The 28-spotted potato ladybird beetle, Henosepilachna vigintioctopunctata (Fabricius), is a notorious foliar pest of solanaceous and cucurbitaceous plants in Asian countries (Kawazu, 2014, Sharma et al., 2012). At present, the control of this pest is mainly based on the application of chemical insecticides (Jamwal et al., 2017). Currently, increasing experimental evidence demonstrates that RNA interference (RNAi) can be exploited in agriculture to control insect pests (Christiaens et al., 2020, Liu et al., 2020). Moreover, previous studies have shown that H. vigintioctopunctata is sensitive to exogenous dsRNA (Chikami et al., 2019, Ohde et al., 2009, Wu et al., 2021). Nevertheless, identification of amenable target genes is the first step for their potential application via RNAi (Liu et al., 2020).

In H. vigintioctopunctata, hardening of the larval, pupal and adult cuticles occurred rapidly after molting (Casari and Teixeira, 2015). The hard cuticles provide protection against physical injury and water loss, rigidness for muscle attachment and mechanical support and a shield against damaging UV light and various types of ionizing radiation (Sugumaran, 2010). The process of hardening of the beetle integument involves the development of cross links between protein or protein/chitin and is known as melanization and sclerotization (tanning). Therefore, disruption of melanin biosynthesis pathway genes by RNAi is expected to aid the development of the dsRNA-based pesticide to control H. vigintioctopunctata.

From a biochemical perspective, the precursors to produce melanins and sclerotins are two aromatic amino acids: tyrosine and phenylalanine. Tyrosine is a normal food component. It can also be obtained from phenylalanine by phenylalanine hydroxylase (PAH, EC 1.14.16.1) (Shamim et al., 2014). The first biochemical step to generate melanins and sclerotins is the conversion of tyrosine to 3,4-L-dihydroxyphenylalanine (dopa) by tyrosine hydroxylase (TH, EC 1.14.16.2) (Mun et al., 2020). Dopa is then converted into dopamine. Dopa and dopamine can produce five different molecules: three melanins (dopa-melanin, dopamine-melanin and pheomelanin), and two sclerotins (β-alanyl dopamine, NBAD; N-acetyl dopamine, NADA) (Mun et al., 2020, Shamim et al., 2014).

The presence of a pteridine, tetrahydrobiopterin (BH4), is essential for the production of melanins and sclerotins (Jiang et al., 2020, Schallreuter, 1994). BH4 is an essential cofactor for TH, PAH, tryptophan hydroxylase (TPH, EC 1.14.16.4), nitric oxide synthase (NOS, EC 1.14.13.39) and alkyl-glycerol monooxygenase (AGMO, EC 1.14.16.5), as well as the proliferation of erythroid cells (Lowenstein and Snyder, 1992, Marletta, 1994, Tanaka et al., 1989). BH4 also acts as an essential cofactor for the oxidative cleavage of ether lipid (Thony et al., 2000, Watschinger et al., 2009, Watschinger et al., 2015, Werner et al., 2011). As a result, BH4 deficiency in humans can lead to severe neurological disorders, such as hyperphenylalaninemia, phenylketonuria, Parkinson’s disease, Alzheimer’s disease, and depression (Thony et al., 2000). In insects, the catalytic activity of insect TH requires and is regulated by BH4 in D. melanogaster (Li et al., 2015, Neckameyer and White, 1993).

Pteridines are biosynthesized from guanosine triphosphate (GTP) through a conserved biosynthesis pathway in eukaryotes such as mammals and insects (Figure 1A). In insect, the pathway has initially been elucidated using Drosophila melanogaster mutants (Chen et al., 1994, Kim et al., 2012, Kim et al., 1996, McLean et al., 1990, O'Donnell et al., 1989a, O'Donnell et al., 1989b, Reynolds and O'Donnell, 1987) and has been further confirmed in Bombyx mori mutants (Chen et al., 2015, Fujii et al., 2013, Jiang et al., 2020, Li et al., 2015, Meng et al., 2009, Tong et al., 2018). In D. melanogaster, for instance, the enzyme GTP cyclohydrolase (GTPCH, EC 3.5.4.16), encoded by punch gene, converts GTP into 7,8-dihydro-neopterin 3’-trisphosphate (dihydroneopterin) with the release of formic acid (Chen et al., 1994, McLean et al., 1990, O'Donnell et al., 1989a, O'Donnell et al., 1989b, Reynolds and O'Donnell, 1987). Dihydroneopterin then forms 6-pyruvoyltetrahydropterin (6-PTP) under catalyzation by 6-PTP synthase (PTPS, EC 4.2.3.12), encoded by purple gene (Kim et al., 1996). 6-PTP can be transferred into several pteridines (xanthopterin, drosopterin, isodrosopterin and aurodrosopterin). Importantly, 6-PTP is reduced by the enzyme sepiapterin reductase (SR, EC 1.1.1.153), encoded by spr gene, to form 1’-hydroxy-2’-oxopropyl-tetrahydropterin, which is reduced to 1’-oxo-2’-hydroxypropyl-tetrahydropterin and then to tetrahydrobiopterin (BH4) in two additional reactions also catalyzed by SR (Kim et al., 2012). Alternately, 6-PTP is converted to sepiapterin and then dihydrobiopterin, dihydrobiopterin then forms BH4, under catalyzation of dihydrofolate reductase (DHFR) (Bracher et al., 1998, Kapatos, 2013, Thony et al., 2000) (Figure 1A). In D. melanogaster, most punch mutant embryos are lethal at distinct stages of embryogenesis (Kahsai et al., 2016, McLean et al., 1990). This lethality can be attributed to the deficiency of dopa and dopamine (O'Donnell et al., 1989a, O'Donnell et al., 1989b, Reynolds and O'Donnell, 1987). In Papilio xuthus (Futahashi and Fujiwara, 2006) and B. mori (Chen et al., 2015), the GTPCH inhibitor can prevent pigmentation in cultured integuments. In B. mori, mutation in BH4 biosynthesis genes changes larval body color; the paired black markings in wild larvae are vanished (Jiang et al., 2020, Meng et al., 2009, Tong et al., 2018). However, whether BH4 is involved in melanogenesis in other insect species remains to be determined.

Moreover, several pteridine chromes create various colors. For instance, sepiapterin and xanthopterin are yellow pigments, while drosopterin, isodrosopterin and aurodrosopterin are red chromes (Shamim et al., 2014). These pteridine chromes, along with ommochromes, are involved in eye coloring in several insect species (Brent and Hull, 2019, Dong and Friedrich, 2005, Khan et al., 2017, Liu et al., 2013, Summers et al., 1982). In D. melanogaster, for example, ommochromes are brown and pteridines are red and yellow, and both contribute to the deep red color of the adult eyes (Dreesen et al., 1988). However, the effect of pteridine on eye pigment in Coleoptera insects is rarely reported.

In the present paper, we addressed three questions in H. vigintioctopunctata. 1) Whether disruption of pteridine generation affects melanogenesis in the larvae, pupae and adults. 2) Whether pteridine chromes pigment eyes, especially immature ocelli. 3). Does disruption of melanin biosynthesis pathway genes by RNAi cause severe larval lethality? Four cDNAs (Hvgtpch-a, Hvgtpch-b, Hvptps, Hvsr) involved in melanin biosynthesis were identified. Our results addressed, at least partially, the three issues and unraveled that Hvgtpch may be a potential target for RNAi-based control of H. vigintioctopunctata.