Comparison of preservation techniques for DNA extraction from hymenopterous insects

Two species of parasitic wasp, Venturia canescens and Leptomastix dactylopii, were killed and preserved by various methods used for Hymenoptera and in mass-collecting devices. Total genomic DNA was subsequently extracted and a 524 bp fragment of the mitochondrial 16s ribosomal RNA gene amplified by PCR. Results for these techniques were compared with that for fresh material and museum specimens. Material from -80°C, 100% ethanol, air-drying in a desiccator, and critical-point dried from alcohol all yielded good results after short and long-term storage, as did specimens from ethylene glycol but not formalin (the latter two being commonly used in pitfall and flight intercept traps). Specimens killed in ethyl acetate vapour and air-dried yielded very degraded DNA which did not successfully PCR. The use of this killing agent is a likely reason for previous reports of inconsistent results obtained from museum specimens, and the now widespread use of critical-point drying of wasps and other insects from alcohol is advocated as a potential source of DNA from rare taxa

Identification of screwworm species by polymerase chain reaction-restriction fragment length polymorphism

Restriction fragment length polymorphisms in polymerase chain reaction amplified fragments (PCR-RFLP) of mitochondrial DNA were used to differentiate species of New World screwworms (Diptera : Calliphoridae) . Twenty-seven restriction enzymes were screened on five regions of mtDNA . Eleven restriction fragment length patterns differentiated New World screwworm, Cochliomyia hominivorax (Coquerel), from secondary screwworm, Cochliomyia macellaria (F.) . Five restriction fragment length patterns were polymorphic in C. hominivorax while all fragment patterns were fixed in C. macellaria. Diagnostic restriction fragment length patterns were used for species diagnosis, whereas intraspecific variable patterns were used to characterize field samples and laboratory strains . The PCR-RFLP technique is flexible with regard to developmental stage of the sample and method of preservation . We were able to characterize specimens of all life stages from egg to adult including larvae preserved in alcohol and pinned adults . PCR-RFLP is rapid and inexpensive, enabling specimens to be characterized within 24 h for less than $2 .50