Ladies and Gentlemen,
At earlier conferences here in Lindau, as just mentioned,
I spoke several times about problems in the field of insect biochemistry.
My decision to choose the same topic to talk to you about today
was prompted mainly by a desire to tell you about the progress and achievements
that have been made in areas that, for the most part, could only be discussed in earlier lectures
in terms of the problems they presented.
Unfortunately, in addition to presenting new facts, I cannot avoid covering some ground
that may already be familiar to some of you, for which I beg your indulgence.
For those of you who have not yet come across these problems, please permit me a preliminary remark.
Some of you may question the point of tackling such specific questions as those posed by the insect world.
I’d like to counter that opinion with the following observation.
You will all be aware that the science of biology has long since abandoned its descriptive efforts
and now seeks, with the help of methods borrowed from physics and chemistry,
to analyse fundamental life phenomena that apply universally to all organisms.
In this way, it is doing its part in pursuing the aim of science to explore the nature of humankind itself
and the living world around it.
In so doing, it is free to choose its objects and, depending on the question being addressed and the method used,
uses a wide range of different organisms in the animal, plant, and microbial realms.
And you will also be aware that insects are very often used to solve fundamental biological problems.
We should recall, for example, that the laws of classical genetics that apply to all life forms – including humans –
were discovered for the most part in insects and that animal behaviour laws were investigated in insects to a large extent.
And in particular, the principles discovered in social insects have delighted awestruck researchers over and over again.
Also Biochemistry, which aims to analyse the chemical transformations associated with life processes,
chooses its objects from among all the organisms on Earth.
For over twenty years we at the Max Planck Institute for Biochemistry have been investigating a number of chemical life phenomena
that have been made accessible by studying insects.
And as so often we have found in pursuing this work that very specific questions
that appear meaningful only for the insect world hold the key to explaining generally valid phenomena
or yield findings that can be of practical importance to our own social lives.
I would like to demonstrate this with the help of several examples.
Let’s begin by considering the natural pigments that are characteristic of the insect kingdom.
You all know that the numerous pigments
that embellish the natural world and delight humans were a major focus of interest for many generations of chemists.
Each period, with improvements in analytical methods, introduced new ways
to isolate and identify the structure of natural pigments.
So for a long time scientists believed
that they were well acquainted with all the important and widespread pigments in the natural world,
which often served as models for the technical synthesis of organic dyestuffs.
Surprisingly, however, they had overlooked the red, brown, yellow and purple pigments of the insect world,
including a previously unknown natural pigment type, which we discovered during our research in recent years
and which, as we found, figures among the most widely distributed pigments in nature.
First, let us see in pictures what these pigments are and where they occur.
In the first picture we see a butterfly, the well-known small tortoiseshell, and this butterfly has just shed its pupal case
and has secreted what entomologists call pupal secretion.
Many butterflies secrete a cleansing liquid like this immediately after pupating.
You can see in this case that the secretion, captured here on blotting paper, has a bright red hue.
This is a pigment of the red type in the insect world, and the wing markings,
insofar as they are red, yellow or brown, also fall into the group of pigments we will be talking about.
Wing pigment of our type can also be seen in the next pictures.
In the next picture we again see the wings of the small tortoiseshell.
Here is the peacock butterfly, here the admiral.
All examples of where yellow, red and brown wing pigments occur.
In the next picture we see the silver-washed fritillary, such colours,
as well as the pretty red markings of this large American moth Cecropia platysamia, belong here.
On the epidermis of some insects or larvae are the natural pigments we’re talking about.
In the next picture we see the caterpillar of the puss moth, characterised by the fact
that it turns deep red immediately before pupating.
The pigmentation is just starting here as depicted.
Again this red pigment type.
In the next picture you see the pretty colour pattern of a grasshopper larva Schistocerca,
here we also have the individual pigment patterns, and finally, I would also like to show you the eye of the grasshopper.
Many insect eyes are pigmented red, brown or a darker shade.
Now, all these pigments that I’ve presented to you, I repeat, as red, brown or even darker shades of pupating secretion,
wing pigments, epidermis pigments, and eye pigments in insects are chemically closely related.
And based on their universal occurrence in all insect eyes,
where they were first discovered, they have been given the collective name ommochrome eye pigments.
The occurrence of these substances is not limited to insects.
They are also found as eye and skin pigments in all arthropods,
for example crabs, as well as in cephalopods and more rarely in other animal classes as well.
There is a whole series of ommochromes as chemical entities.
As a characteristic and widespread representative of these ommochromes,
let us consider the structure of just one substance in the next picture, of so-called xanthommatin.
This yellowish brown pigment, xanthommatin, was first obtained in pure crystallised form
from the red pupating secretion of around 10,000 small tortoiseshells, which yielded just 100 mg of the pigment.
Later, 19 mg was isolated from the eyes of 7,800 blowflies,
marking the first time an insect eye pigment was obtained in pure form.
Xanthommatin occurs in very many insect eyes. It is one of the pigments found in wings and skin.
The structure of this substance was elucidated and definitively confirmed by synthesis.
For those of you familiar with the language of chemical formulae, here is the formula of xanthommatin
and you can see that it is what is known as a phenoxazone system, that is a tricyclic system with nitrogen,
to which a quinoline ring is attached, and here there is also an oxoaminocarbonic acid side chain.
This phenoxazone system is found in all ommochromes. We could say that ommochromes are phenoxazone pigments.
Typical of xanthommatin and other ommochromes is a characteristic redox behaviour, which is shown here in the picture.
This yellowish brown pigment stage is reduced by the addition of hydrogen, resulting in a bright red stage.
Here is the structure of hydroxanthommatin with the two attached hydrogen atoms here
and the subsequent rearrangement of the double bonds.
The red hydroxanthommatin, with the release of hydrogen, is converted back to the yellowish brown product.
And this redox behaviour is shown again in the next picture in colour, here the oxidised yellowish brown,
here the reduced bright red colour, this behaviour is, firstly, characteristic of the group and, secondly,
is unusual for biochemists and dye chemists,
because here reduction is accompanied by a deepening of the colour, which is generally not the case.
It is interesting, that nature uses both types, the oxidised and the reduced pigment.
We know, for example, that the previously mentioned change from the brown colour of the puss moth
to the bright red colour before pupation is due to such a reductive process.
All ommochromes are phenoxazone pigments, as I said, we regard xanthommatin as the basic substance,
which can be converted in various ways into other types of ommochromes.
It is interesting to note that the red hydrated pigment stage is not very stable in the presence of oxygen,
instead the oxidised pigment is the stable form.
What does nature do when it wants to fix the red hydrated pigment stage?
It does this by converting xanthommatin into two other pigments of the bright red type,
which can be derived from hydrated xanthommatin, and which we have named rhodommatin and ommatin D.
In the next picture I would like to suggest how the hydrated red stage is fixed.
Here you see again the hydrated form of xanthommatin, but there is only one hydrogen atom here on the nitrogen,
down there on the oxygen it is replaced by a residue R.
This residue R can be either a sugar residue,
in which case it forms a glucoside, or it can be a sulfa residue, a sulphuric acid ester,
and whenever such a hydrogen atom is replaced by such groups – through sugar groups, through sulphuric acid –
the reduced form cannot be converted as easily, namely only after removal of the residue, into the oxidised form,
and in this way nature fixes the hydrated unstable pigments to produce bright red.
They are therefore at the same time substances that show the formula types of other ommochromes.
Thank you. And the following strikes us as being of general interest.
The ommochromes were found to contain a new pigment system
which, as I mentioned, had not previously been observed in nature, the phenoxazone system.
This system has been known for over 40 years in the field of synthetic dyestuffs,
but its much earlier invention by nature has never been copied.
Among these phenoxazone-type synthetic dyes, some are used as photosensitisers on photographic plates.
Are pigments of this type in the eyes of insects also involved in light absorption, in the visual process,
instead of just acting, as assumed, as photoprotection pigments?
This is a question for sensory physiologists.
The ommochromes of insects were the first phenoxazone pigments to be discovered in nature.
Soon after their discovery, other representatives of this pigment type were found in the natural world.
Determining the structure of ommochromes provided the key for analysing the pigmentation system of the so-called actinomycins.
Antibiotically active metabolic products of ray fungi
and also proved to be the key to other representatives of this class in the fungus kingdom.
You again see in the top picture the formula of xanthommatin as a class
and how this basic substance is converted by substitution in the group of actinomycins, investigated by Brockmann,
and in the lower picture you see types of fungal pigments investigated by Gripenberg.
According to very recent observations,
it is likely that xanthommatin is also a metabolic product of mammals and perhaps even humans.
Whether it has any significance in this context is not known. Actinomycins are antibiotics.
This means that they are among the modern weapons doctors use against infectious diseases,
about which, in the presentation by Herr Domagk yesterday, we heard so many fascinating things.
Because the actinomycins include drugs against malignant diseases of the lymphatic system, it is understandable
that the ommochromes – initially merely objects of whimsical research –
are increasingly attracting the attention of the pharmaceutical industry.
The ommochromes however have gained the greatest theoretical importance as traits
which are expressed only under the effect of specific genetic factors.
That was the topic of my very first lecture in Lindau: “What do we know about the effects of genetic factors?”
Entomologists have found that some insect species are unable to produce ommochromes.
And genetic analysis of these ommochrome-free species has shown
that they differ from ommochrome-containing wild species in that there is a change or mutation in individual genes in the genome.
These genes must therefore control the synthesis of ommochromes.
This discovery made it possible to analyse the effects of genes for the first time,
that is to answer the question of how specific eye traits of an organism
are determined by the presence of specific genetic factors located in the cell nucleus.
In the next picture I would like to remind you of the classical experiment by Alfred Kühn, who pioneered this development.
In this diagram we see the caterpillar and head of the flour moth Ephestia kuehniella,
specifically the wild form characterised by dark ommochrome pigmentation.
Here is an ommochrome-free mutant, the caterpillar’s skin is colourless, the eyes red,
and there is no dark pigment, and these two variants differ only in a single genetic trait.
If, as Kühn did, you implant tissue from the wild form into this penultimate instar of the colourless mutant ommochrome,
which is otherwise absent, is synthesised in the host under the influence of this tissue,
and the colourless insects are transformed into insects that are phenotypically indistinguishable from the wild forms.
The implant must transfer a factor that restores the somehow defective process of ommochrome synthesis.
The biochemical analysis of this experiment then led to the recognition of a chain of genetic effects,
from which the following can be concluded:
The ommochromes in our pigments are synthesised from the amino acid tryptophan, a normal product of protein metabolism,
by way of stepwise catabolism via kynurenine and hydroxykynurenine.
Each of these steps is catalysed by a characteristic enzyme.
Those enzymes, however, are present only if certain genes are present in the pool of genetic factors.
We therefore conclude the following: the synthesis of the enzymes that catalyse a given reaction step is dependent upon genes.
Genes act via enzymes, when genes mutate, the associated enzymes are absent or altered,
so that chemical reaction steps, in this case subprocesses of ommochrome synthesis, no longer occur.
This experiment showed, for the first time, that genes act via enzymes.
Enzymes are the first detectable products of genetic factors.
Because enzymes are specific proteins, we also draw the important conclusion from the experimental results
that the information required for the synthesis of specific proteins resides in the structure of genetic factors.
In this way, a basic problem in biology was discovered and then made accessible by a subsequent experiment.
The question as to how genetic factors in a cell convey information for the synthesis of specific proteins
and thus for the production of all specific cellular structures is one of the most exciting problems in biochemistry today,
the answer to which is probably in the offing.
We have insects to thank for teaching us a law that has meanwhile been confirmed on a broad basis in many organisms
and that now allows us to analyse control processes in nature that reside primarily in the genetic material.
Unfortunately, we cannot continue this thread.
We will now turn instead to another control problem in the field of developmental physiology.
Most organisms develop from a single cell - the fertilised egg cell –
and the question of how this development of the individual is controlled,
how each phase of development is causally determined by the preceding phase,
defines the complex of problems addressed by developmental physiology.
Here, too, insects have taught us many fundamental principles.
You will all be aware that the development of many insects proceeds via an interesting metamorphosis.
From the egg of a fly or butterfly hatches the larva or caterpillar.
As it grows, it passes through stages during which it moults its outer layer, or cuticle.
In the process of pupal moulting, the mature larva changes into a pupa.
The pupa undergoes further transformation, at the end of which the sexually mature form, the imago, develops.
It emerges from the pupa in a process known as imago moulting.
It has been found that this sequence of processes, larval moulting, pupal moulting, imago moulting,
is triggered and controlled by three specifically acting hormones, and here we are confronted by the second problem,
which I’ve already spoken about in Lindau in the past.
At the time, it was believed that the three metamorphosis hormones interact according to this scheme,
the first hormone, adenotropic hormone, is produced in neurosecretory cells of the brain,
under whose effect the prothoracic gland produces a second hormone, prothoracic hormone.
Its presence then causes the epidermis, the skin, to moult.
The prothoracic hormone triggers moulting.
The form of moulting is determined by the presence or absence of the third hormone, which is produced in the corpora allata.
When this hormone is present, which is the case during the entire period of larval development,
the insect undergoes larval moulting, if the corpora allata ceases to produce this hormone,
known as juvenile hormone, pupal moulting occurs,
and the same prothoracic hormone is ultimately still required for imago moulting.
All three hormones at work here have meanwhile been isolated.
And we are able to demonstrate their effects on suitable objects.
We will limit our observations today to prothoracic hormone – the actual moulting or pupation hormone.
It is the only one of the three to date to have been isolated in crystallised form.
From one tonne of fresh silk worm pupae, 75 to 100 mg of the hormone can be obtained,
which we have named ecdysone from ecdysis, or moulting.
It is a bicyclic system without nitrogen with the formula C18H30O4.
Unfortunately, we still do not know the structural details.
We now hope to get help from x-ray structural analysis.
Nevertheless, in recent years it has been possible, using the pure hormone,
to study its physiological effects in detail and in this context I would like to discuss two examples.
Here a ligature is being performed on a fly maggot which separates the head end from the rear end.
The result of such an intervention, as shown in the next picture, is a partial pupation.
Only the anterior end pupates; the posterior end remains a permanent larva.
This pattern results because the metamorphosis hormone that induces pupation is produced in the head
and cannot travel past the ligature to the abdominal section of the insect.
If we inject pure ecdysone into the abdominal section of the permanent larva, it too subsequently undergoes pupation.
Here you see how the hormone is injected into the small larva using a microinjection syringe.
We inject 0.01 ml of a solution, equivalent to 0.01 grams, or 10^-8 grams of the hormone, to induce pupation.
The result of such an injection is statistical in nature, as expected in biological experiments.
Here are a number of abdominal sections injected with hormone
and you can see that in addition to total pupations, partial pupations have also occurred,
while some insects did not respond, as we would expect in a biological experiment.
Many of you will recall that this effect forms the basis of the physiological test to detect and isolate ecdysone.
The most elegant biological experiment on the effect of ecdysone was conducted by Carrol Williams,
and in the next picture, from a paper by Carrol Williams, you can see what he did.
Previously we saw the large moth Cecropia platysamia, a beautiful colourful silkmoth, a giant.
And here at the top is the isolated abdomen of a pupa of this moth.
We see how two pieces of organ are inserted using small needles, namely neurosecretory cells from the brain and prothoracic gland.
So the two hormone-secreting glands, under the influence of the other implant,
the prothoracic gland, produces prothoracic hormone.
This hormone, as I said, also controls the final stage of imago development,
and the abdomen of the pupa develops into the abdomen of the moth.
The entire morphology changes, the colour changes and the isolated abdomen even lays eggs. It functions.
Without the intervention, without a supply of hormone, it would have died.
This experiment was repeated with pure crystallised ecdysone instead of the two organ implants.
In the next picture we see this isolated abdomen, coloured yellowish brown, placed on a small agar plate,
before imago development, before imago moulting.
If we did nothing, this isolated abdomen would, of course, die within a short time.
After injection of 6 micrograms of ecdysone, the entire developmental process occurs, this is a photo of the same setup.
You see the change in the shape of the moth’s abdomen.
You see the expression of the colours and the pattern.
So the entire developmental process occurs under the influence of a tiny amount of pupating hormone.
It would be difficult to demonstrate the causal effect and significance of a hormone
in the course of a developmental step more convincingly.
Hormones also act as regulators in the developmental stages of mammals, including humans,
but nowhere can their effects be studied so easily and in such an isolated setting as here.
So how does this hormone actually work?
The activity of hormones is, for the most part, not known in detail.
We are repeatedly struck by the astonishing fact that such tiny quantities of a substance
can trigger such dramatic processes in the organism.
I am delighted to tell you
that Dr Karlsson, in cooperation with Beermann’s department at the Max Planck Institute for Biology in Tübingen,
has recently made an observation with the help of ecdysone which I regard as groundbreaking.
For the first time namely it has been shown that the function of gene loci in the nucleus is influenced by this hormone
– in a highly characteristic manner.
We thank Beermann for demonstrating that those parts of a chromosome
in which active gene loci are located undergo a histological change.
So-called puffs form, which we interpret biochemically as a loosening of the genetic substance
and a transition to its actual function.
Geneticists have always claimed that such puffs cannot occur simply from an internal mechanism of the chromosome
but that the surrounding milieu must induce the gene loci to carry out specific functions
that are required for further development.
But it has never been possible to exert an unambiguous influence on puff formation experimentally.
Only now has it been possible to demonstrate, using ecdysone and giant chromosomes of dipterans,
in which these things can be observed so well,
that minute amounts of hormone can induce puffing in highly specific gene loci, thus activating their function.
Recalling everything I’ve said before, that in some way the production of specific proteins is initiated from these gene loci,
we can imagine that such activation of individual gene loci initiates the production of specific enzymes.
That is really just a start. And we also know how we must interpret this effect.
And as I said, this is a groundbreaking achievement for which I have the greatest admiration.
Ladies and Gentlemen, another example of the control of the development of individual organisms by an active substance
is the honey bee, Apis mellifica, in which the expression of various sexual forms will serve
as an introduction to the problem of assignment within a social colony.
A robust beehive consists of about 50,000 to 80,000 worker bees, 2,000 to 3,000 male bees, the drones, and a single queen.
In the next picture we see the morphological differentiation of these types,
in the middle the queen, on the left the drones, on the right the workers, you will already be familiar with all this.
The drone is nearly as big as the queen but darker.
We know that the drones, the male bees, are produced through parthenogenesis. This means from unfertilised eggs.
Whereas the queen and workers are produced from fertilised eggs.
Whether a fertilised egg gives rise to a worker or a queen is determined solely by how the broods are fed.
Here are the various forms of cells, which you are familiar with.
A fertilised egg raised in the small cells produces a worker.
A fertilised egg raised in a characteristic queen cell becomes a large queen.
It has been observed that larvae hatched from the same eggs are fed differently in the various cells.
Whereas the worker larvae are fed only a pinhead quantity of worker food, the larva in the large queen cell selected as the queen
receives a copious amount of royal jelly, which is produced by glands in the head of nurse bees.
In the next picture you see a cross-section through a bee, and here in the head are a number of glands.
This one here is the gland that produces royal jelly.
Despite much effort - let me reiterate – one gets the impression
that whether an egg develops into a queen is determined by royal jelly, that is by the diet.
If a bee larva is transferred just after it has hatched in a hive from a normal brood cell,
in which it would have developed into a worker, to a queen cell, it is fed royal jelly and becomes a queen.
But despite much effort, all attempts were in vain to transform a young larva into a queen outside the hive
by feeding it royal jelly.
And consequently, even today, many still contend that entirely unknown brood-care factors determine
whether a larva becomes a queen.
The situation has changed dramatically since Dr Hanser at our institute recently succeeded in identifying the conditions
in which just-hatched presumptive worker larvae can be raised in an incubator, outside the hive,
into completely normal queen bees solely by feeding them royal jelly, excluding all other brood-care factors.
Dr Hanser’s experimental setup is shown in this picture.
It is a small wooden board into which, as you can see, small artificial honeycombs are inserted.
They consist of small glass vessels, like this one here shown in detail.
And you can see here in the vessels the developing larvae floating.
They are floating in royal jelly.
If one manages to transfer young larvae, within the first two days of hatching, a period during
which they are particularly sensitive, living and unharmed to the experimental cells,
they will develop in an incubator at a normal hive temperature of 35° and fed with royal jelly, into queens.
Larvae, at the end of the second day of life or later, subjected to the same conditions, always develop into workers.
However, under the influence of royal jelly, they can develop into giant workers, which rarely occur in normal hives, if at all.
The result of the experimental setup is shown here.
You see on the left a normal queen from the hive.
You see on the far right a normal worker from the hive.
This is a queen raised by us in the laboratory, which developed from a small larva that would have produced a worker in the hive.
And here is a so-called giant worker, which develops if you introduce the larva to the experimental setup too late,
namely when the actual determination has already been made.
The queen differs from the workers not only in size,
but above all in the development of ovaries and the morphological shape of the mouth parts.
Moreover, the queen lacks the tools on her posterior legs required to collect pollen, namely the brushes and baskets, in short:
the queen not only differs from the workers in size and the development of ovaries, her entire morphology is different.
That’s why it is important to recognize that a queen produced experimentally in an incubator
shows all the features of a normal queen, she is even accepted as queen by a colony.
In this picture, let me show you the differing morphology of the mouth parts.
On the right by the worker with a long proboscis, on the left by the queen.
And you can clearly see the morphological differentiation.
Now we can therefore draw the following conclusion:
It is clear that the decision about the biological fate of a larva is determined in the first 48 hours of its life
and indeed solely by the food it is fed.
Whether, as we would probably like to assume, royal jelly contains a determinant substance with specific activity
or, also a possibility, whether the determination of a larva to develop into a worker
is due to poor nutrition during the sensitive period remains uncertain.
But the experimental foundation to answer this question has been laid,
and we have begun experiments to fractionate royal jelly and test the individual fractions to see
if they induce queen development in the incubator.
We look forward to next season’s experiments with particular excitement.
Ladies and Gentlemen, I would also like to conclude this problem of determination here.
The time is already advanced, and in the final part of my lecture I would like to return to a problem
that has already been broached here in earlier lectures.
The problem regarding communication between insects through chemical substances.
We call substances that are produced in an individual, from these transferred to others,
and received by those individuals as communication signals pheromones.
Pheromones are therefore chemical messengers conveyed from individual to individual.
There are many of them and we can regard them as substances of social cohesion,
because they are produced in greater numbers in social insects so that individuals can communicate with each other.
Pheromones include species- and gender-specific scents produced by insects
which are released by the male or female in order to attract a partner and advertise the presence of individuals of the same sex.
We have worked with one such gender- and species-specific sexual attractant in the silkmoth.
And last year, after 20 years of effort, we were successful for the first time in isolating such a pheromone,
such a gender-specific sexual attractant, and determining its chemical structure.
In many insects, finding a sexual partner is facilitated by such species-specific attractants.
Detailed knowledge about their activity has been gained mainly from butterflies and moths,
whose extraordinary olfactory performance was noted very early on.
Insect lovers and entomologists have shown through numerous observations that the female of many butterfly
and moth species are able to attract mates from afar.
I would like to describe an elegant experiment from the recent literature which was carried out on Chinese silkmoths.
Males of this moth species were marked individually and released from a moving train at various distances from the home location,
at which a female of the species was kept in a gauze cage.
From a distance of 4.1 kilometres, 40% of the released males, and from a distance of 11 kilometres 26% returned to the female.
By means of a series of easily performed experiments it can be reliably shown
that the female in special scent glands produces gender- and species-specific substances
which can be extracted from the glands with lipoid solvents
and are perceived by the males through sensory fronds on their antennae.
We now know that perception of this scent prompts males to fly against the air current, against the direction of the wind,
and thus get underway on their quest for a sexual mate.
This behaviour explains why males are frequently attracted from large distances.
They do not measure for instance the concentration of the molecules,
but instead a scent molecule is interpreted to mean “fly off against the direction of the wind”,
and we can easily see that in most cases the attractant must have been carried by the wind
so that the male’s chances of finding the female are not bad.
Many experiments have been carried out to isolate such scents in order to determine their chemical makeup.
Usually insect pests such as the nun moth, grapevine moth and gypsy moth are used.
And using extracts from the female scent glands of these species, researchers have tried to attract males in the outdoors.
The number of males captured served as a measure of the potency of each extract.
The knowledge thus gained about the nature of such sexual scents in 40 years of work was extraordinarily poor.
In 1939 we undertook the biochemical identification, as I mentioned, of the sexual attractant of the silkmoth Bombyx mori.
It had, until then, never been used in such experiments and yet it had the great advantage for the laboratory
that it is a domesticated insect which can be easily bred and which is procured in large quantities in the silk industry.
The moths no longer consume food, they can be kept in open trays because they do not fly,
and in the presence of a female silkworm or under the influence of an extract from the abdominal glands of the female,
the practically stationary males become highly excited, beating their wings rapidly and rotating in searching movements.
And that forms the basis for the required chemical identification test.
We have been, since 1939, in the summer and autumn months,
conducting experiments with the aim of identifying this attractant biochemically.
For opening up this interesting field, my coworkers and I are indebted to the suggestion and advice of Walter Schoeller,
the former head of the main laboratory of Schering AG in Berlin, who, I believe, is also among our friends here today.
The choice of the experimental object, this simple experimental object, was made on the advice of the entomologist Görnitz,
who, after orientating experiments conducted jointly with Schotte at Schoeller’s Laboratory in Berlin,
recognized the possibility of learning about the general character of the sexual attractant of lepidopterans
by using the silkmoth as a laboratory model.
Here you see a silkmoth and when sexually excited,
the female extends two glands from the posteriormost abdominal segment, the lateral sacculi.
They are pigmented yellow, and the attractant can be extracted from them with lipoid solvents.
The males sit there very calmly after hatching.
They can be identified by their large antennae. They maintain their idle state.
For the attractant test they can be kept in open trays, where they sit calmly and are used as follows for the test.
We first insert a clean glass rod into the glass vessel, position it in front of the males’ antennae
and expect no change in the insects’ idle state.
When we then dip the tip of the glass rod into an attractant, the insects begin to intensively whirr immediately.
For us it is the clearest sign that the insect is still alive at all.
These insects live for around eight days without eating, as I mentioned.
When you believe that they are outwardly no longer alive, you can determine with this attractant
whether they will attempt the very last movements of this kind.
You can therefore gauge a reaction to the test in this way.
You can define an attractant unit, for example, by saying that one attractant unit of 1 microgram means that the substance,
when present in a concentration of 1 microgram per cubic centimetre,
elicits this excited dancing in 50% of the experimental insects
after we dip the tip of the glass rod, the magic wand, into the solution.
We know from the first experiments in 1939 and 1940, which aimed to concentrate and chemically identify the attractant,
that the attractant must be a lipoid-soluble, neutral and nonsaponifiable alcohol
that is resistant to dilute acids and bases, but sensitive to oxidants.
From 7,000 female butterflies we were able at the time to isolate 100 mg of a waxlike fraction
that contained one attractant unit in 0.01 micrograms, i.e. 10^-2 micrograms.
We believed at the time that we were already pretty close to the goal, but the following years showed
that those products would have to be concentrated by several orders of magnitude to obtain the pure attractant.
After we, on the basis of many years of gradually improving experimentation,
again processed the extract from 500,000 glands the year before last, we finally achieved the goal of purification.
As I said, just about 20 years after the work was commenced.
We succeeded in obtaining pure attractant in the form of 12 mg from those 500,000 glands
and 12 mg of a crystallised coloured ester, from which the colourless attractant itself can be generated.
With this small quantity it was possible to carry out an analysis of the chemical structure.
The formula of this first gender- and species-specific attractant is given here below.
It is a hexadecadienol. That means that we have a straight chain of 16 carbon atoms.
Hexadeca is the basic substance. At the end of this chain we have a primary alcohol group-ol, attached to carbon atom 16.
We have two double bonds between carbon atoms 4 and 5 and between 6 and 7. Hence, a hexadecadienol.
Our picture shows how the essential structural elements were found.
The free alcohol was reduced to a known substance, cetyl alcohol, which was unambiguously identified.
In this way we showed that a straight chain of 16 carbon atoms was present.
The coloured ester was then oxidised using a micromethod specifically developed for this purpose.
And all 16 carbon atoms were detected in the form of hydroxycapric acid,
in the form of butyric acid and in the form of oxalic acid.
Thus, the formula was unequivocally identified.
Now, chemists know that the spatial arrangement around a double bond can always give rise to two isomers,
resulting from the configuration of the atoms in space.
We can have a cis or a trans configuration at the double bonds that carry substituents.
This means that a compound with this formula exists in four isomeric forms.
A cis can occur at both double bonds, and a trans can occur at both.
A cis configuration here, a trans there, or a trans here, a cis there.
Spectroscopic analyses have shown that one of the double bonds has a trans configuration, the other probably a cis configuration.
Thus, the number of 4 possible isomers has been reduced to 2.
In the past year we have attempted to prepare these 4 isomers of hexadecadienol synthetically
and have been successful in doing so.
These isomers are extraordinarily similar to each other, as you might expect.
But the hope of reporting to you today about the physiological testing of the 4 isomers has not been realized,
because we still have no silkmoths.
This conference was scheduled about 14 days too early. We plan to test these 4 isomers.
We are convinced that we have successfully synthesised the attractant itself,
and which of the isomers is the actual attractant
and how greatly the isomers differ quantitatively in their physiological effect is a question
that we are keen to study in the coming weeks.
Remarkable is the extremely potent physiological effect of the attractant.
The solution used to test the efficacy limit contains,
according to the definition of the attractant unit, 10^-10 micrograms per cubic centimetre.
10^-10 micrograms contains on the order of 10^5 molecules of attractant.
The tip of the glass rod, when wetted with the test solution, has around 10^-2 cubic centimetres of test solution adhering to it,
corresponding to around 1,000 molecules on the tip.
The magic wand carrying this small number of molecules causes 50 of 100 individual silkmoths to beat their wings.
When you consider that the vapour pressure of hexadecadienols is not very high
and that the vaporised molecules also have to cross the air space between the glass rod and the insects’ antennae,
probably very few individual molecules actually reach the sensitive elements of the antennae.
Biochemists are astonished by the relatively simple structure of a substance with such specific action,
as this substance is perceived as an attractant only by male silkmoths, not by others.
And this attractant contains no branches of carbon atoms, no asymmetric carbon atom.
Evidently, part of its specificity lies in the configuration of the conjugated double-bond system.
Studying the relationship between structure and activity in this example therefore has particularly interesting prospects.
Of course, knowledge of the structure of the sexual attractant of the silkmoth is only of theoretical interest.
We expect, however, from the outset
that the specific sexual attractants of butterflies and moths all belong to the same substance class,
so that the Bombyx attractant holds the key to unlocking the secrets of other sexual attractants.
This could prove of practical importance in fighting those moths that are feared as plant pests.
We know that the insecticides in use today, we will hear more about them tomorrow, are by no means toxic only to insects,
they also harm other animal species and plants treated with insecticides can also be detrimental to humans.
Unfortunately, along with insect pests, useful insects are also destroyed, and the lack of selectivity in today’s pesticides
can lead to major disruptions in the biological equilibrium of the treated biotope.
In addition, the use of insecticides leads to the development of resistant strains
that are immune to the effects of the agent being used.
The notion of using specific sexual attractants to combat insect pests is old and compelling.
We could conceivably use a synthetically manufactured sexual attractant of an insect pest
to attract only the males out of a district, capture these, and thus interrupt the propagation of the species.
The development of resistance to the attractive scent of the coveted females is, I believe, nothing to fear.
Ladies and Gentlemen, I’m happy to tell you that a first step in this direction has been made in the chemical field.
A working group headed by Haller in Washington, using the methods we developed on the silkmoth,
recently isolated the sexual attractant of the gypsy moth, prepared it in pure form, and analysed it.
The gypsy moth ranks among those insects that cause untold damage.
And here’s the gratifying news for us. What is the chemical nature of this substance?
It also contains 16 carbon atoms in a straight chain. It is also a primary alcohol, like our substance.
But it contains not two double bonds but just one, and in place of the other double bond,
it contains an oxygen function, an acetylated hydroxyl group.
The positions of the double bond and the acetoxy group are still unknown.
But what is known shows that our idea was correct, that the silkmoth holds the key to the secrets of attractants of insect pests.
With the attractant of the gypsy moth experiments conducted outdoors have been successful
insofar as it is now possible, with the help of this substance,
to determine in a biotope whether gypsy moths are present or not, which is important.
And even that would represent a gain if our hope of mounting a real campaign with the attractant is not realized.
Ladies and Gentlemen, I would like to stop here.
and Thank you for your attention and that I hope I have been able to show you that old problems,
which we talked about in the past, are still exciting, and that progress, though unfortunately slow, is being made.