Cloning And Function Characterization Of Ommochromes Synthesis And Transport Related Genes In Henosepilachna Vigintioctopunctata

Henosepilachna vigintioctopunctata is one of the most serious insect pests on nightshades and cucurbits in Asian countries.At present,studies have shown that the beetle is sensitive to RNA interference(RNAi).Also,the transcriptome of this insect has been documented.These facilitate clarification of gene function in the beetle.Insect compound eyes are colorated by two types of pigments,ommochromes and pteridines,or only by ommochromes.These pigments are mainly present in the pigment cells of the compound eyes in pupae and adults,and have an important role in protecting the photoreceptor cells from excessive scattered light and ultraviolet rays.In addition,these pigments are involved in the coloring and hardening of insect epidermis and wings.In the present study,we focused on the biosynthesis and transport pathway genes of ommochromes in H.vigintioctopunctata.Firstly,we identified several representative genes by digging the transcriptomic data of H.vigintioctopunctata.We then determined the temporal expression patterns of these genes.Finally,we knocked down these genes by RNAi,and observed the defects on eye pigmentation in the resultant puape and adults In addition,we examined two behaviors,phototaxis and climbing ability,in the resulting adults.The results were as follows.1.Molecular cloning of these genesBased on the transcriptomic data of H.vigintioctopunctata,eight representative genes were obtained by aligning homologous sequences of Tribolium castaneum,Drosophila melanogaster and Bombyx mori.Among the eight genes,vermilion and cardinal encode a tryptophan 2,3-dioxygenase and a heme peroxidase,which are indispensable for ommochrome biosynthesis.Moreover,karmoisin and five whites code for a monocarboxylate transporter and ATP-binding cassette(ABC)transporter Whites,which are essential for transportation of precursors.Using polymerase chain reaction(PCR)technology,we cloned the full lengths of five genes(with the corresponding Genbank accession numbers),they were vermilion(MN905517),karmoisin(MN905518),cardinal(MN905519),white3(MN848343),and white5(MN848346).Moreover,only incomplete sequences of three other genes,white1(MN848342),white2(MN848344),and white4(MN848345),were verified.2.Temporal transcription patterns and phenoty pes after RNAiThe temporal expression patterns of these eight genes in H.vigintioctopunctata were determined by qRT-PCR.The eight representative genes were transcribed throughout all development stages in this beetle.The results showed that karmoisin was highly expressed in the egg,pupae and adults:vermilion was abundantly transcribed in the early and late stages of the 1st,2nd and 3rd larval instars,but was lowly expressed at the end of 4th instar.cardinal was greatly expressed in the pupae and adults stages.Injection of dsRNAs against the 8 representative genes into the fourth instar larvae successfully knocked down the target transcripts.It was found that knockdown of karmoisin,vermilion,cardinal or white3 decolorated the compound eyes of pupae and adults to some extent.When the mRNA of vermilion or cardinal was depleted,the compound eyes of pupae and adults have the same pale yellow color as the body color,whereas the ocular diaphragm remained as a ring of black pigment surrounding each eye.When karmoisin or white3 were silenced,the compound eyes of the pupae and adults appeared red brown and dark brown.Moreover,higher doses of dswhite3 resulted in a more obvious phenotype of eye color changes in pupae and adults.However,suppression of the expression level of white1,white2,white4,or white5 did not cause any changes in the color of pupal and adult eyes.3.RNAi on phototaxis and climbing abilityDifferent from the positive phototaxis of the control adults,the karmoisin,vermilion,or cardinal RNAi adult beetles showed negative phototaxis to a degree,so it can be speculated that ommochromes are strongly related to phototaxis of insects.Moreover,our results showed that control,karmoisin,vermilion and cardinal RNAi males spent an average of 15.0,22.9,22.9 and 19.0 seconds to climb a distance of 8 cm in a vial,with the later three were significantly more than the former.Similarly,the control,karmoisin,vermilion and cardinal RNAi females used an average of 17.6,22.5,27.6 and 19.3 seconds to finish the climbing,with the middle two significantly more than the control.Similarly,our data revealed that treatment with dswhite2 at the fourth-instar larval stage,rather than others,impaired climbing ability of both male and female adults.Our results support the hypothesis that ommochromes are critical for improvement of eye visual acuity and hardening of cuticle.Also,White2 and White3 exhibit function divergence in H.vigintioctopunctata.