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.     

Involvement of Wingless/Armadillo signaling in the posterior sequential segmentation in the cricket, Gryllus bimaculatus (Orthoptera), as revealed by RNAi analysis

In insects, there are two different modes of segmentation. In the higher dipteran insects (like Drosophila), their segmentation takes place almost simultaneously in the syncytial blastoderm. By contrast, in the orthopteran insects (like Schistocerca (grasshopper)), the anterior segments form almost simultaneously in the cellular blastoderm and then the remaining posterior part elongates to form segments sequentially from the posterior proliferative zone. Although most of their orthologues of the Drosophila segmentation genes may be involved in their segmentation, little is known about their roles. We have investigated segmentation processes of Gryllus bimaculatus, focusing on its orthologues of the Drosophila segment-polarity genes, G. bimaculatus wingless (Gbwg), armadillo (Gbarm) and hedgehog (Gbhh). Gbhh and Gbwg were observed to be expressed in the each anterior segment and the posterior proliferative zone. In order to know their roles, we used RNA interference (RNAi). We could not observed any significant effects of RNAi for Gbwg and Gbhh on segmentation, probably due to functional replacement by another member of the corresponding gene families. Embryos obtained by RNAi for Gbarm exhibited abnormal anterior segments and lack of the abdomen. Our results suggest that GbWg/GbArm signaling is involved in the posterior sequential segmentation in the G. bimaculatus embryos, while Gbwg, Gbarm and Gbhh are likely to act as the segment-polarity genes in the anterior segmentation similarly as in Drosophila.     

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.     

Location of micropyles and early embryonic development of the two-spotted cricket Gryllus bimaculatus (Insecta, Orthoptera)

Early embryogenesis of the two-spotted cricket Gryllus bimaculatus was examined by scanning electron microscopy and several fluorescence staining methods, with special reference to these four issues: (i) the location of micropyles; (ii) the transfer of the female pronucleus following meiosis; (iii) the timing of cellularization; and (iv) the process of the germ primordium formation. Between two and four micropyles lie in the mid-ventral region of the egg. The egg nucleus is at the mid-dorsal periphery of the new laid egg, and meiosis resumes and is completed there. The female pronucleus moves to the mid-ventral side, and fertilization occurs there. Energid starts to proliferate and migrates to the periphery of the egg, initiating blastoderm formation. Actin caps surround each superficial nucleus. Cellularization occurs during the blastoderm stage. At a late blastoderm stage, nuclei aggregate in both the posterolateral patch-like regions of the egg to form a germ primordium. The germ primordium looks like a pair of dumbbells. Both the patches shift towards the ventral side and fuse into a germ primordium. The germ primordium contracts to produce a clearly delineated germ band. Observations on distribution patterns of F-actin indicate that, all through the process, the germ primordium retains that unity, and is not separated into two parts.     

Antenna and all gnathal appendages are similarly transformed by homothorax knock-down in the cricket Gryllus bimaculatus

Our understanding of the developmental mechanisms underlying the vast diversity of arthropod appendages largely rests on the peculiar case of the dipteran Drosophila melanogaster. In this insect, homothorax (hth) and extradenticle (exd) together play a pivotal role in appendage patterning and identity. We investigated the role of the hth homologue in the cricket Gryllus bimaculatus by parental RNA interference. This species has a more generalized morphology than Oncopeltus fasciatus, the one other insect besides Drosophila where homothorax function has been investigated. The Gryllus head appendages represent the morphologically primitive state including insect-typical mandibles, maxillae and labium, structures highly modified or missing in Oncopeltus and Drosophila. We depleted Gb’hth function through parental RNAi to investigate its requirement for proper regulation of other appendage genes (Gb’wingless, Gb’dachshund, Gb’aristaless and Gb’Distalless) and analyzed the terminal phenotype of Gryllus nymphs. Gb’hth RNAi nymphs display homeotic and segmentation defects similar to hth mutants or loss-of-function clones in Drosophila. Intriguingly, however, we find that in Gb’hth RNAi nymphs not only the antennae but also all gnathal appendages are homeotically transformed, such that all head appendages differentiate distally as legs and proximally as antennae. Hence, Gb’hth is not specifically required for antennal fate, but fulfills a similar role in the specification of all head appendages. This suggests that the role of hth in the insect antenna is not fundamentally different from its function as cofactor of segment-specific homeotic genes in more posterior segments.     

Expression patterns of dachshund during head development of Gryllus bimaculatus (cricket)

We report that Gryllus bimaculatus dachshund (Gbdac), a cricket homologue of Drosophila dachshund (Dmdac), is expressed in the developing eye and brain. During brain development, Gbdac was first expressed in the medial head region, corresponding to a part of developing protocephalic region, and expressed in the primordial and adult Kenyon cells. During eye development, Gbdac was first expressed in the lateral head region, becoming to the eye primordium and a part of the deutocerebrum. Then, Gbdac was expressed in the posterior region of the eye primordium, prior to the formation of compound eyes. The expression domain shifted to the anterior domain concomitantly with the movement of morphogenetic furrows. Gbdac was also expressed in the developing optic lobes during differentiation of the retina. These expression patterns were compared with those of Dmdac. We found that although developmental processes of the Gryllus eye and brain differ from those of the Drosophila ones, the expression patterns of Gbdac are essentially similar to those of the Dmdac.     

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 influence of auditory and visual experience on the phonotactic behavior of the cricket,Gryllus bimaculatus

Female crickets lacking experience with phonotaxis to conspecific calling song respond to trains of continuously repeated sound pulses (trill), whereas experienced females do not. In the present study such inexperienced crickets were tested for their responsiveness to trills of pulse repetition periods from 30 to 70 ms on a Y- shaped maze. Stimulation with a repetition period of 30 ms led to unexpectedly low phonotactic and exploratory activity. Initial stimulation with trills of 30- ms repetition period drastically reduced the responsiveness of inexperienced animals to conspecific calling song and other attractive stimuli. Effects of visual stimulation on the phonotactic behavior of female crickets are demonstrated. Threatening visual stimuli changed the behavior of experienced animals to a state that resembles that of inexperienced animals. The relevance of these observations is discussed with respect to the development of the auditory pattern recognition mechanism in crickets.     

even-skipped is not a pair-rule gene but has segmental and gap-like functions in Oncopeltus fasciatus, an intermediate germband insect

The pair-rule gene even-skipped is required for the initiation of metameric pattern in Drosophila. But Drosophila segmentation is evolutionarily derived and is not representative of most insects. Therefore, in order to shed light on the evolution of insect segmentation, homologs of the pair-rule gene even-skipped have been studied in several insect taxa. However, most of these studies have reported the expression eve but not its function. We report the isolation, expression and function of the homolog of Drosophila even-skipped from the intermediate germband insect Oncopeltus fasciatus. We find that in Oncopeltus, even-skipped striped expression initiates in a segmental and not pair-rule pattern. Weak RNAi suppression of Oncopeltus even-skipped shows no apparent pair-rule like phenotype, while stronger RNAi suppression shows deletion of nearly the entire body. These results suggest that in Oncopeltus, even-skipped is not acting as a pair-rule gene. In almost all insects, prior to its striped expression, even-skipped is expressed in a conserved broad gap-like domain but its function has been largely ignored. We find that this early broad domain is required for activation of the gap genes hunchback and Krüppel. Given the large RNAi deletion phenotype and its regulation of hunchback and Krüppel, even-skipped seems to act as an über-gap gene in Oncopeltus, indicating that it may have both upstream and downstream roles in segmentation.     

Sites of ecdysteroid biosynthesis in female adults of Gryllus bimaculatus (Ensifera,Gryllidae)

Summary Ovaries from 4-day-old female adults of Gryllus bimaculatus produce about 5 ng of free and conjugated ecdysteroids per hour during a 16-h incubation in Grace's medium. During incubation of pieces of the abdominal integument together with the adjacent segmental fat body, a net synthesis of moulting hormones is observed (2.3 ng per hour per animal), similar to that in the ovary. Separate incubations of disunited abdominal epidermis and segmental fat body tissue result in much lower rates of ecdysteroid synthesis. Ecdysteroid synthesis in ovarian homogenates is about one-third of that in intact organs. This reduction is due to a lack of conjugate formation in homogenates. Homogenates of the abdominal integument complex are no longer capable of synthesizing ecdysteroids. For both tissues, a de novo synthesis of ecdysteroids is corroborated by following the in vitro incorporation of [¹⁴C]-label from cholesterol and [³H]-label from 2,22,25-trideoxyecdysone (5-ketodiol), respectively, into free ecdysone. The rate of incorporation into ecdysone is only 0.0014% for cholesterol but 0.48% for 5-ketodiol. Both tissues represent primary sources of ecdysteroids in female adult crickets.Abbreviations HPLC high performance liquid chromatography - IU international units - NP normal phase - RIA radioimmunoassay - RP reversed phase - SEM standard error of mean     

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.     

Development of the filiform hairs on the cerci of Gryllus bimaculatus Deg. (Saltatoria,Gryllidae)

Summary The filiform hairs, mechanoreceptors of Gryllus, pass through six developmental stages during the last larval stage. The cytoplasm of their sense cells suggests intensive synthesis of protein for cellular metabolism and intercytoplasmic exchange of material via glial evaginations. Ultrahistochemical tests demonstrated acid phosphatase in the lysosomes as well as in components of the Golgi apparatus. There was no significant change in the appearance of the sense cell cytoplasm, indicating a maintained functional state also during molting. The new cuticular apparatus is formed after apolysis by the three enveloping cells. Formation of the replacement hairs is initiated by a cytoplasmic outgrowth of the trichogen cell. During morphogenesis of the new hair, the microtubules serve as a cytoskeleton and probably control the flow of vesicles, which contain phenol oxidase, also demonstrated in the Golgi apparatus, and are incorporated into the new cuticle. Bundles of microfibrils are involved in the surface sculpturing of the replacement hair. The trichogen cell also forms a number of structural elements, e.g. the cup and strut marked geometric peculiarities of which indicate that they are important in the spatial orientation of the dendrite and thus also in transduction. Reduction of the apical cell membrane of the tormogen cell after apolysis permits unrestricted growth of the new hair into the exuvial space. The tormogen cell participates in the formation of the joint membrane, parts of the socket and the articulation of the hair.Supported by the Deutsche Forschungsgemeinschaft. The author thanks Mrs. G. Thomas for drawing the diagrams, and Miss I. Grossman and Mrs. M. Ullmann for technical assistance     

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.     

The nuclear receptor E75A has a novel pair-rule-like function in patterning the milkweed bug, Oncopeltus fasciatus

Genetic studies of the fruit fly Drosophila have revealed a hierarchy of segmentation genes (maternal, gap, pair-rule and HOX) that subdivide the syncytial blastoderm into sequentially finer-scale coordinates. Within this hierarchy, the pair-rule genes translate gradients of information into periodic stripes of expression. How pair-rule genes function during the progressive mode of segmentation seen in short and intermediate-germ insects is an ongoing question. Here we report that the nuclear receptor Of'E75A is expressed with double segment periodicity in the head and thorax. In the abdomen, Of'E75A is expressed in a unique pattern during posterior elongation, and briefly resembles a sequence that is typical of pair-rule genes. Depletion of Of'E75A mRNA caused loss of a subset of odd-numbered parasegments, as well as parasegment 6. Because these parasegments straddle segment boundaries, we observe fusions between adjacent segments. Finally, expression of Of'E75A in the blastoderm requires even-skipped, which is a gap gene in Oncopeltus. These data show that the function of Of'E75A during embryogenesis shares many properties with canonical pair-rule genes in other insects. They further suggest that parasegment specification may occur through irregular and episodic pair-rule-like activity.     

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.     

Evolutionary flexibility of pair-rule patterning revealed by functional analysis of secondary pair-rule genes, paired and sloppy-paired in the short-germ insect, Tribolium castaneum

In the Drosophila segmentation hierarchy, periodic expression of pair-rule genes translates gradients of regional information from maternal and gap genes into the segmental expression of segment polarity genes. In Tribolium, homologs of almost all the eight canonical Drosophila pair-rule genes are expressed in pair-rule domains, but only five have pair-rule functions. even-skipped, runt and odd-skipped act as primary pair-rule genes, while the functions of paired (prd) and sloppy-paired (slp) are secondary. Since secondary pair-rule genes directly regulate segment polarity genes in Drosophila, we analyzed Tc-prd and Tc-slp to determine the extent to which this paradigm is conserved in Tribolium. We found that the role of prd is conserved between Drosophila and Tribolium; it is required in both insects to activate engrailed in odd-numbered parasegments and wingless (wg) in even-numbered parasegments. Similarly, slp is required to activate wg in alternate parasegments and to maintain the remaining wg stripes in both insects. However, the parasegmental register for Tc-slp is opposite that of Drosophila slp1. Thus, while prd is functionally conserved, the fact that the register of slp function has evolved differently in the lineages leading to Drosophila and Tribolium reveals an unprecedented flexibility in pair-rule patterning.     

Female mating behavior in the field cricket,Gryllus pennsylvanicus (Orthoptera: Gryllidae) at different operational sex ratios

The influence of operational sex ratio on the mating behavior of female field crickets,Gryllus pennsylvanicus, was investigated. Females were predicted to be more discriminating under conditions of high mate availability and show less selectivity when males were rare. Such selectivity was indicated in this study with the proportion of courtships leading to a mating changing with sex ratio. Females accepted almost 70% of all courtships at the female-biased sex ratio, but only about half of all courtships were successful at even or male-biased sex ratios. Females moved least at the female-biased sex ratio. There was also a trend for females to be guarded more under male-biased conditions. Female weight did not influence any of the behaviors examined.