Specific primers used in PCR.
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.
- Apollo butterfly
- deformed wings
- reduced wings
- Yersinia pseudotuberculosis
- isolated butterfly population
Until recently, the cause of the malformations in
2. Materials and methods
Insects used in this work were either withdrawn from a meadow near the city of Cracow, Poland (individuals of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 .
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
|5′ AAG GAA AAA CTG AAT ACG CTG GC|
5′ CGA GAC GCC CCA ACT TTC C
|5′ CTC CGA AGA AGG TCT GCC GCA AG|
5′ AAT TCG TGC TCG TCG TAT TTT C
|5′ TAA GGG TAC TAT CGC GGC GGA|
5′ CGT GAA ATT AAC CGT CAC ACT
2.4. Statistical analysis
Since only a low number of samples could be analyzed (due to restrictions caused by regulations of
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 |
The presence of
The question appears what might be effects of infections of Apollo butterflies with
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.
Erickson DL, Waterfield NR, Vadyvaloo V, Long D, Fischer ER, Ffrench‐Constant R, Hinnebusch BJ. Acute oral toxicity of Yersinia pseudotuberculosisto fleas: implications for the evolution of vector‐borne transmission of plague. Cell Microbiol. 2007; 9:2658–2666.
Waterfield N, Hares M, Hinchliffe S, Wren B, Ffrench‐Constant R. The insect toxin complex of Yersinia. Adv Exp Med Biol. 2007; 603:247–257.
Pinheiro VB, Ellar DJ. Expression and insecticidal activity of Yersinia pseudotuberculosisand Photorhabdus luminescenstoxin complex proteins. Cell Microbiol. 2007; 9:2372–2380.
Champion OL, Cooper IA, James SL, Ford D, Karlyshev A, Wren BW, Duffield M, Oyston PC, Titball RW. Galleria mellonellaas an alternative infection model for Yersinia pseudotuberculosis. Microbiology. 2009; 155:1516–1522.
van Swaay C, Wynhoff I, Verovnik R, Wiemers M, López Munguira M, Maes D, Sasic M, Verstrael T, Warren M, Settele J Parnassius apollo. The IUCN Red List of Threatened Species. 2010. Version 2015.2. <www.iucnredlist.org>
Nakonieczny M, Kędziorski A, Michalczyk K. Apollo butterfly ( Parnassius apolloL.) in Europe – its history, decline and perspectives of conservation. Funct Ecosyst Commun. 2007; 1:56–79.
Łozowski B, Kędziorski A, Nakonieczny M, Łaszczyca P. Parnassius apollolast‐instar larvae development prediction by analysis of weather condition as a tool in the species’ conservation. C R Biol. 2014; 337:325–331.
Witkowski Z, Adamski P. Decline and rehabilitation of the Apollo butterfly Parnassius apollo(Linnaeus, 1758) in the Pieniny National Park (Polish Carpathians). In: Settele J, Margules CR, Poschlod P, Henle K, editors. Species Survival in Fragmented Landscapes. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1996. pp. 7–14.
Witkowski Z, Adamski P, Kosior A, Płonka P. Extinction and reintroduction of Parnassius apolloin the Pieniny National Park (Polish Carpathians). Biologia. 1997; 52:199–208.
Adamski P, Witkowski Z. Wing deformation in an isolated Carpathian population of Parnassius apollo(Papilionidae: Parnassinae). Nota Lepid. 1999; 22:67–73.
Łukasiewicz K, Sanak M, Węgrzyn G. Lesions in the wingless gene of the Apollo butterfly ( Parnassius apollo, Lepidoptera: Papilionidae) individuals with deformed or reduced wings, coming from the isolated population in Pieniny (Poland). Gene. 2016; 576:820–822.
Łukasiewicz K, Węgrzyn G. Changes is genes coding for laccases 1 and 2 may contribute to deformation and reduction of wings in apollo butterfly ( Parnassius apollo, Lepidoptera: Papilionidae) from the isolated population in Pieniny National Park (Poland). Acta Biochim Pol. 2016; 63:177–180.
Łukasiewicz K, Sanak M, Węgrzyn G. A lack of Wolbachia‐specific DNA in samples from apollo butterfly ( Parnassius apollo, Lepidoptera: Papilionidae) individuals with deformed or reduced wings. J Appl Genet. 2016; 57:271–274.
Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001.
Nakajima H, Inoue M, Mori T, Itoh K, Arakawa E, Watanabe H. Detection and identification of Yersinia pseudotuberculosisand pathogenic Yersinia enterocoliticaby an improved polymerase chain reaction method. J Clin Microbiol. 1992;30:2484–2486.
Philbey AW, Glastonbury JR, Links IJ, Matthews LM. Yersiniaspecies isolated from sheep with enterocolitis. Aust Vet J. 1991; 68:108–110.
Slee KJ, Skilbeck NW. Epidemiology of Yersinia pseudotuberculosisand Y. enterocoliticainfections in sheep in Australia. J Clin Microbiol. 1992; 30:712–715.
Severini M, Ranucci D, Miraglia D, Cenci Goga BT. Pseudotuberculosis in sheep as a concern of veterinary public health. Vet Res Commun. 2003; 27(Suppl 1) :315–318.
Magistrali CF, Cucco L, Pezzotti G, Farneti S, Cambiotti V, Catania S, Prati P, Fabbi M, Lollai S, Mangili P, Sebastiani C, Bano L, Dionisi AM, Luzzi I. Characterisation of Yersinia pseudotuberculosisisolated from animals with yersiniosis during 1996‐2013 indicates the presence of pathogenic and Far Eastern strains in Italy. Vet Microbiol. 2015; 180:161–166.
Andrzejewska L. Analysis of a sheep pasture ecosystem in the Pieniny Mountains (the Carpathians). V. Herbivores and their effect on plant production. Ekol Pol. 1974; 22:527–534.
Czerwiński Z, Jakubczyk H, Tatur A, Traczyk T. Analysis of a sheep pasture ecosystem in the Pieniny Mountains (the Carpathians). VII. The effect of penning‐up sheep on soil, microflora and vegetation. Ekol Pol. 1974; 22:547–558.
Jakubczyk H. Analysis of a sheep pasture ecosystem in the Pieniny Mountains (the Carpathians). VIII. Development of microflora in dung and in soil of a spring sheep‐fold. Ekol Pol. 1974; 22:559–568.
Bresolin G, Morgan JA, Ilgen D, Scherer S, Fuchs TM. Low temperature‐induced insecticidal activity of Yersinia enterocolitica. Mol Microbiol. 2006; 59:503–512.
Kapan DD, Flanagan NS, Tobler A, Papa R, Reed RD, Gonzalez JA, Restrepo MR, Martinez L, Maldonado K, Ritschoff C, Heckel DG, McMillan WO. Localization of Müllerian mimicry genes on a dense linkage map of Heliconius erato. Genetics. 2006; 173:735–757.