Since their evolutionary origin some million years ago Zhang et al. Fireflies Coleoptera: Lampyridae rank among the most charismatic beetles, with distinctive bioluminescent courtship displays that make them a potential flagship group for insect conservation. With more than species worldwide, firefly beetles exhibit surprisingly diverse life history traits figure 1 ; Ohba , Lloyd , Lewis , including nonluminous adults with daytime activity periods, glowworm fireflies with flightless females, and lightning bugs that exchange species-specific flash signals.
Fireflies also inhabit ecologically diverse habitats, including wetlands e. Their predaceous larvae, which can be aquatic, semiaquatic, or terrestrial, spend months to years feeding on snails, earthworms, and other soft-bodied prey. In contrast, firefly adults are typically short lived and do not feed. Some taxa are habitat and dietary specialists, whereas others are ecological generalists Reed et al. Fireflies are economically important in many countries, because they represent a growing ecotourist attraction Napompeth , Lewis However, as is true for many invertebrates Cardoso et al.
Firefly beetles Coleoptera: Lampyridae show great diversity in their ecology, behavior and extinction risk factors. Monitoring studies that provide quantitative data on population trends are lacking for almost all firefly species. However, surveys have revealed significant recent declines in the mangrove firefly Pteroptyx tener in Malaysia Jusoh and Hashim , Khoo et al.
Anecdotal reports and expert opinion also suggest reductions in both the occurrence and abundance of many firefly species over recent decades Lewis , Faust , Lloyd In , an international group of firefly experts convened in Malaysia and wrote The Selangor Declaration on the Conservation of Fireflies Fireflyers International Network , recommending actions to preserve these iconic insects.
As part of this effort, in the present article we discuss perceived threats to firefly biodiversity and persistence on the basis of an opinion survey of experts from different geographic regions. We also review the current evidence for the impact of such threats on firefly populations. Finally, for each threat, we discuss associated risk factors sensu Reed et al.
In January , we sent a short Qualtrics survey see box 1 by email to people on the distribution list of the Fireflyers International Network, a scientific organization composed of individuals with interests and expertise in firefly ecology, behavior, taxonomy, or conservation. These survey results should be interpreted with caution, because they reflect only expert opinion concerning perceived threats to firefly species persistence.
What country or biogeographic region does your main firefly expertise cover? In your country or region, how important is each of the following as a current threat to firefly populations? Threats were presented in randomized order, and a 0—5 scale with a scoring resolution of 0. Habitat loss. Are there other current or future threats to firefly populations in your country or region that we have not listed here?
If so, please describe. Thinking about the two most important current threats , can you describe how they affect a particular species or group of fireflies? We had only single respondents each from Australia and South America and none from Africa.
Comments by the respondents about specific threats are summarized below see the supplemental material for detailed respondent comments. We also conducted a literature search for existing evidence concerning how these perceived threats influence firefly survival, reproduction, or population persistence. This information, in addition to comments by respondents about specific threats, is summarized below see the supplemental material for detailed respondent comments.
Habitat loss, artificial light, and pesticide use were identified as the three most serious threats when scores were averaged across the eight regions table 1 , figure 2. More than half of the 49 respondents assigned the highest possible threat score 5 to habitat loss, whereas nearly one-third did so for light pollution, and one-fifth did so for pesticide use. However, their threat scores differed considerably across geographic regions table 2 , figure 2 , with additional threats such as water pollution and tourism ranked as important concerns in some regions.
Below, we provide details of the survey results, review the current evidence concerning the impact of each perceived threat, and propose risk factors that may interact with certain threats to increase the risk of population declines, local extirpation, or global extinction for particular firefly species.
See Table 2 for geographic regions, sample sizes, and scores for additional threats. Note: The threats to fireflies were scored on a scale from 0 to 5 by 49 respondents surveyed in January—February Habitat loss was perceived as the most serious threat to fireflies globally table 1 , as well as within nearly all regions table 2. Habitat loss and fragmentation are predicted to be particularly problematic for habitat specialists Reed et al.
Genome-wide SNP single nucleotide polymorphism analysis of Photinus pyralis , a species widespread and abundant across the eastern United States, demonstrated low gene flow among populations, with Fst fixation index values averaging 0. If this result is applicable to other firefly species, this degree of genetic isolation implies that extirpated populations are unlikely to be rescued by migration.
Dispersal distances are even more limited in species with flightless females; this includes the glowworms Lampyris noctiluca Atkins et al. Dispersal through larval movement may also be low: The estimated dispersal distance for the terrestrial larvae of Luciola parvula is only several meters during the entire larval period Kakehashi et al.
However, dispersal distances may be higher for species with aquatic larvae, because these might be transported along rivers and irrigation channels, as was reported for other aquatic macroinvertebrates e. In Thailand, eggs and larvae of the aquatic firefly Sclerotia aquatilis are often attached to duckweed and so may get transported along with this aquatic vegetation. If females or terrestrial larvae get transported by floods and survive in sufficient numbers, they may be able to colonize new patches of suitable habitat.
In Europe, firefly habitat has been lost through urbanization, industrialization, and agricultural intensification De Cock Agricultural intensification—which entails habitat loss and fragmentation in addition to increased use of pesticides, herbicides, and fertilizer—has been identified as a major driver for declining populations of many insects Wagner , Throughout the United Kingdom, grassland habitats frequented by the glowworm Lampyris noctiluca have been lost to both agricultural intensification and woodland succession following the abandonment of pastureland Gardiner Long-term surveys of L.
In Italy, one survey respondent considered agricultural intensification responsible for declining numbers of Luciola italica , Luciola lusitanica , and Lampyris fireflies on the Padana plain and the northern Appenines. In contrast, another respondent in Mediterranean Spain expressed concern about the abandonment of small orchards and irrigated agricultural plots in which Nyctophila reichii , Lampyris iberica , and Lamprohiza paulinoi often occur.
Once abandoned, these cultivated areas become more xeric and less suitable for snails, which constitute the main prey for certain fireflies. In Japan, an iconic traditional landscape known as satoyama is disappearing in the face of development and rural out-migration Kobori and Primack Composed of farming villages with streams, ponds, rice paddies, and cultivated fields surrounded by forest, this managed habitat once supported considerable biodiversity in the Japanese countryside, including fireflies Oba et al.
In Malaysia, breeding congregations of Pteroptyx tener fireflies declined following conversion of riverbank mangroves to agriculture, aquaculture, and urbanization Jusoh and Hashim , Khoo et al. Throughout Southeast Asia, large areas of riverbank mangroves have been cleared for oil palm plantations, shrimp farms, or flood mitigation, making these sections unsuitable for the growth and development of Pteroptyx firefly larvae and their snail prey Wong , Nada et al.
In addition, Pteroptyx adults gather for nightly courtship displays in specific, prominent trees located along mangrove rivers, and many of these display trees have been removed e. Among the top threats to fireflies globally are habitat loss and artificial light at night. The Atlantic rainforest of Brazil hosts high firefly biodiversity Viviani , Viviani and Santos , Silveira and Mermudes , , but this is among the most threatened and fragmented rainforests worldwide Hoorn et al.
In Tlaxcala, Mexico, populations of Macrolampis palaciosi another species with flightless adult females are restricted to forest remnants fragmented by extensive logging Vance and Kuri In eastern North America, the loss of firefly habitat occurs mainly through urbanization and commercial and residential development e. Globally, increasing human populations along coastlines have caused extensive habitat loss and fragmentation Polidoro et al.
One such coastal habitat specialist in Delaware is Photuris bethaniensis , which is found in freshwater swales between oceanside dunes Heckscher ; its wetland habitat faces imminent threat from extensive residential development Kitt Heckscher, Delaware State University, Dover, Delaware, personal communication, In the western United States and Texas, several fireflies are restricted to habitats adjoining permanent water sources, including rivers, streams, lakes, ponds, springs, and irrigated fields.
Groundwater pumping to meet urban and agricultural water demands has substantially reduced surface water flow and lowered groundwater tables Larry Buschman, Kansas State University, Lawrence, Kansas, personal communication, , and increased drought due to climate change is likely to further diminish suitable firefly habitat in these areas. Globally, artificial light at night ALAN was rated as the second most serious threat to fireflies table 1.
ALAN includes both direct lighting that affects a localized area e. Light pollution was perceived as the top threat to fireflies in East Asia and South America and the second or third most serious threat in most other regions table 2. ALAN is expected to be particularly problematic for nocturnally active firefly taxa, because these adults rely on bioluminescent courtship signals to locate mates Lloyd , Lewis Observational and experimental studies provide evidence that ALAN adversely affects firefly populations for a review, see Owens and Lewis ; also Mbugua et al.
Several studies have shown negative correlations between high levels of ALAN and firefly abundance. For example, surveys showed that populations of Luciola italica were absent from the more brightly lit parts of the city of Turin, Italy Picchi et al. Because ALAN is so tightly correlated with urbanization; however, these observational studies make it difficult to isolate the primary cause of reduced firefly populations.
In this context, local knowledge can be useful; one survey respondent cited increased light pollution around small and medium-size villages as responsible for declines in Spanish glowworms. Owens and Lewis reviewed experimental studies demonstrating that artificial light interferes with the production and reception of firefly courtship signals. Many lightning bug fireflies engage in flash dialogs in which females give flash responses to male courtship signals Lloyd Furthermore, Photinus pyralis females exposed to ALAN responded less often to male courtship signals Firebaugh and Haynes and showed a nonsignificant reduction in mating success Firebaugh and Haynes Therefore, several lines of evidence indicate that ALAN interferes with firefly reproductive behavior and may heighten extinction risk.
Globally, pesticides were rated as the third most serious threat to fireflies table 1 , with some variation among geographic regions table 2. Common agricultural insecticides include various organochlorines, organophosphates and, more recently, neonicotinoids Simon-Delso et al. Although only a few studies have investigated their direct effects on fireflies see below , such broad-spectrum insecticides are known to adversely affect numerous nontarget insects and other taxa reviewed by Sanchez-Bayo , Pisa et al.
Mechanisms of insecticide exposure include aerial spraying, contact with insecticide-containing soil or water, or ingestion of contaminated prey. For fireflies, high insecticide concentrations in water and soil may be particularly harmful, because the larval stage lives and develops for months to years either underwater e. Other firefly life stages may also be exposed, because eggs are laid in soil, moss, or rotting wood, and pupae develop underground or on tree trunks.
Adults may also be exposed to insecticide residues when resting on treated soil or foliage. To date, the effects of direct exposure to pesticides on fireflies has been tested in only two published laboratory studies. In Southeast Asia, agricultural runoff from oil palm plantations and shrimp farms poses hazards to fireflies that are aquatic or semiaquatic during their larval stage. A — study in the Selangor River in Malaysia found levels of several pesticides that sometimes exceeded acceptable limits for freshwater organisms Leong et al.
In Japan, industrial pollution and pesticide contamination of rivers has been implicated in the declining populations of Luciola cruciata and Aquatica lateralis that occurred during the second half of the twentieth century Yuma , Ohba Although the European Union banned outdoor use of neonicotinoids in April , in other regions, including the United States, these compounds continue to be widely used in both agricultural and residential settings Bonmatin et al.
In the United States, nearly all corn and soybean seeds are routinely coated with neonicotinoid insecticides Douglas and Tooker , which are persistent in most soils. In a field test conducted over a single growing season, corn plots planted with clothianidin-treated seed showed a Imidacloprid is a commonly used neonicotinoid marketed to homeowners for killing white grubs larvae of Scarabaeidae beetles ; in a 3-year study, the application of this pesticide as a lawn treatment greatly reduced the abundance of nontarget insects, including a 2.
Insecticides such as pyrethroids are widely used for adult mosquito control but may also affect nontarget insects Davis et al. Fireflies may be particularly at risk, because spraying is generally done at dusk, when mosquitoes and fireflies are both active.
In field bioassays conducted with the beneficial lady beetle Harmonia convergens , ultra-low-volume application of permethrin caused high mortality for beetles contacted by the spray Peterson et al. Pesticides can also affect fireflies indirectly by reducing the availability or increasing toxicity of their larval prey, which include snails and earthworms. Imidacloprid and other neonicotinoids have been shown to be highly toxic to earthworms for a review, see Sanchez-Bayo , Pisa et al.
Earthworms and other prey can also bioaccumulate neonicotinoids Douglas and Tooker , Chevillot et al. Finally, lethal nontarget effects on firefly larvae have been observed for various biological control agents, including the fungi Metarhizium and Beauvaria bassiana and Steinernema sp.
However, the respondents in particular regions did highlight several other perceived threats to fireflies:. Across Asia and in South America, the respondents identified agricultural and industrial runoff containing fertilizers, pesticides, and other water-borne pollutants as the third or fourth most serious threat table 2. In contrast to the mainly terrestrial larvae of Nearctic fireflies, numerous Asian fireflies have aquatic larvae that inhabit freshwater ponds, rivers, and streams, where they feed and develop through several larval instars Lloyd This stage typically lasts for several months, during which both larvae and their snail prey will be exposed to water-borne pollutants.
Firefly tourism has long been popular in Japan, Malaysia, and Taiwan, and similar recreational activity has recently been proliferating within other countries, including Thailand Thancharoen , the United States Faust , , and Mexico Vance and Kuri Collectively, firefly tourism attracts more than , visitors per year Lewis and carries considerable economic benefits. However, if such tourism is not responsibly managed, it can threaten local firefly populations by disturbing larval and adult habitats and interfering with adult reproduction.
Although many different fireflies produce attractive bioluminescence, most at risk are those that spontaneously synchronize; these species, such as Photinus carolinus in the United States, produce stunning displays in which hundreds of males flash rhythmically in unison.
Throughout Southeast Asia, synchronous fireflies in the genus Pteropytx are a major tourist attraction because they congregate in large numbers in mangrove forests along tidal rivers Wong and Yeap ALAN from commercial tour operations, flashlights, and even camera flashes Thancharoen and Masoh can interfere with Pteropytx courtship behavior.
In Thailand, Pteropytx tourism has generated high-speed motorboat traffic along mangrove rivers, resulting in riverbank erosion that topples display trees and destroys larval habitat. Other tourist-attracting fireflies have flightless females that may inadvertently get trampled by tourists as they signal from the ground or low vegetation; these include Phausis reticulata in North Carolina, and Macrolampis palaciosi in Nanacamilpa, Mexico.
Although not currently considered a threat, historically, the extensive harvest of fireflies from wild populations likely caused some population declines Bauer et al. During the late nineteenth and early twentieth centuries, the Genji firefly, Luciola cruciata , was commercially harvested in Japan Lewis In the United States, from until about , Sigma Chemical Company annually harvested about three million North American fireflies to extract luciferase and luciferin light-producing compounds; Lewis In China, between and , millions of fireflies were harvested and sold online for theme park exhibitions and romantic gifts Lewis and Owens Because of protests and letter-writing campaigns mounted by firefly conservation organizations, the commercial harvest of wild fireflies in China has been largely curtailed Lei Ping, Firefly Ecological Alliance, Chengdu, China, personal communication, Although most of the respondents did not rate exotic invasives as a major threat, the spread of Solenopsis fire ants across the southern United State could be an emerging threat, particularly for firefly species such as Pyractomena borealis , whose larvae are active aboveground Lynn Faust, Emory River Land Company, Knoxville, Tennessee, personal communication, Although the effect of anthropogenic climate disruption on firefly populations remains unknown, the restricted ranges and specialized habitat requirements of certain fireflies suggest that they are likely to be threatened by drought and sea level rise Reed et al.
Fireflies require moist conditions throughout their life stages Lloyd , Atkins et al. For example, dry tropical montane regions Wagner , Janzen and Hallwachs may threaten the high diversity of fireflies in moist Neotropical forests. Similarly, in Australia and the western United States, where fireflies are restricted to areas with permanent water, drought may cause mortality either directly or by reducing larval food sources.
In Maryland and Delaware, Photuris salina is a habitat specialist that occupies the drier portion of coastal brackish and salt marshes, a habitat vulnerable to inundation from rising sea levels Heckscher As was noted above, the Delaware coastal species P.
In Southeast Asia, several species of Pteropytx fireflies congregate in tall, visually prominent Sonneratia caseolaris mangrove trees, a species with low salt tolerance Nada et al. This article provides a global perspective concerning threats that may cause firefly population declines and that may increase extirpation or extinction risk. Our survey results elucidate what knowledgeable respondents judged to be the most important threats to firefly species persistence, revealing differences among various geographic regions.
Although apparent that such opinions cannot be used to identify the relative importance or extent of such threats, our results are consistent with other assessments of the multifaceted causes for general declines in insect abundance and biodiversity e.
We believe this information will be useful for future studies aimed at understanding local drivers of firefly diversity and abundance. In addition, our literature review provides a comprehensive summary of the existing evidence about whether and how such threats affect firefly populations and describes risk factors likely to increase the vulnerability of certain firefly species to particular threats.
This perspective also highlights the urgent need to invest in monitoring studies that can provide long-term data to track trends in abundance and diversity for at-risk firefly species and sites. With a few notable exceptions, most evidence about firefly population trends is anecdotal, and work is needed to develop a set of standardized monitoring protocols.
In addition, experimental studies are needed to characterize acute and chronic toxicity of common insecticides on firefly life stages. We need to identify critically endangered species and establish sanctuaries that protect key firefly sites. In so doing, it is essential to consider the distinct habitat requirements of each life stage, thus ensuring suitable habitat for larvae and their prey, pupation sites, adult courtship displays, and female oviposition.
Fireflies have the potential to serve as flagship species for establishing key biodiversity areas. In Malaysia, rapid loss of riverbank mangroves and adjacent land poses an ongoing threat to several species of Pteroptyx fireflies, an economically valuable ecotourist attraction. Therefore, identifying and preserving buffer zones adjacent to the riverbank will help ensure sustainable firefly populations and also support high wildlife diversity, including other invertebrates, plants, reptiles, mammals, and birds.
To encourage successful mating by fireflies that rely on bioluminescent courtship signals, we need to minimize ALAN in and around their habitats. Ongoing studies are aimed at developing specific lighting recommendations, involving the tuning of light color wavelength and intensity, that will provide for public safety while promoting firefly reproduction. However, the diverse visual sensitivities of insects and other animals are likely to limit the effectiveness of color tuning to specific taxa.
Reducing artificial light—both its extent and its duration—should, in contrast, benefit a wide range of culturally and economically important nocturnal animals. Use of insecticides for cosmetic purposes such as on residential gardens, lawns, and public parks should be minimized. Most insecticide exposure occurs during larval stages, because firefly larvae spend months to years living in litter, belowground, or underwater.
Although the direct impacts on fireflies have been examined in few studies, commonly used insecticides have adverse effects on a broad range of nontarget organisms, including other predaceous beetles and the prey consumed by larval fireflies.
Firefly tourism is proliferating worldwide and would benefit from recommendations about best practices for establishing and managing tourist sites. Such guidelines would outline ways to protect both larval habitat and adult display sites from disturbances that include trampling, light pollution, and pesticides. We thank all of the survey respondents for their input, with additional thanks to Larry Buschman, Ben Pfeiffer, and Lynn Faust for sharing their insights in great detail and to Sarah Hoyle for information on pesticide use.
We are grateful to Pedro Cardoso and David Wagner for their detailed and thoughtful comments that greatly improved this manuscript. We are also grateful to Tufts University Department of Biology for administrative support. Sara M. Lewis sara. Owens, and J. Atkins V et al. The status of the glow-worm Lampyris noctiluca L.
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Basic and Applied Ecology 34 : — Fireflyers International Network. Finally, the potential functions of predicted ORF products were explored using these annotations as well as similarity to viral proteins of known function. To characterize Orthomyxoviridae viral diversity in P. Significant hits were retrieved and the target TSA projects further explored with the complete Orthomyxoviridae refseq collection to assess the presence of additional similar viral segments.
To identify P. FastTree accounted for variable rates of evolution across sites by assigning each site to one of 20 categories, with the rates geometrically spaced from 0. Support for individual nodes was assessed using an approximate likelihood ratio test with the Shimodaira-Hasegawa-like procedure. Tree topology, support values and substitutions per site were based on tree resamples. To facilitate taxonomic identification, we complemented BLASTP data with two levels of phylogenetic insights: i Trees based on the complete refseq collection of ssRNA - viruses which permitted a conclusive assignment at the virus family level.
PB1-based trees were complemented independently with phylogenetic studies derived from amino acids of predicted nucleoproteins, hemagglutinin protein, PB2 protein, and PA protein which supported species, genera and family demarcation based on solely on PB1, the standard in Orthomyxoviridae. Where definitive identification was not easily assessed, protein Motif signatures were determined by identification of region of high identity between divergent virus species, visualized by Sequence Logo Crooks et al.
Viral RNA levels in the transcriptome sequences were also examined. All curation, phylogeny construction, and visualization were conducted in Geneious 8. Animal silhouettes in Appendix 5—figure 2 were developed based on non-copyrighted public domain images. Figure compositions were assembled using Photoshop CS5 Adobe. Bar graphs were generated with Excel software Microsoft. RNA levels normalized as mapped transcripts per million per library were visualized using Shinyheatmap Khomtchouk et al.
Finally, to identify endogenous viral-like elements, tentative virus detections and the viral refseq collection were contrasted to the P. Then 15 Kbp genome flanking regions were retrieved and annotated. Lastly, transposable elements TEs were determined by the presence of characteristic conserved domains e. Repeat prediction for P.
To identify tandem repeats, we also ran Tandem Repeat Finder v4. The complex repeats identified by Tandem Repeat Finder were added to this classified list to create the final library of repeats. This repeat library is dubbed the P. Methylation analysis was performed using methylpy Schultz et al. In total, A sodium bisulfite non-conversion rate of 0. Libraries were subjected to identical methylation analysis as P. Weighted DNA methylation was calculated for CG sites by dividing the total number of aligned methylated reads by the total number of methylated plus un-methylated reads Schultz et al.
For genic metaplots, the gene body start to stop codon , base pairs bp upstream, and bp downstream was divided into 20 windows proportional windows based on sequence length bp. Weighted DNA methylation was calculated for each window and then plotted in R v3. We conducted FISH on squashed larval tissues according to previously published methods Larracuente and Ferree, , with some modification. Briefly, we dissected larvae in 1X PBS and treated tissues with a hypotonic solution 0.
This species is a member of the subfamily Luciolinae and had long belonged in the genus Luciola , but was recently moved to the new genus Aquatica with some other Asian aquatic fireflies Fu et al. The life cycle of A. Aquatic larva possesses a pair of outer gills on each abdominal segment and live in still or slow streams near rice paddies, wetlands and ponds. Larvae mainly feed on freshwater snails. They pupate in a mud cocoon under the soil near the water. Adults emerge in early to end of summer.
While both males and females are full-winged and can fly, there is sexual dimorphism in adult size: the body length is about 9 mm in males and 12 mm in females Ohba, Like other firefly larvae, A. Larvae possess a pair of lanterns at the dorsal margin of the abdominal segment 8. Adults are also luminescent and possess lanterns at true abdominal segments 6 and 7 in males and at segment six in females Branham and Wenzel, ; Ohba, ; Kanda, The adult is dusk active. Male adults flash yellow-green for about 1.
Female adults, located on low grass, respond to the male signal with flashes of 1—2 s in duration every 3—6 s. Males immediately approach females and copulate on the grass Ohba, ; Ohba, Like many other fireflies, A.
The contents of the eversible glands is perhaps similar to that reported for A. The geographical range of A. Kawashima et al. For example, in , Japanese Ministry of Environment began efforts to protect the population of A. This population was established from a few individuals collected from rice paddy in Kanagawa Prefecture of Japan in and Ikeya, by Mr.
Haruyoshi Ikeya, a highschool teacher in Yokohama, Japan. Ikeya collected adult A. Because of the small number of individuals used to establish the population and the number of generations of propagation, this population likely represents a partially inbred strain. Under laboratory rearing conditions, the life cycle is reduced to 7—8 months. The original habitat of this strain has been destroyed and the wild population which led to the laboratory strain is now extinct. Unlike P. We determined the genome size of A.
These tissues were chosen to avoid the ovary tissue. Three technical replicates of this sample were performed. Independent runs for extracted Aphid nuclei Acyrthosiphon pisum ; Mbp , and fruit fly nuclei Drosophila melanogaster ; Mbp were performed as calibration standards.
Genome size inference via Kmer spectral analysis estimated a genome size of Mbp Appendix 2—figure 1. Genomic DNA was extracted from the whole body of a single laboratory-reared A. Scaffolds were filtered to remove non-firefly contaminant sequences using blobtools Laetsch and Blaxter, , resulting in the final assembly Alat1. The final assembly Alat1.
Genome sequencing library statistics are available in Appendix 4—table 1. These results are consistent when considering the possible systematic error of kmer spectral analysis and flow cytometry genome size estimates. The heterozygosity is lower than that measured for P. Potential contaminants in Alat1. First, scaffolds were compared to known sequences by performing a blastn v2.
This process removed scaffolds 1. Scaffolds were taxonomically annotated as described in Appendix 2. Live specimens were anesthetized on ice and dissected during the day. The lantern tissue was dissected from the abdomen and contains the cuticle, photocyte layer and reflector layer.
The fragmentation of mRNA was performed for 4 min. The enrichment PCR was done using six cycles. Sequence quality was inspected with FastQC Andrews, A non strand-specific de novo transcriptome assembly was produced with Trinity v2. Peptides were predicted from the de novo transcripts via Transdecoder v5. De novo transcripts were then aligned to the A. A reference guided transcriptome was produced from all available A. Reads were first mapped to the A.
Then StringTie assemblies were performed on each separate bam file corresponding to the original libraries using default parameters. Finally, the produced. GTF files were merged using StringTie --merge. A protein-coding gene reference set for A. For transcripts, we combined reference guided and de novo transcriptome assembly approaches. Notably, these reference guided and de novo transcriptome assembly approaches differed from the current de novo Appendix 2.
In the reference-guided approach applied here, RNA-Seq reads were mapped to the genome assembly with TopHat and assembled into transcripts with Cufflinks parameters: --min-intron-length 30 Trapnell et al. The ORF sequences were mapped to the genome using Exonerate in est2genome mode for splice-aware alignment. We processed homology evidence at the protein level using the reference proteomes of D. These reference proteins were split-mapped to the A. These gene models derived from multiple evidence were merged by the EVM program to obtain the reference annotation for the genomes.
Lastly, gene models for luciferase homologs, Ps, and de novo methyltransferases DNMTs which were fragmented or were incorrect e. The official gene set. A de novo species-specific repeat library for A. This process yielded a final library of interspersed repeats. We then used this library and RepeatMasker v4. This repeat library is dubbed the Aquatica lateralis Official Repeat Library 1.
The Elateridae includes about 10, species Slipinski et al. These luminous species are recorded only from tropical and subtropical regions of Americas and some small Melanesian islands, such as Fiji and Vanuatu Costa, ; Costa et al.
All luminous species are closely related - luminous click beetles belong to the tribes Pyrophorini and Euplinthini Costa, ; Arias-Bohart, of the subfamily Agrypninae, with the single exception of Campyloxenus pyrothorax Chile in the related subfamily Campyloxeninae Stibick, This near-monophyly of bioluminescent elaterid taxa is supported by both morphological Douglas, and molecular phylogenetic analysis Sagegami-Oba et al.
This suggests a single origin of bioluminescence in this family. The genus Ignelater was established by Costa in and I. The genus Ignelater is characterized by the presence of both dorsal and ventral photophores Costa, ; Rosa, An unreviewed report suggested that the adult I.
Phylogenetic analyses based on the morphological characters suggested that the genera Ignelater and Photophorus which contain only two species from Fiji and Vanuatu are the most closely related genera in the tribe Pyrophorini Rosa, The exact function of bioluminescence across different life stages remains unknown for many luminous elaterid species. Bioluminescent elaterid beetles typically have two paired lanterns on the dorsal surface of the prothorax, and a single lantern on the ventral abdomen, which is only exposed during flight.
Several bioluminescent Elateridae produce different colored luminescence from their prothorax and abdominal lanterns Oba et al. Harvey reported that there was not a marked difference in the luminescence color of the dorsal and ventral lanterns of Puerto Rican I. Like fireflies, elaterid larvae often produce light, with the glowing termite mounds of Brazil that contain the predatory larvae of Pyrearinus termitilluminans being a striking example Costa and Vanin, A description of the anatomy of the larval light organ of Pyrophorus is provided by Harvey, , and a more modern photograph of the larval light organ is provided by Bechara and Stevani, Like other bioluminescent elaterid larvae, I.
Adult I. Once a female is observed, the prothorax lanterns of the male go dark, the ventral lantern becomes illuminated, and the male approaches the female via a circular search pattern. Mating is brief, reportedly taking only 5 seconds. Unlike fireflies, bioluminescent elaterid species are not known to have potent chemical defenses.
A defense role for I. Similarly, I. This geographic distribution of Ignelater suggests that Puerto Rico may contain multiple Ignelater species and, given the difficulty of distinguishing different species of bioluminescent Elateridae by morphological characters, a definitive species distribution for I. Individuals were captured at night on April 20th and April 28th during flight on the basis of light production.
Identification to species was performed by comparing antenna and dissected genitalia morphology to published keys Costa, ; Rosa, ; Rosa, Appendix 3—figure 1. Specimens collected at the same time, but not those used for genitalial dissection, were used for sequencing. Although the genitalia morphology of the sequenced specimens was not inspected to confirm their sex, sequenced specimens were inferred to be male, based on the fact that female bioluminescent elaterid beetles are rarely seen in flight Personal communication: S.
Velez and the dissected specimens collected in the same batch as the sequenced specimens were confirmed to be male. A Dorsal and B ventral view of an Ignelater luminosus aedeagus, dissected from the same batch of specimens used for linked-read sequencing and genome assembly. The species identity of this specimen was confirmed as I. The karyotype of male Puerto Rican I. The genome sizes of 5 male I. After a minimum of 30 min staining in the dark and cold, the average fluorescence channel number for the PI red fluorescence of the 2C diploid nuclei of the sample and standard were determined using a CytoFlex Flow Cytometer Beckman-Coulter.
The 1C amount of DNA in each sample was determined as the ratio of the 2C channel number of the sample and standard times Mbp. The genome size of these I. Genome size inference via Kmer spectral analysis of the I.
The resulting library was then sequenced on one HiSeqX lane. A summary of the library statistics for the genomic sequencing is available in Appendix 4—table 1. The draft genome of I. The reported mean molecule size was A Supernova v2. Manual long-read based scaffolding was then applied to produce a final assembly Ilumi1. Before analysis, 10x Chromium barcodes were trimmed off Read1 using cutadapt v1.
The heterozygosity is higher than that measured for P. The read error rate for this library is also significantly higher than the P. We sought to systematically remove assembled non-elaterid contaminant sequence from Ilumi1. A tab delimited text file containing the results of this blobtools annotation is available on FigShare DOI: This approach removed scaffolds Kbp , representing 0. While filtering the Ilumi1.
Upon closer inspection, we found conflicting information as to the most likely taxonomic source of these scaffolds. Although Hymenolepis diminuta infects mammals, it also spends a period of its life cycle in intermediate insect hosts, including beetles, as cysticercoids Center for Disease Control and Prevention, ; Sheiman et al. For a beetle like I. Scaffolds were taxonomically annotated as described in Appendix 3. Manual inspection of the initial gene-models for Ilumi1. In order to run Pilon efficiently, we split the taxonomically filtered Ilumi1.
We determined via manual gene-model annotation of Ilumi1. Reads were mapped to Ilumi1. Inspection of mapped reads with Integrative Genomics Viewer v2. Notably, this repeat unit was present the right edge of Ilumi1. Although our Nanopore sequencing did not unambiguously span this repetitive element and bridge the two scaffolds, we surmised that this information was sufficient to manually merge these scaffolds Appendix 3—figure 5. The long Ilumi1. Finally, the remaining read was reverse complemented, and concatenated to the right edge of Ilumi1.
Alignment performed in in Gepard Krumsiek et al. Diagram of the manual merge of Ilumi1. See Figure 3 in the maintext for explanation of presented genes. This library was multiplexed with the P. Briefly, poly-A mRNA was purified using oligo dT primed magnetic beads and chemically fragmented into smaller pieces. Cleaved fragments were converted to double-stranded cDNA by using N6 primers. Following this, a single strand DNA was separated at a high temperature and then a Splint oligo sequence was used as bridge for DNA cyclization to obtain the final library.
Both de novo Appendix 3. For the de novo transcriptome approach, all available I. A non-strand-specific de novo transcriptome assembly was produced with Trinity v2. De novo transcripts were then aligned to the I. A reference guided transcriptome was produced from all available I. Reads were first mapped to the I. We annotated the coding gene structure of I. Augustus predictions of Ilumi1. In the final version, eight sources of evidence were used for EVM: de novo transcriptome direct coding gene models Ilumi1.
Lastly, gene models for luciferase homologs, Ps, and de novo methyltransferases DNMTs which were fragmented or were incorrectly assembled e. The official gene set models gff3 file was then manually modified based on the observed evidence. A de novo species-specific repeat library for I.
This repeat library is dubbed the Ignelater luminosus Official Repeat Library 1. The mitochondrial genome sequence of I. Although these reads still contain the 16 bp Chromium library barcode on read 1 R1 , Bowtie2 in local mapping mode can accurately map these reads. The R1, R2, and singleton reads in. Given that increased levels of insertions after polynucleotide stretches are a known systematic error of Illumina sequencing, it was concluded that the lower coverage path represented technical error rather than an authentic genetic variant and was deleted.
This produced a single 16, bp circular contig. Small mis-annotations e. Like other reported elaterid beetle genomes, the I. The mitochondrial genome of I. The level of non-eukaryote contamination of the raw read data for each P. Overall contamination levels were low Appendix 4—table 1 , in agreement with a low level of contamination in our final assembly Appendix 1—figure 9 , Appendix 2—figure 2 , Appendix 3—figure 3.
On average, contamination was 3. There was no support for Wolbachia in any of the P. QUAST version 4. BUSCO v3. N : Number of individuals used for sequencing. Date : collection date for wild-caught individuals. Number : number of reads. Cov : Mode of autosomal coverage mode of putative X chromosome, LG3a, coverage , determined from mapped reads with QualiMap v2.
ND: Not Determined. Contamination : Percent contamination as estimated by kraken v1. See Appendix 1. Orthologs were identified by clustering the P. Not all redundant isoforms are removed as there may not have been sufficient evidence to support a particular isoform as the canonical isoform, or there were unusual annotation situations alternative splice variants annotated as separate genes.
Overlaps of number of shared orthogroups across species are shown in Appendix 4—figure 1. Overlaps on a gene-basis only P. Figure produced with InteractiVenn Heberle et al. Intermediate scripts and species specific overlaps are available as Figure 2—source data 1. For differential expression testing, Kallisto transcript expression results for P. Differential expression DE tests for P. These enzyme lists are available as supporting files associated with the official geneset filesets.
Orthogroup membership was determined from the OrthoFinder analysis Appendix 4. These custom scripts and results of the differential expression testing are available on FigShare Levels and patterns of mCG in P. Notably, P. Size of circles at nodes corresponds to bootstrap support bootstrap replicates.
Branch lengths are in amino acid substitutions per site. The multiple sequence alignment and phylogenetic topology are available on FigShare Conversely, a unimodal distribution is suggestive of a set of loci that are mostly low to un-methylated. Initial tree s for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with the best log likelihood value.
The resulting tree was rooted using D. The tree shown in Figure 6C was truncated in Dendroscope v3. The multiple sequence alignment FASTA files and newick files of the full and truncated tree are available in Figure 6—source data 1. The gene for firefly luciferase was first isolated from the North American firefly P. To date, firefly luciferase genes have been isolated from more than 30 lampyrid species in the world.
Two different types of luciferase genes, Luc1 and Luc2 , have been reported from Photuris pennsylvanica Ye et al. Luciferase genes have also been isolated from members of the other luminous beetles families: Phengodidae, Rhagophthalmidae, and Elateridae Wood et al. The chemical structures of the substrates for these enzymes are identical to firefly luciferin. These results that the bioluminescence systems of luminous beetles are essentially the same, supports a single origin of the bioluminescence in elateroid beetles.
Recent molecular analyses based on the mitochondrial genome sequences strongly support a sister relationship between the three luminous families: Lampyridae, Phengodidae, and Rhagophthalmidae Timmermans et al. However, ambiguity in the evolutionary relationships among luminous beetles, including luminous Elaterids, does not yet exclude multiple origins.
Molecular analyses have suggested that the origin of Lampyridae was dated back to late Jurassic McKenna and Farrell, or mid-Cretaceous periods Mckenna et al. Luciolinae and Lampyrinae was diverged at the basal position of the Lampyridae Martin et al. Taken together, the divergence of Luciola and Lampyridae is dated back at least Mya. A Intron-exon structure of P.
Between fireflies and click-beetles, the structure of the luciferase genes are globally similar, with seven exons, similar intron lengths, and identical splice junction locations Appendix 4—figure 5. The intron-exon structure of IlumLuc is consistent with the reported intron-exon structure of Pyrophorus plagiophthalamus luciferase Velez and Feder, Exonic sequence is capitalized, whereas intronic sequence is lowercase.
The final alignment was blocks and sequences. Finally, a maximum likelihood gene phylogeny was estimated using RAxML v8. Supporting files such as multiple sequence alignment, gene accession numbers, and other annotations are available on FigShare DOI: To more closely examine luciferase evolution, an independent maximum likelihood gene tree was constructed for luciferase co-orthologous genes defined above highlighted clade as grey in Appendix 4—figure 6 with well important genes: non-luminescent luciferase homolog from two model insect D.
Then co-orthologous genes were confirmed to be phylogenetically sister to DmelPACS CG and their evolution examined using a maximum likelihood ML gene phylogeny approach. The final alignment was blocks and 67 sequences. The tree was rooted using DmelACS as an outgroup. The peroxisomal targeting signal 1 PST1 was predicted using the regular expressions provided by the Eukaryotic Linear Motif database Dinkel et al. Supporting files such as multiple sequence alignment, gene accession numbers, and other annotation and expression values are available as Figure 3—source data 1.
Members of the clade that includes both known firefly luciferase and CG of D. Branch length represents substitutions per site. Genes found from this study are indicated in blue. Lampyridae Luc1-type and Luc2-type luciferases are highlighted in yellow-green and green.
Rhagophthalmidae and Phengodidae luciferases are highlighted in lime-green. Elateridae luciferases are highlighted in yellow. Genbank accession numbers of luciferase orthologs genes are indicated after the species name. OrthoDB taxon and protein IDs of luciferase co-orthologs are indicated after species name.
Bootstrap values are indicated on the nodes. The genes from Coleoptera are indicated as purple strip. Grey closed circles indicate genes that have PTS1. We performed an ancestral character state reconstruction of luciferase activity on the luciferase homolog gene tree within Mesquite v3. First, the gene tree from Figure 3C in Newick format was filtered using Dendroscope v3.
TcasPACS4 was set as the rooting outgroup. A gene was given the 1-state if it had been previously characterized as having luciferase activity, or was directly orthologous to a gene with previously characterized luciferase activity against firefly D-luciferin. A gene was given the 0-state if it had been previously characterized as a non-luciferase, or was directly orthologous to a gene previously characterized to not have luciferase activity towards firefly D-luciferin.
IlumLuc luciferase activity was inferred via orthology to the P. The luciferase activity of the included phengodid Viviani et al. We then reconstructed the ancestral luciferase activity character state over the tree, using an unordered parsimony model, and a maximum likelihood ML model. ML analyses were performed under the AsymmMk model with default parameters i. Root State Frequencies Same as Equilibrium. Peptide sequences for elaterid luciferase homologs descending from the putative common ancestor of firefly and elaterid luciferase as determined by a preliminary maximum likelihood molecular evolution analysis of luciferase homologs not shown , were selected from Uniprot, whereas their respective CDS sequences were selected from the European Nucleotide Archive ENA or National Center for Biotechnology Information NCBI.
The peptide and CDS sequence of the Pyrearinus termitilluminans luciferase PtermLuc were manually transcribed from the literature Viviani et al. The dorsal AF The CDS sequence of the complete I. The analysis involved 20 nucleotide sequences. There were a total of positions in the final dataset. Initial tree s for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood MCL approach, and then selecting the topology with the superior log likelihood value.
The input MSA contained 20 sequences with sites codons. All 37 branches of the gene phylogeny were formally tested for diversifying selection. The aBSREL analysis found evidence of episodic diversifying selection on 3 out of 37 branches in the phylogeny. Input files and full results are available on FigShare Subsequent Bayes Empirical Bayes BEB estimation identified 31 sites with evidence of selection on these branches, 5 of which were significant. Full results are available on FigShare Nineteen of the overall sites were shared between the MEME analysis, and are shown in Appendix 4—table 5.
The frequency of extant amino acids at these sites are shown in Appendix 4—figure 7. Proportion indicates the proportion of sites in each site class 0, 1, 2a, 2b. Site classes 0 and 1 are those in the constrained and neutral classes, respectively. Site numbering relative to IlumLuc.
Figure produced with seqkit Shen et al. This fatty acid binding protein was previously reported to bind strongly to fatty acids, and weakly to luciferin. Octopamine is known to be the key effector neurotransmitter of the adult and larval firefly lantern and this identified GPCR likely serves as the upstream receptor of octopamine activated adenylate cyclase, previously reported as abundant in P. The neurobiology of flash control, including regulation of flash pattern and intensity, is a fascinating area of behavioral research.
Our data generate new hypotheses regarding the molecular players in flash control. A particularly interesting highly and differentially expressed gene in both P. This protein is intriguing as hemocyanins are typically thought to be oxygen binding. We speculate that this octopamine binding secreted hemocyanin, previous demonstrated to be abundant, octopamine binding, and secreted from the lantern presumably into the hemolymph of the light organ , could be triggered to release oxygen upon octopamine binding, thereby providing a triggerable O 2 store within the light organ under control of neurotransmitter involved in flash control.
As O 2 is believed to be limiting in the adult light reaction, such a release of O 2 could enhance flash intensity or accelerate flash kinetics. Further research is required to test this hypothesis. Peptide sequences from P. As this is a genome-wide analysis where bootstrap replicates would be computationally prohibitive, no bootstrap replicates were performed to evaluate the support of the tree topology. Opsins are G-protein-coupled receptors that, together with a bound chromophore, form visual pigments that detect light, reviewed here Briscoe and Chittka, While opsin genes are known for their expression in photoreceptors and function in vision, they have also been found to be expressed in other tissues, suggesting non-visual functions in some cases.
Insects generally use rhabdomeric opsins r-opsins for vision, while mammals generally use ciliary opsins c-opsins for vision, products of an ancient gene duplication Briscoe and Chittka, ; Porter et al. Both insects and mammals may retain the alternate opsin type, generally in a non-visual capacity.
The ancestral insect is hypothesized to have three visual opsins - one sensitive to long-wavelengths of light LW , one to blue-wavelengths B , and one to ultraviolet light UV. Previously, two opsins, one with sequence similarity to other insect LW opsins and one with similarity to other insect UV opsins, were identified as highly expressed in firefly heads Sander and Hall, ; Martin et al. A likely non-visual c-opsin was also detected, although not highly expressed Sander and Hall, ; Martin et al.
The amino acid substitution model for ML analysis was estimated using Aminosan v1. While LW and UV opsins were highly and differentially expressed in heads of both fireflies, c-opsin was lowly expressed, in P. In contrast, Rh7 was not expressed in the P. The detection of Rh7 in our genomes is unusual in beetles Feuda et al. Rh7 has an enigmatic function - a recent study in Drosophila melanogaster showed that Rh7 is expressed in the brain, functions in circadian photoentrainment, and has broad UV-to-visible spectrum sensitivity Ni et al.
Extraocular opsin expression has been detected in other eukaryotes: a photosensory organ is located in the genitalia at the posterior abdominal segments in butterfly Lepidoptera Arikawa and Aoki, In the bioluminescent Ctenophore Mnemiopsis leidyi, three c-opsins are co-expressed with the luminous photoprotein in the photophores Schnitzler et al. In the bobtail squid, Euprymna scolopes, one of the c-opsin isoforms is expressed in the bacterial symbiotic light organ Tong et al.
Thus, it is possible that Rh7 has a photo sensory function in the lantern of A. Future study will confirm and further explore the biological, physiological, and evolutionary significance of Rh7 expression in the light organ across firefly taxa. We assayed the hemolymph of adult P. We chose to analyze extracted hemolymph from both P. For P. Specific tissues were chosen for extracts to enable a smaller quantity of tissue to go into the metabolite extraction, and to explore possible difference in compound abundance across tissues, but we expected that defense compounds like lucibufagins would be roughly equally abundant present in all tissues.
This centrifugation crushed the specimen on top of the bead, and allowed the hemolymph to collect at the bottom of the tube. For A. For extraction of P. The extract was then centrifuged 20, x g for 10 min , and filtered through a 0. For extraction of A. The extract was sonicated in a water bath sonicator for 30 min, centrifuged 20,xg for 10 min , and filtered through a 0.
For extraction of I. Injections of these filtered extracts P. Chromatographically both methods are identical up to 20 min. All other sample extracts were separated by the slow 44 min reversed-phase chromatography method, using a C18 column with a gradient of Solvent A 0. Positive mode and negative mode MS 1 and MS 2 data were obtained in a single run via polarity switching for the optimized method, or in separate runs for the slow method.
Data was collected as profile data. The instrument was always used within 7 days of the last mass accuracy calibration. Within MZmine2, data were from all five samples on positive mode, and were first cropped to 20 min in order to compare data which was obtained with the same LC gradient parameters. The five peaklists P. These aligned peaklists were then gap-filled.
We first performed a MS 2 similarity search within P. This search was performed through the MS 2 similarity search module of MZmine2 v2. Chemical formula prediction was assigned to each precursor ion using the Chemical formula search module of MZmine2, whereas chemical formula predictions for product ions was performed within MZmine2 using SIRUIS v3. The structural identity of the nine putative lucibufagins detected via the MS 2 spectra similarity search was easily interpreted in light that the different chemical formula represented the core lucibufagins that had undergone acetylation COCH 3 or propylation COCH 2 CH 3 , in different combinations.
We then performed a MS 2 similarity search within P. This MS 2 similarity search revealed 14 putative lucibufagin isomers with highly similar MS 2 spectra Appendix 4—table 7. Complexes and fragments were manually removed from the analysis.
Nodes represent MS 2 spectra from the initial dataset, whereas edges represent an MS 2 similarity match between two MS 2 spectra. MS 1 adducts and complexes of the presented ions were manually removed. To address this, we performed a MS 2 similarity search against the A. None of these features were detected in P. The notable degree of MS 2 similarity may be due to the A. That being said, the identity and role of the compound giving rise to ion The complete genome of the molicute Phylum: Tenericutes Entomoplasma luminosum subsp.
A single 1. Inspection of the Canu produced assembly graph with Bandage v0. Together this data suggested that this contig represented a complete Mycoplasmal genome. The resulting consensus sequence was restarted with seqkit Shen et al. This mapping, consensus calling, and rotation process was repeated three times total, after which no additional nucleotide changes occurred.
Protein overlap comparisons using the OrthoFinder pipeline v1. The next most abundant category at 1. The next most abundant category at 0. The reads were then split into three partitions: P. We inspected the M. Notably, although an inspection of the contig was circular, and showed a high degree of similarity upon blastn to the M.
Similarly, the. GFA output of Canu noted an overlap of 29,, indicating that the assembler was unable to determine an appropriate overlap, other than the entire contig. This mtDNA was taxonomically identified in a separate analysis to originate from A. Tandem repetitive regions were manually annotated. The complete A. The mitochondrial genome of A.
After the successful metagenomic assembly of the mitochondrial genome of an unknown Phorid fly species from the P. We planned to achieve this by collecting the Phorid flies which emerged from adult P. We successfully obtained phorid fly larvae emerging from P. Two adults from this batch were identified as A.
We conclude that this is sufficient evidence to denote that our assembled Phorid mitochondrial genome is the mitochondrial genome of A. Notably, A. To our knowledge, this is the first report of a mitochondrial genome which was first assembled via an untargeted metagenomic approach and then later correlated to its species of origin. We identified the first two viruses associated to P. Genomic and phylogenetic studies suggest that the detected viruses correspond to a new lineage within the Orthomyxoviridae family ssRNA - Appendix 5—figure 2A-I.
The concomitant occurrence in the P. This tentative interface is correlated to low viral RNA levels, persistence and no apparent phenotypes associated with infection. We suggest that the identified viruses are potential endophytes of high prevalence as a result of potential evolutionary modulation of viral levels associated to EVEs. They are multipartite linear ssRNA negative strand viruses, conformed by five genome segments generating a ca.
Genome segments one through three ca. Genome segment four 1. In addition, HA as expected, presents an N-terminal signal domain, a C terminal transmembrane domain, and a putative glycosylation site. Lastly, genome segment five ca. Despite sharing genome architecture and structural and functional domains of their predicted proteins, PpyrOMLV1 and PpyrOMLV2 pairwise identity of ortholog gene products range between These primary and secondary sequence cues are associated to polymerase binding and promotion of both replication and transcription.
Given that the presence of those additional segments varies among diverse OMV genera, and that 35 related tentative new virus species identified in TSA did not present any additional segments, we believe that these lineages of viruses are conformed by five genome segments. Further experiments based on specific virus particle purification and target sequencing could corroborate our results.
These are the first viruses that have been associated with the Lampyridae beetle family, which includes over species. The OMV virus members share diverse structural, functional and biological characters that define and restrict the family. OMV virions are 80— nm in diameter, of spherical or pleomorphic morphology. The virion envelope is derived from the host cell membrane, incorporating virus glycoproteins and eventually non-glycosylated proteins one or two in number.
Typical virion surface glycoprotein projections are 10—14 nm in length and 4—6 nm in diameter. The virus genome is multisegmented, has a helical-like symmetry, consisting of different size ribonucleoproteins RNP , 50— nm in length. Influenza RNPs can perform either replication or transcription of the same template. Virions of each genus contain different numbers of linear ssRNA - genome segments King et al.
Johnston Atoll virus JAV genome is still incomplete, and only two segments have been described. Segment lengths range from to nt. Genome size ranges from In addition, a non-glycosylated matrix protein M is present in most species. There are some species-specific divergence in some structural OMVs proteins. The HA protein of FLUCV, besides its hemagglutinating and envelope fusion function, has an esterase activity that induces host receptor enzymatic destruction King et al.
For instance, human infecting Influenza viruses selectively bind to glycolipids that contain terminal sialyl-galactosyl residues with a 2—6 linkage, in contrast, avian influenza viruses bind to sialyl-galactosyl residues with a 2—3 linkage King et al.
As an illustration, M2 and BM2 function during un-coating and fusion by equilibrating the intralumenal pH of the trans-Golgi apparatus and the cytoplasm. OMV share replication properties, which have been studied mostly in Influenza viruses. It is important to note that gene reassortment has been described to occur during mixed OMV infections, involving viruses of the same genus, but not between viruses of different genera Kimble, This is used also as a criteria for OMV genus demarcation.
These remarkable dynamic multifunction characters of OMV polymerases are associated with its complex tertiary structure, of this modular heterotrimeric replicase Te Velthuis and Fodor, We explored in detail the putative polymerase subunits of the identified firefly viruses. The generated structure shared structural cues related to its multiple role of RNA nucleotide binding, endonuclease, cap binding, and nucleotidyl transferase Appendix 5—figure 2G-H.
The engendered subunit structures suggest a probable conservation of PpyrOMLV1 POL, that could allow the predicted functional enzymatic activity of this multiple gene product. The overall polymerase rendered structure presents a typical U shape with two upper protrusions corresponding to the PA endonuclease and the PB2 cap-binding domain. The PB1 subunit appears to plug into the interior of the U and has the distinctive fold of related viral RNA polymerases with fingers, palm and thumb adjacent to a tentative central active site opening where RNA synthesis may occur Reich et al.
For Influenza, sialic acid bound to glycoproteins or glycolipids function as receptor determinants of endocytosis. Fusion between viral and cell membranes occurs in endosomes. The infectivity and fusion of influenza is associated to the post-translational cleavage of the virion HA. Cleavability depends on the number of basic amino acids at the target cleavage site King et al.
In thogotoviruses, no requirement for HA glycoprotein cleavage have been demonstrated Leahy et al. Integral membrane proteins migrate through the Golgi apparatus to localized regions of the plasma membrane. New virions form by budding, incorporating matrix proteins and viral RNPs. Another tentative function of the NP could be associated to the potential interference of the host immune response in the nucleus mediated by capsid proteins of some RNA virus, which could inhibit host transcription and thus liberate and direct it to viral RNA synthesis Wulan et al.
Protein synthesis of influenza viruses occurs in the cytoplasm. Partially complementary vRNA molecules act as templates for new viral RNA synthesis and are neither capped nor polyadenylated. Given the diverse hosts of OMV, biological properties of virus infection diverge between species. Influenzaviruses A infect humans and cause respiratory disease, and they have been found to infect a variety of bird species and some mammalian species.
Interspecies transmission, although rare, is well documented. Influenza B virus infect humans and cause epidemics, and have been rarely found in seals. Influenzaviruses C cause limited outbreaks in humans and have been occasionally found on dogs. Influenza spreads globaly in a yearly outbreak, resulting in about three to five million cases of severe illness and about , to , human deaths Thompson et al.
Influenzavirus D has been recently reported and accepted and infects cows and swine Hause et al. Natural transmission of influenzaviruses is by aerosol human and non-aquatic hosts or is water-borne avians. In contrast, Thogoto and Dhori viruses which also infect humans, are transmitted by, and able to replicate in ticks. Thogoto virus was identified in Rhipicephalus sp.
Dhori virus infection in humans causes a febrile illness and encephalitis. Serological evidence suggests that cattle, camel, goats, and ducks might be also susceptible to this virus. Experimental hamster infection with THOV may be lethal. Unlike influenzaviruses, these viruses do not cause respiratory disease. The transmission of fish infecting isaviruses ISAV is via water, and virus infection induces the agglutination of erythrocytes of many fish species, but not avian or mammalian erythrocytes Mjaaland et al.
Quaranfil and Johnston Atoll are transmitted by ticks and infect avian species Presti et al. We have limited biological data of the firefly detected viruses. Besides relying on the OMV structural and functional signatures determined by virus genome annotation, we explored the evolutionary clustering of the detected viruses by phylogenetic insights. We generated MAFFT alignments and phylogenetic trees of the predicted viral polymerase of firefly viruses and the corresponding replicases of all proposed and accepted species of ssRNA - virus.
The generated trees consistently clustered the diverse sequences to their corresponding taxonomical niche, at the level of genera. Furthermore, it appears that virus genomic sequence data, while it has been paramount to separate species, in the case of genera, there are some contrasting data that should be taken into consideration.
It is worth noting that similarity thresholds and phylogenetic clustering based in genomic data have been used differently to demarcate OMV genera, hence there is a need to eventually re-evaluate a series of consensus values, which in addition to biological data, would be useful to redefine the OMV family. Perhaps, these criteria discrepancies are more related to a historical evolution of the OMV taxonomy than to pure biological or genetic standards.
As RNA was isolated from independent P. Wild caught individuals were collected in period spanning six years, and locations separated as much as miles New Jersey — Georgia, USA. Interestingly PpyrOMLV1 and 2 were found in individuals of both location, and the corresponding assembled isolate virus sequences presented negligible differences, with an inter-individual variability equivalent to that of isolates 0.
A similar result was observed for virus sequences identified in RNA libraries generated from samples collected in different years. We were not able to identified fixed mutations associated to geographical or chronological cues.
Further experiments should explore the mutational landscape of PpyrOMLV1 and 2, which appears to be significantly lower than of Influenzaviruses, specifically Influenza A virus, which are characterized by high mutational rate ca. Overall virus RNA levels were generally low, with an average of 9. When the expression levels are scrutinized by genome segment, HA and NP encoding segments appear to be, for both viruses, at higher levels, which would be in agreement with other OMV such as Influenzaviruses, in which HA and NP proteins are the most expressed proteins, and thus viral mRNAs are consistently more expressed King et al.
Nevertheless, these preliminary findings related to expression levels should be taken cautiously, given the small sample size. Perhaps, the more remarkable allusion derived from the analyses of virus presence is related to tissue and organ deduced virus tropism. Strikingly, we found virus transcripts in samples exclusively obtained from light organs, complete heads, male or female thorax, female spermatheca, female spermatophore digesting glands and bursa, abdominal fat bodies, male reproductive spiral gland, and other male reproductive accessory glands Appendix 5—table 3 , S5.
For instance, influenza viruses present a epithelial cell-specific tropism, restricted typically to the nose, throat, and lungs of mammals, and intestines of birds. Tropism has consequences on host restriction. Human influenza viruses mainly infect ciliated cells, because attachment of all influenza A virus strains to cells requires sialic acids. Differential expression of sialic acid residues in diverse tissues may prevent cross-species or zoonotic transmission events of avian influenza strains to man Zeng et al.
Tropism has also influence in disease associated effects of OMV. Some influenza A virus strains are more present in tracheal and bronchial tissue which is associated with the primary lesion of tracheobronchitis observed in typical epidemic influenza. Other influenza A virus strains are more prevalent in type II pneumocytes and alveolar macrophages in the lower respiratory tract, which is correlated to diffuse alveolar damage with avian influenza Mansfield, The presence of PpyrOMLV1 and 2 virus RNA in reproductive glands raises some potential of the involvement of sex in terms of prospective horizontal transmission.
Interestingly, we did not observe in any of the 24 virus positive samples evidence of reassortment. Reassortment is a common event in OMV, a process by which influenza viruses swap gene segments. Genetic exchange is possible due to the segmented nature of the OMV viral genome and may occur during mixed infections. Reassortment generates viral diversity and has been associated to host gain of Influenzavirus Steel and Lowen, Reassorted Influenzavirus have been reported to occasionally cross the species barrier, into birds and some mammalian species like swine and eventually humans.
These infections are usually dead ends, but sporadically, a stable lineage becomes established and may spread in an animal population Kimble, In light of the presence of virus RNA in reproductive glands, we further explored the potential life style of PpyrOMLV1 and 2 related to eventual vertical transmission.
Vertical transmission is extremely exceptional for OMV, and has only been conclusively described for the Infectious salmon anemia virus Isavirus Marshall et al. Moreover, virus RNA levels fluctuated among the different developmental stages of the samples. The average RNA levels of the adult female were Interestingly, virus RNA levels appear to drop in first instar larvae, in the sequenced library average virus RNA levels were of Future experiments should focus on PpyrOMLV1 and 2 virus titers at extended developmental stages to complement these preliminary results.
However, it is interesting to note that the tissue specific library corresponding to female spermatheca, where male sperm are stored prior to fertilization, presented relatively high levels of both PpyrOMLV1 and 2 virus RNAs, suggesting that perhaps during early reproductive process and during egg development virus RNAs tend to raise.
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GOP lawmakers invited to WH today Greater Boston: Vindman testifies about Trump's Zelensky call and Volker revises his testimony Group of former intelligence community watchdogs call for protecting whistleblowers Heginbotham speaks about war games at inaugural Applied IR Speaker Series Hot new Japan book releases for the sweltering summer How North Korea got away with the assassination of Kim Jong-nam How Pakistan is playing Washington—again Impeachment spotlights vulnerability of acting federal watchdogs Increasing threats against mobile devices force HHS, others to rethink protections India claims successful test of anti-satellite weapon India says committed to 'no first use' of nuclear weapons for now India slips further behind China during first five years of Modi India weighs military options against Pakistan as Kashmir tensions rise India, Pakistan exchange fire India—Pakistan nuclear escalation: where could it lead?
Is the US about to lower the bar for North Korea denuclearisation? This perspective also highlights the urgent need to invest in monitoring studies that can provide long-term data to track trends in abundance and diversity for at-risk firefly species and sites. With a few notable exceptions, most evidence about firefly population trends is anecdotal, and work is needed to develop a set of standardized monitoring protocols. In addition, experimental studies are needed to characterize acute and chronic toxicity of common insecticides on firefly life stages.
We need to identify critically endangered species and establish sanctuaries that protect key firefly sites. In so doing, it is essential to consider the distinct habitat requirements of each life stage, thus ensuring suitable habitat for larvae and their prey, pupation sites, adult courtship displays, and female oviposition. Fireflies have the potential to serve as flagship species for establishing key biodiversity areas.
In Malaysia, rapid loss of riverbank mangroves and adjacent land poses an ongoing threat to several species of Pteroptyx fireflies, an economically valuable ecotourist attraction. Therefore, identifying and preserving buffer zones adjacent to the riverbank will help ensure sustainable firefly populations and also support high wildlife diversity, including other invertebrates, plants, reptiles, mammals, and birds. To encourage successful mating by fireflies that rely on bioluminescent courtship signals, we need to minimize ALAN in and around their habitats.
Ongoing studies are aimed at developing specific lighting recommendations, involving the tuning of light color wavelength and intensity, that will provide for public safety while promoting firefly reproduction. However, the diverse visual sensitivities of insects and other animals are likely to limit the effectiveness of color tuning to specific taxa. Reducing artificial light—both its extent and its duration—should, in contrast, benefit a wide range of culturally and economically important nocturnal animals.
Use of insecticides for cosmetic purposes such as on residential gardens, lawns, and public parks should be minimized. Most insecticide exposure occurs during larval stages, because firefly larvae spend months to years living in litter, belowground, or underwater. Although the direct impacts on fireflies have been examined in few studies, commonly used insecticides have adverse effects on a broad range of nontarget organisms, including other predaceous beetles and the prey consumed by larval fireflies.
Firefly tourism is proliferating worldwide and would benefit from recommendations about best practices for establishing and managing tourist sites. Such guidelines would outline ways to protect both larval habitat and adult display sites from disturbances that include trampling, light pollution, and pesticides.
We thank all of the survey respondents for their input, with additional thanks to Larry Buschman, Ben Pfeiffer, and Lynn Faust for sharing their insights in great detail and to Sarah Hoyle for information on pesticide use. We are grateful to Pedro Cardoso and David Wagner for their detailed and thoughtful comments that greatly improved this manuscript.
We are also grateful to Tufts University Department of Biology for administrative support. Sara M. Lewis sara. Owens, and J. Atkins V et al. The status of the glow-worm Lampyris noctiluca L. Coleoptera: Lampyridae in England. Lampyrid 4 : 20 — Google Scholar. Modeling effects of harvest on firefly population persistence. Ecological Modelling : 43 — Bek RJ. Bachelor of science thesis, University of Leeds. Google Preview.
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Environmental Science and Technology 49 : — Adult firefly abundance is linked to weather during the larval stage in the previous year. Ecological Entomology 44 : — The new world atlas of artificial night sky brightness. Science Advances 2 : e Faust L. Pages — in Napompeth B , ed. Queen Sirikit Botanic Garden. University of Georgia Press. Firebaugh A , Haynes KJ.
Experimental tests of light-pollution impacts on nocturnal insect courtship and dispersal. Oecologia : — Light pollution may create demographic traps for nocturnal insects. Basic and Applied Ecology 34 : — Fireflyers International Network.
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Encyclopedia of Entomology. Molecular variation across populations of a widespread North American firefly, Photinus pyralis, reveals that coding changes do not underlie flash color variation or associate visual sensitivity. BMC Evolutionary Biology 18 : Conservation efforts for the synchronous fireflies of the Selangor River in Malaysia. Napompeth B , ed. Effects of artificial light on the larvae of the firefly Lamprigera sp. Journal of Asia-Pacific Entomology 23 : 82 — The terrestrial bioluminescent animals of Japan.
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It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Sign In. Advanced Search. Search Menu. Skip Nav Destination Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract.
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Model of a Firefly Swarm — Wolfram Alpha : This very cool demonstration of swarm modeling math, it is possible to make a somewhat unrealistic model of firefly behavior with the following equations. Tufts University : Scientific resource on fireflies, covering Nitric Oxide and the bioscience behind firefly flashes.
How to Photograph Fireflies : Informative how-to guide by Terry Priest on how to photograph fireflies Photinus pyralis in flight. A very interesting video of a firefly lampyrid beetle larva glowing at night in Khao Yai National Park, Thailand. Note the brushes on the prolegs. A rather entertaining Peugeot car commercial with fireflies.
Close-up video of Firefly flashing. Fireflies appear in everything from song lyrics such as Owl City — Fireflies song, car commercials, or movies such as Avatar. At least one chemical company pays youngsters to supply fireflies that they catch and mail to the company's headquarters, using cans containing a drying agent. But for some work, Professor Branchini said, he needs what are called ''freshly sacrificed'' fireflies, which must be kept in special laboratory freezers. Also, he needs the protein, or enzyme, not only from Photinus pyralis, the kind of firefly commonly caught and supplied, but also from Photinus photuris, a kind that is very difficult to capture it flies high and fast and is not supplied commercially.
Thus, Professor Branchini and his students often must head out with their nets. Sometimes, he said, they go to remote areas where there are few onlookers. But one year he took his University of Wisconsin students to a state park in Tennessee where, he said, ''We had to explain ourselves.
He noted that he got information on good spots in which to find needed fireflies from various local people such as high school science teachers. Last fall, he traveled to Brownsville, Tex. The firefly season in Connecticut is just getting under way, he noted, displaying a jar with about 10 fireflies thoughtfully given to him by the child of a colleague.
The summer research work is also getting under way in a new addition to Hale Laboratory, which houses the chemistry department at Connecticut College. Recently moved in are computers and such laboratory equipment as luminescence photometers which will be used for the summer study of bioluminescence by Professor Branchini and the three participating students, Sarah Bamford of the University of Connecticut and Matthew Hayward and Anita McNeill of Connecticut College.
Professor Branchini said that the highly efficient firefly bioluminescense system is a perfect topic for the undergraduate research. That is partly because, he said, ''the basic science of what's involved is interesting. That's one of the most important aspects. Sometimes that gives people the spark they need to continue in science. On a recent day Miss Bamford, who will be working with a computerized system of light measurements, and Mr.
Hayward, who will be synthesizing analogues of luciferin, spent most of the afternoon hauling supplies from a basement room known as ''the black hole'' into the new laboratory. Miss Bamford spoke of her plans to work with a related project at UConn, and Mr. Hayward noted that his work this summer will probably become part of his honor's thesis during the coming school year.
He also noted that he'd spent the previous evening unsuccessfully searching for the needed Photinus photuris in the college arboretum.