A New Molecular Technique for Determining the Sex of Harmonia Axyridis
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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 be- tween 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-deter- mination 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.