TiLIA: a software package for image analysis of firefly flash patterns.

As flash signaling patterns of fireflies are species specific, signal-pattern analysis is important for understanding this system of communication. Here, we present time-lapse image analysis (TiLIA), a free open-source software package for signal and flight pattern analyses of fireflies that uses video-recorded image data. TiLIA enables flight path tracing of individual fireflies and provides frame-by-frame coordinates and light intensity data. As an example of TiLIA capabilities, we demonstrate flash pattern analysis of the fireflies Luciola cruciata and L. lateralis during courtship behavior.

Expression of the nos gene and firefly flashing: a test of the nitric-oxide-mediated flash control model.

Fireflies (Coleoptera: Lampyridae) emit various types of light that differ among species and populations of the same species. Their lights are assumed to be biological properties that play important ecological and evolutionary roles. Some species in the Lampyridae emit periodic luminescence, the patterns of which are characterized by species-specific intervals. In previous work, it was predicted that the nitric oxide (NO) regulates the oxygen supply required for the bioluminescence reaction of fireflies. Here, the expression of the NO synthase (NOS) mRNA in some fireflies was examined to verify the predictive model of nitric-oxide-mediated flash control in these insects. The expression of the nos gene in the lantern organ was observed not only in nocturnal flashing species but also in diurnal non-flashing species. It was shown that the expression levels of nos were higher in the lantern of Luciola cruciata (Motschulsky) larvae, which that emits continuous light, than in other body parts, although expression in the lantern of the adults, who flash periodically, was not high. Furthermore, there was no significant difference in expression levels among adults of Luciola cruciata characterized by different flashing intervals. The data do not support the model of an NO-mediated flash control mechanism, during which oxygen becomes available for the luciferin-luciferase reaction through NO-mediated inhibition of mitochondrial respiration. It is also indicated that flash patterns do not co-vary with NOS production. However, high nos expression in the larval lantern suggests that NO may play a role in producing continuous light by functioning as a neurotransmitter signal for bioluminescence.

Cloning and characterization of luciferase from a Fijian luminous click beetle.

Luminous click beetle is distributed almost exclusively in Central and South America with a single genus in Melanesia. Among these click beetles, the description of Melanesian species has been fragmentary, and its luciferase gene and phylogenetic relation to other click beetles still remain uncertain. We collected a living luminous click beetle, Photophorus jansonii in Fiji. It emits green-yellow light from two spots on the pronotum and has no ventral luminous organ. Here, we cloned a luciferase gene from this insect by RT-PCR. The deduced amino acid sequence showed high identity of ~85% to the luciferases derived from other click beetle species. The luciferase of the Fijian click beetle was produced as a recombinant protein to characterize its biochemical properties. The Km for D-luciferin and ATP were 173 and 270 µm, respectively. The luciferase was pH-insensitive and the spectrum measured at pH 8.0 showed a peak at 559 nm, which was in the range of green-yellow light as seen in the luminous spot of the living Fijian click beetle. The Fijian click beetle luciferase was assigned to the Elateridae clade by a phylogenetic analysis, but it made a clearly different branch from Pyrophorus group examined in this study.

Nitric oxide synthase (NOS) in the Japanese fireflies Luciola lateralis and Luciola cruciata.

Species-specific flash patterns in firefly species are important for the investigation of the evolution of Lampyridae. Since nitric oxide synthase (NOS) is one of the key enzymes controlling flash patterns, we determined the cDNA sequences of NOS in the Japanese fireflies Luciola lateralis and L. cruciata. The identity of the NOS sequences was very high between these 2 species. Firefly NOS also exhibited a high identity with those of other insect species, and the cofactor-binding domains were particularly well conserved. Many negatively selected sites were detected throughout the NOS sequences; however, no positive selection was detected. The phylogenetic relationship of insect NOS was different from that of the general classification system, although the lineages corresponded to the major recognized taxonomic groups.

Mitochondrial genomes of two luminous beetles, Rhagophthalmus lufengensis and R. ohbai (Arthropoda, Insecta, Coleoptera).

We determined the mitochondrial DNA (mtDNA) sequences of two luminous beetles (Arthropoda, Insecta, Coleoptera), Rhagophthalmus lufengensis from Yunnan, China and Rhagophthalmus ohbai from Yaeyama Island, Japan. We identified all the 37 mtDNA genes of R. lufengensis (15,982 bp) and the 34 genes of R. ohbai (15,704 bp). R. lufengensis and R. ohbai genomes have higher A + T contents than other coleopteran genomes although the gene arrangements are similar. Interestingly, in a study of the evolutionary relationship among R. lufengensis, R. ohbai and the firefly Pyrocoelia rufa, the phylogenetic tree inferred from lrRNA genes from mitochondrial genomes indicates a biogeographic relationship among the bioluminescent insects in East Asia and the phylogenetic tree inferred from luciferase-related genes from nuclear genomes shows an appropriate relationship among coleopterans, reflecting the evolutionary origin of bioluminescence. Thus, the mtDNAs of luminescent beetles can provide an insight into their evolutionary origin and biogeography.

Phylogeographic analysis of the firefly, Luciola lateralis, in Japan and Korea based on mitochondrial cytochrome oxidase II gene sequences (Coleoptera: Lampyridae).

Luciola lateralis is widely distributed throughout the Korean Peninsula, northeast China, Sakhalin, and Japan. Two ecological types are recognized in Japan based on flash and hatching time characteristics. The mitochondrial cytochrome oxidase II gene was surveyed by restriction fragment length polymorphism analysis for Japan (46 populations) and Korea (two populations). Eleven haplotypes were detected. Gene trees revealed that haplotypes between Japan and Korea are much more differentiated in nucleotide sequences (8.1%) than those within Japan (0.3-1.4%) and Korea (0.7%). Haplotypes between Honshu and Hokkaido are not separated as clades, and the two ecological types cannot be segregated from each other phylogenetically. We suggest that the Japanese populations of this species may have dispersed within one million years ago and that ecological differences may be the result of physiological adaptation to cold climates.

Flash communication systems of Japanese fireflies.

Japanese fireflies range from nocturnal luminescent species to diurnal non-luminescent species. Their communication systems are classified into 6 types based on the following criteria: 1) Female responds to male's flashes after a fixed delay; 2) Male is directly attracted by female's light signal, the male perches on a leaf near the female, then the male changes his flashes with twinkling, and copulation behavior is released. However, the female may not respond to the male; 3) Male seeks female calling signal during the male's flying and synchronous flashing, then the male approaches the female, emitting flashes with various patterns, displaying walking-luminescing, sedentary signaling, chasing, and copulating; 4) Male is attracted by continuous luminescent signals of the female, and male perches near the female, then the male distinguishes the female's light organs shape. Thereafter, the male copulation behavior is released by her sex pheromone; 5) Male and female flight occurs in the daytime; when the male approaches the female, copulation is released by the female's pheromone; weak luminescent signals may be fulfilling the function of supplementary communication signals; 6) Luminescent signals have nothing to do with communication between male and female, and copulation is released by a sex pheromone.

Embryogenesis of the glowworm Rhagophthalmus ohbai Wittmer (Insecta: Coleoptera, Rhagophthalmidae), with emphasis on the germ rudiment formation.

The early embryonic development and features of the developing embryo of the glowworm Rhagophthalmus ohbai are described chiefly by light microscopy, with emphasis on the germ rudiment formation and its phylogenetic implication. The egg period is 30-34 days at about 23 degrees C. The newly laid egg is a short ellipsoid, 1.09 by 0.78 mm in size, and the size increases to 1.15 by 0.95 mm by 17 days after oviposition. Cleavage is of the typical superficial type. The germ disk is formed by cell aggregation of the embryonic area at the anterior end of the egg. The central part of the germ disk then sinks into the yolk and the spherical germ rudiment is formed by fusion of the amnioserosal folds extended from all margins of the germ disk. The inner region of the germ rudiment soon becomes slender and develops into the short embryo, whereas the outer region facing the anterior end is extended to form the thin amnion. The embryo then rapidly elongates, the elongation being accompanied by embryo segmentation and formation of appendages. The submerged condition of the embryo persists until about 17 days after oviposition (about 1 day before embryonic revolution) and thereafter the embryo becomes superficial in position. The presence of the following embryonic characters in R. ohbai supports the molecular data placing it within the Lampyridae: 1) formation of a spherical germ rudiment near the anterior end of the egg, and 2) the submerged condition of the developing embryo persists until shortly before revolution.

Gene diversity and geographic differentiation in mitochondrial DNA of the Genji firefly, Luciola cruciata (Coleoptera: Lampyridae).

The Genji firefly, Luciola cruciata, is divided into two ecological types, the fast-flash and slow-flash types, on the basis of the interflash interval of mate-seeking males. To evaluate the evolutionary origin of the two types, 62 populations were examined by restriction fragment length polymorphism analysis of the mitochondrial cytochrome oxidase (CO) II gene. As a result, 19 haplotypes were detected, and their distributions were indigenous to local areas. Phylogenetic trees constructed from sequence comparison of the haplotypes revealed three major clades (I, II, and III). The boundary of haplotypes between clades I and II is approximately concordant with the geological structure of the Japanese Islands, which is a great rupture zone called the Fossa Magna, and the distribution of haplotypes in clades III and I-II corresponds to the Kyushu and Honshu-Shikoku Islands, respectively. The results suggest a vicariant scenario in which current L. cruciata diversity would have arisen from phylogenetic separations subsequent to the formation process of the Japanese Islands based on the molecular clock. The CO II gene trees also suggested that the fast-flash type should be considered an ancestral form, while the slow-flash type would be a derived one. The divergence time between the slow- and the fast-flash types is estimated to be about 4.6 to 2.0 mya (the Pliocene epoch).