Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels (Bivalvia: Unionida)

Our protocol standardizes methodologies for sampling the gills of gravid freshwater mussels and characterizing the different stages of larval development while also providing an online database for data submission and exploration of the Gravidity Almanac. The technique is nonlethal and does not substantially affect a female mussel's ability to remain gravid after sampling the gills, therefore the method can be applied to both common and protected species. This methodology can be applied to organisms characterized in the Bivalvia families of Hyriidae, Margaritiferidae, and Unionidae, which are distributed in freshwater ecosystems on every continent except Antarctica.

It is important to open mussels with care, collect location information, and make sure data are collected properly. If identifications are questionable, one should take pictures and contact the appropriate experts. Start by collecting live mussels from the field using tactile visual methods.

Evaluate female gravidity by visual inspection either during or after collection by gently prying open the valves just enough to look inside and see whether the gills are inflated. If using a preserved specimen from a museum, perform visual inspection by opening the valves and inspecting the gills. To sample the gill content, prepare a 1.5-milliliter plastic microcentrifuge collection tube with approximately one milliliter of either water or ethanol, depending on how soon evaluation will take place.

Unwrap a sterile 30-gauge bevel-tip needle on a 10-milliliter syringe, unscrew the cap to expose the needle, and push the handle of the syringe down so the stopper is at the zero-milliliter line. Pick up the gravid female and gently pry open the two valves using the tips of the thumbs. Use the needle tip to gently penetrate a single water tube of the inflated marsupial gill, then gently scoop the gill contents out with the beveled tip of the needle.

Store them in the prepared microcentrifuge tube for later evaluation. Record relevant information, such as genus-species identification, collection location information, and gravidity status on the data sheet and record a unique identifier on each collection vessel to ensure accurate data records during transport. Properly orient the mussel to photograph the right valve for identity validation, lay it down, and take the photograph, making sure to include the labeled sample tube in the picture.

To evaluate the gill contents, transfer them to a Petri dish and fill the bottom of the dish with water. Gently swirl the dish to collect contents in the center for a more concentrated view of the sample. Place the Petri dish under a dissection microscope to evaluate the sample and, if possible, take a photograph of the sample under the microscope.

Record the results of which developmental stages are present in each gill sample. If the females are brooding larvae at multiple developmental stages, report every developmental stage observed in a given sample. If fully developed glochidia are identified and ethanol was not used for preservation, evaluate their viability by adding a crystal of sodium chloride to a subset droplet of the gill sample.

Viable glochidia will respond by opening and closing their valves or simply closing them from a open position. Report any salt-tested glochidia with a T at the end of the designation when data are recorded. To report the data, access the FMGA page or mobile app and record the results in the data entry form using dropdown menus and text entry fields.

For large pre-existing data sets, contact the authors for the template spreadsheet. Enter the recorded data under the appropriate column headings, keeping in mind that each row on the spreadsheet represents observations of a gill sample from one gravid individual, then submit the results and they will be added to the FMGA database after being validated by an administrator, who may contact the collector to request further details or photos. The FMGA database compiles all the information submitted and allows the user to visualize temporal gravidity data for all species in the database.

The Freshwater Mussel Gravidity Almanac is a research tool designed to standardize the collection of gravidity data while facilitating future research, conservation, and recovery efforts. The gill-sampling protocol was evaluated by tagging and recapturing females at monthly intervals throughout the year. Although sample sizes in the study are unequal across species, the results highlight the beneficial and practical applications of this protocol.

The gravidity calendar for Villosa lienosa shows gravid females brooding fully developed glochidia were found in almost every month of the year except August, when only females brooding egg masses were found. Female Hamiota australis were found not-gravid in July, August, and December. A larger proportion of females were brooding fully developed glochidia in January and February and in October and November.

No individuals of Elliptio pullata were found brooding fully developed glochidia, although females were brooding egg masses from May to June. Only one Strophitus williamsi female was found and recaptured three times. This female was found brooding fully developed glochidia in March, not gravid in May, reported gravid in June, and brooding eggs in August.

Gravid females of Villosa vibex brooding fully developed glochidia were found between February and June. Freshwater mussels are a highly imperiled group of organisms. This methodology is important because it provides a nonlethal way to evaluate reproductive characteristics of imperiled species.

It is critical to handle mussels with care. Environmental conditions, such as water temperature and stream flows can be measured in the field to determine what conditions trigger spawning and larval development in freshwater mussels. The compilation of temporal gravidity data for all species of freshwater mussels can assist studies in epidemiology, glochidial morphology, life history, phylogenetics, propagation, and translocation.