Gene transfer into conchospores of Porphyra haitanensis (Bangiales, Rhodophyta) by glass bead agitation

Genetic transformation by electroporation of protoplasts is a standard procedure for many plants. However, for the genus Porphyra, the method is not effective because of low viability of protoplasts and is a time-consuming and expensive procedure. Based on the life history of Porphyra, a spore-targeted strategy of genetic transformation was developed, that is, using fresh conchospores of Porphyra haitanensis Chang & Zheng transformed by agitation with glass beads. A SV40 promoter-driven lacZ reporter gene was expressed in conchospores 48 h after the agitation. More transformants were obtained by increasing the agitation time from 10 to 25 s. The maximum number of transformants was more than six out of 1 million agitated conchospores. Transfer of a SV40 promoter-driven egfp gene into conchospores resulted in significant green GFP fluorescence. The expression of lacZ and egfp revealed that this strategy of spore-targeted transformation using glass bead agitation is feasible in P. haitanensis and that the SV40 promoter is effective for monitoring expression of foreign genes in this red algal species.

Transformation of Platymonas (Tetraselmis) subcordiformis (Prasinophyceae, Chlorophyta) by agitation with glass beads

A transient transformation system for the unicellular marine green alga, Platymonas subcordiformis, was established in this study. We introduced the pEGFP-N1 vector into P. subcordiformis with a glass bead method. P. subcordiformis was incubated in cell wall lytic enzymes (abalone acetone powder and cellulase solutions) to degrade the cell wall. The applicable conditions for production of viable protoplasts were pH 6.5, 25 degrees C, and 3 h of enzyme treatment. The protoplast yield was 61.2% when P. subcordiformis cells were added to the enzyme solution at a concentration of 10(7) cell ml(-1). The protoplasts were immediately transformed with the pEGFP-N1 vector using glass-bead method. The transformation frequency was about 10(-5), and there was no GFP activity observed in either the negative or the blank controls. This study indicated that GFP was a sensitively transgenic reporter for P. subcordiformis, and the method of cell wall enzymolysis followed by glass bead agitation was applicable for the transformation of P. subcordiformis.

Observations on the nocturnal activity and feeding behavior of Anguilla japonica glass eels under laboratory conditions

Glass eels of the temperate anguillid species, Anguilla japonica, clearly showed a nocturnal activity rhythm under laboratory conditions. Light-dark cycle was a determinant factor affecting their photonegative behavior, nocturnal locomotor activity, and feeding behavior. Under natural light conditions, glass eels remained in shelters with little daytime feeding, but came out to forage during darkness. They moved and foraged actively in the following dark, and then their activity gradually declined possibly because of food satiation. They finally buried in the sand or stayed in tubes immediately after the lights came on. Under constant light, glass eels often came out of the shelters to forage in the lights but spent little time moving outside the shelters (e.g. swimming or crawling on the sand). Glass eels took shelter to avoid light and preferred tubes to sand for shelter possibly because tubes were much easier for them to take refuge in than sand. Feeding and locomotor activities of the glass eels were nocturnal and well synchronized. They appeared to depend on olfaction rather than vision to detect and capture prey in darkness. Feeding was the driving force for glass eels to come out of sand under constant light. However, in the dark, some glass eels swam or crept actively on sand even when they were fully fed. The lunar cycles of activity rhythms of glass eels that have been observed in some estuarine areas were not detected under these laboratory conditions.