Neurogenesis in directly and indirectly developing enteropneusts: of nets and cords Author Wanninger, Sabrina Kaul-Strehlow Makoto Urata Takuya Minokawa Thomas Stach Andreas text Organisms Diversity & Evolution 2015 2015-01-31 15 2 405 422 http://dx.doi.org/10.1007/s13127-015-0201-2 journal article 10.1007/s13127-015-0201-2 1618-1077 PMC4514687 26225120 12764142 Balanoglossus misakiensis (Kuwano, 1902) Adult B. misakiensis were collected at a depth of 1 to 2 m at Sunset beach, Aomori-Bay, Asamushi, Aomori , Japan , in June 2012 . Specimens were maintained in aquaria with running seawater at ambient water temperature (24–26 °C) at the Research Center for Marine Biology Tohoku University in Asamushi. Spawning of gravid females was induced by elevating the temperature to 31 °C, and insemination was carried out artificially ( Urata and Yamaguchi 2004 ). Embryos were cultured in membrane-filtered seawater in an incubator at 24 °C in Petri dishes without agitation. Hatched larvae were transferred into 5-l beakers at a concentration of 2 larvae /10 ml and were cultured with gentle stirring. From 2-day postfertilization (pf) onward, they were daily fed a mixture of the single-celled algae Chaetoceros gracilis , Dunaliella sp. , and Pavlova luteri . Most of the water in the beaker was replaced by fresh membrane-filtered seawater every day, and streptomycin ( 50 mg /l) was added to prevent fungal growth. Agassiz-stage larvae (14 days pf) were cultured with sand grains from their natural habitat in Petri dishes to induce metamorphosis.