Specialized Terms
allelochemicals Plant chemicals, sometimes called secondary plant compounds because they are produced as by-products of intermediary metabolism, that may function in defense against insect herbivory.antifeedant A chemical, often toxic, that prevents or reduces feeding.
complementary foods Foods that individually are nutritionally unbalanced, but when combined can provide a balanced diet. heterotrophic A life condition in which an organism obtains organic chemicals in a preformed state by consuming other organisms or their by-products.
nutritional rail A means of representing a food in a nutrient space as a line which moves from the origin into the space. intake target The optimal nutrient intake over a given period in development that can be represented as a point in multidimensional nutrient space.
phagostimulant A chemical that induces and maintains
feeding.
required nutrients Nutrients, including essential nutrients, that enhance or optimize growth, development, and/or reproduction.
The nutrition of animals reflects their heterotrophic character, and the need for animals to obtain preformed organic substances that they lack the ability to synthesize from simpler carbon sources. Nutrients are environmental factors that connect and intersect an animal's physiology and ecology. In the broadest sense, nutrition refers to the ingestion and processing of substances that fuel the organism's energy and structural needs for growth, maintenance, and reproduction. A strict definition of nutrition, however, is not possible and any specific view of nutrition depends on perspective. Regarding nutritional requirements, insects share much in common with other animals. Studies with insects have contributed significantly to our general understanding of nutrition, both through development of new ways of thinking about the complexity of nutrition, and through the use of molecular genetics to establish Drosophila as a model system for the study of nutritional homeostasis.
HISTORICAL OVERVIEW
Early interest in insect nutrition aimed to understand the dietary requirements of insects, an investigative focus often referred to as dietetics. This involves identifying and characterizing the food that insects eat to satisfy their nutritional needs, as well as the feeding behaviors and sensory physiology associated with obtaining foods. A principal purpose of this research was to rear insects in the laboratory on artificial foods, thereby avoiding the often-costly effort necessary to maintain natural foods. Among the first successes was E. Bogdanow's 1908 report on rearing of a dipteran Calliphora vomitoria on a diet containing meat extract, starch, and peptone. Similar accounts followed, describing the rearing of many insect species representing a variety of taxa. Numerous scientists are associated with this early work, including S. Beck, R. Craig, G. Fraenkel, W. Trager, and B. Uvarov and others. Subsequent research attempted to refine diets by using more purified natural products, or even pure nutritional chemicals. The effort was greatly aided by new information gleaned from research on the basic nutritional requirements of insects. A major advance was the discovery by E. Hobson in 1935 that insects require dietary sterol. This clearly distinguished the nutrition of insects from that of most mammals and other higher taxa, whose nutritional requirements were far better known. The discovery quickly led to advances in the study of insect dietetics and nutrition.ARTIFICIAL SYNTHETIC DIETS
Despite the diversity of insect dietary habits, insect nutritionists have been remarkably successful at developing artificial diets for rearing insects. Published compilations of insect diets representing the collective effort of over one thousand scientific contributors indicate that several hundred species can be reared partially, or through their entire life cycles, in the absence of their natural foods. Literature is available describing various applications of this nutritional technology. The catalog of a well-known commercial concern lists for sale diets for rearing over 50 species of insects, and artificial diets have been employed for mass culture of several species. A United States Department of Agriculture laboratory in Phoenix, AZ, for example, is currently rearing on a completely artificial diet, 6-7 million pink bollworm moths, Pectinophora gossypiella, daily, for use in an autocidal pest management control program in California.QUALITATIVE NUTRITIONAL
REQUIREMENTS
The nutritional requirements of insects were at first inferred from knowledge of the chemical compositions of natural foodstuffs. Insects were categorized as carnivorous, omnivorous, or phytophagous, with the appropriate inferences regarding the nutritional content of the animals, plants, and other foods eaten. Studies of food utilization, and the analysis and comparison of foodstuff eaten and excreted or unabsorbed, allowed further assessment of the relative importance of various nutrients for insects with different feeding habits. A more detailed and complete understanding of insect nutritional requirements finally emerged following the successful development of artificial diets, particularly chemically defined diets. The essentiality of many nutrients was established by dietary deletion, the sequential elimination of individual chemicals, potentially nutritious, from a diet. These advancements involved many scientists now recognized for their contributions to the foundation of insect nutrition—J. S. Barlow, R. H. Dadd, S. Friedman, H. T. Gordon, H. L. House, J. G. Rodriquez. T. Ito, E. S. Vanderzant, G. P. Waldbauer, and others.A nutrient is deemed essential if, when deleted from the diet, further growth, development, and/or reproduction are prevented. Almost all insects have a common set of essential nutritional requirements. Nutrients demonstrated to be necessary for optimizing growth, development, and/or reproduction are required nutrients. The value of specific nutrients, however, often depends on total dietary content. Dietary carbohydrate, for example, is often unnecessary, but may be required or even become essential, for providing energy in the absence of dietary fat, or protein in excess of the amount required for normal growth. The following summarizes the essential qualitative requirements of most insects.
Nitrogen
Insects consume and utilize a wide variety of proteins to satisfy their nutritional requirements for amino acids. Of the commonly occurring amino acids that comprise most proteins, 10 are not synthesized by insects, or by most animals, and are essential nutrients. These include arginine, histidine, isoleucine, leucine, lysine, methionine, phen-yalanine/tyrosine, threonine, tryptophan, and valine. Beyond the bulk requirement for protein synthesis, these essential amino acids serve additional physiological functions of particular note in insects, and other invertebrates. Arginine, for example, is a precursor for the principal invertebrate muscle phosphagen, phosphoargininine. Tyrosine is important for production of phenolic and quinone metabolites, critical components for cross-linking of protein during sclerotization.The other 10 commonly occurring proteinaceous amino acids are generally non-essential, but may nevertheless be required to some degree for normal growth and development. Some amino acids, however, may be uniquely essential for some species. Several mosquitoes and one tachinid species, for example, may be unable to synthesize asparagine which is an essential dietary amino acid. An essential requirement for proline by several taxonomically disparate insect species may be related to a limited activity of the urea cycle. For unknown reasons, glutamic acid or aspartic acid, normally non-essential, have been reported as essential for a few species.
Few insects develop on diets containing only the 10 essential amino acids. Moreover, many insects do poorly on diets containing amino acids as the sole source of nitrogen, and require protein or a mixture of protein and amino acid for normal growth and development.
Insects generally synthesize their own nucleosides, nucleotides, and nucleic acids, although several species, particularly various dip-terans, have been shown to benefit by inclusion of nucleic acid or some constituents of nucleic acids. In a few cases, nucleic acid is considered essential for completion of development.
Vitamins
Vitamins, particularly water-soluble B vitamins, including biotin (Vitamin H), folic acid (B11), niacin, pantothenic acid, pyridoxine (B6), riboflavin (B2), and thiamine (B1), or close chemical relatives of these, are essential nutrients for all insects. They serve principally as precursors for the coenzymes of intermediary metabolism. Regarding the fat-soluble vitamins, only tocopherol (E) and retinol (A) have proven beneficial for reproduction and vision, respectively, by some insects. Tocopherol also plays an important role as a lipid antioxidant.Carbohydrate
Carbohydrate nutrients, although often required as an energy source, are rarely essential. An exception may be the case of some adult lepidopterans that feed solely on plant nectars. Indeed, some of these insects are thought to be capable of digesting sucrose alone. The same may hold true for some adult dipterans and hymenopterans. In addition to their role as nutrients, sugars, particularly sucrose, are powerful phagostimulants, without which some insects feed poorly or not at all.Sterols
Insects have an essential requirement for a dietary sterol. Cholesterol appears to be widely acceptable, but a number of other plant sterols, particularly (3-sitosterol are equally acceptable. Among a few exceptions, is the interesting example of Drosophila pachea, the senita cactus fly, that requires A7 dietary sterols, metabolic derivatives of schottenol, a A7 sterol found in its natural food, the senita cactus. In addition to the bulk requirement for sterol utilization in membrane formation, sterol is also important for the production of ecdysone and other molting hormones.Fatty Acids
Fatty acids are considered non-essential for most insects, but several mosquitoes and some lepidopterans require a polyunsaturated fatty acid. This requirement is associated with one of only a few known nutritional diseases or syndromes—the "crumpled wings" syndrome. Absence of a polyunsaturated fatty acid results in adult insects that fail to fully expand their wings following eclosion, and are thus unable to fly. The chemical nature of this requirement is poorly understood. In the case of mosquitoes, arachidonic acid or some closely related fatty acid with an lo-6 double bond is essential. In those Lepidoptera requiring a polyunsaturated fatty acid, however, some species utilize to6 fatty acids, whereas others utilize lo-3 fatty acids such as a-linolenic acid. Moreover, in those Lepidoptera requiring an lo-6 fatty acid, a-linoleic is generally preferred, whereas arachidonic acid is unsuitable. Nothing is known of the physiological basis for this requirement, but in the mosquitoes it may be linked to the synthesis of prostaglandins, local regulators that target a wide variety of cellular functions in vertebrate animals.Inorganics
A complex of mineral ions is essential for insects. The balance of minerals, however, is often dramatically different from the well-known salt requirements established for mammals. Many insects, for example, require much greater proportions of potassium, magnesium, and phosphate, relative to sodium, calcium, and chloride.Ascorbic Acid and Other Water-Soluble
Growth Factors
L-ascorbic acid, vitamin C in vertebrate animals, is an essential growth factor for many phytophagous insects. In its absence, these insects generally fail to grow and/or develop. The pattern of this requirement among insects is thus similar to that of the higher animals where species that have adapted to a diet of fruit and/or vegetables have apparently lost the ability to synthesize ascorbic acid. In contrast to the vitamin C requirement of vertebrates, ascorbic acid is required by insects in relatively large amounts, although this may in part reflect the necessity for a high antioxidant activity in synthetic artificial diets employed for testing. Moreover, unlike vertebrates requiring L -ascorbic acid, some insects utilize dehydroascorbate as effectively as L -ascorbate, and may use the D geometric forms of closely related lactones, although these are generally not as effective. Although there is little understood about the role of ascorbic acid in insects, beyond its potential antioxi-dant action, it may play the same role as in vertebrates, that is, as a factor necessary for enzyme activities involving hydroxylation. Deficiency in insects is often associated with abnormalities of molting, possibly due to the absence of the effects of ascorbic acid on diphenyloxidases.The lipogenic growth factors, choline and inositol, principal components of phospholipids, are required by many insects. Choline may be essential nutrient for most insects.
A unique and essential requirement for carnitine is known for several tenebrionid beetles, where this usually non-essential nutrient is called vitamin BT. Normally derived from choline, carnitine plays an important role in fatty acid transport.
Essential and non-essential nutrients are required in specific amounts, but as implied above, optimal levels of individual nutrients often depend on the presence and concentrations of other nutrients. Early studies with artificial diets purported to formulate optimal levels of nutrients, based on the relative concentrations found in natural foodstuffs. Diets employing these concentrations of nutrients often produced adequate results, but subsequent studies have demonstrated that the quantitative aspects of insect nutrition were far more complex than that approach suggested. Moreover, ecological, behavioral, and physiological factors are also important for optimal nutrition.
QUANTITATIVE NUTRITION
If an insect is to survive, grow, and reproduce, it must ingest several dozen different types of nutrient molecules. These molecules come packaged in varying amounts and ratios within foods, along with non-nutritive (and sometimes toxic) compounds. Foods in turn are distributed through time and space, and their finding, ingestion, and processing engender metabolic and ecological costs. Moreover, the nutritional needs of insects change with age, stage of development, reproductive status, etc. Matching the multidimensional and changing nutritional demands of the insect against the complex and changing composition of the nutritional environment has posed one of the greatest challenges to evolution—and to scientists who study insect nutrition. The problem is particularly difficult for herbivorous insects, where the nutritional composition of host plants may be highly variable and there is the added complexity of secondary plant metabolites serving as antifeedants and toxins. There is a growing realization, however, that predators too may face considerable variation in food quality, and may therefore have to regulate their intake and use of multiple nutrients rather than relying on more general food properties.A powerful way of defining and exploring nutritional regulation that has arisen from work on insects has been to represent the animal, its intake and growth requirements, and the foods in its environment using multinutrient plots. This "geometric framework" has enabled the identification of the key elements in complex nutritional systems, and the quantification of the interactions among them. These include interactions among the multiple constituents of the food, as well as between behavioral and physiological regulatory mechanisms. The resulting descriptions provide a powerful means to study the mechanisms, ecology, and evolution of nutritional systems.
Quantifying Intake Requirements
Estimating the intake requirements (intake target) is a primary aim of any nutritional study. One way to do this is to allow the insect to demonstrate whether it is able to regulate its nutrient intake, and if so in which nutrient dimensions. A well-documented study system is the locust, which has been shown to regulate its intake of both protein and carbohydrate under several challenges:(a) Pairs of complementary foods. When locusts were provided with one of four complementary food pairings (28:14 or 14:7, % protein:% digestible carbohydrate vs. either 14:28 or 7:14), they adjusted the amount and ratio of the two foods eaten to maintain a constant point of protein and carbohydrate intake (Fig. 1, upper panel).
(b)Food dilution. When locusts were given one of five foods containing a near-optimal ratio of protein to digestible carbohydrate (1:1), but diluted up to fivefold with indigestible cellulose (35:35, 28:28, 21:21, 14:14, or 7:7, % protein:% digestible carbohydrate), they adjusted their consumption across all dilutions to maintain a constant point of nutrient intake (Fig. 1, middle panel).
(c)Food frequencies. When two complementary foods (31:11 and 7:35) were provided in relative abundances of 1:3, 2:2, or 3:1 dishes of one vs. the other food type, locusts precisely selected a
point of protein to carbohydrate intake by adjusting their distribution of consumption between dishes (Fig. 1, lower panel).
These remarkable feats of homeostasis were found to extend to regulation of salt vs. macronutrient intake. Moreover, other studies indicate that such capabilities are by no means restricted to locusts.
Changes in Intake Requirements with Time
The intake requirements of insects are not static. They change with recent nutritional experience and level of activity. For instance, locusts and caterpillars select a protein-rich food following a short experience (only one meal in the case of the locust) of a protein-deplete food, and show a similar preference for carbohydrate-rich food after a 4 h period of carbohydrate deprivation. Nutrient requirements also vary with stage of development, reproduction, and diapause. Over a longer time scale, nutritional requirements evolve to track changing nutritional environments and life histories.Mechanisms Regulating Intake
Regulating nutrient intake requires two sources of information, the first being the composition of the food and the second the nutritional state of the animal. The responsiveness of an animal to a food of given composition should reflect, through feedbacks, the animal's nutritional state. Insects are able to taste certain key nutrients, notably sugars, amino acids, salts, and water. Studies on locusts, caterpillars, and blow flies have shown that the responsiveness of taste receptors to sugars and amino acids varies with nutritional state, as represented by concentrations of these nutrients in the hemolymph. Such nutrient-specific feedbacks enable insects to make sophisticated behavioral decisions about what foods to eat to regulate nutrient intake. In addition, learning of various sorts, including aversion, learned specific appetites, and induced neophobic responses, also plays an important role in regulating food selection in insects. An impressive example is the ability of locusts to learn to associate the odor of a food with its protein content and to be attracted by odors previously paired with high-protein food, but only when in a state of protein deficit.Nutrient Balancing on Suboptimal Foods
If an insect is restricted to suboptimal foods and is unable to reach its intake target, it must balance undereating some nutrients against overeating others, reaching some "point of compromise." Such a situation exposes whether and how the mechanisms regulating intake of different nutrient groups interact. A simple means of exploring the interactions between regulatory systems for different nutrients is to provide insects with one of an array of foods of varying composition and to measure intake. Collectively, the resulting points of nutrient intake across the array of foods form a pattern that describes the relationship between the mechanisms regulating intake of the nutrients concerned.Various such relationships have been described to date in insects. The simplest outcome is where the insect abandons regulation of one nutrient when forced to balance it against regulation of another. For instance, locusts regulated macronutrient (protein and carbohydrate) intake and let salt intake vary passively when fed single foods containing suboptimal salt levels, even though they regulated both salt and the macronutrients when allowed to switch between complementary foods. In other cases, one nutrient does not overwhelm the other. When locusts were fed foods varying in protein and carbohydrate content they balanced over- and undereating the two nutrients, with the precise balancing rule depending on the species of locust. The grass-feeding specialist, Locusta migratoria, was less able to overeat unbalanced foods to gain more of the limiting nutrient
FIGURE 1 Three experiments indicating regulation of intake to a point (intake target) in a carbohydrate-protein plane. The plots are of the form used in the geometric framework. Foods are shown as lines (rails) radiating out from the origin. An insect feeding from a single food is constrained to move along that food rail in intake space, while two foods provide the opportunity to move anywhere in between by food mixing. In the upper panel locusts were provided with one of four food choices (14:28 or 7:14, % protein:% carbohydrate vs. 28:14 or 14:7) and altered relative amounts eaten from the two foods, thus reaching the same place in nutrient space. Open symbols indicate where insects would have arrived were they to have eaten indiscriminately between the two foods provided. The middle panel shows locusts that were given one of five foods containing a 1:1 ratio of protein to digestible carbohydrate, but diluted to various degrees with indigestible cellulose. Those on food 7:7 ingested five times more food than those on 35:35, thus achieving the same intake of both protein and digestible carbohydrate. Open symbols show points of intake were insects not to have compensated for dilution but eaten the same amount of food on each treatment. Locusts in the lower panel were given four food dishes, containing either 7:35, % protein:% carbohydrate (food C), or 31:11 (food P). The two food types were provided at different frequencies (all four dishes contained C, all four contained P, or two contained C and two P). Locusts adjusted the amounts eaten from each dish and regulated nutrient intake. Open symbols indicate points of intake were locusts to have distributed feeding equally among dishes and not shown frequency-dependent food selection.
FIGURE 2 Arrays of nutrient intake shown by locusts when fed single foods, and hence forced to balance their intake of two nutrient groups. The two panels show how the grass-specialist Locusta migratoria and the host-plant generalist Schistocerca gregaria have different balancing rules for protein and carbohydrate. L. migratoria minimized the distance from the intake target in nutrient space when unable to reach its intake target (the CD rule), while in comparison S. gregaria ate more of the nutritionally unbalanced foods (the ED rule).
than was the host-plant generalist, Schistocerca gregaria ( Fig. 2 ). It appears that the generalist species is better able to capitalize on excess ingested nutrients than is the specialist.
Regulation of Growth and Metabolism
Whereas an insect may be constrained from reaching its intake requirements by available foods, it may still be able to regulate post-ingestive processing and thus achieve its growth target (Fig. 3) . Regulation of growth and body composition involves differentially using ingested nutrients: ridding nutrients in excess and conserving those in deficit. Locusts are extremely effective at regulating growth, excreting excess ingested nitrogen, and respiring excess ingested
FIGURE 3 Patterns of intake and growth in locusts fed 1 of 25 diets varying in protein and digestible carbohydrate content. Intake data are across the entire fifth stadium. Note that protein- and carbohydrate-derived growth were regulated despite insects on the different diets having eaten widely different amounts of protein and carbohydrate. Locusts on all but the most extremely unbalanced diets regulated growth to a high degree in both nutrient dimensions. Regulation of growth involved differential utilization of ingested protein and carbohydrate. On diets with a very low protein-to-carbohydrate ratio (upright triangles), development was extended and body lipid content was high, whereas on foods with a very high protein-to-carbohydrate ratio (inverted triangles), there was no lipid deposition. Insects able to regulate their intake by selecting between complementary foods are indicated with the open square symbol.
carbohydrate or converting it to lipid. Deamination of excess ingested protein, principally by oxidation or transamination to the corresponding keto acid, may serve to augment limiting carbohydrate for energetic purposes.
Studies on Drosophila are beginning to uncover the molecular and cellular mechanisms involved in metabolic homeostasis and coming years promise a rapid expansion in knowledge about the roles of different tissues and signaling and sensing systems.
NUTRIENT-ALLELOCHEMICAL
INTERACTION
The foods of many animals contain, in addition to primary nutrients, harmful or unpalatable non-nutritive materials. This is especially the case for plants, which contain an abundance of defensive secondary metabolites (often termed allelochemicals). In some cases, insects make use of such compounds either as cues for host-plant recognition or as resources in their own right for defense or communication. In the main, however, allelochemicals are an impediment to nutrient regulation. A key point is that the effectiveness of allelochemicals as anti-nutritive compounds depends on the nutritional context in which they occur in the food. For example, locusts are not affected adversely by the presence of tannic acid in their food, even up to 10% dry weight, if the protein and carbohydrate ratio and concentration are near optimal. When foods contain less than an optimal protein to carbohydrate ratio, tannic acid serves as a powerful feeding deterrent, and thus causes high mortality and extended development. However, at higher than optimal protein-to-carbohydrate ratios tannic acid does not reduce intake
FIGURE 4 General effects of symbionts on nutrient intake. Data summarize the protein (or amino acid)-to-carbohydrate ratios in diets that support good larval development in a selection of insect species. Note how insects with microbial endosymbionts develop best on diets with a lower protein (or amino acid)-to-carbohydrate ratio (steep rails) than do other species. Dotted lines indicate endop-terygote species, while unbroken lines are for exopterygotes.
but instead results in high mortality by disrupting protein utilization. Dietary nutrient ratio has also been shown to moderate the toxic effects of nicotine on growth of tobacco hornworm caterpillars, Manduca sexta.
MICROBIAL ASSOCIATIONS
Symbionts, principally actinomycete fungi and bacteria, play a critical role in insect nutrition, enabling many species to develop normally on foods of limited nutritional value. Well-known examples of such foods include wood, blood, phloem, and plant litter. Many insects would quickly perish in the absence of these symbiotic relationships. Symbionts often provide nutrients directly as a result of synthetic capabilities that their insect hosts lack, and/or allow, through the production of gut enzymes, insects to digest otherwise indigestible foodstuff. Alternately, the symbiont itself may serve as food. Many symbiotic relationships are casual, involving ectosymbionts, usually comprising a rich gut microflora. Endosymbiosis is also common, and many insects have developed highly specialized anatomical and behavioral features for optimizing the benefits of the relationship and for efficiently transmitting their symbionts between generations.Among the most significant endosymbioses are those involving mycetocytes and bacteriocytes, host cells specialized for housing symbiotic microorganisms—fungi or bacteria. Often these polyploid cells are associated with the midgut epithelium, although in the case of fungal-infected cells, aggregates called mycetomes may sometimes be found in the hemocoel. The genomes of the symbiont and the host cell are closely coordinated, forming a functional unit known as the symbiocosm. Such an endosymbiont is found in the rice weevil,
Sitophilus oryzae, where it is referred to as the SOPE or S. oryzae principal endosymbiont. This bacterium (family Enterobacteriaceae), whose expression is partly regulated by the host, occurs in the cytoplasm of the bacteriocyte (2 X 103 bacteria/host cell) and is known to be critically important in the insect's biology. It is, for example, the source of several vitamins, including riboflavin, pantothenic acid, and biotin. Moreover, the presence of the symbiont alters the balance of amino acid metabolism and mitochondrial phosphorylation, thereby affecting flight ability and performance.
The impact of symbionts on the quantitative nutrition of insects was recently made apparent by an analysis of the optimal dietary protein: carbohydrate ratio for 117 insect species. Insects housing symbionts displayed very steep intake target rails (Fig. 4), strongly suggesting that symbionts add considerably to the nitrogen nutrition of such species and, moreover, that this may generally be the case.