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Here I present an overview of the basics of what is now known about the hormonal control of development in insects. I do this using the model organism Drosophila Melanogaster as an example and illustrate how three hormones; prothoracicotropic Hormone (PTTH), Juvenile hormone (JH) and moulting hormone (20-hydroxyecdysone) regulate its development. I also recreate one of the first fundamental experiments in insect development, which showed that a hormone from the brain was responsible for regulating development.
As early as 1917 metamorphosis and moulting in holometabolous insects were shown to be controlled by hormones and though the extensive studying of model systems we now know that three hormones; prothoracicotropic Hormone (PTTH), Juvenile hormone (JH) and moulting hormone (20-hydroxyecdysone) are involved. The mechanisms by which these hormones control the development of insects have been the subject of many reports.
Holometabolous insects develop in four main stages; an embryonic stage, a larval stage, a pupa stage and an adult stage. In Drosophila Melanogaster the developmental period typically lasts around 9 days at 25°C but can be dramatically affected by different environmental conditions, such as temperature. In Drosophila, after the egg has been fertilized, the embryo becomes completely developed in less than a day, and then hatching takes place. The larva then feeds for three instars, moulting between each one. Metamorphosis then occurs at the end of the third instar. After 5 days in its pupal case, an adult fly emerges. A summary of the life cycle of drosophila is shown in figure one.
Figure one - The life cycle of Drosophila Melanogaster
But how do the three hormones control the development of drosophila? The first step in this process is the release of prothoracicotropic Hormone (PTTH) from the corpora cardiacum (although it is made in the brain). It is thought that neural, hormonal, and environmental factors can all trigger the release of PTTH. For example it has been shown in several insect groups that weight gain and photoperiod both control the release of PTTH. It is reasonable to assume that there is several factors (including weight gain and photoperiod) that determine whether PTTH is released or not, and that these factors each as a checkpoint that must be passed in order for release to go ahead. Truman et al (1972) demonstrated that not only does a larva have to achieve a critical weight to trigger PTTH to be released, but there is also a photoperiodic restraint on the larva that means that the release can only happen within an eight hour time frame within each day. If the minimal weight isn't reached in this time frame then the larva will carry on feeding until the next 8 hour unrestrained period.
Once PTTH has been released, it acts on the prothoracic glands, which are a pair of endocrine glands, located just behind the brain. In response to PTTH the prothoracic glands release ecdysone which is a prohormone of 20-hydroxyecdysone. Once in the body tissues, ecdysone is converted into 20-hydroxyecdysone by heme-containing oxidase in the mitochondria. Due to there being different stages of development in insects, ecdysone is released into the body in pulses, with each pulse triggering a moult. The 20-hydroxyecdysone is recognised by the ecdysone receptor in the nuclei of the insect cells. The ecdysone receptor binds to 20-hydroxyecdysone and this binding is stabilised by the protein ultraspiracle (USP) [i] . The ecdysone receptor once bound to 20-hydroxyecdysone and USP is then able to bind to a promoter region within the DNA to bring about a cascade of gene transcription. This cascade of promotional activity was first evidenced as by puffing of the polytene chromosomes of the salivary glands. This is one of the factors that makes drosophila such a good model system for the study of hormonal control of development; the salivary glands of drosophila posses large chromosomes that puff when ecdysone is present, therefore the transcription of genes in drosophila in response to the pulses of ecdysone can be seen occurring in waves of puffing in the chromosomes. The titre of 20-hydroxyecdysone in the blood of drosophila is one factor that determines whether the larva moults or forms a pupa. Figure two shows the 20-hydroxyecdysone level throughout the drosophila life cycle.
Figure 2 - The level of 20-hydroxyecdysone throughout the drosophila life cycle.
There is a high level in the drosophila embryo as there is a huge amount of development occurring. Shortly after a diploid zygote has been formed, the nucleus within the zygote is replicated so that a cell with several nuclei is formed; this is followed by the nuclei shifting to the periphery. This process keeps occurring until the result is a layer of roughly 6,000 nuclei enclosing a yolk. Cell membranes then begin to form between the nuclei. These
nuclei are each assigned a route of differentiation. Some follow the dorsal ventral route and others the anterior posterior. Four regions are then formed along the dorsal ventral axis; the mesoderm, ventral ectoderm, dorsal ectoderm and the amnioserosa. Regions that will go on to form the head thorax and abdomen are formed along the anterior axis. Figure three shows a first instar larva.
Figure 3 - A diagram of a first instar larva, showing the head thorax and abdomen.
It is the 20-hydroxyecdysone hormone that causes the initial transcriptional cascade that leads to this development from the embryo, to a first instar larva.
As figure two shows, each of the moults that the larva undergoes, is preceded by a rise in the level of 20-hydroxyecdysone. In fact there are two pulses before each moult. The first pulse leads triggers the cells to take on different roles in terms of development and the second pulse triggers the events that will lead to a moult; the 20-hydroxyecdysone causes epidermal cells to synthesise the enzymes to digest the cuticle.
After the second to third instar moult, the level of 20-hydroxyecdysone drops, and remains at a low level until roughly five hours before the formation of the puparium, when it starts to increase, and continues to do so until it reaches a peak just before a white prepupa is formed. During this peak of 20-hydroxyecdysone there is numerous significant developmental changes that occur. For instance the breakdown of some larval muscles occurs, the muscle cells that will eventually power the wings begin to appear and the structure of the gut become much more like the gut structure of an adult fly. After the prepupa is formed, the 20-hydroxyecdysone level then falls for roughly 12 hours and then over the next day (the 6th to 7th day) will begin to rise until it reaches its highest level. This huge pulse of 20-hydroxyecdysone brings out the remaining main metamorphical changes that have yet to take place. For example larval salivary glands break down, and the many of the remaining larval muscle cells begin to break down. After this large peak in 20-hydroxyecdysone level gradually fall and upon completion of metamorphosis an adult fly emerges from the pupa case.
Juvenile hormone (JH) is the third hormone that is involved in the regulation of insect development. JH is secreted from the corpora allata. It is JH that determines the result of the moult by modulating ecdysone activity. During larval moults the corpora allata secretes JH, but secretes much less of it at the end of the larval stage. When large amounts of JH are present, 20-hydroxyecdysone triggers moulting that results in larger larva. In comparison to this when JH levels are low 20-hydroxyecdysone triggers the pupal stage and metamorphosis can take place. During the third larval instar the corpora allata is prevented from secreting JH and in addition to this the rate of degradation of JH in the body is increased and this leads to the low JH level that allows the pupa stage to begin. Figure four provides an overall summary of the hormonal control of development in drosophila.
Figure 4 - Hormonal control of drosophila development
Methods and results
I recreated one of the first experiments that were used to show that a hormone that came from the brain controls the development of insects. The protocol for the experiment was exquisitely simple; I took several calliphora erythrocephela (blow fly) larva that were in the wandering stage and tied a piece of thread tightly around their heads. The larva along with several control larva were then left for a week.
Figure - 6
The ligature had the effect of restricting the pulses of ecdysone from reaching the rest of the body. Therefore after one week just the head of the larva formed a pupal case. In comparison, the abdomen and tail regions of the larva stayed in the larval stage and there was only a small amount of pupa development just beneath the ligature. The control larva that were not ligated had formed a complete pupal case. My observations are shown in the figures below.
Figure 5 - Sketch of a calliphora erythrocephela larva in the wandering stage.
Figure - 7http://www.livingwithbugs.com/Images/fly_pupae.jpg
Figure - 9
Figure - 8
Figure 6 and 7 - The control larva that did not have the ligature applied, all entered the pupal stage. The larva gets much shorter and slightly wider as a pupa case forms. Figure 8 - In the larva that had the ligature applied only the head progressed into the pupal stage. Figure 9 - Shows a sketch of a pharate adult drosophila through the pupal case.
As you can see from figures 6, 7 and 8 the experiment worked just as intended and the pulses of ecdysone were prevented from reaching the body of the larva where the ligature had been applied and as a result, only the head entered the pupal stage. The control larva on the other hand completely entered the pupal stage, as expected. This and other basic experiments, such as those carried out by the likes of Wigglesworth in 1934 and Kopec in 1917, although extremely simple provided the foundations for research into insect development. Many studies now are focused on learning about the molecular mechanisms by which these hormones act
and understanding just how wide ranging the effects of juvenile hormone are (Wheeler 2003 provides a good review of this). It is through studies of insect development that we are able to better understand development in humans and develop pesticides that work by preventing the development of insects.
My sketch of the pharate adult drosophila through the pupal case shows the main external features of the drosophila adult and when considering that the time from the wondering stage to the pharate adult stage can be just four days the sheer amount of development that the animal has undergone both internally and externally, shows that these three hormones must trigger a phenomenal amount of transcription.