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.     

Gene expression reveals evidence for EGFR‐dependent proximal‐distal limb patterning in a myriapod

Evolution of segmented limbs is one of the key innovations of Arthropoda, allowing development of functionally specific specialized head and trunk appendages, a major factor behind their unmatched evolutionary success. Proximodistal limb patterning is controlled by two regulatory networks in the vinegar fly Drosophila melanogaster, and other insects. The first is represented by the function of the morphogens Wingless (Wg) and Decapentaplegic (Dpp); the second by the EGFR‐signaling cascade. While the role of Wg and Dpp has been studied in a wide range of arthropods representing all main branches, that is, Pancrustacea (= Hexapoda + Crustacea), Myriapoda and Chelicerata, investigation of the potential role of EGFR‐signaling is restricted to insects (Hexapoda). Gene expression analysis of Egfr, its potential ligands, and putative downstream factors in the pill millipede Glomeris marginata (Myriapoda: Diplopoda), reveals that—in at least mandibulate arthropods—EGFR‐signaling is likely a conserved regulatory mechanism in proximodistal limb patterning.     

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.     

Role for the Epidermal Growth Factor Receptor in Chemotherapy-Induced Alopecia

Treatment of cancer patients with chemotherapeutics like cyclophosphamide often causes alopecia as a result of premature and aberrant catagen. Because the epidermal growth factor receptor (EGFR) signals anagen hair follicles to enter catagen, we hypothesized that EGFR signaling may be involved in cyclophosphamide-induced alopecia. To test this hypothesis, skin-targeted Egfr mutant mice were generated by crossing floxed Egfr and Keratin 14 promoter-driven Cre recombinase mice. Cyclophosphamide treatment of control mice resulted in alopecia while Egfr mutant skin was resistant to cyclophosphamide-induced alopecia. Egfr mutant skin entered catagen normally, as indicated by dermal papilla condensation and decreased follicular proliferation, but did not progress to telogen as did Egfr wild type follicles. Egfr mutant follicles responded with less proliferation, apoptosis, and fewer p53-positive cells after cyclophosphamide. Treatment of control mice with the EGFR inhibitors erlotinib or gefitinib similarly suppressed alopecia and catagen progression by cyclophosphamide. Secondary analysis of clinical trials utilizing EGFR-targeted therapies and alopecia-inducing chemotherapy also revealed evidence for involvement of EGFR in chemotherapy-induced alopecia. Taken together, our results demonstrated the involvement of EGFR signaling in chemotherapy-induced alopecia, which will help in the design of novel therapeutic regimens to minimize chemotherapy-induced alopecia.     

Targeted gene delivery in the cricket brain,using in vivo electroporation

The cricket (Gryllus bimaculatus) is a hemimetabolous insect that is emerging as a model organism for the study of neural and molecular mechanisms of behavioral traits. However, research strategies have been limited by a lack of genetic manipulation techniques that target the nervous system of the cricket. The development of a new method for efficient gene delivery into cricket brains, using in vivo electroporation, is described here. Plasmid DNA, which contained an enhanced green fluorescent protein (eGFP) gene, under the control of a G. bimaculatus actin (Gb′-act) promoter, was injected into adult cricket brains. Injection was followed by electroporation at a sufficient voltage. Expression of eGFP was observed within the brain tissue. Localized gene expression, targeted to specific regions of the brain, was also achieved using a combination of local DNA injection and fine arrangement of the electroporation electrodes. Further studies using this technique will lead to a better understanding of the neural and molecular mechanisms that underlie cricket behaviors.     

Cricket body size is altered by systemic RNAi against insulin signaling components and epidermal growth factor receptor

A long-standing problem of developmental biology is how body size is determined. In Drosophila melanogaster, the insulin/insulin-like growth factor (I/IGF) and target of rapamycin (TOR) signaling pathways play important roles in this process. However, the detailed mechanisms by which insect body growth is regulated are not known. Therefore, we have attempted to utilize systemic nymphal RNA interference (nyRNAi) to knockdown expression of insulin signaling components including Insulin receptor (InR), Insulin receptor substrate (chico), Phosphatase and tensin homologue (Pten), Target of rapamycin (Tor), RPS6-p70-protein kinase (S6k), Forkhead box O (FoxO) and Epidermal growth factor receptor (Egfr) and observed the effects on body size in the Gryllus bimaculatus cricket. We found that crickets treated with double-stranded RNA (dsRNA) against Gryllus InR, chico, Tor, S6k and Egfr displayed smaller body sizes, while Gryllus FoxO nyRNAi-ed crickets exhibited larger than normal body sizes. Furthermore, RNAi against Gryllus chico and Tor displayed slow growth and RNAi against Gryllus chico displayed longer lifespan than control crickets. Since no significant difference in ability of food uptake was observed between the Gryllus chico(nyRNAi) nymphs and controls, we conclude that the adult cricket body size can be altered by knockdown of expressions of Gryllus InR, chico, Tor, S6k, FoxO and Egfr by systemic RNAi. Our results suggest that the cricket is a promising model to study mechanisms underlying controls of body size and life span with RNAi methods.     

Involvement of hedgehog,wingless, and dpp in the initiation of proximodistal axis formation during the regeneration of insect legs,a verification of the modified boundary model

To understand the mechanism of regeneration, many experiments have been carried out with hemimetabolous insects, since their nymphs possess the ability to regenerate amputated legs. We first succeeded in observing expression patterns of hedgehog, wingless (wg), and decapentaplegic (dpp) during leg regeneration of the cricket Gryllus bimaculatus. The observed expression patterns were essentially consistent with the predictions derived from the boundary model modified by Campbell and Tomlinson (CTBM). Thus, we concluded that the formation of the proximodistal axis of a regenerating leg is triggered at a site where ventral wg-expressing cells abut dorsal dpp-expressing cells in the anteroposterior (A/P) boundary, as postulated in the CTBM.     

A problem of taxonomic status of “banana cricket” from culture of the Moscow Zoo insectarium

The “banana cricket” is one of the preferred laboratory objects used for studying general and applied biological problems. However, the exact identity of this form remains obscure. Correct identification of the insects maintained in culture is vital for the correct prediction of the properties of the object in question and comparative studies. Analysis of acoustic signals showed that the banana cricket from the Moscow Zoo culture did not belong to the Gryllus assimilis (F.) group as it was assumed earlier. Analysis of acoustic signals and genitalia revealed similarity between the banana cricket and insects from the culture maintained at the Institute of Evolutionary Physiology and Biochemistry (IEPhB, St. Petersburg), which were supposed to be Gryllus argentinus (Sauss.). The calling songs and genitalia of crickets from both cultures differed from those of G. argentinus. Thus, the banana cricket and Gryllus sp. from the IEPhB culture belong to the same species but the exact identity of that species has not been yet determined.     

brachyenteron is necessary for morphogenesis of the posterior gut but not for anteroposterior axial elongation from the posterior growth zone in the intermediate-germband cricket Gryllus bimaculatus

In the long-germband insect Drosophila, all body segments and posterior terminal structures, including the posterior gut and anal pads, are specified at the blastoderm stage. In short- and intermediate-germband insects, however, posterior segments are sequentially produced from the posterior growth zone, a process resembling somitogenesis in vertebrates, and invagination of the posterior gut starts after anteroposterior (AP) axial elongation from the growth zone. The mechanisms underlying posterior segmentation and terminal patterning in these insects are poorly understood. In order to elucidate these mechanisms, we have investigated the roles of the Brachyury/brachyenteron (Bra/byn) homolog in the intermediate-germband cricket Gryllus bimaculatus. Loss-of-function analysis by RNA interference (RNAi) revealed that Gryllus byn (Gb'byn) is not required for AP axial elongation or normal segment formation, but is required for specification of the posterior gut. We also analyzed Gryllus caudal (Gb'cad) RNAi embryos using in situ hybridization with a Gb'byn probe, and found that Gb'cad is required for internalization of the posterior gut primordium, in addition to AP axial elongation. These results suggest that the functions of byn and cad in posterior terminal patterning are highly conserved in Gryllus and Drosophila despite their divergent posterior patterning. Moreover, because it is thought that the progressive growth of the AP axis from the growth zone, controlled by a genetic program involving Cdx/cad and Bra/byn, might be ancestral to bilaterians, our data suggest that the function of Bra/byn in this process might have been lost in insects.     

Matrix metalloproteinases (MMPs) are required for wound closure and healing during larval leg regeneration in the flour beetle,Tribolium castaneum

Regenerative abilities are found ubiquitously among many metazoan taxa. To compare mechanisms underlying the initial stages of limb regeneration between insects and vertebrates, the roles of matrix metalloproteinases (MMPs) and fibroblast growth factor (FGF) signaling were investigated in the red flour beetle, Tribolium castaneum. RNA interference-mediated knockdown of MMP2 expression delayed wound healing and subsequent leg regeneration. Additionally, pairwise knockdown of MMP1/2 and MMP2/3, but not MMP1/3, resulted in inhibition of wound closure. Wound healing on the dorsal epidermis after injury was also delayed when MMPs were silenced. Our findings show that functionally redundant MMPs play key roles during limb regeneration and wound healing in Tribolium. This MMP-mediated wound healing is necessary for the subsequent formation of a blastema. In contrast, silencing of FGF receptor did not interfere with the initial stages of leg regeneration despite the alterations in tanning of the cuticle. Thus, insects and vertebrates appear to employ similar developmental processes for the initial stages of wound closure during limb regeneration, while the role of FGF in limb regeneration appears to be unique to vertebrates.     

Positive Relationship between Signalling Time and Flight Capability in the Texas Field Cricket, Gryllus texensis

A trade‐off between dispersal ability and reproduction is generally thought to explain the persistence of wing dimorphism in insects, although this trade‐off has received minimal attention in male insects. Research on male sand cricket, Gryllus firmus, supports the trade‐off hypothesis insofar as flight capable cricket’s spend significantly less time signalling for potential mates than their flightless counterparts. By contrast, here I show that this expected trade‐off between signalling time and wing dimorphism does not exist in a male congener, the Texas field cricket (Gryllus texensis). In G. texensis, flight capable males signal twice as often as flightless males. Thus, unless male G. texensis express trade‐offs between dispersal ability and other, presently unmeasured components of reproduction, the trade‐off hypothesis may not explain the persistence of wing dimorphism in all male insects.     

Placental and Embryonic Growth Restriction in Mice With Reduced Function Epidermal Growth Factor Receptor Alleles

Embryos lacking an epidermal growth factor receptor (EGFR) exhibit strain-specific defects in placental development that can result in mid-gestational embryonic lethality. To determine the level of EGFR signaling required for normal placental development, we characterized congenic strains homozygous for the hypomorphic Egfr^wa2 allele or heterozygous for the antimorphic Egfr^Wa5 allele. Egfr^wa2 homozygous embryos and placentas exhibit strain-dependent growth restriction at 15.5 days post-coitus while Egfr^Wa5 heterozygous placentas are only slightly reduced in size with no effect on embryonic growth. Egfr^wa2 homozygous placentas have a reduced spongiotrophoblast layer in some strains, while spongiotrophoblasts and glycogen cells are almost completely absent in others. Our results demonstrate that more EGFR signaling occurs in Egfr^Wa5 heterozygotes than in Egfr^wa2 homozygotes and suggest that Egfr^wa2 homozygous embryos model EGFR-mediated intrauterine growth restriction in humans. We also consistently observed differences between strains in wild-type placenta and embryo size as well as in the cellular composition and expression of trophoblast cell subtype markers and propose that differential expression in the placenta of Glut3, a glucose transporter essential for normal embryonic growth, may contribute to strain-dependent differences in intrauterine growth restriction caused by reduced EGFR activity.EPIDERMAL growth factor receptor (EGFR) is the prototypical member of the ERBB family of receptor tyrosine kinases and is known to regulate many aspects of cellular biology including cell proliferation, survival, differentiation, and migration (reviewed in Yarden and Sliwkowski 2001). Eleven known ligands bind the extracellular region of ERBB-family receptors, and activation of the tyrosine kinase domain occurs following receptor homo- or heterodimerization. The resulting biological responses are dependent upon specific signaling cascades initiated by ERBBs and can be influenced by the particular ligand–ERBB combination (Yarden and Sliwkowski 2001). Studies using cultured cells have underscored the importance of EGFR in modulating various cellular processes, while animal models have been able to demonstrate that EGFR is required for numerous developmental and physiological processes (Casalini et al. 2004). In vivo studies have shown that EGFR is particularly important for normal placental development in mice; placentas from Egfr nullizygous (Egfr^{tm1Mag/tm1Mag}) embryos exhibit strain-specific defects that result in differential embryonic lethality (Sibilia and Wagner 1995; Threadgill et al. 1995). Two additional Egfr alleles result in reduced EGFR signaling in mice: the recessive hypomorphic Egfr^wa2 and dominant antimorphic Egfr^Wa5 alleles (Luetteke et al. 1994; Fowler et al. 1995; Du et al. 2004; Lee et al. 2004). These alleles can provide insight into the level of EGFR signaling required for normal placental development.Egfr^wa2 is a classical spontaneous mutation that arose in 1935 that causes a distinct wavy coat phenotype in the homozygote (Figure 1; Keeler 1935). This recessive mutation was subsequently found to be a single nucleotide transversion resulting in a valine → glycine substitution in the highly conserved kinase domain of EGFR (Luetteke et al. 1994; Fowler et al. 1995). Since mice homozygous for the Egfr^tm1Mag null allele die before or shortly after birth depending on genetic background, the hypomorphic Egfr^wa2 allele has been the primary model used to study the effect of attenuated EGFR signaling in a variety of adult physiological and disease states. In addition to eye and hair phenotypes, the adult Egfr^wa2 homozygous mouse exhibits delayed onset of puberty, abnormal ovulation, enlarged aortic valves and cardiac hypertrophy, decreased body size, defects in mammary gland development and lactation, increased susceptibility to colitis, and impaired intestinal adaptation following small bowel resection (Fowler et al. 1995; Helmrath et al. 1997; Chen et al. 2000; Egger et al. 2000; O''Brien et al. 2002; Prevot et al. 2005; Hsieh et al. 2007). Despite the widespread use of the Egfr^wa2 allele, there are limitations in using Egfr^wa2 homozygous mice to clearly define the physiological roles of EGFR. Egfr^wa2 has traditionally been maintained in cis, tightly linked with a hypomorphic Wnt3a allele, Wnt3a^vt (vestigal tail), making phenotypic analysis of reduced EGFR signaling by itself difficult. Furthermore, Egfr^wa2 has also typically been maintained on a mixed genetic background and since the Egfr nullizygous phenotype is similarly influenced by genetic modifiers, a mixed background could mask phenotypes that become evident when Egfr^wa2 mice are inbred.Open in a separate windowFigure 1.—Congenic 129 Egfr allelic series. Wild-type (left), Egfr^wa2 homozygote (middle), and Egfr^wa5 heterozygote (right) mice. As weanlings and adults, the Egfr^wa2 homozygotes and Egfr^wa5 heterozygotes are grossly indistinguishable.The Egfr^Wa5 allele arose in a large, genomewide N-ethyl-N-nitrosourea mutagenesis screen for dominant visible mutations in the mouse. Egfr^Wa5 heterozygous mice were first identified by their open eyelids at birth and by development of a wavy coat, similar to the phenotype of Egfr^wa2 homozygous mice (Figure 1). Egfr^Wa5 failed to complement the Egfr^tm1Mag null allele and was shown to function as an antimorph since Egfr^Wa5, but not Egfr^tm1Mag, heterozygotes exhibit eyelid and coat phenotypes (Lee et al. 2004). A single nucleotide missense mutation was found in the Egfr^Wa5 allele that results in an Asp → Gly substitution in the highly conserved DFG domain of the EGFR kinase catalytic loop (Du et al. 2004; Lee et al. 2004). Although Egfr^Wa5 heterozygotes are viable, Egfr^Wa5 homozygotes die prenatally and exhibit placental defects identical to those from Egfr^tm1Mag homozygous null embryos. Placentas from Egfr^Wa5 heterozygotes on a mixed background show variable reduction in the spongiotrophoblast layer and minor abnormalities in the labyrinth region, but no effects on embryo survival have been reported.In vitro studies with Egfr^Wa5 suggest that it encodes a kinase-dead EGFR since no phosphorylation of EGFR^Wa5 is detected following stimulation with ligands. In agreement with the genetic data showing that Egfr^Wa5 is an antimorph, in vitro studies have demonstrated that the EGFR^Wa5 receptor can inhibit phosphorylation of EGFR and MAPK in a dose-dependent manner (Lee et al. 2004). In Chinese hamster ovary cells expressing an equimolar ratio of EGFR and EGFR^Wa5 receptors, <10% of wild-type phosphorylation levels were observed by Western blot analysis.The Egfr allelic series available in the mouse has high utility for studying gene function since EGFR is involved in a multitude of developmental processes and human diseases. Although both Egfr^wa2 and Egfr^Wa5 alleles result in reduced EGFR signaling, the activity and phenotypic consequences of Egfr^wa2 homozygosity has not been compared to that of Egfr^Wa5 heterozygosity when both are on the same genetic backgrounds. Adult Egfr^Wa5 heterozygous mice appear highly similar to Egfr^wa2 homozygotes, but crosses with the Apc^Min intestinal tumor model have shown that a more substantial reduction in tumor number occurs when the Apc^Min mutation is bred onto the Egfr^wa2 homozygous vs. Egfr^Wa5 heterozygous background (Roberts et al. 2002; Lee et al. 2004). These results suggest that Egfr^Wa5 heterozygous mice retain higher levels of EGFR activity than Egfr^wa2 homozygous mice; however, the data are confounded by the fact that the crosses were performed using different mixed genetic backgrounds.This study reports a comprehensive genetic analysis of reduced EGFR signaling in Egfr^wa2 homozygotes and Egfr^Wa5 heterozygotes in placental development and embryonic growth for three congenic backgrounds, C57BL/6J (B6), 129S1/SvImJ (129), and BTBR/J-T+, tf/tf (BTBR). Wild-type placenta weight, embryo weight, and mRNA levels of genes selected for their trophoblast-specific expression were found to be highly strain dependent. Egfr^wa2 homozygous placentas are reduced in size in all three strains, and a proportion of 129-Egfr^wa2 homozygotes die before 15.5 days post-coitus (dpc). Egfr^wa2 homozygous embryos also display background-dependent intrauterine growth restriction (IUGR) in late gestation, which is most severe on 129 and BTBR backgrounds and models EGFR-associated IUGR in humans. Egfr^Wa5 heterozygous placentas exhibit a minor reduction in size on all three backgrounds with no impact on embryonic growth. These results suggest that reduced levels of EGFR signaling can interfere with normal placental development and that embryo development is affected only after placental size is sufficiently reduced. In addition, our data show that the level of EGFR signaling in Egfr^Wa5 heterozygous mice is higher than in Egfr^wa2 homozygotes and suggests that different Egfr allele combinations can be generated to “genetically titer” total EGFR activity in vivo.     

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.     

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.     

Ancestral functions of Delta/Notch signaling in the formation of body and leg segments in the cricket Gryllus bimaculatus

Delta/Notch signaling controls a wide spectrum of developmental processes, including body and leg segmentation in arthropods. The various functions of Delta/Notch signaling vary among species. For instance, in Cupiennius spiders, Delta/Notch signaling is essential for body and leg segmentation, whereas in Drosophila fruit flies it is involved in leg segmentation but not body segmentation. Therefore, to gain further insight into the functional evolution of Delta/Notch signaling in arthropod body and leg segmentation, we analyzed the function of the Delta (Gb'Delta) and Notch (Gb'Notch) genes in the hemimetabolous, intermediate-germ cricket Gryllus bimaculatus. We found that Gb'Delta and Gb'Notch were expressed in developing legs, and that RNAi silencing of Gb'Notch resulted in a marked reduction in leg length with a loss of joints. Our results suggest that the role of Notch signaling in leg segmentation is conserved in hemimetabolous insects. Furthermore, we found that Gb'Delta was expressed transiently in the posterior growth zone of the germband and in segmental stripes earlier than the appearance of wingless segmental stripes, whereas Gb'Notch was uniformly expressed in early germbands. RNAi knockdown of Gb'Delta or Gb'Notch expression resulted in malformation in body segments and a loss of posterior segments, the latter probably due to a defect in posterior growth. Therefore, in the cricket, Delta/Notch signaling might be required for proper morphogenesis of body segments and posterior elongation, but not for specification of segment boundaries.     

Regeneration in the auditory system of nymphal and adult bush crickets Tettigonia viridissima

Regeneration and reestablishment of synaptic connections is an important topic in neurobiological research. In the present study, the regeneration of auditory afferents and the accompanying effects in the central nervous system are investigated in nymphs and adults of the bush cricket Tettigonia viridissima L. (Orthoptera: Tettigoniidae). In all animals in which the tympanal nerve is crushed, neuronal tracing shows a regrowth of the afferents into the prothoracic ganglion. This regeneration is seen in both adult and nymphal stages and starts 10–15 days after nerve crushing. Physiological recordings from the leg nerve indicate a recovery of tympanal fibres and a formation of functional connections to interneurones in the same time range. Electrophysiological recordings from the neck connective suggest additional contralateral sprouting of interneurones and the formation of aberrant connections. The regeneration processes of the tympanal nerve in nymphal stages and adults appear to be similar.     

A conserved genetic mechanism specifies deutocerebral appendage identity in insects and arachnids

The segmental architecture of the arthropod head is one of the most controversial topics in the evolutionary developmental biology of arthropods. The deutocerebral (second) segment of the head is putatively homologous across Arthropoda, as inferred from the segmental distribution of the tripartite brain and the absence of Hox gene expression of this anterior-most, appendage-bearing segment. While this homology statement implies a putative common mechanism for differentiation of deutocerebral appendages across arthropods, experimental data for deutocerebral appendage fate specification are limited to winged insects. Mandibulates (hexapods, crustaceans and myriapods) bear a characteristic pair of antennae on the deutocerebral segment, whereas chelicerates (e.g. spiders, scorpions, harvestmen) bear the eponymous chelicerae. In such hexapods as the fruit fly, Drosophila melanogaster, and the cricket, Gryllus bimaculatus, cephalic appendages are differentiated from the thoracic appendages (legs) by the activity of the appendage patterning gene homothorax (hth). Here we show that embryonic RNA interference against hth in the harvestman Phalangium opilio results in homeonotic chelicera-to-leg transformations, and also in some cases pedipalp-to-leg transformations. In more strongly affected embryos, adjacent appendages undergo fusion and/or truncation, and legs display proximal defects, suggesting conservation of additional functions of hth in patterning the antero-posterior and proximo-distal appendage axes. Expression signal of anterior Hox genes labial, proboscipedia and Deformed is diminished, but not absent, in hth RNAi embryos, consistent with results previously obtained with the insect G. bimaculatus. Our results substantiate a deep homology across arthropods of the mechanism whereby cephalic appendages are differentiated from locomotory appendages.     

Correlation of diversity of leg morphology in Gryllus bimaculatus (cricket) with divergence in dpp expression pattern during leg development

Insects can be grouped into mainly two categories, holometabolous and hemimetabolous, according to the extent of their morphological change during metamorphosis. The three thoracic legs, for example, are known to develop through two overtly different pathways: holometabolous insects make legs through their imaginal discs, while hemimetabolous legs develop from their leg buds. Thus, how the molecular mechanisms of leg development differ from each other is an intriguing question. In the holometabolous long-germ insect, these mechanisms have been extensively studied using Drosophila melanogaster. However, little is known about the mechanism in the hemimetabolous insect. Thus, we studied leg development of the hemimetabolous short-germ insect, Gryllus bimaculatus (cricket), focusing on expression patterns of the three key signaling molecules, hedgehog (hh), wingless (wg) and decapentaplegic (dpp), which are essential during leg development in Drosophila. In Gryllus embryos, expression of hh is restricted in the posterior half of each leg bud, while dpp and wg are expressed in the dorsal and ventral sides of its anteroposterior (A/P) boundary, respectively. Their expression patterns are essentially comparable with those of the three genes in Drosophila leg imaginal discs, suggesting the existence of the common mechanism for leg pattern formation. However, we found that expression pattern of dpp was significantly divergent among Gryllus, Schistocerca (grasshopper) and Drosophila embryos, while expression patterns of hh and wg are conserved. Furthermore, the divergence was found between the pro/mesothoracic and metathoracic Gryllus leg buds. These observations imply that the divergence in the dpp expression pattern may correlate with diversity of leg morphology.