RNA interference of timeless gene does not disrupt circadian locomotor rhythms in the cricket Gryllus bimaculatus

Molecular studies revealed that autoregulatory negative feedback loops consisting of so-called “clock genes” constitute the circadian clock in Drosophila. However, this hypothesis is not fully supported in other insects and is thus to be examined. In the cricket Gryllus bimaculatus, we have previously shown that period (per) plays an essential role in the rhythm generation. In the present study, we cloned cDNA of the clock gene timeless (tim) and investigated its role in the cricket circadian oscillatory mechanism using RNA interference. Molecular structure of the cricket tim has rather high similarity to those of other insect species. Real-time RT-PCR analysis revealed that tim mRNA showed rhythmic expression in both LD and DD similar to that of per, peaking during the (subjective) night. When injected with tim double-stranded RNA (dstim), tim mRNA levels were significantly reduced and its circadian expression rhythm was eliminated. After the dstim treatment, however, adult crickets showed a clear locomotor rhythm in DD, with a free-running period significantly shorter than that of control crickets injected with Discosoma sp. Red2 (DsRed2) dsRNA. These results suggest that in the cricket, tim plays some role in fine-tuning of the free-running period but may not be essential for oscillation of the circadian clock.     

A Comparative Study of the Level of Locomotor Activity Throughout Postembryonic Development of Two Cricket Species: Acheta domesticus L. and Gryllus bimaculatus De Geer (Ensifera: Gryllidae)

The level of locomotor activity throughout the entire postembryonic development of two crickets, Acheta domesticus (L.) and Gryllus bimaculatus De Geer, has been investigated under controlled laboratory conditions. In both species the level of activity was subject to changes in the course of their development. Activity was reduced during the molting periods, creating a fluctuating pattern, yet in both species it was significantly higher at the end of development than at the beginning. Although the general trend of changes occurring was similar in both species, there were significant between-species differences in their pattern. The activity of A. domesticus larvae seemed to increase more gradually and it was significantly higher than the activity of G. bimaculatus larvae.     

Morph-specific diurnal variation in allatostatin immunostaining in the corpora allata of Gryllus firmus: Implications for the regulation of a morph-specific circadian rhythm for JH biosynthetic rate

Previous studies have documented a circadian cycle in juvenile hormone (JH) biosynthesis in the long-winged, flight-capable morph, but not in the short-winged flightless morph of the cricket Gryllus firmus. One rapid and reversible inhibitor of in vitro JH biosynthesis by the corpora allata (CA) in crickets is the neuropeptide Phe-Gly-Leu/Ile-amide type of allatostatins (ASTs). To investigate the possible role of allatostatin regulation of the morph-specific circadian cycle of JH production, the quantity of this type of AST in the nerves within the CA was determined by the density of anti-AST-immunostaining in confocal images using the Image J program. The density of immunostaining was inversely related to the rate of JH biosynthesis: Immunostaining in the CA was high and did not differ between morphs early in the photophase when the in vitro rate of JH biosynthesis is low and equivalent in the morphs. However, during the end of the photophase, when the rate of JH biosynthesis rises dramatically in the flight-capable morph, but not in the flightless morph, immunostaining was significantly lower in the flight-capable compared to the flightless morph. These results indicate that morph-specific differences in delivery of AST to the CA and its probable release likely regulate the morph-specific circadian pattern of JH biosynthesis. Also, the negative correlation between AST density and JH production provides evidence for predicting the periods of altered release of these rapid-acting paracine regulators of JH biosynthesis.     

Phylogenetic evaluation of Geomyces and allies reveals no close relatives of Pseudogymnoascus destructans, comb. nov., in bat hibernacula of eastern North America

White-nose syndrome (WNS)  of bats, caused by the fungus previously known as Geomyces destructans, has decimated populations of insectivorous bats in eastern North America. Recent work on fungi associated with bat hibernacula uncovered a large number of species of Geomyces and allies, far exceeding the number of described species. Communication about these species has been hindered by the lack of a modern taxonomic evaluation, and a phylogenetic framework of the group is needed to understand the origin of G. destructans and to target closely related species and their genomes for the purposes of understanding mechanisms of pathogenicity. We addressed these issues by generating DNA sequence data for the internal transcribed spacer (ITS) region, nuclear large subunit (LSU) rDNA, MCM7, RPB2, and TEF1 from a diverse array of Geomyces and allies that included isolates recovered from bat hibernacula as well as those that represent important type species. Phylogenetic analyses indicate Geomyces and allies should be classified in the family Pseudeurotiaceae, and the genera Geomyces, Gymnostellatospora, and Pseudogymnoascus should be recognized as distinct. True Geomyces are restricted to a basal lineage based on phylogenetic placement of the type species, Geomyces auratus. Thus, G. destructans is placed in genus Pseudogymnoascus. The closest relatives of Pseudogymnoascus destructans are members of the Pseudogymnoascus roseus species complex, however, the isolated and long branch of P. destructans indicates that none of the species included in this study are closely related, thus providing further support to the hypothesis that this pathogen is non-native and invasive in eastern North America. Several conidia-producing isolates from bat hibernacula previously identified as members of Pseudeurotium are determined to belong to the genus Leuconeurospora, which is widespread, especially in colder regions. Teberdinia hygrophila is transferred to Pseudeurotium as Pseudeurotium hygrophilum, comb. nov., in accordance with the one name per fungus system of classification, and two additional combinations are made in Pseudogymnoascus including Pseudogymnoascus carnis and Pseudogymnoascus pannorum. Additional sampling from other regions of the world is needed to better understand the evolution and biogeography of this important and diverse group of fungi.     

Functional analysis of the circadian clock gene period by RNA interference in nymphal crickets Gryllus bimaculatus

The circadian clock gene period (Gryllus bimaculatus period, Gb’per) plays a core role in circadian rhythm generation in adults of the cricket Gryllus bimaculatus. We examined the role of Gb’per in nymphal crickets that show a diurnal rhythm rather than the nocturnal rhythm of the adults. As in the adult optic lobes, Gb’per mRNA levels in the head of the third instar nymphs showed daily cycling in light-dark cycles with a peak at mid night, and the rhythm persisted in constant darkness. Injection of Gb’per double-stranded RNA (dsRNA) into the abdomen of third instar nymphs knocked-down the mRNA levels to 25% of that in control animals. Most Gb’per dsRNA injected nymphs lost their circadian locomotor activity rhythm, while those injected with DsRed2 dsRNA as a negative control clearly maintained the rhythm. These results suggest that nymphs and adults share a common endogenous clock mechanism involving the clock gene Gb’per.     

Divergent and conserved roles of extradenticle in body segmentation and appendage formation, respectively, in the cricket Gryllus bimaculatus

The cricket Gryllus bimaculatus is a typical hemimetabolous intermediate germ insect, in which the processes of segmentation and appendage formation differ from those in Drosophila, a holometabolous long germ insect. In order to compare their developmental mechanisms, we have focused on Gryllus orthologs of the Drosophila developmental regulatory genes and studied their functions. Here, we report a functional analysis of the Gryllus ortholog of extradenticle (Gb′exd) using embryonic and parental RNA interference (RNAi) techniques. We found the following: (1) RNAi suppression of Gb′exd results in the deletion or fusion of body segments. Especially the head was often very severely affected. This gap-like phenotype may be related to reduced expression of the gap genes hunchback and Krüppel in early RNAi germbands. (2) In the appendages, several segments (podomeres) were fused. (3) Head appendages including the antenna were transformed to a leg-like structure consisting of at least one proximal podomere as well as several tarsomeres. The defects in appendages are reminiscent of the phenotype caused by large exd clones in Drosophila antennal discs. These findings led us to the conclusion that (1) Gb′exd is required for segment patterning in the gnathal to abdominal region, acting in a gap gene-like manner in the anterior region. (2) Gb′exd plays important roles in formation of the appendages and the determination of their identities, acting as a regulatory switch that chooses between the fates of head appendages versus the appendage ground state. Although functions of Gb′exd in appendage patterning appear fundamentally conserved between Gryllus and Drosophila, its role in body segmentation may differ from that of Drosophila exd.     

Complex innervation of three neck muscles by motor and dorsal unpaired median neurons in crickets

Summary In the crickets, Gryllus campestris and Gryllus bimaculatus, the innervation of the dorso-ventral neck muscles M62, M57, and M59 was examined using cobalt staining via peripheral nerves and electrophysiological methods. M62 and M57 are each innervated by two motoneurons in the suboesophageal ganglion. The four motoneurons project into the median nerve to bifurcate into the transverse nerves of both sides. M62 and M57 are the only neck muscles innervated via this route. These bifurcating axon-projections are identical to those of the spiracular motoneurons in the prothoracic ganglion innervating the opener and closer muscle of the first thoracic spiracle in the cricket. The morphology of their branching pattern is described. The neck muscle M57 and the opener muscle of the first thoracic spiracle are additionally innervated by one mesothoracic motoneuron each, with similar morphology. These results suggest, that in crickets, the neck muscles M57 and M62 are homologous to spiracular muscles in the thoracic segments. The two neck muscles M62 and M59 (the posterior neighbour of M57) receive projections from a prothoracic dorsal unpaired median (DUM) neuron that also innervates dorsal-longitudinal neck muscles but not M57. In addition, one or two mesothoracic DUM neurons send axon collaterals intersegmentally to M59. This is the first demonstration of the innervation of neck muscles by DUM neurons.     

Analysis of DNA sequences of six chloroplast and nuclear genes suggests incongruence, introgression, and incomplete lineage sorting in the evolution of Lespedeza (Fabaceae)

The genus Lespedeza (Fabaceae) consists of 40 species disjunctively distributed in East Asia and eastern North America. Phylogenetic relationships of all Lespedeza species and closely related genera were reconstructed using maximum parsimony, maximum likelihood, and Bayesian analyses of sequence data from five chloroplast (rpl16, rpl32-trnL, rps16-trnQ, trnL-F, and trnK/matK) and one nuclear (ITS) DNA regions. All analyses yielded consistent relationships among major lineages. Our results suggested that Campylotropis, Kummerowia, and Lespedeza are monophyletic, respectively. Lespedeza is resolved as sister to Kummerowia and these two together are further sister to Campylotropis. Neither of the two subgenera, subgen. Lespedeza and subgen. Macrolespedeza, in Lespedeza based on morphological characters, is recovered as monophyletic. Within Lespedeza, the North American clade is retrieved as sister to the Asian clade. The nuclear and chloroplast markers showed incongruent phylogenetic signals at shallow-level phylogeny, which may point to either introgression or incomplete lineage sorting in Lespedeza. The divergence times within Lespedeza and among related genera were estimated using Bayesian approach with BEAST. It is assumed that following the divergence between Kummerowia and Lespedeza in Asia in the late Miocene, the ancestor of Lespedeza diverged into the North American and the Asian lineages. The North American ancestor quickly migrated to North America through the Bering land bridge in the late Miocene. The North American and Asian lineages started to diversify almost simultaneously in the late Miocene but resulted in biased numbers of species in two continents.     

AFLPs detect low genetic diversity for Phytophthora nemorosa and P. pseudosyringae in the US and Europe

In California and Oregon, two recently described oomycete forest pathogens, Phytophthora nemorosa and P. pseudosyringae, overlap in their host and geographic ranges with the virulent P. ramorum, causal agent of “sudden oak death.” Epidemiological observations, namely broader geographic distribution and lack of landscape-level mortality, led to the hypothesis they are native to this region, whereas multiple lines of evidence indicate P. ramorum is exotic to North America. We used AFLP analysis to measure genetic variability in the homothallic P. nemorosa and P. pseudosyringae and to evaluate the hypothesis of endemism. We analysed 39 P. nemorosa and 48 P. pseudosyringae isolates (29 American and 19 European) from throughout their geographic and host ranges. In the US, both P. nemorosa and P. pseudosyringae have a dominant AFLP clone with several closely related variants. There is no evidence that genetic diversity is partitioned by host or location in P. nemorosa, but the US P. pseudosyringae clonal lineage is largely nested within a more genetically variable European group. Though the absence of highly variable sampled source populations does not allow us to determine whether each species is native or introduced in the western US with certainty, the results are most consistent with the hypothesis that both are introduced — P. pseudosyringae perhaps from Europe. Invasive Phytophthora species are increasingly being implicated in emergent forest diseases, highlighting the need to identify and characterize both native and previously unknown introduced forest Phytophthoras.     

Mitochondrial DNA phylogeny of North American field crickets: perspectives on the evolution of life cycles,songs, and habitat associations

North American field crickets (genus Gryllus) exhibit a diversity of life cycles, habitat associations, and calling songs. However, patterns of evolution for these ecological and behavioral traits remain uncertain in the absence of a robust phylogenetic framework. Analyses of morphological variation have provided few clues about species relationships in the genus Gryllus. Here we use comparisons of mitochondrial DNA restriction site maps for 29 individuals representing 11 species (including potential outgroups) to examine relationships among eastern North American field crickets. Initially chosen as likely outgroup taxa, the two European species of Gryllus do not obviously fall outside of an exclusively North American clade and (based on amount of sequence divergence) appear to have diverged from North American lineages at about the same time that major North American lineages diverged from each other. The egg-overwintering crickets comprise a strongly supported monophyletic group, but relationships among these three closely related species cannot be resolved. The mtDNA data are consistent with a single origin of egg diapause and do not support a model of recent life cycle divergence and allochronic speciation for Gryllus pennsylvanicus and G. veletis. The two crickets are not sister species, despite remarkable similarity in morphology, habitat, and calling song. This conclusion is consistent with published data on allozyme variation in North American field crickets. The habitat associations of eastern North American field crickets have been labile, but calling songs sometimes have remained virtually unchanged across multiple speciation events.     

Dominance is not always an honest signal of male quality,but females may be able to detect the dishonesty

Females prefer dominant males as mating partners in numerous species. Male dominance rank is considered as an honest signal of male quality, because only healthy males in good condition are thought to be able to win fights with other males. Here, we tested whether activation of the immune system influences the success of males in male–male competition and mating in the field cricket, Gryllus integer. We activated the immune system of males with a nylon monofilament (to mimic a parasitoid larva), and arranged fights between male pairs to assess male dominance and associated mating success. Activation of the immune system with nylon monofilament substantially enhanced the fighting success of males during male–male competition but had no effect on mating success. However, sham-manipulation (a wound only) did not have any effect on fighting success although females mated more often with dominant males. Our study suggests that when male crickets meet an apparent survival threat they may behave more dominantly, probably owing to terminal investment. Male success during male–male competition is not always an honest signal of males’ quality, but females may be able to detect this dishonesty.     

Analysis of rhizobial strains nodulating Phaseolus vulgaris from Hispaniola Island,a geographic bridge between Meso and South America and the first historical link with Europe

Hispaniola Island was the first stopover in the travels of Columbus between America and Spain, and played a crucial role in the exchange of Phaseolus vulgaris seeds and their endosymbionts. The analysis of recA and atpD genes from strains nodulating this legume in coastal and inner regions of Hispaniola Island showed that they were almost identical to those of the American strains CIAT 652, Ch24-10 and CNPAF512, which were initially named as Rhizobium etli and have been recently reclassified into Rhizobium phaseoli after the analysis of their genomes. Therefore, the species R. phaseoli is more abundant in America than previously thought, and since the proposal of the American origin of R. etli was based on the analysis of several strains that are currently known to be R. phaseoli, it can be concluded that both species have an American origin coevolving with their host in its distribution centres. The analysis of the symbiovar phaseoli nodC gene alleles carried by different species isolated in American and European countries suggested a Mesoamerican origin of the α allele and an Andean origin of the γ allele, which is supported by the dominance of this latter allele in Europe where mostly Andean cultivars of common beans have been traditionally cultivated.     

Diversity,host associations,and phylogeography of temperate aurofusarin-producing Hypomyces/Cladobotryum including causal agents of cobweb disease of cultivated mushrooms

Temperate species of Hypomyces and Cladobotryum that produce the red pigment aurofusarin are common on agaricoid and polyporoid basidiomata of species from five orders of Agaricomycetes. Several cause cobweb disease of commercially cultivated mushrooms resulting in serious losses. We sequenced rpb1, rpb2, tef1, and FG1093 regions in 90 wild strains and 30 strains from mushroom farms, isolated from Europe, North America, Africa, Asia, Australia, and New Zealand. Multigene analyses support the distinctness of five species but reveal Hypomyces rosellus to be paraphyletic, comprising several cryptic lineages. Hypomyces rosellus s. str. is characterised by wide dispersal and gene flow across Eurasia but does not occur in North America. Instead, the lineages from the West and the East Coast appear distinct, having given rise to species inhabiting the Southern Hemisphere. Our results reveal wide misuse of the name H. rosellus, especially for cobweb isolates. The majority of these belong to Hypomyces odoratus, including a weakly supported group of fungicide-resistant strains from Europe and North America sharing identical sequence data. New collections are presented for Cladobotryum rubrobrunnescens and Cladobotryum tenue as well as Cladobotryum multiseptatum and Hypomyces dactylarioides, all previously known only from their type material. The former species pair occurs in Europe and the latter in Australia and New Zealand. Separate lineages appear to be maintained by geographic isolation in North America and temperate Australasia but by host specialisation in the species occurring sympatrically in Europe and Asia. Both specialist and generalist host use strategies have evolved in the group. Although teleomorphs are known in most of the species and unnamed lineages, analyses of the five-gene regions suggest the prevalence of clonal reproduction in H. odoratus. This can be the reason for its success in mushroom farms, also facilitating the spread of fungicide resistance. While tef1 and rpb2 can be recommended for species delimitation, low variation, not exceeding 1 % in the whole ingroup, impeaches the use of ITS as a barcoding gene region in this group of fungi.     

The use of insects as a bioassay for Penaeus merguiensis densovirus (PmergDNV)

The lack of available cell lines has hampered the study of viral diseases in crustaceans. This is particularly important for aquaculture which has been plagued by viral diseases since its rapid expansion to meet with the growing demand for seafood products. This study was designed to find an alternative bioassay to cell lines by investigating the use of insects as potential animal models for Penaeus merguiensis densovirus (PmergDNV). Acheta domesticus (house cricket) and Tenebrio molitor (mealworms) were challenged with approximately 1 × 10⁶ virions of PmergDNV by inoculation. PmergDNV was detected in 20% of Tenebrio molitor and 86.6% of Acheta domesticus challenged with PmergDNV. During a subsequent time course experiment, there was a non significant increase in PmergDNV titres (10^4-5 virions), reaching a maximum peak at day 5 (10⁶ copies). A threshold of PmergDNV DNA level equal to or greater than 10³ virions was necessary for mortality in Acheta domesticus. As the inoculum increased from 10³ DNA copies to 10⁴, 10⁵, 10⁶, mortality increased from 20% to 60%, 80% and 100%, respectively. This is the first evidence that insects may be directly used to study viruses from crustaceans and concludes Acheta domesticus may be used as a potential model to study Penaeus merguiensis densovirus.     

Immune-Enhancing Activity Screening on Extracts from Two Crickets, Gryllus bimaculatus and Teleogryllus emma

This study was performed to explore novel and valuable uses of insect resources, important subjects of the natural compound used in bio‐industries. The whole bodies of two crickets, Gryllus bimaculatus and Teleogryllus emma, selected from medicinal insect species, were carefully ground and treated with 80% EtOH. The insect extracts were solubilized and separated by hexane, butanol, and D.W according to their polarities. Three types of extracts, a D.W fraction (G1) and a boiling extract (G2) of an introduced cricket, G. bimaculatus, and a D.W fraction (T1) of a Korean local cricket, T. emma, were prepared to assay immune stimulating activity of cricket originated compounds. The all of three treated cricket extracts showed to increase IL‐4, IFN‐, and TNF‐α. Among those extract, extract G2, boiled extract from G. bimaculatus, was the best immune–enhancing fraction. The results of this study could be fundamental information for further works to use insects as natural resources having plenty of potentials and varieties.     

New adapiform primate of Old World affinities from the Devil's Graveyard Formation of Texas

Most adapiform primates from North America are members of an endemic radiation of notharctines. North American notharctines flourished during the Early and early Middle Eocene, with only two genera persisting into the late Middle Eocene. Here we describe a new genus of adapiform primate from the Devil’s Graveyard Formation of Texas. Mescalerolemur horneri, gen. et sp. nov., is known only from the late Middle Eocene (Uintan) Purple Bench locality. Phylogenetic analyses reveal that Mescalerolemur is more closely related to Eurasian and African adapiforms than to North American notharctines. In this respect, M. horneri is similar to its sister taxon Mahgarita stevensi from the late Duchesnean of the Devil’s Graveyard Formation. The presence of both genera in the Big Bend region of Texas after notharctines had become locally extinct provides further evidence of faunal interchange between North America and East Asia during the middle Eocene. The fact that Mescalerolemur and Mahgarita are both unknown outside of Texas also supports prior hypotheses that low-latitude faunal assemblages in North America demonstrate increased endemism by the late middle Eocene.     

蟋蟀对召唤声识别的趋声性定位行为

为了探究蟋蟀种内个体间的声音通讯原理,本研究通过回放利用Cool-edit2000声音采集软件采集的双斑蟋Gryllus bimachlatus召唤声,分别对雌、雄蟋蟀识别召唤声的趋声性定位行为进行了观测研究。实验结果表明:雌、雄双斑蟋对回放的召唤声均有较强的识别反应,但雌蟋蟀的趋声定位性较强,能在较短的时间内积极寻找到声源,路径成"s"型;雄蟋蟀反应相对较弱,路径较长,找到声源时间比雌蟋蟀长。本研究结果揭示召唤声在蟋蟀种内通讯中不仅对个体间的联系有作用,对异性求偶也有重要作用。     

Genetic variation and relationships in Laetiporus sulphureus s. lat., as determined by ITS rDNA sequences and in vitro growth rate

The aim of this study was to characterise the genetic variation and molecular relationships of the brown rot polypore, Laetiporus sulphureus s. lat., from Europe, South America, Africa, and Asia, using ITS sequences of the nu-rDNA and by comparing the growth rate in vitro. In a NJ analysis of the sequences of 130 individuals of L. sulphureus s. lat., eight distinct clusters emerged, supported by BS values of 70-100 %. Within each cluster, the ITS rDNA sequence variation was below 3 %. The sequences were also analysed together with Laetiporus sequences available from GenBank. Results demonstrated the possible presence of L. huroniensis in Europe (invalidly named L. montanus) and L. gilbertsonii in South America, and provided more information on the Pan-American and European distribution of one of the clades, currently known in North America as L. sulphureus. L. conifericola formed a separate distinct clade. Moreover, the analysis revealed two unknown Laetiporus taxa in Korea, one in South Africa, and one in Europe. As L. sulphureus is described from Europe (France), and we show that more than one taxon exist here, it is presently not possible to define L. sulphureus s. str. Certain biological differences between some of the clades (in vitro growth rates, chemical composition, and pigmentation) were demonstrated and discussed.     

PATTERNS OF VARIATION AND LINKAGE DISEQUILIBRIUM IN A FIELD CRICKET HYBRID ZONE

The distribution of multilocus genotypes found within a natural hybrid zone is determined by the sample of genotypes present when the hybrid zone first formed, by subsequent patterns of genetic exchange between the hybridizing taxa, and by drift and selection within each of the hybrid zone populations. We have used anonymous nuclear DNA restriction fragment polymorphisms (RFLPs) to characterize the array of multilocus genotypes present within a well-studied hybrid zone between two eastern North American field crickets, Gryllus pennsylvanicus and Gryllus firmus. These crickets hybridize along a zone of contact that extends from New England to Virginia. Previous studies have shown that both premating and postmating barriers exist between the two cricket species, but the absence of diagnostic morphological and allozyme markers has made it difficult to assess the consequences of these barriers for genetic exchange. Analyses based on four diagnostic anonymous nuclear markers indicate that hybrid zone populations in Connecticut contain few F₁ hybrids, and that nonrandom associations persist among nuclear gene markers, between nuclear and cytoplasmic markers, and between molecular markers and morphology. Field cricket populations within the hybrid zone are not “hybrid swarms” but consist primarily of crickets that are very much like one or the other of the parental species. Despite ample opportunity for genetic exchange and evidence for introgression at some loci, the two species remain quite distinct. Such a pattern appears to be characteristic of many natural hybrid zones.