Comprehensive phylogeny of Pieridae butterflies reveals strong correlation between diversification and temperature.

Temperature is thought to be a key factor influencing global species richness patterns. We investigate the link between temperature and diversification in the butterfly family Pieridae by combining next generation DNA sequences and published molecular data with fine-grained distribution data. We sampled nearly 600 pierid butterfly species to infer the most comprehensive molecular phylogeny of the family and curated a distribution dataset of more than 800,000 occurrences. We found strong evidence that species in environments with more stable daily temperatures or cooler maximum temperatures in the warm seasons have higher speciation rates. Furthermore, speciation and extinction rates decreased in tandem with global temperatures through geological time, resulting in a constant net diversification.

To and fro in the archipelago: Repeated inter-island dispersal and New Guinea's orogeny affect diversification of Delias, the world's largest butterfly genus.

The world's largest butterfly genus Delias, commonly known as Jezebels, comprises ca. 251 species found throughout Asia, Australia, and Melanesia. Most species are endemic to islands in the Indo-Australian Archipelago or to New Guinea and nearby islands in Melanesia, and many species are restricted to montane habitats over 1200 m. We inferred an extensively sampled and well-supported molecular phylogeny of the group to better understand the spatial and temporal dimensions of its diversification. The remarkable diversity of Delias evolved in just ca. 15-16 Myr (crown age). The most recent common ancestor of a clade with most of the species dispersed out of New Guinea ca. 14 Mya, but at least six subsequently diverging lineages dispersed back to the island. Diversification was associated with frequent dispersal of lineages among the islands of the Indo-Australian Archipelago, and the divergence of sister taxa on a single landmass was rare and occurred only on the largest islands, most notably on New Guinea. We conclude that frequent inter-island dispersal during the Neogene-likely facilitated by frequent sea level change-sparked much diversification during that period. Many extant New Guinea lineages started diversifying 5 Mya, suggesting that orogeny facilitated their diversification. Our results largely agree with the most recently proposed species group classification system, and we use our large taxon sample to extend this system to all described species. Finally, we summarize recent insights to speculate how wing pattern evolution, mimicry, and sexual selection might also contribute to these butterflies' rapid speciation and diversification.

Ontogenetic progression of individual head size in the larvae of the beetle Trypoxylus dichotomus (Coleoptera: Scarabaeidae): catch-up growth within stages and per-stage growth rate changes across stages.

The ontogenetic progression of insect larval head size has received much attention due to its fundamental and practical importance. However, although previous studies have analyzed the population mean head size, such an approach may not be appropriate for developmental studies of larval head sizes when the trajectory of individual head size growth is correlated with pre-molt head size and developmental stage. Additionally, there is covariation between the head and body sizes; however, few studies have compared the ontogenetic progression of individual head sizes with that of individual body sizes. In this investigation, the per-stage growth rates (PSGRs) for head width (HW) and cubic-rooted body mass at the beginning of each instar (body size, BS) were assessed in Trypoxylus dichotomus. Linear models were used to test the size- and instar-dependence of the ontogenetic progression of individual HW and BS. The individual PSGRs of the HW (iPSGRH) and BS (iPSGRB) were then compared. In addition, the allometric relationship between HW and BS was examined. The iPSGRH was negatively correlated with the pre-molt HW at every instar (i.e., head catch-up growth). Furthermore, the mean iPSGRH at L2 was relatively higher than that at L1 when the pre-molt HW was used as covariate in the analysis (i.e., instar-effect), whereas the mean iPSGRH decreased ontogenetically. The iPSGRB showed catch-up growth and instar-effects similar to those of iPSGRH; however, iPSGRH was found to be lower than iPSGRB. Due to the differences between the PSGRs for the larval head and body, the larval head size showed negative ontogenetic allometry against body size.

Oviposition in the onion fly Delia antiqua (Diptera: Anthomyiidae) is socially facilitated by visual cues.

Ovipositional decisions in herbivorous insects may be affected by social information from conspecifics. Social facilitation of oviposition has been suggested for the onion fly Delia antiqua. In the current study, we found that D. antiqua oviposition was unequal between paired oviposition stations of equal quality and that more eggs were laid on an oviposition station baited with decoy flies than on the control. The increased oviposition toward the decoys continued over time >8 h. When decoys were placed upside down, the number of eggs laid did not differ between the decoy and control sides of oviposition stations, suggesting that social facilitation of oviposition is mediated by visual cues. Based on these findings, mechanisms of social facilitation of oviposition in D. antiqua were discussed.

Insights into How Longicorn Beetle Larvae Determine the Timing of Metamorphosis: Starvation-Induced Mechanism Revisited.

Larvae of holometabolous insects must determine the timing of their metamorphosis. How they determine this timing has only been studied in detail for a few insect species. In a few species of Coleoptera, starvation is known to be a cue for metamorphosis, leading to the formation of smaller adults (starvation-induced pupation, SiP). We investigated the occurrence of SiP in the beetle Psacothea hilaris. When P. hilaris larvae were starved late in the feeding phase of the last (5th) instar, they exhibited typical SiP characterized by constancy of the time from food deprivation to pupation (TTP) irrespective of the body weight upon food deprivation or the length of prior feeding. In contrast, when larvae were starved early in the feeding phase, TTP decreased by roughly 1 day as the feeding became 1 day longer. The change in the response to starvation was estimated to occur on day 5.9 in the last instar. A series of refeeding experiments suggested that whereas SiP occurred readily in the larvae starved in the late feeding phase, activation of SiP was suspended until day 5.9 in the larvae starved early in the feeding phase. When P. hilaris larvae were fed continuously, they eventually ceased feeding spontaneously and pupated. The time length between spontaneous cessation of feeding and pupation was approximately equal to the TTP in SiP. This suggests that the same mechanism was activated by food deprivation in the late feeding phase and by spontaneous cessation of ad libitum feeding.

Cloning, phylogeny, and expression analysis of the Broad-Complex gene in the longicorn beetle Psacothea hilaris.

Seven isoforms of Broad-Complex (PhBR-C), in which the sequence of the zinc finger domain differed (referred to as Z1, Z2, Z3, Z2/Z3, Z4, Z5/Z6, and Z6, respectively), were cloned from the yellow-spotted longicorn beetle Psacothea hilaris. The Z1-Z4 sequences were highly conserved among insect species. The Z5/Z6 isoform was aberrant in that it contained a premature stop codon. Z6 had previously only been detected in a hemimetabola, the German cockroach Blattella germanica. The presence of Z6 in P. hilaris, and not in other holometabolous model insects such as Drosophila melanogaster or Tribolium castaneum, suggests that Z6 was lost multiple times in holometabolous insects during the course of evolution. PhBR-C expression levels in the brain, salivary gland, and epidermis of larvae grown under different feeding regimens were subsequently investigated. PhBR-C expression levels increased in every tissue examined after the gut purge, and high expression levels were observed in prepupae. A low level of PhBR-C expression was continuously observed in the brain. An increase was noted in PhBR-C expression levels in the epidermis when 4th instar larvae were starved after 4 days of feeding, which induced precocious pupation. No significant changes were observed in expression levels in any tissues of larvae starved immediately after ecdysis into 4th instar, which did not grow and eventually died.

Workers select mates for queens: a possible mechanism of gene flow restriction between supercolonies of the invasive Argentine ant.

Some invasive ants form large networks of mutually non-aggressive nests, i.e., supercolonies. The Argentine ant Linepithema humile forms much larger supercolonies in introduced ranges than in its native range. In both cases, it has been shown that little gene flow occurs between supercolonies of this species, though the mechanism of gene flow restriction is unknown. In this species, queens do not undertake nuptial flight, and males have to travel to foreign nests and cope with workers before gaining access to alien queens. In this study, we hypothesized that male Argentine ants receive interference from workers of alien supercolonies. To test this hypothesis, we conducted behavioral and chemical experiments using ants from two supercolonies in Japan. Workers attacked males from alien supercolonies but not those from their own supercolonies. The level of aggression against alien males was similar to that against alien workers. The frequency of severe aggression against alien males increased as the number of recipient workers increased. Cuticular hydrocarbon profiles, which serve as cues for nestmate recognition, of workers and males from the same supercolony were very similar. Workers are likely to distinguish alien males from males of their own supercolony using the profiles. It is predicted that males are subject to considerable aggression from workers when they intrude into the nests of alien supercolonies. This may be a mechanism underlying the restricted gene flow between supercolonies of Argentine ants. The Argentine ant may possess a distinctive reproductive system, where workers participate in selecting mates for their queens. We argue that the aggression of workers against alien males is a novel form of reproductive interference.

Heat treatment of the Adzuki bean borer, Ostrinia scapulalis infected with wolbachia gives rise to sexually mosaic offspring.

A maternally inherited intracellular bacterium, Wolbachia, causes reproductive alterations in its arthropod hosts. In the adzuki bean borer, Ostrinia scapulalis (Walker) (Lepidoptera: Crambidae), naturally-occurring Wolbachia selectively kills male progeny. This Wolbachia strain appears to have a feminizing effect, since antibiotic treatment of infected female moths gives rise to male progeny with sexually mosaic phenotypes. It is proposed that male-specific death occurs through the feminizing effect, and sexual mosaics are produced when this effect is incompletely exerted. Here we examined whether the treatment of infected female moths with high temperatures (34 degrees C, 36 degrees C, or 38 degrees C), which is likely to suppress the activity of Wolbachia, induces sexually mosaic progeny. It was found that eggs laid within 24 h after treatment of Wolbachia-infected mothers at 36 degrees C gave rise to seven sexual mosaics along with 54 normal females. The time lag between treatment and the appearance of mosaic progeny was much shorter with heat treatment than antibiotic treatment, suggesting that heat treatment is more useful for spotting developmental timing when Wolbachia exerts its feminizing effect on O. scapulalis embryos.

Upregulation of a desaturase is associated with the enhancement of cold hardiness in the onion maggot, Delia antiqua.

Cold-acclimated non-diapause pupae, and summer- and winter-diapause pupae of the onion maggot, Delia antiqua (Diptera: Anthomyiidae), show marked cold hardiness as compared with intact non-diapause pupae. Homeoviscous adaptation of cellular membranes is crucial to enhance the cold hardiness of organisms, and Delta9-acyl-CoA desaturases have been assumed, albeit without experimental evidence in insects, to play a key role in the adaptation. We cloned the cDNA of a desaturase gene (Dadesat) from D. antiqua, which is most likely to encode Delta9-acyl-CoA desaturase. Expression of Dadesat mRNA in the brain, midgut, and Malpighian tubules of cold-acclimated and diapause pupae was upregulated 2-10 fold, correlating well with the increase in cold hardiness. In the pupae with enhanced cold hardiness, palmitoleic and oleic acids, the presumed products of Dadesat, in the phospholipids were significantly increased. These findings suggest that the increase in the expression of Dadesat contributes to enhanced cold hardiness in D. antiqua through the production of these unsaturated fatty acids.

Sex-specific death in the Asian corn borer moth (Ostrinia furnacalis) infected with Wolbachia occurs across larval development.

Maternally inherited endosymbiotic bacteria of the genus Wolbachia induce various kinds of reproductive alterations in their arthropod hosts. In a Wolbachia-infected strain of the adzuki bean borer moth, Ostrinia scapulalis (Lepidoptera: Crambidae), males selectively die during larval development, while females selectively die when Wolbachia are eliminated by antibiotic treatment. We found that naturally occurring Wolbachia in the congener O. furnacalis caused sex-specific lethality similar to that in O. scapulalis. Cytogenetic analyses throughout the entire larval development clarified that the death of males (when infected) and females (when cured) took place mainly during early larval stages. However, some individuals also died after complete formation of larval bodies but before egg hatching, or at late larval stages, even in the penultimate instar. Although the specific timing was highly variable, death of males and females occurred before pupation without exception. The potential association of sex-specific lethality with the sex determination mechanism was also examined and is discussed.

Transinfection reveals the crucial importance of Wolbachia genotypes in determining the type of reproductive alteration in the host.

Wolbachia , a group of endosymbiotic bacteria in arthropods, alter the reproduction of their hosts in various ways. A Wolbachia strain (wSca) naturally infecting the adzuki bean borer moth Ostrinia scapulalis induces male killing, while another strain (wKue) infecting the Mediterranean flour moth Ephestia kuehniella induces cytoplasmic incompatibility (CI) in the resident host. Transinfection of Wolbachia can be a powerful tool to elucidate the relative importance of Wolbachia and the host in determining the type of reproductive alterations. Recently, male killing was shown to occur in E. kuehniella transinfected with w Sca. In the present study, we transferred w Kue to O. scapulalis by embryonic microinjection. In the O. scapulalis transinfected with wKue, CI, but not male killing occurred. Thus, in addition to wSca, wKue was shown to induce the same type of alteration in a foreign host as in its natural host. These results demonstrate the crucial role of the Wolbachia genotype in determining the type of reproductive alteration. However, the present study also revealed the involvement of host factors. First, the degree of incompatibility induced by wKue in O. scapulalis was stronger than that in E. kuehniella , indicating that host factors can affect the level of CI. Second, the vertical transmission rate of wKue in O. scapulalis was generally low, suggesting that the host affects the dynamics of Wolbachia transmission.

DaTrypsin, a novel clip-domain serine proteinase gene up-regulated during winter and summer diapauses of the onion maggot, Delia antiqua.

Diapause prepares insects and other arthropods to survive in harsh environments. To explore the molecular basis of winter (WD) and summer diapauses (SD), we screened for diapause-specific genes in the onion maggot, Delia antiqua, that diapauses as a pupa in both summer and winter. A diapause-induced transcript, DaTrypsin, was identified through differential display, and examined by Northern blot, quantitative real-time PCR and sequence analyses. The full-length cDNA, 1379 bp long, encodes 384 a.a. with a molecular mass of 43,005 Da. The protein contains a 20-a.a. secretion peptide, followed by an amino-terminal clip domain and a carboxyl-terminal serine proteinase domain. With Ser, His and Asp as catalytic residues and Asp, Gly and Ser as specificity determinants, DaTrypsin is anticipated to be a trypsin-like enzyme. DaTrypsin transcription is up-regulated in both SD and WD pupae with higher mRNA levels during WD than SD. Heat shock further elevated gene transcription in both SD and WD pupae, whereas cold shock reduced DaTrypsin expression in SD pupae and had no significant effect on WD pupae. In SD pupae, DaTrypsin transcripts gradually build up during diapause, and after temperature shocks, whereas in WD pupae DaTrypsin mRNA levels are high at the beginning of diapause and immediately after a temperature shock and then gradually decrease with time. DaTrypsin represents the first serine proteinase gene expressed during diapause as well as the first gene up-regulated in both SD and WD. It may participate in the host's immune defense and/or maintain the developmental status in the diapausing pupae.

Two kinds of sex ratio distorters in a moth, Ostrinia scapulalis.

We surveyed female-biased sex ratio traits (SR traits) in a moth, Ostrinia scapulalis (Lepidoptera: Crambidae) from six geographic locations in Japan. A small proportion (8.3%) of females showed the SR trait. About half of them were associated with a Wolbachia infection (SRw+), whereas the remaining ones were not (SRw-). No significant differences in frequencies were detected among locations and years for either SRw+ or SRw- females. In SRw+ matrilines, the SR trait was inherited. Curing the Wolbachia infection by tetracycline at the larval stages resulted in all-male broods in the next generation. These characteristics were identical to the Wolbachia-induced SR trait observed previously in the related species Ostrinia furnacalis. The Wolbachia strain in O. scapulalis was indistinguishable from that in O. furnacalis in terms of the wsp and ftsZ gene sequences, suggesting either that infection originated from a common ancestor of the two hosts or that Wolbachia was transmitted horizontally between the host species. In SRw- matrilines, on the other hand, the SR trait was not due to bacterial infection but some other maternally inherited element, since antibiotic treatment had no effect and bacterial PCR did not give the characteristic product. In these matrilines, the SR trait occasionally failed to show, suggesting the presence of nuclear factor(s) suppressing the SR trait.

Sexual mosaics induced by tetracycline treatment in the Wolbachia-infected adzuki bean borer, Ostrinia scapulalis.

Wolbachia-infected Ostrinia scapulalis (Lepidoptera: Crambidae) females generate all-female or nearly all-female broods. Curing the infection by tetracycline treatment during larval stages results in the generation of all-male broods in the next generation. Here we show that sexually mosaic offspring are produced by Wolbachia-infected females treated with tetracycline at the adult stage. The sexual mosaics had wings that were composed of distinctive female and male sectors. Besides wings, the sexually dimorphic mid tibiae displayed an intermediate morphology in some of the mosaics. Many of the mosaic individuals had an abnormal structure of the external genitalia as well, a combination of the male uncus and the female ovipositor. We assume that Wolbachia has a feminizing effect on O. scapulalis genetic males and, hence, incomplete curing of the Wolbachia infection results in the generation of sexually mosaic individuals.

Female sex pheromone polymorphism in adzuki bean borer, Ostrinia scapulalis, is similar to that in European corn borer, O. nubilalis.

Individual analysis of the female sex pheromone of the adzuki bean borer, Ostrinia scapulalis, has shown that the sex pheromone of this species comprised (E)-11-tetradecenyl acetate (E11-14:OAc) and (Z)-11-tetradecenyl acetate (Z11-14:OAc) at variable blend ratios. The pheromone blend could be tentatively categorized into three types with respect to the proportion of E11-14:OAc: E type (94-100%, median 99.2%), Z type (0-16%, median 3.0%), and intermediate type (I type, 48-85%, median 63.7%). In addition to the identity of components, the blend ratios in the three types were similar to those of the E strain, Z strain, and hybrid of the European corn borer, O. nubilalis, respectively. This finding suggests that two closely related but morphologically distinct species, O. scapulalis and O. nubilalis, share almost the same sex pheromone communication systems. The significance of this similarity in the two sibling species is discussed.