Degeneration Of Prothoracic Glands Research Articles

The hormonal pathway controlling cell death during metamorphosis in a hemimetabolous insect

Metamorphosis in holometabolous insects is mainly based on the destruction of larval tissues. Intensive research in Drosophila melanogaster, a model of holometabolan metamorphosis, has shown that the steroid hormone 20-hydroxyecdysone (20E) signals cell death of larval tissues during metamorphosis. However, D. melanogaster shows a highly derived type of development and the mechanisms regulating apoptosis may not be representative in the insect class context. Unfortunately, no functional studies have been carried out to address whether the mechanisms controlling cell death are present in more basal hemimetabolous species. To address this, we have analyzed the apoptosis of the prothoracic gland of the cockroach Blattella germanica, which undergoes stage-specific degeneration just after the imaginal molt. Here, we first show that B. germanica has two inhibitor of apoptosis (IAP) proteins and that one of them, BgIAP1, is continuously required to ensure tissue viability, including that of the prothoracic gland, during nymphal development. Moreover, we demonstrate that the degeneration of the prothoracic gland is controlled by a complex 20E-triggered hierarchy of nuclear receptors converging in the strong activation of the death-inducer Fushi tarazu-factor 1 (BgFTZ-F1) during the nymphal–adult transition. Finally, we have also shown that prothoracic gland degeneration is effectively prevented by the presence of juvenile hormone (JH). Given the relevance of cell death in the metamorphic process, the characterization of the molecular mechanisms regulating apoptosis in hemimetabolous insects would allow to help elucidate how metamorphosis has evolved from less to more derived insect species.

Functions of the ecdysone receptor isoform-A in the hemimetabolous insect Blattella germanica revealed by systemic RNAi in vivo

The molecular basis of ecdysteroid function during development has been analyzed in detail in holometabolous insects, especially in Drosophila melanogaster, but rarely in hemimetabolous. Using the hemimetabolous species Blattella germanica (German cockroach) as model, we show that the ecdysone receptor isoform-A (BgEcR-A) mRNA is present throughout the penultimate and last nymphal instars in all tissues analyzed (prothoracic gland, epidermis and fat body). To study the functions of BgEcR-A, we reduced its expression using systemic RNAi in vivo, and we obtained knockdown specimens. Examination of these specimens indicated that BgEcR-A during the last nymphal instar is required for nymphal survival, and that reduced expression is associated with molting defects, lower circulating ecdysteroid levels and defects in cell proliferation in the follicular epithelium. Some BgEcR-A knockdown nymphs survive to the adult stage. The features of these specimens indicate that BgEcR-A is required for adult-specific developmental processes, such as wing development, prothoracic gland degeneration and normal choriogenesis.

Adhesion of hemocytes to desialylated prothoracic glands of Galleria mellonella (Lepidoptera) in the larval stage

Summary Earlier work showed the existence of sialic acid (SA) in the prothoracic glands (PGs) of Galleria mellonella (Karaçali et al., 1997). In this paper we investigated the role of SA during the degeneration process of PGs. Neuraminidase-digested larval PGs were incubated within the hemolymph collected from same age larvae. Light and electron microscopic observations showed incomplete capsule formation by the accumulation of hemocytes around desialylated gland cells, but not in the control group. Desialylated larval PG cells were recognized as a foreign structure or non-self by hemocytes. The result indicates that SA acts as a mask for hemocytic receptors during the larval period under normal conditions. However, in the absence of SA, the glands start to degenerate. We provide an explanation for the role of SA in the recognition process of hemocytes that initiates the degeneration of PGs in pupal cells and show the functionality of SA in insects, confirming that SA is a universal molecule.

Degeneration of moulting glands in male crickets

The degeneration of the prothoracic glands of the male cricket, Gryllus bimaculatus, was analyzed by using an in vitro assay for ecdysteroid release from the moulting glands in last instar nymphs as well as in adult animals, and correlated with light and transmission electron microscopy. Apoptosis was examined by the TUNEL-reaction. The ability to synthesize ecdysteroids reached a peak at the 8th day of the last larval instar, identified as the moulting peak. After adult ecdysis it decreased to barely measurable values. Prothoracic gland degeneration was initiated at the time of the moulting peak, characterized by TUNEL positive reactions, nuclear and cytoplasmatic condensation, a striking abundance of residual basal laminae; besides a great amount of autophagic vacuoles are observed. The results reveal that apoptosis and autophagy are the basic mechanisms for programmed cell death in the prothoracic gland of Gryllus bimaculatus.

Juvenile Hormone Prevents the Onset of Programmed Cell Death in the Prothoracic Glands ofManduca sexta

During normal pupal–adult development, programmed cell death of the prothoracic gland ofManduca sextaproceeds via apoptosis. By employing the DNA laddering technique, the earliest sign of DNA fragmentation in the cells comprising the gland occurred on Day 5 of pupal–adult development and DNA fragmentation peaked 1 day later. Since juvenile hormone (JH) is known to prevent adult development and since prothoracic gland degeneration occurs during the initial stages of adult development, we wished to determine the possible role of JH in prothoracic gland maintenance. JH was injected into pupae and the glands analyzed by DNA laddering andin situTUNEL labeling. The administration of JH prevented apoptosis of the prothoracic gland even 11 days after JH injection into young pupae. The prothoracic glands remained intact and their ability to synthesize ecdysteroids was maintained at a fairly active level as ascertained by radioimmunoassay afterin vitroincubation. The control glands had degenerated by this time and were almost devoid of ecdysteroidogenesis capability. Ultrastructural analysis confirmed that JH-treated prothoracic gland cells were rescued from the apoptotic sequence, i.e., nuclear condensation, cytoplasmic budding, and cell fragmentation. They exhibited a preserved smooth endoplasmic reticulum and intercellular channel system typical of active prothoracic gland cells. The composite data suggest that JH can both maintain and stimulate the prothoracic gland. Therefore, during normal pupal–adult metamorphosis the absence of JH is prerequisite for both the initiation and completion of prothoracic gland degeneration.

Programmed cell death of the prothoracic glands of Manduca sexta during pupal-adult metamorphosis

The degeneration of the prothoracic glands of Manduca sexta during pupal-adult metamorphosis was analyzed by both light and transmission electron microscopy, and correlated with a functional analysis of the ability of the prothoracic glands to synthesize ecdysteroids in vitro. Apoptosis of degenerating glandular cells was also examined by in situ indirect immunofluorescein detection of digoxigenin-labeled genomic DNA cleavage (the TUNEL method). The ability of the prothoracic gland to synthesize ecdysteroids reached a peak 4 days after pupation, decreased drastically on day 6 and then decreased further to the basal level 2 days later. Prothoracic gland degeneration was initiated on day 6 and was characterized by nuclear condensation, cytoplasmic budding, giant autophagic vacuoles, the disappearance of the smooth endoplasmic reticulum and intercellular channels, and the fragmentation of the cytoplasm into membrane-bound bodies. The cell debris of these degenerating cells was then engulfed by numerous phagocytic hemocytes. The results of the analysis of apoptosis by immunofluorescence detection are in complete accord with the histological, ultrastructural and radioimmunoassay data, suggesting that apoptosis is the basic mechanism for programmed cell death of the cells comprising these vital glands.

Stadium-specific degeneration of host prothoracic glands by Campoletis sonorensis calyx fluid and its association with host ecdysteroid titers

Campoletis sonorensis is a polydnavirus-carrying wasp that parasitizes Heliothis virescens larvae. Previous studies indicated that C. sonorensis polydnavirus (CsPDV) induces degeneration of host prothoracic glands when injected into last-stadium hosts. Degeneration also occurs when last instars are naturally parasitized, but it is not observed in hosts parasitized as penultimate instars. This study investigated the effect of CsPDV on the development of penultimate-stadium hosts and the temporal sensitivity of H. virescens prothoracic glands to virally-induced degeneration. We found that calyx fluid (containing CsPDV) caused degeneration of host prothoracic glands when injected into newly-ecdysed, penultimate-stadium hosts, but degeneration was delayed until injected larvae reached the critical head capsule size for the last stadium, even when injected larvae exhibited supernumerary molts. Host prothoracic glands became sensitive to virally-induced gland degeneration just prior to apolysis to the last larval larval molt , simultaneous with an ecdysteroid peak. Prothoracic gland degeneration in response to calyx fluid was also noted in two other hosts of C. sonorensis, Helicoverpa zea and Spodoptera exiguae.

Lack of Prothoracic Gland Degeneration in Developmentally Arrested Host Larvae of Manduca sexta Parasitized by the Braconid Wasp Cotesia congregata

Although 5th (last) instar parasitized Manduca sexta larvae undergo developmental arrest and do not wander, they exhibit a small hemolymph ecdysteroid peak (300–400 pg/μl) which begins one day prior to the parasitoid's molt to the 3rd (last) instar and concomitant emergence from the host. Ecdysteroids present in this peak were 20-hydroxyecdysone, 20,26-dihydroxyecdysone and one or more very polar ecdysteroids, as well as small amounts of 26-hydroxyecdysone and ecdysone. In parasitized day-1 and -2 5th instars ligated just behind the 1st abdominal proleg, hemolymph ecdysteroid levels increased in both anterior and posterior portions (100–500 pg/μl), while in unparasitized larvae, hormone levels only increased in the anterior portion (100–350 pg/μl). Thus, the ecdysteroid peak observed in host 5th instars was probably produced, at least in part, by the parasitoids. It may serve to promote Cotesia congregata's molt from the second to the third instar and/or to facilitate parasitoid emergence from the host. In parasitized day-1 and -2 5th instars ligated between the last thoracic and 1st abdominal segments, hemolymph ecdysteroid titers reached much higher levels (500–3500 pg/μl) in the anterior portion (no parasitoids present) than in the posterior portion (150–450 pg/μl). Therefore, it appears that the parasitoid's regulation of hemolymph ecdysteroid titers occurs at two levels. First, parasitization neutralizes the host's ability to maintain its normal hemolymph ecdysteroid levels. Second, in a separate action, the parasitoid manipulates the ecdysteroid-producing machinery so that hemolymph levels are maintained at the 200–400 pg/μl characteristic of day 3–4 hosts. This is the first report of a parasitoid's ability to interfere with the normal inhibitory mechanisms which prevent prothoracic gland production of ecdysteroid at inappropriate periods of insect growth and development.

Metamorphosis of the corpus allatum and degeneration of the prothoracic glands during the larval-pupal-adult transformation of Drosophila melanogaster: A cytophysiological analysis of the ring gland

The degeneration of the prothoracic glands of Drosophila melanogaster during pupal-adult metamorphosis was analyzed by light microscopy, scanning, and transmission electron microscopy. The ultrastructural observations were correlated with the ability of the ring gland to synthesize ecdysteroids in vitro. The ring gland is prominent during larval life and is identifiable until just before adult eclosion but undergoes dramatic changes in location, shape, size, ultrastructure, and function during pupal-adult development. Prothoracic gland degeneration is characterized by: a gradual decrease in its ability to synthesize ecdysteroids; a decreasing quantity of smooth endoplasmic reticulum (SER) and mitochondria; the absence of intercellular channels; cytoplasmic fragmentation; and the separation of the prothoracic gland from the corpus allatum and corpus cardiacum. An ultrastructural analysis of the corpus allatum during larval-pupal-adult metamorphosis and adult life was also correlated with function, i.e., juvenile hormone biosynthesis, using a radiochemical assay of ring glands and adult corpora allata in vitro. A relatively high concentration of SER, mitochondria, and mitochondrion-scalariform junction complexes are typical features of an active corpus allatum cell. The migration of the corpus allatum from the ring gland to its position as a separate gland in the adult fly was studied in detail. The capacity of the corpus allatum to synthesize juvenile hormone is at its peak in the ring gland of the early wandering third instar larva, whereas the corpus allatum of 2-day-old female adults displayed the greatest synthetic activity during adult life. The physiological significance of the alterations in gland activity is discussed.

Stage‐specific effects of Campoletis sonorensis parasitism on Heliothis virescens development and prothoracic glands

Abstract The ichneumonid endoparasitoid Campoletis sonorensis Cameron (Hymenoptera: Ichneumonidae) injects a polydnavirus when it oviposits into a host. We compared the development of Heliothis virescens (F.) (Lepidoptera: Noctuidae) larvae parasitized in the penultimate (fourth) stadium with those parasitized in the last (fifth) stadium by C.sonorensis and show that hosts stung in the fifth stadium exhibited arrested or delayed development compared to the controls. Parasitoids developed normally to the point of emergence in larvae stung in the fifth stadium but most did not successfully emerge from the host. The prothoracic glands in all successfully parasitized fifth stadium hosts and most unsuccessfully parasitized fifth stadium hosts showed some degree of virally‐induced degeneration. Larvae stung in the fourth stadium developed more slowly than controls and either did not moult or developed to a fifth and sometimes a supernumerary sixth stadium before parasitoid emergence. Unsuccessfully parasitized hosts were delayed in their development but eventually moulted to the fifth and, in some cases, a supernumerary sixth stadium before pupating. Hosts stung in the fourth stadium showed no signs of prothoracic gland degeneration whether successfully parasitized or not. In addition, calyx fluid injections into early fourth stadium hosts did not cause prothoracic gland degeneration even after these hosts moulted to the fifth stadium, suggesting that degeneration induced by polydnavirus is specific to the last stadium of the host.

Degeneration of last instar Heliothis virescens prothoracic glands by Campoletis sonorensis polydnavirus

The development of last instar Heliothis virescens larvae was experimentally arrested by ligation between the head and thorax or by injection with calyx fluid from the endoparasitoid Campoletis sonorensis. Prothoracic glands were removed from these larvae at various times after treatment and the gland cells were either measured or prepared for electron microscopy. At 4 days postligation, gland cell ultrastructure was similar to that of cells from unligated larvae and cell size was not significantly reduced until 7 days after ligation. In contrast to ligated controls, cells from calyx fluid-injected larvae were significantly smaller 24 hr after injection. Significant degeneration in gland cell ultrastructure was observed 2 days after injection and by 7 days postinjection, prothoracic glands showed gross degeneration. Similarly, injection of purified polydnavirus caused gross gland degeneration by 7 days after injection. Irradiating calyx fluid abolished its ability to cause gland degeneration. Our results indicate that C. sonorensis polydnavirus stimulates prothoracic gland degeneration. This virally induced degeneration of the prothoracic glands may account for our previously reported observation that injection of calyx fluid causes a drastic reduction in hemolymph ecdysteroid titer and consequent developmental arrest.

Conversion of radiolabelled 2,22,25-tri-deoxyecdysone in Tenebrio pupae

The conversion of a radiolabelled precursor of ecdysteroids 2,22,25-tri-deoxyecdysone has been studied in the pupae of Tenebrio molitor: this insect has a peculiar metamorphosis characterized by the degeneration of prothoracic glands, the classical source of moulting hormone, several days before the ecdysteroid peak, thus raising the question of the origin of the hormone. Injections of 2,22,25-tri-deoxyecdysone into intact pupae, as well as into isolated abdomens of Tenebrio, demonstrated that these animals are able to transform the radiolabelled precursor into 20-hydroxyecdysone (identified by TLC, direct and reverse phase HPLC and co-acetylation with reference substances), thus providing evidence that four hydroxylations of ecdysteroid synthesis can take place in the absence of prothoracic glands. In vitro experiments showed that epidermal explants are able to transform 2,22,25-tri-deoxyecdysone into 2-deoxyecdysone and ecdysone (TLC and HPLC analysis; co-acetylation). This is an argument, but of course not a complete demonstration, in favour of the hypothesis considering the epidermis as an alternative source of ecdysteroids.

Contrôle plurifactoriel de l'évolution postimaginale des glandes ventrales chez Locusta migratoria L. Données expérimentales et infrastructurales

In Locusta migratoria direct experimental and infrastructural data confirm correlations between the fate of post-imaginal prothoracic glands and corpora allata activity. The presence of juvenile hormone in haemolymph during metamorphosis and corpora allata hyperactivity allow the prothoracic glands to persist. Cyclic activity of these glands is synchronous with ovarian cyclic activity. These physiological conditions are obtained by isolation, short day-length, high relative humidity, by CO 2 periodic anaesthesis (1 min by day), or experimentally by the implantation of supernumerary active corpora allata. These conditions also produce the solitaria form. The absence of juvenile hormone in the haemolymph during the first three days of adult life, or precocious corpora allata ablation, prevents prothoracic gland degeneration. The prothoracic gland shows some chief cytological features during the first three imaginal days (‘periode de latence’). Thus, lytic structures with phosphatase acid activity always appear and suggest, according to Wigglesworth's hypothesis, that a cerebral degenerative factor is liberated during metamorphosis. This factor is inhibited by juvenile hormone and the prothoracic glands persist.