Haemolymph ecdysteroid titres in diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis

The ecdysteroid titres of last-instar prediapausing, diapausing and nondiapausing larvae of Ostrinia nubilalis were determined by radioimmunoassay. In the nondiapause larvae a major peak of ecdysteroid activity preceded pupation by 24 h and continued through the pupal ecdysis. This peak was correlated with head and thorax critical periods as well as with changes in behaviour and physiology marking the transition from feeding larva to prepupa. Nondiapause larvae also displayed a rise in ecdysteroid titre during the feeding phase of development. This rise was approx one tenth that of the major peak and lasted 32 h. It was not correlated with any overt changes in larval physiology or behaviour. The diapause ecdysteroid profile was distinctive in that the levels measured were all lower than the lowest of the nondiapause curve. No peaks were observed in the diapause titres. Prepupal changes such as spinning and the cessation of feeding were not correlated with any increase in ecdysteroid levels. During diapause termination, under a long-day photoperiod, no increases in ecdysteroid titre were observed for the first 10 days. After 12 days individuals began to show ecdysteroids above the diapause levels. Pupation started after 16 long days.     

Regulation and significance of ecdysteroid titre fluctuations in lepidopterous larvae and pupae

The last larval moult of Galleria mellonella is induced by an elevation of ecdysteroid titre to more than 200 ng/g. After ecdysis the titre remains very low until 70 hr of the last-instar when a slight elevation in ecdysteroid concentration initiates the onset of metamorphosis. An ecdysteroid peak (275 ng/g), which occurs between 108 and 144 hr, is associated with wandering and cocoon spinning. Pupal ecdysis follows about 20 hr after a large ecdysteroid peak (780 ng/g) with a maximum in slowly-mobile prepupae (160 hr of the last larval instar). The ecdysteroid decrease between the two peaks coincides with the period when the larvae exposed to unfavourable conditions enter diapause. The pupal-adult moult is initiated by a high ecdysteroid peak (1500–2500 ng/g) in early pupae and imaginal cuticle is secreted in response to a smaller peak (ca. 500 ng/g) in the middle of pupal instar.Until early pupae, the ecdysteroid content is regulated by the prothoracic glands. In decapitated larvae the glands become spontaneously active after 30–40 days and the body titre of ecdysteroids undergoes an increase; the glands revert to inactivity when the insects accomplish secretion of pupal cuticle. A similar ecdysteroid increase occurs within 10 days when the decapitated larvae receive implants of brains releasing the prothoracicotropic neurohormone (PTTH). In either case, the pupation-inducing increase of ecdysteroids is 3 times higher than the large ecdysteroid peak in the last-instar of intact larvae. This indicates that the function of prothoracic glands in intact larvae is restrained, probably by the juvenile hormone (JH). Exogenous JH suppresses the spontaneous activation of the prothoracic glands in decapitated larvae and reduces the ecdysteroid concentration in those larvae (both decapitated and intact), whose glands were activated by PTTH. Furthermore, JH influences the PTTH release from the brain in situ: depending on JH concentration and the age and size of treated larvae, the PTTH liberation is either accelerated or delayed.Neither in G. mellonella larvae, nor in the diapausing pupae of Hyalophora cecropia and Celerio euphorbiae, does JH directly activate the prothoracic glands. It is suggested that the induction of the moult by JH in decerebrate insects, which has been observed in some species, is either due to indirect stimulation of ecdysteroid production or to increased sensitivity of target tissues to ecdysteroids. In G. mellonella, a moult occurs at a 5–15 times lower than usual ecdysteroid concentration when the last-instar larvae are exposed to JH.     

Influence of leg regeneration on ecdysteroid titres in Acheta larvae

The effects of regeneration on ecdysteroid levels and duration of the 10th larval instar of female house crickets were studied. Three experimental groups were used. The first consisted of larvae regenerating two legs cut off on the first day of this stage. The second group was made up of insects that underwent either an epidermal incision (sham-operated group I) or bleeding (sham-operated group II) at the same time as amputation in the first group. The last group was composed of normal insects.Larval stage length: in the first two groups, the duration of the 10th larval stage was significantly modified. It increased for regenerating animals as well as for sham-operated group I, but decreased for sham-operated group II.Ecdysteroid production: controls showed two periods of intense ecdysteroid activity. The first, which took place between days 4 and 6, was assumed to induce apolysis of the tegument and to render regeneration impossible (it coincided with the so-called critical period for regeneration). The second, which occurred between days 7 and 8 initiated cuticle synthesis. For sham-operated insects neither epidermal injury nor a loss of blood appear to change this hormonal profile significantly. Regenerating crickets however exhibited drastically reduced ecdysteroid peaks: the total apparent production of ecdysteriods was 50% below normal. Moreover, relative concentration of ecdysone to 20-hydroxy-ecdysone was greatly increased. Thus, regeneration obviously has a great and specific influence on ecdysteroid release in insect larvae. This effect may be related to changes observed in the prothoracic gland cycle which suggest that it plays a complex role in the regulation of the regenerative process.     

Haemolymph ecdysteroid titre and epidermal cell commitment of the female southwestern corn borer larvae

The epidermal cell commitment (to pupation or formation of immaculate larvae) and related haemolymph ecdysteroid titres of the southwestern corn borer, Diatraea grandiosella were studied in both nondiapause-bound and diapause-bound last-instar female larvae. Cell commitment was estimated by examining the characteristics of new cuticle secreted in response to an injection of 20-hydroxyecdysone. Haemolymph ecdysteroid titres were determined by radioimmunoassay. Juvenile hormone effect on epidermal cell commitment was studied by applying a juvenile hormone mimic (ZR-515) to last-instar non-diapause-bound larvae and examining the resulting cuticle.In non-diapause-bound larvae, the epidermis of different body regions was committed to pupal development at different times. When pupal cuticular characteristics were evaluated by a scoring system, it appeared that the development of normal pupal cuticle is discontinuous. Three sudden increases in pupal characteristics were observed at 1.67, 2.67 and 3.67 days into the last-larval instar. Haemolymph ecdysteroid titre changes were correlated with the sudden increases in pupal characteristics. Peak ecdysteroid titres were found at 1.67, 2.33, and 3.33 days into the final instar. A fourth ecdysteroid peak (138.8 ng/ml of haemolymph) occurred in pharate pupae. In contrast, the commitment of diapause-bound larvae to produce immaculate integument was made in a fast and continuous fashion. Full commitment was made by 50% of the individuals 4 days (ca. first quarter) into the stadium. Haemolymph ecdysteroid titres fluctuated during the first 2 weeks of the stadium but no significant peaks were observed prior to pharate stage. An ecdysteroid peak (29.8 ng/ml of haemolymph) was identified in pharate immaculate larvae.Pupal development could be completely prevented in 26.7% of nondiapause-bound larvae as late as 4 days into the last instar by topical application of ZR-515. This indicates that the commitment to pupation as revealed by 20-hydroxyecdysone injection is reversible.     

The photoperiodic clock in the flesh-fly, Sarcophaga argyrostoma

Larval cultures of the flesh-fly, Sarcophaga argyrostoma, were raised in experimental light cycles with periods (T) of 21 to 72 hr, each cycle containing a photoperiod of 4 to 20 hr of white light. This ‘resonance’ technique revealed periodic maxima (～24 hr apart) of pupal diapause, thereby demonstrating an endogenous circadian component in the photoperiodic clock. The positions of these maxima of pupal diapause suggested that the oscillation, like that controlling the pupal eclosion rhythm in Drosophila pseudoobscura, is ‘damped out’ by photoperiods longer than about 11 to 12 hr, but restarts at dusk whereupon it runs with circadian periodicity in a protracted dark period. With photoperiods shorter than 12 hr, however, the two diapause maxima were less than 24 hr apart, suggesting that an additional component, possibly a ‘dawn hour-glass’, was modifying the position of the first peak.Both photoperiod and the period of the driving light cycle (T) were shown to affect the length of larval development (the sensitive period) and the number of calendar days needed to raise the incidence of pupal diapause to 50 per cent (the required day number, RDN). Peaks of diapause induction were shown to be the result of an interaction between a long sensitive period (slow development) and a low RDN, whereas troughs in diapause induction were the result of an interaction between a short sensitive period (fast development) and a higher RDN.Larvae of S. argyrostoma are unable to distinguish (in a photoperiodic sense) between 12 and 18 hr of red light (600 nm).     

鞭角华扁叶蜂蜕皮甾类激素滴度的变化

用放射免疫分析法测定了鞭角华扁叶蜂Chinolyda flagellicornis末龄幼虫及滞育预蛹血淋巴中蜕皮甾类激素滴度。结果表明,末龄幼虫血淋巴中蜕皮甾类激素滴度在第2和4天各有一高峰;滞育预蛹血淋巴中保持一定滴度的蜕皮甾类激素,并随发育时期的不同有所波动;预蛹化蛹前一周血淋巴中蜕皮甾类激素滴度存在两个与变态相对应的峰值。表明鞭角华扁叶蜂的滞育与蜕皮甾类激素相关。     

Haemolymph ecdysteroid titres at metamorphosis in the fruit fly Ceratitis capitata: multiple peaks not apparent in whole body extracts

Abstract. The titre profile of haemolymph ecdysteroids in the fruit fly Ceratitis capitata was constructed from determinations of haemolymph taken from single animals and compared with ecdysteroid levels in whole body extracts. Larvae were sampled throughout the period from the late third instar larva until eclosion and were synchronized using a conspicuous behavioural marker ('leaping') that occurs at the end of the wandering period. Extracts of the whole body exhibit two peaks of ecdysteroids, one associated with pupariation ('pupariation peak' of other authors) and the other with pharate adult development. However, the 'pupariation peak' corresponded with three temporally distinct peaks in the titre of haemolymph ecdysteroids. The first peak occurs at the time of head retraction (lh before formation of the white puparium) and may represent the initial response to release of prothoracicotropic hormone. The second peak occurs 2–3 h later at the onset of tanning and may be responsible for induction of dopa-decarboxylase. These peaks are brief ( c . 2h each), whereas the third is much longer (16 h) and occupies most of the 'pupariation peak'. The first two peaks have not been reported previously in any dipteran. Their existence illustrates that extremely brief pulses of ecdysteroids occur in vivo , apparently with profound developmental consequences. The fact that brief pulses in the haemolymph titre are not apparent in whole body extracts emphasizes that results obtained using the latter method must be interpreted with caution.     

Haemolymph ecdysteroid titres during larval-adult development in Rhodnius prolixus: Correlations with moulting hormone action and brain neurosecretory cell activity

A haemolymph ecdysteroid titre of the fifth (last)-larval instar of the hemipteran, Rhodnius prolixus has been determined by radioimmunoassay. During the last-larval stadium the ecdysteroid titre increases from a negligible level in the unfed insect to a detectable level within minutes following a blood meal. The titre reaches a plateau of ～50–70 ng/ml at 3–4 hr and this level is maintained until day 5–6, the time of the head-critical period in Rhodnius. At the head-critical period the titre begins to increase again, this time dramatically, reaching a peak of ～ 3500 ng/ml at day 13. From day 14 to ecdysis (day 21) the titre declines to a low level, ～ 30 ng/ml. Basal levels of ecdysteroids, ～ 15 ng/ml, were detectable in young adult males and females. A survey of haemolymph volumes during the last-larval instar indicates that the changes in the ecdysteroid titre reflect changes in the rates of ecdysteroid synthesis, and not changes in haemolymph volume. Excretion of ecdysteroids varies systematically during the instar, suggesting that control of ecdysteroid excretion may be important in regulation of the haemolymph titre. Qualitative analysis of the haemolymph ecdysteroid RIA activity revealed the presence of only ecdysone and 20-hydroxy-ecdysone. For the large peak preceding larval-adult ecdysis, 20-hydroxy-ecdysone was the predominant hormone. These results indicate that there may be two periods of release of prothoracicotropic hormone (PTTH) from the brain in Rhodnius, one immediately following the blood meal and the second on day 5 or 6. The significance of these times of PTTH release is discussed in relation to classical evidence of the timing of moulting hormone action, the response of target tissues, and with more recent findings on the timing of release of neurosecretory material from the brain of Rhodnius during moulting.     

The timing of larval wandering and puparium formation in the flesh-fly Sarcophaga argyrostoma

ABSTRACT. In mixed-age cultures of the flesh-fly, Sarcophaga argyrostoma (Robineau-Desvoidy), the initiation of larval wandering (exodus behaviour) occurs as a gated circadian rhythm. In light-dark (LD) cycles, most of this activity occurs in the dark, except in very short nights, or in certain phase relationships between the rhythm and light cycle. When transferred from series of LD cycles into continuous darkness (DD), cultures show a weakly persistent free-running rhythm with a period of about 21 h. However, after transfer of first instar larvae from continuous light (LL) to DD, no such rhythm is observed. In contrast to larval exodus, formation of the puparia occurs at any stage of the LD cycle. The physiological mechanisms underlying this gated exodus behaviour, and its possible selective advantages, are discussed.     

Haemolymph ecdysone concentrations in Hyalophora cecropia pupae,dauer pupae and adults

Haemolymph ecdysone concentrations were determined by radioimmunoassay in diapausing pupae, pharate adults, adults, and chilled dauer pupae. The concentration in diapausing pupae after 6 months chilling (5.35 pg/μl) increased dramatically after 3 days at 27°C (>200 pg/μl) and then decreased to low levels in adult females (1.63 pg/μl). In adult males ecdysone was undetectable in all except one animal. Dauer pupae showed a decrease from 6.1 to 1.7 pg/μl 1 day after being transferred from 6 to 27°C. Over a 3-day period the value increased to 3.19 pg/μl and remained constant for more than a year. These results suggest that diapausing pupae with and without brain neurosecretory cells maintain a low concentration of ecdysone in the haemolymph.     

Comparative studies on ecdysone metabolism between mature larvae and pharate pupae in the fleshfly,Sarcophaga peregrina

Metabolites of radioactive ecdysone or 20-hydroxyecdysone in larvae and pharate pupae of Sarcophaga peregrina were separated and identified by using thin-layer chromatography, high-performance liquid chromatography, and chemical methods. At the larval stage ecdysone was metabolized to biologically less active ecdysteroids predominantly through 20-hydroxyecydsone, at the pharate pupal stage, to other ecdysteroids which were tentatively identified as 26-hydroxyecdysone, 3-epi-26-hydroxyecdysone, and 3-epi-20,26-dihydroxyecdysone. Ecdysteroid acids were found in the polar metabolites during pharate pupal-pupal transformation, but scarcely detected in the larval metabolites. These acids were presumed to be ecdysonoic acid, 20-hydroxyecdysonoic acid, and their epimers. The conjugates of ecdysteroid that released the free ecdysteroids by enzymatic hydrolysis were produced more in larvae than in pupae, whereas the very polar ecdysteroids that were not affected by the enzyme were found more in pupae. Therefore, there are different metabolic pathways of ecdysone between these two successive developmental stages, and the alteration of the metabolic pathway may serve as one of the important factors in a regulatory mechanism of molting hormone activity which is responsible for normal development of this insect.     

Juvenile hormone and moulting hormone titres in diapause- and non-diapause destined flesh flies

Failure of the brain to stimulate the prothoracic gland to release ecdysone has been widely regarded as the basis for diapause in insect pupae. In diapause-destined flesh flies, the absence of a peak of moulting hormone around the time of pupal head eversion supports this contention, but in addition, major pulses of juvenile hormone (JH) activity with a rhythmicity of 24 hr are unique to flesh flies destined for pupal diapause. JH activity persists during diapause, and a pulse of JH precedes the rise of moulting hormone that initiates adult development.     

Influence of temperature on the survival and infectivity of Trichinella spiralis larvae in Sarcophaga argyrostoma (Diptera, Sarcophagidae) maggots

To investigate the role of fleshfly maggots as a paratenic host for Trichinella spiralis larvae, maggots of Sarcophaga argyrostoma (Muscidae, Sarcophagidae) kept at different temperatures (26, 22, 20, 16, 12, 8, and 4 C) were allowed to feed on T. spiralis-infected mouse meat. Trichinella larvae found in maggots kept at 8-26 C were able to cause infection when inoculated in mice. Infective larvae survived in maggots up to 5 days postinfection at 8 C and for shorter periods of time at higher temperatures. The survival time in maggots was negatively related to the temperature of maggot breeding. The results suggest that the role of S. argyrostoma in the dissemination of Trichinella larvae in nature is limited in comparison to the role played by mammals with scavenger and cannibalistic behavior.     

Histogenesis of the cuticle of the adult flies,Sarcophaga bullata and S. argyrostoma

Sequential patterns of cuticle deposition and “melanization” in the imaginal cuticle of Sarcophaga argyrostoma in parts of the body darkening before or after emergence are examined on a histological basis. The patterns in the cuticles examined range from a simple absence of “melanization” to a complex of histological changes involving “melanization” and deposition. Ultrastructural changes in the post-emergent cuticle of Sarcophaga bullata during the hardening and darkening process and cuticle deposition are described.     

Difference in nutrient accumulation patterns between diapause-destined and non-diapause-destined larvae of Hyphantria cunea

Nutrient accumulation is crucial in insect diapause preparation because insufficient nutrient accumulation can shorten the diapause period, interfere with diapause development completion, and decrease the probability of surviving the overwintering period. The amounts of lipids and carbohydrates stored in diapausing pupae of Hyphantria cunea (Drury) (Lepidoptera: Erebidae, Arctiinae) are greater than those in the non-diapausing pupae. In this study, we tested the hypothesis that diapause-destined (DD) and non-diapause-destined (NDD) larvae of H. cunea have different nutrient accumulation patterns in penultimate and final instars. The body mass, as well as lipid, carbohydrate, and soluble protein contents, and the efficiency of converting digested food and ingested food into body matter were greater in the DD penultimate and final instars than in the NDD penultimate and final instars. Larger amounts of lipids, carbohydrates, and proteins were absorbed by DD penultimate and final instars and the final instar development period in the DD larvae was prolonged relative to NDD larvae. The activities of fatty acid synthase and glycogen synthase of DD penultimate and final instars were significantly higher than those of NDD larvae. These results suggest that the changes in nutrient accumulation patterns between DD and NDD larvae occur in penultimate and final instars, and that the DD larvae increase their nutrient accumulation during diapause preparation by the combined effect of extending their final-instar development period and improving their digestive efficiency; they increase their lipid and carbohydrate stores by increasing the activities of fatty acid synthase and glycogen synthase in the fat body.     

Haemolymph protein patterns in developing tobacco hornworm larvae

Quantitative changes in haemolymph proteins from each physiological phase of the last three larval instars of the tobacco hornworm, Manduca sexta, were studied by means of disk electrophoresis. Twelve anodical migrating protein bands were found, six of which occurred only sporadically. Total protein concentration increased from pharate third instar to late fifth instar larvae, then decreased slightly in the pharate pupal stages. Some individual bands showed cyclic patterns within each instar similar to the overall cyclic pattern of total protein, whereas other bands showed different patterns or no pattern.