Proales similis Mass Culture Guide

The ultra-small rotifer Proales simiis averages 88 µm long X 48 µm wide and is useful for the first-feeding of many marine fish and invertebrates (Hoff & Snell 2004, Wullur et al. 2009, 2011, Snell et al. 2018, Hagiwara & Marcial 2019). P. similis primarily eat phytoplankton (single-celled algae) and bacteria. They require well-oxygenated water, consistent temperature (22-28°C), and quality food in sufficient quantities to keep them reproducing rapidly. The following protocol is for growing P. similis in batch cultures in plastic bags of 5-125 L volumes on the green algae Tetraselmis suecica or Nannochloropsis oculata and natural bacteria. After inoculation, rotifers soon consume all of the algae and require supplemental feeding. We recommend Amplifeed Replete, an advanced formulation diet specifically designed for rotifer growth and enrichment. Once the population reaches high density (>1000 rotifers/ml), the bags are harvested so the rotifers can be used for feeding fish larvae or to inoculate subsequent cultures. We have also grown P. similis in continuous cultures with daily replacement of seawater.

The following is a general guide for maintaining P. similis stock cultures, inoculating, feeding, and harvesting mass cultures. Adjustments may need to be made depending upon the rotifer and algae species, bag size, and culture conditions.

Maintaining P. similis stock cultures

We maintain P. similis stock populations at 22-28°C, 12 g/L Instant Ocean salt, in three or four 250 ml flasks on a diet of Tetraselmis suecica in constant fluorescent light. We initiate a new culture by preparing 100 ml of fresh seawater and adding 2.5 ml from a dense Tetraselmis culture. Each culture is inoculated with 50-100 Proales females. These cultures require feeding about once a week with 2.5 ml fresh algae and can be maintained for a month without transfer to new media. Stock cultures are not aerated, but are swirled by hand a few times per week.

Inoculating P. similis 12L bag cultures

To start a 12 L bag culture, we add 3 L of 12 g/l artificial seawater and 150 ml from a dense Tetraselmis culture. Proales are added at an initial density of about one rotifer/ml. Rotifer density is checked daily to confirm good population growth. An important feature of P. similis cultures is that they should be lightly aerated (~60 bubbles/min). After 3 days, culture volume is doubled to 6 L with fresh seawater and Tetraselmis. After 3 more days, volume is doubled again to 12 L and supplemental feeding with Amplifeed Replete is started with 100 mg/L as needed. It is easy to overfeed Proales cultures, so be mindful of the dosing. Harvests can begin after a few days by filtering a fraction of the culture with a 25 µm net which will retain the Proales. The culture can be topped up with fresh media and Tetraselmis or used to inoculate larger bags.