Illuminating patterns of firefly abundance using citizen science data and machine learning models.

As insect populations decline in many regions, conservation biologists are increasingly tasked with identifying factors that threaten insect species and developing effective strategies for their conservation. One insect group of global conservation concern are fireflies (Coleoptera: Lampyridae). Although quantitative data on firefly populations are lacking for most species, anecdotal reports suggest that some firefly populations have declined in recent decades. Researchers have hypothesized that North American firefly populations are most threatened by habitat loss, pesticide use, and light pollution, but the importance of these factors in shaping firefly populations has not been rigorously examined at broad spatial scales. Using data from >24,000 surveys (spanning 2008-16) from the citizen science program Firefly Watch, we trained machine learning models to evaluate the relative importance of a variety of factors on bioluminescent firefly populations: pesticides, artificial lights at night, land cover, soil/topography, short-term weather, and long-term climate. Our analyses revealed that firefly abundance was driven by complex interactions among soil conditions (e.g., percent sand composition), climate/weather (e.g., growing degree days), and land cover characteristics (e.g., percent agriculture and impervious cover). Given the significant impact that climactic and weather conditions have on firefly abundance, there is a strong likelihood that firefly populations will be influenced by climate change, with some regions becoming higher quality and supporting larger firefly populations, and others potentially losing populations altogether. Collectively, our results support hypotheses related to factors threatening firefly populations, especially habitat loss, and suggest that climate change may pose a greater threat than appreciated in previous assessments. Thus, future conservation of North American firefly populations will depend upon 1) consistent and continued monitoring of populations via programs like Firefly Watch, 2) efforts to mitigate the impacts of climate change, and 3) insect-friendly conservation practices.

Identification of a Female-Produced Sex Attractant Pheromone of the Winter Firefly, Photinus corruscus Linnaeus (Coleoptera: Lampyridae).

Firefly flashes are well-known visual signals used by these insects to find, identify, and choose mates. However, many firefly species have lost the ability to produce light as adults. These "unlighted" species generally lack developed adult light organs, are diurnal rather than nocturnal, and are believed to use volatile pheromones acting over a distance to locate mates. While cuticular hydrocarbons, which may function in mate recognition at close range, have been examined for a handful of the over 2000 extant firefly species, no volatile pheromone has ever been identified. In this study, using coupled gas chromatography - electroantennographic detection, we detected a single female-emitted compound that elicited antennal responses from wild-caught male winter fireflies, Photinus corruscus. The compound was identified as (1S)-exo-3-hydroxycamphor (hydroxycamphor). In field trials at two sites across the species' eastern North American range, large numbers of male P. corruscus were attracted to synthesized hydroxycamphor, verifying its function as a volatile sex attractant pheromone. Males spent more time in contact with lures treated with synthesized hydroxycamphor than those treated with solvent only in laboratory two-choice assays. Further, using single sensillum recordings, we characterized a pheromone-sensitive odorant receptor neuron in a specific olfactory sensillum on male P. corruscus antennae and demonstrated its sensitivity to hydroxycamphor. Thus, this study has identified the first volatile pheromone and its corresponding sensory neuron for any firefly species, and provides a tool for monitoring P. corruscus populations for conservation and further inquiry into the chemical and cellular bases for sexual communication among fireflies.

Inferring the demographic history of the North American firefly Photinus pyralis.

The firefly Photinus pyralis inhabits a wide range of latitudinal and ecological niches, with populations living from temperate to tropical habitats. Despite its broad distribution, its demographic history is unknown. In this study, we modelled and inferred different demographic scenarios for North American populations of P. pyralis, which were collected from Texas to New Jersey. We used a combination of ABC techniques (for multi-population/colonization analyses) and likelihood inference (dadi, StairwayPlot2, PoMo) for single-population demographic inference, which proved useful with our RAD data. We uncovered that the most ancestral North American population lays in Texas, which further colonized the Central region of the US and more recently the North Eastern coast. Our study confidently rejects a demographic scenario where the North Eastern populations colonized more southern populations until reaching Texas. To estimate the age of divergence between of P. pyralis, which provides deeper insights into the history of the entire species, we assembled a multi-locus phylogenetic data covering the genus Photinus. We uncovered that the phylogenetic node leading to P. pyralis lies at the end of the Miocene. Importantly, modelling the demographic history of North American P. pyralis serves as a null model of nucleotide diversity patterns in a widespread native insect species, which will serve in future studies for the detection of adaptation events in this firefly species, as well as a comparison for future studies of other North American insect taxa.

Toxicity of clothianidin to common Eastern North American fireflies.

BACKGROUND: Previous research suggests that fireflies (Coleoptera: Lampyridae) are susceptible to commonly used insecticides. In the United States, there has been a rapid and widespread adoption of neonicotinoid insecticides, predominantly used as seed coatings on large-acreage crops like corn, soy, and cotton. Neonicotinoid insecticides are persistent in soil yet mobile in water, so they have potential to contaminate firefly habitats both in and adjacent to application sites. As a result, fireflies may be at high risk of exposure to neonicotinoids, possibly jeopardizing this already at-risk group of charismatic insects. METHODS: To assess the sensitivity of fireflies to neonicotinoids, we exposed larvae of Photuris versicolor complex and Photinus pyralis to multiple levels of clothianidin-treated soil and monitored feeding behavior, protective soil chamber formation, intoxication, and mortality. RESULTS: Pt. versicolor and Pn. pyralis larvae exhibited long-term intoxication and mortality at concentrations above 1,000 ng g-1 soil (1 ppm). Under sub-lethal clothianidin exposure, firefly larvae fed less and spent less time in protective soil chambers, two behavioral changes that could decrease larval survival in the wild. DISCUSSION: Both firefly species demonstrated sub-lethal responses in the lab to clothianidin exposure at field-realistic concentrations, although Pt. versicolor and Pn. pyralis appeared to tolerate higher clothianidin exposure relative to other soil invertebrates and beetle species. While these two firefly species, which are relatively widespread in North America, appear somewhat tolerant of neonicotinoid exposure in a laboratory setting, further work is needed to extend this conclusion to wild populations, especially in rare or declining taxa.

Molecular Evolution of Phototransduction Pathway Genes in Nocturnal and Diurnal Fireflies (Coleoptera: Lampyridae).

Most organisms are dependent on sensory cues from their environment for survival and reproduction. Fireflies (Coleoptera: Lampyridae) represent an ideal system for studying sensory niche adaptation due to many species relying on bioluminescent communication; as well as a diversity of ecologies. Here; using transcriptomics; we examine the phototransduction pathway in this non-model organism; and provide some of the first evidence for positive selection in the phototransduction pathway beyond opsins in beetles. Evidence for gene duplications within Lampyridae are found in inactivation no afterpotential C and inactivation no afterpotential D. We also find strong support for positive selection in arrestin-2; inactivation no afterpotential D; and transient receptor potential-like; with weak support for positive selection in guanine nucleotide-binding protein G(q) subunit alpha and neither inactivation nor afterpotential C. Taken with other recent work in flies; butterflies; and moths; this represents an exciting new avenue of study as we seek to further understand diversification and constraint on the phototransduction pathway in light of organism ecology.

Comparative Transcriptomics Reveals Gene Families Associated with Predatory Behavior in Photuris femme fatale Fireflies.

Identifying the basis of phenotypic variation is a key objective of genetics. This work has been mostly limited to model systems with a plethora of genetic manipulation and functional characterization tools. With the development of high-throughput sequencing and new computational tools, it is possible to identify candidate genes related to phenotypic variation in non-model organisms. Fireflies are excellent for studying phenotypic variation because of their diverse and well-characterized behaviors. Most adult fireflies emit a single mating flash pattern and do not eat. In contrast, adult females of many species in the genus Photuris employ multiple flash patterns and prey upon mate-seeking males of other firefly species. To investigate the genetic basis for this variation, we used comparative transcriptomics to identify positively selected genes between a predatory firefly, Photuris sp., and a non-predatory relative, Photuris frontalis, controlling for genes generally under selection in fireflies by comparing to a Photinus firefly. Nine gene families were identified under positive selection in the predatory versus non-predatory Photuris comparison, including genes involved in digestion, detoxification, vision, reproduction, and neural processes. These results generate intriguing hypotheses about the genetic basis for insect behavior and highlight the utility of comparative transcriptomic tools to investigate complex behaviors in non-model systems.

Special Issue: Repetitive DNA Sequences.

Repetitive DNAs are ubiquitous in eukaryotic genomes and, in many species, comprise the bulk of the genome. Repeats include transposable elements that can self-mobilize and disperse around the genome and tandemly-repeated satellite DNAs that increase in copy number due to replication slippage and unequal crossing over. Despite their abundance, repetitive DNAs are often ignored in genomic studies due to technical challenges in identifying, assembling, and quantifying them. New technologies and methods are now allowing unprecedented power to analyze repetitive DNAs across diverse taxa. Repetitive DNAs are of particular interest because they can represent distinct modes of genome evolution. Some repetitive DNAs form essential genome structures, such as telomeres and centromeres, that are required for proper chromosome maintenance and segregation, while others form piRNA clusters that regulate transposable elements; thus, these elements are expected to evolve under purifying selection. In contrast, other repeats evolve selfishly and cause genetic conflicts with their host species that drive adaptive evolution of host defense systems. However, the majority of repeats likely accumulate in eukaryotes in the absence of selection due to mechanisms of transposition and unequal crossing over. However, even these "neutral" repeats may indirectly influence genome evolution as they reach high abundance. In this Special Issue, the contributing authors explore these questions from a range of perspectives.

Firefly genomes illuminate parallel origins of bioluminescence in beetles.

Fireflies and their luminous courtships have inspired centuries of scientific study. Today firefly luciferase is widely used in biotechnology, but the evolutionary origin of bioluminescence within beetles remains unclear. To shed light on this long-standing question, we sequenced the genomes of two firefly species that diverged over 100 million-years-ago: the North American Photinus pyralis and Japanese Aquatica lateralis. To compare bioluminescent origins, we also sequenced the genome of a related click beetle, the Caribbean Ignelater luminosus, with bioluminescent biochemistry near-identical to fireflies, but anatomically unique light organs, suggesting the intriguing hypothesis of parallel gains of bioluminescence. Our analyses support independent gains of bioluminescence in fireflies and click beetles, and provide new insights into the genes, chemical defenses, and symbionts that evolved alongside their luminous lifestyle.

Molecular variation across populations of a widespread North American firefly, Photinus pyralis, reveals that coding changes do not underlie flash color variation or associated visual sensitivity.

BACKGROUND: Genes underlying signal production and reception are expected to evolve to maximize signal detection in specific environments. Fireflies vary in their light signal color both within and between species, and thus provide an excellent system in which to study signal production and reception in the context of signaling environments. Differences in signal color have been hypothesized to be due to variation in the sequence of luciferase, the enzyme that catalyzes the light reaction. Similarly, differences in visual sensitivity, which are expected to match signal color, have been hypothesized to be due to variation in the sequence of opsins, the protein component of visual pigments. Here we investigated (1) whether sequence variation in luciferase correlates with variation in signal color and (2) whether sequence variation in opsins correlates with inferred matching visual sensitivity across populations of a widespread North American firefly species, Photinus pyralis. We further tested (3) whether selection has acted on these loci by examining their population-level differentiation relative to the distribution of differentiation derived from a genome-wide sample of loci generated by double-digest RADseq. RESULTS: We found virtually no coding variation in luciferase or opsins. However, there was extreme divergence in non-coding variation in luciferase across populations relative to a panel of random genomic loci. CONCLUSIONS: The absence of protein variation at both loci challenges the paradigm that variation in signal color and visual sensitivity in fireflies is exclusively due to coding variation in luciferase and opsin genes. Instead, flash color variation within species must involve other mechanisms, such as abdominal pigmentation or regulation of light organ physiology. Evidence for selection at non-coding variation in luciferase suggests that selection is targeting luciferase regulation and may favor differ expression levels across populations.

Rates and Patterns of Mutation in Tandem Repetitive DNA in Six Independent Lineages of Chlamydomonas reinhardtii.

The mutational patterns of large tandem arrays of short sequence repeats remain largely unknown, despite observations of their high levels of variation in sequence and genomic abundance within and between species. Many factors can influence the dynamics of tandem repeat evolution; however, their evolution has only been examined over a limited phylogenetic sample of taxa. Here, we use publicly available whole-genome sequencing data of 85 haploid mutation accumulation lines derived from six geographically diverse Chlamydomonas reinhardtii isolates to investigate genome-wide mutation rates and patterns in tandem repeats in this species. We find that tandem repeat composition differs among ancestral strains, both in genome-wide abundance and presence/absence of individual repeats. Estimated mutation rates (repeat copy number expansion and contraction) were high, averaging 4.3×10-4 per generation per single unit copy. Although orders of magnitude higher than other types of mutation previously reported in C. reinhardtii, these tandem repeat mutation rates were one order of magnitude lower than what has recently been found in Daphnia pulex, even after correcting for lower overall genome-wide satellite abundance in C. reinhardtii. Most high-abundance repeats were related to others by a single mutational step. Correlations of repeat copy number changes within genomes revealed clusters of closely related repeats that were strongly correlated positively or negatively, and similar patterns of correlation arose independently in two different mutation accumulation experiments. Together, these results paint a dynamic picture of tandem repeat evolution in this unicellular alga.

The evolution of sexual signal modes and associated sensor morphology in fireflies (Lampyridae, Coleoptera).

Animals employ different sexual signal modes (e.g. visual, acoustic, chemical) in different environments and behavioural contexts. If sensory structures are costly, then evolutionary shifts in primary signal mode should be associated with changes in sensor morphology. Further, sex differences are expected if male and female signalling behaviours differ. Fireflies are known for their light displays, but many species communicate exclusively with pheromones, including species that recently lost their light signals. We performed phylogenetically controlled analyses of male eye and antenna size in 46 North American taxa, and found that light signals are associated with larger eyes and shorter antennae. In addition, following a transition from nocturnal light displays to diurnal pheromones, eye size reductions occur more rapidly than antenna size increases. In agreement with the North American taxa, across 101 worldwide firefly taxa in 32 genera, we found light displays are associated with larger eye and smaller antenna sizes in both males and females. For those taxa with both male and female data, we found sex differences in eye size and, for diurnal species, in antenna size.

Variable Rates of Simple Satellite Gains across the Drosophila Phylogeny.

Simple satellites are tandemly repeating short DNA motifs that can span megabases in eukaryotic genomes. Because they can cause genomic instability through nonallelic homologous exchange, they are primarily found in the repressive heterochromatin near centromeres and telomeres where recombination is minimal, and on the Y chromosome, where they accumulate as the chromosome degenerates. Interestingly, the types and abundances of simple satellites often vary dramatically between closely related species, suggesting that they turn over rapidly. However, limited sampling has prevented detailed understanding of their evolutionary dynamics. Here, we characterize simple satellites from whole-genome sequences generated from males and females of nine Drosophila species, spanning 40 Ma of evolution. We show that PCR-free library preparation and postsequencing GC-correction better capture satellite quantities than conventional methods. We find that over half of the 207 simple satellites identified are species-specific, consistent with previous descriptions of their rapid evolution. Based on a maximum parsimony framework, we determined that most interspecific differences are due to lineage-specific gains. Simple satellites gained within a species are typically a single mutation away from abundant existing satellites, suggesting that they likely emerge from existing satellites, especially in the genomes of satellite-rich species. Interestingly, unlike most of the other lineages which experience various degrees of gains, the lineage leading up to the satellite-poor D. pseudoobscura and D. persimilis appears to be recalcitrant to gains, providing a counterpoint to the notion that simple satellites are universally rapidly evolving.