Hemolymph ecdysteroid titer and ecdysteroid-dependent developmental events in the last-larval stadium of the silkworm,Bombyx mori: role of low ecdysteroid titer in larval-pupal metamorphosis and a reappraisal of the head critical period

The endocrine regulation of larval-pupal metamorphosis was studied in the silkworm, Bombyx mori, by measuring the following changes: hemolymph ecdysteroid titer, the secretory activity of prothoracic glands and the responsiveness of larvae to ecdysteroids and prothoracicotropic hormone (PTTH), with regard to developmental events such as the occurrence of spinneret pigmentation, initiation of cocoon spinning and onset of wandering stage as indicated by gut purge. These measurements were concentrated especially on the time before and after the head critical period (HCP) which falls 3-4 days before the gut purge ([Sakurai, 1984]). A small increase in the hemolymph ecdysteroid titer was first found during the HCP, and then the titer increased with daily fluctuations. Small but significant titer peaks were found prior to the occurrence of both spinneret pigmentation and gut purge, indicating that an individual titer peak could possess a specific role in development. Responsiveness of larvae to exogenous 20-hydroxyecdysone (20E) after the HCP was markedly higher than that before the HCP. The sensitivity of the prothoracic gland to PTTH also changed during the HCP. The results thus showed that the HCP is not the period after which an additional PTTH release is not required for the developmental events occurring on schedule, but rather it is the period during which complex events occur not only in the endocrine glands but also in the peripheral tissues. In addition, various developmental phenomena before gut purge are brought about by the hemolymph ecdysteroid whose concentration gradually increased with daily fluctuations, and these precise changes in the titer appeared to be important for the sequential occurrence of developmental events in the larval-pupal metamorphosis.     

Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca sexta

The titer of ecdysone in whole animal extracts of Manduca sexta was determined by radioimmunoassay during the fifth (last) larval instar, pharate pupal development and pupation. A subtle peak in ecdysone concentration was noted at day 4 (just prior to the onset of the wandering stage) and a second and greater peak at day 8.5 (coincident with pharate pupal development). The titer fluctuations during development were a result of changes in tissue ecdysone and not of alterations in the ecdysone content of the gut. When prothoracic gland secretory activity was analyzed in vitro at the same stages, the most rapid rate of α-ecdysone secretion was shown to occur on day 7 (one day prior to the peak in whole-animal ecdysone concentration). An earlier peak in prothoracic gland activity may occur at day 4–5. Thin layer and gas-liquid chromatographic analyses revealed developmental changes in the ratio of β:α-ecdysone in hemolymph and whole-animal extracts. It is suggested that the steroid-hydroxylating capacity of the insect increases during the instar.     

Steroidal regulation of hydrolyzing activity of the dietary carbohydrates in the silkworm, Bombyx mori

Blood sugar is an essential energy source for growth and development and is maintained at a constant level through precise regulation of formation and utilization. Sugars are produced from dietary carbohydrates by enzymatic hydrolysis in the digestive tract, which are under the homeostatic control of paracrine and prandial mechanisms in mammals. Here, we show that dietary carbohydrates hydrolyzing activity of the digestive tract is developmentally regulated by the steroid hormone ecdysone in the silkworm, Bombyx mori. The dietary carbohydrates hydrolyzing activity remained high throughout the last larval period and then decreased to negligible levels until the pupal period. However, dietary carbohydrates digestive activities were constitutively high when the steroidogenic organ, prothoracic glands were ablated. The prothoracic glands produced and released a large amount of ecdysone at the end of the larval period, suggesting that ecdysone is responsible for the decrease in dietary carbohydrates hydrolyzing activity. In fact, ecdysone decreased the activity to negligible levels in silkworms lacking the prothoracic glands. The present results indicate that the dietary carbohydrates hydrolyzing activity is regulated by ecdysone and that an increase in ecdysone titer decreases that activity at the end of the larval period, suggesting that ecdysone is essential for metabolic coordination during development.     

Characterization of limb autotomy factor-proecdysis (LAF(pro)), isolated from limb regenerates,that suspends molting in the land crab Gecarcinus lateralis

Molting and limb regeneration are tightly coupled processes, both of which are regulated by ecdysteroid hormone synthesized and secreted by the Y-organs. Regeneration of lost appendages can affect the timing and duration of the proecdysial, or premolt, stage of the molt cycle. Autotomy of all eight walking legs induces precocious molts in various decapod crustacean species. In the land crab Gecarcinus lateralis, autotomy of a partially regenerated limb bud before a critical period during proecdysis (regeneration index <17) delays molting so that a secondary limb bud (2 degrees LB) forms and the animal molts with a complete set of walking legs. It is hypothesized that 2 degrees LBs secrete a factor, termed limb autotomy factor-proecdysis (LAF(pro)), that inhibits molting by suppressing the Y-organs from secreting ecdysone. Molting was induced by autotomy of eight walking legs; autotomy of primary (1 degrees ) LBs reduced the level of ecdysteroid hormone in the hemolymph 73% by one week after limb bud autotomy (LBA). Injection of extracts from 2 degrees LBs, but not 1 degrees LBs, inhibited 1 degrees LB growth in proecdysial animals, thus having the same effect on molting as LBA. The inhibitory activity in 2 degrees LB extracts was stable after boiling in water for 15 min, but was destroyed by boiling 15 min in 0.1 N acetic acid or incubation with proteinase K. These results support the hypothesis that LAF(pro) is a peptide that resembles a molt-inhibiting hormone.     

Restricted dietary protein in pregnant beef cows: II. Effect on the immune response

Cows fed 0.37 Kg crude protein per day for four months before calving showed no decrease in colostral IgG, IgA, or IgM from cows fed a normal ration. Similarly, no difference in serum immunoglobulin concentration was detected in their calves. Cows in the low protein group developed significantly lower (P < 0.01) titers to Salmonella pullorum antigen than did the normal group. The data show significant positive linear regressions of calf agglutination titer to S. pullorum on the dam's colostral titers. Serum levels of IgG measured at 24 hours were significantly lower in calves from second-calf heifers than from these of other age groups. The IgA levels of serums from calves from the six-year-old cows were significantly higher than the serum levels of calves from younger dams (P < 0.01).     

Bipolar head regeneration induced by artificial amputation in Enchytraeus japonensis (Annelida, Oligochaeta)

The Enchytraeida Oligochaeta Enchytraeus japonensis propagates asexually by spontaneous autotomy. Normally, each of the 5-10 fragments derived from a single worm regenerates a head anteriorly and a tail posteriorly. Occasionally, however, a head is formed posteriorly in addition to the normal anterior head, resulting in a bipolar worm. This phenomenon prompted us to conduct a series of experiments to clarify how the head and the tail are determined during regeneration in this species. The results showed that (1) bipolar head regeneration occurred only after artificial amputation, and not by spontaneous autotomy, (2) anesthesia before amputation raised the frequency of bipolar head regeneration, and (3) an extraordinarily high proportion of artificially amputated head fragments regenerated posterior heads. Close microscopic observation of body segments showed that each trunk segment has one specific autotomic position, while the head segments anterior to the VIIth segment do not. Only the most posterior segment VII in the head has an autotomic position. Examination just after amputation found that the artificial cutting plane did not correspond to the normal autotomic position in most cases. As time passed, however, the proportion of worms whose cutting planes corresponded to the autotomic position increased. It was suspected that the fragments autotomized after the artificial amputation (corrective autotomy). This post-amputation autotomy was probably inhibited by anesthesia. The rate at which amputated fragments did not autotomize corresponded roughly to the rate of bipolar regeneration. It was hypothesized then that the head regenerated posteriorly if a fragment was not amputated at the precise autotomic position from which it regenerated without succeeding in corrective autotomy.     

Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. VI. Inhibition by juvenile hormones.

In the salivary gland chromosomes of late-third instar larvae and in late (8- to 12-hr) prepupae of Drosophila melanogaster, there are ecdysone-induced sequences of puffing patterns which can be reproduced in vitro. These two sequences are separated by a period when the glands are thought to be exposed to a low titer of β-ecdysone and during which they acquire the competence to respond to ecdysone at the late prepupal puff sites. Attempts to modify either the late larval or the late prepupal responses to ecdysone in vitro by the simultaneous addition of juvenile hormone (JH) with ecdysone, to larval or prepupal glands, respectively, are unsuccessful. If, however, JH (ca. 10^?6M) is added to larval glands cultured 6 hr in ecdysone and then 3 hr in JH alone, the subsequent induction of prepupal ecdysone puffs is inhibited. Thus the role of JH appears to lie in modifying the acquisition of competence to respond to ecdysone rather than in a direct antagonism between the two hormones.     

Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster,: IV. The mid prepupal period

In the mid prepupal period of development of Drosophila melanogaster two major changes in gene activity occur in the salivary glands: (a) The mid prepupal puffs (e.g., 63E, 75CD) are induced and (b) the late prepupal puffs (e.g., 62E, 74EF, 75B, and 93F) acquire the competence to respond to ecdysone. These events can be studied in vitro. Both require that the ecdysone titre be very low (<5 × 10^?9, M) and both events depend upon protein synthesis.     

Control of muscle degeneration following autotomy of a hindleg in the grasshopper,Barytettix humphreysii

When the grasshopper, Barrytettix humphreysii, sheds a hindlimb during autotomy, certain thoracic muscles degenerate although they are neither directly damaged nor denervated. Muscle degeneration is induced when a leg nerve (N5) that does not innervate the thoracic muscles is severed. Together these results suggest that transneuronal mechanisms influence muscle survival. To further characterize this autotomy-induced process, we studied the degeneration of a thoracic tergotrochanteral muscle (M#133b,c) following autotomy or experimental manipulation in adult animals. Its degeneration is correlated with reduced activity of its neural input and occurs by programmed cell death (PCD). PCD onset is variable between individual muscle fibers, indicating that the trigger of degeneration is fiber specific. Muscle degeneration appears to be triggered by the loss of proprioceptive input from the autotomized limb, since severing of axons from proprioceptive organs, but not exteroceptive chemo- or mechanoreceptors, leads to muscle degeneration. Muscle disuse, neuronal degeneration, or changes in juvenile hormone titer do not appear to play a role in autotomy-induced degeneration. We propose that the loss of proprioceptive input from proximal campaniform sensilla on the tibia deafferents the thoracic muscle motor neurons and leads to a decrease in their activity. Muscle degeneration is ultimately triggered by the loss of normal neural activity.     

Thyroid hormone autoantibodies (THAA) in two cases of Graves' disease: effects of antithyroid drugs, prednisolone, and subtotal thyroidectomy

Changes in titers of serum thyroid hormone autoantibodies (THAA) and anti-thyroglobulin (Tg) antibodies during treatment with antithyroid drugs (methimazole and propylthiouracil) were examined in two cases of Graves' disease. Effects of prednisolone and subtotal thyroidectomy were also investigated in one case (case 1). Initially both cases had only anti-T4 autoantibodies in their serum. During methimazole therapy, the titer of anti-T4 autoantibodies increased in both cases, and anti-T3 autoantibodies became detectable and their titer increased in case 2. The influence of propylthiouracil on the titer of THAA was not clear. Both prednisolone plus methimazole therapy and subtotal thyroidectomy decreased the level of anti-T4 autoantibodies in case 1. There was a significant correlation between titers of THAA and anti-Tg antibodies in both cases, although titers of anti-Tg antibodies in case 1 stayed within the normal range throughout the investigation period. These results indicate that methimazole treatment could induce and/or enhance the production of THAA and THAA are antibodies against thyroid hormone-containing Tg molecule.     

Effect of bovine viral diarrhea virus infection on fertility of dairy heifers

A prospective field study in heifers from birth to first breeding was undertaken on two commercial dairies to assess the effect of bovine viral diarrhea virus (BVDV) congenital and post-natal infection (PNI) on fertility. A high BVDV Type 2 antibody titer (1:4096) at 10 months of age was associated with 32 more days to conceive, compared with a low titer (1:128). Conversely, infection with BVDV by 5-6 months of age and high BVDV Type 2 titers 1 month before conception or breeding was associated with improved fertility. Heifers with evidence of congenital BVDV infection had lower fertility than non-infected heifers (15-42 days longer time-to-first AI), which depended on BVDV Type 2 titers at 10 months of age. Neospora caninum infection was associated with additional services per conception (SPC) and Leptospira interrogans infection was associated with a delay in the time-to-first breeding. It appears that under field conditions, the effect of subclinical BVDV infection on subsequent heifer fertility may be due to a complex of interrelationships among multiple BVDV infections that depend on the type and timing of infection relative to reproductive development and events.     

Effects of culture parameters on the production of retroviral vectors by a human packaging cell line

The use of retroviral vectors for human gene therapy requires the production of large quantities of high titer vector stocks. Maintaining high titers during the prolonged culture of packaging cells will require that critical parameters be controlled. The aim of this study was to determine which culture parameters critically affect the production/decay of retroviral vectors produced by the human packaging cell line FLYRD18/LNC-hB7. The stability of retroviral vectors released by this cell line was found to be temperature dependent (half-life of 6.9, 11.0, and 64.3 h when incubated at 37, 32, and 0 degrees C, respectively). Titers increased up to 10-fold when the packaging cells were cultured at 32 degrees C, compared to 37 degrees C, despite a decrease in cell yield (cell-specific titers were 20-fold higher). Virus titers were also over 10-fold higher when the packaging cells were cultured in a reduced serum concentration (1%) compared to 5%. Retrovirus production at a range of pH levels revealed a significant decrease in virus titer at pH levels below 6.8 and above 7.2, optimum titers being achieved in cultures at pH 7.2. Dissolved oxygen levels in the range 20-80% did not significantly affect titers under the conditions tested. Finally, a packed bed system containing the packaging cells immobilized on porous microcarriers was shown to sustain the production of active retroviral vectors for over 1 month, in relatively large volumes.     

Coordinated changes in JH biosynthesis and JH hemolymph titers in Aedes aegypti mosquitoes

Juvenile hormone III (JH) is synthesized by the corpora allata (CA) and plays a key role in mosquito development and reproduction. A decrease in JH titer during the last instar larvae allows pupation and metamorphosis to proceed. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa once again synthesizes JH, which plays an essential role in orchestrating reproductive maturation. In spite of the importance of Aedes aegypti as a vector, a detailed study of the changes of JH hemolymph titers during the gonotrophic cycle has never been performed. In the present studies, using a high performance liquid chromatography coupled to a fluorescent detector (HPLC–FD) method, we measured changes in JH levels in the hemolymph of female mosquitoes during the pupal and adult stages. Our results revealed tightly concomitant changes in JH biosynthesis and JH hemolymph titers during the gonotrophic cycle of female mosquito. Feeding high sugar diets resulted in an increase of JH titers, and mating also modified JH titers in hemolymph. In addition these studies confirmed that JH titer in mosquitoes is fundamentally determined by the rate of biosynthesis in the CA.     

Experience of Vaccination against Porcine Parvovirus in Pig-Breeding Herds: Serological Status and Reproductive Performance

Blood samples from 77 gilts were examined for HI-antibody titers to PPV, all gilts belonged to the same herd and PPV-induced reproductive failure had previously occurred in the herd. Thirty-three gilts were vaccinated twice 5 and 2 weeks before mating while 44 gilts served as non-vaccinated controls. Only 3 % of the vaccinated gilts were seronegative at the time of mating compared to 14 % of the non-vaccinated gilts and 32 % of the non-vaccinated gilts had a serum titer lower than 1:64. The second part of the study comprised 4 herds with 50–70 sows in each herd. All of the herds had previously had reproductive problems caused by PPV infection. During the last 2 years, all gilts in these herds were vaccinated against PPV at 6.5 months of age with a revaccination 3–4 weeks later. There was a marked variation in serum titer levels among the 4 herds. In two herds the titers were overall rather low. In the third herd all had high PPV-titers at both sampling occasions and in the fourth herd the titers varied among animals but were rather consistent within animals at the two sampling occasions. In the herd with high titer, a PPV-outbreak was confirmed during January-March 1984. During that period all sows, vaccinated as gilts, farrowed normal litters. The results indicate that even in PPV-infected herds a large number of gilts are seronegative at the time of breeding and vaccination of gilts is therefore recommended. Furthermore it does not seem necessary to revaccinate sows, vaccinated as gilts, in herds where PPV is still present.     

Hemolymph concentrations of host ecdysteroids are strongly suppressed in precocious prepupae of Trichoplusia ni parasitized and pseudoparasitized by Chelonus near curvimaculatus.

Regulation of ecdysteroid production in lepidopteran prepupae was studied using a parasitic wasp (C. near curvimaculatus) which specifically suppresses host prepupal ecdysteroid production after the induction of precocious host metamorphosis. At the developmental stage at which the hemolymph of the unparasitized metamorphosing host has its maximum titer of prepupal ecdysteroids, the hemolymph of 4th instar "truly parasitized" hosts (hosts with a surviving endoparasite) had a strongly reduced ecdysteroid titer. However, during the photophase about 12 h later, just prior to emergence of the parasite larva, an ecdysteroid peak was observed in the host hemolymph. Fourth instar pseudoparasitized prepupal hosts (in which the endoparasite was not present or died early in development) exhibited a sustained suppression in the hemolymph ecdysteroid titer. Small 5th instar pseudoparasitized hosts, which normally would molt to a 6th instar prior to metamorphosis, but which precociously attained the prepupal stage, also had a strongly reduced ecdysteroid titer. The late increase observed in truly parasitized hosts could be completely prevented by surgical removal of the parasite 24 h earlier, resulting in a titer similar to that in pseudoparasitized hosts. HPLC analysis of ecdysteroids in normal, truly parasitized, and 4th or 5th instar pseudoparasitized prepupae showed that both ecdysone and 20-OH ecdysone* were suppressed in truly and pseudoparasitized prepupae, with ecdysteroid levels being lowest in pseudoparasitized hosts. These data, and those of Brown and Reed-Larsen (Biol Contr 1, 136 [1992]), showing endoparasite secretion of ecdysteroids just prior to its emergence from the host, strongly indicate that: (1) the prepupal peak in truly parasitized hosts originates from the endoparasite, and (2) the low level of ecdysteroids in pseudoparasitized hosts results from the host's intrinsic inability to express a normal level of prepupal ecdysteroid titer. While precocious 4th or 5th instar prepupae of similar size had similarly suppressed ecdysteroid titers, smaller 4th instar prepupae had a lower ecdysteroid titer than larger, precocious 5th instar prepupae. Rare 5th instar pseudoparasitized prepupae that were of nearly normal size showed a prepupal ecdysteroid titer distinctly greater than those of the usual smaller, precocious 5th instar prepupae. The data suggest that the competence of the host to express a normal hemolymph titer of prepupal ecdysteroids is more closely correlated with the size of the prepupae than with the instar attained.     

Effect of an ecdysone agonist,RH-5849, on wandering behaviour in Spodoptera littoralis

1. Wandering behaviour of the fall armyworm, Spodoptera littoralis has been examined using a video-digitizing computer system. 2. Ecdysteroid titers prior to and during wandering have been determined using an enzyme immunoassay. 3. RH 5849, an agonist of 20-hydroxyecdysone (20E) applied at various times before wandering to last instar larvae of S. littoralis caused a delay in the onset of wandering, varying from 0.5 to 4.5 hr. This delay was dose dependent. 4. We suggest that wandering in S. littoralis may be induced by a decrease in ecdysteroid titers.     

Ecdysteroid titers and changes in chromosomal activity in the salivary glands of Rhynchosciara americana

Titers of ecdysone and 20-OH ecdysone were measured separately in both hemolymph and salivary glands of metamorphosing Rhynchosciara larvae. Gland titers were consistently higher than hemolymph titers. Although 20-OH ecdysone was the most prominent form of the hormone, measurable quantities of ecdysone were also observed throughout development in both tissues. Changes in salivary gland replication and puffing activity could be correlated with changes in gland 20-OH ecdysone titers. This was true for both developmentally changing RNA puffs and DNA puffs, which occur during the prepupal period. The DNA puffs are tied to the final DNA replication cycle, and both this cycle and the period of amplification can be correlated with increases in gland 20-OH ecdysone content. Various aspects and possible interpretations of the above correlations are discussed.This work is dedicated to the memory of Prof. Hans D. Berendes     

Tissue Damage Disrupts Developmental Progression and Ecdysteroid Biosynthesis in Drosophila

In humans, chronic inflammation, severe injury, infection and disease can result in changes in steroid hormone titers and delayed onset of puberty; however the pathway by which this occurs remains largely unknown. Similarly, in insects injury to specific tissues can result in a global developmental delay (e.g. prolonged larval/pupal stages) often associated with decreased levels of ecdysone – a steroid hormone that regulates developmental transitions in insects. We use Drosophila melanogaster as a model to examine the pathway by which tissue injury disrupts developmental progression. Imaginal disc damage inflicted early in larval development triggers developmental delays while the effects are minimized in older larvae. We find that the switch in injury response (e.g. delay/no delay) is coincident with the mid-3rd instar transition – a developmental time-point that is characterized by widespread changes in gene expression and marks the initial steps of metamorphosis. Finally, we show that developmental delays induced by tissue damage are associated with decreased expression of genes involved in ecdysteroid synthesis and signaling.