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.     

even-skipped has gap-like, pair-rule-like, and segmental functions in the cricket Gryllus bimaculatus, a basal, intermediate germ insect (Orthoptera)

Developmental mechanisms of segmentation appear to be varied among insects in spite of their conserved body plan. Although the expression patterns of the segment polarity genes in all insects examined imply well conserved function of this class of genes, expression patterns and function of the pair-rule genes tend to exhibit diversity. To gain further insights into the evolution of the segmentation process and the role of pair-rule genes, we have examined expression and function of an ortholog of the Drosophila pair-rule gene even-skipped (eve) in a phylogenetically basal insect, Gryllus bimaculatus (Orthoptera, intermediate germ cricket). We find that Gryllus eve (Gb'eve) is expressed as stripes in each of the prospective gnathal, thoracic, and abdominal segments and as a broad domain in the posterior growth zone. Dynamics of stripe formation vary among Gb'eve stripes, representing one of the three modes, the segmental, incomplete pair-rule, and complete pair-rule mode. Furthermore, we find that RNAi suppression of Gb'eve results in segmentation defects in both anterior and posterior regions of the embryo. Mild depletion of Gb'eve shows a pair-rule-like defect in anterior segments, while stronger depletion causes a gap-like defect showing deletion of anterior and posterior segments. These results suggest that Gb'eve acts as a pair-rule gene at least during anterior segmentation and also has segmental and gap-like functions. Additionally, Gb'eve may be involved in the regulation of hunchback and Krüppel expression. Comparisons with eve functions in other species suggest that the Gb'eve function may represent an intermediate state of the evolution of pair-rule patterning by eve in insects.     

Involvement of canonical Wnt/Wingless signaling in the determination of the positional values within the leg segment of the cricket Gryllus bimaculatus

The cricket Gryllus bimaculatus is a hemimetabolous insect whose nymphs possess the ability to regenerate amputated legs. Previously, we showed that Gryllus orthologues of Drosophila hedgehog (Gb'hh), wingless (Gb'wg) and decapentaplegic (Gb'dpp) are expressed during leg regeneration and play essential roles in the establishment of the proximal-distal axis. Here, we examined their roles during intercalary regeneration: when a distally amputated tibia with disparate positional values is placed next to a proximally amputated host, intercalary growth occurs in order to regenerate the missing part. In this process, we examined expression patterns of Gb'hh and Gb'wg. We found that expressions of Gb'hh and Gb'wg were induced in a regenerate and the host proximal to the amputated region, but not in the grafted donor distal to the regenerate. This directional induction occurs even in the reversed intercalation. Because these results are consistent with a distal-to-proximal respecification of the regenerate, Gb'wg may be involved in the re-establishment of the positional values in the regenerate. Furthermore, we found that no regeneration occurs when Gb'armadillo (the orthologue of beta-catenin) was knocked down by RNA interference. These results indicate that the canonical Wnt/Wingless signaling pathway is involved in the process of leg regeneration and determination of positional information in the leg segment.     

Characterization of stretch-activated calcium permeable cation channels in freshly isolated myocytes of the cricket (Gryllus bimaculatus) lateral oviduct

Stretch-activated channels (SACs) were investigated in myocytes isolated from the lateral oviduct in cricket Gryllus bimaculatus using the cell-attached or excised inside-out patch clamp technique. Application of both negative and positive pressure (10-100 cm H(2)O) into the patch pipettes induced the unitary channel current openings. The open probability (NPo) of the channel increased when negative pressure applied into the patch pipettes increased. The single channel conductance for this channel was approximately 20 pS with 140 mM Na(+), K(+), or Cs(+) in the patch pipettes and was approximately 13 pS with 100mM Ca(2+) or Ba(2+) in the patch pipettes. External application of Gd(3+), La(3+), Cd(2+) and Zn(2+)inhibited the channel with the IC(50) values of 14, 15, 28, and 18 microM respectively. Interestingly external application of TEA, a specific blocker of K(+) channel, also inhibited this channel with IC(50) value of 8.8mM. These results show for the first time the presence of stretch activated Ca(2+)-permeable nonselective cation channel in myocytes isolated from the cricket lateral oviduct. The physiological significance of this channel in oviposition behavior is discussed.     

Establishment of the new genus Paranosema based on the ultrastructure and molecular phylogeny of the type species Paranosema grylli Gen. Nov., Comb. Nov. (Sokolova, Selezniov, Dolgikh, Issi 1994), from the cricket Gryllus bimaculatus Deg

The ultrastructure of the microsporidian parasite Nosema grylli, which parasitizes primarily fat body cells and haemocytes of the cricket Gryllus bimaculatus (Orthoptera, Gryllidae) is described. All observed stages (meront, meront/sporont transitional stage ("second meront"), sporont, sporoblast, and spore) are found in direct contact with the host cell cytoplasm. Nuclei are diplokaryotic during almost all stages of the life cycle, but a brief stage with one nucleus containing an abundance of electron-dense material is observed during a "second merogony." Sporogony is disporous. Mature spores are ovocylindrical in shape and measure 4.5+/-0.16micromx2.2+/-0.07 microm (n=10) on fresh smears and 3.3+/-0.06 micromx1.4+/-0.07 microm (n=10) on ultrathin sections. Spores contain 15-18 coils of an isofilar polar filament arranged in one or two layers. Comparative phylogenetic analysis using rDNA shows N. grylli to be closely related to another orthopteran microsporidian, Nosema locustae, and to Nosema whitei from the confused flour beetle, Tribolium confusum. Antonospora scoticae, a parasite of the communal bee Andrena scotica, is a sister taxon to these three Nosema species. The sequence divergence and morphological traits clearly separate this group of "Nosema" parasites from the "true" Nosema clade containing Nosema bombycis. We therefore propose to change the generic name of N. grylli and its close relative N. locustae to Paranosema n. comb. We leave N. whitei in former status until more data on fine morphology of the species are obtained.     

Acoustic communication in the trilling field cricket,Gryllus rubens (Orthoptera: Gryllidae)

Male field crickets produce calling songs that are used for attracting conspecific females for mating. Acoustic communication was studied in the trilling field cricket, Gryllus rubens.A trilled calling song consists of a continuous train of sound pulses, each of which was repeated at a stereotyped rate. Singing males were recorded at different temperatures. The pulse period of the calling song decreased with increasing temperature; there was less effect of temperature on pulse duration. Female phonotaxis was studied on a noncompensating, spherical treadmill. In two-stimulus (choice) playback experiments, females preferred the conspecific trilled song over the chirped calling song of a sympatric species, Gryllus fultoni.This preference persisted even when the song of G. fultoniwas 6 dB louder. Females also discriminated between synthetic trills having different pulse periods; females chose trills with the conspecific pulse period over trills having lower and higher pulse periods.     

Putative neurohemal areas in the peripheral nervous system of an insect,Gryllus bimaculatus,revealed by immunocytochemistry

The morphology and position of putative neurohemal areas in the peripheral nervous system (ventral nerve cord and retrocerebral complex) of the cricket Gryllus bimaculatus are described. By using antisera to the amines dopamine, histamine, octopamine, and serotonin, and the neuropeptides crustacean cardioactive peptide, FMRFamide, leucokinin 1, and proctolin, an extensive system of varicose fibers has been detected throughout the nerves of all neuromeres, except for nerve 2 of the prothoracic ganglion. Immunoreactive varicose fibers occur mainly in a superficial position at the neurilemma, indicating neurosecretory storage and release of neuroactive compounds. The varicose fibers are projections from central or peripheral neurons that may extend over more than one segment. The peripheral fiber varicosities show segment-specific arrangements for each of the substances investigated. Immunoreactivity to histamine and octopamine is mainly found in the nerves of abdominal segments, whereas serotonin immunoreactivity is concentrated in subesophageal and terminal ganglion nerves. Immunoreactivity to FMRFamide and crustacean cardioactive peptide is widespread throughout all segments. Structures immunoreactive to leucokinin 1 are present in abdominal nerves, and proctolin immunostaining is found in the terminal ganglion and thoracic nerves. Codistribution of peripheral varicose fiber plexuses is regularly seen for amines and peptides, whereas the colocalization of substances in neurons has not been detected for any of the neuroactive compounds investigated. The varicose fiber system is regarded as complementary to the classical neurohemal organs.     

Responsiveness of the adult cricket (Gryllus bimaculatus andAcheta domesticus) retrocerebral complex to allatostatin-1 from a cockroach,Diploptera punctata

Brain-retrocerebral complexes of female crickets,Gryllus bimaculatus andAcheta domesticus, treated with antibody to allatostatin-1 from a cockroach,Diploptera punctata, show extensive immunoreactivity. The results suggest that allatostatins or allatostatin-like molecules are produced in neurosecretory cells of the brain and are delivered to the corpora allata through nervous connections and/or via haemolymph. Radiochemical measurements of juvenile hormone III biosynthesis by isolated corpora cardiaca-corpora allata complexes from adultG. bimaculatus have been used to demonstrate an in vitro sensitivity of these glands to allatostatin-1 fromD. punctata. Allatostatin-1 is a relatively potent inhibitor of juvenile hormone III biosynthesis in corpora allata of both young adult females and males. In glands taken from 3-day virgin females, 50% inhibition of hormone biosynthesis is reached at ca. 3 nmol·l^-1 allatostatin-1. The inhibitory action of allatostatin-1 is rapid, dose-dependent and reversible. Addition of 200 mol·l^-1 farnesol to the incubation medium prevents inhibition of juvenile hormone III biosynthesis by allatostatin-1. Juvenile hormone III biosynthesis by isolated corpora allata of 3-day female house crickets,A. domesticus, is also susceptible to inhibition by 1 mol·l^-1 allatostatin-1.Abbreviations ASB2 Diploptera punctata allatostatin-5 - CA corpora allata - CC corpora cardiaca - Dip A-1 Diploptera punctata allatostatin-1 - HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid - JH juvenile hormone(s) - Mas-AS Manduca sexta allatostatin - MF methyl farnesoate - NCA nervus corporis allati - NCC nervus corporis cardiaci - SEM standard error of mean - TRIS Tris(hydroxymethyl)aminomethane     

the effect of pair formation on diel calling patterns in two cricket species,Gryllus veletis andGryllodes sigillatus (Orthoptera: Gryllidae)

After pair formation, male crickets should reduce calling to minimize the risks to males of attracting predators and/or rivals. We tested this hypothesis in two cricket species, one in which males exhibit a high mating propensity (Gryllus veletis)and another in which the mating propensity of males is constrained by the manufacture of elaborate, bipartite spermatophores (Gryllodes sigillatus).Calling durations of male G. veletisdeclined precipitously after the introduction of females but remained unchanged in G. sigillatus.We attribute the asymmetric effect of female proximity on male calling to differences in the mating propensity of males of the two species. Male G. veletisabstain from further calling in favor of repeated matings with the same female. Male G. sigillatusprobably lack the opportunity to mate repeatedly with the same female and, thus, resume calling shortly after mating to increase their probability of attracting additional mates.     

Exploring myriapod segmentation: the expression patterns of even-skipped,engrailed, and wingless in a centipede

Segment formation is critical to arthropod development, yet there is still relatively little known about this process in most arthropods. Here, we present the expression patterns of the genes even-skipped (eve), engrailed, and wingless in a centipede, Lithobius atkinsoni. Despite some differences when compared with the patterns in insects and crustaceans, the expression of these genes in the centipede suggests that their basic roles are conserved across the mandibulate arthropods. For example, unlike the seven pair-rule stripes of eve expression in the Drosophila embryonic germband, the centipede eve gene is expressed strongly in the posterior of the embryo, and in only a few stripes between newly formed segments. Nonetheless, this pattern likely reflects a conserved role for eve in the process of segment formation, within the different context of a short-germband mode of embryonic development. In the centipede, the genes wingless and engrailed are expressed in stripes along the middle and posterior of each segment, respectively, similar to their expression in Drosophila. The adjacent expression of the engrailed and wingless stripes suggests that the regulatory relationship between the two genes may be conserved in the centipede, and thus this pathway may be a fundamental mechanism of segmental development in most arthropods.     

Functional analyses in the milkweed bug Oncopeltus fasciatus (Hemiptera) support a role for Wnt signaling in body segmentation but not appendage development

Specification of the proximal-distal (PD) axis of insect appendages is best understood in Drosophila melanogaster, where conserved signaling molecules encoded by the genes decapentaplegic (dpp) and wingless (wg) play key roles. However, the development of appendages from imaginal discs as in Drosophila is a derived state, while more basal insects produce appendages from embryonic limb buds. Therefore, the universality of the Drosophila limb PD axis specification mechanism has been debated since dpp expression in more basal insect species differs dramatically from Drosophila. Here, we test the function of Wnt signaling in the development of the milkweed bug Oncopeltus fasciatus, a species with the basal state of appendage development from limb buds. RNA interference of wg and pangolin (pan) produce defects in the germband and eyes, but not in the appendages. Distal-less and dachshund, two genes regulated by Wg signaling in Drosophila and expressed in specific PD domains along the limbs of both species, are expressed normally in the limbs of pan-depleted Oncopeltus embryos. Despite these apparently paradoxical results, Armadillo protein, the transducer of Wnt signaling, does not accumulate properly in the nuclei of cells in the legs of pan-depleted embryos. In contrast, engrailed RNAi in Oncopeltus produces cuticular and appendage defects similar to Drosophila. Therefore, our data suggest that Wg signaling is functionally conserved in the development of the germband, while it is not essential in the specification of the limb PD axis in Oncopeltus and perhaps basal insects.     

Morphology and taxonomy of the microsporidium Liebermannia covasacrae n. sp. from the grasshopper Covasacris pallidinota (Orthoptera, Acrididae)

During a survey for grasshopper pathogens in Argentina in 2005-2006, individual Covasacris pallidinota from halophylous grasslands in Laprida, Buenos Aires province were found to be infected with a microsporidium. Infection was restricted to the salivary gland epithelial cells. The microsporidium produced ovocylindrical spores averaging 2.6 ± 0.28 × 1.4 ± 0.12 μm (range 2.2-3.4 × 1.1-1.7 μm), which resembled in size and shape the spores of Liebermannia patagonica and L. dichroplusae, two recently described species that also parasitize Argentine grasshoppers. The life cycle of the microsporidium included the formation of polynucleate, diplokaryotic, moniliform, merogonial plasmodia wrapped in flattened cisterns of the host endoplasmic reticulum (ER). Plasmodia divided to produce diplokaryotic cells. The latter underwent elongation, dissociation of diplokarya counterparts, vacuolization, dismantling of the host ER envelope, and deposition of electron-dense material outside the plasma membrane. The resultant binucleate sporogonial plasmodia divided into two uninucleate sporoblasts, which eventually transformed into spores. Uninucleate spores contained a lamellar polaroplast, embraced by an elongated polar sac, anchoring disc, 3-5 polar filament coils, and a cluster of anastomizing tubules (sporoblast trans-Golgi, posterosome) at the posterior end. Sequence similarity of the SSU rDNA of the newly discovered microsporidium (Genbank accession no. EU709818) to L. patagonica and L. dichroplusae was 99% and 97%, respectively, suggesting that the three species belong to one genus. All three species fell into one clade in SSU rDNA-based phylogenetic trees produced by neighbor joining, maximum parsimony, and maximum likelihood analyses with 100% statistical support. We assign the name Liebermannia covasacrae to this microsporidium. It can be easily differentiated from both congeners by host species, tissue tropism, type of sporogony, and several features of morphology. Comparison of the three Liebermannia spp. demonstrates that the nuclear phase (dikaryotic versus monokaryotic spores) and type of sporogony (polysporous versus disporous) may vary in closely related species.     

A new fossil cricket of the genus Proanaxipha in Miocene amber from the Dominican Republic (Orthoptera,Gryllidae, Pentacentrinae)

A new species of the cricket genus Proanaxipha Vickery & Poinar (Orthoptera: Gryllidae: Pentacentrinae) from Early Miocene Dominican amber is described and illustrated. Proanaxipha madgesuttonae sp. n. is distinguished from congeners by: (1) head capsule bearing a distinctive posteriorly bilobed colour spot on the vertex; (2) presence of crossveins in the proximal part of the mediocubital area; (3) apical field of tegmen entirely dark; and (4) median process of epiphallus short. The poorly known Proanaxipha bicolorata Vickery & Poinar, of questionable affinity and status, is herein regarded as a nomen inquirendum.     

JWA, a novel signaling molecule, involved in the induction of differentiation of human myeloid leukemia cells

Dysregulation of hematopoietic cellular differentiation contributes to leukemogenesis. Unfortunately, relatively little is known about how cell differentiation is regulated. JWA (AF070523) is a novel all-trans retinoic acid (ATRA) responsible gene that initially isolated from ATRA-treated primary human tracheal bronchial epithelial cells. For the notable performance achieved by ATRA in the differentiation induction therapy, we investigated the role of JWA in the induction of differentiation of human myeloid leukemia cells. Our results showed that JWA was not only regulated by ATRA but also by several other differentiation inducers such as phorbol-12-myristate-13-acetate (TPA), arabinoside (Ara-C), and hemin, involved in the mechanisms of differentiation along different lineages of myeloid leukemia cells arrested at different stages of development. Generally, JWA was up-regulated by these inducers in a time-dependent manner. Inhibition of JWA by RNA interference decreased the induced cellular differentiation. However, in NB4 cells treated with ATRA, dissimilar with others, the expression of JWA was down-regulated, and the induced cellular differentiation could be enhanced by silencing of JWA. Collectively, JWA works as a potential critical molecule, associated with multi-directional differentiation of human myeloid leukemia cells. In NB4 cells, JWA may function as a lineage-restricted gene during differentiation along the monocyte/macrophage-like or granulocytic pathway.     

Tow (Target of Wingless), a novel repressor of the Hedgehog pathway in Drosophila

Hedgehog (Hh) signalling plays a crucial role in the development and patterning of many tissues in both vertebrates and invertebrates. Aberrations in this pathway lead to severe developmental defects and cancer. Hh signal transduction in receiving cells is a well studied phenomenon; however questions still remain concerning the mechanism of repression of the pathway activator Smoothened (Smo) in the absence of Hh. Here we describe a novel repressor of the Hh pathway, Target of Wingless (Tow). Tow represents the Drosophila homolog of a conserved uncharacterised protein family. We show that Tow acts in Hh receiving cells, where its overexpression represses all levels of Hh signalling, and that this repression occurs upstream or at the level of Smo and downstream of the Hh receptor Patched (Ptc). In addition, we find that like Ptc, overexpression of Tow causes an accumulation of lipophorin in the wing disc. We demonstrate that loss of tow enhances different ptc alleles in a similar manner to another pathway repressor, Suppressor of Fused (SuFu), possibly through mediating Ptc dependant lipophorin internalisation. Combined, these results demonstrate that Tow is an important novel regulator of the Hh pathway in the wing imaginal disc, and may shed light on the mechanism of Ptc repression of Smo.