Synthesis of lipids in the fat body of Gryllus bimaculatus: age-dependency and regulation by adipokinetic hormone.

The free abdominal fat body of adult female Mediterranean field crickets, Gryllus bimaculatus, synthesizes lipids from [1-(14)C]-acetate in vitro. Up to an age of 12h, the incorporation of acetate into lipids is very low and then rises to a maximum 24h after adult emergence. Thereafter, the incorporation of acetate decreases to moderate levels at day 2 and then slowly decreases until day 30. The adipokinetic hormone of G. bimaculatus (Grb-AKH) significantly inhibits the incorporation of acetate at a concentration of 10(-11) M; maximum inhibition (approximately 95%) is reached at 10(-8) M. The inhibiting effect of Grb-AKH is fast, dose-dependent, and reversible. The periovaric fat body shows a similar pattern of acetate incorporation, although rates of incorporation are lower; the incorporation can be inhibited by Grb-AKH as well. The segmental abdominal fat body and the fat body from the head both incorporate acetate into lipids at low rates that cannot be inhibited significantly by AKH. Prepurified brain extracts significantly inhibit acetate incorporation by free abdominal fat bodies at a concentration of 0.1 brain equivalent. Allatostatins and crustacean cardioactive peptide, which are both present in cricket brains, are not responsible for this inhibiting effect. Octopamine causes a dose-dependent inhibition of acetate incorporation whereas synephrine had no such effect. The inhibiting effect of Grb-AKH on the formation of lipid stores in the fat body and its consequences for reproductive processes are discussed.     

Age-dependent cyclic locomotor activity in the cricket, Gryllus bimaculatus, and the effect of adipokinetic hormone on locomotion and excitability

Excitability and locomotor activity of male and female last instar larvae and adults of the two-spotted cricket are measured under crowded conditions, allowing the animals to interact with conspecifics during observations. Male and female last instar larvae display age-dependent cyclic patterns of activity with maxima during early to mid scotophase and minima during early photophase. A period of low locomotor activity without time of day-dependent cyclic changes starts 1 day before the final moult and lasts until 1 day after the moult. Then, both excitability and locomotor activity increase and become cyclic again within 2 or 3 days. The cyclic changes gradually dampen in adult females older than 6 days and finally cease. When injected into photophase larvae and adults, adipokinetic hormone (AKH) increases excitability and locomotor activity in a dose-dependent manner, whereas it has no such effect when injected into scotophase animals. Other behaviours (jumping, hind wing trembling) that mostly occur in scotophase crickets are also increased by injecting AKH into photophase crickets. We argue that AKH could be responsible for linking the endogenous clock output with the cyclic changes in locomotor activity. Furthermore, AKH may serve to synchronise metabolism and behaviour to optimise larval development and reproduction.     

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.     

Effects of adipokinetic hormone and its related peptide on maintaining hemolymph carbohydrate and lipid levels in the two-spotted cricket,Gryllus bimaculatus

Adipokinetic hormone (AKH) regulates energy homeostasis in insects by mobilizing lipid and carbohydrate from the fat body. Here, using RNA sequencing data, we identified cDNAs encoding AKH (GbAKH) and its highly homologous hormone AKH/corazonin-related peptide (GbACP) in the corpora cardiaca of the two-spotted cricket, Gryllus bimaculatus. RT-PCR revealed that GbAKH and GbACP are predominantly expressed in the corpora cardiaca and corpora allata, respectively. Phylogenetic analysis confirmed that the identified GbAKH and GbACP belong to the clades containing other AKHs and ACPs, respectively. Injection of synthetic GbAKH and GbACP elevated hemolymph carbohydrate and lipid levels and reduced food intake significantly. In contrast, knockdown of GbAKH and GbACP by RNA interference increased the food intake, although hemolymph lipid level was not altered. Collectively, this study provides evidence that ACP regulates hemolymph carbohydrate and lipid levels in cricket, possibly collaborative contribution with AKH to the maintenance of energy homeostasis.     

Adipokinetic hormone inhibits the formation of energy stores and egg production in the cricket Gryllus bimaculatus

Adipokinetic hormones (AKHs) mobilise lipids, carbohydrates and/or proline from insect fat body stores. In addition, AKHs inhibit lipid and protein synthesis in the fat body. In the current study, 100 pmol homologous Grybi-AKH was injected twice daily into adult female crickets, Gryllus bimaculatus, starting on the day of adult emergence. The effects of the injected AKH on the formation of energy reserves in the fat body and on egg production were measured on day 4 after adult emergence. In comparison to water-injected control animals, lipid and protein content in the fat body of the AKH-injected crickets was significantly reduced, suggesting an inhibitory effect of AKH on the formation of lipid reserves and protein stores. The content of glycogen and free carbohydrate in the fat body was significantly higher in the AKH-injected animals. The most pronounced effect of the AKH-injections was a significant reduction of ovary mass, due to the retarded maturation of the oocytes and the significantly lower number of terminal oocytes produced. It is concluded that AKH inhibits egg production indirectly by interference with the formation of energy stores in the fat body that are mobilised to fuel egg production.     

Lipid mobilization and locomotor stimulation in Gryllus bimaculatus by topically applied adipokinetic hormone

Abstract.  Walking activity of 3-day-old adult female Gryllus bimaculatus (de Geer) (Ensifera, Gryllidae) was measured over 24 h. A high level of locomotor activity during the scotophase was found, which was two- to three-fold higher than that during the photophase. The titre of lipid in the haemolymph was relatively low 2 h after lights on, increased significantly 2 h after lights off, although, 2 h after lights on in the next photophase, the lipid titre had decreased to the basal level. Topical application of homologous Grybi-adipokinetic hormone (AKH) (100 pmol in 20% 2-propanol) led to a significant increase in haemolymph lipids, comparable with the maximal increase caused by injection of AKH (3 pmol in water). Topical application of AKH also stimulated locomotor activity in crickets (maximal stimulation 1.8-fold with 100 pmol Grybi-AKH). The results suggest that AKH penetrates the cuticle quickly. It is assumed that AKH stimulates locomotory activity at least in part via the increase of haemolymph lipid titres; however, the stimulation of locomotor activity via a direct neuromodulatory effect of AKH cannot be excluded.     

Diel rhythms of adipokinetic hormone, fat body response, and haemolymph lipid and sugar levels in the house cricket

Abstract. The adipokinetic hormone (Grb-AKH) content in the corpora car-diaca of the house cricket, Acheta domesticus , varied during a day with two peaks in the scotophase and one peak in the photophase. There were two distinct peaks of total lipid concentration in the blood, one early in the photophase and the other early in the scotophase. Fat body sensitivity to adipokinetic hormone also varied in close synchrony with the lipid rhythm. It was not possible to attribute the rhythm of blood lipid titre unequivocally to either the rhythm of adipokinetic hormone content in the corpora cardiaca or to the rhythm of sensitivity of the fat body to the hormone. In adult crickets the blood carbohydrate titre had two peaks in adult females, one towards the end of scotophase and another in the late photophase (c. 12h apart), but a single peak at the end of scotophase was apparent in last instar larvae. The blood carbohydrate rhythm persists in DD and is therefore endogenous. Carbohydrates were not mobilized by the Grb-AKH, therefore could not be involved in the blood carbohydrate rhythm. Exposure to various day lengths caused shifts in the patterns of the carbohydrate rhythms, but imposition of a cyclic temperature regime had no effect on the rhythm.     

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.     

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.     

Isolation and amino acid composition of the adipokinetic neuropeptide from the corpus cardiacum of the cricket, Gryllus bimaculatus

ABSTRACT. The corpora cardiaca of the cricket, Gryllus bimaculatus De Geer, contain peptidic material which elicits hyperlipaemia in migratory locusts and in crickets. Appreciable amounts of this adipokinetic factor are found only in the corpora cardiaca of the cricket, and not in other parts of the nervous system. The factor is heat-stable and retains its activity after incubation with the exopeptidases leucine aminopeptidase and carboxypeptidase A. Fractionation of a methanolic extract of corpora cardiaca from Gryllus bimaculatus by RP-HPLC shows that the adipokinetic activity is confined to a single absorbance peak. The amino acid composition of the purified adipokinetic neuropeptide was determined after acid hydrolysis with HCI and with methanesulfonic acid. The factor is an octapeptide containing the following amino acid residues in equimolar amounts: Asx, Thr, Ser, Glx, Gly, Val, Phe and Trp. This composition closely resembles the AKHI II molecule from Schistocerca species , and the Gryllus peptide can be assigned to the AKH/RPCH family of neuropeptides.     

piggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus

Transgenic insects have been artificially produced to study functions of interesting developmental genes, using insect transposons such as piggyBac. In the case of the cricket, however, transgenic animals have not yet been successfully artificially produced. In the present study, we examined whether the piggyBac transposon functions as a tool for gene delivery in embryos of Gryllus bimaculatus. We used either a piggyBac helper plasmid or a helper RNA synthesized in vitro as a transposase source. An excision assay revealed that the helper RNA was more effective in early Gryllus eggs to transpose a marker gene of eGFP than the helper plasmid containing the piggyBac transposase gene driven by the G. bimaculatus actin3/4 promoter. Further, only when the helper RNA was used, somatic transformation of the embryo with the eGFP gene was observed. These results suggest that the piggyBac system with the helper RNA may be effective for making transgenic crickets.     

Physics of directional hearing in the cricket Gryllus bimaculatus

In the cricket ear, sound acts on the external surface of the tympanum and also reaches the inner surface after travelling in at least three pathways in the tracheal system. We have determined the transmission gain of the three internal sound pathways; that is, the change of amplitude and phase angle from the entrances of the tracheal system to the inner surface of the tympanum. In addition, we have measured the diffraction and time of arrival of sound at the ear and at the three entrances at various directions of sound incidence. By combining these data we have calculated how the total driving force at the tympanum depends on the direction of sound. The results are in reasonable agreement with the directionality of the tympanal vibrations as determined with laser vibrometry.At the frequency of the calling song (4.7 kHz), the direction of the sound has little effect on the amplitudes of the sounds acting on the tympanum, but large effects on their phase angles, especially of the sound waves entering the tracheal system at the contralateral side of the body. The master parameter for causing the directionality of the ear in the forward direction is the sound wave entering the contralateral thoracic spiracle. The phase of this sound component may change by 130–140° with sound direction. The transmission of sound from the contralateral inputs is dominated by a very selective high-pass filter, and large changes in amplitude and phase are seen in the transmitted sounds when the sound frequency changes from 4 to 5 kHz. The directionality is therefore very dependent on sound frequency.The transmission gains vary considerably in different individuals, and much variation was also found in the directional patterns of the ears, especially in the effects of sounds from contralateral directions. However, the directional pattern in the frontal direction is quite robust (at least 5 dB difference between the 330° and 30° directions), so these variations have only little effect on how well the individual animals can approach singing conspecifics.Abbreviations CS contralateral spiracle - CT contralateral tympanum - IS ipsilateral spiracle - IT ipsilateral tympanum - P the vectorial sum of the sounds acting on the tympanum     

The precision of auditory lateralization in the cricket, Gryllus bimaculatus

ABSTRACT. The precision of auditory lateralization was determined behaviourally for the cricket, Gryllus bimaculatus L. A forced-choice Y-maze test was devised in which the cricket, on entering the test arena, could not — in contrast to free phonotactic approaches — change its walking direction until after it had passed through a narrow wire-mesh tunnel. For a sound frequency of 4.7 kHz, matching the species' calling frequency, the minimum audible angle for correct side discrimination was 15°. For stimulus angles smaller than 15° from the longitudinal body axis, the crickets walked randomly to either side; stimulus angles greater than 25° resulted in all crickets turning correctly. These data reveal a sharply tuned lateral sensitivity for the auditory pathway of crickets, with an optimum at the species' calling frequency of 4.7 kHz (when compared with 3.5 and 6.0 kHz). The results for the forced-choice test are compared with the walking pattern during free phonotactic approaches, in order to determine the possible strategy underlying the acoustic orientation behaviour of the cricket.     

Hypertrehalosaemic and hyperlipaemic responses to adipokinetic hormone in fifth larval instar locusts,Locusta migratoria

In fifth instar larvae of Locusta migratoria the haemolymph lipid concentration is elevated after injection of adipokinetic hormone (AKH). This hyperlipaemic response in larvae remains substantially lower than in adults; over 75% of the mobilized lipid consists of diacylglycerol. In addition, unlike adult locusts, fifth instar larvae also exhibit a consistent, though moderate, hypertrehalosaemic response to AKH. The increases of both lipid and carbohydrate concentrations in larvae are dose-dependent, showing a significant linear regression on log dose in the range 0.2–20 pmol AKH.Glycogen phosphorylase in the fat body of fifth instar larvae as well as young adults is activated on injection of AKH, the percentage active phosphorylase increasing linearly with log dose in the range 0.04–20 pmol AKH. For a given response, a somewhat higher dose of AKH is needed in larvae than in young adults.Fat body glycogen phosphorylase is strongly activated during the period of the larval-adult ecdysis, when active phosphorylase accounts for almost half of the total enzyme, which is approximately ten times more than it is two days before, and two days after the ecdysis.The corpora cardiaca of fifth larval instar locusts already possess the potencies to elevate carbohydrate and lipid concentrations in larval haemolymph, and to activate fat body glycogen phosphorylase.     

Photoperiodic modulation of circadian rhythms in the cricket Gryllus bimaculatus

The waveform and the free-running period of circadian rhythms in constant conditions are often modulated by preceding lighting conditions. We have examined the modulatory effect of variable length of light phase of a 24h light cycle on the ratio of activity (alpha) and rest phase (rho) as well as on the free-running period of the locomotor rhythm in the cricket Gryllus bimaculatus. When experienced the longer light phases, the alpha/rho-ratio was smaller and the free-running period was shorter. The magnitude of changes in alpha/rho-ratio was dependent on the number of cycles exposed, while the free-running period was changed by a single exposure, suggesting that there are separate regulatory mechanisms for the waveform and the free-running period. The neuronal activity of the optic lobe showed the alpha/rho-ratio changing with the preceding photoperiod. When different photoperiodic conditions were given to each of the two optic lobe pacemakers, the alpha/rho-ratio of a single pacemaker was rather intermediate between those of animals treated with either of the two conditions. These results suggest that the storage of the photoperiodic information occurs at least in part in the optic lobe pacemaker, and that the mutual interaction between the bilateral optic lobe pacemakers is involved in the photoperiodic modulation.     

Changes in the chemical composition of fat body during the last larval instar of Manduca sexta.

The fat body increases in weight throughout the last larval instar in spite of the loss in total body weight during the wandering and prepupal stages. The protein content of fat body increases dramatically and is greatly responsible for the increase in fat body weight in the wandering and prepupal stages. Lipids do not contribute significantly to the fat body weight gain except during the feeding stage. The amount of total fat body RNA increases until wandering then drops abruptly in the prepupal stage. The total amount of fat body DNA peaks before the onset of wandering and prepupal stages.     

Mobilities of the cercal wind-receptor hairs of the cricket, Gryllus bimaculatus

The deflection sensitivities of cercal filiform hairs of the cricket, Gryllus bimaculatus, were determined by direct measurement. The tangential velocity of deflecting hair shafts in response to stimulus air motion was measured in situ by a laser-Doppler velocimeter with surface scattering of the shaft. The velocity of the stimulus air motion in a small wind tunnel was calibrated by the same velocimeter with smoke from a joss-stick. The mobility of the hair was obtained from former measurements with reference to the latter calibration of the single apparatus. A Gaussian white noise signal was employed as a stimulus waveform, and the stimulus-response transfer function was calculated through a cross-correlation method, which provides greater precision and wider frequency for a longer period of measurement. The mobility of hair was expressed in deflection amplitudes and phase shifts in reference to the velocity sinusoid of a stimulus at various frequencies. The measurements established the following conclusions. The wind receptor hairs comprise an array of mechanical band-pass filters whose best frequencies are inversely proportional to the length. The motion dynamics of the wind-receptor hairs have strong damping. Accepted: 24 February 1998     

Postembryonic changes in circadian photo-responsiveness rhythms of optic lobe interneurons in the cricket Gryllus bimaculatus

The photo-responsiveness of 2 groups of interneurons responding to light in the protocerebrum was investigated at 2 developmental stages, the last instar nymphs and adults, in the cricket Gryllus bimaculatus. The cricket is diurnally active during the nymphal stage but becomes nocturnal as an adult. In both adults and nymphs, light-induced responses of optic lobe light-responding interneurons that conduct light information from the optic medulla to the lobula and the cerebral lobe showed a circadian rhythm peaking during the subjective night. Amplitudes of the rhythms were not significantly different between adults and nymphs, but adults showed more stable day and night states than did nymphs. The medulla bilateral neurons that interconnect the bilateral medulla areas of the optic lobe also showed circadian rhythms in their light-induced responses in both adults and nymphs. The rhythm had a clear peak and a trough in adults, and its amplitude was significantly greater than that of nymphs. These results suggest that the 2 classes of interneurons are differentially controlled by the circadian clock. The difference might be related to their functional roles in the animal's circadian behavioral organization.     

Activity, release and flow of digestive enzymes in the cricket, Gryllus bimaculatus

Abstract.  In the cricket Gryllus bimaculatus , proventricular pressure forces a nutrient fluid from the ground food-brei through a sieve at the base of the caecae, and the combination of secreted enzymes and water causes a rapid inflation of the caecae on the first day after imaginal ecdysis. The ceacal region of the midgut is the primary site for the secretion of digestive enzymes. Proteases and amylase flow from the caecae into the mostly empty crop on day 1, and carbohydrate and protein digestion starts as soon as food is present (6 h). Thereafter, much of the amylase activity (but not protease) in the crop is synthesized and released by the crop tissues themselves. Regurgitating proteases and amylases from the caecae into the crop after day 1 is most likely accomplished by temporarily halting proventricular peristalsis and allowing the caecal muscles to contract, forcing caecal contents, including enzymes, forward. The total activity of digestive enzymes in the caecae is virtually identical in 2-day-old fed and unfed females, indicating little or no secretagogue (prandial) regulation of enzyme secretion. Most of the digestive enzymes in the ventricular endoperitrophic space may originate from the mucus dragged from the caecae. Lipase activity is low in all gut regions in both starved and fed females. Head ligation or injection of trypsin modulating oostatic factor, allatostatin A or B fails to indicate any involvement of nerves or hormones in the release of digestive enzymes in the caecae. Gryllus bimaculatus appears to secrete digestive enzymes continuously, and a considerable loss of enzymes may occur at certain times through egestion.