for sibling larvae reared in ventilated 800 mL cultures designed for equilibration to ambient air or to defined atmospheric gas mixtures. Contrasting with these laboratory culture conditions; growth and competence data were also collected for larvae reared in a 15 L flow-through ambient seawater mesocosm located in a field population of reproductive adults. Growth rates and timing of metamorphic competence in the laboratory cultures were similar to those reported in previously published studies. Larvae reared in the field mesocosm grew much faster and metamorphosed sooner than reported for any laboratory studies. Together, these methods are suited for exploring larval development under predetermined controlled conditions in the laboratory as well as under naturally occurring conditions in the field.

Author Spotlight: Bridging the Gap Between Field Observations and Lab Manipulations in Larval Ecology Research

The paper presents methods for larval culture of the gastropod Crepidula fornicata in a small-volume laboratory-scale system and in an ambient-seawater mesocosm system that can be deployed in the field.

The calyptraeid gastropod mollusk, Crepidula fornicata, has been widely used for studies of larval developmental biology, physiology, and ecology. Brooded ...

Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica

Summary

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Effective Techniques for the Feeding and Ex Situ Culture of a Brooding Scleractinian Coral, Pocillopora acuta

Laboratory and Field Culture of Larvae of The Slipper Limpet, Crepidula fornicata