A New Molecular Technique for Determining the Sex of Harmonia Axyridis

Journal of Insect Biotechnology and Sericology 84, 9-15 (2015)

A new molecular technique for determining the sex of Harmonia axyridis

Hiroki Gotoh1＊, Hideto Nishikawa1＊, Ken Sahara2, Toshinobu Yaginuma1 and Teruyuki Niimi1＊＊

1 Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan 2 Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, 020-8550, Japan (Received November 25, 2014; Accepted January 30, 2015)

We have developed a fast and efficient PCR-based technique to sex all developmental stages of the ladybird beetle Harmonia axyridis. Previously, we established a male-specific fluorescent expressing strain of H. axyridis with the piggyBac vector. FISH analyses revealed that the transgene inserted into the H. axyridis genome in the male Y-chromosome. Because only males expressed the fluorescent signal, we were able to differentiate between the sexes of this transgenic strain even in newly hatched larvae. We took advantage of this insertion and designed an inverse PCR reaction and genome walking surrounding the inserted region to identify 3,781 bp of the male Y-chromosome genomic sequence. From this male specific region, we designed a PCR-based protocol to identify sexes from all H. axyridis developmental stages. Key words: Transgenic strain, male-specific transgene expression, ladybird beetle, Coleoptera, Y specific marker

aphid pests worldwide. Recently our group has developed INTRODUCTION a suite of molecular tools in H. axyridis such as larval The molecular developmental mechanisms of insect RNAi (Niimi et al., 2005; Ohde et al., 2009) and trans- sex-determination have been successfully identified in re- genesis techniques (Kuwayama et al., 2006; Kuwayama et cent years (Salz, 2011; Gempe and Beye, 2011). What is al., 2014). Considering those characteristics of H. axyri- interesting is that while the initial sex-determination fac- dis, this species is also an important new model system tors are diverse among taxa, most downstream sex deter- for investigating molecular mechanisms of the sex-determination cascade genes are highly conserved (Burtis and mination pathway. Baker, 1989; Williams and Carrol, 2009; Kopp, 2012; As a first step in establishing H. axyridis as a suitable Kiuchi et al., 2014). In Coleoptera, the sex-determination model for investigating molecular sex determination gene doublesex (dsx) has been examined in five species mechanisms, it was necessary for us to establish a proto- which include Tribolium castaneum (Tenebrionidae, Tene- col to sex eggs and larvae. Here we describe how we brionoidea; Shukla and Palli, 2012), Onthophagus taurus used a transgenic H. axyridis strain with male-specific flu- and O. sagitarius (Scarabaeidae, Scarabaeoidea; Kijimoto orescent marker expression by means of piggyBac vector et al., 2012), Trypoxylus dichotomus (Scarabaeidae, Scara- system (Kuwayama et al., 2014) to design a PCR-based baeoidea; Ito et al., 2013), and Cyclommatus metallifer sexing technique. We tested this PCR assay on several (Lucanidae, Scarabaeoidea; Gotoh et al., 2014). Despite different populations of H. axyridis and six additional spe- interesting works on the downstream sex-determination cies of ladybird beetle. As results, we found that this genes in the Coleoptera, the primary molecular signal of method is widely available in H. axyridis but is not in sex determination has not identified in any beetle species. other ladybird species for sexing. Because these primary sex-determination signals function at very-early embryonic stages (Bopp et al., 2013; Sakai MATERIALS AND METHODS et al., 2014), being able to accurately sex individuals at these early embryonic stages is necessary. Insects Harmonia axyridis is a ladybird beetle (Coccinellidae, The ladybird beetle species used in this experiment Cucujoidea) which is easy to rear and breed by using arti- were kept in the laboratory according to Niimi et al. ficial diet. It has a short generation time (approximately (2005). The transgenic strain of H. axyridis with male 1 month at 25°C incubation), and is a well-known and of- specific fluorescence used in this study was established by ten used natural predator and biological control agent of using the transgenesis method reported in Kuwayama et al. (2014). Henosepilachna vigintioctopunctata was kept * Contributed equally according to Ohde et al. (2009). Beetle sampling sites in **To whom correspondence should be addressed. Japan are listed in Table 1. Fax: +81-52-789-4036. Tel: +81-52-789-4038. Email: [email protected] 10 Gotoh et al.

Table 1. Species name and sampling locations of ladybird beetles used in the study Species name Sampling site Name of population Harmonia axyridis Sapporo, Hokkaido Hokkaido Koriyama, Fukushima Fukushima Ueda, Nagano Nagano Nagoya, Aichi Aichi Fukuyama, Hiroshima Hiroshima Fukuoka, Fukuoka Fukuoka Aso, Kumamoto Kumamoto Harmonia octomaculata Kunigami, Okinawa Coccinella septempunctata Nagoya, Aichi Menochilus sexmaculatus Nagoya, Aichi Propylea japonica Nagoya, Aichi Calvia muiri Gifu, Gifu Henosepilachna vigintioctopunctata Nagoya, Aichi

Production of a transgenic ladybird beetle strain in-12-dCTP (Perkin Elmer, Boston, USA) by BioNick The transgenic strain of H. axyridis with male specific Labeling System (GibcoBRL, Life technologies Inc., Karl- fluorescence used in this study was established by piggyBac sruhe, Germany). The labeled DNAs (300 ng Cy3- and vector in our previous study, Kuwayama et al. (2014). In 500 ng Fluorescein-labeled probes) were mixed with brief, we established a composite vector named pBac(hsp70- 25 μg salmon sperm DNA and sonicated with 3 μg H. transposase)::(3xP3-ECFP)::hsp27-EGFP::(3xP3-DsRed) axyridis female genomic DNA in 10% dextran sulfate and which contains DsRed and ECFP as transformation mark- 50% formamide to make the probe cocktail. The cocktail ers. Transgenic ladybird beetles were generated by micro- was incubated for three days with the chromosome prepa- injection of the composite vector into ladybird embryos. ration and followed by washing with a solution of 1% tri- In this study, we only used DsRed as the marker because tonX-100 and 0.1x SSC. The hybridized preparations were DsRed signal was much stronger than ECFP. We have mounted by DABCO (1,4-Diazabicyclo[2.2.2]octane) with kept this transgenic strain with male-specific fluorescent DAPI (4’,6-diamidino-2-phenylindole) nuclear staining marker expression for more than twelve years. reagent and observed with a fluorescent microscope (DM6000B, Leica). The fluorescent signals captured Chromosome preparations through B&W CCD camera were processed by Adobe We made chromosome preparations according to the Photoshop (version 7). We applied green and red pseudo method described in Sahara et al. (1999) with slight mod- colors to signals from genomic and plasmid probes. Gray ifications. Briefly, adult testes of transgenic ladybird bee- pseudo color was applied to DAPI fluorescence for the tles were dissected in Ephestia’s saline solution (0.9% chromosome images.

NaCl, 0.042% KCl, 0.025% CaCl2, 0.02% NaHCO3, pH 7.8) and fixed with Carnoy’s fixative (Ethanol : Chlo- Inverse PCR and genome walking roform : Acetic acid = 6:3:1) for 15 min. On slide glasses Inverse PCR for identifying the genome sequence of cells were dissociated in 60% acetic acid at 55°C. Then the inserted transgene region was performed as described the preparations were washed with a graded ethanol series by Kuwayama et al. (2006). In brief, genomic DNA was (70%, 80%, 98%) for 30 seconds (each wash) and pre- digested with Hae III or Msp I. PCR was performed using served at −30°C until used. the following primers (Hediger et al., 2001), PLF; 5′- CTT GAC CTT GCC ACA GAG GAC TAT Fluorescence in situ hybridization (FISH) TAG AGG -3′ The FISH procedure we used here followed Yoshido et PLR; 5′- CAG TGA CAC TTA CCG CAT TGA CAA al. (2005). Instead of BAC probes, we used the composite GCA CGC -3′ vector (piggyBac plasmid) DNA carrying the DsRed gene For identifying longer sequences, we performed PCR (see above) and genomic probes. Briefly, the plasmid using the GenomeWalker library as a template with prim- DNA harboring the inserted sequences and H. axyridis ers designed for the 126 bp sequence we initially identi- male genomic DNA extracted from adults were labeled fied. Preparation of genome library was performed by with Cy3-dCTP (Amersham, Tokyo, Japan) and fluoresce- using BD GenomeWalker Universal Kit (BD Biosciences Molecular sexing technique in Harmonia axyridis 11

Clontech, San Jose, CA, USA) according to the manufac- natant portion was mixed with 1 μl of 20 mg/ml glycogen turer’s protocol. Sequence specific outer primers (SSP1) and 24 μl of isopropanol and centrifuged again (20,950 g, and nested primers (SSP2) were designed as follows, 10 min). Precipitated DNA pellet was washed with 75% SSP1 ethanol and dried up. Then, 100 μl DNA resuspension in Ha-Ytg-1; 5′- CCA GGA TGA TCG CCA ATA TTC distiled water was boiled for 10 min and stored in −20°C GGA ACG -3′ until use. PCR was performed using AmpliTaq Gold 360 Ha-Ytg-3; 5′- CTC CGA TTC TTC TTC TTC TTC Master Mix (Applied Biosystems, Branchburg, NJ, USA) TTC GGG -3′ with PCR program as follows: 95°C for 9 min, 45 cycles SSP2 of 94°C 1 min, 60°C 30 sec and 72°C 30 sec. For positive Ha-Ytg-2; 5′- CCC GAA GAA GAA GAA GAA control of gDNA PCR, we used 28S rRNA gene sequence GAA TCG GAG -3′ by PCR program 95°C for 9 min, 45 cycles of 94°C Ha-Ytg-4; 5′- CGT TCC GAA TAT TGG CGA TCA 1 min, 50°C 30 sec and 72°C 60 sec. Primer sequences TCC TGG -3′ used for PCR were according to Kim et al. (2000) as fol- Using those two primer pairs (HA-Ytg-1 and HA- lows, Ytg-2, HA-Ytg-3 and HA-Ytg-4), the target region was 28S-F; 5′- GAC TAC CCC CTG AAT TTA AGC AT -3′ amplified according to the manufacturer’s protocol. All 28S-R; 5′- GAC TCC TTG GTC CGT GTT TCA AG amplified PCR products were purified with MagExtractor- -3′ PCR & Gel-Cleanup- (Toyobo, Osaka, Japan) and subcloned into pBlueScript KS+ vector (Stratagene, La Jolla, RESULTS & DISCUSSION CA, USA). Subcloned inserts were sequenced using the dideoxy chain-termination method by an automatic DNA FISH for detection of the transgene sequencer (DNA sequencer 3130 genetic analyser; Ap- DsRed expression in the transgenic strain is male spe- plied Biosystems, Foster City, CA, USA). Sequence anal- cific (Kuwayama et al., 2014) (Fig. 1). The exclusive ysis was carried out using a DNASIS system (Hitachi DsRed expression in male transgenic individuals was ob- Software Engineering, Tokyo, Japan). Database searches served in 427 individuals examined over 8 generations by for identified Y-chromosome sequences homology were the cross between transgenic males and wild-type females. performed using BlastX and BlastN at the NCBI server To identify where the transgene inserted into the chromo- (http://blast.ncbi.nlm.nih.gov/Blast.cgi). some, we performed a cytogenetic analysis. In H. axyridis, Y chromosomes can be easily distinguished by its PCR dependent sexing method in Harmonia axy- smaller size from the X chromosome during meiotic meta- ridis phase I (Fig. 2A). Genomic in situ hybridization (GISH) To develop PCR-based sexing method in this species using male gDNA probes also revealed this difference we designed primers on our identified genome sequence (Fig. 2B). The probes of piggyBac plasmid DNA carrying (Fig. 3) as follows, the DsRed gene clearly hybridized to a peripheral region Ha-Y-1; 5′- ATA TTC GGA ACG AAT AGG CA -3′ of the Y chromosome (Fig. 2C). At anaphase I (Fig. 2D), Ha-Y-2; 5′- AAG ATC TAG GCT CAT GGA AG -3′ green GISH signals clearly differentiated Y or X carrying Ha-Y-3; 5′- TAT GGT AAG AGA GGA CCA GG -3′ chromosome masses (Fig. 2E). The red transgene signals Ha-Y-4; 5′- CGT TCC GAA TAT TGG CGA TCA TCC were observed only on the Y chromosome (Fig. 2F). The TGG -3′ spermatid divided into two types with or without the red Ha-Y-5; 5′- CCG TTT TTA GGT TTC CTA TTG AT signal at a 1:1 ratio (Fig. 2G, H). These cytogenetic re- -3′ sults explain cytogenetic mechanisms why only the trans- Ha-Y-6; 5′- CGG AGA AAA AAT GAA TTT CCT GA genic males exhibit the DsRed signals from the cross -3′ between wild type females (XX) and the transgenic males We tested the accuracy of the primer pairs for sexing in (XYDsRed). Since the insertion of the transgene into highly seven different natural populations of H. axyridis. Genom- heterochromatic Y chromosome has known to be difficult ic DNA (gDNA) was extracted from frozen or fresh ani- (Nakanishi et al., 2002; Ma et al, 2013), the insertion to mal legs from each population or species (see Table 1) for small Y chromosome in our strain is likely to be a rare PCR template. Whole body or dissected legs of ladybird event in this species. beetles were washed by water and homogenized with 50 μl of TES (0.1 M Tris-HCl (pH 9.0), 0.1 M EDTA, 1% Identification of genomic sequences around the SDS) and incubated at 70°C for 30 min. Then 7 μl of 8 M transgene inserted region Acetate was added and samples were incubated on ice for To identify Y specific sequences, inverse PCR and ge- 30 min. After centrifugation (20,950 g, 10 min), the super- nome walking were carried out. Inverse PCR identified 12 Gotoh et al.

Fig. 1. Male specific transgene expressing strain. In this strain, DsRed expression is under the control of the3xP3 element, which promotes expression in CNS and visual systems (Kuwayama et al., 2014). DsRed expression as a transformation marker in central nerve system (CNS) and visual systems was only detected in males. (A) Ventral view of first instar larvae. CNS of male larvae express DsRed, while in female DsRed is not expressed. (B) Frontal view of adult heads. Compound eyes of male adult express DsRed, while not in females.

Fig. 2. Cytogenetic proof of Y insertion of the transgene. FISH (fluorescence in situ hybridation) with the composite vector (piggyBac plasmid) DNA (red signals) probes and green signals from GISH (genomic in situ hybridization) clearly showed that the transgene has inserted into peripheral region of the Y chromosome and transmit through Y carrying sperm. Meiotic metaphase I (A-C), anaphase I (D-F) and spermatids (G, H) of the transgenic specimens. DAPI stained image (A, D, G); GISH signals (B, E); FISH signals (C, F, H). X, X chromosomes; Y, Y chromosomes; Ar- rows point to signals from piggyBac probes. Bar represents 5 μm for A-F and 2 μm for G and H. Molecular sexing technique in Harmonia axyridis 13

Fig. 3. Nucleotide sequence identified by inverse PCR and genome walking. Arrows indicate primer positions used for sexing. Nucleotides represented in red are determined by inverse PCR. The dotted line indicates undetermined sequences. Characteristic piggyBac TTAA target sequence is highlighted by the blue box. Sequences with high similarity to Dendroctonus ponderosae are indicated by the green line (see text for detail).

126 bp of Y chromosome sequence including the TTAA Because the PCR products were amplified only when piggyBac target site located at the 5′ side of the inserted male genomic DNA was used as template in our transgen- transgene sequence (Fig. 3). Subsequent genome walking ic population (Fig. 3), we tested this primer pair in seven successfully identified total 3,781 bp (Fig. 3). Homology different populations of H. axyridis (Fig. 4A). In all seven searches showed that transgene was inserted into non-cod- populations, PCR products were amplified only from male ing region; some of the sequence has high similarity genomic DNAs (Fig. 4B). Amplified PCR products were (289 bp/380 bp, 76% homology; e-value = 5e−44) with non- appeared as single band and the size was also correspond- coding genome region of APGK01048852 in Dendrocto- ed with expected product size (125 bp). Thus, this primer nus ponderosae. This homology indicates that at least a pair Ha-Y-1 and Ha-Y-2 can be used as PCR dependent part of identified region found in H. axyridis might be sexing marker in H. axyridis populations. We also de- conserved in distant family. But its chromosomal position signed two additional primer pairs (Ha-Y-3, Ha-Y-4 and and function is unknown. Ha-Y-5, Ha-Y-6) on identified region (Fig. 3) and performed PCR. However, those primer pairs amplified PCR Establishment of a male-specific PCR marker in products both in females and males (data not shown). Harmonia axyridis We designed a PCR primer pair, Ha-Y-1 and Ha-Y-2 (Fig. 3) for establishing our PCR-based sexing method. 14 Gotoh et al.

Fig. 4. PCR amplification patterns of genomic DNA from different geographic populations of H. axyridis and other ladybird beetle species. (A) Sampling sites of H. axyridis populations in Japan. (B) PCR amplification patterns of genomic DNA from different geographic populations of H. axyridis. DNA was amplified using the H. axyridis Y-chromosome specific primer pair (YS: Ha-Y-1 and Ha-Y-2). In all of population, PCR product was amplified only from male genomic DNA. The 28S rRNA gene sequence was used as a positive control. (C) PCR amplification patterns of genomic DNA from different ladybird beetle species. DNA was amplified using the H. axyridis Y-chromosome specific primer pair. Contrary to H. axyridis, PCR amplification was not male-specific in all species tested. The 28S rRNA genese- quence was used as a positive control. Ho: Harmonia octomaculata, Cs: Coccinella septempunctata, Ms: Menochilus sexmaculatus, Pj: Propylea japonica, Cm: Calvia muiri, Hv: Henosepilachna vigintioctopunctata.

Validation of a male-specific PCR marker in other collecting H. axyridis in Japan and Dr. R. Futahashi for Coccinellid species kindly providing originally drawn white map of Japan. We We further tested the potential sexing ability of this thank Dr. L. Lavine for her comments and English correc- PCR marker in six additional species of ladybird beetles. tions on the manuscript. We thank Drs. Y. Matsuda, M. Although size of amplified PCR product was correspond- Kobayashi and M. Ikeda for helpful discussions of this ed with expected size (125 bp), amplification of the PCR work. Finally, we are especially grateful to Ms. H. product was not sex-specific in any species even in a spe- Kawaguchi for careful maintenance of the ladybird bee- cies of the same genus (H. octomaculata) (Fig. 4C). These tles. This work was in part supported by JSPS and MEXT results indicate that this PCR marker cannot be used for KAKENHI Grant number 25292202 and 26113708, re- molecular sexing in other species. Considering that same spectively. size of products were amplified in all of tested species, the identified sequence is potentially conserved, but locat- REFERENCES ed in either autosome or X chromosome in those species. Bopp, D., Saccone, G. and Beye, M. (2013) Sex determination Conclusion in insects: variations on a common theme. Sex Dev., 8, 20- Here, we described how we designed a molecular sex- 28. Burtis, K.C. and Baker, B.S. (1989) Drosophila doublesex ing technique in H. axyridis. The molecular sexing tech- gene controls somatic sexual differentiation by producing nique in H. axyridis enables us to compare gene expression alternatively spliced mRNAs encoding related sex-specific pattern between sexes at very early embryonic stages, polypeptides. Cell, 56, 997-1010. which are critical stages of initial sex-determination cues. Gempe, T. and Beye, M. (2011) Function and evolution of sex This technique will allow us to identify the genetic sex of determination mechanisms, genes and pathways in insects. Bioessays, 33, 52-60. an individual when analyzing sex-determination genes, Gotoh, H., Miyakawa, H., Ishikawa, A., Ishikawa, Y., Sugime, even when their morphological sex is mutated by func- Y., Emlen, D.J., Lavine, L.C. and Miura, T. (2014) Devel- tional knockdown of putative sex-determination genes. In opmental link between sex and nutrition; doublesex regu- conclusion, this transgenic strain and PCR marker offer a lates sex-specific mandible growth via juvenile hormone unique new tool for elucidating the molecular sex-deter- signaling in stag beetles. PLoS Genet., 10, e1004098. Hediger, M., Niessen, M., Wimmer, E.A., Dübendorfer, A. and mination mechanisms of ladybird beetle H. axyridis. Bopp, D. (2001) Genetic transformation of the housefly Musca domestica with the lepidopteran derived transposon piggyBac. Insect Mol. Biol., 10, 113-119. ACKNOWLEDMENTS Ito, Y., Harigai, A., Nakata, M., Hosoya, T., Araya, K., Oba, Y., We thank Drs. S. Asano, K. Miura and T. Kusakabe for Ito, A., Ohde, T., Yaginuma, T. and Niimi, T. (2013) The Molecular sexing technique in Harmonia axyridis 15

role of doublesex in the evolution of exaggerated horns in A., Tairaka, A., Hayashi, T., Takagi, T., Ikawa, M., Matsuda, the Japanese rhinoceros beetle. EMBO Rep., 14, 561-567. Y. and Okabe, M. (2002) FISH analysis of 142 EGFP trans- Kijimoto, T., Moczek, A.P. and Andrews, J. (2012) Diversifi- gene integration sites into the mouse genome. Genomics, cation of doublesex function underlies morph-, sex-, and 80, 564-574. species-specific development of beetle horns. Proc. Natl. Niimi, T., Kuwayama, H. and Yaginuma, T. (2005) Larval Acad. Sci. USA, 109, 20526-20531. RNAi applied to the analysis of postembryonic development Kim, C.G, Zhou, H.Z., Imura, Y., Tominaga, O., Su, Z.H. and in the ladybird beetle, Harmonia axyridis. J. Insect Biotech. Osawa, S. (2000) Pattern of morphological diversification in Sericol., 74, 95-102. the Leptocarabus ground beetles (Coleoptera: Carabidae) as Ohde, T., Masumoto, M., Morita-Miwa, M., Matsuura, H., deduced from mitochondrial ND5 gene and nuclear 28S Yoshioka, H., Yaginuma, T. and Niimi, T. (2009) Vestigial rDNA sequences. Mol. Biol. Evol., 17, 137-145. and scalloped in the ladybird beetle: a conserved function in Kiuchi, T., Koga, H., Kawamoto, M., Shoji, K., Sakai, H., wing development and a novel function in pupal ecdysis. Arai, Y., Ishihara, G., Kawaoka, S., Sugano, S., Shimada, T., Insect Mol. Biol., 18, 571-581. Suzuki, Y., Suzuki, M.G. and Katsuma, S. (2014) A single Sahara, K., Marec, F. and Traut, W. (1999) TTAGG telomeric female-specific piRNA is the primary determiner of sexin repeats in chromosomes of some insects and other arthro- the silkworm. Nature, 509, 633-636. pods. Chromosome Res., 7, 449-460. Kopp, A. (2012) Dmrt genes in the development and evolution Sakai, H., Fugaku, A. and Suzuki, M.G. (2014) Identification of sexual dimorphism. Trends Genet., 28, 175-184. of the key stages for sex determination in the silkworm, Kuwayama, H., Yaginuma, T., Yamashita, O. and Niimi, T. Bombyx mori. Dev. Genes Evol., 224, 119-123. (2006) Germ-line transformation and RNAi of the ladybird Salz, H.K. (2011) Sex determination in insects: a binary deci- beetle, Harmonia axyridis. Insect Mol. Biol., 15, 507-512. sion based on alternative splicing. Curr. Opinion Genet. Kuwayama, H., Gotoh, H., Konishi, Y., Nishikawa, H., Dev., 21, 395-400. Yaginuma, T. and Niimi, T. (2014) Establishment of trans- Shukla, J.N. and Palli, S.R (2012) Doublesex target genes in genic lines for jumpstarter method using a composite trans- the red flour beetle, Tribolium castaneum. Sci. Rep., 2, 00948. poson vector in the ladybird beetle, Harmonia axyridis. Yoshido, A., Bando, H., Yasukochi, Y. and Sahara, K. (2005) PLoS ONE, 9, e100804. The Bombyx mori karyotypeand the assignment of linkage Ma, S., Wang, X., Fei, J., Liu, Y., Duan, J., Wang, F., Xu, H., groups. Genetics, 170, 675-685. Zhao, P. and Xia, Q. (2013) Genetic marking of sex using a Williams, T.M. and Carroll, S.B. (2009) Genetic and molecu- W chromosome-linked transgene. Insect Biochem. Mol. Biol., lar insights into the development and evolution of sexual 43, 1079-1086. dimorphism. Nat. Rev. Genet., 10, 797-804. Nakanishi, T., Kuroiwa, A., Yamada, S., Isotani, A., Yamashita,