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, 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 µm mesh plankton nets from a drifting ship. Cultures are most reliably established when water temperatures are below 21 °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.
Zooplankton account for the largest animal biomass in the ocean, are key components in marine food webs, and play important roles in ocean biogeochemical cycles1,2. Zooplankton, although comprised of a huge diversity of organisms, can be grossly distinguished into two categories: gelatinous and non-gelatinous with few intermediate taxa3,4. Compared to the non-gelatinous zooplankton, gelatinous zooplankton are especially difficult to study because of their complex life histories5, and their delicate tissues are easily damaged during capture and handling. Gelatinous zooplankton species are, therefore, notoriously difficult to culture in the laboratory and generally less studied compared to non-gelatinous species6.
Among gelatinous zooplankton groups, one abundant and of ecological importance in the world’s ocean are the Thaliaceans. Thaliaceans are a class of pelagic tunicates that include the orders Salpida, Pyrosomida, and Doliolida7. Doliolida, collectively referred to as doliolids, are small barrel-shaped free-swimming pelagic organisms that can reach high abundances in productive neritic regions of subtropical oceans. Doliolids are among the most abundant of all the zooplankton groups4,8. As suspension feeders, doliolids collect food particles from the water column by creating filter currents and capturing them on mucus nets9. Taxonomically, doliolids are classified in the phylum Urochordata10. Ancestral to the chordates, and in addition to their ecological significance as key components of marine pelagic systems, Thaliaceans are of significance to understanding the origins of colonial life history10,11 and the evolution of the chordates5,7,10,12,13,14.
The life history of doliolids is complex and contributes to the difficulty in culturing and sustaining them through their life cycle. A review of the doliolid life cycle and anatomy can be found in Godeaux et al.15. The doliolid life cycle, which involves an obligatory alternation between sexual and asexual life-history stages, is presented in Figure 1. Eggs and sperm are produced by the hermaphroditic gonozooids, the only solitary stage of the life cycle. Gonozooids release sperm to the water column and eggs are internally fertilized and released to develop into larvae. Larvae hatch and metamorphose into oozooids that can reach 1-2 mm. Presuming conducive environmental conditions and nutrition, oozooids become early nurses within 1-2 days at 20 °C and initiate the colonial stages of the life cycle. Oozooids asexually produce buds on their ventral stolon. These buds leave the stolon and migrate to the dorsal cadophore where they line up in three paired rows. The central double rows become phorozooids and the outer two double rows become trophozooids. The latter provide food to both the nurse and the phorozooids16,17. The trophozooids supply the nurse with nutrition as she loses all internal organs. As the abundance of trophozooids increases, the size of the nurse can reach 15 mm in the laboratory. As the phorozooids grow, they increasingly ingest planktonic prey and reach ~ 1.5 mm in size prior to being released as individuals17. A single nurse may release > 100 phorozooids during its lifespan18. After the phorozooids are released from the cadophore, they continue to grow and are the second colonial stage of the life cycle. Once they reach ~ 5 mm in size, each phorozooid develops a cluster of gonozooids on their ventral peduncle. These gonozooids can ingest particles when they reach ~1 mm in length. After the gonozooids have reached ~ 2 to 3 mm in size they are released from the phorozooid and become the only solitary stage of the life cycle. Once they reach ~ 6 mm in size, gonozooids become sexually mature17. Gonozooids can reach 9 mm or greater in length. Gonozooids are hermaphroditic, sperm is released intermittently while the fertilization of the eggs occurs internally16,17. When the gonozooid is ≥ 6 mm in size, it releases up to 6 fertilized eggs. Successful culturing requires supporting the specific needs of each of these unique life history stages.
Due to the ecological and evolutionary significance of Thaliaceans, including doliolids, there is a need for the cultivation methodologies to advance the understanding of this organism’s unique biology, physiology, ecology, and evolutionary history19. Doliolids have considerable promise as experimental model organisms in developmental biology and functional genomics because they are transparent and likely have streamlined genomes20,21. The lack of reliable cultivation methods, however, impedes their usefulness as laboratory models. Although a handful of laboratories have published results based on cultured doliolids, to our knowledge cultivation approaches and detailed protocols have not been previously published. Based on years of experience, and trial and error cultivation attempts, the purpose of this study was to review experiences and to share protocols for the collection and cultivation of doliolids, specifically the species Dolioletta gegenbauri.
1. Preparing culturing facilities for rearing D. gegenbauri
NOTE: All materials and equipment required are listed in the Table of Materials.
2. Phytoplankton culture
3. Collection of wild doliolids and seawater for culture
NOTE: An overview of collection and cultivation approaches is outlined in Figure 3. A description of the specialized collection plankton net and cod-end is provided in Figure 4.
4. Maintaining D. gegenbauri cultures
Following the described procedures for collecting and culturing the doliolid, D. gegenbauri outlined in Figure 3, it is possible to maintain a culture of D. gegenbauri throughout its complex life history (Figure 1) and sustain it for many generations. Although cultivation of D. gegenbauri is described here, these procedures should also be relevant for the cultivation of other doliolid species.
Capturing healthy and undamaged doliolid zooids requires the application of specialized nets and towing procedures (Figure 4). As delicate animals with no hard structures, care should be taken to minimize procedures that may result in any physical damage. These factors can include turbulence, pressure, and interactions with surfaces including the net, air and air bubbles. Despite their delicate nature, however, undamaged doliolid zooids can be collected using a conical plankton net with an opening diameter to length ratio of 1:5 and equipped with a relatively large weighted non-filtering cod-end. Routinely we have used a 202 µm mesh 2.5 m (length) plankton net with a 0.5 m opening mounted in a swivel harness and equipped with a 4 L weighted non-filtering cod-end (Figure 4). Although the effect of plankton mesh size on the capture of cultivatable D. gegenbauri zooids has not been systematically investigated, theoretically, the use of a net with a larger mesh size may result in further improvement as larger mesh size would reduce the pressure field generated during towing. Alternatively, greater mesh size will result in greater water flow through the net, potentially damaging doliolid zooids. Towing speeds and net angle should be optimized to minimize tow time and damage during collection. In our experience, we have found that sufficiently gentle towing conditions can be achieved by towing the net obliquely at an angle of 15-25° from a drifting ship with vertical deployment and retrieval speeds not greater than 15 m/min. To orient the net to the direction of water flow, the plankton net is mounted in a swivel harness. It is usually the case that the distribution of doliolids in the water column is not random and generally greatest in the region with the highest particulate loads24. Therefore, the water column from below the subsurface chlorophyll maximum to the surface should be sampled. In the shallow SAB mid-continental shelf (20 – 45 m), the water column from ~ 1 m above the bottom to the surface is sampled.
Once healthy zooids have been collected, it is critical to maintaining them in a manner that minimizes exposure to surfaces. To minimize encounters with surfaces doliolids are kept in rounded jars filled with seawater and gently tumbled on a slowly rotating plankton wheel (Figure 2).
Although it is theoretically possible to start a culture with zooids of any life stage, exploration of successes and failures at establishing new cultures of D. gegenbauri from 6 attempts between 2015 – 2018 in the South Atlantic Bight suggest that success is most often achieved when zooids are collected from waters that are < 21°C, and when life stages other than large mature gonozooids are utilized to start a new culture (Table 1 and Table 2). In practice, it is helpful, or at least not detrimental, to include multiple life stages of doliolid zooids when initiating a new culture.
Success in sustaining a culture of D. gegenbauri, as has been described for other pelagic tunicate species20, depends on providing sufficient, but not excessive, food and food diversity required to support each life stage. As diet requirements vary throughout the life cycle, the amount of algae provided at each feeding time must be varied to maintain food concentrations at the desired target levels (40 – 95 µg C/L) (Table 3). Concentrations above or below these levels can result in increased mortality rates (G.A. Paffenhöfer pers. comm.). Although the natural diet of D. gegenbauri remains poorly understood6, cultures can be maintained by supplying relatively simple mixtures of cultured algae and utilizing procedures that allow diverse microbial communities to establish in the culture. Increasing the potential diversity of the prey field is achieved by retaining a fraction of particle laden-water from older cultures and the inclusion of a small number of living copepods and large doliolids at each water change or transfer. Presumably, these organisms process algae and detrital material and serve to diversify the particle size and quality spectrum available for doliolid nutrition, but additional studies are required to confirm this hypothesis.
The availability of doliolid cultures provides the means to investigate, under controlled experimental conditions, many important aspects of doliolid biology, physiology, ecology, and molecular biology. For example, although doliolids are abundant in numerous regions of the coastal ocean and are major planktonic grazers25, data on rates of feeding and growth remain scarce26. Utilizing cultures of D. gegenbauri, a focus of culture-based research has been to quantify feeding and growth rates in response to critical environmental parameters including temperature and food concentrations26. Results from these studies have indicated that clearance rates are similar at concentrations from 20 to 60 μg C/L and decrease as the food concentrations increase (Figure 5A). Clearance rates increase proportionally over temperature ranges supportive of D. gegenbauri growth (Figure 5B). Growth rates (k) range from 0.1 – 0.7/day as a function of temperature and food availability (Figure 6). These studies, in addition to providing practical information for culturing, have allowed the determination of quantitative relationships between doliolid feeding and growth rates as a function of environmental parameters and provide critical insights into the biology and ecology of doliolids required for including this important zooplankton group into modeling frameworks27.
Figure 1: The Life cycle of D. gegenbauri at 20 °C.
The life cycle drawing (1A) has been modified after Walters et al. 20186 and re-drawn with permission. Please click here to view a larger version of this figure.
Figure 2: Plankton wheel used to culture D. gegenbauri. Please click here to view a larger version of this figure.
Figure 3: Schematic overview of D. gegenbauri collection and cultivation approach.
Collection at sea (A), transfer from concentrated buckets to small glass beaker in small batches (B), isolation of doliolid zooids into cultivation jars containing particle-rich seawater (C), maintenance on the plankton wheel throughout the life cycle (D,E). Please click here to view a larger version of this figure.
Figure 4: Plankton net and deployment.
Deployment (top left), retrieval (top right), and schematic of net and cod end (bottom). Please click here to view a larger version of this figure.
Figure 5: Algal clearance rates of D. gegenbauri gonozooids.
(A) relationship between (A) Mean (± S.E.) clearance rates (mL/zooid/day) versus phytoplankton concentration (µg C/L) for three sizes of D. gegenbauri gonozooids. Each point represents 4–11 observations. (B) Mean (± S.E.) clearance rates (mL/zooid/day) versus temperature (°C) for three sizes of D. gegenbauri gonozooids. Each point represents 4–12 observations. Gonozooids sizes are 2.5 mm (), 4.5 mm (), and 6.5 mm (). Figures have been re-drawn with permission26. Please click here to view a larger version of this figure.
Figure 6: Growth rates of D. gegenbauri gonozooid.
Relationship between (A) Mean (± S.E.) growth rates (k) versus phytoplankton concentration (μg C/L) for three sizes of Dolioletta gegenbauri gonozooids. Each point represents 4–11 observations. (B) Mean (± S.E.) growth rates (k) versus temperature (°C) for three sizes of Dolioletta gegenbauri gonozooids. Each point represents 4–12 observations. Gonozooids sizes are 2.5 mm (), 4.5 mm (), and 6.5 mm (). Figures have been re-drawn with permission from Gibson and Paffenhöfer26. Please click here to view a larger version of this figure.
Table 1. Oceanographic Conditions and Doliolid Abundance | |||||||||||
Surface | Bottom | Surface | Bottom | Surface | Bottom | Doliolid Abundance | |||||
Date | Cruise ID | Latitude (N) | Longitude (W) | Depth (m) | Temperature (⁰C) | Temperature (⁰C) | Salinity (PSU) | Salinity (PSU) | Chla (µg/L) | Chla (µg/L) | zooids/m3 |
20/05/2015 | SAV-15-10 | 31.1889 | 80.1527 | 41.30 | 25.26 | 22.43 | 33.58 | 36.96 | NA | 0.20 | NA |
04/08/2015 | SAV-15-19 | 29.5687 | 80.3269 | 40.00 | 26.40 | 21.75 | 36.26 | 36.32 | 1.04 | 1.35 | 218 |
02/12/2015 | SAV-15-31 | 31.1674 | 80.1249 | 40.80 | 23.24 | 22.60 | 35.91 | 35.81 | 1.06 | 1.70 | 13 |
02/02/2017 | SAV-17-03 | 31.2139 | 80.1823 | 41.00 | 18.72 | 18.84 | 36.00 | 36.12 | 0.83 | 1.50 | 3 |
07/11/2017 | SAV-17-23 | 31.2144 | 80.1822 | 42.00 | 24.19 | 23.85 | 36.00 | 36.04 | 0.63 | 1.30 | 254 |
01/02/2018 | SAV-18-02 | 31.1835 | 80.1466 | 43.00 | 16.85 | 16.45 | 36.50 | 36.48 | 0.56 | 0.89 | NA |
NA: data Not Available |
Table 1: Oceanographic conditions and doliolid abundance on the South Atlantic Bight mid-continental shelf at the time and location where D. gegenbauri zooids were collected and used to initiate new cultures.
Table 2. Outcomes of D. gegenbauri Culturing Attempts | ||||
Date | Cruise ID | Zooids Collected | Outcome | Comments |
20/05/2015 | SAV-15-10 | Sexually mature large (6-7 mm) gonozooids | Failed | All gonozooids had died after 4 days. Oozooid and early nurse life stages were produced but failed to thrive. |
04/08/2015 | SAV-15-19 | Sexually mature large (8-10 mm) gonozooids | Failed | Gonozooids died shortly after collection. Oozooids and early nurses were produced but failed to thrive. |
02/12/2015 | SAV-15-31 | Mixed collection including late nurse (4-5 mm) with attached trophozooids, sexually mature large (6 mm) gonozooids, and oozooids (2 mm) | Successful | Cultured for 4 full generations, additional gonozooids and nurses collected in January and March 2016 were added to the culture. Laboratory was evacuated for 4 days during Hurricane Matthew in October 2016 and the culture did not survive. |
02/02/2017 | SAV-17-03 | Mixed collection including gonozooids (1.5-5 mm) and large phorozooids (6 mm) with attached gonozooid clusters | Successful | Cultured for 4 full generations, additional gonozooids collected in April 2017 were added to the culture. Terminated culture in September 2017 in advance of Hurricane Irma. |
07/11/2017 | SAV-17-23 | Gonozooids (3-6 mm) | Failed | Large gonozooid died after 1 day. The immature gonozooid survived in culture for 14 days. Eggs were released by both gonozooids. Oozooids were produced but failed to develop into nurse stages. Culture failed after 1 month. |
01/02/2018 | SAV-18-02 | Large (6-7 mm) late nurse without trophozooids | Successful | In culture the nurse produced trophozooids. Culture was maintained for 3 generations and was terminated at the end of June 2018 when experiments were concluded. |
Table 2: Outcome of attempts to establish laboratory cultures of D. gegenbauri collected from the South Atlantic Bight mid-continental shelf.
Dolioletta gegenbauri | zooid number per | zooid number per | |||
life stage | 3.9 L jar | 1.9 L jar | Isochrysis galbana | Rhodomonas sp. | Thalassiosira weissflogii |
oozooid | 20 | 10 | INCLUDE | INCLUDE | DO NOT INCLUDE |
early nurse | 20 | 10 | INCLUDE | INCLUDE | DO NOT INCLUDE |
late nurse with 8 trophozooids | 4 | 2 | INCLUDE | INCLUDE | INCLUDE |
late nurse with 20 trophozooids | 2 | 1 | INCLUDE | INCLUDE | INCLUDE |
late nurse with 30 trophozooids | 1 | 1 | INCLUDE | INCLUDE | INCLUDE |
phorozooid (1 to 3 mm) | 30 | 15 | INCLUDE | INCLUDE | INCLUDE |
phorozooid gonozooid cluster (> 5 mm) | 2 | 1 | INCLUDE | INCLUDE | INCLUDE |
gonozooid (1 to 3 mm) | 30 | 15 | INCLUDE | INCLUDE | INCLUDE |
gonozooid (> 5 mm) | 2 | 1 | INCLUDE | INCLUDE | INCLUDE |
Target concentrations of algae should be maintained between 40 – 95 µg C/L with equal mixtures (by carbon content) of each algal species |
Table 3: Target culture conditions for each D. gegenbauri life cycle phase.
Supplementary Figure 1: Detailed description of the custom plankton wheel. Please click here to download this figure.
The capacity to culture doliolids has been established over the past several decades and has been used to support research in several areas. Experimental studies in our laboratories have supported the publication of at least 15 scientific studies focused on the feeding and growth18,26, reproduction18,28, diet6,29, physiology30, ecology31, and ecological modelling27 of doliolids.
Although the culture of these delicate animals is currently labor-intensive and time consuming, the cultivation of doliolids is feasible and, if undertaken by the broader community will foster the advancement of the understanding of this ecologically and evolutionarily important group of animals. The objective of this study was to describe current approaches for collecting, rearing, and maintaining D. gegenbauri in culture for the purpose of conducting laboratory-based studies.
The establishment of a doliolid culture requires the collection of healthy and undamaged animals and, once captured, gentle treatment, appropriate nutrition, and husbandry. Doliolids, specifically the species D. gegenbauri, occurs circumglobally on subtropical continental shelves but abundance can be highly variable. For example, in a recent study focused on the mid-shelf region of the SAB, although the abundance varied dramatically from <1/m3 to > 20,000/m3, doliolids were present throughout the year6. Because of the high variability of doliolids in space and time and the relative difficulty involved with sampling continental shelf margin environments, reliable knowledge of doliolid community dynamics where studies are being conducted is an important prerequisite to the successful establishment of the culture.
Once doliolid zooids have been located and captured, it can be difficult to determine whether the animals have been damaged. Animals may appear to be undamaged and exhibit active swimming and escape behaviors, but even the smallest injury can result in their failure to thrive. One characteristic that is especially pertinent to the health assessment of captured doliolid zooids is their ability to feed. Feeding activity can be assessed simply by providing pigmented algae to freshly captured animals. If an animal is feeding, the gut will become colored within a short period of time. In our experience, we have found that adding a small amount of the red pigmented algae, Rhodomonas sp., quickly provides information about feeding activity. If feeding is not observed, it is highly unlikely that a culture can be established.
Vigilance and good husbandry are critical for establishing and sustaining doliolids throughout their complex life cycle. Perhaps the most problematic stage is the development of a viable nurse from the larval stage and the production (sprouting) of the feeding trophoozoids. At this life stage, we speculate that the food requirements, with respect to quantity, quality, and particle size is most limited. To our knowledge, there have been no previous studies that have investigated the feeding activity of D. gegenbauri larvae and oozooids. For example, although the developing gonozooids and phorozooids are able to ingest particles over a wide range of sizes, the capacity of larvae, oozooids, and small nurses is likely more limited. In practice, we find that successful cultivation at these life stages can be achieved by omitting diatoms from the algal food mixture, by maintaining food concentrations at moderate levels, by conducting frequent feedings at lower concentrations, by maintaining a single larger gonozooid and a few copepods with the culture, and by manually removing large aggregates of detritus.
Although we have maintained cultures of D. gegenbauri for multiple generations originating from a single collection, when possible we routinely supplement existing cultures with freshly collected animals to increase genetic diversity and robustness of the culture. A potential danger of this practice is the introduction of parasites or diseases into the culture, but to our knowledge, we have never encountered this problem. Although there have been few reports of parasites of doliolids32, undoubtedly, they do exist. Interestingly, in a recent study comparing the diet of cultured D. gegenbauri gonozooids exposed to natural waters with field-caught D. gegenbauri gonozooids, presumptive Apicomplexa parasites were detected in the wild population that were absent in the cultured animals6.
An existing limitation of the described culture technology is the limitation of zooid production volume. Particularly because the described techniques involve cultivation in sealed jars at low densities on a rotating plankton wheel, it is unclear if this approach could be scaled-up or that the work flow would be amenable to automation. Larger scale cultivation systems, however, for another delicate small gelatinous marine zooplankton species, the larvacean Oikopleura dioica, have been described20,33,34, suggesting that it may be possible to design similar systems for doliolids in the future. However, the complex life history of D. gegenbauri compared to the simpler life history of O. dioica will remain a significant challenge to large scale cultivation.
In conclusion, following the protocols described here, D. gegenbauri can be reliably cultivated under controlled laboratory conditions throughout its complex life history. This capacity makes the species amenable to a variety of controlled experimental studies, and perhaps to develop doliolids as a new animal model in developmental biology and evolution. Limitations of production scale, however, will need to be overcome before this goal can be achieved.
The authors have nothing to disclose.
We are grateful to the many persons who have contributed accumulated knowledge to this project over the years including G.-A. Paffenhöfer and D. Deibel who originally developed these protocols. M. Köster, and L. Lamboley have also contributed significantly to the development of these procedures. N.B. López-Figueroa and Á.E. Rodríguez-Santiago generated the estimates of doliolid abundance provided in Table 1. This study was supported in part by the US National Science Foundation awards OCE 082599, 1031263 to MEF, collaborative projects OCE 1459293 and OCE 14595010 to MEF and DMG and, the National Oceanic and Atmospheric Administration award NA16SEC4810007 to DMG. We are grateful to the hardworking and professional crew of the R/V Savannah. Lee Ann DeLeo prepared the figures, Charles Y. Robertson proofread the manuscript and, James (Jimmy) Williams manufactured the plankton wheel
Algal culture tubes (55 mL sterile disposable glass culture tubes) | Any | NA | For algal cultures |
Autoclave | Any | NA | For sterilizing equipment and seawater for algal cultures |
Beakers (2 L glass) | Any | NA | For sorting diluted plankton net tow contents |
Buckets (5 gallon, ~20L) | Any | NA | For diluting contents of planton net tow – should be seawater conditioned before first use |
Carboys (20 L) | Any | NA | For storing seawater |
Doliolid glass culturing jar (1.9 L narrow mouth glass jar with cap) | Qorpak | GLC-01882 | Container for culture |
Doliolid glass culturing jar (3.8 L narrow mouth glass jar with cap) | Qorpak | GLC-01858 | Container for culture |
Environmental Chamber (Temperature controlled enviromental chamber) | Any | NA | To accommodate plankton wheel and culture maintenance |
Filtration apparatus for 47 mm filters | Any | NA | For filtering seawater for cultures |
Glass microfiber filters, 47 mm | Whatman | 1825-047 | For filtering seawater for cultures |
Glass pipette (borosillicate glass pipette (glass tubing), OD 10mm, ID 8 mm, wall thickness 1mm) | Science Company | NC-10894 | Custom cut and edges polished |
Hose clamps, stainless steel, #104 (178 mm) | Any | NA | For holding culturing jars to the plankton wheel |
Isochrysis galbana strain CCMP1323 | National Center for Marine Algae and Microbiota (NCMA) | strain CCMP1323 | For feeding doliolid cultures |
L1 Media Kit, 50 L | National Center for Marine Algae and Microbiota (NCMA) | MKL150L | For culturing algae |
Lamp (Fluorescent table lamp with an adjustable arm) | Any | NA | For illuminating doliolids in the jars and beakers |
Lighted temperature controlled incubator | Any | NA | For algal cultures |
Micropipettes and sterile tips (0-20 µl, 20-200 µl, 200-1000 µl) | Any | NA | For algal cultures |
Plankton Net (202 µm 0.5 m, 5:1 length) with cod end ring and 4 L aquarium cod-end | Sea-Gear Corporation | 90-50×5-200-4A/BB | For collecting living doliolids (see Figure 4) |
Plankton Wheel | NA | NA | Custom built (see Figure 2) |
Plastic wrap | Any | NA | To cover inside of lid of doliolid culture jars |
Potassium Permanganate | Fisher Scientific | P279-500 | Reagent for cleaning jars and glassware |
Rhodomonas sp. strain CCMP740 | National Center for Marine Algae and Microbiota (NCMA) | strain CCMP740 | For feeding doliolid cultures |
Rubber Tubing | NA | NA | For holding culturing jars to the plankton wheel (can be made from tygon tubing) |
Sodium Bisulfite | Fisher Scientific | S654-500 | Reagent for cleaning jars and glassware |
Sodium Hydroxide | Fisher Scientific | BP359-212 | Reagent for cleaning jars and glassware |
Sterile serological pipettes (1 mL, 5 mL, 10 mL, 25 mL) | Any | NA | For algal cultures |
Thalassiosira weissflogii strain CCMP1051 | National Center for Marine Algae and Microbiota (NCMA) | strain CCMP1051 | For feeding doliolid cultures |
Tissue culture flasks (250 mL) | Any | NA | For algal cultures |