Effects of artificial light on growth, development, and dispersal of two North American fireflies (Coleoptera: Lampyridae).

Holometabolous insects exhibit complex life cycles in which both morphology and ecological niche change dramatically during development. In the larval stage, many insects have soft, slow-moving bodies and poor vision, limiting their ability to respond to environmental threats. Artificial light at night (ALAN) is an environmental perturbation known to severely impact the fitness of adult insects by disrupting both temporal and spatial orientation. The impact of ALAN on earlier life stages, however, is largely unknown. We conducted a series of laboratory experiments to investigate how two distinct forms of ALAN affect the development and movement of immature Photuris sp. and Photinus obscurellus fireflies. Although long-term exposure to dim light at night (dLAN), akin to urban skyglow, did not impact overall survivorship or duration of egg, larval, and pupal stages in either species, it did accelerate weight gain in early-instar Photuris larvae. Late-instar Photuris exposed to point sources of ALAN at the start of their nightly foraging period were also significantly more likely to burrow beneath the soil surface, rather than disperse across it. ALAN may therefore impede dispersal of firefly larvae away from illuminated areas, which could have downstream consequences for the reproductive fitness of adults.

Do holometabolous insects molt spontaneously after adulthood? An exceptional case report in fireflies (Coleoptera: Lampyridae), with discussion of its inferred endocrine regulation especially in relation to neoteny.

It has been a traditionally held view that winged insects stop molting after they reach adulthood. We observed a fascinating phenomenon of a post-imago molt occurring in the neotenic females of a firefly species in Taiwan over the last two years. By rearing Lamprigera minor larvae to adults, four out of the five unmated females studied were found undergoing an extra molt 8-18 days after adult eclosion. They were reproductively mature when the post-imago molt occurred, as evidenced by the eggs inside their bodies. The four females died without oviposition whereas the only normal female laid eggs. A comparison of exuviae of different stages confirmed the existence of post-imago ecdysis. The adult skin differed from the pupal one mainly in the mouthparts and leg structures. No mix of pupal and adult traits was seen in the adult skin. The females retained the same morphology after the extra molt. A close examination of the post-imago molting females revealed that their oviduct openings were all blocked by larval or pupal skin and thus unable to lay eggs. The reproductive stress may invoke an endocrine disorder and lead to an extra molt. We propose that L. minor females retain their prothoracic glands even as adults, allowing them to molt as adults under certain environmental or physiological conditions. Thus, neoteny of L. minor is reflected in both the external morphology as well as the internal physiology. The possible developmental changes associated with the evolution of neoteny are discussed.

2-S-cysteinylhydroquinone is an intermediate for the firefly luciferin biosynthesis that occurs in the pupal stage of the Japanese firefly, Luciola lateralis.

Firefly luciferin is a natural product that is well-known to function as the substrate of the bioluminescence reaction in luminous beetles. However, the details of the biosynthetic system are still unclear. In this study, we showed by LC-MS/MS analysis that stable isotope-labeled 2-S-cysteinylhydroquinone was incorporated into firefly luciferin in living firefly specimens. Comparison of the incorporation efficiency among the developmental stages suggested that firefly luciferin is biosynthesized predominantly in the pupal stage. We also accomplished the in vitro biosynthesis of firefly luciferin using 2-S-cysteinylhydroquinone and the crude buffer extract of firefly pupae, suggesting the presence of a biosynthetic enzyme in the pupal extract.

Biochemical characteristics and gene expression profiles of two paralogous luciferases from the Japanese firefly Pyrocoelia atripennis (Coleoptera, Lampyridae, Lampyrinae): insight into the evolution of firefly luciferase genes.

Two paralogous genes of firefly luciferase, Luc1 and Luc2, have been isolated from the species in two subfamilies, Luciolinae and Photurinae, of the family Lampyridae. The gene expression profiles have previously been examined only in the species of Luciolinae. Here we isolated Luc1 and Luc2 genes from the Japanese firefly Pyrocoelia atripennis. This is the first report of the presence of both Luc1 and Luc2 genes in the species of the subfamily Lampyrinae and of the exon-intron structure of Luc2 in the family Lampyridae. The luminescence of both gene products peaked at 547 nm under neutral buffer conditions, and the spectrum of Luc1, but not Luc2, was red-shifted under acidic conditions, as observed for Luc2 in the Luciolinae species. The semi-quantitative reverse transcription-polymerase chain reaction suggested that Luc1 was expressed in lanterns of all the stages except eggs, while Luc2 was expressed in the non-lantern bodies of eggs, prepupae, pupae, and female adults. These expression profiles are consistent with those in the Luciolinae species. Considering the distant phylogenetic relationship between Lampyrinae and Luciolinae in Lampyridae, we propose that fireflies generally possess two different luciferase genes and the biochemical properties and gene expression profiles for each paralog are conserved among lampyrid species.

Identification and characterization of the Luc2-type luciferase in the Japanese firefly, Luciola parvula, involved in a dim luminescence in immobile stages.

Nocturnal Japanese fireflies, Luciola parvula, emit from their lanterns a yellow light, one of the most red-shifted colors found among fireflies. Previously, we isolated and characterized two different types of luciferase gene, Luc1 and Luc2, from the fireflies Luciola cruciata and Luciola lateralis; Luc1 is responsible for the green-yellow luminescence of larval and adult lanterns, whereas Luc2 is responsible for the dim greenish glow of eggs and pupal bodies. The biological role of firefly lanterns in adults is related to sexual communication, but why the eggs and pupae glow remains uncertain. In this study, we isolated the gene Luc2 from L. parvula, and compared its expression profiles and enzymatic characteristics with those of Luc1. A semi-quantitative reverse transcription polymerase chain reaction showed that Luc1 was predominantly expressed in larvae, prepupae, pupae and adults, whereas Luc2 was expressed in eggs, prepupae, pupae and adult females. Enzymatic analyses showed that the luminescent color of Luc1 matches the visual sensitivity of L. parvula eyes, whereas that of Luc2 is very different from it. These results suggest that the biological role of Luc2 expressed in immobile stages is not intraspecific communication.

Further studies on south eastern Asian Luciolinae: 1. Sclerotia Ballantyne, a new genus of fireflies with back swimming larvae 2. Triangulara Pimpasalee, a new genus from Thailand (Coleoptera: Lampyridae).

Sclerotia Ballantyne gen. nov. is established for seven species. Sclerotia aquatilis (Thancharoen, 2007), Scl. brahmina (Bourgeois, 1890), Scl. carinata (Gorham, 1880), Scl. flavida (Hope, 1845), Scl. seriata (Olivier, 1891), and Scl. substriata (Gorham, 1880) are transferred from Luciola Laporte. Sclerotia fui sp. nov. from China is described as new. Luciola cingulata Olivier, 1885 is synonymised with Luciola substriata Gorham. Luciola formosana Pic, 1916 is removed from synonymy with Luciola substriata and newly synonymised with Luciola flavida (Hope). Males are characterised by linear elytral punctation, a set of three sclerites surrounding the aedeagal sheath and a light organ in ventrite 7 with emarginated anterior margin. Associated females lack bursa plates. Larvae associated for three species are back swimmers, having heavily sclerotised exoskeleton and a metapneustic respiratory system. Males and associated females are keyed. Analysis of molecular evidence for Chinese populations of three species indicates monophyly of Sclerotia Ballantyne gen. nov. and distinct separation from other genera.Triangulara Pimpasalee gen. nov. is described from Tri. frontoflava Pimpasalee gen. et sp. nov. from Thailand. It is superficially similar to Sclerotia gen. nov. but larger, and specimens in collections appear to have been confused with the similarly coloured Scl. substriata (Gorham) comb. nov. Males differ in having an elongate triangular light organ in ventrite 7 and are without sclerites around the aedeagal sheath. Larvae are not reliably associated but may be aquatic.

Revisional studies on the Luciolinae fireflies of Asia (Coleoptera: Lampyridae): 1. The genus Pyrophanes Olivier with two new species. 2. Four new species of Pteroptyx Olivier and 3. A new genus Inflata Boontop, with redescription of Luciola indica (Motsch.) as Inflata indica comb. nov.

The Luciolinae firefly genus Pyrophanes is redescribed and a key is provided for 7 species, viz. appendiculata Olivier, beccarii Olivier, quadrimaculata Olivier, semilimbata (Olivier) and similis Olivier, with Py. elongata Ballantyne sp. nov. and Py. similisimma Ballantyne sp. nov. described as new. Macropterous females associated for several species have 2 pairs of broad bursa plates. Following an enlarged phylogenetic analysis of morphological characters, an expanded generic description of Pteroptyx Olivier now accommodates four new species with metafemoral combs. Three of these have an entire light organ in ventrite 7 but are without the generic hallmark of deflexed elytral apices in the male. The first species of Pteroptyx to possess these features are Pt. sayangia Ballantyne sp. nov., Pt. galbina Jusoh sp. nov. and Pt. surabayia Ballantyne sp. nov. Based on a single unusual specimen Pt. gombakia Ballantyne sp. nov. is here described with deflexed elytral apices and bipartite light organ in ventrite 7, but with exceptional incurving lobes along the posterior margin of ventrite 7, which is a characteristic of Pyrophanes. The description of Luciola testacea Motschulsky is discussed and many morphological characters of its type specimen are correctly outlined. Its place as the type for Pteroptyx is addressed. Inflata gen. nov. Boontop is erected for specimens of Luciola indica Motschulsky as Inflata indica (Motsch.) comb. nov., and is described from males and reliably associated females. Discussion overviews the present situation regarding Luciolinae taxonomy of southeast Asian and the Australopacific regions.

A review of the genus Lamellipalpodes Maulik (Coleoptera: Lampyridae).

The Ototretinae genus Lamellipalpodes Maulik, 1921 is reviewed. Four species are proposed as new to science, L. bajhangensis Bocakova sp. nov., L. holzschuhi Bocakova sp. nov. and L. godawarensis Bocakova sp. nov., all from Nepal, and L. yunnanensis Bocakova sp. nov. from China (Yunnan) and northern Laos. Six species are redescribed. Illustrations of diagnostic characters are included, with a checklist and a key to Lamellipalpodes species. The genus is newly recorded from China and Laos.

Ybytyramoan, a new genus of fireflies (Coleoptera: Lampyridae, Lampyrinae, Photinini) endemic to the Brazilian Atlantic Rainforest, with description of three new species.

Here we describe a new Photinina genus with three species endemic to Serra dos Órgãos Mountains in Brazil. Ybytyramoan gen. nov. occurs in high altitudes, from 980m up to 2000m, and has the following unique set of characters: head abruptly depressed at vertex; lanterns not fully developed, somewhat rounded or anteriorly rounded, straight posteriad, with posterolateral rounded projections (billycock-shaped), at the middle of the abdominal sterna VI and VII; abdominal sternum VIII not covered by VII; phallus and parameres apically teethed. We provide illustrations and a key to the three species in this genus: Ybytyramoan praeclarum sp. nov. (type-species), Y. diasi sp. nov., Y. monteirorum sp. nov.

The function of Hox and appendage-patterning genes in the development of an evolutionary novelty, the Photuris firefly lantern.

Uncovering the mechanisms underlying the evolution of novel traits is a central challenge in biology. The lanterns of fireflies are complex traits that lack even remote homology to structures outside luminescent beetle families. Representing unambiguous novelties by the strictest definition, their developmental underpinnings may provide clues to their origin and offer insights into the mechanisms of innovation in developmental evolution. Lanterns develop within the context of abdominal Hox expression domains, and we hypothesized that lantern formation may be instructed in part by these highly conserved transcription factors. We show that transcript depletion of Abdominal-B in Photuris fireflies results in extensive disruption of the adult lantern, suggesting that the evolution of adult lanterns involved the acquisition of a novel regulatory role for this Hox gene. Using the same approach, we show that the Hox gene abdominal-A may control important secondary aspects of lantern development. Lastly, we hypothesized that lantern evolution may have involved the recruitment of dormant abdominal appendage-patterning domains; however, transcript depletion of two genes, Distal-less and dachshund, suggests that they do not contribute to lantern development. Our results suggest that complex novelties can arise within the confines of ancestral regulatory landscapes through acquisition of novel targets without compromising ancestral functions.

Bioluminescence of a firefly pupa: involvement of a luciferase isotype in the dim glow of pupae and eggs in the Japanese firefly, Luciola lateralis.

We isolated cDNA for a luciferase isotype, LlLuc2, from the ovary of the Japanese firefly, Luciola lateralis. The gene product LlLuc2 showed 59% amino acid identity with LlLuc1, which had been isolated from the adult L. lateralis lantern. Molecular phylogenetic analysis indicated that LlLuc2 is an orthologue of LcLuc2 from Luciola cruciata. The spectral maxima of the luminescence by recombinant LlLuc1 and LlLuc2 were 550 and 539 nm, respectively. Quantitative PCR analysis revealed that LlLuc1 was expressed predominantly in larvae and adults, and LlLuc2 was expressed in eggs and pupae, which glow dimly, and we found that the in vivo luminescence spectra of the egg and pupa in L. lateralis were in good agreement with the in vitro luminescence spectrum by LlLuc2. These results suggest that, in L. lateralis, LlLuc1 is responsible for the yellowish luminescence of larval and adult lanterns, and LlLuc2 is responsible for the dim, greenish glow of eggs and whole pupae. Similar results were obtained in L. cruciata.

Biosynthesis of firefly luciferin in adult lantern: decarboxylation of L-cysteine is a key step for benzothiazole ring formation in firefly luciferin synthesis.

BACKGROUND: Bioluminescence in fireflies and click beetles is produced by a luciferase-luciferin reaction. The luminescence property and protein structure of firefly luciferase have been investigated, and its cDNA has been used for various assay systems. The chemical structure of firefly luciferin was identified as the D-form in 1963 and studies on the biosynthesis of firefly luciferin began early in the 1970's. Incorporation experiments using (14)C-labeled compounds were performed, and cysteine and benzoquinone/hydroquinone were proposed to be biosynthetic component for firefly luciferin. However, there have been no clear conclusions regarding the biosynthetic components of firefly luciferin over 30 years. METHODOLOGY/PRINCIPAL FINDINGS: Incorporation studies were performed by injecting stable isotope-labeled compounds, including L-[U-(13)C3]-cysteine, L-[1-(13)C]-cysteine, L-[3-(13)C]-cysteine, 1,4-[D6]-hydroquinone, and p-[2,3,5,6-D]-benzoquinone, into the adult lantern of the living Japanese firefly Luciola lateralis. After extracting firefly luciferin from the lantern, the incorporation of stable isotope-labeled compounds into firefly luciferin was identified by LC/ESI-TOF-MS. The positions of the stable isotope atoms in firefly luciferin were determined by the mass fragmentation of firefly luciferin. CONCLUSIONS: We demonstrated for the first time that D- and L-firefly luciferins are biosynthesized in the lantern of the adult firefly from two L-cysteine molecules with p-benzoquinone/1,4-hydroquinone, accompanied by the decarboxylation of L-cysteine.

Memoan ciceroi gen. et sp. nov., a remarkable new firefly genus and species from the Atlantic Rainforest (Coleoptera: Lampyridae).

A species of firefly discovered in a fragile and rapidly disappearing Atlantic Rainforest biome in Brazil does not fit into any of the existing subfamilies nor described generic categories in the Lampyridae and is described here as Memoan ciceroi gen. et sp. nov. and classed as Lampyridae Incertae sedis, as it exhibits features of both the Amydetinae and Lampyrinae. An overview of subfamily arrangements and relevant generic characters is given to support this action. Memoan gen. nov. can be distinguished by its alveolate pronotum and elytra; subserrate antennae, antenommeres II-IX compressed, antennal sockets obliquely inserted on tubercles; labial palp one-segmented and obconic, and by its conspicuous pleuroventral suture.

Studies on South-east Asian fireflies: Abscondita, a new genus with details of life history, flashing patterns and behaviour of Abs. chinensis (L.) and Abs. terminalis (Olivier) (Coleoptera: Lampyridae: Luciolinae).

Abscondita, a new genus of fireflies from South-east Asia, is described from males and females of Abs. anceyi (Olivier 1883), Abs. cerata (Olivier 1911), Abs. chinensis (L. 1767), Abs. perplexa (Walker 1858), Abs. promelaena (Walker 1858) and Abs. terminalis (Olivier 1883), all transferred from Luciola Laporte. Both L. dubia Olivier 1903 and L. dejeani Gemminger 1870 are synonymised with Luciola perplexa (Walker), and L. aegrota Olivier 1891 and L. melaspis Bourgeois 1909 with L. promelaena Walker. Females are characterised by their bursa plates. Larvae are associated and described for Abs. anceyi (Olivier), Abs. chinensis (L.) and Abs. terminalis (Olivier). Taxonomic issues regarding the identification of species with very similar colouration of pale dorsum and black tipped elytra are addressed and in some cases resolved. A neotype for Luciola chinensis (L.) is erected and Luciola praeusta (Kiesenwetter 1874) is synonymised with L. chinensis (L.). Descriptions of life histories, biology and flashing patterns of populations of Abs. chinensis and Abs. terminalis from central China are included. A bs. terminalis is the first Asian firefly known to possess multiple flash trains where males are documented to display with repeating flash trains.

Aspisoma lineatum (Gyllenhal) (Coleoptera: Lampyridae) firefly: description of the immatures, biological, and ecological aspects.

Aspisoma lineatum (Gyllenhal) is a common firefly in the Southeastern region of Brazil. Adults and larvae were collected in the municipality of Campinas, state of São Paulo, Brazil, and the immature stages were described and reared in the laboratory. Four generations were reproduced in the laboratory, and a method for its rearing was established. The life cycle usually lasts 6 months, but under optimal laboratory conditions, it lasted from 2 to 4 months. Larvae were fed with Bradybaena similaris and Bulimulus tenuissimus snails since the beginning of the larval stage. This species was found to be easily adapted to environments under anthropic influence, such as urban areas and farms.

Degree-day prediction of adult emergence of Photinus carolinus (Coleoptera: Lampyridae).

The synchronous display of the firefly Photinus carolinus Green in the Great Smoky Mountains National Park has been enjoyed by park visitors and studied by scientists and naturalists for the past 17 yr. A degree-day model is presented offering a means of more accurately predicting the display season of P. carolinus to facilitate advance planning required for researchers and the management of the thousands of nightly visitors who come to witness the peak firefly display. A modified growing degree-day (mGDD) model (base 50 degrees F, starting date 1 March) (centigrade equivalent base 10 degrees C, denoted mGDDC) provided the best fit to phenology data collected over a 15-yr period. The predictive model yielded the following values for P. carolinus phenology: male emergence, 838.6 mGDD (range, 776-922)/465.9 mGDDC (range, 431-512); "good" display, 992.5 mGDD (range, 931-1075)/551.4 mGDDC (range, 517-597); female emergence, 1068.8 mGDD (range, 956-1224)/593.8 mGDDC (range, 531-680); and peak male display, 1094.2 mGDD (range, 1020-1182)/607.9 mGDDC (range, 567-657). The accuracy and range limits of using calendar dates versus degree-day values are presented. Additional degree-day values are provided for 14 other local firefly and 1 phengodid species to broaden the applicability of using degree-days to aid in prediction of adult firefly seasons.

Description of Photuris fulvipes (Blanchard) immatures (Coleoptera, Lampyridae, Photurinae) and bionomic aspects under laboratory conditions

Larvae and pupae of the cryptic firefly Photuris fulvipes (Blanchard,1837) are described. They were reared to adult stage from eggs laid by a female collected in São Luiz do Paraitinga, São Paulo, Brazil. Additionally, adult diagnosis and some bionomic data observed in the laboratory are presented. The bionomical and morphological characteristics of P. fulvipes were compared to other unidentified species of the genus and to Bycellonycha sp. from literature. At moment, the data are insufficient to propose a diagnosis for the species, but some characteristics appear to define species group or, at least, they may have generic value.

Larva e pupa de Photuris fulvipes (Blanchard, 1837), criadas a partir de ovos de uma fêmea coletada em São Luiz do Paraitinga, São Paulo, são descritas. São apresentados uma diagnose do adulto e alguns dados bionômicos observados durante a criação em laboratório. Esses dados e a morfologia dos imaturos foram comparados aos de outras espécies não identificadas do gênero e a Bycellonycha sp., descritos na literatura. Nesse momento, os dados são insuficientes para propor uma diagnose para a espécie, mas algumas características parecem ser importantes para definir grupos de espécies ou, pelo menos, para diagnosticar o gênero.

Stereoisomeric bio-inversion key to biosynthesis of firefly D-luciferin.

The chirality of the luciferin substrate is critical to the luciferin-luciferase reaction producing bioluminescence. In firefly, the biosynthetic pathway of D-luciferin is still unclear, although it can be synthesized in vitro from D-cysteine. Here, we show that the firefly produces both D- and L-luciferin, and that the amount of active D-luciferin increases gradually with maturation stage. Studies of firefly body extracts indicate the possible conversion of L-cysteine via L-luciferin into D-luciferin, suggesting that the biosynthesis is enzymatically regulated by stereoisomeric bio-inversion of L-luciferin. We conclude that the selection of chirality in living organisms is not as rigid as previously thought.