Collecting, Isolating, and Hatching Oncomelania hupensis ssp. eggs

 

Authors   Yung-san Liang, Mitzi Sereno, Laksiri Karunaratne, Amy Chang, and Fred Lewis

 

Introduction

Unlike Biomphalaria and Bulinus snails, Oncomelania hupensis ssp. snails do not lay their eggs in a clutch (group), but lay their eggs individually on surfaces. The eggs are most often covered with mud, grains of sand or other debris, making them more difficult to see, isolate and harvest in large numbers.

 

Equipment

Dissecting microscope

Fine sieve (approx. 0.5 mm pore size)

 

Materials and reagents

Aged tap water

Plastic petri dishes

Pasteur pipettes

Small spatula

 

Procedure

·         Once the snails (male and female) are placed in their containers and maintained at ~ 24°C, the containers should be changed once every 10-14 days. With a fresh container of mature snails and under the above conditions, one should begin to see eggs in about 1 month, although some O.h. subspecies (e.g., O. h. hupensis) typically takes about two months to lay their eggs than other subspecies.

           One standard way to maximize the collection of Oncomelania eggs is given in the procedures below:

·         Pour the contents of the container through a fine sieve or household strainer (approximately 0.5 mm pore size), so that the adult snails will be collected in the sieve. All the mud, eggs, and other inclusions will pass through.

·         Spray the container and the surface of the mud mounds with a stream of water to dislodge the upper mud surface. Pass it once again through the sieve, spray the remaining mud, and disperse it sufficiently so that all the mud can be passed through the sieve.

·         If the snails are laying eggs, several to many eggs may be attached to the bottom of the container (appearing as small soil-colored specks approximately 1 mm in diameter), and these can be removed gently with a small spatula and placed in a petri dish containing water alone.

·         The adult snails should be placed into another, fresh container with algae and diatoms.

·         With the dispersed mud in the filtrate, pass the filtrate through a sieve with pore size 0.5 mm, so that the eggs and mud particles of approximately the same size and larger will be trapped.

·         Empty the contents of the sieve into a petri dish and add aged tap water.

·         Many of the Oncomelania hupensis ssp. eggs will be covered with a fine layer of mud and are sometimes difficult to distinguish from other particulates in the preparation (photo – mud mixture). However, there are some key ways to distinguish them from the surrounding particles. The eggs covered in mud are less dense than other particulate material and will move more readily on slight agitation of the petri dish. If the eggs were recovered by spatula from the surface of the container, they will have one flat side, instead of being entirely oval, as are those deposited on the mud mound. The photo included (Onco eggs) shows a typical egg recovered from a mud mound and one recovered from the hard surface of a container. Eggs will also move more readily than grains of sand toward the center of the petri dish upon gentle swirling. The eggs should be removed with a glass pasteur pipette and placed in a petri dish, with aged tap water only.

 

Follow-up comments/recommendations

Oncomelania hupensis ssp. eggs will hatch approximately 16 days after they are laid. Therefore, in the petri dish described above, eggs will be of different ages, so the dish should be examined at least once every day or two. Once eggs have hatched, the juvenile snails* should be transferred to another petri dish and fed a small amount of algae and diatoms. A pasteur pipette can be used to pick up the freshly hatched snails. Care should be taken with the amount of algae and diatoms used in the petri dish to prevent their overgrowth in the dish. One to two weeks later, the juvenile snails should be transferred into a fresh dish with algae and fresh diatoms.

Provided that the conditions for growth are optimal, one can obtain adult Oncomelania hupensis ssp. snails by 1-2 months after hatching.

*Juvenile snails are very small and transparent and may be easily overlooked, particularly if mud and debris have accumulated in one area of the petri dish. One may use the pasteur pipette to very gently swirl the water to move the solid material around the petri dish, thus revealing any newly hatched juvenile snails.

 

References

Bruce, J.I., Radke, M.G., and Davis, G.M. 1971. Culturing Biomphalaria and Oncomelania (Gastropoda) for large-scale studies of schistosomiasis. Biomedical Report No. 19, 406th Medical Laboratory, U.S. Army.

 

Liang, Y-S., Bruce, J.I., and Boyd, D.A. 1987. Laboratory cultivation of schistosome vector snails and maintenance of schistosome life cycles. Proceedings of the First Sino-American Symposium 1: 34-48.

Tucker, M. S., Karunaratne, L. B., Lewis, F. A., Frietas, T. C., and Liang, Y-S. 2013. Schistosomiasis, in Current Protocols in Immunology 19.1.1-19.1.57, John Wiley and Sons, Inc., (R. Coico, Ed).  Published online November 2013 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142735.im1901s103.