Genomic and experimental data provide new insights into luciferin biosynthesis and bioluminescence evolution in fireflies

Abstract

Fireflies are among the most charismatic insects for their spectacular bioluminescence, but the origin and evolution of bioluminescence remain elusive. Especially, the genic basis of luciferin (d-luciferin) biosynthesis and light patterns is largely unknown. Here, we present the high-quality reference genomes of two fireflies Lamprigera yunnana (1053 Mb) and Abscondita terminalis (501 Mb) with great differences in both morphology and luminous behavior. We sequenced the transcriptomes and proteomes of luminous organs of two species. We created the CRISPR/Cas9-induced mutants of Abdominal B gene without luminous organs in the larvae of A. terminalis and sequenced the transcriptomes of mutants and wild-types. Combining gene expression analyses with comparative genomics, we propose a more complete luciferin synthesis pathway, and confirm the convergent evolution of bioluminescence in insects. Using experiments, the function of the firefly acyl-CoA thioesterase (ACOT1) to convert l-luciferin to d-luciferin was validated for the first time. Comparisons of three-dimension reconstruction of luminous organs and their differentially expressed genes among two species suggest that two positive genes in the calcium signaling pathway and structural difference of luminous organs may play an important role in the evolution of flash pattern. Altogether, our results provide important resources for further exploring bioluminescence in insects.

Luciferins Under Construction: A Review of Known Biosynthetic Pathways

“…Current evidence suggests that at the final step L-luciferin undergoes CoA esterification catalyzed by luciferase, followed by epimerization and thioester hydrolysis thus generating the D-form (Niwa et al, 2006;Maeda et al, 2017). These results were further supported by expression analysis of firefly luciferin biosynthetic pathway candidate genes (polyphenol oxidase, β-glucosidase, luciferase, and acyl-CoA thioesterase) in the luminous organs of Lamprigera yunnana and Abscondita terminalis and L-luciferin enzymatic deracemization experiments in vitro (Zhang et al, 2020), demonstrating that acyl-CoA thioesterases can efficiently convert L-luciferin to D-luciferin. An earlier hypothesis of possible luciferin storage mechanism in the form of sulfoluciferin (Fallon et al, 2016) was also corroborated by sulfotransferase expression analysis in the luminous organs of both species (Zhang et al, 2020).…”

Draft genome of the Brazilian railroad worm Phrixothrix hirtus E.Olivier (Phengodidae: Coleoptera)

“…They also showed the existence of two distinct luciferase genes in P. pyralis firefly located in two chromosomes, suggesting events of gene duplication associated with a translocation between the chromosomes, which corroborates the presence of two luciferases isozymes in the lanterns and fat body in distinct life stages of fireflies (Strause and DeLuca, 1981;Viviani et al, 2008;Oba et al, 2010;Bessho-Uehara and Oba, 2017;Carvalho et al, 2020). In addition, genomic and transcriptomic analyses of two Palearctic firefly species, Abscondita terminalis and Lamprigera yunnana, indicated putative luciferin biosynthesis pathways in fireflies (Zhang et al, 2020), involving several gene products, which were also described in RNA-Seq analyses in other Elateroidea species (Vongsangnak et al, 2016;Amaral et al, 2017a,b;2018). Such studies brought important contributions and insights about the genome organization within Elateroidea, however, the functional genomic information remains limited to Lampyridae and Elateridae.…”

“…The SOD enzyme seems to be associated with the protection of lanterns during hyperoxia and oxidative stress and is recovered in high abundance in lanterns of firefly species (Barros and Bechara, 1998;Amaral et al, 2017b). The other two gene families are associated with cysteine availability, which is a precursor of luciferin biosynthesis in Elateroidea (Viviani et al, 2013;Kanie et al, 2016;Amaral et al, 2017aAmaral et al, , 2017bAmaral et al, , 2019bZhang et al, 2020).…”

“…Bioluminescence emission control and antioxidant enzymes. Recent studies using transcriptome and genomic data described possible gene products associated with the bioluminescent control in Elateridae, Lampyridae, and Phengodidae (Amaral et al, 2017a;2017b;2019b;Zhang et al, 2020). Trimmer et al (2001) and Amaral et al (2017b) suggested the importance of nitric oxide synthase and octopamine/dopamine receptors to firefly flash control, mainly in the adult stage, in which the flash pattern and duration are fundamental for intraspecific communication.…”

“…Luciferin biosynthesis. In P. hirtus genome, several gene products that were previously potentially associated with luciferin biosynthesis in luminescent Elateroidea were found (Niwa et al, 2006;Viviani et al, 2013;Vongsangnak et al, 2016;Amaral et al, 2017aAmaral et al, , 2017bAmaral et al, , 2019bZhang et al, 2020). Among them, the adenosylhomocysteinase and cysteine sulfinic acid decarboxylase are associated with the conversion of homocysteine to cysteine, which may spontaneously react with p-benzoquinone to generate luciferin (Kanie et al, 2016).…”