ANALYSIS OF CYTOCHROME OXYDASE I SEQUENCES OF ELACHISTINAE (LEPIDOPTERA: ELACHISTIDAE) SPECIES

Abstract

The aim of this study was to analyse mtDNA sequences of the mtDNA cytochrome c oxidase I (COI) gene as a tool for investigating the genetic polymorphism of Elachistinae. The polymorphism of the sequenced COI gene was assessed in 31 specimens of 11 species. PCR amplification of genomic DNA with the COI primer in each of the samples yielded a specific fragment corresponding to position 2239–2944 in the sequence. The tree was constructed using the Neighbor-joining (NJ) method with the Kimura 2-parameter model. Analysis of the COI sequenced 31 samples of 11 Elachistinae species produced 640bp sequence alignment. The nucleotide diversity was obtained for Elachistinae moths. The sequences contrasted with 229 polymorphic nucleotides. The maximum parsimony analysis revealed 182 parsimony informative characters.

Full text

52
NEW AND RARE FOR LITHUANIA INSECT SPECIES. Volume 22
A
NALYSIS OF CYTOCHROME OXYDASE I SEQUENCES OF
ELACHISTINAE (LEPIDOPTERA: ELACHISTIDAE) SPECIES
BRIGITA PAULAVIČIŪTĖ
1,2
, ALGIMANTAS PAULAUSKAS
1
, VIRGINIJUS
SRUOGA
3
1
Vytautas Magnus University, Kaunas, Lithuania.
E-mail: b.paulaviciute@gmf.vdu.lt; a.paulauskas@gmf.vdu.lt
2
Kaunas T. Ivanauskas Zoological Museum, Kaunas, Lithuania
3
Vilnius Pedagogical University, Vilnius, Lithuania. E-mail: virginijus.sruoga@vpu.lt
Abstract. The aim of this study was to analyse mtDNA sequences of the mtDNA
cytochrome c oxidase I (COI) gene as a tool for investigating the genetic polymorphism
of Elachistinae. The polymorphism of the sequenced COI gene was assessed in 31
specimens of 11 species. PCR amplification of genomic DNA with the COI primer in
each of the samples yielded a specific fragment corresponding to position 2239–2944 in
the sequence. The tree was constructed using the Neighbor-joining (NJ) method with the
Kimura 2-parameter model. Analysis of the COI sequenced 31 samples of 11
Elachistinae species produced 640bp sequence alignment. The nucleotide diversity was
obtained for Elachistinae moths. The sequences contrasted with 229 polymorphic
nucleotides. The maximum parsimony analysis revealed 182 parsimony informative
characters.
Key words: Elachista, COI gene, sequencing, polymorphism
Introduction
Molecular tools are a standard part of many conservation studies and can be
informative at many different levels of analysis, although there are inherent limitations
and strengths of different genes or parts of genes to find answers to specific questions.
Insect DNA barcodes, 600- to 800-base-pair segments of the mitochondrial gene
cytochrome c oxidase I (COI), have been proposed as a means to quantify global
biodiversity. Mitochondrial (mt) DNA has a long history of use at the species level.
Recent analyses suggested that the use of a single gene, particularly mitochondrial, is
sufficient in taxonomic scope to recognize many species lineages. A mitochondrial
genome can result in very different assessments of biodiversity (Rubinoff, 2006). The
study of mitochondrial DNA sequences has become the method for a wide range of
taxonomic, population and evolutionary investigations in Lepidoptera (Lunt et al., 1996).
We used this method for investigating of Elachistinae moths. There are still very few
publications on the investigations of mtDNA of this subfamily (Kaila & Ståhls, 2006;
Sruoga et al., 2009; Paulavičiūtė & Paulauskas, 2010).
Elachistinae is a rather small subfamily in comparison to many other groups of
Lepidoptera. Moths are small with the wingspan of 6 to 14 mm. The forewing pattern
mainly consists either of a white fascia and spots on a dark background, or dark marks
on a light background, or moths are unicolorous (white, yellowish or creamy). Larvae of

NAUJOS IR RETOS LIETUVOS VABZDŽIŲ RŪŠYS. 22 tomas
53
Elachistinae are typical leaf-miners, trophically connected mainly with
Monocotyledonous grasses.
The aim of this study was to analyze the mtDNA sequences of the COI gene as a tool
for investigating the genetic polymorphism of Elachistinae.
Material and Methods
Study area
Elachistinae moths were collected in eight places of Lithuania: Neringa and Palanga
municipalities, Jurbarkas, Kaišiadorys, Kaunas, Panevėžys, Šakiai, Tauragė, Trakai, and
Vilnius administrative districts (Fig. 1).
Fig. 1. Sampling sites of Elachistinae specimens in Lithuania: 1 – Paneriai, Vilnius env.
(54°38'08,0"N, 25°11'30,61"E); 2 – Mickūnai env., Vilnius district (54°42'56,0"N,
25°32'42,1"E); 3 – Kalveliai, Vilnius district (54°38'6,4"N, 25°41'06,5"E); 4 –
Aukštadvaris, Trakai district (54°34'48,9"N, 24°31'39,4"E); 5 – Čižiūnai, Trakai district
(54°35'51,1"N, 24°33'50,4"E); 6 – Rumšiškės, Kaišiadorys district (54°52'38,1"N,
24°10'44,7"E); 7 – Baniškės, Kaišiadorys district (54°52'31,5"N, 24°14'03,3"E); 8 –
Strėvininkų Miškas f., Kaišiadorys district (54°48'42,6"N, 24°21'37,2"E); 9 – Jiesia
landscape reserve, Kaunas district (54°49'00,2"N, 23°55'02,4"E); 10 – Juškinė forest,
Šakiai district (55°01'26,3"N, 23°26'54,0"E); 11 – Eičiai, Tauragė district (55°09'39,6"N,
22°28'40,7"E); 12 – Viešvilė, Jurbarkas district (55°05'10,5"N, 22°24'20,8"E); 13 –
Palanga (55°55'17,5"N, 21°03'47,9"E); 14 – Kupstai, Panevėžys district (55°49'31,0"N,
24°16'04,8"E)
Sampling methods
Material was sampled from early spring to late autumn in 2004–2008 using an
entomological net and during light trapping at night (160W DRL type bulb lamp was
used).
Species were identified by the external appearance and genitalia of moths, using a
Motic SMZ 168 stereomicroscope.

54
NEW AND RARE FOR LITHUANIA INSECT SPECIES. Volume 22
DNA extraction
We used pinned specimens and specimens which were stored in 96% ethanol. DNA
was extracted from head or thorax using the Nucleospin Tissue Kit (Machery-Nagel,
Düren, Germany) according to manufacturer’s protocols.
DNA amplification
Primers used to amplify COI fragments were: LCO1490 (5'-GGT CAA CAA ATC
ATA AAG ATA TTG G-3') and HCO2198 (5'-TAA ACT TCA GGG TGA CCA AAA
AAT CA-3') (Folmer et al., 1994; Herbert et al., 2003).
PCR reactions were carried out in 25 µl reaction mixtures containing 2 µl of DNA
extract, 2 µl of each primer (at 10 pmol/µl) (MBI Fermentas, Lithuania), 0.5 µl of
Amplitaq DNA polymerase (5 U/µl), 2.5 µl 25 mM of MgCl
2
, 2.5 µl of 10X buffer
(Fermentas) and 1 µl 10 mM of dNTP (Fermentas), and water. Termocycler conditions
were the initial denaturing at 94°C for 2 min, 35 cycles of 30 s denaturing at 94°C, 45 s
annealing at 50°C, 1 min extension at 72°C, followed by a final extension of 4 min at
72°C.
After amplification, PCR products were separated by electrophoresis in 1.5% agarose
gel. Agarose gel was stained with ethidium bromide and photographed under UV light
(EASY Win32, Herolab, Germany). DNA fragment sizes were assessed by comparison
with GeneRuler
TM
100bp DNA Lader Plus (MBI Fermentas, Lithuania) (Fig. 2).
Fig. 2. PCR-amplified products of COI gene of Elachista moths. Lane M: 100 bp DNA
ladder. Lanes 1–9: amplified products (about 700 bp)
Sequencing
PCR products were purified with the Nucleospin Extract Kit (Machery-Nagel, Düren,
Germany). Cycle sequencing was done using the ABI PRISM BigDye terminator version
3.1 cycle sequencing kit. One to eight reactions were used to produce 20 µl of a cycle-
sequencing product, using 8 µl of the ABI reaction mix. Products were separated and
visualized using an ABI PRISM 310 Genetic analyzer. All fragments were sequenced in
both directions.
Data analysis
The editing of DNA sequences, counting assembly, and the alignment of consensus
sequences was performed using Bioedit 5.0.9 software. Phylogenetic analysis was
performed using MEGA 4 (Tamura et al., 2007).

NAUJOS IR RETOS LIETUVOS VABZDŽIŲ RŪŠYS. 22 tomas
55
Results and discussion
Cytochrome c oxidase I partial sequences of 31 specimens were amplified and
sequenced. Sequences of 640 bp (correspond to position 2239–2944) were aligned.
GenBank accession numbers are presented in Table 1.
Table 1. List of specimens used for DNA sequencing and GenBank accession numbers
Examined specimens GenBank accession number
Perittia herrichiella(1) HM034446
Perittia herrichiella(2) HM034447
Elachista albifrontella(1) GU248251
Elachista albifrontella(2) GU248252
Elachista alpinella(1) GU248254
Elachista alpinella(2) GU248255
Elachista alpinella(3) GU248256
Elachista alpinella(4) GU248257
Elachista alpinella(5) GU248258
Elachista alpinella(6) GU248259
Elachista argentella(1) DQ666137
Elachista argentella(2) DQ666138
Elachista argentella(3) HM034449
Elachista argentella(4) HM034448
Elachista canapennella GU248260
Elachista consortella GU248261
Elachista humilis(1) GU248253
Elachista humilis(2) HM034450
Elachista maculicerusella(1) DQ666141
Elachista maculicerusella(2) DQ666142
Elachista maculicerusella(3) DQ666143
Elachista pollinariella(1) DQ666140
Elachista pollinariella(2) DQ666139
Elachista pollinariella(3) DQ666144
Elachista pollinariella(4) GU248246
Elachista pollinariella(5) GU248247
Elachista pullicomella(1) GU248248
Elachista pullicomella(2) GU248249
Elachista pullicomella(3) GU248250
Elachista utonella(1) HM034451
Elachista utonella(2) HM034452
Analysis of partial COI gene sequences demonstrated a different distributional rate of
nucleotides (Table 2); 38.7% of T, 16.7% of C, 30.4% of A and 14.1% of G nucleotides
were obtained in the sequences. The Elachista canapennella sequence has 41.5 % of T
nucleotides. The E. alpinella(4) sequence has the maximum number of C nucleotides –
18.6%. The E. maculicerusella(3) sequence has the largest number of A nucleotides –
31.4%. The largest number of G nucleotides – 14.8 % has been obtained in the E.
humilis(2) sequence.

56
NEW AND RARE FOR LITHUANIA INSECT SPECIES. Volume 22
Table 2. Elachistinae specimens and the rate of nucleotides of mtDNR COI gene
fragments
Specimens T (%) C (%) A (%) G (%) Total number
of nucleotides
Perittia herrichiella(1) 38.7 17.5 29.7 14.1 576
Perittia herrichiella(2) 38.5 17.5 29.9 14.1 576
Elachista albifrontella(1) 38.0 16.7 30.9 14.4 576
Elachista albifrontella(2) 38.0 16.7 30.9 14.4 576
Elachista alpinella(1) 36.8 18.4 30.7 14.1 576
Elachista alpinella(2) 37.3 18.4 30.2 14.1 576
Elachista alpinella(3) 36.8 18.4 30.7 14.1 576
Elachista alpinella(4) 37.0 18.6 30.4 14.1 576
Elachista alpinella(5) 36.8 18.4 30.7 14.1 576
Elachista alpinella(6) 36.8 18.4 30.7 14.1 576
Elachista argentella(1) 39.0 16.7 30.3 14.1 575
Elachista argentella(2) 39.0 16.7 30.3 14.1 575
Elachista argentella(3) 38.8 16.7 30.0 14.5 640
Elachista argentella(4) 38.8 17.0 29.8 14.4 631
Elachista canapennella 41.5 14.6 30.0 13.9 576
Elachista consortella(1) 39.4 15.6 30.6 14.4 576
Elachista humilis(1) 39.4 15.6 30.6 14.4 576
Elachista humilis(2) 39.8 15.4 30.0 14.8 623
Elachista maculicerusella(1) 39.1 16.3 30.4 14.2 576
Elachista maculicerusella(2) 39.1 16.3 30.4 14.2 576
Elachista maculicerusella(3) 39.1 15.6 31.4 13.9 576
Elachista pollinariella(1) 39.5 16.0 30.8 13.7 575
Elachista pollinariella(2) 39.4 16.1 30.7 13.7 576
Elachista pollinariella(3) 39.5 16.0 30.8 13.7 575
Elachista pollinariella(4) 39.8 15.4 31.1 13.7 570
Elachista pollinariella(5) 39.1 16.5 30.7 13.7 576
Elachista pullicomella(1) 39.9 15.5 30.6 14.1 576
Elachista pullicomella(2) 40.2 15.8 30.3 13.7 575
Elachista pullicomella(3) 39.9 15.5 30.7 13.9 574
Elachista utonella(1) 37.2 18.1 30.3 14.3 623
Elachista utonella(2) 37.6 18.4 29.5 14.4 630
Average
38.7 16.7 30.4 14.1 584
COI sequences of Elachistinae moths have 63 codons (Table 3). The most
common codon in mtDNA COI sequences of this subfamily is AUU(I) which amount to
17.3%. Codons GUC(V), GUG(V), GCA(A), GAU(D) and GGA(G) are most rarely
detected in the sequences; they accounted for as little as 0.1 % only. The CUA(L) codon
was not detected in COI sequences of Elachistinae moths.
The analysis of COI sequences of 11 Elachistinae species and 31 samples produced a
640 bp sequence alignment. The total nucleotide diversity and genetic divergence
obtained for the Elachistinae moths contrasted with 229 polymorphic nucleotides.
Molecular analysis results were summarized and the neighbor-joining (NJ) tree was
constructed (Fig. 3).

NAUJOS IR RETOS LIETUVOS VABZDŽIŲ RŪŠYS. 22 tomas
57
Table 3. Elachistinae specimen codons and their number
Codon Number
(%)
Codon Number
(%)
Codon Number
(%)
Codon Number
(%)
UUU(F) 15.8(1.50) UCU(S) 2.6(0.65) UAU(Y) 16.9(1.56) UGU(C) 4.6(1.22)
UUC(F) 5.2(0.50) UCC(S) 1.7(0.44) UAC(Y) 4.8(0.44) UGC(C) 2.9(0.78)
UUA(L) 1.2(0.99) UCA(S) 3.3(0.84) UAA(*) 8.5(1.65) UGA(*) 5.6(1.09)
UUG(L) 2.3(1.88) UCG(S) 2.0(0.51) UAG(*) 1.4(0.26) UGG(W) 7.7(1.00)
CUU(L) 2.0(1.62) CCU(P) 1.3(0.59) CAU(H) 1.5(1.12) CGU(R) 0.6(0.30)
CUC(L) 1.1(0.91) CCC(P) 5.8(2.56) CAC(H) 1.2(0.88) CGC(R) 0.8(0.42)
CUA(L) 0.0(0.00) CCA(P) 1.1(0.49) CAA(Q) 0.3(1.23) CGA(R) 0.4(0.20)
CUG(L) 0.7(0.60) CCG(P) 0.8(0.37) CAG(Q) 0.2(0.77) CGG(R) 0.6(0.30)
AUU(I) 17.3(2.26) ACU(T) 3.8(1.54) AAU(N) 14.1(1.45) AGU(S) 4.3(1.08)
AUC(I) 5.5(0.71) ACC(T) 4.4(1.79) AAC(N) 5.4(0.55) AGC(S) 9.7(2.47)
AUA(I) 0.2(0.03) ACA(T) 0.9(0.36) AAA(K) 4.5(1.10) AGA(R) 2.1(1.08)
AUG(M) 1.1(1.00) ACG(T) 0.8(0.32) AAG(K) 3.7(0.90) AGG(R) 7.2(3.71)
GUU(V) 0.3(1.03) GCU(A) 0.2(1.00) GAU(D) 0.1(0.44) GGU(G) 0.2(0.42)
GUC(V) 0.1(0.31) GCC(A) 0.2(1.17) GAC(D) 0.2(1.56) GGC(G) 0.8(2.17)
GUA(V) 0.7(2.36) GCA(A) 0.1(0.33) GAA(E) 0.3(0.95) GGA(G) 0.1(0.25)
GUG(V) 0.1(0.31) GCG(A) 0.3(1.50) GAG(E) 0.4(1.05) GGG(G) 0.5(1.17)
The NJ tree contained two big clusters: A and B. Cluster A has two subclusters (A1,
A2). The A1 subcluster includes genetically similar species: Elachista alpinella, E.
humilis, E. maculicerusella, E. albifrontella and E. consortella. A total of 122
polymorphic nucleotides were detected in COI gene fragments in these species. The
maximum parsimony analysis revealed 109 parsimony-informative characters.
Genetically most similar were E. humilis and E. consortella species. In total 530
conservative sites were detected in their COI gene fragments. The most genetically
different species in subcluster A1 are E. alpinella and E. maculicerusella: 73
polymorphic sites were detected in the sequences of these species. The A2 subcluster
contains genetically similar species: Elachista argentella, E. canapennella, E.
pullicomella and E. pollinariella. Genetically most similar are E. argentella and E.
canapennella species. Their sequences have 578 conservative sites and 53 polymorphic
nucleotides. Genetically most different species in subcluster A2 are E. argentella and E.
canapennella, E. argentella and E. pullicomella. COI gene fragments of these moths
have 60 polymorphic nucleotides from 640.
Cluster B includes Elachistinae species from two genera: Perittia herrichiella and
Elachista utonella. COI gene fragments of this species differ in 102 polymorphic
nucleotides. The largest intraspecific differences were found in the Elachista utonella
species. Two specimens of this species have 39 variable nucleotides in COI sequences.
Conclusions
1. The investigated fragment of citochrome c oxidase I gene of Elachistinae moths
has 229 polymorphic sites out of 640.

58
NEW AND RARE FOR LITHUANIA INSECT SPECIES. Volume 22
Fig. 3. The neighbor-joining tree of Elachistinae species. The tree was constructed by
Kimura2-parameter model (bootstrap replications = 10000, complete deletion), based on
analysis of 640 sites of COI gene fragment. Bootstrap values are shown above branches.
The sequence of Mythimna phaea (GQ353295.1) was used as the outgroup.
2. COI gene fragments of Elachistinae sequences demonstrate a different
distributional rate of nucleotides: 38.7% of T, 16.7% of C, 30.4% of A and 14.1%
of G nucleotides.
3. COI sequences of Elachistinae moths have 63 codons, the most frequent codon
being UAU(Y) (16.9%). Codon CUA(L) was not detected in the sequences of
Elachistinae moths.
4. The greatest intraspecific differences were observed in Elachista utonella: two
specimens of this species have 39 polymorphic nucleotides, whereas no
A1
A
2
A
B

NAUJOS IR RETOS LIETUVOS VABZDŽIŲ RŪŠYS. 22 tomas
59
intraspecific differences were detected in E. argentella, E. albifrontella and E.
humilis sequences.
References
Folmer O., Black M., Hoeh W., Lutz R., Vrijenhoek R. 1994. DNA primers for
amplification of mitochondrial cytochrome c oxidase subunit I from diverse
metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294–297.
Hebert P.D.N., Cywinska A., Ball S.L., De Waard J.R. 2003. Biological identifications
through DNA barcodes. Proceedings of the Royal Society B: Biological Sciences
270: 313–321.
Kaila L., Ståhls G. 2006. DNA barcodes: Evaluating the potential of COI to diffentiate
closely related species of Elachista (Lepidoptera: Gelechioidea: Elachistidae) from
Australia. Zootaxa 1170: 1–26.
Lunt D.H., Zhang D.X., Szymura J.M., Hewitt G.M. 1996. The insect cytochrome
oxidase I gene: evolutionary patterns and conserved primers for phylogenetic studies.
Insect Molecular Biology 5:153–165.
Paulavičiūtė B., Paulauskas A. 2010. DNA diagnostics to identify Elachista
(Lepidoptera: Elachistidae: Elachistinae) species. Ecology & Safety 4(1): 47–52.
Rubinoff D. 2006. Utility of mitochondrial DNA barcodes in species conservation.
Conservation Biology 20(4): 1026–1033.
Sruoga V., Stunžėnas V., Paulavičiūtė B. 2009. COI gene as a molecular marker of
Elachista species (Lepidoptera: Elachistidae: Elachistinae) from different Lithuanian
populations. Proceedings of the Latvian Academy of Sciences, Section B 63(1/2): 21–
24.
Tamura K., Dudley J., Masatoshi M., Kumar S. 2007. MEGA4: Molecular Evolutionary
Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution
24(8): 1596–1599.
Elachistinae (Lepidoptera: Elachistidae) drugių rūšių citochromo oksidazės I geno
sekų analizė
B. PAULAVIČIŪTĖ, A. PAULAUSKAS, V. SRUOGA
Santrauka
Molekulinių tyrimų metu buvo ištirta 11 Elachistinae pošeimio drugių rūšių. Buvo
amplifikuoti 31 šios šeimos individo Citochromo c oksidazės I (COI) geno fragmentai.
Gauti 640 bazių porų fragmentai, esantys 2239–2944 nukleotidų pozicijos atkarpoje.
Elachistinae pošeimio drugių COI geno fragmentų sekų analizė parodė skirtingą
nukleotidų pasiskirstymo dažnį. Užregistruoti 63 nukleotidų kodonai. Gauti rezultatai
buvo apibendrinti ir artimiausių kaimynų jungimo metodu, naudojant Kimura 2
parametrų modelį, sudarytas Elachistinae pošeimio drugių mtDNR COI geno medis.
Received: October 15, 2010