18-19 July 1996
University of Illinois at Urbana-Champaign

The meeting convened at 8 AM with Michael Irwin chairing. Present were Ev and Marion Schlinger (Schlinger Foundation); Brian Wiegmann (North Carolina State University, and co-PI) and his graduate student, Longlong Yang; Chris Thompson (USDA Smithsonian Institution, consultant) Don Webb (Illinois Natural History Survey, collaborator); Steve Gaimari, Kevin Holston, and Mark Metz (graduate students with Mike Irwin, UIUC); Gail Kampmeier (INHS/UIUC, collaborator); Maya Patel (graduate student at UIUC, illustrator); also visiting were Ed Armbrust (Director, Center for Economic Entomology, INHS), May Berenbaum (Head, Department of Entomology, UIUC), and Christine Armer (graduate student, UIUC). Unable to attend were David Yeates (co-PI) and his graduate student, Shaun Winterton, from the University of Queensland, Australia. Additions to the agenda were solicited after introductions were completed.

Accomplishments on the PEET Grant

Database Capture and Management. Gail Kampmeier gave a presentation of the databases that were being used to capture label information on therevid specimens from museums and collections from around the world. Thus far 20,650 specimens in over 4,200 lots have been recorded in the database, with the initial concentration being those specimens from Australia. The specimen-related databases require minor fine-tuning, and participants were encouraged to think of the types of outputs they would like to see from the databases for their use and for queries on the WWW. Needing major input for revamping were those databases primarily concerned with literature. The existing databases focusing on literature were demonstrated and discussion on the second day will result in a major overhaul in the way these databases function.

All of the databases feature on-line context sensitive (both field- and database-specific) and general help. Questions about anything in the databases are tracked on-line, date and time-stamped, and marked with whether the question has been resolved. Unanswered questions were circulated to attendees in an attempt to resolve them.

An outline of the presentation appears in Appendix A.

Cladistics of the Therevid Project.
Morphology Mike Irwin gave an overview of family and subfamily phylogeny from the point of view of morphology. He and David Yeates have completed a detailed morphological analysis of the head and female terminalia including the spermatheca, which is not sclerotized in the Therevidae. They plan on completing a survey of characters of the adult, in preparation for a cladistic analysis. Maya Patel is working on the drawings for this aspect of the project and showed examples of her work to the group.

Molecular Analysis Brian Wiegmann explained the steps taken when he receives a fly in 95-100% ethanol from someone in the group.

1) The entire fly is ground in ca. 50 µl of buffer to extract the DNA. Many types of DNA and RNA are extracted, all of the following are potential sources of variation for phylogeny: nuclear DNA, mitochondrial DNA, mRNA, rRNA, and tRNA.

2) extract is then preserved at -80° and used a few µl at a time in various tests. It is important to keep everything cold as RNA degrades more easily than DNA

3) use primers to tag gene sequences and PCR (polymerase chain reaction) to amplify the gene sequence of choice. This is the most critical stage, and work is ongoing to determine which gene holds the most promise for the Therevidae, although ideally it is hoped that more than one marker will be identified. Genes examined or under consideration include:

18s rDNA -- has been useful for the oldest lepidopterans, but thus far not found to work with therevids. Diptera as a whole are different from other orders at this gene, but families of flies are little different from one another (only 2%).
28s rDNA -- looks more divergent than 18s
EF1 a = elongation factor 1 a -- used for noctuids in Maryland
DDC = dopadecarboxylase -- used for drosophila and Aedes in the gene bank
PEPCK = phosphoenolopyruvate carboxykinase -- used with families of lower Brachycera; very conserved
CO I and CO II = cytochrome oxydase I & II
16s rDNA

By the end of summer, Brian expects to know the utility of the above genes to delineation of the Therevidae. He is searching for a gene or genes with about a 25% mutation rate (1 in 4 of the bases making up DNA could change) that would allow a maximum amount of change before saturation occurs.

4) run agarose gels to clean up the system and isolate one or more genes

5) sequencing step: no longer using radiolabelling but fluorescent marking that can be read by a new ABI automated sequencer, on which data are read and collected directly onto a computer.

These basic steps can be learned in a week or so. Now the researchers can spend more time looking at the systematics rather than at the biochemistry of the process.

Preservation of Material for Molecular Studies. Brian Wiegmann's lab has been conducting experiments to find best methods for preservation of material for subsequent molecular analysis. What works best is followed by less successful methods in descending order:

1) live fly into liquid nitrogen or in -80° freezer, if used soon; add cold 95-100% ethanol if not to be used right away

2) live fly into 95-100% cold ethanol (do not bake in car, put in cooler on ice if possible)

3) live fly into 70% ethanol

4) recently dead fly into 70% ethanol

5) museum specimens in 70% ethanol -- only a small percentage of these work

6) dried specimen -- works only for some abundant genes (mitochondrial and ribosomal genes) when specimens have been handled well (humidity is not high)

They have also experimented with cyanide-killed specimens. If the specimens are left in the cyanide tube, they got great gene amplification because the cyanide acted as a desiccant. They have yet to test killing with cyanide and quickly moving the specimen to 95-100% ethanol.

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