Tribolium is a widely utilized model organism for evolutionary and developmental biology questions and understanding regulatory mechanisms for agricultural pests (Adamski et al., 2019; Brown et al., 2009; Pointer et al., 2021; Rosner et al., 2020). Tribolium was the first Coleoptera genome to be sequenced (Tribolium Genome Sequencing Consortium, 2008), and an updated genome sequence based upon long read sequencing (icTriCast1.1 - (Childers et al., 2021) is now available. Tribolium is amenable to both forward and reverse genetics, and in particular transgenesis is well established (Campbell et al., 2022; Klingler & Bucher, 2022). For transgenics, the 3XP3 fluorescent marked transposable elements is an efficient marker (Berghammer et al., 1999; Horn et al., 2000) and easily detected in Tribolium mutant retinas that lack pigmentation, e.g. pearl , platinum and white . Lorenzen et al. demonstrated that the Tribolium white mutation (Eddleman & Bell, 1963) is a null mutation of the Tribolium homolog of vermilion (Lorenzen et al., 2002a). Moreover, given the absence of pigment in vermilion mutant retinas, a set of transposable elements have been generated that result in the expression of vermilion and thus restores pigmentation to the retina (Lorenzen et al., 2002b). As a result, many Tribolium transgenics are generated in the v ^w mutant background.

 

The initial characterization of vermilion ^white demonstrated that the mutant is a deletion that removes the first five exons and extends into the last sixth exon of vermilion ( Figure 1A ).  The upstream breakpoint was not mapped. To map the upstream breakpoint, we took advantage of PacBio long read sequence that contained the v ^w mutation. Our sequencing revealed a 4,434 bp deletion. The sequence confirmed the break point that lies within vermilion and identified the upstream second breakpoint ( Figure 1B ). The deletion eliminates the entire gustatory receptor candidate 58 locus and extends into the Tribolium norpA locus. norpA encodes a Phospholipase C which is critical for phototransduction and in its absence vision is disrupted (Bloomquist et al., 1988). The deletion results in a 3’ prime deletion of exon 9 of the norpA locus resulting in the truncation of the C2 domain of the phospholipase and an addition of 15 unrelated amino acids Figure 1C, D). The C2 domain is 91 amino-acid residues and thought to be involved in calcium-dependent phospholipid binding and membrane targeting (Davletov & Sudhof, 1993). The loss of the C2 domain can have multiple effects. It can prevent the protein from binding to the lipid bilayers (Croessmann et al., 2018) or it can impair the protein’s ability to respond to the calcium signals (Corbalan-Garcia & Gomez-Fernandez, 2014). The truncation can also reduce the stability of the protein, leading to premature degradation (Buetow & Huang, 2016). Thus the truncation of the C2 domain would suggest that the mutated norpA allele in the v ^w mutation is a loss of function allele.

 

With respect to the generation of transgenics, one will need to account for the additional mutations in v ^w but this concern can be alleviated using CRISPR/Cas9 generated vermilion alleles (Adrianos et al., 2018; Markley et al., 2024). Whereas the existence of two additional mutations may decrease the usefulness of v ^w with respect to transgenics, the existence of defined deletions has been critical for mapping of genes and confirming the nature of alleles. Moreover, the v ^w mutation now adds a key mutation to the toolbox of understanding sensory perception. The uncovering of the norpA mutation can now help define the dynamics of phototransduction in Tribolium and permit an investigation in the role of vision in Tribolium behaviors, e.g. circadian rhythms. Overall due to the elimination of two other loci we propose that the v ^w mutation is now referred as Deficiency vermilion ^white , Df ( v ^w ).

 

 

Tribolium lines and husbandry: All animals were raised at 28 ^o C on a standard flour yeast mix. The following strains were utilized: vermilion ^white ( v ^w ), and a strain heterozygous for vermilion ^white ( v ^w ) (Lorenzen et al., 2002a) and Lucifer ( Lu ) (Haas & Beeman, 2012).

Sequencing and genome assembly: 40 < 1 day old vermilion ^white ( v ^w )&nbsp; and Lucifer ( Lu ) heterozygote pupae were isolated and immediately frozen in liquid nitrogen and stored at –80 ^o C. Pupae were shipped on dry ice to Psomagen ( https://www.psomagen.com/ ) for processing. DNA extraction, library construction, and PacBio sequencing were all performed by Psomagen. The PacBio hifi reads were assembled with hifiasm (v0.25.0-r726) (Cheng et al., 2024; Cheng et al., 2021; Cheng et al., 2022). The resulting assemblies as well as the input reads were mapped to the reference genome Tribolium castaneum strain icTriCast1.1 using minimap2 (v2.28) (Li, 2018) with default parameters. This identified a 4434 bp deletion. Among reads that span the deleted region, we identified 139 reads without the deletion and 149 reads that contain the deletion. The sequencing reads have been deposited in the SRA database and have the following accession numbers: SRR37271882 : wild-type vermilion locus reads&nbsp;and SRR37271883 : v ^w deletion locus reads.

norpA protein structure : The protein domains were compiled using InterPro (Blum et al., 2025).