ecd¹ homozygotes shifted to the restrictive temperature of 29[o]C from day 3 after eclosion are short lived; their posterior midguts are thinner, contain fewer enterocytes, as well as more apoptotic progenitors (ISCs and enteroblasts) and enteroendocrine cells.

The majority of ecd¹/ecd¹ homozygous clones in mosaic third instar larval eye-antennal discs are eliminated after shifting individuals to the restrictive temperature of 29[o]C.

ecd¹ pupae, reared since beginning of pupation at the restrictive 30[o]C temperature, show full pupal lethality. Also, they show persistence of the posterior seven pairs of abdominal segmental dorsomedian (Scer\GAL4^sev.EP-positive) neurons, and a less compact arrangement of Malpighian tubules cells, as compared to controls.

Temperature sensitive ecd¹ mutants grown at 29[o]C display smaller larvae with transparent bodies, low adipose tissue, reduced mobility and improperly developed Malpighian tubules, which have reduced numbers of and irregular organization of principal cells and stellate cells. These mutants also show reduced uric acid deposition in the Malpighian tubules at 29[o]C. These phenotypes are not seen in larvae reared at 25[o]C.

ecd¹ mutants do not show significant changes in the density of circulating hemocytes in larvae, as compared to controls.

Mutant animals kept at 29[o]C at the pupal stage have defects in the mushroom body α/β lobes: the lobes may appear slim, or the β lobes may be fused.

Mutant females raised at the permissive temperature and then transferred to the restrictive temperature as 3-day old adults contain germaria filled with supernumerary germline cells containing a single spectrosome (SSCs) after 5 days. After 7 days at the restrictive temperature the number of undifferentiated SSCs is further increased, causing somatic cell defects affecting cysts pinching off from the germarium. Approximately 50% of the mutant germaria have dumbbell-shaped fusomes after 7 days at the restrictive temperature.

Mutant adults (raised at 18[o]C and shifted to the semi-restrictive temperature of 28[o]C at the start of the wandering larva stage) have reduced total body triglyceride levels (normalised to total body protein) compared to controls.

The stellate cells of the Malpighian tubules are clustered in homozygous larvae raised at the restrictive temperature (instead of being evenly distributed as in wild type).

ecd¹ homozygotes die between L2 and mid L3. Most ecd¹/Df(3L)R-R2, ecd¹/ecd² or ecd¹/ecd^l(3)23 animals die during the L2/L3 molt, displaying typical molting defects such as double mouth hooks, but some survive into L3. Larval lethality of ecd¹ homozygous larvae is rescued by feeding with 20-hydroxyecdysone at 29^oC.

ecd¹ animals maintained at the non-permissive temperature show defects in germ band retraction, head involution and cuticle deposition.

ecd¹/Df(3L)R-G7 larvae show lymph gland hypertrophy and failure to pupariate when raised at 27^oC. ecd¹ homozygotes and ecd¹/Df(3L)R-G7 mutants exhibit a total and near total loss of encapsulation capacity when challenged with L. boulardi G486. Lamellocytes appearing in response to parasitization are reduced to zero or near zero. ecd¹/Df(3L)R-G7 mutants also show a significant reduction in the frequency of lymph gland dispersal in parasitized individuals. Parasitised mutant lymph glands fail to show the hemocyte mitotic burst characteristic of wild type. Lymph glands of older, developmentally arrested mutant larvae are larger and show a temperature dependent hypertrophy. The greatest degree of hypertrophy is seen in the posterior lobe. Parasitisation does not disperse the lymph glands. Parasitised mutant lymph glands show a near normal increase in population size of crystal cells.

Mutant larvae transferred to 29^oC (the restrictive temperature) for 7 hours at 6 days after egg laying at 22^oC and then maintained at 29^oC for 18 hours show larval clusters of vesicles and tubules in the Golgi areas of leg and wing discs. Mutant larvae maintained at 29^oC for 7 hours and then transferred to 22^oC for 18 hours show neatly stacked cisternae in the Golgi areas of the discs. Discs dissected from larvae maintained at 29^oC for 6 hours and incubated at 22^oC in the absence of ecdysone show larval clusters in the Golgi areas, whereas those incubated in the presence of ecdysone show stacked cisternae.

Homozygotes show a developmental delay; the time taken for 50% of the population to form pupae is increased compared to wild-type controls.

Homozygous larvae have a slower mean rate in body wall contraction and mouth hook movement compared to controls.

The composite excitatory junctional potential (EJP) of the Ib and Is axons that innervate muscle m6 has a larger amplitude in late third instar homozygous larvae compared to controls, although there is no significant difference in the EJP amplitude between mutant and wild-type early third instar larvae. Late third instar homozygous larvae show a substantial decrease in EJP amplitude upon direct application of 20-hydroxyecdysone on exposed neuromuscular junctions (this effect is not seen in wild-type controls).

The total length of the Is axon terminal on muscle m6 is higher in homozygous late third instar larvae than in controls. The number of varicosities on the Is terminals is increased in homozygous late third instar larvae compared to controls.

Homozygous females are sterile if held at the nonpermissive temperature for 5 days, and egg chambers in these flies arrest development at stage 8 and subsequently degenerate. Border follicle cells fail to develop in these arrested egg chambers. When mutant females are held at the nonpermissive temperature for 2 days, some stage 10 egg chambers develop, in which border cells differentiate. More than 50% of these egg chambers show delayed border cell migration.

The temperature-sensitive lethality of ecd¹ animals can be partially rescued by exogenous application of the compounds 20-hydroxyecdysone, RH-5849 and RH-5992; the compounds can stimulate abortive pupariation in the animals maintained at the restrictive temperature from the third larval instar stage.

Homozygotes show eye defects when shifted to 30^oC for 24 hours during the third larval instar stage. ecd¹/Df(3L)R-G7 larvae exposed to 30^oC for 24 hours during the third larval instar stage and then shifted to the permissive temperature (18^oC) develop into adults with defects in the eye and anterior nicks in the retinal tissue. Progression of the morphogenetic furrow is disrupted; the furrow is either slowed or stopped and is also dysmorphic. Cell proliferation ahead of the furrow is not visibly altered, but cell-cycle synchrony in the furrow is lost. No excess cell death is seen. Animals exposed to 30^oC for 24 hours during the second larval instar stage have more severe eye defects, with intrusions of cuticle into the eye field.

Mutant larvae develop relatively normally at the restrictive temperature of 29^oC, but are unable to pupariate.

Larvae fail to pupate when transferred to 30^oC midway through third instar. They remain as larvae for an extended period of time and develop enlarged type III synaptic boutons.

Homozygotes fail to produce the precursor of 20-hydroxyecdysone at the non-permissive temperature (29^oC).

The mutant shows changes in the amounts, and differences in ratios of particular, of cuticular hydrocarbons compared to the wild-type (at both permissive and restictive temperatures).

Homozygotes reared continuously at the restrictive temperature survive to the third larval instar stage. These larvae appear to lack both eye-antennal and mesothoracic discs, and leg discs are reduced in size. Hemizygous larvae survive through to the late second instar stage, but die suddenly before moulting. ecd¹/ecd² transheterozygotes die as they moult to the third larval instar; most die during ecdysis, with the old cuticle often remaining attached to the posterior end of the abdomen. Only 0.7% of eggs from a cross between ecd^3D/+ females and ecd¹/+ males hatch as ecd¹/ecd^3D larvae at 29^oC. These infrequent survivors die shortly after hatching. 7.3% of eggs from a cross between ecd^3D/+ males and ecd¹/+ females hatch as ecd¹/ecd^3D larvae at 29^oC. The number of macrochaetae on the thorax is reduced in homozygous and hemizygous ecd¹ flies, and in ecd¹/ecd², ecd¹/ecd^g4, ecd¹/ecd^g6, ecd¹/ecd^g5 and ecd¹/ecd^3D flies kept at 29^oC in the hours that follow prepupal formation.

Temperature-sensitive allele. Histolysis of the larval fat body at the restrictive temperature of 29^oC is the same in heterozygotes and homozygotes.

The prothoracic gland cells of homozygous larvae shifted to 29^oC show a number of ultrastructural abnormalities, including a decrease in the invaginations of the plasma membrane, an accumulation of lipid droplets in the cytoplasm, lack of smooth endoplasmic reticulum, an increase in the amount of rough endoplasmic reticulum and highly electron-dense mitochondria. The prothoracic glands produce only about 16% of the level of ecdysteroids synthesised by wild-type glands at 29^oC. The corpus allatum appears normal at 29^oC. The prothoracic gland defects are also seen in prothoracic glands dissected from larvae reared at 18^oC and then cultured in vitro for 4 hours at 29^oC.

Homozygous females develop multiform ovarian dysfunctions within a few days of being transferred to 29^oC. The first defects are detected well before vitellogenesis, and include egg chambers which lack the oocyte, although the nurse cells grow normally, abnormal numbers of nurse cells and a flattened follicular epithelium. Follicle polarity may be abnormal, with the oocyte being lateral instead of posterior. Mid-stage follicles are also abnormal, with pyknosis in the nuclei of follicular and nurse cells, followed by gradual disorganisation of the somatic and germline derivatives. Live cells may be close to degenerated ones in the same egg chamber. About half of stage 13-14 follicles or newly laid eggs appear highly disorganised, with irregular thickness of the yolk-free peripheral cytoplasm and/or abnormal distribution of the yolk bodies. The newly laid eggs often appear flaccid and translucent, with the proportion increasing as the time after the temperature shift of the females to 29^oC increases. The eggs are reduced in volume and length compared to wild-type. Stage 12 to 14 oocyte envelopes are permeable to Fe³⁺ ions. Multiform defects are seen over the first half of embryogenesis; pseudocleavage may occur during the first 2 hours - cytoplasmic islands devoid of nulcei or chromosomes emerge within the yolk mass. Invasion of the peripheral cytoplasm by cleavage nuclei is incomplete and/or there may be local degeneration of some blastoderm nuclei. Abnormal morphogenetic movements are seen during gastrulation, formation of the midgut invaginations, germband elongation and segmentation.

At 29^oC the prepupal activity peak of Argk is not seen. At 20^oC the activity peak reappears. At the restrictive temperature imaginal discs progressively lose Argk gene product activity.

Ovarian development becomes severely disturbed and oogenesis gradually declines in a few days in newly eclosed homozygous females transferred to 29^oC. Follicles begin to degenerate, mainly at stage 9 and enlarged agametic chambers become conspicuous (in the absence of the oocyte, the nurse cells keep on growing until they degenerate, while some follicle cells die). In other egg chambers the yolk deposition starts then slows down and stops, and local cell death followed by general degeneration of the follicle occurs. Five days after the temperature shift to 29^oC some females lay a few eggs which are flaccid and/or translucent. When oviposition has stopped, the degenerating ovarian chambers are piled up within the tubular sheath of each ovariole. Mature homozygous flies transferred to 29^oC also show disruptions in ovarian functioning; the frequency of aberrant previtellogenic follicles steadily increases, cell death occurs in increasing numbers of follicles, both germinal and somatic cells die and disintegrate, follicles become disorganised, and eggs have defective eggshell layers, appearing flaccid and translucent.

Defective imaginal disc development.

Most homozygous larvae transferred to the restrictive temperature of 29^oC at the end of the second larval instar die before pupariation without reaching full growth. Larvae transferred to 29^oC during the 30 hours after the second molt show variable behaviour; some form a normally tanned puparium but pupal development is arrested, some die as fully elongated larvae with partial darkening of the cuticle, and some remain as active larvae. Larvae transferred to 29^oC immediately at the second molt remain as active, permanent larvae. These larvae do not leave the food, reach a maximum weight greater than wild-type larvae and contain low levels of ecdysteroids compared to wild-type. Returning the larvae to a permissive temperature (20^oC) does not induce normal pupariation. Feeding with a 20-hydroxyecdysone-yeast mixture triggers abortive pupariation. Larvae maintained at 27.5^oC have nearly normal levels of ecdysteroids.

Homozygotes shifted to 29^oC at puparium formation die at emergence, although the mid-pupal peak of ecdysteroids is unaffected in these animals. Homozygotes shifted to 29^oC 24 hours before pupariation exhibit a significant decrease in the mid-pupal peak of ecdysteroids, although the pupariation peak is unaffected. These animals die before emergence, but after the mid-pupal ecdysteroid peak.

temperature-sensitive recessive lethal. At 29^oC,

the nine-fold increase in β-ecdysone content during

embryogenesis occurs normally in ecd homozygotes.

Accompanied by normal increases in dopa-decarboxylase

and dopamine acetyltransferase activity (Marsh and

Wright, 1980). The four-fold

increase during the first larval instar is reduced to

40% of normal; the additional twelve-fold increase

normally occurring at pupariation eliminated.

embryonic lethal. Embryonic development of ecd at 29^oC normal, but

first larval molt delayed and death usually occurs by

end of second instar; shift down to 20^oC at mid

second instar produces full yield of adult progeny.

Larvae shifted from 20^oC to 29^oC midway through

third instar fail to pupate and survive as larvae for

up to 3 weeks; ring gland, salivary glands and brain

of nonpupariating larvae are smaller than wild type;

such ring glands cultured in vitro secrete ecdysone at

lower than normal levels. Effects of 29^oC on third

instar reversible by ecdysone feeding or by shift down

to 20^oC within 3-5 days of shift up; after that

imaginal discs cannot differentiate; ecd imaginal

discs develop normally at 29^oC when implanted in a

wild-type host. A heat pulse during larval development

results in cell death with consequent abnormalities in

emerging adults. At 29^oC, third instar larvae

exhibit abnormally low dopadecarboxylase activity

(Kraminsky, Clark, Estelle, Gietz, Sage, O'Connor and

Hodgetts, 1980); Marsh and Wright, 1980) and high urate oxidase

activity (Krase and Friedman, 1979); ecdysone feeding effects normal levels.

Transfer to restrictive conditions at the beginning of

the pupal stage leads to death as pharate adults and

to the elimination of mechanosensory chaetae;

Non-sensory chaetae and other sensilla not affected.

Chaetae loss is autonomous as seen in homozygous

clones produced by somatic exchange and in ecd discs

passed through metamorphosis in wild type hosts under

restrictive conditions (Sliter, 1989). Sensitivity to restrictive temperature

disappears in 24 hour pupae. ecd fails to block

midpupal increase in ecdysone titer (Marsh and

Wright, 1980). Shifting newly emerged ecd adults to 29^oC

results in drastically reduced ecdysone titers and

sterilizes both males and females; reversible by shift

back to 20^oC; temperature-sensitive periods and

therefore the times that ecdysone is required for

embryonic development, chorion formation, and

vitellogenesis are 1-2 days before oviposition, 24 hr

prior to oviposition, and prior to stage 7,

respectively (Audit-Lamour and Bussin, 1981).

ecd¹ is a suppressor of abnormal immune response | heat sensitive phenotype of hop^Tum

ecd¹ is a suppressor of melanotic mass phenotype | heat sensitive phenotype of hop^Tum

Eip78C^Δ31, ecd¹ has female germline cyst phenotype

Bar¹, ecd¹ has eye phenotype

ecd¹, l(2)gl^ts3 has wing disc phenotype

ecd¹, l(2)gl^ts3 has larva phenotype