Research Training Program

Dawn Southard
University of Maryland
College Park, Maryland

Jonathan Coddington, Ph.D.
Project Advisor
Department of Entomology

"The RTP is an excellent opportunity to learn new skills and excellent exposure to a whole new world."

A Method to Freeze Spiders In-Copula

A process by which to freeze spiders in-copula is described, enabling study of the functional morphology of the genitalia. The method consists of submerging a specially designed caged, containing the copulating pair of spiders, into liquid nitrogen. The process of freeze drying is then used to fix the spiders in this position, which will allow them to be prepared for photographing using the scanning electron microscope.

The project was to find a method by which spiders could be frozen in-copula. If this could be accomplished, the spiders would then be fixed permanently and dried using a freeze-dryer. With these two processes completed, the spiders would then be prepared for photographing with the Scanning Electron Microscope (SEM). The functional morphology of the genitalia could then be studied and better understood. Every aspect of the project was successfully completed. Male and female spiders wre collected in the field and brought into the lab. They were set up individually in temporary homes of Dixie cups. Sand and paper towels were used to retain moisture and Saran-Wrap was used as the lid was held in place by a rubber band. Spiders were fed Drosophila, smaller spiders or crickets and watered on a daily basis. Cages were designed out of 1/16" wire mesh screening and fastened with metal staples. The cage diameter was 4.6cm and the depth was 3.8cm. A wire coat hanger was attached to the side for use as a handle. The top and bottom of the cage were covered by petri dishes. The spiders used were Linyphiids due to the amount of males and females collected, and their adaptability to the lab. The females were placed in these cages for one week prior to the introduction of the male. In that time they established webs, and ate regularly. A male was introduced so the courtship and copulation process could be observed. It was discovered that they could be disturbed quite a bit before they would separate. These spiders underwent 2 phases - the first courtship and pseudocopulation, each of which lasted 6 hours, and then phase 2 with the building of the sperm web, charging of the palps, short period of quick insertions, then another charging of the palps, then a period of much longer insertions. The cage was picked up and moved across the room without the spiders coming apart. Liquid nitrogen was the chosen medium for freezing. Another male was placed into a different female's cage and when pseudocopulation began, the cage was placed onto the lid of the liquid nitrogen thermos. When the second phase began and the insertions were increasing in length, the cage was submerged into the liquid nitrogen. This procedure worked three of three times. A freeze dryer using a vacuum pump was employed, along with a vacuum plate, bell jar, high vacuum hosing, and high vacuum silicon grease. The spiders remained frozen suspended from their web in the cage; this was placed on a petri dish of Drierite, CaSO4, and put into the chamber. The vacuum remained at a constant 30" Hg for 3 days. The spiders were removed from the chamber - they were dry but they were no longer fixed in-copula. They were prepared and scanned and photographed. A lot was learned about the male's terminal apophysis, as well as other important structures. Why they came apart could be due to several different variables, all currently under investigation.

This research was supported by a grant from the Smithsonian Women's Committee.