INTERGENIC EXCHANGE, GEOGRAPHIC ISOLATION, AND THE EVOLUTION OF BIOLUMINESCENT COLOR FOR PYROPHORUS CLICK BEETLES

Gene duplication is an evolutionary process in which the emergent property of the whole can become greater and different than the sum of its parts. One potential outcome for gene duplication is for loci to evolve different, yet related functions. In this case, intergenic exchange can shuffle blocks of differentiated nucleotides between paralogues to create new alleles and phenotypes rather than simply homogenize loci. Bioluminescent click beetles in the genus Pyrophorus (Coleoptera: Elateridae) provide an opportunity to explore the creative potential of intergenic exchange for gene family evolution. Pyrophorus beetles bioluminesce different light colors from a pair of dorsal light organs and a ventral light organ. The light organs are under the separate genetic control of dorsal and ventral luciferase loci. Here, we report that intergenic exchange is common between dorsal and ventral loci for beetles from Jamaica ( P. plagiophthalamus ), the Dominican Republic ( P. mellifluous ), Belize ( P. luscus ), and Trinidad ( P. noctilucus ). We also present evidence that periods of past geographic isolation for beetles on Jamaica, probably acting in concert with selection, built differentiated blocks of substitutions within dorsal and ventral P. plagiophthalamus luciferase loci. Gene flow and intergenic exchange subsequently shuffled these substitutions between dorsal and ventral loci to produce new color phenotypes on Jamaica, including a yellow–green polymorphism. We discuss the possibility of a previously unrecognized emergent evolutionary property of intergenic exchange for luciferase involving cycles of bioluminescent color change related to differences in selective constrains acting on dorsal versus ventral loci. We also explore whether intergenic exchange may commonly create novel variation and the potential for cyclic evolution in other multigene family systems.     

Isolation and characterization of novel dinucleotide repeat microsatellites in the Jamaican click beetle Pyrophorus plagiophthalamus (Coleoptera: Elateridae)

We report 10 polymorphic microsatellite loci for the Jamaican click beetle, Pyrophorus plagiophthalamus. A survey of 36 individuals from three populations in Jamaica showed that these are highly variable, with three to 17 alleles per locus. Observed heterozygosity ranged from 0.250 to 0.917, and mean heterozygosity from 0.601 to 0.747. Most loci were in Hardy–Weinberg equilibrium, although excess of homozygotes was observed in four tests (out of 20), suggesting the possibility of null alleles. Significant linkage disequilibrium was observed for only one pair. These newly developed markers will be useful in understanding the population structure of click beetles in Jamaica, and in identifying possible selective factors responsible for bioluminescent colour variation.     

The evolution of the adenylate-forming protein family in beetles: multiple luciferase gene paralogues in fireflies and glow-worms

Bioluminescence in beetles is dependent upon the enzyme luciferase. It has been hypothesised luciferase evolved from a fatty acyl-CoA synthetase gene deriving a novel bioluminescent function (neofunctionalization) after a gene duplication event. We evaluated this hypothesis within a phylogenetic framework using independent evidence obtained from the genome of Tribolium castaneum, published luciferase genes and novel luciferase and luciferase-like sequences. This phylogenetic study provides evidence for a large gene family of luciferase and luciferase-like paralogues in bioluminescent and non-bioluminescent beetles. All luciferase sequences formed a clade supporting a protoluciferase existing prior to the divergence of the Lampyridae, Elateridae and Phengodidae (Elateroidea). Multiple luciferase genes were identified from members of the Photurinae and the Luciolinae indicating complex gene duplication events within lampyrid genomes. The majority of luciferase residues were identified to be under purifying selection as opposed to positive selection. We conclude that beetle luciferase may have arisen from a process of subfunctionalization as opposed to neofunctionalization early on in the evolution of the Elateroidea.     

Use of Controlled Luciferase Expression To Monitor Chemicals Affecting Protein Synthesis

In this article, we present a new bioluminescent test system for the screening of chemical compounds with an inhibitory effect on protein synthesis. The test is based on the measurement of real-time in vivo light production by Escherichia coli strains expressing different luciferase genes. The eukaryotic lucGR gene from Pyrophorus plagiophthalamus was found to be the best of three types of luciferase genes tested. Chemicals with known inhibitory effects on protein synthesis were used as test chemicals together with some general toxicants. The incubation of a test chemical with cells was performed either prior to or after the induction of protein synthesis, and the difference in the results of the two methods distinguishes the possible influence on protein synthesis from direct metabolic inhibition. Using lyophilized bacteria, the test is performed in less than an hour without any bacterial cultivation, which makes the test suitable for rapid and sensitive screening of chemicals or environmental samples. Compared with the standardized 50% inhibitory concentration calculation method of the bioluminescent cytotoxicity test, the more direct approach of calculation developed in this study proved to be more convenient than and as reliable as the standard method.     

The first report of luminescent liver tissue in fishes: Evolution and structure of bioluminescent organs in the deep‐sea naked barracudinas (Aulopiformes: Lestidiidae)

Bioluminescent organs that provide ventral camouflage are common among fishes in the meso‐bathypelagic zones of the deep sea. However, the anatomical structures that have been modified to produce light vary substantially among different groups of fishes. Although the anatomical structure and evolutionary derivation of some of these organs have been well studied, the light organs of the naked barracudinas have received little scientific attention. This study describes the anatomy and evolution of bioluminescent organs in the Lestidiidae (naked barracudinas) in the context of a new phylogeny of barracudinas and closely related alepisauroid fishes. Gross and histological examination of bioluminescent organs or homologous structures from preserved museum specimens indicate that the ventral light organ is derived from hepatopancreatic tissue and that the antorbital spot in Lestrolepis is, in fact, a second dermal light organ. In the context of the phylogeny generated from DNA‐sequence data from eight gene fragments (7 nuclear and 1 mitochondrial), a complex liver with a narrow ventral strand running along the ventral midline evolves first in the Lestidiidae. The ventral hepatopancreatic tissue later evolves into a ventral bioluminescent organ in the ancestor of Lestidium and Lestrolepis with the lineage leading to the genus Lestrolepis evolving a dermal antorbital bioluminescent organ, likely for light‐intensity matching. This is the first described hepatopancreatic bioluminescent organ in fishes. J. Morphol. 276:310–318, 2015. © 2014 Wiley Periodicals, Inc.     

Diversity and divergence patterns in regulatory genes suggest differential gene flow in recently derived species of the Hawaiian silversword alliance adaptive radiation (Asteraceae)

The impact of gene flow and population size fluctuations in shaping genetic variation during adaptive radiation, at both the genome-wide and gene-specific levels, is very poorly understood. To examine how historical population size and gene flow patterns within and between loci have influenced lineage divergence in the Hawaiian silversword alliance, we have investigated the nucleotide sequence diversity and divergence patterns of four floral regulatory genes (ASAP1-A, ASAP1-B, ASAP3-A, ASAP3-B) and a structural gene (ASCAB9). Levels and patterns of molecular divergence across these five nuclear loci were estimated between two recently derived species (Dubautia ciliolata and Dubautia arborea) which are presumed to be sibling species. This multilocus analysis of genetic variation, haplotype divergence and historical demography indicates that population expansion and differential gene flow occurred subsequent to the divergence of these two lineages. Moreover, contrasting patterns of allele- sharing for regulatory loci vs. a structural locus between these two sibling species indicate alternative histories of genetic variation and partitioning among loci where alleles of the floral regulatory loci are shared primarily from D. arborea to D. ciliolata and alleles of the structural locus are shared in both directions. Taken together, these results suggest that adaptively radiating species can exhibit contrasting allele migration rates among loci such that allele movement at specific loci may supersede genetic divergence caused by drift and that lineage divergence during adaptive radiation can be associated with population expansion.     

Bioluminescent organs of two deep-sea arrow worms, Eukrohnia fowleri and Caecosagitta macrocephala, with further observations on Bioluminescence in chaetognaths

Bioluminescence in the deep-sea chaetognath Eukrohnia fowleri is reported for the first time, and behavioral, morphological, and chemical characteristics of bioluminescence in chaetognaths are examined. Until this study, the only known species of bioluminescent chaetognath was Caecosagitta macrocephala. The luminescent organ of that species is located on the ventral edge of each anterior lateral fin, whereas that of E. fowleri runs across the center of the tail fin on both dorsal and ventral sides. Scanning electron microscopy showed that the bioluminescent organs of both species consist of hexagonal chambers containing elongate ovoid particles-the organelles holding bioluminescent materials. No other luminous organism is known to use hexagonal packing to hold bioluminescent materials. Transmission electron microscopy of particles from C. macrocephala revealed a densely packed paracrystalline matrix punctuated by globular inclusions, which likely correspond to luciferin and luciferase, respectively. Both species use unique luciferases in conjunction with coelenterazine for light emission. Luciferase of C. macrocephala becomes inactive after 30 min, but luciferase of E. fowleri is highly stable. Although C. macrocephala has about 90 times fewer particles than E. fowleri, it has a similar bioluminescent capacity (total particle volume) due to its larger particle size. In situ observations of C. macrocephala from a remotely operated vehicle revealed that the luminous particles are released to form a cloud. The discovery of bioluminescence in a second chaetognath phylogenetically distant from the first highlights the importance of bioluminescence among deep-sea organisms.     

Mutations in the melanocortin 1 receptor (MC1R) gene are associated with coat colours in the domestic rabbit (Oryctolagus cuniculus)

We sequenced almost the complete coding region of the MC1R gene in several domestic rabbits (Oryctolagus cuniculus) and identified four alleles: two wild-type alleles differing by two synonymous single nucleotide polymorphisms (c.333A>G;c.555T>C), one allele with a 30-nucleotide in-frame deletion (c.304_333del30) and one allele with a 6-nucleotide in-frame deletion (c.280_285del6). A polymerase chain reaction-based protocol was used to distinguish the wild-type alleles from the other two alleles in 263 rabbits belonging to 37 breeds or strains. All red/fawn/yellow rabbits were homozygous for the c.304_333del30 allele. This allele represents the recessive e allele at the extension locus identified through pioneering genetic studies in this species. All Californian, Checkered, Giant White and New Zealand White rabbits were homozygous for allele c.280_285del6, which was also observed in the heterozygous condition in a few other breeds. Black coat colour is part of the standard colour in Californian and Checkered breeds, in contrast to the two albino breeds, Giant White and New Zealand White. Following the nomenclature established for the rabbit extension locus, the c.280_285del6 allele, which is dominant over c.304_333del30, may be allele E(D) or allele E(S).     

Spectral correspondence between visual spectral sensitivity and bioluminescence emission spectra in the click beetle Pyrophorus punctatissimus (Coleoptera: Elateridae)

The presence of two spectral mechanisms, near-ultraviolet and green (lambda(max)=545nm), is strongly suggested by electroretinographic visual spectral sensitivity curves obtained under dark and red chromatic adaptation conditions in the compound eyes of the click beetle Pyrophorus punctatissimus. The bioluminescence emission of the dorsal prothoracic lanterns is deep green (lambda(max)=543nm) and that of the ventral abdominal lantern is lime green (lambda(max)=556nm) in colour in P. punctatissimus. A broad green visual receptor would detect both deep green and lime green bioluminescent optical signals.     

Engineering the C-terminus of firefly luciferase as an indicator of covalent modification of proteins

Protein kinase recognition sequences and proteinase sites were engineered into the cDNA encoding firefly luciferase from Photinus pyralis in order to establish whether these modified proteins could be developed as bioluminescent indicators of covalent modification of proteins. Two key domains of the luciferase were modified in order to identify regions of the protein in which peptide sequences may be engineered whilst retaining bioluminescent activity; one between amino acids 209 and 227 and the other at the C-terminus, between amino acids 537 and 550. Mutation of amino acids between residues 209 and 227 reduced bioluminescent activity to less than 1% of wild-type recombinant. In contrast engineering peptide sequences at the C-terminus resulted in specific activities ranging from 0.06–120% of the wild-type recombinant. Addition of cyclic AMP dependent protein kinase catalytic subunit, to a variant luciferase incorporating the kinase recognition sequence, LRRASLG, with a serine at amino-acid position 543 resulted in a 30% reduction in activity. Alkaline phosphatase treatment restored activity. The bioluminescent activity of a variant luciferase containing a thrombin recognition sequence, LVPRES, with the cleavage site positioned between amino acid 542 and 543, decreased by 50% when incubated in the presence of thrombin. The results indicate regions within luciferase where peptide sequences may be engineered while retaining bioluminescent activity and have shown changes in bioluminescent activity when these sites are subjected to covalent modification. Changes in secondary structure, charge and length at the C-terminus of luciferase disrupt the microenvironment of the active site, leading to alterations in light emission. This has important implications both in understanding the evolution of beetle bioluminescence and also in the development of bioluminescent indicators of the covalent modification of proteins.     

Microsatellites and allozymes as the genetic memory of habitat fragmentation and defragmentation in populations of the ground beetle Carabus auronitens (Col., Carabidae)

Aim This paper has three aims: (1) to reconstruct the population history of a flightless silvicolous (forest) ground beetle in a region where strong anthropogenic activity has altered the connectivity of the landscape; (2) to estimate the effects of both contemporary and historical landscape structure on the genetics of populations; and (3) to investigate the reasons for clinal variation in one gene locus found in an earlier study carried out in the same geographical location. Location Münster (Westphalia), north‐west Germany. Methods We investigated 26 populations of the ground beetle Carabus auronitens Fabricius, 1792 by analysing seven polymorphic microsatellite loci and an allozyme locus. Samples of at least 16 individuals per site were studied. These were obtained from dry pitfall traps placed at 23 sites and from three additional (refuge) populations. We used regression and correlation analyses to investigate the effects of both historical and contemporary landscape structure on the allele frequency distributions of the investigated loci. Spatial autocorrelation analysis was used to study possible clinal variations, and admixture rates were calculated in order to assess the genetic influence of populations from possible refuges. Possible reasons for the development of the cline were examined using simulation models. Results The allele frequency distributions at the investigated loci could not be explained by selection or isolation by distance. We found clinal variation in 50% of the investigated loci and our simulations indicated that this was unlikely to have developed by chance. Admixture rates show significant influences of the investigated refuge populations on the populations under study. Main conclusions The findings strongly suggest that the clinal variation is secondary. It results from recolonization of the area by C. auronitens from multiple refuges after anthropogenic landscape changes caused forest fragmentation and led to species isolation.     

Orthologous gene of beetle luciferase in non-luminous click beetle, Agrypnus binodulus (Elateridae), encodes a fatty acyl-CoA synthetase

A homologous gene of beetle luciferase, AbLL (Agrypnus binodulusluciferase-like gene) was isolated from a Japanese non-luminous click beetle, A. binodulus, and its gene product was characterized. The identity of amino acid sequence deduced from AbLL with the click beetle luciferase from the Jamaican luminous click beetle, Pyrophorus plagiophthalmus, is 55%, which is higher than that between click beetle luciferase and firefly luciferase (approximately 48%). Phylogenetic analysis indicated that AbLL places in a clade of beetle luciferases, suggesting that AbLL is an orthologous gene of beetle luciferase. The gene product of AbLL (AbLL) has medium- and long-chain fatty acyl-CoA synthetase activity, but not luciferase activity. The fatty acyl-CoA synthetic activity was slightly inhibited in the presence of beetle luciferin, suggesting that AbLL has poor affinity for beetle luciferin. By comparing the amino acid residues of the catalytic domains in beetle luciferases with AbLL, the key substitutions for the luminescence activity in beetle luciferase will be proposed.     

Bioluminescent reporters for identification of gene allelic variants

A method for single nucleotide polymorphism identification was developed, which was based on the primer extension reaction (PEXT) followed by bioluminescent solid-phase microassay. Recombinant Ca²⁺-regulated photoprotein obelin and coelenterazine-dependent Renilla muelleri luciferase were used as reporters. The study was performed as an example of SNP genotyping of the human F5 gene encoding human Factor V Leiden polymorphism 1691 G??A (R506Q). Genomic DNA was amplified by PCR using primers flanking polymorphic site of 140 base pairs. PCR products were used as templates for two PEXT reactions using two primers containing 3??-terminal nucleotides, which were complementary to either normal or mutant alleles. If the template and allele-specific primer were completely complementary, the latter was elongated with DNA polymerase. The resulting extension product contained biotin residue due to the presence of biotinylated deoxyuridine triphosphate (B-dUTP) in the reaction mixture. The products were analyzed using obelin-streptavidin conjugates. The optimal PEXT-reaction conditions were found, which ensured a high reliability of SNP genotyping. A new approach to simultaneously revealing both alleles in one well was developed using two bioluminescent reporters. The efficiency of the proposed approach was shown in the study of clinical DNA samples.     

Coat colours in the Massese sheep breed are associated with mutations in the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes

Massese is an Italian dairy sheep breed characterized by animals with black skin and horns and black or apparent grey hairs. Owing to the presence of these two coat colour types, this breed can be considered an interesting model to evaluate the effects of coat colour gene polymorphisms on this phenotypic trait. Two main loci have been already shown to affect coat colour in sheep: Agouti and Extension coding for the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes, respectively. The Agouti locus is affected by a large duplication including the ASIP gene that may determine the Agouti white and tan allele (A(Wt)). Other disrupting or partially inactivating mutations have been identified in exon 2 (a deletion of 5 bp, D(5); and a deletion of 9 bp, D(9)) and in exon 4 (g.5172T>A, p.C126S) of the ASIP gene. Three missense mutations in the sheep MC1R gene cause the dominant black E(D) allele (p.M73K and p.D121N) and the putative recessive e allele (p.R67C). Here, we analysed these ASIP and MC1R mutations in 161 Massese sheep collected from four flocks. The presence of one duplicated copy allele including the ASIP gene was associated with grey coat colour (P = 9.4E-30). Almost all animals with a duplicated copy allele (37 out of 41) showed uniform apparent grey hair and almost all animals without a duplicated allele (117 out of 120) were completely black. Different forms of duplicated alleles were identified in Massese sheep including, in almost all cases, copies with exon 2 disrupting or partially inactivating mutations making these alleles different from the A(Wt) allele. A few exceptions were observed in the association between ASIP polymorphisms and coat colour: three grey sheep did not carry any duplicated copy allele and four black animals carried a duplicated copy allele. Of the latter four sheep, two carried the E(D) allele of the MC1R gene that may be the cause of their black coat colour. The coat colour of all other black animals may be determined by non-functional ASIP alleles (non-agouti alleles, A(a)) and in a few cases by the E(D) Extension allele. At least three frequent ASIP haplotypes ([D(5):g.5172T], [N:g.5172A] and [D(5):g.5172A]) were detected (organized into six different diplotypes). In conclusion, the results indicated that coat colours in the Massese sheep breed are mainly derived by combining ASIP and MC1R mutations.     

Bioluminescent imaging: applications to cancerology and endocrinology

Our laboratory has been using various bioluminescent imaging systems for more than 20 years to visualize and quantify bioluminescent and chemiluminescent reactions. This equipment allowed us to establish numerous cell lines expressing bioluminescent reporter genes to study the mechanism of action of nuclear receptors. Cells expressing the luciferase gene under the control of a constitutive promoter were used to follow in vivo proliferation of cancer cells. Intensities of in vitro and in vivo bioluminescent signals were compared and the conditions of bioluminescent reaction measurements were determined. These bioluminescent models are new tools for evaluating cancer treatment efficiencies and the role of hormone receptors in invasion. Cells expressing the luciferase gene under the control of hormones are used as in vivo biosensors for studying analog bioavailabilities and in vivo response kinetics. They are complementary models to in vitro models that have been developed in our laboratory for several years. In the future, targeting reporter gene (luciferase and GFP) expression to specific tissues should allow the detailed localisation of the action of nuclear receptor ligands.     

CCD imaging of basal bioluminescence in larval fireflies: clues on the anatomic origin and evolution of bioluminescence.

The anatomic and biochemical origin of beetle bioluminescence is still poorly understood. Through CCD imaging we report that larvae and pupae of the Brazilian fireflies Aspisoma lineatum and Cratomorphus sp emit a continuous weak glow throughout the entire body during all stages. This luminescence is especially developed after feeding, ecdysis and in the pupal stage, gradually disappearing as the cuticle becomes sclerotized and the adult emerges. This weak glow arises from the fat body, which consists of small lobes spread all over the body cavity. According to their pigmentation, these lobes can be divided in whitish and pinkish, and display different luciferase isozymes. Morphological studies suggest that the jelly-like ventral lanterns in the 8th abdominal segment evolved from these white lobes, providing a rationale for the widespread location of lanterns in larvae of different bioluminescent beetles. The biological and biochemical function of this weak diffuse bioluminescence is discussed in the context of the larval life-history.     

Cretophengodidae,a new Cretaceous beetle family,sheds light on the evolution of bioluminescence

Bioluminescent beetles of the superfamily Elateroidea (fireflies, fire beetles, glow-worms) are the most speciose group of terrestrial light-producing animals. The evolution of bioluminescence in elateroids is associated with unusual morphological modifications, such as soft-bodiedness and neoteny, but the fragmentary nature of the fossil record discloses little about the origin of these adaptations. We report the discovery of a new bioluminescent elateroid beetle family from the mid-Cretaceous of northern Myanmar (ca 99 Ma), Cretophengodidae fam. nov. Cretophengodes azari gen. et sp. nov. belongs to the bioluminescent lampyroid clade, and would appear to represent a transitional fossil linking the soft-bodied Phengodidae + Rhagophthalmidae clade and hard-bodied elateroids. The fossil male possesses a light organ on the abdomen which presumably served a defensive function, documenting a Cretaceous radiation of bioluminescent beetles coinciding with the diversification of major insectivore groups such as frogs and stem-group birds. The discovery adds a key branch to the elateroid tree of life and sheds light on the evolution of soft-bodiedness and the historical biogeography of elateroid beetles.     

Controlled expression of click beetle luciferase using a bacterial operator-repressor system

Abstract The bioluminescent phenotype conferred by luciferase genes in a particular bacterium has demonstrated to be one of the most versatile and useful methods to detect microorganisms. Genetic constructions derived from miniTn5 vectors have been constructed for the introduction and stable maintenance of the click beetle luciferase gene, lucOR , in various Gram-negative bacteria. To attenuate the expression in the environment where the marked strain has to survive (and to allow sensitive detection when desired) a DNA fragment containing the repressor gene lacI ^q and a P _trc:: lucOR fusion was cloned onto a suicide plasmid. This construction is able to express high luciferase levels only when induced by IPTG. Matings between Escherichia coli containing the suicide transposoon vector and different recipient bacteria gave transposition frequencies from 10⁻⁷ to 10⁻⁵. Strains with miniTn5- lucOR insertions showed luciferase activity induced by IPTG addition. The stringency of the regulation and the intensity of light emission depended on the tagged strain. This system allows stable maintenance of the marker and tight control of luciferase expression in the environment.     

Selection of the sex‐linked inhibitor of apoptosis in mountain pine beetle (Dendroctonus ponderosae) driven by enhanced expression during early overwintering

The mountain pine beetle (Dendroctonus ponderosae) is an insect native to western North America; however, its geographical range has recently expanded north in BC and east into Alberta. To understand the population structure in the areas of expansion, 16 gene‐linked microsatellites were screened and compared to neutral microsatellites using outlier analyses of F_st and F_ct values. One sex‐linked gene, inhibitor of apoptosis (IAP), showed a strong signature of positive selection for neo‐X alleles and was analyzed for evidence of adaptive variation. Alleles of IAP were sequenced, and differences between the neo‐X and neo‐Y alleles were consistent with neutral evolution suggesting that the neo‐Y allele may not be under functional constraints. Neo‐Y alleles were amplified from gDNA, but not effectively from cDNA, suggesting that there was little IAP expression from neo‐Y alleles. There were no differences in overall IAP expression between males and females with the common northern neo‐X allele suggesting that the neo‐X allele in males compensates for the reduced expression of neo‐Y alleles. However, males lacking the most common northern neo‐X allele thought to be selected for in northern populations had reduced overall IAP expression in early October—at a time when beetles are preparing for overwintering. This suggests that the most common allele may have more rapid upregulation. The reduced function of neo‐Y alleles of IAP suggested by both sequence differences and lower levels of expression may foster a highly selective environment for neo‐X alleles such as the common northern allele with more efficient upregulation.