Disruption of tetrahydrobiopterin (BH4) biosynthesis pathway affects cuticle pigmentation in Henosepilachna vigintioctopunctata.

Tetrahydrobiopterin (BH4) is produced from guanosine triphosphate (GTP) under catalyzation of GTP cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS) and sepiapterin reductase (SR), among others. In Drosophila melanogaster, BH4 and other pteridines are required for cuticle tanning and eye pigmentation. In this study, two Hvgtpch (Hvgtpch-a and Hvgtpch-b), an Hvptps and an Hvsr transcripts were identified in a serious defoliator Henosepilachna vigintioctopunctata. Hvgtpch-a and Hvgtpch-b were highly expressed just before and/or right after the molt, in contrast to Hvptps and Hvsr. RNA interference (RNAi) by injection of a dsgtpch targeting the common fragment of Hvgtpch-a and Hvgtpch-b into the third instar larvae caused albino fourth-instar larvae and pupae. Around 80% of the Hvgtpch RNAi larvae failed to pupate. The remaining 20% of Hvgtpch RNAi pupated beetles did not completely remove the larval/pupal exuviae after emerged as adults and eventually died. Depletion of Hvgtpch at the fourth instar stage resulted in under-pigmented pupae and adults, with significantly low pupation and emergence rates. The Hvgtpch RNAi adults rarely moved and fed on plant leaves; they died within a week after emergence. Silence of Hvptps or Hvsr at the third- and fourth-instar stages led to similar but less serious phenotypes, with lowest influence in the Hvsr RNAi ladybirds. Moreover, RNAi of Hvgtpch, Hvptps or Hvsr did not affect coloration of the larval ocelli and pupal/adult compound eyes. Therefore, our results demonstrated that pteridines are involved in melanin formation but not in eye pigmentation in H. vigintioctopunctata. Moreover, our findings will enable the development of a dsgtpch-based pesticide to control H. vigintioctopunctata larvae.

RNAi of vacuolar-type H+-ATPase genes causes growth delay and molting defect in Henosepilachna vigintioctopunctata.

Henosepilachna vigintioctopunctata is one of the most serious insect pests to a large number of nightshades and cucurbits. RNA interference (RNAi) triggered by double-stranded RNA (dsRNA) offers a reduced risk approach to control the beetle. Identification of amenable target genes and determination of appropriate life stage for dsRNA treatment are two critical steps in order to improve RNAi efficiency. In the present paper, we identified three vATPase genes, namely HvvATPaseC, HvvATPaseE and HvvATPaseH. We found that the three transcripts were widely expressed in the eggs, first- to fourth-instar larvae, prepupae, pupae and adults. They were abundantly transcribed in the hindgut and Malpighian tubules, in contrast to the epidermis and fat body. Three days' ingestion of dsvATPaseC, dsvATPaseE and dsvATPaseH by the fourth-instar larvae significantly decreased corresponding transcript level by 90.1, 88.9 and 97.2%, greatly reduced larval fresh weight by 28.0, 29.9 and 28.0%, and caused 66.7, 100 and 78.7% larval lethality respectively. Comparably, 3 days' exposure of the third-instar larvae to dsvATPaseC significantly reduced HvvATPaseC mRNA level by 89.5%, decreased approximately 80% of the larval fresh weight, and killed 100% of the treated larvae. Therefore, the three vATPase genes, especially HvvATPaseE, are potential amenable target genes and young larvae are more susceptible to dsRNA. Our findings will enable the development of the dsRNA-based pesticide to control H. vigintioctopunctata.