TITLE: Isolation of green algal symbionts from freshwater sponges and subsequent reinfection of sponge tissues
AUTHORS: April Hill, Ivy Nguyen,  Malcolm Hill

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>A variety of green algal species form intracellular symbioses with freshwater sponges. These sponges and their algal symbionts play important roles in freshwater ecosystems and provide a model for asking questions about freshwater endosymbioses involving heterotrophic animal hosts in mutalistic relationships with photosynthesizing algae. The freshwater sponge </span><span style = "font-style:italic;">Ephydatia muelleri</span><span>, for example, has a fully sequenced chromosomal level genome as well as many features that make it ammenable to cellular and molecular studies. Here, we provide a simple protocol for isolating green algae from freshwater sponge host tissues. In most cases, microalgae can be cultured outside of the host in commericially available algal medium and on agar substrates. We also describe methods for infecting algal-free sponges hatched from gemmules to establish stable symbioses in juvenile sponges. </span></div></div>

[STEPS]
?. [Algal cell isolation]
Identify algal-bearing sponges in the field.
?. [Algal cell isolation]
Wash the tissue or gemmules in 1% H2O2 by shaking gently for several seconds at room temperature followed by at least 5 washes in sterile distilled water, M-medium, or Strekal’s medium.
?. [Algal cell isolation]
Place tissue or gemmules in a clean mortar & pestle.
?. [Algal cell isolation]
Grind tissue/gemmules in a small volume (~0.5 mL) of sterile algal culturing media. We have had the most success with Modified Bold 3N Medium (UTEX), but many strains also grow fine in Bold Modified Basal Freshwater Nutrient Solution (Sigma Life Science).
?. [Algal cell isolation]
Transfer the solution of ground sponge tissue with algae to a microfuge tube.
?. [Algal cell isolation]
Tap spin for 5 seconds in a mini-centrifuge to remove spicules and large debris.
?. [Algal cell isolation]
Transfer the supernatant to new microcentrifuge tube. Tap spin again for 10 seconds to remove excess animal cells.
?. [Algal cell isolation]
Transfer the supernatant to a new microcentrifuge tube and spin the remaining solution at 5000 rpm for 1 minute to pellet the algal cells. Some sponge cells will remain in the mixture, but these cells will not grow in the algal culture media.
?. [Algal cell isolation]
Resuspend the pellet (it should be green), add 0.5 mL of media, spin again at 5000 rpm for 1 min, and remove supernantant.
?. [Algal cell isolation]
Resuspend the algal cell pellet in ~0.5mL of media and transfer to a 125 mL sterile culture flask containing ~30 mL of sterile media.
?. [Algal cell isolation]
An alternative method to isolate algae from freshwater sponge hosts involves obtaining green gemmules if they are available in the host tissue. Not all gemmules harbor algae, but algae may be isolated from green gemmules if they can be found. Separate approximately 20 green gemmules from the adult tissue for subsequent steps. Finally, it is also possible to hatch green gemmules and isolate algae from juvenile sponges. Protocols for gemmule seperation and hatching can be found at dx.doi.org/10.17504/protocols.io.863hzgn.
?. [Algal cell isolation]
Place algae under grow lights (we use plant grow lights on a 16 hr light; 8 hr dark cycle) at room temperature. Shake/mix cultures once per day or place on a rotating platform.
?. [Algal cell isolation]
Algal grow should be obvious within one week. Depending on the state of the initial inoculate, contaminating organisms may be present. Further purification of algal cultures can be achieved using the plating technique (see below).
?. [Purification of Algal Cultures]
To obtain pure algal cultures, initial cultures can be streaked on agar plates made with Modified Bold 3N Medium (15 g agar/L).
?. [Purification of Algal Cultures]
Prepare a dilution series of algal culture (log phase stock, 1:10, 1:100) in culture media and spread 100 μL of each on three plates. Grow under lights at room temperature for several days to a week.
?. [Purification of Algal Cultures]
Alternatively, use a sterile loop to streak algae onto agar plates. Grow as above.
?. [Purification of Algal Cultures]
Use a disecting microscope to visualize algal colonies. In some cases, there will be bacteria growing alongside or even around the algae. In this case, it may be necessary to use antibiotics if pure algal strains are needed, however, some prefer not to eliminate the commensual bacteria. We find that many strains of algae will grow well with and without the bacteria.
?. [Purification of Algal Cultures]
Select individual colonies to innoculate into 10 mL of Modified Bold 3N Medium (UTEX) in glass culture vials.
?. [Purification of Algal Cultures]
Grow cultures at room temperature under grow lights (as above) with occasional agitation. After these cultures reach log phase, DNA can be isolated for species identification using SSU and ITS rDNA molecular barcoding markers.
?. [Infection of freshwater sponges with microalgae]
Refer to "Hatching and freezing gemmules from the freshwater sponge Ephydatia muelleri"  dx.doi.org/10.17504/protocols.io.863hzgn for protocols on cleaning and hatching gemmules. This protocol will assume that Stage 5 juvenile sponges can be obtained.
?. [Determination of algal cell densities]
Algal cell densities from cultures can be empirically determined from liquid media by direct cell counts using a hemacytometer. A faster method of determining cell density involves the use of OD (i.e., absorbance) readings at 425 nm or 675 nm (depending on algal pigment properties). To use OD readings, one must first create a standard curve for each algal isolate by regressing OD against known cell densities. Once a standard curve is created for each algal strain, a simple linear equation can be used to estimate cell density based on OD.
?. [Infection of freshwater sponges with microalgae]
Hatch gemmules in 6 or 12 well tissue culture microplates or in individual tissue culture dishes in the dark at room temperature for two days (or longer depending on gemmule hatching). Replace half of culture media (Strekal's or M-Med) daily after hatching. Sponges should always be immersed/covered by culture media and monitored for growth stages until Stage 5.
?. [Infection of freshwater sponges with microalgae]
When sponges have reached Stage 5 juveniles with functioning oscula, they can be infected with microalgae. Refresh the media right before infection and then carefully pipette the determined number of algal cells around the base and above  sponges in each culture dish or well. Obtain early log phase microalgal cultures for the algal line of interest.
?. [Infection of freshwater sponges with microalgae]
Place infected sponges under grow lights (e.g., 16 hours light:8 hours dark) for at least 4 hours and up to 24 hours to establish the infection.
?. [Infection of freshwater sponges with microalgae]
Infected sponges can be grown for up to a week post infection under grow lights. Sponges can be re-infected every 24 hours after gentle washing in Strekal's or M-medium if desired.
?. [Infection of freshwater sponges with microalgae]
Wells/dishes can be washed several times with culture media to remove excess and dead algae after the infection is established. Sponges not infected with algae can continue to grow in the dark to inhibit growth of any possible algae  that were present in the gemmules. We routinely grow sponges that are free of algae (as determined by fluorescent microscopy and/or qPCR).
?. [Post-infection experimentation]
Algal infected sponges can be used for a variety of experiments including DNA and RNA isolation for qRT-PCR or transcriptome sequencing, confocal and other fluorescent microscopy, or TEM.
?. [Post-infection experimentation]
Symbiotic sponges to be used for subsequent RNA isolation can be gently scraped off the bottom of the culture dish using the end of a p1000 filtered tip and transferred to a microfuge tube. Excess media should be carefully removed and replaced with RNAlater storage solution. Sponges can be held at 4oC overnight and then excess RNAlater is removed before storage at -80oC until further use.
?. [Post-infection experimentation]
For imaging by confocal or other fluorescent microscopy, media should be replaced with 4% paraformaldehyde made in 1/4 Holfreter's solution (HS) directly into culture dishes (sponges can be grown on sterile coverslips or on 35 mm MatTek dishes with mounted coverslips. Fix tissue overnight at 4oC followed by at least 3 washes in 1/4 HS the next day before staining and viewing.
?. [Algal cell isolation]
Obtain a small piece of freshwater sponge containing green algal symbionts. Excise a small piece (~0.5 cm) for subsequent work. Keep a small piece of sponge tissue for DNA barcoding the sponge and algal species. We recommend using CTAB to isolate genomic DNA and then barcoding with ITS1 and ITS2 sequences of rDNA. Identification of the sponge species is also possible if gemmoscleres from the gemmule are available.