Specimen collection and preservation for barcoding
Fresh and dry samples of Lepidoptera perform ideally in DNA amplification. Papered and most pinned specimens are perfectly suitable for DNA barcoding, though we encourage sampling before spreading to avoid DNA degradation caused by the relaxing process. If specimens are less than 10 years old and never were exposed to excessively wet conditions, moistures or rotting either while collecting in the field or later when relaxed, then their DNA is usually recovered with very high success. Global campaigns on large-bodied moths (e.g. saturniids or sphingids which usually need a longer relaxing time) mostly based on the use of pinned dry specimens have shown more than 80% amplification and sequencing success for specimens up to 10 years old. Amplification success then decreases rapidly with age, and we recommend not sampling specimens older than 15 years old unless they are of critical importance and hardly available in fresher condition.
We have developed a special protocol to recover DNA barcodes from decades or centuries old specimens. As it remains costly and has to be applied off the DNA barcoding core pipeline, this protocol is reserved to types or similarly unique specimens. Additional funding sources may be requested to process these ancient samples.
Generally, a single leg (or 2 for the smallest Microlepidoptera) or a leg fragment (2 to 3 mm long) only is sufficient for DNA extraction. Other body parts like antennae or wing veins/membrane are also suitable for that purpose, but legs are usually preferred because they are paired appendices and their removal does not affect the general aspect of the specimen. The posterior end of the abdomen may also be used and the genitalia can be recovered after DNA extraction. This is only applicable for small individuals since the size of the tissue fragment used for extraction should not exceed 3 or 4 mm. Because of the additional recovery step, special notice and agreement should be made if participants wish to use this method.
Whole specimens can also be sent directly for sampling. We will proceed to leg pulling and will return the voucher or, if donated, it will be integrated in the natural history collection of the Biodiversity Institute of Ontario.
Sampling of preimaginal stages
Eggs, caterpillars or pupae can be used as well for DNA barcoding as long as the specimens are preserved as vouchers (eggs will then require a tissue recovery step). The head capsule, a transversal section of a segment or a thoracic leg (or a fragment of it) can be used for DNA extraction from a caterpillar, as well as any epidermis tissue sample from a pupa. These tissues can be either collected fresh and allowed to dry rapidly, or preserved in 95% ethanol. Specimens preserved in high concentration ethanol usually remain suitable for DNA extraction for 5 to 10 years, possibly more if the storage conditions are optimal (ethanol/sample volume ratio, ethanol renewal, protection from UVs, samples frozen or refrigerated).
While specimens sampled and processed in the context of a global campaign usually come with species identification under the supervision of expert taxonomists, the continental campaigns frequently gather records for non-identified specimens within a broad range of families. If you are a taxonomist and wish to be involved and contribute to the identification of some samples on BOLD, your help is very welcome and we invite you to contact the campaign coordinator. You can have a detailed overview of the status of species identification in your group of expertise by using the taxonomy browser of BOLD.
Tissue samples submitted to the Biodiversity Institute of Ontario core analytical facility are analyzed using standard high-throughput molecular protocols. Therefore it is critical that tissues destined for DNA barcoding analysis and associated specimen data arrive at BIO in a compliant lab-ready format. To ensure this, standard sampling kits developed at BIO are being supplied to external collaborators complete with detailed instructions for sampling and data submission. All prospective donors are encouraged to observe these sampling guidelines and resolve any questions with their project/campaign coordinator. BIO has developed several types of sample submission formats which allow direct compatibility with the high-throughput lab molecular protocols, particularly, the 96-well format of all analytical stages. The sample storage medium most advocated by the Lepidoptera campaign is the 96-well microplate. This medium is cheap and compact and allows submitting the minimum amount of tissue required for one DNA extraction (ca. 2-3 mm in dimension or 5 mg), thereby avoiding the necessity to manage residual tissue. It also allows the samples to enter directly the lab DNA extraction pipeline, bypassing the intermediate subsampling stage. To facilitate proper tracking of each sample throughout the analytical chain, it is critical that each specimen is assigned a unique individual identifier (e.g., collection catalogue number prefixed by the museum acronym), or Sample ID, which is recorded in the CCDB data record spreadsheet, providing a map of locations for each sample inside a 96-well plate. This number should exactly correspond to the Sample ID submitted to BOLD together with specimen provenance information and images, thereby linking the sequence and the specimen record. See also section on specimen numbering conventions.
It is well-known that provenance data and biological information are an integral part of any collection voucher specimen and constitute a large portion of its research value. Therefore it is critical that any data associated with each specimen used for populating the reference library of DNA barcodes are recorded and digitized. There are several vital data fields required by BOLD (i.e., detailed locality with coordinates and elevation, collection date, name(s) of collector(s), sex and age), however, there are additional pieces of information that should be recorded as a part of best practices. These include standard measurements, weight, reproductive data, and habitat. Field collecting of insects usually also produces ancillary information on community structure, parasite infestations, habitat preferences, etc., which may pose significant synecological value. Although collecting these data falls outside the scope of DNA barcoding per se, prospective donors are encouraged to consult specialized literature on how to obtain and log this valuable information. The Lepidoptera barcoding campaign has a set of standard protocols for detailed data capture (in both digital and analog format) which can be distributed among interested participants, upon request.
It is imperative that any prospective contributor to the mammal barcoding campaign is well aware of the national, international, and institutional regulations pertaining to the collection and shipping of insect specimens and any derivative biomaterials and conducts his/her operations in a compliant manner. This particularly concerns locally or globally endangered species and those listed under the Convention of International Trade of Endangered Species (CITES). It is preferred that any institution hosting or facilitating such collecting activities has a nationally recognised collection and is registered with the national CITES authority. Regulations differ drastically from nation to nation, and it is important that they are followed by all campaign participants as they apply to their research activities. The Biodiversity Institute of Ontario is a CITES-registered institution and will gladly assist in obtaining Canadian import documentation for samples intended for processing at the core sequencing facility at Guelph, Ontario.