Research Article |
Corresponding author: Attila Takács ( molyirto@gmail.com ) Academic editor: Lauri Kaila
© 2022 Attila Takács, Csaba Szabóky, Gusztáv Boldog, Sándor Jordán, Miklós Bozsó, Dávid Fülöp, Balázs Tóth.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Takács A, Szabóky C, Boldog G, Jordán S, Bozsó M, Fülöp D, Tóth B (2022) Biology and DNA barcode analysis of Coleophora lessinica Baldizzone, 1980 and Coleophora impalella Toll, 1961 (Lepidoptera, Coleophoridae) with description of their larval cases. Nota Lepidopterologica 45: 191-205. https://doi.org/10.3897/nl.45.80106
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Host plants and cases of several species in the genus Coleophora Hübner, 1822 have remained unknown until now, while the latter structures provide important characters for identification. Host plants and cases of Coleophora lessinica Baldizzone, 1980 and Coleophora impalella Toll, 1961 were discovered by the authors and are presented here for the first time. New data on the distribution and biology of the two species in Hungary are given. Coleophora lessinica is recorded as new for the fauna of Romania. DNA barcode sequencing was performed for both Coleophora species and loaded into the BOLD System and to GenBank.
Members of the family Coleophoridae are distributed worldwide but the number of described species is highest in the Palaearctic region. The family currently includes more than 1500 described species in total (
Based on the latest phylogenetic research (
Knowledge of the biology of Coleophora species is well-known in northern Europe but rather limited in southern Europe. The spectrum of host plants is wide and includes Asteraceae, Betulaceae, Caryophyllaceae, Cistaceae, Chenopodiaceae, Ericaceae, Juncacae, Fabaceae Lamiaceae, Poaceae, Rhamnaceae, Rosaceae, but the host plants of many species are still unknown (
Coleophora lessinica Baldizzone, 1980 is a southern European species described on the basis of specimens collected in the Lessini mountain near Verona (
Coleophora impalella Toll, 1961 was described from Southern Russia (
DNA based identification is often useful, not just for species identification by non-specialists, but also to reveal association between different life stages and sexes of the same species which otherwise might be a very time demanding task. Here we give some details about the biology of two Coleophora species and describe their larval cases.
All field work was undertaken in Hungary and in Romania near the Hungarian border (Map
Larvae of the studied species, including overwintering stages, were reared on their host plants planted in the gardens of the authors. Escape of larvae was prevented with a tulle bag attached around the host plant. Shortly before the expected time of emergence each case was put in a separate plastic vial to maintain appropriate humidity.
Specimens are deposited in the private collections of G. Baldizzone, Cs. Szabóky and A. Takács. Three specimens of Coleophora impalella Toll, 1961 are to be deposited in the Hungarian Natural History Museum after the publication of this paper.
Bldz: genitalia preparation of Giorgio Baldizzone (Asti, Italy)
IgR: genitalia preparation of Ignác Richter (Malá Čausa, Slovakia)
TB: genitalia preparation of Balázs Tóth (
Images of intact larvae, adults and larval damage were prepared with a Canon 450 D camera, applied to a Carl Zeiss Stemi-2000 binocular stereomicroscope; they were edited with software Adobe Photosop CS6. The habitat image was taken with a Sony A7R2 camera.
Methods for preparation of the genitalia follow van
Images were adjusted with the software Adobe Photoshop CS6.
Four larvae of the two species were selected for molecular analyses (Table
Species | Host plant | Locality | GPS coordinates | Date of collection | Collector | Stage | GenBank Accession number |
---|---|---|---|---|---|---|---|
C. lessinica | Artemisia alba | Csákberény | 47°20'49"N, 18°21'19"E | 12.x.2018 | Attila Takács | Larva | MZ662875 |
C. lessinica | Artemisia alba | Gánt-Gránás | 47°21'11"N, 18°22'17"E | 09.x.2018 | Attila Takács | Larva | MZ664319 |
C. lessinica | Artemisia alba | Nagyharsány | 45°51'24"N, 18°24'52"E | 04.xi.2018 | Attila Takács | Larva | MZ662874 |
C. impalella | Galatella sedifolia | Szabadkígyós, Róka kaszinó | 46°34'54"N, 21°05'00"E | 17.viii.2021 | Attila Takács | Larva | OK329945 |
Amplification of the standard COI barcode region was performed with the primers LCO-1490 and HCO-2198 (
To identify the larvae, two independent approaches were used. First nblast search was carried out in GenBank (www.ncbi.nlm.nih.gov/Genbank) optimized for highly similar sequences (megablast) (Accessed on 11/12/2021). Also, the identification engine of the BOLD homepage (www.barcodinglife.org) was used to search in the species level barcode library (Accessed on 11/12/2021). The graphical results obtained were visualised and edited with FigTree 1.4.4 (
Rearing young larvae is difficult and cumbersome but has the advantage of limiting parasitism compared with starting with more advanced larvae. However, young larvae are very sensitive to the quality and quantity of food as well as to disturbance. Larvae in closed containers without ventilation are easily killed by mould, whereas in an open container the food plant easily becomes too dry to be palatable.
Perhaps the most sensitive period is when the larva changes its case. Natural conditions should be simulated as much as possible to enhance rearing success. The best approximation is to grow the host plants in a garden setting, place cases on them, and cover them with tulle bags to prevent larvae from escaping and to protect them from parasitoids. Success is not assured even when larvae reach the final L5 stage at which point they need to make a U-turn inside the case in preparation for pupation; many larvae fail to perform this turn and die. After overwintering, the larval stage lasts for an additional 4–5 months, with pupation taking place only 3–4 weeks before emergence of the adult. Despite of these difficulties, rearing is still the most efficient way to study the biology of these species.
1 ♀, Hungary: Fejér county, Kőszárhegy, 16.viii.2020, light trap. 1 ♀, same locality, 17.viii.2020, light trap; det. Giorgio Baldizzone (Asti), slide No. Bldz 17234 (Figs
Larval morphology of Coleophora lessinica. 1. Head and thorax, dorsal view; 2. Head and thorax, lateral view; 3. Head and thorax, ventral view; 4. Prolegs, ventral view; 5. Anal plate, dorso-lateral view. Scale bar: 1 mm, cranial direction top, caudal direction bottom, photos by A. Takács.
Hungary: 11 cases, Fejér county, Gánt-Gránás 09.x.2018, on Artemisia alba Turra, leg. A. Takács. 23 cases, same locality but 15.x.2019, on Artemisia alba, leg. A. Takács. 15 cases, same locality but 22.x.2020, on A. alba, leg. A. Takács. 25 cases, Fejér county, Csákberény, Bucka-hegy 10.x.2018, on A. alba, leg. A. Takács. 45 cases, same locality but 16.x.2019, on A. alba. leg. A. Takács. 10 cases, same locality but 22.x.2020, on A. alba, leg. A. Takács. 5 cases, Csákvár, Haraszt-hegy, 15.x.2021, on A. alba, leg. A Takács. 35 cases, Baranya county, Nagyharsány, Szársomlyó, 11.xi.2018, on A. alba subsp. canescens, leg. Cs. Szabóky, B. Tóth. 5 cases, same locality but 05.x.2019, on A. alba subsp. canescens, leg. Cs. Szabóky, B. Tóth. 3 cases, same locality but 22.x.2020, on A. alba subsp. canescens, leg. Cs. Szabóky, B. Tóth. 3 cases, Veszprém county, Balatonfüred, Nagymező, 10.x.2020, on A. alba, leg. Cs. Szabóky. 12 cases, Várpalota, Barbély-völgy, 18.x.2018, on A. alba, leg. Cs. Szabóky.
1 ♂, Hungary: Békés county, Bélmegyer, Fás puszta 29.iii.2021, ex larva, on Galatella sedifolia (L.), leg. A. Takács. 1 ♀, same data but 31.iii.2021. 1 ♀, same data but 13.iv.2021. 1 ♂, same data but 03.v.2021. 3 ♂ and 5 ♀, same locality but 12.v.2021, leg. Cs. Szabóky and A. Takács. 2 ♂ and 8 ♀, Békés county, Szabadkígyós, Kígyósi puszta, 12.v.2021, leg. Cs. Szabóky and A. Takács, slide Nos IgR31860 (Fig.
Genitalia of C. impalella and C. lessinica. 6. Male genitalia of C. impalella, slide No. IgR 31860 (phallotheca right); 7. Male genitalia of C. lessinica, slide No. IgR 23392 (phallotheca right); 8. Female genitalia of C. impalella, signum broken, flattened, slide No. TB2148f; 8a. Intact signum of C. impalella (enlarged), slide No. TB2149f; 9. Abdominal segments of C. impalella, slide No. TB2149f; 10. Female genitalia of C. lessinica, slide No. Bldz 17234; 11. Abdominal segments of C. lessinica. Scale bars: 0.5 mm. Figs
Hungary: 10 cases, Bélmegyer, Fáspuszta 24.ix.2020, on G. sedifolia. 3 cases, Békés county, Szabadkígyós, Kígyósi puszta, 17.viii.2021, on G. sedifolia, leg. A. Takács. 1 case, Békés county, Elek, 06.x.2021, on G. sedifolia, leg. G. Farkas. Romania: 1 case, Arad county, Grăniceri, 06.x.2021, on G. sedifolia, leg. G. Farkas.
Case and larval behaviour .
Initial case is made of a dry inflorescence of A. alba. The larva consumes the centre of the inflorescence, remaining parts are strengthened with silk (Fig.
Cases of C. lessinica and C. absinthii. 12. Flower case of C lessinica. Csákberény, Bucka-hegy, 20.x.2020, on Artemisia alba; 13. Sheath case before overwintering of C. lessinica. Csákberény, Bucka-hegy, 27.x.2020, on A. alba; 14. Sheath case before overwintering of C. lessinica. Csákberény, Bucka-hegy, 20.x.2020, on A. alba; 15. Flower case of C. absinthii. Pákozd, 25.viii.2021, on A. absinthium. The larva is feeding in the third inflorescence, which will be incorporated to its case; 16. Flower case of C. absinthii. Pettend, 30.viii.2021, on A. absinthium; 17. Sheath case before overwintering of C. absinthii. Pettend, 04.ix.2021, on A. absinthium. Scale bars: 0.5 mm, photos by Attila Takács.
The mouth of case is at a right angle to the longitudinal axis of the case. Anal end of case is trivalved. Colour of case is light brown before overwintering, becoming dark brown after winter.
Overwintering happens at the base of the host plant. The larva climbs on the stem up to the basal-most leaves during August and September. There it attaches its case permanently to the stem, and this is the place where the larva turns around inside the case.
The early case, as well as the overwintering one, is very similar to that of Coleophora absinthii Wocke, 1877 (Figs
(Figs
The habitats of C. lessinica differ from each other in terms of bedrock. The bedrock in the habitats of Eastern Bakony Mountains (Kőszárhegy, Várpalota), the Balaton Uplands (Litér, Balatonfüred) and the Villány Mountains (Nagyharsány) is limestone, that of the Vértes Mountains (Csákvár, Csákberény, Gánt-Gránás) is dolomite. The bedrock affects the species ofplant associations, but the association-forming species everywhere in these habitats is A. alba (
Construction of the case begins in late August or early September from the leaves of the host plant, Galatella sedifolia. This finding does not support
The case is made of 2–6 mm long and 2–4 mm wide strips spun to each other with overlap, resulting in a sheath case (Figs
Adults emerge 10–12 days after the permanent attachment of the case. They fly in daytime and remain close to the host plant. Mating was observed between 11 h and 13 h.
(Figs
Larval morphology of Coleophora impalella. 18. Head and thorax, dorsal view; 19. Head and thorax, lateral view; 20. Head and thorax, ventral view; 21. Prolegs on segments A3–A6, ventral view; 22. Anal prolegs, ventral view; 23. Anal plate, dorsal view. Scale bar: 1 mm, cranial direction top, caudal direction bottom, photos by A. Takács.
Ten cases of C. impalella were collected in Bélmegyer, Fáspuszta (Figs
Some stages and habitat of C. impalella. 24. L5 case of C. impalella before overwintering, Bélmegyer, 13.x.2020; 25. L4 case of C. impalella before overwintering, Bélmegyer 24.ix.2020; 26. Imago of C. impalella from Szabadkígyós, Kígyósi puszta, 12.v.2021; 27. Habitat of C. impalella in Szabadkígyós, Kígyósi puszta, 20.v.2021. Scale bar: 3 mm (24); 2 mm (25); 2.1 mm (26). Figs
In addition to the Hungarian locality already known, the species was also found in Szabadkígyós, Kígyósi puszta on 12.v.2021 (Fig.
Coleophora impalella feeds on Asteretum sedifolii Soó 1947 corr. Borhidi 1996 (Pannonic salt steppes and salt marshes), a species that is highly influenced by dry continental climate with extreme temperatures and uneven distribution of precipitation. This habitat type was formed by secondary salinisation (Fig.
No cases of C. impalella and C. lessinica specimens have given parasitoid specimens in our rearings to date.
The specimens from Artemisia alba all belong to the same haplotype. The alignment of the COI region numbered 536 nucleotide positions. The sequence length of the specimen from Galatella sedifolia was 595 nucleotides in the final data set.
No exact matches were found in GenBank (Table
First five hits in GenBank using MEGABlast search on the COI sequences of the Hungarian specimen identified as Coleophora impalella. Accessed on 11/12/2021.
Scientific Name | Max Score | Total Score | Query Cover | E value | Percent identity | Accession Length | Accession number |
---|---|---|---|---|---|---|---|
Coleophora impalella | 1083 | 1083 | 100% | 0.00 | 99.5 | 658 | KX048258 |
Coleophora striolatella | 911 | 911 | 100% | 0.00 | 94.29 | 658 | KX049535 |
Coleophora striolatella | 900 | 900 | 100% | 0.00 | 93.95 | 658 | KX047871 |
Coleophora thurneri | 889 | 889 | 100% | 0.00 | 93.62 | 652 | KX048570 |
Coleophorasp. JFL051 | 889 | 889 | 100% | 0.00 | 93.62 | 658 | HM406336 |
In case of the specimens from Artemisia alba the closest matches in GenBank were three specimens of Coleophora artemisicolella with 97.57% similarity (Table
First five hits in GenBank using MEGABlast search on the COI sequences of the Hungarian specimens later identified as Coleophora lessinica. Accessed on 11/12/2021.
Scientific Name | Max Score | Total Score | Query Cover | E value | Percent identity | Accession Length | Accession number |
---|---|---|---|---|---|---|---|
Coleophora artemisicolella | 918 | 918 | 100% | 0.00 | 97.57 | 672 | JN248884 |
Coleophora artemisicolella | 918 | 918 | 100% | 0.00 | 97.57 | 658 | HM871573 |
Coleophora artemisicolella | 918 | 918 | 100% | 0.00 | 97.57 | 658 | HM875737 |
Coleophora fasciella | 913 | 913 | 100% | 0.00 | 97.39 | 658 | MT394458 |
Coleophora nubivagella | 907 | 907 | 100% | 0.00 | 97.2 | 658 | KX042273 |
The examination of DNA barcodes confirmed that C. lessinica and C. impalella are present with at least one population in Hungary. Based on our analysis, the barcodes of “lessinica” and “impalella” populations form monophyletic clusters. The studied DNA sequences of the Macedonian and Hungarian populations of C. lessinica were identical. The intraspecific distance between studied Russian and Hungarian C. impalella populations was 0.5% (3 nucleotide positions). Identification was confirmed by genitalia dissection (Figs
Top 20 matches from the BOLD System identification tool of the specimens later identified as Coleophora lessinica.
Genus | Species | Specimens | Similarity | Remarks |
---|---|---|---|---|
Coleophora | lessinica | 2 | 100% | |
Coleophora | magyarica | 1 | 99.06% | private |
Coleophora | microdon | 1 | 97.94% | early-release |
Coleophora | artemisicolella | 8 | 97.57% | |
Coleophora | nubivagella | 8 | 97.57% | private |
We were able to collect C. lessinica specimens from every location where its host plant, Artemisia alba is known to occur in Hungary. This implies that the species is more widespread than the published records suggest. To clarify the distribution of the species the host plant populations should be investigated during the flowering period. A disjunct distribution of C. impalella is unlikely, due to the widespread occurrence of its host plant, G. sedifolia. With more comprehensive searches, it is likely that more populations will be discovered in the future.
Our results support that identifying material based on DNA barcodes is often highly reliable for this group and provides an alternative to morphology-based identification. DNA-based identifications are very useful and could be widely used in species identification when different life stages are difficult to connect. Nevertheless, morphological confirmations are still needed, especially when species have highly similar DNA barcodes.
We are grateful to those who helped us in searching for cases: Péter Finy, Diána Győri, István Harta and Kristóf Antal (Fejér County Government Office, Velence), Gábor Farkas (Békés County Government Office, Békéscsaba), Péter Gábor Sulyán (ÖMKI, Budapest). We thank Ignác Richter (Malá Čausa, Slovakia) for the genital preparations, and János Babics for genitalia photos. Special thanks to Giorgio Baldizzone (Asti, Italy) and Jean-François Landry (Ottawa, Canada) for their advice and professional help. We are indebted to Colin W. Plant (Bishops Stortford, UK) and the editor of Nota Lepidopterologica (David Lees) for linguistic corrections.