Journal of Applied Ichthyology J. Appl. Ichthyol. 31 (2015), 1–9 Received: April 5, 2014 © 2014 Blackwell Verlag GmbH Accepted: June 3, 2014 ISSN 0175–8659 doi: 10.1111/jai.12645 Improving nematode culture techniques and their effects on amino acid profile with considerations on production costs By B. H. Buck1,2,J.Bruggemann€ 3, M. Hundt4, A. A. Bischoff5, B. Grote1, S. Strieben1 and W. Hagen6 1Alfred Wegener Institute, Helmholtz Center for Polar and Marine Research (AWI), Bremerhaven, Germany; 2University of Applied Science, Bremerhaven, Germany; 3Institute for Marine Resources (IMARE), Bremerhaven, Germany; 4Institute for Environmental Sciences, University of Koblenz-Landau, Landau, Germany; 5Lehrstuhl fur€ Aquakultur und Sea-Ranching, Agrar- und Umweltwissenschaftliche Fakultat€ der Universitat€ Rostock, Rostock, Germany; 6Marine Zoology, University of Bremen, Bremen, Germany Summary ing diversification of aquaculture and the lack of knowledge The purpose of this study was to investigate the effectiveness about new species concerning their nutrient requirements. of 11 different culture media for production of the free-living Aquaculture thus still depends on live food for the initial nematode Turbatrix aceti. Several other harvesting methods feeding period. Although substantial efforts have been were tested in addition to mass production. A further focus invested in the development of small particle-sized formu- was the investigation of amino acid alterations caused by the lated feeds for the culture of fish and crustacean larvae, application of various media during the culture of T. aceti numerous species still depend on live food to meet their and two additional nematode species, Panagrellus redivivus nutritional requirements during the onset of exogenous feed- and Caenorhabditis elegans. Finally, a cost analysis for the ing or even to a large extent their larval-rearing period (Zam- production of T. aceti was generated and its outcome com- bonino-Infante and Cahu, 2001; Holt et al., 2011) The use of pared to the production of conventional live feed organisms. live food in aquaculture is currently limited to a relatively Altogether 11 liquid culture media were tested for mass pro- small number of species, of which nauplii of the brine shrimp duction of the nematode Turbatrix aceti using a minimum of Artemia salina and rotifers such as Brachionus sp. are the effort in terms of labour and costs. Six harvesting methods, most common (Lavens and Sorgeloos, 1996). Rotifer cultiva- including filtration as well as active swimming of T. aceti tion is somewhat risky due to a possible collapse of the cul- were evaluated. Additional to the culture of T. aceti in four tures (Støttrup and McEvoy, 2003) as well as the difficulty in of the above-mentioned media, the nematodes P. redivivus keeping them free of contamination (Reguera, 1984), poten- and C. elegans were cultured on two different solid media. tially leading to increased production costs. Although Cost analysis for the production of T. aceti includes those of Artemia nauplii are, in principle, convenient in hatchery the media, the equipment, as well as the labour costs for cul- operations since the eggs can be stored over long time peri- ture and harvest. An average density of approx. ods and are readily available when needed, their limited sup- 30 9 106 Æ 8.13 9 106 nematodes LÀ1 was achieved for ply can lead to relatively high prices (Lavens and Sorgeloos, T. aceti. The most efficient method (20 lm filtration) allowed 2000). Due to the rapid growth demands of the aquaculture harvesting 85.3 Æ 2.7% of the nematodes from the medium industry, which are barely satisfied by brine shrimp cyst pro- without disturbing the particles. Lowest efficiency was ducers, the search for non-Artemia alternatives must be an achieved by combining sedimentation and filtration, accom- integral part of future aquaculture research (Sorgeloos et al., plishing a harvest of 42.1 Æ 5.8%. The amino acid profile of 2001). all three nematode species turned out to be both stable and In recent years several species of nematodes have been very similar. Amino acid enrichment had little effect. The identified as possibly suitable alternatives to Artemia nauplii € costs for producing one million T. aceti individuals ranged (Bruggemann, 2012). The non-parasitic soil nematode Pana- between 5.39 and 6.19 €, where labour costs accounted for grellus redivivus has received particular attention. This nema- 73 to 84% of the total production costs. In conclusion, tode can reproduce rapidly and provide large biomass yields T. aceti appears to be very robust, easy to handle, as well as (Ricci et al., 2003). According to Wilkenfeld et al. (1984), cheaper to cultivate compared to other live-feed organisms. the production is about 30% cheaper compared to Artemia Therefore, its use in commercial aquaculture should be given culture. Additionally, the fatty acid profiles and the lipid future consideration. contents are extremely variable, depending on the culture media used (Schlechtriem et al., 2004a,b), in P. redivivus between 2.7 and 39.8% with regard to the total lipid content Introduction (Rouse et al., 1992; Schlechtriem et al., 2005). The amino The production of fish, invertebrates and plants in aquacul- acid profile of this nematode species is similar to Artemia ture has rapidly increased in recent years due to the increas- (Biedenbach et al., 1989; Lavens and Sorgeloos, 1996; U.S. Copyright Clearance Centre Code Statement: 0175-8659/2015/3101–1$15.00/0 2 B. H. Buck et al. Santiago et al., 2003) and to frozen zooplankton (Schlecht- tion of nematodes on the tank bottom. The apparent suit- riem et al., 2004b). ability of T. aceti as live feed, as demonstrated for decades These advantages prompted aquaculturists to evaluate the in ornamental fish culture, and the apparent advantages in suitability of P. redivivus as a live food for feeding to differ- terms of its swimming ability, led us to conduct a series of ent types of fish and/or shrimp. It has been shown that the experiments in order to improve the culture conditions of use of P. redivivus in co-feeding regimes of the common carp this species and evaluate the nutritional quality. Two differ- (Cyprinus carpio) and silver carp (Hypophthalmichthys moli- ent experiments regarding T. aceti cultivation were carried trix) can enhance larval growth in comparison to larvae fed out, fostering several research topics: a dry diet. Kahan et al. (1980) and Schlechtriem et al. 1 Cultivating T. aceti in different media in order to identify (2004a) found that P. redivivus led to decent survival rates in suitable liquid media for mass production, with a focus larvae of common carp (C. carpio). However, it must be on reproduction rates and costs. noted that although P. redivivus seems to reach acceptable 2 Testing six types of harvesting methods to evaluate the performance rates in feeding regimes of some fish species, effectiveness of harvesting nematodes using these different most studies show that the survival and growth is lower than methods, with the explicit goal to develop and identify a € in fish fed with Artemia nauplii (see Bruggemann 2012, for simple and effective method. details). 3 Comparing amino acid profiles of three different nema- Regarding the feeding of shrimp larvae in contrast to fish tode species (T. aceti, P. redivivus, Caenorhabditis elegans) larvae, nematode performance is shown to be equal or even to Artemia nauplii and rotifers, with the results giving an superior compared to Artemia nauplii. Biedenbach et al. overview of potential similarities and differences in the (1989) raised Pacific white shrimp (Litopenaeus vannamei) lar- amino acid composition of the aforementioned species. vae on nematodes and showed that the larvae fed with differ- The results can be used to estimate the potential of nema- ent nematode densities grew faster or were at least similar in todes to provide the required amino acids to larvae as comparison to an Artemia diet. Also in Pacific white shrimp conventional live food organisms. larvae, Focken et al. (2006) showed that there was little dif- ference in survival when fed either nematodes or Artemia and that larvae grew almost as fast as with Artemia in high Materials and methods enough nematode densities (100–150 Ind. mlÀ1). Wilkenfeld et al. (1984) found nematodes to be an excellent food for lar- Nematode cultivation for identifying suitable liquid media vae of northern brown shrimp (Farfantepenaeus aztecus), Turbatrix aceti was grown in horizontal Corningâ cell cul- northern white shrimp (Litopenaeus setiferus), as well as Paci- ture bottles (volume 200 ml) each filled with 80 ml medium. fic white shrimp. Culture performance of shrimp larvae could The bottles were sealed with caps and with holes for a con- also be improved by improving the fatty acid profiles of stant air supply. Inoculation was approx. 5000 nematodes nematodes by enrichment with different types of oils (Kumlu per bottle. The cultures were kept at room temperature et al., 1998). Nematodes (+algae) led to higher survival rates (20°C), with five replicas for each medium. Once a week in Indian prawn (Fenneropenaeus indicus) than with a diet of nematode density was evaluated by counting a subsample algae and Artemia. (between 0.1 and 1 ml, depending on the nematode density) Although there are a variety of free-swimming nematodes, using a microscope. Evaporated water was replaced on a aquaculture research has so far focused mainly on P. redivi- weekly basis. Tests were scheduled for 70 days or were vus. The free-living nematode species Turbatrix aceti in par- aborted earlier when cultures collapsed. Altogether eleven ticular seems to be suitable as a live food in aquaculture, media (media ID A – K) were used, the media compositions since it is very similar in character to P. redivivus. These selected either from the literature or adapted according to nematodes are approximately the same size (1–2 mm long, suggestions from the literature as well as our own observa- ~50 lm diameter) and are very tolerant against numerous tions.