SUMMARY Variations in body pigmentation, encompassing both the range of specific colors as well as the spatial arrangement of those colors, are among the most noticeable and lineage-specific insect features. However, the genetic mechanisms responsible for generating this diversity are still limited to several model species that are primarily holometabolous insects. To address this lack of knowledge, we utilize Oncopeltus fasciatus, an aposematic hemimetabolous insect, as a new model to study insect pigmentation. First, to determine the genetic regulation of black pigment production in Oncopeltus, we perform an RNAi analysis on three core genes involved in the melanin pathway, tyrosine hydroxylase (TH), dopa decarboxylase (DDC), and laccase 2 (lac2). The black pigmentation is affected in all instances, showing that the black pigments in this species are derived from the melanin pathway. The results of the DDC RNAi are particularly informative because they reveal that it is Dopamine melanin, not DOPA melanin, which is the predominant component of black pigments in Oncopeltus. Second, we test whether pigmentation follows a two-step model where the spatial pre-mapping of enzymatic activity is followed by vein-dependent transportation of melanin substances. We confirm the existence of the first step by observing that premature wings develop black pigmentation when exposed to melanin precursors. In addition, we provide evidence for the second step by showing that wing melanin patterning is disrupted when vein transportation is halted. These findings bring novel insights from a hemimetabolous species and establish a framework for subsequent studies on the mechanisms of pigment production and patterning responsible for variations in insect coloration.

Diversity in insect pigmentation, encompassing a wide range of colors and spatial patterns, is among the most noticeable features distinguishing species, individuals, and body regions within individuals. In holometabolous species, a significant portion of such diversity can be attributed to the melanin synthesis genes, but this has not been formally assessed in more basal insect lineages. Here we provide a comprehensive analysis of how a set of melanin genes (ebony, black, aaNAT, yellow, and tan) contributes to the pigmentation pattern in a hemipteran, Oncopeltus fasciatus. For all five genes, RNA interference depletion caused alteration of black patterning in a region-specific fashion. Furthermore, the presence of distinct nonblack regions in forewings and hindwings coincides with the expression of ebony and aaNAT in these appendages. These findings suggest that the region-specific phenotypes arise from regional employment of various combinations of the melanin genes. Based on this insight, we suggest that melanin genes are used in two distinct ways: a "painting" mode, using predominantly melanin-promoting factors in areas that generally lack black coloration, and, alternatively, an "erasing" mode, using mainly melanin-suppressing factors in regions where black is the dominant pigment. Different combinations of these strategies may account for the vast diversity of melanin patterns observed in insects.KEYWORDS Oncopeltus fasciatus; insect pigmentation; melanin patterning; melanin-promoting factors; melanin suppressors P IGMENT patterns are among the most striking and variable features of insect morphology. An extraordinary diversity in coloration distinguishes species, populations within species, individuals within populations, and different body regions (Wittkopp and Beldade 2009). Most insights into the mechanisms underlying such diversity have come from studies on melanization in Drosophila (Wittkopp et al. 2003;Wittkopp and Beldade 2009). Melanization is the pigmentation process wherein precursors (catecholamines) are converted into pigment molecules that are incorporated into the cuticle (Wittkopp and Beldade 2009). These studies have helped to identify a network of melanin genes and their roles in body color patterning (Wright 1987;Wittkopp et al. 2003;Wittkopp and Beldade 2009). The core part of this proposed pathway is shown in Figure 1. The pathway begins with the conversion of tyrosine to dihydroxyphenylalanine (DOPA). DOPA can then be used in two different manners: to produce DOPA melanin (black) or to be converted to dopamine, another precursor of black melanin. In the conversions from DOPA/dopamine to black melanin, the yellow gene is thought to play an essential role in promoting these processes. However, it is still unclear whether yellow plays a role in producing DOPA melanin, dopamine melanin, or both (question marks in Figure 1). Alternatively, production of dopamine melanin can be suppressed by converting dopamine to N-b-alanyldopamine (NBAD) or N-acetyldopamine (NADA). The NBAD bran...

The most prominent colors observed in insects are black or brown, whose production is attributed to the melanin pathway. At present, though, the contribution of this pathway to overall body pigmentation throughout ontogenesis is still lacking. To address this question we examined the roles of 2 key melanin genes (TH and DDC), in embryonic and postembryonic development of the American cockroach, Periplaneta americana. Our results show that the melanin pathway does not contribute to the light brown coloration observed in the first nymphs. However, the dark brown coloration in mid nymphs and adults is produced solely from the melanin pathway. In addition, the DDC RNAi results reveal that it is dopamine melanin, not DOPA melanin, acts as the main contributor in this process. Overall, present study provides a new insight into insect pigmentation suggesting that genetic mechanisms of coloration can change during ontogenesis. Future studies of additional basal insect lineages will be required to assess in more details the generality of this phenomenon.