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     

Ecdysteroid release by the prothoracic gland of Gryllus bimaculatus (Ensifera: Gryllidae) during larval-adult development

The in vitro secretion of ecdysteroids from the prothoracic glands of larvae of Gryllus bimaculatus was analysed by HPLC-RIA. The primary product was identified as 3-dehydroecdysone (65-93%), with lesser amounts of ecdysone (7-35%). Production and release of ecdysteroids from the prothoracic glands are calcium-dependent. The rate of ecdysteroid release was low during the beginning and the end of the last two larval stages and high in between. Prothoracic glands from young adult females produced only minor amounts of ecdysteroids and ceased hormone production around day 4 after the moult.     

Expression of allatostatins in the Mediterranean field cricket, Gryllus bimaculatus de Geer (Ensifera, Gryllidae)

The allatostatin (AST) type A gene of the cricket Gryllus bimaculatus encodes a hormone precursor including at least 14 putative peptides with a common C-terminus Y/FXFGL/Iamide. By RT-PCR we have analyzed the expression of the allatostatin precursor in various tissues of 0-21 days old adult virgin and mated females. In 3-day-old virgin females, the gene is strongly expressed in the brain (oesophageal ganglion), the suboesophageal ganglion and the caecum, but to a lower extent in other parts of the digestive tract (ileum, midgut, colon), and in various other tissues such as the fat body, ovaries and female accessory reproductive glands. In the brain and ovaries of virgin females, the AST expression is rather constant throughout adult life, whereas in brains of mated animals, expression is low until day 7, but increases sharply from day 8 onwards to reach values triple those before day 7. In ovaries of mated animals AST gene expression is also age-dependent, with high expression rates during the first 4 days after imaginable moult, a second but smaller peak from day 15 to 21, and very low values in between. In the fat body of virgin crickets allatostatin expression is high during the first 9 days after ecdysis and declines thereafter, whereas in mated animals two peak values, day 1 and day 6, are observed, and a third peak in older animals.     

Characterization of an iridescent virus isolated from Gryllus bimaculatus (Orthoptera: Gryllidae)

We have isolated an iridescent virus from commercially produced colonies of Gryllus bimaculatus in Germany, which showed apparent mortality. Transmission electron microscopy studies on adult cricket specimens revealed the paracrystalline assembly of icosahedral virus particles in the cytoplasm of hypertrophied abdominal fat body cells. The infecting agent could be cultivated in the lepidopteran cell line sf-9, where it caused cytopathogenic effects such as cell hypertrophy, cytoplasmic vacuolization, and cell death within 8 days postinfection. Infection titers of the first virus passage reached 10(7.5) TCID(50)/ml. Negatively stained virus particles (n = 100) had dimensions of 172 +/- 6 nm (apex to apex) and 148 +/- 5 nm (side to side). SDS-polyacrylamide gel electrophoresis of virus proteins showed more than 20 distinct polypeptides with a major species of approximately 50 kDa. Analysis of the restriction fragment length profiles from digestion of purified viral DNA with the endonucleases EcoRI, BamHI, and HindIII showed marked differences from the profiles of iridoviruses of lower vertebrates (genus Ranavirus), e.g., Rana esculenta Iridovirus and Frog virus 3. Restriction enzyme digests with the endonucleases MspI and HpaII indicated the lack of methylation of viral DNA. Polymerase chain reaction led to the amplification of a 420-bp gene fragment with 97% sequence homology to the major capsid protein gene of Chilo iridescent virus. These data indicate that this new isolate, which we propose to be termed Gryllus bimaculatus iridescent virus, belongs to the genus Iridovirus of the family Iridoviridae.     

Postembryonic development of mating behavior in the male cricket Gryllus bimaculatus DeGeer

Summary The intact male nymph cricket, Gryllus bimaculatus DeGeer, was found to show mating-like behavior, that is, courtship-like behavior (CSLB) and copulation-like behavior (CPLB), in the 7th and 8th (last) instars. The 8th instar nymph exhibited less CSLB and CPLB than the adult but much more than the 7th instar nymph. The movement patterns of CSLB and CPLB were essentially the same as those of adults except for motor acts requiring the use of the genitalia. CSLB was short and often ceased spontaneously before it switched to CPLB. CPLB also ended earlier than in adults. The occurrence of CSLB and CPLB was almost zero the few days around ecdysis. The nymph was very sensitive to disturbance, so that he often stopped courtship for more than 30 min after stimulation. CSLB was similarly induced in the male nymph (8th instar) by pairing with a female adult, male adult, female nymph (8th) and male nymph (8th). The female nymph (8th) was observed to mount not only the male adult but also the male nymph (8th). A fixed time sexual refractoriness forming a basis of cyclical mating activity was not present after CPLB in the nymph. It appeared in association with the emergence of spermatophore protrusion behavior around day 3 after the imaginal molt. In fledglings, there were some transitions during the sexual maturation process, such as failures in hook hanging, spermatophore extrusion, and spermatophore transfer to the female. The decerebration experiments on nymphs and fresh adults agreed with behavioral observations. These results suggest that the development of mating behavior in the male cricket is a process of enhancement of basic motor patterns but not a process of addition of new movements by changes in pattern generation circuits in the central nervous system.Abbreviations CPLB copulation-like behavior - CPPT interval between copulation and spermatophore protrusion - CSCP interval between calling song and copulation - CSLB courtship-like behavior - CSS courtship song - PTCS interval between spermatophore protrusion and calling song - SPE spermatophore extrusion     

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     

Immunological evidence for FGLamide- and W2W9-allatostatins in the ovary of Gryllus bimaculatus (Ensifera, Gryllidae)

Using polyclonal antisera to Gryllus bimaculatus allatostatin A1 (FGLamide) and B1 (W²W⁹‐amide), two sensitive and specific competitive enzyme‐linked immunosorbent assays (ELISAs) were developed, which allowed detection of A‐ and B‐allatostatin‐like immunoreactivity in extracts of ovaries and partially purified chromatography (HPLC) fractions. Immuno‐positive HPLC fractions inhibited biosynthesis of Juvenile Hormone in vitro in the rapid partition assay. Both antisera were also used to immunolocalize A‐ and B‐allatostatin epitopes in the ovary of larval and adult crickets. A‐ and B‐allatostatin immunoreactivity appeared in the cortical cytoplasm of the oocytes at the anterior cell poles. Controls treated with the pre‐immune sera or with antisera preadsorbed onto the respective peptides coupled to a solid phase support did not stain. There was no staining in any neural structures within the ovary.     

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.     

Calling song and selective phonotaxis in the field crickets,Gryllus firmus andG. pennsylvanicus (Orthoptera: Gryllidae)

The field cricket species, Gryllus firmusand G. pennsylvanicus,occur in a mosaic hybrid zone that roughly parallels the eastern slope of the Appalachian mountains in the northeastern United States. It is important to know what role, if any, the calling song plays in mate choice in sympatric and allopatric populations. In this report, we present results on the variability of calling song properties along transects across this hybrid zone. We also present the results of experiments on phonotactic selectivity of females from an allopatric population of G. firmus.The male calling song of allopatric G. firmuswas significantly slower in temporal rhythm (i. e., chirp and pulse repetition rates) and lower in pitch (i.e., dominant frequency) than that of allopatric G. pennsylvanicus.Calling song properties of males recorded in the hybrid zone varied considerably in temporal and spectral properties. In two-stimulus (choice) phonotaxis experiments, allopatric females of G. firmuspreferred synthetic calling songs with conspecific pulse repetition rates over songs that had lower and higher pulse rates. This preference persisted even when the sound pressure levels of alternative stimuli were unequal. Therefore, allopatric females of G. firmuscan discriminate between conspecific and heterospecific calling songs. Whether or not this same selectivity is present in sympatric populations remains unclear. Investigations of phonotactic selectivity in other allopatric and sympatric populations of both species are currently under way.     

Neurohormonal control of the mating interval in the male cricket,Gryllus bimaculatus DeGeer

Summary Between two mating acts of the male cricket (Gryllus bimaculatus), spermatophore protrusion (SP) and courtship stridulation (CS), there is a fixed time interval. This interval lasts about 1 h. During the period from SP to CS, the male cricket does not stridulate nor make any type of mating sound (post-spermatophore protrusion silence: PSPS) and tolerates external sensory stimuli. We examined the effects of injections of hemolymph and ganglia extracts on the interval. Extracts obtained from crickets which had just started CS (CS crickets) and those which had finished SP (SP crickets) were effective. The extracts were fractionated by ul trafiltration. Fractions with a molecular weight of less than 1 kdalton affected the length of the PSPS. The fractions from both the hemolymph and the mesothoracic ganglion of CS crickets shortened the PSPS. On the other hand, the fractions from the hemolymph and the brain of SP crickets lengthened the PSPS. We estimated, by gel filtration, the molecular weight of the effective fractions from the mesothoracic ganglion and the brain to be 100–200 daltons. We also examined the effects of biogenic amines on the PSPS. Octopamine shortened the PSPS, whereas serotonin lengthened it. The results in dicate that at least two neurohormones from the brain and the mesothoracic ganglion reciprocally control the elicitation of CS and provide an appropriate interval in the mating sequence of the male cricket. Octopamine and serotonin are possible candidates for these neurohormones.Abbreviations CP copulation - CS courtship stridulation - SP spermatophore protrusion - PSPS post-spermatophore protrusion silence     

Detailed developmental morphology of the spermatophore of the Mediterranean field cricket, Gryllus bimaculatus (De Geer) (Orthoptera: Gryllidae)

The detailed developmental morphology of the spermatophore of a cricket (Gryllus bimaculatus, De Geer) is described for the first time. A specific behavioural reference point is identified from which time developmental processes proceed in a more or less robotic manner. The structures previously identified as forming part of the fully formed spermatophore, that is, the ampulla, the sperm tube and the attachment plate are all identified in detail during their development. Interestingly the attachment plate material is extruded as an amorphous mass from the ejaculatory duct prior to its insertion into the dorsal pouch, the sperm do not enter the sperm sac until the end of spermatophore formation, and the sperm tube itself is formed from a flattened layer of material which rolls up into a tube and involves the use of a die mechanism.     

Male responses to conspecific advertisement signals in the field cricket Gryllus rubens (Orthoptera: Gryllidae)

In many species males aggregate and produce long-range advertisement signals to attract conspecific females. The majority of the receivers of these signals are probably other males most of the time, and male responses to competitors' signals can structure the spatial and temporal organization of the breeding aggregation and affect male mating tactics. I quantified male responses to a conspecific advertisement stimulus repeatedly over three age classes in Gryllus rubens (Orthoptera: Gryllidae) in order to estimate the type and frequency of male responses to the broadcast stimulus and to determine the factors affecting them. Factors tested included body size, wing dimorphism, age, and intensity of the broadcast stimulus. Overall, males employed acoustic response more often than positive phonotactic response. As males aged, the frequency of positive phonotactic response decreased but that of the acoustic response increased. That is, males may use positive phonotaxis in the early stages of their adult lives, possibly to find suitable calling sites or parasitize calling males, and then later in life switch to acoustic responses in response to conspecific advertisement signals. Males with smaller body size more frequently exhibited acoustic responses. This study suggests that individual variation, more than any factors measured, is critical for age-dependent male responses to conspecific advertisement signals.     

The characteristics of karyotype and telomeric satellite DNA sequences in the cricket, Gryllus bimaculatus (Orthoptera, Gryllidae)

The chromosomes derived from the Japanese population of Gryllus bimaculatus were characterized by C-banding and Ag-NOR staining. The chromosome number, 2n = 28 + XX (female)/XO (male), corresponded with that of other populations of G. bimaculatus, but the chromosome configuration in idiograms varied between the populations. NORs were carried on one pair of autosomes and appeared polymorphous. The positive C-bands located at the centromere of all chromosomes and the distal regions of many chromosome pairs, and the size and the distribution pattern of the distal C-heterochromatin showed differences among the chromosomes. In addition, this paper reports on the characteristics of HindIII satellite DNA isolated from the genome of G. bimaculatus. The HindIII repetitive fragments were about 0.54 kb long, and localized at the distal C-bands of the autosomes and the interstitial C-bands of the X chromosome. Molecular analysis showed two distinct satellite DNA sequences, named the GBH535 and GBH542 families, with high AT contents of about 67 and 66%, respectively. The two repetitive families seem to be derived from a common ancestral sequence, and both families possessed the same 13-bp palindrome sequence. The results of Southern blot hybridization suggest that the sequence of the GBH535 family is conserved in the genomic DNAs of Gryllus species, whereas the GBH542 family is a species-specific sequence.