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

Summary

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.

Abstract

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.

Introduction

Zooplankton account for the largest animal biomass in the ocean, are key components in marine food webs, and play important roles in ocean biogeochemical cycles^1,2. Zooplankton, although comprised of a huge diversity of organisms, can be grossly distinguished into two categories: gelatinous and non-gelatinous with few intermediate taxa^3,4. 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 dur....

Protocol

1. Preparing culturing facilities for rearing D. gegenbauri

NOTE: All materials and equipment required are listed in the Table of Materials.

1. Prepare 1 M Sodium Hydroxide (NaOH), 0.06 M Potassium Permanganate (KMnO₄) solution. To prepare this solution, dissolve 400 g of NaOH into 10 L deionized water. Add 100 g of KMnO₄ to the NaOH solution and mix well.
2. Prepare a 0.1 M Sodium bisulfite (NaHSO₃) solution by dissolving 100 g of NaHSO₃ into 10 L deionized water and mix well.
   CAUTION: These reagents are irritants that may cause respiratory problem....

Results

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 heal.......

Discussion

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 growth^18,26, reproduction^18,28, diet^6,29, physiology³⁰

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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	90-50x5-200-4A/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