CHAPTER TWENTY THREE. NUTRITIONAL METABOLISM:

DIETARY REQUIREMENTS

We want to regard the natural connections between the bulky foods of insects, feeding behaviors, and digestive physiology. The foods of insects are either other animals, or plants or decomposed products these organisms. As such, these comprise the preformed organic compounds that are nutritionally required by all animals, including insects. The study of the particular organic forms, that is, the organisms or their natural byproducts, that in the ordinary way of things make up the usual food of a particular animal species is known as dietetics. Although this work can be of enormous importance in ecological arenas, it is the most basic and least sophisticated form of nutritional inquiry. Study of nutrition depends upon some knowledge of the chemical composition of dietary components. The intent of nutrition is the chemical delineation of essential, beneficial and deleterious constituents of food.

Feeding behavior is a practical concern in studies of the nutrition of organisms because many species will feed either not at all or only poorly in the absence of specific stimuli. In these situations there is the problem of not knowing if the resulting poor growth is due to a failure of the diet or due to starvation. For many omnivorous insects the common nutrients are themselves enough stimulus to release feeding behavior. On the other hand, many insects, those consuming only a very narrow range of plants (a behavior known as oligophagy) release full feeding activity only in response to certain token stimuli. Token stimuli do not provide nutrients, but they signal the presence of them. Perhaps we can make a connection to token stimuli which herald coming seasons. In the arena of dietetics and nutrition, token stimuli are often secondary products of plant metabolism and they have nothing to do with nutrition in the rigorous chemical sense.

Digestion is the enzymic hydrolysis of macromolecules into their component molecules preparatory to absorption across the midgut epithelium and into the body proper. Digestion is pertinent to nutrition because many of the macromolecules in food can not be absorbed across the epithelium. Moreover, digestion efficiencies vary across insect species and across various stages in the life of a given species. This variance depends upon a number of factors, including (1) how finely food is ground, or triturated, during ingestion, (2) the availability within a given macromolecule of active sites for digestive enzymes, (3) the rate of food passage through the alimentary tract and hence the amount of time available for digestion and absorption, and (4) the production of particular digestive enzymes by a particular life stage. The interplay of these and other factors regulates the relative amounts of various macromolecules that can be made available for absorption.

Recalling that nutrition has as a goal the chemical delineation of essential, beneficial and deleterious constituents of food, we can pose a core question in the science of nutrition. How well can we know the nutrition of any insect species? To move from the level of dietetics to a physiological study of chemical needs, it is necessary to devise an artificial diet that supports at least moderate growth. This often begins by preparing a diet from crude organic fractions of the insect's natural diet. Diets made of crude organic fractions are called oligidic. If this crude diet supports even moderate growth and development, chemical analysis of particular nutrient classes might allow certain crude organic fractions to be replaced by individually defined chemicals. This is generally an iterative procedure, in which, for example, the crude proteins are eventually replaced by pure amino acids. The evolving diet becomes at least partly defined at a chemical level, and is termed meridic. Diets refined to this level can very useful in nutritional research. I used a meridic diet to study nutritional metabolism of fatty acids in waxmoths. The diet was chemically defined with respect to all lipid inputs, but not in terms of proteins or other classes of nutrients.

Of course, the ultimate aim would be to replace all crude organic inputs by specific chemicals. This would be an entirely defined synthetic diet, and such diets are called holidic - please note that this is a rare correct spelling: the word is not holistic. A good question would be, can we prepare a chemically defined diet? Yes. There are diets of as completely chernically defined composition as the purity of available chemicals allows. One such diet was devised for a wild-type strain of the fruitfly Drosophila melanogaster. Fruitflies are routinely cultured on what is called a yeast diet, shown here:

42.2 grams cane sugar

20.0 grams baker's yeast

15.0 grams agar

Add to the 1000 ml of media

15.0 mls of 10% methyl-phydroxybenzoate as mold inhibitor

This would be reckoned a crude culture medium. Let us compare this to a completely defined medium.

The study of nutrition is aimed at understanding the meaning of these chemical constituents of the medium. When this diet is put together it comes out as a liquid medium. Fruitflies can not eat a purely liquid diet, and a thickening agent is required simply to facilitate movement of food through the animal. This is an example of the importance of studying feeding behavior in nutritional research. To meet the feeding behavioral requirement, agarose was added to provide a minimum viscosity, or a sort of gel, to the medium. After we got the medium a little bit thicker, the larvae could eat.

The larvae also needed a minimum of carbohydrate for energy input, and that is what the sucrose provides. An accepted generalization in insect biochemistry is that insects are not able to biosynthesize sterols. Hence the cholesterol was included to meet this universal insect requirement. The next 18 components are amino acids. These are of the highest chemical purity and serve to replace the protein requirements. The next five components are actually nucleotides, that is they are the monophosphates of each of the bases. These are also of highest chemical purity and they are the chemical analogue of dietary nucleic acids. Now, all organisms need a modicum of salts and phosphates. These are provided by the next five ingredients. The EDTA complexes are known as sequesterines, and they serve to provide known amounts of trace ions.

The last nine items are a list of vitamin requirements. We will become familiar with some of the metabolic roles each of these play out in metabolism, except for carnitine and choline chloride. The carnitine acts as an intracellular carrier molecule, and is involved in fatty acid metabolism. Choline serves as a lipogenic factor in synthesis of phosphatidyl-choline and as a component of the neurotransmitter acetylcholine. It is provided in the diet as its chloride salt.

When properly assembled, these components comprise a chemically defined diet. This is one of the two or three most highly refined diets produced for any animals. Various microbes can provide nutritional requirements to insects. To maintain the chemical definition, this medium must be sterilized by autoclaving it, and the fruitfly eggs must be surface sterilized to prevent inoculating the medium with microbes. Sterilization is verified by inoculating the infusion broth with samples of the medium. These points are important for two reasons. First, we have seen that microbes provide micronutrients to insects, and second, heavy microbial infections can retard normal growth and development.

Yes, they can. Moreover, they can mate and produce viable progeny. This point should be taken with a dose of care, however, because, the carry over of certain micronutrients by way of the eggs may provide essential materials. The absence of such maternally-provided nutrients will not become apparent until a second generation. This concern is often addressed by rearing consecutive generations on the chemically defined medium. When this fruitfly medium was developed, the flies were reared through a number of consecutive generations. As a point of history, at the tenth generation the sterile conditions were inadvertently broken. That is why the experiment ended at generation ten.

Diets of similarly highly refined chemical definition, that is, holidic diets, have been devised for certain mosquito species. At one point the mosquito Culex pipiens was brought through 14 unbroken generations on a holidic medium. Several other mosquito species have been reared through a single generation on similar medium, also holidic. I brought the mosquito Aedes aegypti through four generations on a holidic medium.

In the first case, it is a direct objective of nutritional studies to understand the dietary requirements of an animal at a chemically defined level. Understanding at this level gives insight into basic metabolic capabilities of the organism. A clear example of this in insect nutrition is the universal requirement for exogenous sterol. Since insect nutrition takes its roots and intellectual paradigms from the older mammalian nutrition, sterol was not recognized as dietarily essential in the early years. In metabolic terms, this finding indicates that one or several steps in the biosynthesis of sterol can not be carried out by insects. Sterols are formed along an

acetate--->mevalonate--->farnesyl phosphosphate pathway. Since insects can biosynthesize other isoprenoid chemicals, including juvenile hormone, ubiquinone, defense substances and squalene, that share this pathway, it is thought that the cyclization of squalene is not possible in insects. Hence, the nutritional requirement for sterol provided a basic insight into lipid metabolism in insects. Again, these sorts of insights into metabolism arise from knowledge of dietary needs at the chemical level.

From this point of view, then, a chemically defined diet can be exploited as a research tool. Once formulated, a totally defined diet can be manipulated in the first instance by simply leaving individual chemicals out, one at a time. This technique has been often used to learn which of the 20 or so amino acids were essential. A slightly more sophisticated manipulation is to replace a known chemical with a substitute, then observe the resulting effects on growth. Such substitution techniques have been used in study of sterol nutritional metabolism. Although sterol of some sort is required by all insects, plant feeding insects are able to utilize a broad range of plant sterols by converting them into cholesterol. Carnivorous insects are not able to make use of a range of plant sterols. We may consider an overview of metabolism of plant sterols into cholesterol, and conversion of cholesterol into the molting hormone. The key point to be taken is that nutritional analyses can move beyond yes/no questions of dietary require-meets to more subtle work on metabolism with a class of chemicals.

We gain still more information when observations on the nutritional efficacy of a compound are coupled with chemical analyses of insect tissues. I used such nutritional studies on the waxmoth to show that the absence of particular polyunsaturated fatty acids, namely 18:2 or 18:3, from larval growth media produced adults that were either monstrously deformed or else dead upon adult emergence. When 18:2n-6 was added to the medium, normal adults emerged. When the analogous fatty acid 20:2n-6 was substituted for 18:2n-6, normal adults similarly obtained. I concluded 18:2n-6 and 20:2n-6 were nutritionally equivalent. When the tissue fatty acids were analyzed, though, both groups of adults had the same proportions of 18:2n-, with no 20:2n-6 in the tissues of either group. The insight into metabolism is clear: this insect is capable of controlled fatty acid chain shortening - that is, the 20:2 was shortened to exactly 18:2, and not to other chain lengths. This remains one of the few examples of controlled chain shortening metabolism in insects. The key point, for our purposes, is recognizing the ability to exploit a defined diet as a strategy to gain insights into the biochemistry of insects.

Aside from a greater appreciation of the details of biochemistry of a particular class of molecules, nutritional studies can also give insight into their biological significance or at least when in the life cycle the absence of the chemical is most acutely evident. For example, the absence of vitamin E from the diets of crickets does not produce a measurable effect until the fecundity of the adults is considered. The observation that vitamin E deficient females produced normal numbers of viable eggs when mated with males fed on complete diets and sub-normal numbers when mated to vitamin E deficient males focused attention upon features of male fecundity. Vitamin E deficient males, it was later observed, do not produce normal sperm.