The assassin fly Scleropogon duncani photographed in the field near Parker, AZ feeding on a Heteroptera species.
I have collected flies for my research for almost 20 years now and over the past 15 preserved specimens for different research applications. The standard for morphological taxonomic and phylogenetic research on asiloid flies is to hand-net specimens, preserve them in ethyl acetate, and pin them in the field. For internal morphological study, I have preserved specimens in Kahle's Fluid (see information here) and for molecular phylogenetic studies in 95% ethanol. It has always been my aim to collect a series of specimens of a particular species at the same site, pin several of them for proper species identification, and preserve a few in 95% ethanol and Kahle's fluid to utilize the specimens in combined morphological and molecular phylogenetic analyses. In our genomics age, my collecting efforts have changed slightly in that I strive to preserve specimens in liquid nitrogen to achieve the highest quality of preservation of RNA and DNA for transcriptome and genome sequencing or other next-generation sequencing applications in addition to specimens for morphological study.
Below is an outline of a workflow developed during my first trip to southern Arizona and California, in which I used a liquid nitrogen dry shipper and databased every single specimen in the field, conducted in April 2015. This trip was a good test scenario because I visited collecting sites I had visited in previous years and had an idea which genera and species of asiloid flies I could expect. The field sites were mostly inland sand dunes or sandy habitats such as shown here.
Vegetated sand dunes east of Parker, AZ.
Vegetated sand dunes and interdune valleys east of Parker, AZ.
The Smithsonian Institution's National Museum of Natural History (NMNH) has recently established a liquid nitrogen repository called Biorepository that is a new "type of collection" not restricted to taxonomic groups and which will store vials with tissues or whole organisms of disparate taxa adjacent in a liquid nitrogen tank. The Biorepository is integrated in the Global Genome Initiative (GGI) and the Global Genome Biodiversity Network (GGBN), which are both headquartered at the NMNH.
A few words about specimen data
Collecting specimens today involves keeping track of associated data right from the beginning, i.e., directly in the field. In order to associate the pinned fly, which will serve as an exemplar in the NMNH Diptera collection for future verification of identification or morphological study, with the specimen(s) of the same species preserved in liquid nitrogen, it needs to have a unique specimen identifier assigned to it right away. Likewise, each specimen preserved in liquid nitrogen will need a unique identifier. In the case of the NMNH Biorepository, a second identifier is necessary to track the vial in the liquid nitrogen tanks, which is captured in a FreezerPro database. Once the record of the pinned fly is in the NMNH institutional KE Emu database, both records can be associated and the information forwarded to the GGBN data portal. At the NMNH, the field data can either be captured in a Field Information Management System (FIMS) Excel spreadsheet or, as in my case, in a personal research database from where they will be exported following the FIMS template.
Prior to field work
I prepared the vials by affixing two specimen identifiers (UNMENT and Bioreposiory identifier) to each of them so that I won't have to do this step in the field. (I brought a hand-held label scanner into the field to scan the identifier labels and enter them into the correct field to avoid typing errors.) I also sent the liquid nitrogen dry shipper, such as the one below, to the FedEx office at the Phoenix airport from where I picked it up and had it filled with liquid nitrogen at a welding supply store.
In the field
I am collecting asiloid flies (chiefly my research taxa Apioceridae, Asilidae, and Mydidae) with a hand-net by collecting one specimen at a time. These flies are big enough so that they can be identified to at least the genus in the field. Since I needed to make a decision of which specimens will be preserved in liquid nitrogen and which one in ethyl acetate in the field, I decided to keep individual flies at a specific site alive in separate vials. Once I had collected several specimens of the same species, I preserved the next few in ethyl acetate for pinning. The flies destined for liquid nitrogen were placed in a small cooler with ice packs and later transferred to a fridge in the hotel room before processing. This had the added advantage that the flies slowed down and could be moved easily to one of the pre-labeled vials.
Databasing specimen records in the field
In the evening back at the hotel, specimens were processed. The specimens preserved in ethyl acetate were pinned, received a unique specimen identifier (USNMENTXXXXXXXX), and were added to my specimen-level database.
Screen shot of custom research database (in FileMaker Pro) capturing occurrence data for pinned specimen.
Next, the genomic sample specimen that was kept alive and which is vouchered by the above pinned specimen was placed in a pre-labeled vial with both the USNMENT unique identifier and the unique Biorepository identifier (such as AA8IQ05) and added to the database.
Screen shot of research database capturing occurrence data for genomic sample specimen.
These two records need to be connected, which is done by adding the USNMENT identifier of the exemplar and genomic sample to the respective records. A pinned exemplar can have several genomic samples, but each genomic sample refers to only one pinned exemplar in this case.
Screen shot of research database showing relationship of pinned exemplar specimen to genomic sample specimens (one-to-many relationship).
Screen shot of research database showing relationship of genomic sample specimen to pinned exemplar specimen (one-to-one relationship).
Specimens to be preserved in liquid nitrogen were placed in the vials, wrapped in aluminum foil (to prevent the labels getting disassociated form the vial), and then dropped into the dry shipper containing liquid nitrogen.
Pre-labeled cryovials with two identifiers (USNMENT and Biorepository).
Cryovials wrapped in aluminum foil before being dropped in liquid nitrogen.
From each pinned exemplar and genomic sample specimen, a hind leg was placed in a buffer plate so that a genetic barcode (mitochondrial COI) can be sequenced to provide a second means of identification of the specimens.
96-well plate with buffer for preservation of DNA from leg for sequencing of COI gene.
This leg gets it's own record and unique specimen identifier and likewise is connected to the voucher specimen (exemplar or genomic sample).
Screen shot of research database showing record for leg removed for sequencing COI gene.
After the field trip
Since I sent the liquid nitrogen dry shipper back to the Biorepository from Phoenix via FedEx, the GGI team moved the specimens from the dry shipper to the liquid nitrogen tanks and added the vial numbers to FreezerPro. The data of all specimens I had collected and the legs for COI sequencing were exported from my database to the FIMS spreadsheet and uploaded into EMu by the Department of Entomology data management team.
The pinned specimens needed to be labeled and I use my database to generate the locality and identification labels. I added a script that would include a label indicating whether a pinned specimen is an exemplar for a specimen in the Biorepository and whether a leg has been removed for COI barcoding.
Example labels for a pinned exemplar specimen.
Below is the set of pinned Scleropogon duncani specimens with two of them being exemplars for specimens in the Biorepository as well as vouchers for a leg removed for COI sequencing.
Unit tray with assassin fly Scleropogon duncani specimens labeled and ready to go into main USNM Diptera collection.
Today, the data of the flies I collected are publicly accessible. For example, the exemplar specimen can be found on the NMNH Entomology collection database: USNMENT01115108. The associated specimen in the Biorepository can't be found in the same database yet (NMNH doesn't publish genomic samples to the public directly yet), but it is accessible through the GGBN record identified by the unique NMNH Biorepository number AA8IQ05.
The assassin fly Albibarbefferia albibarbis photographed in the field near Portal, AZ feeding on a Heteroptera species and ovipositing in crevices in the bark.
Now I am all set for a GGI-funded field trip to the Eastern Cape in South Africa to collect flies for genomics research in December 2015.
Did we succeed in preserving RNA and DNA properly? Yes! The largest assassin fly I collected on this trip was Proctacanthus coquilletti Hine, 1911 and we are now sequencing the genome of this species based on a single specimen (see record at GGBN) using the HiSeq 2500 platform and DISCOVAR de novo assembly. We have also sequenced the transcriptomes of the imago of the apiocerid fly Apiocera parkeri Cazier, 1941, the mydas fly Messiasia californica (Cole, 1969), and the assassin flies Laphystia limatula Coquillett, 1904 and Scleropogon duncani Bromley, 1937 on the HiSeq 2000 platform in a single lane. The results are good and watch this space for updates on the progress of these projects in particular the novel genome.
The assassin fly Proctacanthus coquilletti photographed in the field.
I need to thank Patricia Gentile-Poole and Michael Lloyd from the Department of Entomology for help with uploading the specimen data from the FIMS into EMu. Chris Huddleston and his GGI team at the Biorepository helped in preparing this trip and took care of the vials shipped to them.
Posted by Torsten Dikow
update 2016-03-09: Erroneously, the location of some of the images was given as Portal, AZ whereas it is Parker, AZ.