Gradients1-KOK1606-DiazotrophAbundance_2020-03-04_v1.2

Cruise Name: Gradients 1.0 / KOK1606

Project Name: SIMONS Foundation NPSG Gradient

At select stations and depths (ranging from 5 to 45m), the entire volume of a Niskin bottle was drained directly into clean ~10L carboys fitted with spigots. Carboys were covered in dark cloth and the volume was gravity filtered through a 47-mm diameter, 3 micron pore size and 10 micron pore size, black polycarbonate filter with a polyester drain disk as a backing filter. If the volume was not filtered after 2 hours, filtration was terminated and the remaining volume of the carboy was measured in order to calculate volume filtered.  Following filtration, filter holders were fit with a short section of tubing and a syringe luer fitting on once side and a 2-way valve on the outflow side. For each filter, 5-ml of 2% glutaraldehyde was slowly injected onto the filter and samples were allowed to fix for 30 minutes. Fixative was drained after this time and 60ml of air was used to flush all filters. Polycarbonate filters were then mounted onto 3x2” glass slides with immersion oil, cover slides were added and the edges of each cover slip was sealed with quick dry nail polish. All slides were stored at -20°C and counted within 30 days. Enumeration of diazotrophic taxa was performed using epifluorescence microscopy.The entire slide was counted for DDAs and Trichodesimum abundance. Endosymbiont bearing diatoms of the following genus were enumerated: Rhizosolenia, Hemiaulus, Climacodium and Chaetoceras. Free Richelia intracellularis were also counted. Trichodesmium filaments were counted and the length of each filament was recorded. Trichodesmium cell number was then calculated by dividing the filament length by the mean cell length (9.9 micron ±2.5 micron).