Ecdysteroid titres in a tenebrionid beetle, Zophobas atratus: effects of grouping and isolation

doi:10.1016/S0022-1910(97)00029-2

Journal of Insect Physiology

Volume 43, Issue 9, September 1997, Pages 815-821

https://doi.org/10.1016/S0022-1910(97)00029-2 Get rights and content

Abstract

Metamorphosis in Zophobas atratus is dependent on isolation: when kept in grouped conditions, larvae undergo numerous supernumerary moults, growing in size, without pupating. This beetle thus represents an interesting model for the analysis of possible differences in the endocrine regulation of normal vs. supernumerary larval moults. In this study, the ecdysteroid titres have been analysed in this species, using enzyme immunoassay. The hormonal variations of larvae undergoing normal or supernumerary larval cycles were particularly examined, in either grouped or isolated conditions. Normal larval cycles presented very similar ecdysteroid variations in grouped as well as isolated conditions, showing a single hormonal peak (at about 1000 pg/μl). Supplementary larval cycles, occurring in grouped conditions, also showed a similar single ecdysteroid peak, but after a longer period of basal levels. Isolation of such larvae triggered their larval–pupal transformation, which was characterized by more complex hormonal fluctuations, including a small ecdysteroid peak before the main one. Interestingly, the isolation of big larvae during a large part of their cycle induced this peculiar hormonal pattern synchronously, confirming the involvement of a complex neuroendocrine control between external conditions and ecdysteroid titres.

Introduction

Crowding has been shown to delay metamorphosis in several insect species and more particularly in the family Tenebrionidae (Tschinkel and Willson, 1971). Such a group effect, which has been confirmed by several studies in Tribolium freemani (Nakakita, 1982, Nakakita, 1990; Kotaki et al., 1993; Kotaki and Fujii, 1995) or in Tenebrio molitor (Weaver and McFarlane, 1989, Weaver and McFarlane, 1990; Connat et al., 1991) takes a major importance in Zophobas atratus Fab. (=Z. rugipes Kirsh; Tschinkel, 1984), a large neotropical beetle (Tschinkel and Willson, 1971; Tschinkel and van Belle, 1976; Tschinkel, 1978, Tschinkel, 1993). In this species, indeed, metamorphosis is completely dependent on isolation and never occurs in grouped conditions: although animals become able to pupate after having reached approximately 12 larval moults (about 4 months old) and a critical weight of about 400 mg (Quennedey et al., 1995), they undertake metamorphosis only if they become isolated from other larvae (or if they are experimentally isolated). In natural conditions, such fully grown larvae leave their original habitat to escape from the contacts with other larvae and they wander until a quiet protected place is found to undertake pupation (Tschinkel, 1981). However, such animals, maintained in grouped conditions, undergo several supplementary larval moults (up to eight to 10 extra instars), alternating feeding and wandering periods at the surface of the mass rearing during the most part of their remaining life, reaching about 1500 mg at 1 year of age, and generally die after 18 months, without pupating (Quennedey et al., 1995).

Thus, Z. atratus is an interesting model for the analysis of possible differences in the endocrine regulations of normal or supernumerary larval moults. In this study, our aim was to examine the ecdysteroid titres in the function of isolated or grouped conditions.

Section snippets

Insects

Zophobas atratus was reared at 25°C ±1°C and about 70% r.h. on bran, as previously described (Quennedey et al., 1995). Most experiments were made from animals taken out of the mass rearing as white, unsclerotized larvae (i.e. animals less than 3h after their last larval ecdysis), provided they had a body weight within either of the two following classes: 200–300 mg (`small' larvae) or 500–600 mg (`big' larvae). They were either isolated in plastic compartmented boxes (about 3.5×3.5×2.5 cm per

`Small' larvae

Larvae of Z. atratus weighing between 200 and 300 mg corresponded to eighth or ninth larval instars (Quennedey et al., 1995). They underwent a larval–larval cycle that lasted 15–16 days, on average, with approximately 10 days of feeding and activity (A-period). The profile of ecdysteroid titre (Fig. 1) showed a single hormonal peak occurring on the 12th day, i.e. 3–4 days before larval ecdysis and reaching 1190±380 pg/μl.

`Big' larvae

Big grouped larvae of Z. atratus, weighing 500–600 mg, underwent supernumerary

Discussion

Ecdysteroid titres in Zophobas atratus present characteristic variations, showing a major hormonal peak during every moult cycle, as generally observed in numerous other insects [review in Smith (1985)]. Hormonal titres culminate at significantly higher values in pupae (about 4000 pg/μl or 8×10⁻⁶ m) than in larvae (about 1000 pg/μl, i.e. 2×10⁻⁶ m), as also noticed in numerous other holometabolous insects [review in Smith (1985]. Interestingly, the peak values are very similar to those of the

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Control of the ecdysteroid level plays a crucial role in density-dependent metamorphosis in the giant mealworm beetle Zophobas atratus
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However, more detailed experiments are required to explain this phenomenon clearly. JH inhibits metamorphosis, and increases in ecdysteroid levels are crucial for inducing metamorphosis in species whose metamorphosis is controlled by crowding (Kotaki et al., 1993; Quennedey et al., 1995; Aribi et al., 1997; Ruang-Rit and Park 2018). However, hormonal responsiveness to the density changes has been largely unknown so far.
Metamorphic transition in some tenebrionid beetles is dependent on population density. This phenomenon is useful for pupae that are vulnerable to cannibalism. The physiological mechanism of this adaptive developmental phenomenon remains unclear. In Zophobas atratus, which show density-dependent metamorphosis, larval isolation can induce metamorphosis. We herein demonstrated that the return of isolated larvae to a crowded condition (re-crowding) inhibited their metamorphosis. The timing of metamorphic initiation was slightly extended according to the duration of re-crowding experienced by the isolated larvae. Therefore, the re-crowding induced physiological changes needed for metamorphic inhibition. We investigated whether hormone-related genes involved in signaling of metamorphic inhibitor (juvenile hormone, JH) and molting hormone (ecdysteroid) responded to the re-crowding. An expression analysis showed that gene expression of ecdysteroid signaling was maintained at low levels under the re-crowded condition. Actually, ecdysteroid levels decreased responding to re-crowding. Ecdysteroid injections induced metamorphosis in re-crowded larvae. In contrast, the JH signaling gene showed little fluctuation in both isolated and re-crowded conditions, and knockdown of JH signaling factors did not affect inhibition of metamorphosis under the re-crowded condition. The present study suggests that regulation of ecdysteroid level rather than JH is more crucial in the density dependent metamorphosis in Z. atratus.
Evaluation of ecdysteroid antisera for a competitive enzyme immunoassay and extraction procedures for the measurement of mosquito ecdysteroids
2017, General and Comparative Endocrinology
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Our laboratory had also previously used the L2 and H22 antisera to measure E and 20E titers in A. aegypti by RIA (Jenkins et al., 1992; Sieglaff et al., 2005; Telang et al., 2007). For the EIAs, the L2 antiserum (Aribi et al., 1997; Blais et al., 2010; donated by J.-P. Delbecque, Université Bordeaux 1, Talence, France) was diluted 1:14,000. The H22 antiserum (Warren et al., 2009; donated by L. I. Gilbert, University of North Carolina) was diluted 1:7000.
Ecdysteroid hormones regulate several aspects of insect development and reproduction. The predominant ecdysteroids produced by insects including mosquitoes are ecdysone (E) and 20-hydroxyecdysone (20E). The ability to measure E and 20E titers is essential for many studies, but few sensitive, low cost options are currently available for doing so. To address this deficiency, we developed a new enzyme-linked immunoassay (EIA). In the first part of the study, we compared the affinity of two new antisera named EAB25 and EAB27 to other available ecdysteroid antisera. EAB25 had a 27-fold higher affinity for 20E than E, while EAB27 had a four-fold higher affinity for 20E. In the second part of the study, EIA protocols were developed for analyzing E and 20E produced by the mosquito Aedes aegypti. Results indicated that pelts from fourth instar larvae and ovaries from blood-fed, adult females produced E and 20E. Methanol extraction in the presence of magnesium from whole body samples altered antibody recognition of E and 20E by EIA. However, extraction with 1-butanol and two organic/water phase separations eliminated this problem and improved assay performance. We conclude the new antisera used in the EIA provide a low-cost, flexible, and sensitive method for measuring E and 20E in insects.
Neuroendocrine disruption in the shore crab Carcinus maenas: Effects of serotonin and fluoxetine on chh- and mih-gene expression, glycaemia and ecdysteroid levels
2016, Aquatic Toxicology
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Results are expressed either as concentrations or as relative changes of glucose concentrations compared to controls at t0. Ecdysteroids were quantified using EIA (Porcheron et al., 1989; modified by De Reggi et al., 1992; Pascual et al., 1995; Aribi et al., 1997). 50 μL of sample were extracted with 300 μL of ethanol and centrifuged (6000g, 5 min, room temperature).
Serotonin, a highly conserved neurotransmitter, controls many biological functions in vertebrates, but also in invertebrates. Selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, are commonly used in human medication to ease depression by affecting serotonin levels. Their residues and metabolites can be detected in the aquatic environment and its biota. They may also alter serotonin levels in aquatic invertebrates, thereby perturbing physiological functions. To investigate whether such perturbations can indeed be expected, shore crabs (Carcinus maenas) were injected either with serotonin, fluoxetine or a combination of both. Dose-dependent effects of fluoxetine ranging from 250 to 750 nM were investigated. Gene expression of crustacean hyperglycemic hormone (chh) as well as moult inhibiting hormone (mih) was assessed by RT-qPCR at 2 h and 12 h after injection. Glucose and ecdysteroid levels in the haemolymph were monitored in regular intervals until 12 h. Serotonin led to a rapid increase of chh and mih expression. On the contrary, fluoxetine only affected chh and mih expression after several hours, but kept expression levels significantly elevated. Correspondingly, serotonin rapidly increased glycaemia, which returned to normal or below normal levels after 12 h. Fluoxetine, however, resulted in a persistent low-level increase of glycaemia, notably during the period when negative feedback regulation reduced glycaemia in the serotonin treated animals. Ecdysteroid levels were significantly decreased by serotonin and fluoxetine, with the latter showing less pronounced and less rapid, but longer lasting effects. Impacts of fluoxetine on glycaemia and ecdysteroids were mostly observed at higher doses (500 and 750 nM) and affected principally the response dynamics, but not the amplitude of glycaemia and ecdysteroid-levels. These results suggest that psychoactive drugs are able to disrupt neuroendocrine control in decapod crustaceans, as they interfere with the normal regulation of the serotonergic system.
Periodic abdominal pumping supports leg development during metamorphosis in tenebrionid beetle Zophobas atratus
2008, Comparative Biochemistry and Physiology - A Molecular and Integrative Physiology
Rhythmic abdominal pumping movements in a pupa of giant mealworm beetle Zophobas atratus caused large hemolymph pressure pulses of approximately 20 mmHg. The abdominal pumping movements were completely blocked by transecting the ventral nerve cord (VNC) between the first and second abdominal ganglia. Transection of the VNC until 2 days after pupation caused a developmental defect of adult legs: morphogenesis of the tibial and tarsal segments was severely retarded, and the segments remained covered with a thick pupal cuticle. The developmental defect was rescued by artificially inducing rhythmic abdominal bending for 3 days after transection of VNC. Blocking of the abdominal pump did not increase the amount of water loss during the pupal period. The transplanted tibial segments lacking active tracheal ventilation could form a thick adult cuticle. The results suggest that abdominal pumping movements during the pupal period support the development of adult legs by facilitating hemolymph circulation.
Effects of pyriproxyfen, a juvenile hormone analog, on development of the mealworm, Tenebrio molitor
2006, Pesticide Biochemistry and Physiology
Pyriproxyfen, an analog of the juvenile hormone (JH) in insects, has been evaluated in vivo and in vitro on an important stored product pest, the mealworm Tenebrio molitor. On newly ecdysed pupae, this insect growth regulator (IGR) was applied topically (at 0.10 and 0.20 μg/insect). Enzyme immunoassay measurements showed a decrease of the ecdysteroid titer in the hemolymph of treated specimens. In addition, the treatment induced an increase of the protein concentration in the hemolymph, however, there was no significant effect on the protein band pattern. Using in vitro pupal integument explant cultures, pyriproxyfen (at 1 and 10 μM in the medium) caused a significant inhibition of ecdysteroid amounts and of the thickness of new cuticle secreted. These new results are discussed in relation to the physiology of this IGR with a JH action.
Endocrine changes associated with the starvation-induced premature metamorphosis in the yellow-spotted longicorn beetle, Psacothea hilaris
2005, General and Comparative Endocrinology
Under 25 °C and a long day photoperiod, about half of the fourth instar Psacothea hilaris larvae molt to the fifth instar on day 13 and pupate about 18 days later; the rest pupate without a further larval molt with a mean fourth instar period of 24 days. However, starvation of fourth instar larvae exceeding a threshold weight induces premature pupation, resulting in the formation of small but morphologically normal adults. To clarify the endocrine basis for this premature pupation, hemolymph juvenile hormone (JH) and ecdysteroid titers were quantified during the fed and the starved periods. Normally fed fourth instar larvae exhibited two populations with regard to JH and ecdysteroid titers, one having JH titers ranging from 1.2 to 2.1 ng/ml through to day 13, the other, similarly high titers in the early part of the instar but low titers reaching 0.1 ng/ml on day 13. One population had ecdysteroid titers with a peak of 43 ng/ml on day 10, coinciding well with the period when some larvae normally molt to the fifth instar (day 13), the other, a small peak of 14 ng/ml on day 14 and a large peak of 70 ng/ml on day 17 coinciding well with the period just before the prepupa stage. When fourth instar larvae were starved after 4 days of feeding, JH titers decreased sharply in the next 24 h and never recovered, and a small but significant increase (to 21 ng/ml) in ecdysteroid titers was observed on day 6, followed by a large peak of 63 ng/ml on day 11. Altogether, these results suggest that starvation induces a rapid decline in the JH titer, and this cues the early occurrence of a small ecdysteroid peak that commits larvae to early metamorphosis.

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¹: Permanent address: Laboratoire de Biologie Animale, Institut des Sciences de la Nature, Université de Annaba, BP 12, El-Hadjar, 23000 Annaba, Algérie.

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Journal of Insect Physiology

Abstract

Introduction

Section snippets

Insects

`Small' larvae

`Big' larvae

Discussion

References (25)

The onset of metamorphosis in Tenebrio molitor L.: effects of a juvenile hormone analogue and of 20-hydroxyecdysone

Journal of Insect Physiology

The onset of metamorphosis in Tenebrio molitor larvae (Insecta, Coleoptera) under grouped, isolated and starved conditions

Journal of Insect Physiology

Cuticular cycle and molting hormone levels during the metamorphosis of Tenebrio molitor (Insecta Coleoptera)

Developmental Biology

Metamorphosis, activity of juvenile-hormone esterase and alteration of ecdysteroid titres: effects of larval density and various stress on the red flour beetle, Tribolium freemani Hinton (Coleoptera: Tenebrionidae)

Journal of Insect Physiology

Development of an enzyme immunoassay for ecdysteroids using acetylcholinesterase as label

Insect Biochemistry

Postembryonic development of Zophobas atratus Fab. (Coleoptera: Tenebrionidae) under crowded or isolated conditions and effects of juvenile hormone analogue applications

Journal of Insect Physiology

Larval dispersal and cannibalism in a natural population of Zophobas atratus (Coleoptera: Tenebrionidae)

Animal Behaviour

The effect of larval density on growth and development of Tenebrio molitor

Journal of Insect Physiology

External factors and ecdysone release in Calliphora erythrocephala

Experientia

Crowding inhibits pupation in Tribolium freemani: contact chemical and mechanical stimuli are involved

Entomology and Experimental Applications

Cited by (19)

Control of the ecdysteroid level plays a crucial role in density-dependent metamorphosis in the giant mealworm beetle Zophobas atratus

Evaluation of ecdysteroid antisera for a competitive enzyme immunoassay and extraction procedures for the measurement of mosquito ecdysteroids

Neuroendocrine disruption in the shore crab Carcinus maenas: Effects of serotonin and fluoxetine on chh- and mih-gene expression, glycaemia and ecdysteroid levels

Periodic abdominal pumping supports leg development during metamorphosis in tenebrionid beetle Zophobas atratus

Effects of pyriproxyfen, a juvenile hormone analog, on development of the mealworm, Tenebrio molitor

Endocrine changes associated with the starvation-induced premature metamorphosis in the yellow-spotted longicorn beetle, Psacothea hilaris