RESUMO
The Phoridae are a family of small, hump-backed flies, dominating in post-fire areas. Some of these flies are probably able to survive a fire as an egg, larva, or pupa, and may be adapted to the fire-altered environment at the genomic level. In this study, we describe the influence of short-term temperature treatment on the expression of seven heat shock protein genes in the third-instar larvae and imagoes of a scuttle fly Megaselia scalaris-one of the cosmopolitan and polyphagous phorids. In terms of the response to temperature treatment, these genes tested against tubulin as a reference split into three classes. The first class consists of hsp22 (larvae), hsp23 (larvae), and hsp26 (both larvae and imagoes), and is upregulated at the lowest temperature (33°C). The second class consists of hsp22 (imagoes), hsp23 (imagoes), hsp40 (larvae and imagoes), and hsp70 (larvae and imagoes), and is upregulated or induced at 37°C. Expression of genes of the third class (hsp27 and hsp83-larvae and imagoes) increased after treatment at 41°C temperature. Expression of the first two classes of genes occurred at a temperature lower than the LT50 of larvae and imagoes. The fact that there is a gap between the temperature upregulating hsp genes and the temperature leading to the loss of viability suggests that not only the level of hsp gene expression but also the temperature at which gene expression increased is important in an adaptation of M. scalaris to harsh environment. J. Exp. Zool. 323A: 704-713, 2015. © 2015 Wiley Periodicals, Inc.
RESUMO
We describe here the successful coupling of real-time polymerase chain reaction (PCR) and high-resolution melting (HRM) analysis to rapidly identify 15 forensically important species of blowfly from the family Calliphoridae (Diptera), which occur in Poland. Two short regions (119 and 70 base pairs, respectively) of cytochrome oxidase gene subunit I with sufficient sequence diversity were selected. In the case of lacking taxa (e.g., reference species) these amplicons can be synthesized using sequences deposited in gene banks. The technique utilizes low template DNA concentration and is highly reproducible. The melting profile was not altered up to a 10,000-fold difference in DNA template concentration (ranging from 5 pg to 50 ng). The several HRM runs performed on different specimens from Poland belonging to the same species and on different days resulted in only minor variations in the amplification curves and in melting temperatures of the peaks. Intraspecific variation in a larger scale was tested using synthesized oligonucleotides from cosmopolitan Lucilia illustris originating from Poland, France, Great Britain, India, and USA. As HRM PCR analysis is sensitive to even single base changes, all geographic variants of this species were identified. This technique is also cost-effective and simple, and it may even be used by non-geneticists. A working protocol was ultimately constructed for the purpose of rapid and accurate species identification in most countries in Europe regardless of which stage or which part of a blowfly was collected.