- Email: [email protected]
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 576 (2016) 820–822
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Research paper
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) Kinga Łukasiewicz a,b, Marek Sanak a, Grzegorz Węgrzyn b,⁎ a b
Division of Molecular Biology and Clinical Genetics, Department of Medicine, Jagiellonian University Medical College, Skawińska 8, 31-066 Cracow, Poland Department of Molecular Biology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland
a r t i c l e
i n f o
Article history: Received 13 October 2015 Received in revised form 7 November 2015 Accepted 11 November 2015 Available online 12 November 2015 Keywords: Apollo butterfly Deformed wings Reduced wings Wingless gene
a b s t r a c t Parnassius apollo (Lepidoptera: Papilionidae) is a butterfly species which was common in Europe in 19th century, but now it is considered as near threatened. Various programs devoted to protect and save P. apollo have been established, between others the one in Pieniny National Park (Poland). An isolated population of this butterfly has been restored there from a small group of 20–30 individuals in early 1990s. However, deformations or reductions of wings occur in this population in a relatively large number of insects, and the cause of this phenomenon is not known. In this report, the occurrence of lesions in the wingless (wg) gene is demonstrated in most of tested butterflies with deformed or reduced wings, but not in normal insects. Although the analyses indicated that wg lesion(s) cannot be the sole cause of the deformed or reduced wings in the population of P. apollo from Pieniny, the discovery that this genetic defect occurs in most of malformed individuals, can be considered as an important step in understanding this phenomenon. © 2015 Elsevier B.V. All rights reserved.
1. Introduction A century ago, Apollo butterfly, Parnassius apollo (Lepidoptera: Papilionidae), was a relatively common species in Europe. However, its distribution and population size have declined severely during the 20th century, and currently this species is considered as near threatened (van Swaay et al., 2010). The history of this butterfly and the problem of its extinction have been analyzed and summarized previously (Nakonieczny et al., 2007). It appears that changes in weather conditions, and particularly weather anomalies, might have major impact on the decline of the Apollo butterfly populations (Łozowski et al., 2014). Various programs devoted to protect and save P. apollo have been established. Some of them were certainly successful. They can be exemplified by the conservation program in Pieniny National Park (Poland) which led to saving an Apollo butterfly population that had declined to about 20–30 individuals in the early 1990s (Witkowski and Adamski, 1996; Witkowski et al., 1997). Nevertheless, restitution of a population from a low number of individuals may be risky due to a relatively high probability of genetic defects in the offspring. This is a phenomenon known as a bottleneck effect which, apart from theoretical calculations, could also be tested experimentally (Jarvis et al., 2011). However, such an experimental evolution methods have also their ⁎ Corresponding author. E-mail address: [email protected] (G. Węgrzyn).
http://dx.doi.org/10.1016/j.gene.2015.11.011 0378-1119/© 2015 Elsevier B.V. All rights reserved.
limitations (Kawecki et al., 2012), and it is important to find how often such processes occur in the nature and what are their real consequences. In the current, restituted population of P. apollo in Pieniny National Park, there are serious problems with often appearance of insects with deformed or severely reduced wings (Adamski and Witkowski, 1999). Deformed wings are of the size similar to that found in normal individuals (an individual with normal wings is depicted in Fig. 1A), but their shape and arrangement are changed (an example is shown in Fig. 1B). Reduced wings are of smaller size than normal ones, sometimes with different morphology (Fig. 1C). There are also insects with extremely reduced wings which are very small, resembling buds rather than a mature organ (Fig. 1D). Although various mutations affecting wings were reported in Apollo butterfly (Descimon, 1988; Pierrat and Descimon, 2011), none of them resulted in phenotypes occurring in the malformed individuals from Pieniny. Therefore, we aimed to test if the wing abnormalities in these butterflies may be caused by mutation(s) present in the large proportion of the population. 2. Materials and methods 2.1. Insects All insects used in this work were from the collection of Pieniny National Park (specimens were collected in years 1991–2007). Butterflies were collected from the natural environment, in the course of the
K. Łukasiewicz et al. / Gene 576 (2016) 820–822
821
Synthesis, Institute of Biochemistry and Biophysics of the Polish Academy of Sciences (Warsaw, Poland). 3. Results
Fig. 1. Examples of P. apollo individuals with normal (panel A), deformed (panel B), reduced (panel C) and extremely reduced (panel D) wings. Photographs made by the authors.
monitoring program, being a part of the P. apollo protection and saving procedures, as well as from the population reared in semi-natural conditions. The permission for the use of this material have been obtained from the Director of Pieniny Natonal Park (permission no. PB-523224/07, topic ID: p0748). For experiments, 7 specimens of Pieris rapae, 5 of Pieris napi, and 32 of P. apollo were studied. Among the P. apollo individuals, 9 had normal wings, 10 had deformed wings, 5 had reduced wings, and 8 had extremely reduced wings. Photographs of examples of individuals from each group are shown in Fig. 1. 2.2. DNA isolation and amplification DNA was isolated from a material withdrawn from legs of investigated insects. The Sherlock AX Purification Kit (A&A Biotechnology) was used according to the manufacturer's instruction. Fragments of genomic DNA, corresponding to particular tested genes, were amplified by PCR with the use of primers which are listed in Table 1. Amplified DNA was separated by agarose gel electrophoresis and analyzed according to Sambrook and Russell (2001). 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 Escherichia coli (Invitrogen). DNA sequencing was conducted commercially in the Laboratory of DNA Sequencing and Oligonucleotide Table 1 Primers used in PCR. Reference
dpp
Kapan et al. (2006)
hh ptc wg
4. Discussion There is a peculiar phenomenon of appearance, with a high frequency, of Apollo butterflies with deformed or reduced wings in the isolated population of this species in Pieniny (southern Poland) (Adamski and Witkowski, 1999). This population has been restituted from a relatively small number (20–30) of individuals (Witkowski et al., 1997) which might suggest the existence of genetic defect(s) leading to such a phenomenon. The results presented in this report strongly suggest the presence of mutations in the wg gene of the malformed P. apollo individuals. On the basis of this analysis, we are not able to determine what kind of mutations there are, as no specific wg product could be found. However, since loss-of-function alleles of wg were found to be embryonic lethal, at least in Drosophila (Nusslein-Volhard and Wieschaus, 1980; Nusslein-Volhard et al., 1984), it is likely that the observed lack of the Table 2 Results of PCR-mediated DNA amplification with the use of indicated templates and primers specific to the wg gene. Species and characteristics
Gene Primers (forward and reverse) 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′ GAG/A TGC/T AAR TGY CAY GGY ATG TCT GG 5′ ACT ICG CRA ACC ART GGA ATG TRC A
In order to analyze potential genetic defects in P. apollo individuals with deformed or reduced wings, total DNA was isolated from samples of legs of either normal (9 butterflies) or malformed (23 butterflies) insects. In addition, for external control experiments, DNA was isolated analogously from wild-type P. rapae (7 butterflies) and P. napi (5 butterflies) individuals. The quality of DNA templates were proved by PCR reactions with primers for amplification of fragments of dpp, hh and ptc genes (Table 1). The product of amplification of the fragment of the wingless (wg) gene, obtained with the wg primers and using DNA from wildtype P. apollo was cloned into a plasmid vector, and nucleotide sequence of the clone has been determined. The obtained sequence (455 bp long) was found (on the basis of Blast search) to be 99% identical to a fragment of the wg gene of both Hyposcada illinissa (Lepidoptera: Papilionidae) and Oleria cyrene (Lepidoptera: Papilionidae). The P. apollo wg gene fragment sequence has been deposited in GenBank (Accession no. HM213842). We have analyzed the presence of the PCR product after reactions with the wg primers when DNA templates were derived from wildtype P. rapae or P. napi (external controls), wild-type P. apollo, and malformed P. apollo with either deformed wings, reduced wings or extremely reduced wings. In all tested samples obtained from P. rapae and P. napi, the specific reaction product has been detected (Table 2). This was the case also in 8 out of 9 samples obtained from wild-type P. apollo. However, completely different results were obtained for malformed P. apollo. No specific wg amplification product could be observed in all samples derived from butterflies with reduced or extremely reduced wings, and such a product was detected only in 1 out of 10 samples from individuals with deformed wings (Table 2).
Kapan et al. (2006) Kapan et al. (2006) Brower and DeSalle (1998)
Number of individuals used for DNA isolation All tested
P. rapae (normal) P. napi (normal) P. apollo (normal) P. apollo (with deformed wings) P. apollo (with reduced wings) P. apollo (with extremely reduced wings)
7 5 9 10 5 8
With wg specific PCR product
Without wg specific PCR product
7 5 8 1 0 0
0 0 1 9 5 8
K. Łukasiewicz et al. / Gene 576 (2016) 820–822
822
wg fragment amplification may result from either a small deletion or point mutation(s). Such lesions could prevent specific annealing of the primer(s) in appropriate DNA site(s), and a lack of PCR-mediated amplification. Nevertheless, it appears that dysfunction of the wg gene may be responsible, at least partially, for the observed deformation and reduction of wings in P. apollo individuals. Since wg has been discovered due to isolation of mutants in the regulatory region of this gene which caused a transformation of the adult wings into thoracic notum (Sharma and Chopra, 1976), and further studies confirmed that the wg gene is required to pattern the Drosophila wings and other adult body structures (for a review, see Swarup and Verheyen, 2012), it is not a surprise that lesions in wg might affect formation of wings in P. apollo. If this is the case, mutation(s) in this gene can be transmitted to the butterfly offspring, resulting in inheritance of the deformation or reduction of wings. Since P. apollo population in Pieniny has been restituted from a small number of individuals, the presence of the mutation in one or a few of them could cause the genetic problem in the total population. On the other hand, it appears unlikely that a single mutation in the wg gene could be responsible for all the phenotypes observed in the investigated Apollo butterfly population. First, the observed malformations are highly variable, from mild deformation of the wings, through their reduction, to almost total absence (Fig. 1). Thus, even if the mutation is the major cause of these developmental problems, their variability strongly suggest that other genetic, epigenetic or environmental factors are still important in modulation of the phenotypes. Second, in one sample derived from a wild-type Apollo butterfly, we could not detect the wg specific amplification product, while the presence of such a product was evident in one sample derived from an individual with deformed wings. These results are against the hypothesis that a single wg lesion might be the cause of the full spectrum of observed phenotypes. In fact, very recent studies indicated that among malformed P. apollo individuals, relatively large proportion lacks a prokaryotic symbiont, Wolbachia sp. (Łukasiewicz et al., 2015). Moreover, mutations is genes coding for laccases (the enzymes catalyzing single-electron oxidations of phenolic or other compounds, and playing roles in cuticle sclerotization and detoxification of phenolderived compounds present in food) were detected in some Apollo butterflies with deformed or reduced wings (Łukasiewicz and Węgrzyn, 2015). Therefore, contribution of other genetic and environmental factors to the phenomenon observed in the population of P. apollo from Pieniny in likely. One cannot also exclude that deformation of wings occurs in individuals which “get stuck” during eclosion. Nevertheless, despite further studies are necessary to identify other factors, agents or conditions affecting development of wings in the population of P. apollo from Pieniny, and to propose a specific mechanism for the observed changes, the discovery that a lesion in the wg gene (preventing specific amplification of the corresponding DNA fragment) occurs in most of malformed individuals, can be considered as an important step in understanding this interesting phenomenon. Conflict of interest The authors declare no conflict of interest.
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-D242-15-1A). The funding agencies had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. References Adamski, P., Witkowski, Z., 1999. Wing deformation in an isolated Carpathian population of Parnassius apollo (Papilionidae: Parnassinae). Nota Lepid. 22, 67–73. Brower, A.V., DeSalle, R., 1998. Patterns of mitochondrial versus nuclear DNA sequence divergence among nymphalid butterflies: the utility of wingless as a source of characters for phylogenetic inference. Insect Mol. Biol. 7, 73–82. Descimon, H., 1988. A mutant affecting wing pattern in Parnassius apollo (Linne) (Lepidoptera: Papilionidae). J. Res. Lepid. 26, 161–172. Jarvis, J.P., Cropp, S.N., Vaughn, T.T., Pletscher, L.S., King-Ellison, K., Adams-Hunt, E., Erickson, C., Cheverud, J.M., 2011. The effect of a population bottleneck on the evolution of genetic variance/covariance structure. J. Evol. Biol. 24, 2139–2152. Kapan, D.D., Flanagan, N.S., Tobler, A., Papa, R., Reed, R.D., Gonzalez, J.A., Restrepo, M.R., Martinez, L., Maldonado, K., Ritschoff, C., Heckel, D.G., McMillan, W.O., 2006. Localization of Müllerian mimicry genes on a dense linkage map of Heliconius erato. Genetics 173, 735–757. Kawecki, T.J., Lenski, R.E., Ebert, D., Hollis, B., Olivieri, I., Whitlock, M.C., 2012. Experimental evolution. Trends Ecol. Evol. 27, 547–560. Łozowski, B., Kędziorski, A., Nakonieczny, M., Łaszczyca, P., 2014. Parnassius apollo lastinstar larvae development prediction by analysis of weather condition as a tool in the species' conservation. C. R. Biol. 337, 325–331. Łukasiewicz, K., Węgrzyn, G., 2015. 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. in press, DOI: 10.18388/abp.2015_1174. Łukasiewicz, K., Sanak, M., Węgrzyn, G., 2015. A lack of Wolbachia-specific DNA in samples from Apollo butterfly (Parnassius Apollo, Lepidoptera: Papilionidae) individuals with deformed or reduced wings. J. Appl. Genet. in press, DOI: 10.1007/s13353-0150318-1. Nakonieczny, M., Kędziorski, A., Michalczyk, K., 2007. Apollo butterfly (Parnassius apollo L.) in Europe — its history, decline and perspectives of conservation. Funct. Ecosyst. Commun. 1, 56–79. Nusslein-Volhard, C., Wieschaus, E., 1980. Mutations affecting segment number and polarity in drosophila. Nature 287, 795–801. Nusslein-Volhard, C., Wiechaus, E., Kluding, H., 1984. Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. I. Zygotic loci on the second chromosome. Rouxs Arch. Dev. Biol. 193, 267–282. Pierrat, V., Descimon, H., 2011. A new wing pattern mutant in the Apollo butterfly, Parnassius apollo (L. 1758) (Lepidoptera: Papilionidae). Ann. Soc. Entomol. Fr. 47, 293–302. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sharma, R.P., Chopra, V.L., 1976. Effect of the wingless (wg1) mutation on wing and haltere development in Drosophila melanogaster. Dev. Biol. 48, 461–465. Swarup, S., Verheyen, E.M., 2012. Wnt/wingless signaling in Drosophila. Cold Spring Harb. Perspect Biol. 4, a007930. van Swaay, C., Wynhoff, I., Verovnik, R., Wiemers, M., López Munguira, M., Maes, D., Sasic, M., Verstrael, T., Warren, M., Settele, J., 2010. Parnassius apollo. The IUCN Red List of Threatened Species. Version 2015.2. bwww.iucnredlist.orgN. Witkowski, Z., Adamski, P., 1996. Decline and rehabilitation of the Apollo butterfly Parnassius apollo (Linnaeus, 1758) in the Pieniny National Park (Polish Carpathians). In: Settele, J., Margules, C.R., Poschlod, P., Henle, K. (Eds.), Species Survival in Fragmented Landscapes. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 7–14. Witkowski, Z., Adamski, P., Kosior, A., Płonka, P., 1997. Extinction and reintroduction of Parnassius apollo in the Pieniny National Park (Polish Carpathians). Biologia 52, 199–208.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Research paper
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) Kinga Łukasiewicz a,b, Marek Sanak a, Grzegorz Węgrzyn b,⁎ a b
Division of Molecular Biology and Clinical Genetics, Department of Medicine, Jagiellonian University Medical College, Skawińska 8, 31-066 Cracow, Poland Department of Molecular Biology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland
a r t i c l e
i n f o
Article history: Received 13 October 2015 Received in revised form 7 November 2015 Accepted 11 November 2015 Available online 12 November 2015 Keywords: Apollo butterfly Deformed wings Reduced wings Wingless gene
a b s t r a c t Parnassius apollo (Lepidoptera: Papilionidae) is a butterfly species which was common in Europe in 19th century, but now it is considered as near threatened. Various programs devoted to protect and save P. apollo have been established, between others the one in Pieniny National Park (Poland). An isolated population of this butterfly has been restored there from a small group of 20–30 individuals in early 1990s. However, deformations or reductions of wings occur in this population in a relatively large number of insects, and the cause of this phenomenon is not known. In this report, the occurrence of lesions in the wingless (wg) gene is demonstrated in most of tested butterflies with deformed or reduced wings, but not in normal insects. Although the analyses indicated that wg lesion(s) cannot be the sole cause of the deformed or reduced wings in the population of P. apollo from Pieniny, the discovery that this genetic defect occurs in most of malformed individuals, can be considered as an important step in understanding this phenomenon. © 2015 Elsevier B.V. All rights reserved.
1. Introduction A century ago, Apollo butterfly, Parnassius apollo (Lepidoptera: Papilionidae), was a relatively common species in Europe. However, its distribution and population size have declined severely during the 20th century, and currently this species is considered as near threatened (van Swaay et al., 2010). The history of this butterfly and the problem of its extinction have been analyzed and summarized previously (Nakonieczny et al., 2007). It appears that changes in weather conditions, and particularly weather anomalies, might have major impact on the decline of the Apollo butterfly populations (Łozowski et al., 2014). Various programs devoted to protect and save P. apollo have been established. Some of them were certainly successful. They can be exemplified by the conservation program in Pieniny National Park (Poland) which led to saving an Apollo butterfly population that had declined to about 20–30 individuals in the early 1990s (Witkowski and Adamski, 1996; Witkowski et al., 1997). Nevertheless, restitution of a population from a low number of individuals may be risky due to a relatively high probability of genetic defects in the offspring. This is a phenomenon known as a bottleneck effect which, apart from theoretical calculations, could also be tested experimentally (Jarvis et al., 2011). However, such an experimental evolution methods have also their ⁎ Corresponding author. E-mail address: [email protected] (G. Węgrzyn).
http://dx.doi.org/10.1016/j.gene.2015.11.011 0378-1119/© 2015 Elsevier B.V. All rights reserved.
limitations (Kawecki et al., 2012), and it is important to find how often such processes occur in the nature and what are their real consequences. In the current, restituted population of P. apollo in Pieniny National Park, there are serious problems with often appearance of insects with deformed or severely reduced wings (Adamski and Witkowski, 1999). Deformed wings are of the size similar to that found in normal individuals (an individual with normal wings is depicted in Fig. 1A), but their shape and arrangement are changed (an example is shown in Fig. 1B). Reduced wings are of smaller size than normal ones, sometimes with different morphology (Fig. 1C). There are also insects with extremely reduced wings which are very small, resembling buds rather than a mature organ (Fig. 1D). Although various mutations affecting wings were reported in Apollo butterfly (Descimon, 1988; Pierrat and Descimon, 2011), none of them resulted in phenotypes occurring in the malformed individuals from Pieniny. Therefore, we aimed to test if the wing abnormalities in these butterflies may be caused by mutation(s) present in the large proportion of the population. 2. Materials and methods 2.1. Insects All insects used in this work were from the collection of Pieniny National Park (specimens were collected in years 1991–2007). Butterflies were collected from the natural environment, in the course of the
K. Łukasiewicz et al. / Gene 576 (2016) 820–822
821
Synthesis, Institute of Biochemistry and Biophysics of the Polish Academy of Sciences (Warsaw, Poland). 3. Results
Fig. 1. Examples of P. apollo individuals with normal (panel A), deformed (panel B), reduced (panel C) and extremely reduced (panel D) wings. Photographs made by the authors.
monitoring program, being a part of the P. apollo protection and saving procedures, as well as from the population reared in semi-natural conditions. The permission for the use of this material have been obtained from the Director of Pieniny Natonal Park (permission no. PB-523224/07, topic ID: p0748). For experiments, 7 specimens of Pieris rapae, 5 of Pieris napi, and 32 of P. apollo were studied. Among the P. apollo individuals, 9 had normal wings, 10 had deformed wings, 5 had reduced wings, and 8 had extremely reduced wings. Photographs of examples of individuals from each group are shown in Fig. 1. 2.2. DNA isolation and amplification DNA was isolated from a material withdrawn from legs of investigated insects. The Sherlock AX Purification Kit (A&A Biotechnology) was used according to the manufacturer's instruction. Fragments of genomic DNA, corresponding to particular tested genes, were amplified by PCR with the use of primers which are listed in Table 1. Amplified DNA was separated by agarose gel electrophoresis and analyzed according to Sambrook and Russell (2001). 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 Escherichia coli (Invitrogen). DNA sequencing was conducted commercially in the Laboratory of DNA Sequencing and Oligonucleotide Table 1 Primers used in PCR. Reference
dpp
Kapan et al. (2006)
hh ptc wg
4. Discussion There is a peculiar phenomenon of appearance, with a high frequency, of Apollo butterflies with deformed or reduced wings in the isolated population of this species in Pieniny (southern Poland) (Adamski and Witkowski, 1999). This population has been restituted from a relatively small number (20–30) of individuals (Witkowski et al., 1997) which might suggest the existence of genetic defect(s) leading to such a phenomenon. The results presented in this report strongly suggest the presence of mutations in the wg gene of the malformed P. apollo individuals. On the basis of this analysis, we are not able to determine what kind of mutations there are, as no specific wg product could be found. However, since loss-of-function alleles of wg were found to be embryonic lethal, at least in Drosophila (Nusslein-Volhard and Wieschaus, 1980; Nusslein-Volhard et al., 1984), it is likely that the observed lack of the Table 2 Results of PCR-mediated DNA amplification with the use of indicated templates and primers specific to the wg gene. Species and characteristics
Gene Primers (forward and reverse) 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′ GAG/A TGC/T AAR TGY CAY GGY ATG TCT GG 5′ ACT ICG CRA ACC ART GGA ATG TRC A
In order to analyze potential genetic defects in P. apollo individuals with deformed or reduced wings, total DNA was isolated from samples of legs of either normal (9 butterflies) or malformed (23 butterflies) insects. In addition, for external control experiments, DNA was isolated analogously from wild-type P. rapae (7 butterflies) and P. napi (5 butterflies) individuals. The quality of DNA templates were proved by PCR reactions with primers for amplification of fragments of dpp, hh and ptc genes (Table 1). The product of amplification of the fragment of the wingless (wg) gene, obtained with the wg primers and using DNA from wildtype P. apollo was cloned into a plasmid vector, and nucleotide sequence of the clone has been determined. The obtained sequence (455 bp long) was found (on the basis of Blast search) to be 99% identical to a fragment of the wg gene of both Hyposcada illinissa (Lepidoptera: Papilionidae) and Oleria cyrene (Lepidoptera: Papilionidae). The P. apollo wg gene fragment sequence has been deposited in GenBank (Accession no. HM213842). We have analyzed the presence of the PCR product after reactions with the wg primers when DNA templates were derived from wildtype P. rapae or P. napi (external controls), wild-type P. apollo, and malformed P. apollo with either deformed wings, reduced wings or extremely reduced wings. In all tested samples obtained from P. rapae and P. napi, the specific reaction product has been detected (Table 2). This was the case also in 8 out of 9 samples obtained from wild-type P. apollo. However, completely different results were obtained for malformed P. apollo. No specific wg amplification product could be observed in all samples derived from butterflies with reduced or extremely reduced wings, and such a product was detected only in 1 out of 10 samples from individuals with deformed wings (Table 2).
Kapan et al. (2006) Kapan et al. (2006) Brower and DeSalle (1998)
Number of individuals used for DNA isolation All tested
P. rapae (normal) P. napi (normal) P. apollo (normal) P. apollo (with deformed wings) P. apollo (with reduced wings) P. apollo (with extremely reduced wings)
7 5 9 10 5 8
With wg specific PCR product
Without wg specific PCR product
7 5 8 1 0 0
0 0 1 9 5 8
K. Łukasiewicz et al. / Gene 576 (2016) 820–822
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wg fragment amplification may result from either a small deletion or point mutation(s). Such lesions could prevent specific annealing of the primer(s) in appropriate DNA site(s), and a lack of PCR-mediated amplification. Nevertheless, it appears that dysfunction of the wg gene may be responsible, at least partially, for the observed deformation and reduction of wings in P. apollo individuals. Since wg has been discovered due to isolation of mutants in the regulatory region of this gene which caused a transformation of the adult wings into thoracic notum (Sharma and Chopra, 1976), and further studies confirmed that the wg gene is required to pattern the Drosophila wings and other adult body structures (for a review, see Swarup and Verheyen, 2012), it is not a surprise that lesions in wg might affect formation of wings in P. apollo. If this is the case, mutation(s) in this gene can be transmitted to the butterfly offspring, resulting in inheritance of the deformation or reduction of wings. Since P. apollo population in Pieniny has been restituted from a small number of individuals, the presence of the mutation in one or a few of them could cause the genetic problem in the total population. On the other hand, it appears unlikely that a single mutation in the wg gene could be responsible for all the phenotypes observed in the investigated Apollo butterfly population. First, the observed malformations are highly variable, from mild deformation of the wings, through their reduction, to almost total absence (Fig. 1). Thus, even if the mutation is the major cause of these developmental problems, their variability strongly suggest that other genetic, epigenetic or environmental factors are still important in modulation of the phenotypes. Second, in one sample derived from a wild-type Apollo butterfly, we could not detect the wg specific amplification product, while the presence of such a product was evident in one sample derived from an individual with deformed wings. These results are against the hypothesis that a single wg lesion might be the cause of the full spectrum of observed phenotypes. In fact, very recent studies indicated that among malformed P. apollo individuals, relatively large proportion lacks a prokaryotic symbiont, Wolbachia sp. (Łukasiewicz et al., 2015). Moreover, mutations is genes coding for laccases (the enzymes catalyzing single-electron oxidations of phenolic or other compounds, and playing roles in cuticle sclerotization and detoxification of phenolderived compounds present in food) were detected in some Apollo butterflies with deformed or reduced wings (Łukasiewicz and Węgrzyn, 2015). Therefore, contribution of other genetic and environmental factors to the phenomenon observed in the population of P. apollo from Pieniny in likely. One cannot also exclude that deformation of wings occurs in individuals which “get stuck” during eclosion. Nevertheless, despite further studies are necessary to identify other factors, agents or conditions affecting development of wings in the population of P. apollo from Pieniny, and to propose a specific mechanism for the observed changes, the discovery that a lesion in the wg gene (preventing specific amplification of the corresponding DNA fragment) occurs in most of malformed individuals, can be considered as an important step in understanding this interesting phenomenon. Conflict of interest The authors declare no conflict of interest.
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-D242-15-1A). The funding agencies had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. References Adamski, P., Witkowski, Z., 1999. Wing deformation in an isolated Carpathian population of Parnassius apollo (Papilionidae: Parnassinae). Nota Lepid. 22, 67–73. Brower, A.V., DeSalle, R., 1998. Patterns of mitochondrial versus nuclear DNA sequence divergence among nymphalid butterflies: the utility of wingless as a source of characters for phylogenetic inference. Insect Mol. Biol. 7, 73–82. Descimon, H., 1988. A mutant affecting wing pattern in Parnassius apollo (Linne) (Lepidoptera: Papilionidae). J. Res. Lepid. 26, 161–172. Jarvis, J.P., Cropp, S.N., Vaughn, T.T., Pletscher, L.S., King-Ellison, K., Adams-Hunt, E., Erickson, C., Cheverud, J.M., 2011. The effect of a population bottleneck on the evolution of genetic variance/covariance structure. J. Evol. Biol. 24, 2139–2152. Kapan, D.D., Flanagan, N.S., Tobler, A., Papa, R., Reed, R.D., Gonzalez, J.A., Restrepo, M.R., Martinez, L., Maldonado, K., Ritschoff, C., Heckel, D.G., McMillan, W.O., 2006. Localization of Müllerian mimicry genes on a dense linkage map of Heliconius erato. Genetics 173, 735–757. Kawecki, T.J., Lenski, R.E., Ebert, D., Hollis, B., Olivieri, I., Whitlock, M.C., 2012. Experimental evolution. Trends Ecol. Evol. 27, 547–560. Łozowski, B., Kędziorski, A., Nakonieczny, M., Łaszczyca, P., 2014. Parnassius apollo lastinstar larvae development prediction by analysis of weather condition as a tool in the species' conservation. C. R. Biol. 337, 325–331. Łukasiewicz, K., Węgrzyn, G., 2015. 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. in press, DOI: 10.18388/abp.2015_1174. Łukasiewicz, K., Sanak, M., Węgrzyn, G., 2015. A lack of Wolbachia-specific DNA in samples from Apollo butterfly (Parnassius Apollo, Lepidoptera: Papilionidae) individuals with deformed or reduced wings. J. Appl. Genet. in press, DOI: 10.1007/s13353-0150318-1. Nakonieczny, M., Kędziorski, A., Michalczyk, K., 2007. Apollo butterfly (Parnassius apollo L.) in Europe — its history, decline and perspectives of conservation. Funct. Ecosyst. Commun. 1, 56–79. Nusslein-Volhard, C., Wieschaus, E., 1980. Mutations affecting segment number and polarity in drosophila. Nature 287, 795–801. Nusslein-Volhard, C., Wiechaus, E., Kluding, H., 1984. Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. I. Zygotic loci on the second chromosome. Rouxs Arch. Dev. Biol. 193, 267–282. Pierrat, V., Descimon, H., 2011. A new wing pattern mutant in the Apollo butterfly, Parnassius apollo (L. 1758) (Lepidoptera: Papilionidae). Ann. Soc. Entomol. Fr. 47, 293–302. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sharma, R.P., Chopra, V.L., 1976. Effect of the wingless (wg1) mutation on wing and haltere development in Drosophila melanogaster. Dev. Biol. 48, 461–465. Swarup, S., Verheyen, E.M., 2012. Wnt/wingless signaling in Drosophila. Cold Spring Harb. Perspect Biol. 4, a007930. van Swaay, C., Wynhoff, I., Verovnik, R., Wiemers, M., López Munguira, M., Maes, D., Sasic, M., Verstrael, T., Warren, M., Settele, J., 2010. Parnassius apollo. The IUCN Red List of Threatened Species. Version 2015.2. bwww.iucnredlist.orgN. Witkowski, Z., Adamski, P., 1996. Decline and rehabilitation of the Apollo butterfly Parnassius apollo (Linnaeus, 1758) in the Pieniny National Park (Polish Carpathians). In: Settele, J., Margules, C.R., Poschlod, P., Henle, K. (Eds.), Species Survival in Fragmented Landscapes. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 7–14. Witkowski, Z., Adamski, P., Kosior, A., Płonka, P., 1997. Extinction and reintroduction of Parnassius apollo in the Pieniny National Park (Polish Carpathians). Biologia 52, 199–208.