The W chromosome Detection in Several Lepidopteran Species byGenomic in situ Hybridization (GISH)

123Atsuo Yoshido1 , Yasuhiro Yamada2 and Ken Sahara1 *4567891

Laboratory of Applied Molecular Entomology, Graduate School of Agriculture,
Hokkaido University, N9, W9, Kita-ku, Sapporo 060-8589, Japan, and2 Field Science
Center for Northern Biosphere, Hokkaido University, N11 W10, Kita-ku, Sapporo060-0811, Japan

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16*To whom Correspondence should be addressed,
Dr. Ken Sahara

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Fax: +81-11-706-2402. Tel: +81-11-706-2402
E- mail: sahara@abs.agr.hokudai.ac.jp
Phone & Fax: 011-706-24021

21(Abstract)

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We newly identified the W chromosome in eight lepidopteran species andconfirmed the absence of W chromosome in Caligula japonica females with the Z0 sex
chromosome constitution by means of genomic in situ hybridization (GISH). In
pachytene oocytes of species with the WZ/ZZ sex chromosome system, female-derived
genomic probes of the respective species highlighted the whole W chromosome threadin the WZ bivalent and also stained specific autosomal region(s) with telomeric and/or
subtelomeric signals. On the basis of GISH results obtained in this study and earlier
published, we classified karyotypes of lepidopteran species into three types.

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Key words: FISH, heterochromatin, Lepidoptera, nucleolar organizer region, pachytenechromosome, sex chromosome2

INTRODUCTION

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Moths and butterflies (Lepidoptera) possess a WZ/ZZ (female/male) sex
chromosome system, or its numerical variations such as Z/ZZ, W1 W2 Z/ZZ andWZ1 Z2 /Z1 Z1 Z2 Z2 (Suomalainen, 1969; Nilsson et al., 1988; Traut and Marec, 1996,1997; Rishi et al., 1999; Traut, 1999; Yoshido et al., 2005b). Pachytene mapping in
Lepidoptera can detect the W chromosome in pachytene oocytes (Traut and Marec,1997). However, this method fails in species which the W does not form aheterochromatin thread. The problem of W chromosome identification can be overcome
with the help of comparative genomic hybridization (CGH) or genomic in situ
hybridization (GISH) providing that there is a sufficient level of moleculardifferentiation between the W and Z sex chromosomes (Traut et al., 1999; Mediouni etal., 2004; Sahara et al., 2003b; Fuková et al., 2005; Yoshido et al., 2005b). With CGH,
it is also possible to estimate gross sequence composition of the W chromosome (Saharaet al., 2003a).
In Lepidoptera, CGH and GISH helped so far to detect W chromosome in 11species (belonging to 5 families) out of 12 species examined using these methods. In
this paper, we carried out GISH in nine other lepidopteran species using respective
female-derived genomic probes and in eight of them identified the W chromosome. Our
results confirmed the easiness of W chromosome identification in Lepidoptera by GISHand extended the practically applicable range to 21 species from 10 families.

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MATERIALS AND METHODS

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Insects

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Specimens of Spilarctia seriatopunctata (Arctiidae), Papilio xuthus(Papilionidae), Inachus io geisha (Nymphalidae), and Pieris brassicae (Pieridae) werecollected in Sapporo in 2003. Caligula japonica (Saturniidae), Orgyia recens(Lymantriidae), and Artaxa subflava (Lymantriidae) were collected in Sunagawa,Naganuma, and Ishikari, Hokkaido, respectively, in 2005. Mamestra brassicae(Noctuidae) was provided by Dr. Hayakawa (Saga University). Antheraea pernyi(Satur niidae) was obtained from Dr. Kajiura (Shinsyu University) through National
Bioresource Project (NBRP).

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Chromosome preparation
Spread chromosome preparations were done as described in Yoshido et al.
(2005a) with a slight modification of the procedure deve loped by Traut (1976). Briefly,ovaries of the matured last instar larvae or young pupae were dissected in Ephestia’ssaline solution (Marec and Traut, 1993) and fixed with Carnoy’s fluid (ethanol,chloroform, acetic acid, 6:3:1). In some species, the ovaries were incubated in ahypotonic solution (83 mM KCl and 17 mM NaCl) before fixing. Chromosomes werespread in 60% acetic acid at 50°C using a heating plate. Preparations were passed
through a graded ethanol series (70%, 80%, and 98%) and stored at -20o C until use.

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FISH and image processing
We carried out GISH according to the procedure of Sahara et al. (2003b). Briefly,
female genomic DNAs were labeled by a Nick Translation System (Invitrogen, Tokyo,Japan) with Cy3-dCTP (Amersham, Tokyo, Japan). Chromosome preparations passed
through an ethanol series and air-dried were denatured at 72°C for 3.5 min in 70%
formamide, 2×SSC. Probe cocktail contained 500 ng of labeled respective female
genomic DNA (Cy3; red), 25 µg of sonicated salmon sperm DNA (Sigma-Aldrich,Tokyo, Japan), and 3 µg of sonicated male genomic DNA in 10 µl of hybridization
solution (50% formamide, 10% dextran sulfate, 2×SSC). Hybridization in moist
chamber was carried out at 37°C for 3 days. Afterwards, the slides were washed at 62°C
in 0.1×SSC containing 1% TritonX-100. Mount ing and counterstaining was done withantifade [0.233g 1,4-diazabicyclo(2.2.2)-octane, 1 ml 0.2 M Tris-HCl, pH 8.0, 9 mlglycerol] containing 0.5 µg/ml of DAPI (4’, 6-diamidino-2-phenylindole;
Sigma-Aldrich, Tokyo, Japan). A Leica DMRE HC fluorescence microscope equippedwith a Photometrics CoolSNAP CCD camera was used for observation and image
capturing. Digital image processing and pseudocolouring was carried out with Adobe
Photoshop, Version 7.0. Routinely, red coloring was used for Cy3 and light blue for
DAPI images.

979899RESULTS AND DISCUSSION

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CGH or GISH represents a powerful tool for sex chromosome identification insome organisms (Traut et al., 1999; Traut and Winking, 2001). So far, these methods
have been used to identify sex chromosome s in 12 lepidopteran species (Table 1). In
this study, we extended the list in Table 1 by nine lepidopteran species. In eight species,

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GISH painted the whole chromosome thread of a bivalent (Figs. 2-9). The thread
obviously represented the W chromosome forming the sex chromosome bivalent with
its pairing partner, the Z chromosome. Thus, the species have a WZ/ZZ (female/male)
sex chromosome system. On the other hand, the whole-chromosome painting was notobserved in C. japonica (Fig. 1; Table 1). In this species, one of the longest
chromosomes formed a univalent in female pachytene nuclei. As meiosis progressed,
the univalent preferentially condensed and became deeply stained with DAPI (notshown). In accordance with the known haploid chromosome number of n=31 (Robinson,1971), pachytene complements consisted of 31 elements, but 30 were bivalents and one
univalent, obviously the Z chromosome. Accordingly, female mitotic complements
consisted of 2n=61 chromosomes. Our results clearly showed that females of C.
japonica have a ZO sex chromosome constitution, which was not known yet.
The Z chromosome surrounded the W in pachytene nuclei of A. subflava (Fig.
3). The characteristic nature of the sex chromosome bivalent in this species mostprobably results from the large difference between the size of sex chromosomes, thelong Z chromosome and very short W chromosome. In S. seriatopunctata (Fig. 5), wedetermined for the first time the haploid chromosome number, which is n=31.
GISH signals were not restricted to the W chromosomes only but they werefound also in autosomes. The autosomal signals stained specific segments in somespecies besides the predominant but fainter spots in telomeric and/or subtelomericregions. After distribution of the autosomal signals we classified species examined into
3 groups: (i) without autosomal signals except for telomeric and/or subtermeric regions,(ii) with highlighted nucleolar organizer region (NOR) –chromosomes, and (iii) withstrong signal only in non NOR-chromosome except for telomeric and/or subtermericregions. The NOR consists of ribosomal gene cluster (Shaw and Doonan, 2005) andsometimes associates heterochromatic region near by depending on lepidopteran species(Yoshido et al., 2005b). The first group involved A. subflava (Fig. 3), O. recens (Fig. 4),
Spilarctia seriatopunctata (Fig. 5), Papillio xuthus (Fig. 7), Cydia pomenella (Fukova etal., 2005), O. antiqua, and O. thyellina (Yoshido et al., 2005b), and Galleria mellonella(Traut et al., 1999). The second group involved A. pernyi (Fig. 2), P. brassicae (Fig. 8),
I. i. geisha (Fig. 9), A. yamamai, Samia cynthia walkeri, S. c. ricini, and S. c. indet. ssp.
(Yoshido et al., 2005b), and Ephestia kuehniella (Traut et al., 1999). Among them A.
pernyi, P. brassicae, and I. i. geisha also have highlighted signals in the telomericand/or subtelomeric regions of the bivalents. The signals appeared in most bivalents ofA. pernyi, in two bivalents of P. brassicae, and in five bivalents of I. i. geisha (Figs. 2, 8,9, respectively). It is notable that I. i. geisha displayed at least five nucleoli in a haploid

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genome, and three of them were strongly labeled by GISH. The last group included only
B. mori (Sahara et al., 2003b; Yoshido et al., 2005a). Finally, we were not able todetermine, whether the autosome with a GISH signal in M. brassicae (Fig. 6) is or is not
the NOR-chromosome.
Our results confirmed the potential of GISH to identify the lepidopteran W
chromosome as shown in representatives of 10 different families. GISH is also a very
useful technique to resolve the sex chromosome constitution in females of Lepidopterawith multiple sex chromosomes (Yoshido et al., 2005b). GISH enables us to detect theW chromosome even in a single female specimen using the female genomic DNA as aprobe and simultaneously as a competitor (A. Yoshido, unpublished). This method isparticularly well suited for obtaining the first and fast information on sex chromosomes
in species with a small population size and/or in unidentified species.

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ACKNOWLEDGMENTS

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We thank Dr. Zenta Kajiura (Shinsyu University, Japan) and Dr. Yoichi
Hayakawa (Saga University, Japan) for providing lepidopteran specimens. Our thanks
are also due to Prof. František Marec for critical reading of this manuscript. This study
was partially supported by a Grant- in-Aid for Scientific Research no s. 15380227,18380037 and 18658020 (K.S.) from the Japan Society for the Promotion of Science(JSPS), and the Insect Technology Project from the Ministry of Agriculture, Forestryand Fisheries in Japan, no. 2013 (K.S.).

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REFERENCES

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Figure legends

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Figs. 1-9. GISH identification of the W chromosome in lepidopteran species.
W-chromosome painting together with hybridization signals of various intensities inother chromosomes are obvious in all figures except for Caligula japonica, which has aZO sex chromosome constitution. Fig. 1, C. japonica; Fig. 2, Antheraea pernyi; Fig. 3,Artaxa subflava; Fig. 4, Orgyia recens; Fig. 5, Spilarctia seriatopunctata; Fig. 6,Mamestra brassicae; Fig. 7, Papilio xuthus; Fig. 8, Pieris brassicae; Fig. 9, Inachus iogeisha. Light blue, chromosomes counterstained with DAPI; red, GISH signals. N,nucleolus. Arrow indicates a Z chromosome univalent in C. japonica. Arrowheads
indicate autosomal heterochromatic segments highlighted by GISH. Bar = 10 µm.

8Table 1. Summary of data on the identification of sex chromosome constitution by CGH or GISH in Lepidoptera
Collection siteCollection Sex chromosome system CGH or GISH done by/in
FamilySpeciesyear
Arctiidae
WZ/ZZ
Sapporo2003
Spilarctia seriatopunctata
This study: Fig. 5
Bombycidae
Bombyx mori
Bombyx mandarina

Sakado

1982

WZ/ZZ
WZ/ZZ

Traut et al. (1999)
Yoshido et al. (2006)

Lymantriidae
Artaxa subflava
Orgyia thyellina
Orgyia antiqua
Orgyia recens

Ishikari
Naganuma
Germany
Naganuma

2005
2000
1999
2005

WZ/ZZ
W1W2 neo-Z /neo-Z neo-Z
neo-W neo-Z/neo-Z neo-Z
WZ/ZZ

This study: Fig. 3
Yoshido et al. (2005b)
Yoshido et al. (2005b)
This study: Fig. 4

NoctuidaeMamesta brassicae

Hirosaki and Shimonoseki

1990s

WZ/ZZ

This study: Fig. 6

Nymphalidae
Inachus io geisha

Sapporo

2003

WZ/ZZ

This study: Fig. 9

Papilionidae
Papilio xuthus

Sapporo

2003

WZ/ZZ

This study: Fig. 7

Pieridae
Pieris brassicae

Sapporo

2003

WZ/ZZ

This study: Fig. 8

Pyralidae
Ectomyelois eratoniae
Ephestia kuehniella
Galleria mellonella

Tunisia
Czech RepublicCzech Republic

1999

WZ/ZZ
WZ/ZZ
WZ/ZZ

Mediouni et al. (2004)
Traut et al. (1999)
Traut et al. (1999)

Saturniidae
Antheraea peryni
Antheraea yamamaiCaligula japonica
Samia cynthia ricini
Samia cynthia ssp. indet
Sa mia cynthia walkeri

NaganoNaganoSunagawa
Vietnam
NaganoSapporo

1880s
2005
1990s
1990s
2003

WZ/ZZ
WZ/ZZ
Z/ZZ
Z/ZZ
neo-WZ1Z2/Z1Z1Z2Z2
neo-W neo-Z/neo-Z neo-Z

This study: Fig. 2
Yoshido et al. (2005b)
This study: Fig. 1
Yoshido et al. (2005b)
Yoshido et al. (2005b)
Yoshido et al. (2005b)

Tortricidae
Cydia pomonella

Russia

1961

WZ/ZZ

Fuková et al. (2005)