Vol.7, No.1 pages 49-57 Science Technology and Engineering Journal (STEJ) Research Article

A New Report on Karyological Analysis of the Jewel-, ruficornis (Coleoptera, ) from Thailand

Nawarat Muanglen1, Kamika Sribenja2, Patcharaporn Chaiyasan2, Alongklod Tanomtong2 and Chadaporn Nuchjangreed3*

1 Department of Fisheries, Faculty of Agricultural Technology, Sakon Nakhon Rajabhat University, Sakon Nakhon, 47000, Thailand 2 Toxic Substance in Livestock and Aquatic Research Group, Department of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand 3 Department of Biological Sciences, Faculty of Allied Health Sciences, Burapha University, Chon Buri, 20131, Thailand * Corresponding author: E-mail: [email protected]

(Received: 22nd January 2021, Revised: 11th May 2021, Accepted: 12th May 2021)

Abstract - The first karyological analysis of theSternocera ruficornis (Coleoptera, Buprestidae) was achieved by using the squash technique collected from the male gonad. Furthermore, the chromosomes were stained by conventional staining and the Ag-NOR banding techniques. The results showed the diploid number of chromosomes was 2n=26, comprising 4 metacentric, 6 submetacentric, 6 acrocentric and 8 telocentric chromosomes. The sex chromosome system was XY system with X and Y chromosomes being metacentric and acrocentric chromosomes, respectively. Heteromorphism (1a1b) nucleolar organizer regions (NORs) are clearly observed near the centromere of chromosome pair one and the subcentromeric region of chromosome pair nine. The karyotype formula demonstrated as follows: 2n (26) =Lm2+Sm2+Ssm6+Sa6+St8+XY. Keywords: Jewel-, Sternocera ruficornis, karyotype, Ag-NOR banding

50 Chadaporn Nuchjangreed et al. Science Technology and Engineering Journal (STEJ) quite popular among collectors. species 2n NORs Karyotype references Stenocera 26 - 24 Dasgupta (Pinkaew 2001) sp autosomes+XY (1977) 1. Introduction Sternocera 26 - 24 Karagyan and laevigata autosomes+XY Kuznetsova (2000) The family Buprestidae (Coleoptera) Sternocera 26 - 24 Karagyan and is one of the largest group of Polyphagan nitidicollis autosomes+XY Kuznetsova (2000) beetles comprising approximately 15,500 Sternocera 26 2 pair 24 This study species in 775 genera. In addition, almost ruficornis autosomes+XY 100 fossil species have been described (Bellamy 2008a-d, 2009). The jewel-beetles 2. Materials and Methods (Sternocera ruficornis) are classified into 2.1 Chromosome preparation the order Coleoptera, family Buprestidae, The specimens were collected from the subfamily , genus Sternocera. In Kalasin province, northeastern Thailand Thailand, there are two species of buprestid FigureFigure 1. 1 . GeneralGeneral characteristics characteristics of the of the Jewel-Beetles, Sternocera ruficornis, and identified according to the categories belonging to the genus Sternocera. They (A) ventralJewel view,- Beetles,and (B) dorsal viewSternocera of Ek-Amnuay (2008). Chromosomes were are red-legged Sternocera ruficornis and (scale bar ruficornisindicate 1 cm)., (A) ventral view, green legged Sternocera aequisignata which prepared directly in vivo. Firstly, and (B) dorsal view (scale bar can be which can be found throughout colchicine was injected into the abdominal Karyotype studies of jewels-beetles Thailand (Ohmomo & Akiyama, 1972). indicate 1 cm). have been published for 92 species (34 cavity of each insect and left for 1 hour. The characteristics of S. ruficornis are a from Armenia) in 22 genera, 14 tribes and Gonad cells were collected by cutting rounded metallic green body, reddish legs, Karyotype studies of jewels-beetles 5 subfamilies (Julodinae, Polycestinae, tissue into small pieces, mixed and and an average body length of between have been published for 92 species (34 Chrysochroinae, Buprestinae and Agrilinae) incubated with 0.075 M KCl for 30-45 30-45 mm (Figure 1). There are widely from Armenia) in 22 genera, 14 tribes and (Smith & Virkki 1978 ; 1991 ; Karagyan minutes. The fresh and cool fixative (3 overspread in Laos, Vietnam, and Thailand, 5 subfamilies (Julodinae, Polycestinae, & Kuznetsova 2000 ; Karagyan 2001 ; methanol: 1 glacial acetic acid) terminated especially in the northeast of Thailand Chrysochroinae, Buprestinae and Karagyan et al., 2004 ; Karagyan & KCl activities then centrifuged at 3,000- (Ek-Amnuay, 2008). Agrilinae) (Smith and Virkki 1978;1991; Lachowska 2007 ; Moura et al., 2008 ; 3,200 rpm for 10 minutes. The Jewel-Beetles are edible, then, KaragKaragyanyan and et Kuznetsova al., 2012). Recently, 2000; Karagyan there are supernatants were separated and discarded. their wings are made into jewelry and 2001;only Karagyan three cytogenetics et al., 2004; studies Karagyan on genus and Fixative solutions were added and other marketable objects. Moreover, their LachowskaSternocera 2007; as Sternocera Moura sp.,et S.al. laevigata, 2008; centrifuged again to wash the cell numbers have declined because they are Karagyanand S. nitidicollis et al., 2012 (Karagyan). Recently & Kuznetsova,, there are quite popular among insect collectors. 2000), demonstrated by conventional sediments for 2-3 times. The obtained cell only three cytogenetics studies on genus (Pinkaew 2001) staining technique suggesting that 2n=26 sediments were kept at -20°C for Sternocera as Sternocera sp., S. laevigata with XY sex chromosome system (Table 1). chromosome studies. and S. nitidicollis (Karagyan and Our work aimed to analyze the chromosomal Kuznetsova, 2000), demonstrated by information of S. ruficornis by conventional 2.2 Conventional staining and Ag-NOR conventionalstaining and staining Ag-NOR technique staining suggesting techniques. banding technique thatFurthermore, 2n=26 with the XY cytogenetic sex chromosome data of S. The cell suspension was dropped on to systemruficornis (Table will 1). be Ourapplied work for aimedstudies to of completely clean slides and fixed with a analyzebreeding, the conservationchromosomal and information chromosomal of evolution among related beetle species. fixative solution. The slides were infused S. ruficornis by conventional staining and in a mixture of 20% Giemsa’ s for 15 Ag-NOR staining techniques. Furthermore, minutes and rinsed with distilled water the cytogenetic data of S. ruficornis will be (Tanomtong et al., 2014). applied for studies of breeding, Ag-NOR banding was performed by conservation and chromosomal evolution applying two drops of 2% gelatin on the among related beetle species. slides, followed by four drops of 50%

silver nitrate, respectively. The slides were Table 1. Cytogenetic data review for genus sealed with cover glasses and incubated at Stenocera.

Volume 7, Number 1, January-June 2021 A New Report on Karyological Analysis of the Jewel-Beetle, 51 Sternocera ruficornis (Coleoptera, Buprestidae) from Thailand

Table 1. Cytogenetic data review for genus Stenocera.

species 2n NORs Karyotype references Stenocera sp 26 - 24 autosomes+XY Dasgupta (1977) Sternocera laevigata 26 - 24 autosomes+XY Karagyan and Kuznetsova (2000) Sternocera nitidicollis 26 - 24 autosomes+XY Karagyan and Kuznetsova (2000) Sternocera ruficornis 26 2 pair 24 autosomes+XY This study

2. Materials and Methods and rinsed with distilled water (Tanomtong et al., 2014). 2.1 Chromosome preparation Ag-NOR banding was performed by applying two drops of 2% gelatin on the The specimens were collected from the slides, followed by four drops of 50% silver Kalasin province, northeastern Thailand nitrate, respectively. The slides were sealed and identified according to the categories with cover glasses and incubated at 60 °C of Ek-Amnuay (2008). Chromosomes were for 5-10 minutes, then soaked in distilled prepared directly in vivo. Firstly, colchicine water until cover glasses are separated. was injected into the abdominal cavity of The slides were then kept for examination each insect and left for 1 hour. Gonad cells with a light microscope (Chaiyasan et al., were collected by cutting tissue into small 2018). pieces, mixed and incubated with 0.075 M KCl for 30-45 minutes. The fresh and cool fixative (3 methanol: 1 glacial acetic acid) 2.3 Data Analysis terminated KCl activities then centrifuged at 3,000-3,200 rpm for 10 minutes. The Twenty clearly observable cells with well supernatants were separated and discarded. spread chromosomes were selected and Fixative solutions were added and photographed. The length of the short centrifuged again to wash the cell sediments arm chromosome (Ls) and the long arm for 2-3 times. The obtained cell sediments chromosome (Ll) were measured and the were kept at -20°C for chromosome studies. length of the total arm chromosome (LT, LT = Ls + Ll) was calculated (Chaiyasut, 1989). The relative length (RL), the 2.2 Conventional staining and centromeric index (CI), and standard Ag-NOR banding technique deviation (SD) of RL and CI were estimated. The CI (q/p + q) between 0.50–0.59, The cell suspension was dropped on to 0.60–0.69, 0.70–0.89, and 0.90–1.00 are completely clean slides and fixed with a described as metacentric, submetacentric, fixative solution. The slides were infused in acrocentric and telocentric chromosomes, a mixture of 20% Giemsa’s for 15 minutes respectively. 52 Chadaporn Nuchjangreed et al. Science Technology and Engineering Journal (STEJ)

Table 2. Mean of length of short arm chromosome (Ls), length of long arm chromosome (Ll), length of total arm chromosome (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) of RL, CI from metaphase chromosome in 20 cells of male Jewel-Beetles (Sternocera ruficornis, 2n=26)

Chro. Ls Ll LT RL±SD CI±SD Size Type pairs (µm) (µm) (µm) 1a 6.021 6.761 12.782 0.269±0.006 0.530±0.024 Large Metacentric 1b 5.476 6.237 11.713 0.247±0.008 0.533±0.031 Large Metacentric 2 1.246 1.708 2.955 0.061±0.007 0.581±0.046 Small Metacentric 3 1.152 1.744 2.896 0.061±0.003 0.604±0.024 Small Submetacentric 4 1.106 1.694 2.799 0.058±0.004 0.605±0.039 Small Submetacentric 5 0.823 1.823 2.646 0.055±0.002 0.687±0.061 Small Submetacentric 6 0.726 1.835 2.561 0.054±0.002 0.717±0.053 Small Acrocentric 7 0.548 1.867 2.415 0.051±0.003 0.772±0.046 Small Acrocentric 8 0.522 1.711 2.234 0.047±0.004 0.773±0.149 Small Acrocentric 9 0.000 1.973 1.973 0.041±0.002 1.000±0.000 Small Telocentric 10 0.000 1.864 1.864 0.039±0.003 1.000±0.001 Small Telocentric 11 0.000 1.844 1.844 0.039±0.004 1.000±0.002 Small Telocentric 12 0.000 1.671 1.671 0.035±0.004 1.000±0.003 Small Telocentric X 2.706 1.671 5.813 0.122±0.004 0.534±0.028 Small Metacentric Y 0.615 3.075 3.689 0.077±0.003 0.836±0.081 Small Acrocentric

* Large chromosome (LT>6.141), Small chromosome (LT<6.132), ** Chro. = Chromosome 3. Results occurs as the XX/XY sex-determination system (Figure 2 and 3). 3.1 Diploid chromosome number (2n) and karyotype of Sternocera ruficornis 3.2 Chromosome marker of Sternocera ruficornis The diploid chromosome number of S. ruficornis was 2n=26 (24+XY) for male and The determination of chromosome marker 2n=26 (24+XX) for female. The karyotype for this species was firstly obtained by composed of 2 large metacentric, 2 small using the Ag-NOR banding technique. The metacentric, 6 small submetacentric, 6 small nucleolar organizer regions (NORs) were acrocentric, 8 small telocentric (Table 2). observed on the centromere of chromosome In addition, the male karyotype showed an one and subcentromeric region of exclusive small acrocentric chromosome chromosome nine (Figure 3 and 4). identified as the Y chromosome. Hence, it

ruficornis, we found morphologically differentiated sex chromosomes of the type XX/XY, the X chromosome represented by Volume 7, Number 1, January-June 2021 A New Report on Karyological Analysis of the Jewel-Beetle, 53 a small sized metacentric and the Y Sternocera ruficornis (Coleoptera, Buprestidae) from Thailand chromosome by a small sized acrocentric. This result consistent with the previous Figure 2. Metaphase chromosome plate study about the sex chromosome system in and karyotypes of male Jewel- Buprestidae suggested that most species Beetles (Sternocera ruficornis) wereruficornis XY, we system found (Karagyanmorphologically and 2n= 26 by a conventional Kuznetsova,differentiated 2000). sex chromosomes of the type staining technique (scale bar XX/XY,The theAg X-NOR chromosome banding represented technique by indicate 5 µm) showinga small thatsized themetacentric Nucleolar and Organizer the Y Regionschromosome (NORs by) aband small appeared sized acrocentric at 2 sites. Figure 2. Metaphase chromosome plate and karyotypes of male Jewel-Beetles (Sternocera ruficornis) 2n =26 by a conventional staining technique (scale bar indicate 5 µm) nThisear theresult centromere consistent of with chromosome the previous one Figure 2. Metaphase chromosome plate andstudy subcentromeric about the sex chromosomeregion of chromosome system in and karyotypes of male Jewel- nineBuprestidae. This suggestedis the thatfirst mostreport species on Beetles (Sternocera ruficornis) polymorphismwere XY of system NORs (Karagyanin S. ruficornis and . 2n= 26 by a conventional AsKuznetsova, commonly 2000). understood, in Coleoptera staining technique (scale bar NORs The may Ag be-NOR located banding in some autosomal technique indicate 5 µm) pairshowing and/or that sex chromosomesthe Nucleolar (AOrganizerlmeida et alRegions., 2000; ( NORsMoura) etband al. , appeared2003; Bione at 2 etsites al. , 2005;near theHolecová centromere et alof., chromosome2008). The mostone and subcentromeric region of chromosome Figure 3. Metaphase chromosome plate and karyotypes of male Jewel-Beetles common pattern in Coleoptera is the (Sternocera ruficornis) 2n=26 by Ag-NOR banding technique. nine. This is the first report on Nucleolar Organizer Regions/NORs near centromere of chromosome pair one. location of the nucleolus organizer region A subcentromeric region of chromosome pair nine (arrows) and scale bar indicates 5 µm. Figure 3. Metaphase chromosome plate inpolymorphism one autosomal of pairNORs (Virkki in S .1983; ruficornis Virkki. As commonly understood, in Coleoptera 4. Discussions and Conclusionsand karyotypesIn a comparative of male karyotypic Jewel analysis- of et al., 1984; Vitturi et al., 1990; Colomba Beetles males(Sternocera and females ruficin S. ruficornisornis) , we etNORs al., 2000; may Mourabe located et al in., 2003;some Almeidaautosomal et All of the specimens showed the diploid found morphologically differentiated sex pair and/or sex chromosomes (Almeida et number (2n) of Sternocera ruficornis2n= was26 chromosomesby Ag-NOR of the typebanding XX/XY, the X al., 2006; Schneider et al., 2007). The 26, similarality with chromosome number chromosome represented by a small sized al., 2000; Moura et al., 2003; Bione et al., (2n) of Sternocera laevigata, S. nitidicollistechnique metacentric. Nucleolar and the OrganizerY chromosome by results showed heteromorphism in 2005; Holecová et al., 2008). The most and most published species in subfamilyRegions/NORs a small sized near acrocentric. centromere This result chromosome pair one with a different size Julodinae (Karagyan & Kuznetsova, 2000). consistent with the previous study about common pattern in Coleoptera is the of chromosome pair one. A of NORs. The purpose of this technique is location of the nucleolus organizer region subcentromeric region of to detect NORs, the location where genes Figure 3. Metaphase chromosome plate in one autosomal pair (Virkki 1983; Virkki chromosome pair nine (arrows) encoding ribosomal RNA were located. and karyotypes of male Jewel- et al., 1984; Vitturi et al., 1990; Colomba and scale bar indicates 5 µm. The idiograms of S. ruficornis showed Beetles (Sternocera ruficornis) et al., 2000; Moura et al., 2003; Almeida et 2n=26 by Ag-NOR banding sizeal., polymorphism2006; Schneider of etchromosome al., 2007). 1 Thedue technique. Nucleolar Organizer toresults size differentiationsshowed heteromorphism of NOR. Thein 4. DiscussionsRegions/NORs and Conclusions near centromere hchromosomeeteromorphic pair size one ofwith this a differentregion sizewas All of the specimensof chromosome showed pair the onediploid. A detectedof NORs . The purposebetween of this techniquehomologous is number (2n) subcentromericof Sternocera ruficornisregion wasof chromosomesto detect NORs,. Thus, the thislocation variation where in genes NOR 26, similaralitychromosome with chromosome pair nine number(arrows) sizeencoding in S. ribosomalruficornis RNA was weredue to located. a variable (2n) of Sternoceraand scale laevigata bar indicates, S. nitidicollis 5 µm. number The idiogramsof rDNA ofgenes S. ruficornis. This could showed be and most published species in subfamily explainedsize polymorphism by mechanisms of chromosome such as unequal 1 due Julodinae (Karagyan and Kuznetsova, crossinto sizeg- over,differentiations transpositions of ,NOR. or otherThe 20004. Discussions). In a andcomparative Conclusions karyotypic rearrangementsheteromorphic includingsize of this deletions region and was/or analysisAll of theof specimensmales and showed females the diploidin S. duplications,detected betweeninvolve segmentshomologous of number (2n) of Sternocera ruficornis was chromosomes. Thus, this variation in NOR 26, similarality with chromosome number size in S. ruficornis was due to a variable (2n) of Sternocera laevigata, S. nitidicollis number of rDNA genes. This could be and most published species in subfamily explained by mechanisms such as unequal Julodinae (Karagyan and Kuznetsova, crossing-over, transpositions, or other 2000). In a comparative karyotypic rearrangements including deletions and/or analysis of males and females in S. duplications, involve segments of

54 Chadaporn Nuchjangreed et al. Science Technology and Engineering Journal (STEJ)

the sex chromosome system in Buprestidae NORs, the location where genes encoding suggested that most species were XY system ribosomal RNA were located. (Karagyan & Kuznetsova, 2000). The idiograms of S. ruficornis The Ag-NOR banding technique showed size polymorphism of chromosome showing that the Nucleolar Organizer 1 due to size differentiations of NOR. The Regions (NORs) band appeared at 2 sites heteromorphic size of this region was detected near the centromere of chromosome one between homologous chromosomes. Thus, and subcentromeric region of chromosome this variation in NOR size in S. ruficornis nine. This is the first report on polymorphism was due to a variable number of rDNA genes. of NORs in S. ruficornis. As commonly This could be explained by mechanisms such understood, in Coleoptera NORs may be as unequal crossing-over, transpositions, located in some autosomal pair and/or or other rearrangements including deletions sex chromosomes (Almeida et al., 2000; and/or duplications, involve segments of Moura et al., 2003; Bione et al., 2005; homologous chromosomes, thus, frequently Holecová et al., 2008). The most common attributed to mechanisms related to pattern in Coleopterahomologous is the locationchromosomes, of structural modificationsthus, of NORs.has shown that the size heteromorphism of the nucleolus organizer region in one The present study is the first report autosomal pairfrequently (Virkki 1983; attributed Virkki et al to., mechanisms related NOR observed represents a true on size heteromorphism (1a1b) of the 1984; Vitturi et al., 1990; Colomba et al., to structural modificationsAg-NOR of NORs in .the Buprestidae polymorphism,species. since it involves variations 2000; Moura et al., 2003; Almeida et al., The result has shown that the size 2006; Schneider etThe al., 2007).present The study results is the first report on in the copy number of genes in heteromorphism of NOR observed showed heteromorphism in chromosome size heteromorphism (1a1brepresents) of a thetrue polymorphism,Ag- h sinceomologous it chromosomes. pair one with a different size of NORs. involves variations in the copy number of The purpose NORof this techniquein the Buprestidae is to detect species. The result 1 genes in homologous chromosomes.2

Figure 4. Idiograms showing lengths and shapes of chromosomes of Jewel-Beetles (Sternocera ruficornis, 2 n=26) by (A) conventional staining technique and (B) Ag-NOR banding technique. Figure 4. Idiograms showing lengths and shapes of chromosomes of Jewel-Beetles (Sternocera ruficornis, 2n= 26) by (A) conventional staining technique and (B) Ag-NOR banding technique.

5. Acknowledgements This work was financially supported in part by a full scholarship from the Science Achievement Scholarship of Thailand. I would like to thank the Department of Biology, Faculty of Science, Khon Kaen University and Sakon Nakhon Rajabhat University.

Volume 7, Number 1, January-June 2021 A New Report on Karyological Analysis of the Jewel-Beetle, 55 Sternocera ruficornis (Coleoptera, Buprestidae) from Thailand

5. Acknowledgements Bellamy, C.L. (2008d). A world catalogue and bibliography of the jewel beetles This work was financially supported (Coleoptera: Buprestoidea) volume in part by a full scholarship from the 4. Pensoft Publishers. Science Achievement Scholarship of Bellamy, C.L. (2009). A world catalogue Thailand. I would like to thank the and bibliography of the jewel beetles Department of Biology, Faculty of Science, (Coleoptera: Buprestoidea) volume Khon Kaen University and Sakon Nakhon 5. Pensoft Publishers. Rajabhat University. Bione, E., Moura, R.C., Carvalho, R. & Souza, M.J. (2005). Karyotype, 6. References C-and fluorescence banding pattern, NOR location and Almeida, M.C., Campaner, C. & Cella, D.M. FISH study of five Scarabaeidae (2006). Karyotype characterization, (Coleoptera) species. Genetics and constitutive heterochromatin and Molecular Biology, 28, 376-381. nucleolus organizer regions of Paranaita opima (Coleoptera, Chaiyasan, P., Supiwong, W., Saenjundaeng, P., Chrysomelidae, Alticinae). Genetics Seetapan, K., Pinmongkhonkul, S. and Molecular Biology, 29, 475-481. and Tanomtong, A. (2018). A Report on classical cytogenetics of highfin Almeida, M.C., Zacaro, A.A. & Cella, barb fish, Cyclocheilichthys D.M. (2000). Cytogenetic analysis armatus (Cypriniformes, Cyprinidae). of Epicauta atomaria (Meloidae) Cytologia, 83, 149-154. and Palembus dermestoides (Tenebrionidae) with Xyp sex Chaiyasut, K. (1989). Cytogenetics and determination system using Cytotaxonomy of the Family standard staining, C-bands, NOR Zephyranthes. Department of Botany, and synaptonemal complex Faculty of Science, Chulalongkorn microspreading techniques. University. Hereditas, 133, 147-157. Colomba, M.S., Vitturi, R. & Zunino, M. Bellamy, C.L. (2008a). A world catalogue (2000). Karyotype analyzes, banding, and bibliography of the jewel beetles and fluorescent in situ hybridization (Coleoptera: Buprestoidea) volume in the Scarab beetle Gymnopleurus 1. Pensoft Publishers. sturmi McLeady (Coleoptera, Scarabaeoidea, Scarabaeidae). The Bellamy, C.L. (2008b). A world catalogue Journal of Heredity, 91, 260-264. and bibliography of the jewel beetles (Coleoptera: Buprestoidea) volume Dasgupta, J. (1977). Comparative 2. Pensoft Publishers. cytology of seven families of Indian Coleoptera. Nucleus, 20, 294-301. Bellamy, C.L. (2008c). A world catalogue and bibliography of the jewel beetles Ek-Amnuay, P. (2008). Beetles of Thailand. (Coleoptera: Buprestoidea) volume 2nd ed. Siam Insect-Zoo & Museum 3. Pensoft Publishers. Chiang Mai. 56 Chadaporn Nuchjangreed et al. Science Technology and Engineering Journal (STEJ)

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