On the cultivation of free-living marine and estuarine nematodes

Abstract

Although a large body of literature exists on the systematics and ecology of free-living marine and brackish-water nematodes, key questions on the nature and magnitude of interactions between nematodes and other organisms in the benthos remain unanswered. Relatively few authors have investigated live nematodes in food web studies or in experiments dealing with the nematodes' response to a varying environment. It is mainly for the latter purpose that attempts have been made to maintain, rear and cultivate selected species. This paper describes the methodology used for the maintenance, rearing, and eventual permanent agnotobiotic cultivation of a variety of estuarine nematodes. Spot plates, where small samples of sediment or macrophyte material are inoculated on a sloppy agar layer, have been used for the purpose of maintenance and initial cultivation. Those species that reproduce on spot plates are then selected for monospecific cultivation on agar layers with different nutrient enrichments and with micro-organisms cotransferred from the spot plates as food. Mixtures of bacto and nutrient agar prepared in artificial seawater were specifically suitable for the xenic cultivation of nine bacterivorous and, when supplied with Erdschreiber nutrients, two algivorous/bacterivorous nematode species. Up to three generations of five other nematode species have been reared under laboratory conditions, and several more were kept alive and active for variable periods of time on agar. Generation times observed on spot plates for Adoncholaimus fuscus and Oncholaimus oxyuris were substantially shorter than previously published estimates and suggest a correspondingly higher predatory and scavenging potency for these and related enoplids. A procedure for the long-term storage of nematodes at -80°C with glycerol as a cryoprotectant was successfully used for Diplolaimella dievengatensis, Panagrolaimus sp. 1, and Pellioditis marina, but not for Diplolaimelloides meyli. The authors have also summarized the existing literature on the cultivation of marine and brackish-water nematodes. Continuous cultivation appears to have been successful mainly for Aufwuchs and epiphytic nematodes; only few sediment-dwellers have been established in permanent culture. Of only just over 30 species that have ever been cultivated, more than half belong to one family (Monhysteridae) and three are Rhabditida, an order poorly represented in the marine environment. Four species have been grown in monoxenic and one in axenic culture, the latter though with limited success. It is concluded that our understanding of the basic nutritional requirements of marine nematodes is as yet insufficient, and that the culture techniques which have so far mainly deployed agar or liquid substrates, while being suitable for the cultivation of Aufwuchs and epiphytic nematodes, do not accurately enough mimic gradients specific of the natural habitat of many sediment-dwellers.