Electroporation-mediated RNA interference reveals a role of the multicopper oxidase 2 gene in dragonfly cuticular pigmentation

Okude, Genta; Futahashi, Ryo; Kawahara-Miki, Ryouka; Yoshitake, Kazutoshi; Yajima, Seishi; Fukatsu, Takema

doi:10.1007/s13355-017-0489-9

Cited by 27 publications

(32 citation statements)

References 42 publications

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“…Part of long isoform would include region 2 because region 2 was shown in reverse transcription PCR of gynomorphic females (data not shown). siRNAs for the multicopper oxidase 2 ( MCO2 ) gene that is essential for melanin pigmentation (Okude, Futahashi, Kawahara-Miki, et al, 2017), were used as positive controls for targeting 5’-GAGCACTTTCCGTTATCAATATA-3’ and 5’-TCCTCTTGATGCTATCTGTAATG-3’. As a negative control, siRNA for the enhanced green fluorescent protein ( EGFP ) gene was used for targeting 5’-CGG CAT CAA GGT GAA CTT CAA GA-3’ (Ando & Fujiwara, 2013).…”

Section: Methodsmentioning

confidence: 99%

“…Electroporation was used to introduce siRNA into larvae (Okude, Futahashi, Kawahara-Miki, et al, 2017). Early-stage, final instar larvae (stage 1 in Okude, Futahashi, Tanahashi, & Fukatsu, 2017) were anesthetized on ice for 1 min, approximately 1 μl of 100 μM siRNA solution was injected into the left side of the thorax using a glass needle and an injector (Nanoject II; Drummond Scientific Company).…”

Section: Methodsmentioning

confidence: 99%

“…Positive and negative platinum electrodes were attached to injection and opposite sites, respectively (Fig. 1b, Okude, Futahashi, Kawahara-Miki, et al, 2017). Electroporation was conducted with five or ten pulses for 25 V (each 280 ms pulses/s) using an electroporator (Cure-Gene; CellProduce).…”

Section: Methodsmentioning

confidence: 99%

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Involvement of thedoublesexgene in body color masculinization of the blue-tailed damselfly,Ischnura senegalensis

Takahashi

Okude

Futahashi

et al. 2020

Preprint

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20Odonata (dragonflies and damselflies) display remarkable color pattern diversity 21 including sexual dimorphism and intrasexual polymorphism. We previously found that 22 expression of a sex-determining transcription factor, the doublesex (dsx) gene, is 23 associated with female color polymorphism (gynomorph for female-specific color and 24 andromorph for male mimicking color) in the blue-tailed damselfly, Ischnura 25 senegalensis. Here we investigate the function of dsx gene on thoracic coloration by 26 electroporation-mediated RNA interference (RNAi). RNAi of the dsx common region 27 changed color patterns of males and andromorphic females to patterns of gynomorphic 28 females. Further, gynomorphic color pattern was not affected by dsx RNAi. The long 29 isoform of dsx RNAi produced no effects, suggesting that the short isoform of dsx is 30 important for body color masculinization in both males and andromorphic females.31Expression pattern changes were also examined in five genes with different expression 32 levels between sexes and female morphs. Among these genes are two melanin 33 suppressing genes, black and ebony, that were upregulated in the dsx-RNAi region 34 compared to a control region. Upregulation coincides with a gynomorphic orange color 35 instead of the black stripe observed in males and andromorphic females. dsx may 36 regulate male color differentiation by suppressing black and ebony in the thoracic 37 region of I. senegalensis. Results add to the understanding of molecular mechanisms 38 underlying the evolution of female polymorphism in Odonata.39 3

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Involvement of thedoublesexgene in body color masculinization of the blue-tailed damselfly,Ischnura senegalensis

Takahashi

Okude

Futahashi

et al. 2020

Preprint

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“…We expect that our method also works with other, non-chemosensory genes regardless of whether they encode soluble proteins or membrane receptors, or whether they are lowly or highly expressed. Moreover, since RNAi-based suppression of Lac2 or TH decreases cuticular pigmentation in different coleopteran 18,[26][27][28] and several other non-coleopteran insect species [29][30][31][32][33][34] , our method can be adapted to many other hexapod species enabling investigations beyond transparent embryos 35 or ovaries 36 . Imaging beneath the RNAi-cleared cuticle may further benefit from light sheet microscopy that minimizes fluorophore bleaching and phototoxic effects.…”

Section: Main Textmentioning

confidence: 99%

Silencing cuticular pigmentation genes enables RNA FISH in intact chemosensory appendagess

Pentzold¹,

Grabe²,

Ogonkov³

et al. 2018

Preprint

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Optical imaging of gene expression by RNA-fluorescent in situ hybridisation (FISH) in whole-mount sensory appendages of insects is often impeded by their highly pigmented cuticle. Since most chemical bleaching agents are incompatible with imaging fluorescentlabelled nucleotides, we developed a RNA interference-based method for clearing cuticular pigmentation that allows imaging of fluorescent mRNA in whole-mount appendages of insects. Silencing key genes of the tyrosine-derived pigmentation pathway by injecting dsRNA of laccase2 or tyrosine hydroxylase in two leaf beetles species (Chrysomela populi, Phaedon cochleariae) resulted in clearance of the highly pigmented cuticle and in significant decreased light absorbance. Intact chemosensory appendages (palps, antennae and legs) from RNAi-cleared individuals were used to image expression and spatial distribution of antisense mRNA of two chemosensory genes (gustatory receptor, odorant-binding protein) via RNA FISH and confocal laser scanning microscopy. Imaging of these genes did neither work for RNAi-controls (dsGfp) due to retained pigmentation, nor for FISH-controls using sense mRNA. Furthermore, we show that several chemical bleaching agents are not feasible with FISH, either due to significant degradation of polynucleotides, lack of clearing efficacy or long incubation times. Overall, silencing pigmentation genes is a significant improvement over bleaching agents allowing fluorescence imaging in whole-mount appendages and organs.. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/318535 doi: bioRxiv preprint first posted online May. 10, 2018; 2 Main textOptical imaging in combination with in situ fluorescent labelling is a powerful method to elucidate spatiotemporal patterns of gene expression and to identify cellular circuits in biological systems 1 . For optimal image sharpness and resolution in fluorescence microscopy, samples should ideally be transparent. However, most tissues or appendages appear opaque and may be coloured by pigments. These obstacles prevent imaging at high depth -or any optical penetration at all -into tissue due to light absorption and scattering, respectively 2 .Thus, sharp imaging, especially deep into a tissue volume such as from whole-mount preparations, becomes strongly limited. Alternatively, physical serial sectioning in ultra-thin slices may be performed; however, correcting single slices for alignment, geometric distortion and staining variation is time-demanding and can be error-prone . Other bleaching protocols for whole insects with brownish bodies require at least three weeks of incubation 16,17 . Since many insect species such as beetles often possess a particular thick, hard and pigmented cuticle, here we present a novel methodological strategy that combines both specific and effective clearing of cuticula...

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“…Recently, effective RNAi-and genome-editing methods have been developed for gene functional analyses in butterflies (Ando and Fujiwara 2013;Nishikawa et al 2015;Li et al 2015;Perry et al 2016;Zhang and Reed 2016;Beldade and Peralta 2017). Applying these methods to dragonflies will be an important step toward future studies in this field (Okude et al 2017).…”

Section: Conclusion and Perspectivementioning

confidence: 99%

Molecular Mechanisms Underlying Color Vision and Color Formation in Dragonflies

Futahashi

2017

Diversity and Evolution of Butterfly Wing Patterns

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Dragonflies are colorful diurnal insects with large compound eyes. Because they visually recognize conspecific and heterospecific individuals, their body color plays essential roles in ecology and reproductive biology. Here I introduce the recent topics of molecular mechanisms underlying color vision and color formation in dragonflies. Complex wing color polymorphism is recognized among the two closely related Japanese Mnais species, presumably due to stepwise character displacement to avoid interspecific mating. We discovered an extraordinary large number of visual opsin genes by RNA sequencing of 12 dragonfly species. Manual correction after de novo assembly was crucial for determining the exact number and sequence of opsin genes. Each opsin gene was differentially expressed between the adult and larva, as well as between dorsal and ventral regions of adult compound eyes, highlighting the behavior, ecology, and adaptation of aquatic larva to terrestrial adult. The repertoire of opsin genes differed among dragonfly species, plausibly involved in the diversity of the habitat and behavior of each species. We also found that sex-specific yellow-red color transition in red dragonflies is regulated by redox changes in ommochrome pigments, which unveils a previously unknown molecular mechanism underlying body color change in animals. Establishment of the methods of gene functional analyses in dragonflies is desired for future studies.

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Electroporation-mediated RNA interference reveals a role of the multicopper oxidase 2 gene in dragonfly cuticular pigmentation

Cited by 27 publications

References 42 publications

Involvement of thedoublesexgene in body color masculinization of the blue-tailed damselfly,Ischnura senegalensis

Involvement of thedoublesexgene in body color masculinization of the blue-tailed damselfly,Ischnura senegalensis

Silencing cuticular pigmentation genes enables RNA FISH in intact chemosensory appendagess

Molecular Mechanisms Underlying Color Vision and Color Formation in Dragonflies

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