Ecdysone metabolism in adult crickets,Gryllus bimaculatus

The fate of injected [³H]ecdysone has been investigated in female and male adults of the Mediterranean field cricket, Gryllus binaculatus (de Geer). The metabolism is similar in both sexes and at various stages of adult life. Several classes of apolar metabolites (A1–A5) represent the major compounds. The amount of polar conjugates is low in all tissues, as are the concentrations of 20-hydroxyecdysone. Ovaries are the only organs capable of storing considerable amounts of ecdysteroids. The amount of radiolabelled ecdysteroid activity (mostly [³H]ecdysone) excreted during the first 24 hr after injection is high.The chemical identity of the apolar metabolites is not yet known. A2, which is the major apolar compound, has recently been identified as a complex of ecdysone conjugates with abundant long-chain fatty acids (Hoffman et al., 1985 Life Sci.37, 185–192). Incubations with tissue homogenates in vitro have shown that several organs are capable of converting ecdysone into apolar compounds. Apolar ecdysteroid acyl esters represent a newly identified class of ecdysone conjugates from insects. Their role in regulation of free ecdysteroid titres during the reproductive period in female crickets is discussed.     

Effects of previous experience on the agonistic behaviour of male crickets, Gryllus bimaculatus

Male solitary animals frequently enter aggressive interactions with conspecific individuals to protect their territory or to gain access to females. After an agonistic encounter, the loser (subordinate individual) changes its behaviour from aggression to avoidance. We investigated agonistic interactions between pairs of male crickets to understand how dominance is established and maintained. Two na?ve males readily entered into agonistic interactions. Fights escalated in a stereotyped manner and were concluded with the establishment of dominance. If individuals were isolated after the first encounter and placed together 15 minutes later, subordinate crickets tended to avoid any further contact with the former dominant opponent. Moreover, subordinate males also avoided unfamiliar dominant and na?ve opponents. They displayed aggressive behaviour only towards unfamiliar subordinate opponents. This suggests that the subordinate male change their behaviour depending on the dominance status of the opponent. Dominant crickets, in contrast, displayed aggressive behaviour towards familiar as well as unfamiliar opponents. If the interval between the first and second encounter was longer than 30 minutes, the former subordinate male showed aggressive behaviour again. However, if the subordinate cricket was paired with the same opponent three consecutive times within 45 minutes, it avoided the former dominant opponent for up to 6 hours following the third encounter. Our results suggest that the maintenance of dominance in male crickets depends largely on the behavioural change of subordinate individuals. Possible mechanisms to maintain dominance are discussed.     

Aggressive behavior of the white-eye mutant crickets, Gryllus bimaculatus

Aggressive behavior of white-eye mutant crickets was investigated and compared with that of wild-type crickets. In the dark, wild-type pairs performed long-lasting fights with significantly higher aggressive levels compared to those in the light. In contrast, fights between two white-eye mutants were not significantly different with those between two wild-type crickets both in duration and the aggressive levels. Ethograms of aggressive behavior showed that the mutants could show typical sequentially escalating fight with the same behavioral categories as the wild-type crickets. These results indicate that the white-eye mutants are able to express normal aggressive behavior.     

Experience-based agonistic behavior in female crickets, Gryllus bimaculatus

Fighting behavior in male crickets is already well described, and some of the mechanisms underlying aggression and aggressive motivation have already been revealed. Much less is known about female/female interactions. Here, we report that adult female crickets that had been isolated for several days readily entered into agonistic interactions with conspecific individuals. Characteristic dyadic encounters between isolated females escalated in a stepwise manner and were concluded with the establishment of a dominant/subordinate relationship. For 15 to 30 minutes following an initial fight, former subordinate females showed a dramatic change in agonistic behavior. If they were paired with the former dominant opponent during this interval, a significant majority did not enter into any aggressive interaction but instead actively avoided the opponent. A similar experience-based and time-dependent increase in avoidance was observed when former subordinate females were paired with unfamiliar na?ve opponents. However, when faced with an unfamiliar subordinate individual in the second encounter, no such increase in avoidance behavior was observed. We propose that the observed changes in the behavior of former subordinate females are the consequence of a change in the general state of arousal and of the recognition of dominance status, but not of individual recognition. The fact that former dominant individuals did not show similar experience-based changes in agonistic behavior suggests that dominant/subordinate relationships between pairs of female crickets are maintained mainly by the behavior of subordinate individuals.     

Free ecdysteroids in adult male crickets, Gryllus bimaculatus

ABSTRACT. Ecdysteroid titres were determined in testes, fat body, muscles, haemolymph, carcass tissue, spermatophores, and faeces of males of the Mediterranean field cricket, Gryllus bimaculatus de Geer, throughout its adult life span. Considerable amounts of free ecdysteroids are concentrated in the testes and the fat body. The ecdysteroid titres were only slightly influenced by environmental temperature. In all tissues except the fat body, ecdysone and 20-hydroxyecdysone were the predominant ecdysteroids present. In faeces, highest ecdysteroid concentrations were found at the time of lowest levels in tissues.     

Apolar ecdysteroid esters in adult female crickets, Gryllus bimaculatus

Six hours after injection of 0.5 microCi 3H-ecdysone into the hemocoele of adult female crickets, several labelled compounds could be separated from hemolymph and tissues by silicic acid column chromatography, TLC, and HPLC. The amount of conjugated, polar ecdysteroids was low in all tissues, whereas apolar metabolites were predominant in all tissues. The apolar compound A2, which is the most abundant in quantity, could be hydrolyzed by porcine liver esterase, yielding ecdysone and various long chain fatty acids. This represents a new class of apolar ecdysteroid conjugates not yet found in other insects.     

Effects of aggression and wing removal on brain serotonin levels in male crickets,Gryllus bimaculatus

When pairs of adult male crickets (Gryllus bimaculatus) that had been housed individually for 7 days were placed together, they fought, and dominant-subordinate relationships were formed within 1min. Aggressive behavior by the dominant male was repeated during the period in which the two males were kept together. Immediately after 10min of aggressive interaction, brain serotonin (5-hydroxytryptamine, 5-HT) levels were unchanged in dominant males and significantly reduced in subordinate males. The emission of aggressive song by dominant males is known to be abolished by removal of the wings. All wings were thus removed from male crickets. After 7 days of isolation, pairs of wingless males were placed together. The wingless males fought and formed dominant-subordinate relationships within 1min. The wingless, dominant males displayed aggressive behavior. Brain 5-HT levels in the wingless males were reduced immediately after 10min of aggressive interaction, and no significant differences in brain 5-HT levels were detected between the dominant and subordinate males, unlike the case for intact males. These data indicate a difference in brain serotonergic activity between dominant and subordinate male crickets during aggressive interaction, and suggest that aggressive behavior by dominant male crickets rapidly reduce brain 5-HT levels in subordinate ones. Furthermore, the data suggest that aggressive song is responsible for the change in brain 5-HT levels.     

Effects of NO/cGMP signaling on behavioral changes in subordinate male crickets, Gryllus bimaculatus

After a loss against an opponent, the aggressiveness of a male cricket is significantly reduced for up to 30 minutes. This depression of aggressiveness is an important factor in the establishment and maintenance of dominance between individuals. In the present study, we investigated the functional roles of nitric oxide (NO) signaling in the depression of aggressiveness in subordinate male crickets. Pairs of male crickets, pre-injected with various NO-related reagents, were allowed to establish dominant/subordinate relationships in dyadic encounters. Opponents were separated for 15 minutes and then paired again. In second encounters, subordinate crickets pre-injected with PTIO (NO scavenger) showed agonistic behavior towards former dominant opponents. A similar effect was observed in crickets pre-injected with L-NAME (NO synthase inhibitor) or ODQ (soluble guanylate cyclase inhibitor). The effects of the latter two drugs were canceled by co-injection of NOR3 (NO donor) with L-NAME or by co-injection of 8-Br-cGMP (cGMP-analog) with ODQ. Injection of NOR3 alone prolonged the inhibition of agonistic behavior in subordinate crickets from 30 minutes to 3 hours. Our results suggest that the change in agonistic behavior observed in subordinate male crickets is closely linked to NO-mediated cGMP signaling.     

双斑蟋的胚胎发育

为了从组织胚胎学角度探究昆虫胚胎发育过程,以双斑蟋Gryllus bimaculatus de Geer的卵为实验材料,通过观察、记录蟋蟀胚胎每一天的形态变化并使用显微摄影方法记录胚胎发育过程,对蟋蟀卵胚胎发育全过程进行系统的观察和研究。根据胚胎形态的发育特点,可将整个胚胎发育过程分为7个阶段:卵裂期、囊胚期、原肠胚、无节幼体期Ⅰ、无节幼体期Ⅱ、无节幼体期Ⅲ、无节幼体期Ⅳ。经历14天,蟋蟀的头部、触角、3对足、尾部、腹部及背部都发育完全,整个胚胎发育随之结束。     

Effects of self-generated wind on compensational recovery of escape direction in unilaterally cercus-ablated crickets, Gryllus bimaculatus

The effects of self-generated wind on the compensational recovery of escape direction were investigated in unilaterally cercus-ablated crickets, Gryllus bimaculatus. To separate walking and self-generated wind during walking, unilaterally cercus-ablated crickets were placed on a styrofoam ball that was easily rotated by leg motion during walking. The stationary walking on the ball did not produce self-generated wind, because no body motion occurred. Crickets that were trained on the ball but given no artificial air puff for 14 days after cercal ablation did not show any compensational recovery of escape direction. Therefore, spontaneous walking itself was not sufficient to compensate the wind-evoked escape direction in the crickets. Artificial air puffs from the anterior direction synchronized with the stationary walking were effective for the compensational recovery of escape direction, but those from the posterior direction were not. As most of the spontaneous walking was directed to the forward direction, only an artificial air puff from the anterior direction coincided well with actual self-generated wind occurring during the onset of normal walking. Therefore, self-generated wind during walking seems essential for the compensational recovery of escape direction in unilaterally cercus-ablated crickets. When artificial air puffs were unsynchronized with walking, no compensational recovery was observed. This result suggests that artificial air puffs should be given just after the onset of spontaneous walking. Otherwise, the artificial air puffs may not be recognized as self-generated wind.     

Changes in the biochemical composition of fat body stores during adult development of female crickets, Gryllus bimaculatus

Age-dependent changes in the fat body composition and aspects of lipogenesis in the free abdominal fat body of female crickets, Gryllus bimaculatus, were studied. Lipid, protein, glycogen, and free carbohydrate content of the fat body, and fat body wet weight increased simultaneously and sharply from day 0 onwards and were doubled/almost doubled by day 2 after adult emergence. Lipogenic activity of the fat body, fat body weight, and the energy stores in the fat body peaked on day 2, except for free carbohydrate, which peaked on day 3. On day 2, the fat body was mainly comprised of lipid (53.8%) and protein (6.6%), while glycogen and free carbohydrate together contributed less than 1% of the fat body wet weight. After peaking, both lipogenesis and energy stores decreased in a synchronous manner. The depletion of the fat body energy stores and the consequent decrease in the fat body weight were concomitant with a fast and massive gain in ovary weight (day 2: 19.5 +/- 1.5 mg; day 4: 332.8 +/- 31.5 mg) due to the vitellogenic oocyte growth that started on day 2. Our data clearly underline the importance of the free abdominal fat body as a source of energy for reproduction in the cricket. Fat body fatty acid synthase activity coincided with lipogenic activity. Adipokinetic hormone inhibits lipid synthesis in the fat body, but treatment of the fat body with adipokinetic hormone in vitro showed no consistent effect on fatty acid synthase activity.     

Effects of a daily temperature cycle on ecdysteroid and cyclic nucleotide titres in adult female crickets, Gryllus bimaculatus

ABSTRACT. Ecdysteroid and cyclic nucleotide titres were determined in ovaries, fat body, muscles, haemolymph and the remaining carcass tissue (cyclic nucleotides only in ovaries and fat body) of females of the Mediterranean field cricket, Gryllus bimaculatus de Geer, during its adult life span. Under a daily temperature cycle 24°: 12°C (16:8h), ecdysteroid levels of the ovaries and fat body reached maximal values 5 times as great and about 10 days earlier than they did under constant 20°C. Under both temperature regimes the highest ecdysone concentrations coincided with the maximum in ovarian fresh weight as well as with the maximum oviposition rate. In the ovaries, titres of c-AMP and c-GMP changed roughly in parallel, the levels of c-GMP, however, were much lower than those of c-AMP. A comparison of the cyclic nucleotide profiles in the ovaries with the ecdysteroid profile shows that the cyclic nucleotide concentrations increase when ecdysteroid titres are still low, and that the highest cyclic nucleotide levels were reached 6–12 days earlier than the highest ecdysteroid titres.     

Sensitivity of larval and adult crickets (Gryllus bimaculatus) to adipokinetic hormone

The transport of lysine has been investigated in epithelial cells isolated from chicken jejunum. The kinetics of lysine transport and the pattern of interaction with zwitterionic amino acids were consistent with system b(0,+) activity, the broad-spectrum and Na(+)-independent amino acid transporter. The half-saturation constant for lysine entry (K(m)+/-S.E.) was 0.029+/-0.002 mM and the flux was not affected significantly by Na(+) replacement with choline. Lysine influx was inhibited by L-leucine both in Na(+) and choline medium with inhibition constants (K(i)+/-S.E.) 0.068+/-0.006 mM (in Na(+)) and 0.065+/-0.009 mM (in choline). Other inhibitory amino acids (K(i)+/-S.E.) were (mM): L-tyrosine (0.073+/-0.018), L-methionine (0.15+/-0.015), L-cystine (0.42+/-0.04), L-cysteine (1.1+/-0.07), L-isoleucine (1.1+/-0.09), L-glutamine (1.8+/-0.16) and L-valine (2.5+/-0.13). Lysine exit was trans-accelerated (approx. 20 fold) by 2 mM L-lysine and L-leucine. The flux was resistant to pretreatment of the cells with p-chloromercuriphenylsulfonate (0.2 mM), which is an inhibitor of system y(+)L, the broad-spectrum and cation-modulated transporter.     

In vivo effects of allatostatins in crickets,Gryllus bimaculatus (Ensifera: Gryllidae)

Two types of cricket allatostatins, Grb-AST A1 and Grb-AST B1, were injected into adult female crickets three times each day on days 0–3 and once on day 4 after adult emergence to test their activity in vivo. On day 4, body weight, ovary weight, number of eggs per ovary, length of the terminal oocytes, ovarian ecdysteroid biosynthesis, and hemolymph titers of ecdysteroids were lower in the allatostatin-injected animals compared with untreated and Ringer-injected controls. Effects of the injected allatostatins on hemolymph juvenile hormone titers were inhomogeneous, and no differences were found in the capacity of the corpora allata to produce juvenile hormone ex vivo. The hemolymph titers of yolk proteins (vitellogenins) were almost twice as high in the allatostatin-injected animals as in the control animals. The effects of the injected allatostatins and their interactions with the endocrine system of the animal are discussed. Arch. Insect Biochem. Physiol. 38:32– 43, 1998. © 1998 Wiley-Liss, Inc.     

Courtship and copulation, but not ejaculates, reduce the longevity of female field crickets (Gryllus bimaculatus)

Females of many taxa incur fitness costs from male sexual coercion and harassment leading to mating. Although male crickets cannot force copulations on females, female Gryllus bimaculatus in this study incurred significant reductions in longevity through being exposed to different levels of male courtship. Virgin females kept in isolation had the longest life spans. Reductions in longevity applied to females in sensory contact with males (without the opportunity to mate), females that courted and mated and females that mated but with fertilization being prevented. Females also incurred significant reductions in longevity when kept with other females, which may have been due to high levels of cannibalism. Consistent with previous studies, females appeared to incur no cost to longevity from receiving sperm or seminal fluid. It is known that female G. bimaculatus benefit genetically from multiple mating. However, this benefit could possibly be offset by the negative effect that male courtship and mating behaviour has on female longevity.     

Neuroplasticity and phonotaxis in monaural adult female crickets (Gryllus bimaculatus de Geer)

Summary One foreleg was amputated at mid-femur in adultGryllus bimaculatus females. In phonotaxis tests these monaural crickets show course deviations and circling towards the intact side (Fig. 1). Mean course stability is best at 60 and 70 dB (Fig. 2). Here it differs significantly from a threshold value for orientated walking in females operated on the day of adult moult, but not in those operated two weeks later. The orientational performance improves with the interval between amputation and test (Fig. 3).Centripetal cobalt backfills reveal degeneration of tympanal nerve fibers on the amputated side (Fig. 4B, C). The mean number of intact afferents crossing the midline of the prothoracic ganglion is increased in monaural versus binaural crickets. Maximum transmidline extension is not correlated with the period of deafferentation (Fig. 5).Intracellular recording and staining of prothoracic auditory interneurons shows some axonal sprouts in ON1i (intact side) and ON2, but no significant physiological changes (Figs. 6A, D; 8A, C, E, G). Apart from axonal sprouts ON1a (amputated side) may show a few dendritic sprouts into the intact auditory neuropil (Figs. 6C, 7). Excitation in some ON1a-cells reveals functional contacts to intact auditory afferents (via crossing dendrites or possibly crossing afferents, Figs. 6e, 7, 8F). Morphological and associated physiological changes start early in AN2a (amputated side). The degree of crossing dendrites and contralateral excitation increases with postoperative age (Figs. 8H, 9).     

Juvenile hormone and reproduction in crickets, Gryllus bimaculatus De Geer: corpus allatum activity (in vitro) in females during adult life cycle

ABSTRACT. Incubation conditions were established for a short-term radiochemical assay of spontaneous juvenile hormone (JH) biosynthesis in vitro by corpora allata from adult female Gryllus bimaculatus. The only JH synthesized was shown by HPLC to be JH III. A further incubation product, predominantly extracted from the corpora allata, was thought to be the JH III precursor, methyl farnesoate. In adult females reared at a constant temperature of 27°C the synthetic activities of the corpora allata-corpora cardiaca complexes in vitro increase from almost zero to a high peak value 4 days after the imaginal moult. Thereafter the activity decreases to varying intermediate levels, but always lower than the first maximum. Two days after the first peak in corpus allatum activity, ovarian fresh weight increases dramatically and the first oviposition occurs 2 days later.
Topical application of JH III to females reared at 20°C, which usually have a low fecundity, causes a dose-dependent stimulation of egg production and oviposition.     

New microsatellite loci isolated from the field cricket Gryllus bimaculatus characterized in two cricket species, Gryllus bimaculatus and Gryllus campestris

We have developed a new set of 27 polymorphic markers for each of two cricket species, Gryllus bimaculatus and Gryllus campestris. Initially, 14 published G. bimaculatus loci were tested in G. campestris; however, only five loci were polymorphic. Therefore, we isolated an additional 50 new microsatellite loci from G. bimaculatus and tested these in both species. In a minimum of 20 individuals, 27 of the new loci were polymorphic in G. bimaculatus and 25 in G. campestris.     

Protein deprivation decreases male survival and the intensity of sexual antagonism in southern field crickets Gryllus bimaculatus

Recent theory predicts that the magnitude of sexual antagonism should depend on how well populations are adapted to their environment. We tested this idea experimentally by comparing intersexual genetic correlations for adult survival in pedigreed populations of southern field crickets (Gryllus bimaculatus) raised on naturally balanced (free‐choice) vs. imbalanced (protein‐deprived) diets. We tested for (1) sex differences in nutritional intake and preference, (2) sex‐specific effects of protein deprivation on survival and (3) diet dependence of the level of sexual antagonism. Adult males and females consumed a similar amount of protein, but protein deprivation decreased male survival but not female survival. Protein deprivation appeared to decrease the degree of sexual antagonism as intersexual genetic correlations were significantly lower than 1 only for the complementary free‐choice diet group but close to 1 for the protein‐deficient diet group. Our findings thereby implied that variation in nutritional environments can alter the magnitude of sexual antagonism. This research represents an important step towards understanding the relationship between sexual antagonism and adaptation in heterogeneous environments.     

The effects of injecting Juvenile Hormone III into the prothoracic ganglion on phonotaxis by female crickets Gryllus bimaculatus

Nanoinjection of Juvenile Hormone III (JH III) into the prothoracic ganglion causes virgin female crickets Gryllus bimaculatus De Geer to become more phonotactically selective for the syllable periods (SPs) of model calling songs. Females responding to all, or almost all, of the SPs presented before JH III injection significantly narrow their responses to a range of SPs that is usually centered on the SPs included in the conspecific males' calling song. Control injections of acetone (i.e. the solvent for JH III) into the prothoracic ganglion do not significantly change the recipient females' phonotactic responses. Injection of JH III into the metathoracic ganglion also has no effect the females' phonotactic choices.