Enzymes for ecdysteroid biosynthesis: their biological functions in insects and beyond.Many advances in arthropod sterol metabolism, as it ecdysteroid biosynthesis to ecdysteroid ecdysteriid, have been made in the past 10 yr. The sterol dealkylation system, present in the midgut of many species, is entirely absent in ecdysteroid biosynthesis members of at least four insect orders ecdysteroid biosynthesis the prothoracic glands of Manduca sexta. The physiological relevance of 3-dehydroecdysteroids in some insects has only recently been appreciated. The sterol 7,8-dehydrogenating enzyme, the sequential terminal hydroxylases and ecdysone monooxygenase all exhibit properties epidural steroid injection effectiveness cytochrome P enzymes. The lack of tissue and substrate specificity reported for the terminal hydroxylations of exogenous substrates suggests that non-specific hydroxylases may be involved.
Ecdysteroid synthesis ‘Black Box’ illuminated | Development
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. 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. Finally, we show that developmental delays induced by tissue damage are associated with decreased expression of genes involved in ecdysteroid synthesis and signaling.
July 13, ; Accepted: October 4, ; Published: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have declared that no competing interests exist. Insects proceed through a series of precisely timed developmental transitions during their life cycle.
In Drosophila melanogaster , after egg hatching, larvae progress through three instars that are separated by a molt during which the cuticle is shed and re-synthesized to accommodate continued growth  , .
The third and final instar is followed by pupariation, the onset of the larval-pupal transition which is characterized by eversion of anterior spiracles, contraction of the larval body and tanning of the larval cuticle to form the puparium  , . It is within this protective casing that metamorphosis to the adult occurs  ,  , .
The timing of these developmental transitions is influenced by nutritional and environmental cues and is regulated by systemic signals such as steroid hormones that direct coordinated developmental responses throughout the insect  ,  , . In insects, localized tissue damage is frequently associated with a systemic injury response resulting in delayed development e.
In Drosophila , damage to imaginal adult precursor tissues via irradiation, induction of cell death clones, or localized activation of apoptosis causes a prolonged third larval instar  ,  ,  ,  ,  , . The mechanism by which localized tissue damage disrupts developmental progression is poorly understood but appears to involve a reduced systemic hemolymph ecdysteroid titer.
In Drosophila , as in most arthropods, the timing of developmental transitions is coordinated by a transient rise in the titer of the steroid hormone ecdysone E  , . Production and release of ecdysteroids is regulated by a small secreted neuropeptide known as prothoracicotropic hormone Ptth  ,  ,  ,  ,  ,  , . Ptth stimulates ecdysone synthesis, at least in part, by regulating transcription of a number of Halloween genes, a family of genes encoding cytochrome P enzymes that are required for ecdysone synthesis in the prothoracic gland cells of the ring gland  ,  ,  ,  ,  , .
Ecdysone is released from the ring gland into the hemolymph and transported to peripheral tissues where it is converted to its active form, hydroxyecdysone 20E , which binds to its receptor comprised of the Ecdysone receptor EcR and Ultraspiracle Usp  ,  , .
It has been suggested that imaginal disc damage triggers developmental delays, possibly by preventing the synthesis or release of ecdysone  ,  ,  , . The mechanism by which injury leads to decreased hemolymph ecdysteroid titers remains unclear but appears to involve delayed release of Ptth  ,  ,  ,  ,  , . The effects of tissue damage on other components required for ecdysone synthesis and signaling are less clear.
The effects of injury on developmental progression are dependent upon the developmental stage of the animal at the time injury is sustained  ,  ,  ,  , . Imaginal tissue damage induced by irradiation or genetic cell ablation only appears to retard pupariation when induced at or before an Injury Response Checkpoint IRC which is reached sometime during the second half of the third larval instar  ,  ,  ,  ,  ,  , . The exact time that the IRC is reached has not been clearly defined, however a number of studies in Drosophila and Lepidoptera Ephestia kuhniella and Lymantria dispar have demonstrated that tissue damage induced early in the last larval instar retards development while injury inflicted closer to pupariation time no longer affects developmental timing  ,  ,  ,  ,  ,  ,  , .
In Drosophila , there are two additional critical developmental time points that are known to occur during the third larval instar.
One of these critical periods is Critical Weight CW , a size-assessment checkpoint reached early in the third instar, after which starvation no longer influences the time to pupariation . The second critical period is the M id-third I nstar T ransition MIT , a developmental time point which marks the initial steps of metamorphosis, is associated with widespread changes in gene expression, and occurs during the middle of the third larval instar .
The possibility that the IRC corresponds with another critical developmental checkpoint e. CW, MIT has not been explored. Here we examine the timing of the IRC and the mechanism by which localized tissue damage triggers developmental delays. We find that imaginal disc damage leads to delayed onset of the MIT, pupariation and adult eclosion.
The effects of injury on developmental timing are minimized or absent closer to pupariation time and the switch from retardation to no response is coincident with the MIT. In addition, we find that tissue damage is associated with 1 reduced ecdysteroid titers, 2 decreased expression of most genes involved in ecdysteroid synthesis and signaling and 3 increased expression of Ecdysone oxidase Eo , a gene involved in ecdysone catabolism.
Together our data suggest that systemic injury response signals act on multiple targets to regulate ecdysteroid titers and ecdysone signaling pathway components. To induce tissue damage, we utilized flies containing a rnGAL4 enhancer trap  , a UAS-eiger transgene, and a temperature sensitive GAL80 variant driven by a tubulin promoter tubGAL80 ts , all recombined onto a single third chromosome Figure 1A .
The rnGAL4 driver is expressed throughout the third larval instar in the wing pouch, the peripodial epithelium overlying the wing pouch, the haltere disc and a ring in the leg discs .
In addition, we observed low but detectable levels of rnGAL4 expression in 1—3 cells in each salivary gland throughout the third larval instar. A Strategy used to produce Ablating and Non-Ablating larvae. B Strategy to induce cell ablation.
The timing of developmental transitions is known to be influenced by temperature as well as genetic background  ,  , . B Fraction of larvae of the indicated genotype that had reached the mid-third transition as measured by Sgs3GFP expression is plotted relative to hours AEL. C Fraction of larvae of the indicated genotype that had undergone pupariation is plotted relative to hours AEL. D Fraction of larvae of the indicated genotype that had eclosed as adults is plotted relative to the time in hours AEL.
E Plot of average larval weight mg at a given time after egg laying for Ablating and Non-Ablating larvae. F Fraction of larvae that underwent pupariation after starvation at a given size for Ablating and Non-Ablating larvae. Cell ablation was induced as described Figure 1 at various time points arrows during the third larval instar. Cell ablation resulted in delayed pupariation Red Arrows , no effect on developmental timing Green Arrows , or a mixed effect Yellow Arrows in which some animals delayed development and others developed at the same time as controls.
The Mid-third Instar Transition MIT is a developmental time point associated with a low titer ecdysteroid pulse and is characterized by widespread changes in gene expression including induction of a glue gene — Salivary gland secretion 3 Sgs3  , . Similarly, we found no significant difference in time to adult eclosion between Ablating and Non-Ablating animals.
Critical Weight CW is the weight at which starvation no longer delays time to pupariation . A second size assessment checkpoint is Minimum Viable Weight MVW which represents the weight at which larvae have enough nutritional stores in the form of fat body to survive the next developmental transition . Third instar larvae of known weights were starved and the proportion of larvae that successfully pupariated was measured.
To examine how localized tissue damage influences the timing of the MIT, we induced cell ablation in the wing imaginal discs Figure 1B at hours AEL and examined larvae for expression of the Sgs3GFP reporter in salivary glands Figure 4. Following the heat-treatment to induce cell death via eiger expression in the wing discs, most Sgs3GFP expression was maintained at high levels throughout the remainder of the third larval instar and was detected in In contrast, following induction of cell death, only High levels of Sgs3GFP expression were detected in only We detected no obvious morphological defects in salivary glands following cell ablation and no signs of cell death within salivary glands at any time following cell ablation suggesting that delayed onset of Sgs3GFP expression is a result of imaginal disc cell ablation data not shown.
A—F Timing of the mid-third instar transition as measured by Sgs3GFP expression in larvae that were heat-treated for 24 hours at hours AEL to induce tissue damage in wing discs. Delay of pupariation was measured as the difference between mean pupariation time of Ablating larvae and Non-Ablating larvae housed in the same vial.
To examine how localized tissue damage influences the timing of pupariation we induced cell ablation in the wing imaginal discs at hours AEL, and then monitored the time to pupariation Figure 5A. A—F Timing of pupariation and adult eclosion following induction of cell ablation in the wing disc at the indicated time. Similar results were obtained with larvae heat-treated at , , or hours AEL data not shown. Mean pupariation times are and hours AEL for ablating and non-ablating larvae, respectively.
Mean eclosion times are and hours AEL for ablating and non-ablating larvae, respectively. Similar results were obtained with larvae heat-treated at hours AEL data not shown. Similar results were obtained with larvae heat-treated at and hours AEL data not shown. To assess the effects of larval age on the systemic injury response we induced tissue damage in the wing imaginal discs in larvae of various ages. Cell ablation in the wing disc at hours AEL delayed pupariation and adult eclosion by 59 and 64 hours, respectively Figure 5A, B.
Similar results were obtained when cell ablation was induced at , or hours AEL Figure 3. Similar results were obtained when cell ablation was induced at hrs AEL Figure 3. Injury induced between — hrs AEL resulted in two groups of Ablating larvae — those that delayed development in response to tissue damage and those that developed at the same time as Non-Ablating controls See Figure 5D.
Larvae that delayed development in response to wing disc ablation typically eclosed as adults with regenerated wings while those that eclosed at the same time as Non-Ablating controls emerged as wingless adults Figure S1.
Wing disc cell ablation induced between — hours AEL resulted in no significant difference in the mean time to pupariation or adult eclosion in Ablating animals compared to Non-Ablating controls Figure 5E, 5F ; Figure 3. None of the Ablating animals showed any evidence of tissue regeneration; all emerged as wingless adults Figure S1. The developmental retardation observed following imaginal disc cell ablation suggested the presence of an underlying ecdysteroid deficiency in injured animals.
To measure the ecdysteroid titers in Ablating and Non-Ablating larvae, we performed an enzyme immunoassay EIA utilizing an ecdysteroid antiserum Cayman Chemical. We examined ecdysteroid levels at four time points Figure 1B: As shown in Figure 6 , just prior to the induction of cell ablation T 0 there was no significant difference in ecdysteroid titers between Ablating 1. At T 1 we detected a small not statistically significant difference between ecdysteroid concentrations in Ablating and Non-Ablating larvae; ecdysteroid concentrations were 1.
At T 2 , ecdysteroid concentrations were 1. Values are expressed as the means of 20E equivalents per mg of tissue. To examine the effects of tissue damage on ecdysteroid signaling, we used qRT-PCR to examine injury-induced changes in expression of genes involved in ecdysone synthesis and signaling. For each genotype Ablating and Non-Ablating , transcript levels in larvae at each time point T 1 —T 3 were compared to transcript levels in larvae at T 0 to determine relative changes in gene expression.
To assess how tissue damage influences ecdysone synthesis, we examined expression of genes including 1 ptth, which encodes the neuropeptide that stimulates ecdysone synthesis in the ring gland  , 2 genes encoding enzymes required for ecdysone synthesis in the ring gland including neverland nvd  , spookier spok  , disembodied dib  , phantom phm  , and shadow sad  , and 3 genes encoding additional components required for ecdysone synthesis including ecdysoneless ecd  , D rosophila a drenodoxin re ductase dare  , and transcription factors molting defective mld  and without children woc .
Graphs show changes in transcript levels 24 hours after heat treatment T 3 ; Figure 2B compared to transcript levels immediately before heat treatment T 0 ; Figure 2B. Conversion of ecdysone to hydroxyecdysone is catalyzed by the Cyp enzyme encoded by shade shd , which is expressed in peripheral tissues . The functional ecdysone receptor is comprised of a heterodimer formed by the Ecdysone Receptor EcR and the RXR homolog encoded by ultraspiracle usp  ,  , .
To further examine the effects of injury on ecdysone signaling, we examined expression of ecdysone inducible genes including Broad br  ,  , Eip74EF  , Eip75B  , Eip71CD  , and Eip78C . Ecdysone oxidase Eo is an enzyme that catalyzes the conversion of ecdysteroids into inactive 3-dehydroecdysteroids .
This ecdysteroid inactivation results in decreased ecdysteroid titers and helps to regulate the sharp ecdysteroid peaks that trigger developmental transitions. To identify potential differences between the early and late response to injury we examined expression of genes involved in ecdysteroid synthesis and signaling at an earlier time point, half-way through the cell ablation treatment T 1 ; Figure 1B. There was no significant difference for most genes examined in Ablating samples compared to Non-Ablating controls at T 1 Figure 8.
Only five genes displayed reduced levels of expression in Ablating larvae compared to Non-Ablating controls at this early time point.