53

Proc. Arthropod. Embryol. Soc. Jpn. 48, 53–54 (2017)Ⓒ 2017 Arthropodan Embryological Society of Japan
ISSN 1341–1527

Identification of Gene Transcripts Using Whole Transcriptome
Shotgun Sequencing (RNA-Seq) in the Sawfly Athalia rosae ruficornis(Hymenoptera)*1, *2
Masatsugu HATAKEYAMA1), Megumi SUMITANI2) and Kazuki SEKINÉ1)
Division of Insect Sciences, National Institute of Agrobiological Sciences, Owashi, Tsukuba, Ibaraki 305–8634, Japan
Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Owashi, Tsukuba, Ibaraki 305–8634,Japan
E-mail: sawfly@affrc.go.jp (MH)1)
2)

The sawfly, Athalia rosae ruficornis, has unique featuresin the aspects of evolution, development and gene function,such that the larva has remarkable abdominal appendages
called prolegs, the general mode of reproduction is haplodiploid, parthenogenetically developed haploid males produce
sperm without reductional divisions (meiosis) and the sex isdetermined by a single locus-multiple allete system (Naitoand Suzuki, 1991; Oishi et al., 1993, 1995; Fujiwara et al., 2004;
Yamamoto et al., 2004). We have demonstrated bymorphological and gene expression analyses that theabdominal proleg of A. rosae ruficornis is homologous to theoutgrowth of coxopodite (endite) resembling that ofmouthparts and not the serial homologue of thoracic leg (Okaet al., 2010). Functional analysis of the genes involved in theregulation of appendicular development, such as Hox genes(Hughes and Kaufman, 2002), will provide evidence to unravel
the molecular mechanisms of proleg formation. In A. rosae
ruficornis as well as other members of Hypmenoptera, theoccurrence of meiosis is closely linked to the sex: meiosis
takes place in females during oogenesis but not in males
during spermatogenesis. It is intriguing what molecularinteractions underlie the sex and the regulation of meiosis.
Analyses of gene functions related to sex determination andmaturation divisions, and their interaction during
spermatogenesis will give clues to understand thecharacteristic mechanisms of Hymenoptera.
One possible approach for gene functional analysis is toinduce the misexpression of candidate genes and examine theconsequent phenotypes. Gene knockdown by RNA
interference (RNAi) is a very useful tool for this purpose ininsects, especially in non-model species (Tomoyasu et al.,2008; Belles et al., 2010; Mito et al., 2011). RNAi by injection of
double-stranded RNA (dsRNA) of the targeted gene transcript
is practicable in most developmental stages of A. rosae
ruficornis (Sumitani et al., 2005; Yoshiyama et al., 2010, 2013;
Yoshiyama and Hatakeyama, 2012). Nevertheless, isolation of

the genes of interests remains laborious by conventional
PCR-based cloning methods. We applied whole transcriptome
shotgun sequencing (RNA-Seq) for efficient identification andisolation of target genes.
We prepared total RNAs to be sequenced from embryos
12 h-144 h after egg activation to obtain Hox gene homologues
and from the testes of males spanning the stages of last instar
larva to PCF 10 pupa (right before adult eclosion) to obtainspermatogenesis-related genes. Libraries for sequencing
were synthesized using a TruSeq RNA sample prep kit(Illumina) as follows: poly (A)-RNAs were selected from totalRNAs, fragmented to about 100 bp, reverse-transcribed tocDNA, adaptors ligated, and PCR-amplified. The libraries
were sequenced by a single read method using a highthroughput DNA sequencer (HiSeq2000, Illumina). The
fragments read were 7.5 x 107 for the embryonic library and 6.3
x 107 for the testicular library. The sequences were assembled
and finally gave rise to transcriptomes of about 25,000transcripts for the embryo and 29,000 transcriptomes for thetestis, respectively.
We searched Hox genes of A. rosae ruficornis based on
the known homologous genes of insects using a Basic localalignment search tool (BLAST) program supplied withGENETYX
bioinformatics
software(GENETYXCorporation). Complete coding sequences of the homologuesof Hox3/zerknullt (zen), Deformed (Dfd), Sex comb reduced(Scr), Antennapedia (Antp), Ultrabithorax (Ubx) andabdominal-A (abd-A), and partial sequences containing
complete homeodomains of proboscipedia (pb) andAbdominal-B (Abd-B) were obtained. The labial (lab) gene
homologue was not found unfortunately. The deduced aminoacid sequences corresponding to the homeodomain of each
Hox gene were well aligned with those of other insects so far
identified with only a few amino acid substitutions. Hox3/zen,which is responsible for the formation of extraembryonictissues, is known to be a functionally changed Hox gene in*1 Abstract of paper read at the 49th Annual Meeting of the Arthropodan Embryological Society of Japan, June 7–8, 2013 (Tsukuba-san, Ibaraki).
*2 This article, which was accepted in 2013 and should have been published in 2014, was printed in 2017 being much delayed due to various circumstances.

54
insects (Falciani et al., 1996; Damen and Tautz, 1998). Lessconservation in the homeodomain of Hox3/zen with severalamino acid substitutions was seen compared to those of other
Hox genes.
We also found some sex-determining gene homologues:
Sex lethal (Sxl), transformer 2 (tra2), doublesex (dsx), fruitless(fru) and intersex (ix), although it remains to be examined
whether sex-specific splicing occurs in these transcripts. In
spite of extensive efforts to isolate dsx by degenerate primer
PCR, we only found two dsx-related transcripts that haveconserved DM domains but are not classified as members ofinsect dsx homologues (Lee et al., 2005). We succeeded in
identifying the authentic dsx with less effort by searching thetranscriptomes.
Moreover, some isoforms of the boule (bol) genetranscripts were identified in addition to the bol homologues
obtained by conventional PCR-based methods. bol is essential
for meiosis in spermatogenesis in animals and is found in thegenomes of hymenopteran species in which meiosis does nottake place during sperm production (Schurko et al., 2010). It isof great interest to unravel whether bol is required forspermatogenesis in Hymenoptera and what is the function ofeach isoform.
In conclusion, searching a transcriptome makes it
efficient and easier to identify and isolate genes of interest,
especially in insects with less genomic information. In thenext step, we will investigate the functions of these genes
with a gene knockdown strategy.

Arthropodan Embryological Society of Japan, 40, 45.
Mito, T., T. Nakamura, T. Bando, H. Ohuchi and S. Noji (2011) The
advent of RNA interference in Entomology. Entomological
Science, 14, 1–8.
Naito, T. and H. Suzuki (1991) Sex determination in the sawfly, Athalia
rosae ruficornis (Hymenoptera): occurrence of triploid males.
Journal of Heredity, 82, 101–104.
Oishi, K., M. Sawa and M. Hatakeyama (1995) Developmental biologyof the sawfly, Athalia rosae (Hymenoptera). Proceedings of theArthropodan Embryological Society of Japan, 30, 1–8.
Oishi, K., M. Sawa, M. Hatakeyama and Y. Kageyama (1993) Geneticsand biology of the sawfly, Athalia rosae (Hymenoptera). Genetica,88, 119–127.
Oka, K., N. Yoshiyama, K. Tojo, R. Machida and M. Hatakeyama (2010)
Characterization of abdominal appendages in the sawfly, Athalia
rosae (Hymenoptera), by morphological and gene expression
analyses. Development Genes and Evolution, 220, 53–59.
Schurko, A. M., D. J. Mazur and J. M. Logsdon Jr (2010) Inventory andphylogenomic distribution of meiotic genes in Nasonia vitripennis
and among diverse arthropods. Insect Molecular Biology, 19(Suppl. 1), 165–180.
Sumitani, M., D. S. Yamamoto, J. M. Lee and M. Hatakeyama (2005)
Isolation of white gene orthologue of the sawfly, Athalia rosae(Hymenoptera)

anditsfunctionalanalysis

usingRNA

interference. Insect Biochemistry and Molecular Biology, 35, 231–240.
Tomoyasu, Y., S. C. Miller, S. Tomita, M. Schoppmeier, D. Grossmannand G. Bucher (2008) Exploring systemic RNA interference ininsects: a genome-wide survey for RNAi genes in Tribolium.

References

Genome Biology, 9, R10.

Belles, X. (2010) Beyond Drosophila: RNAi in vivo and functional

Yamamoto, D. S., M. Sumitani, K. Tojo, J. M. Lee and M. Hatakeyama

genomics in insects. Annual Review of Entomology, 55, 111–128.

(2004) Cloning of a decapentaplegic orthologue from the sawfly,

Damen, W. G. M. and D. Tautz (1998) A hox class 3 orthologue fromAthalia rosae (Hymenoptera), and its expression in the embryonicthe spider Cupiennius salei is expressed in a Hox-gene-likefashion. Development Genes and Evolution, 208, 586–590.

appendages. Development Genes and Evolution, 214, 128–133.
Yoshiyama, N. and M. Hatakeyama (2012) Difference in efficiency ofFalciani, F., B. Hausdorf, R. Schroder, M. Akam, D. Tautz, R. Denell

RNA interference during embryonic development in the sawfly,and S. Brown (1996) Class 3 Hox genes in insects and the origin ofAthalia rosae ruficornis: embryonic RNAi versus parental RNAi.

zen. Proceedings of the National Academy of Sciences of the United

Proceedings of the Arthropodan Embryological Society of Japan, 47,

State of America, 93, 8479–8484.

57–58.

Fujiwara, Y., K. Akita, W. Okumura, T. Kodaka, K. Tomioka and T.

Yoshiyama, N., K. Tojo and M. Hatakeyama (2010) Establishment ofNaito (2004) Estimation of allele numbers at the sex-determining

larval RNAi in the sawfly, Athalia rosae ruficornis Jakovlev

locus in a field population of the turnip sawfly (Athalia rosae).

(Hymenoptera) aiming at analysis of genes involved in appendage

Journal of Heredity, 95, 81–84.
Hughes, C. L. and T. C. Kaufman (2002) Hox genes and the evolutionof the arthropod body plan. Evolution and Development, 4, 459–499.
Lee, J. M., D. S. Yamamoto and M. Hatakeyama (2005) Cloning andcharacterizeation of the DMRT gene homologue in the sawfly,
Athalia rosae ruficornis (Hymenoptera). Proceedings of thedevelopment during larval-adult transition. Proceedings of theArthropodan Embryological Society of Japan, 45, 35–36.
Yoshiyama, N., K. Tojo and M. Hatakeyama (2013) A survey of theeffectivenessofnon-cellautonomousRNAithroughout

development in the sawfly, Athalia rosae (Hymenoptera). Journalof Insect Physiology, 59, 400–407.