Specific primers used in PCR.
Abstract
Yersinia pseudotuberculosis is a bacterium pathogenic to humans and other mammals; however, its insecticidal activity has also been documented in laboratory studies. A small population of Apollo butterfly (Parnassius apollo), reconstituted from less than 30 individuals in 1990s, occurs in Pieniny National Park (Poland). In this report, we demonstrate that a DNA fragment specific to Y. pseudotuberculosis could be detected in 40% of biological samples isolated from insects belonging to the Apollo butterfly population. Although Y. pseudotuberculosis DNA occurred in both normal and malformed insects, the difference between the fractions of infected individuals was statistically significant (p = 0.044 in the Fisher's exact test). No such DNA could be detected in analogous samples from other butterflies (Pieris napi, Pieris rapae, and Zerynthia polyxena) occurring in separate habitats (either a meadow near the city of Cracow, Poland, or in a mountain region of Greece). It is suggested that infection with Y. pseudotuberculosis might weaken the general condition of the P. apollo population from Pieniny and contribute to the appearance of developmental abnormalities of the butterflies. Thus, it appears that Y. pseudotuberculosis infections of insects may be of biological significance in natural environment.
Keywords
- Apollo butterfly
- deformed wings
- reduced wings
- Yersinia pseudotuberculosis
- isolated butterfly population
1. Introduction
Until recently, the cause of the malformations in
2. Materials and methods
2.1. Insects
Insects used in this work were either withdrawn from a meadow near the city of Cracow, Poland (individuals of
2.2. DNA isolation and amplification
A material extracted from legs of investigated insects was used for DNA studies. This material was subjected to wash using deionized water before the procedure to avoid environmental contamination. The procedure was conducted by employing the Sherlock AX Purification Kit (A&A Biotechnology), according to the manufacturer's instruction. Following PCR‐mediated amplification of specific DNA fragments (using primers listed in Table 1), they were separated by agarose gel electrophoresis and analyzed as described previously [14].
2.3. DNA cloning and sequencing
Selected products of DNA amplification were cloned into a plasmid vector by using the TOPO TA Cloning Kit Dual Promoter (with pCR II‐TOPO vector) with One Shot TOPO10F’ Chemically Competent
Gene | Primers (forward and reverse) | References |
---|---|---|
5′ AGA GAA CGT GGC GAG ACA CTG 5′ GAG GAA AGT TGC GTA GGA ACG |
[24] | |
5′ AAG GAA AAA CTG AAT ACG CTG GC 5′ CGA GAC GCC CCA ACT TTC C |
[24] | |
5′ CTC CGA AGA AGG TCT GCC GCA AG 5′ AAT TCG TGC TCG TCG TAT TTT C |
[24] | |
5′ TAA GGG TAC TAT CGC GGC GGA 5′ CGT GAA ATT AAC CGT CAC ACT |
[15] |
2.4. Statistical analysis
Since only a low number of samples could be analyzed (due to restrictions caused by regulations of
3. Results
In the course of our studies on the reasons of deformation and reduction of wings in the population of
Using primers specifically designed to identify
Species and characteristics | Number of individuals used for DNA isolation | ||
---|---|---|---|
All tested | With PCR product |
Without PCR product |
|
3 | 0 | 3 | |
4 | 0 | 4 | |
2 | 0 | 2 | |
12 | 3 | 9 | |
3 | 3 | 0 |
4. Discussion
Pathogenicity of
The presence of
The question appears what might be effects of infections of Apollo butterflies with
Acknowledgments
This work was supported by Ministry of Science and Higher Education (Poland) (project Grant No. N N304 339633 to Kinga Łukasiewicz) and the University of Gdańsk (task Grant No. 530‐L140‐D242‐16‐1A). The authors declare no conflict of interest.
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