Vol. 51 SUMMER 1970 No. 4

SEASONAL CHANGES IN LEAF TANNINS AND NUTRIENTS AS A CAUSE OF SPRING FEEDING BY WINTER CATERPILLARS'

PAUL FEENY2 Department of Zoology, Oxford University, Oxford, England

Abstract. Concentrationin the spring of feeding by caterpillars of the winter moth, Ope- rophtera brumata L., and other species of on oak trees in England is believed to be related to seasonal changes in the texture and chemical composition of the leaves. Increasing leaf toughness is a proximate, though probably not ultimate, factor preventing late larval feed- ing by the winter moth, the commonest spring species on oak. Early feeding coincides with maximum leaf protein content and minimum leaf sugar content, which suggests that avail- ability of nitrogen, rather than of carbohydrate, may be a limiting factor for spring-feeding larvae. The content of oak leaf tannins, which inhibit the growth of winter moth larvae, increases during the summer and may render leaves less suitable for growth by further reducing the availability of nitrogen and perhaps also by influencing leaf palatability. Oak trees are extensively damaged by insect attack, and it is likely that leaf tannins have a defen- sive function against as well as against other herbivores and against pathogens.

INTRODUCTION moth (Lymantria dispar L., Lymantriidae) (Kan- The common or pedunculate oak (Quercu-srobur su 1962). L.) is a dominant species in the deciduous forest Feeding by Lepidoptera species on oak leaves is which is the prevailing climax of much of western largely concentrated in the spring (Fig. 1). In (Jones 1959). It is attacked by the lar- terms of total insect numbers, this concentration vae of more than 200 species of Lepidoptera, most is even more marked. While Lepidoptera larvae of which are widely polyphagous (Scorer 1913, Stovin 1944, Allan 1949, Ford 1949, South 1961). 120 This total, higher than that for any other tree spe- cies in Europe, has been attributed reasonably to 110 onto the considerable age of the oak as a member of the European flora, permitting the development of a ,A 100/ \ large number of oak-insect food-web relation- ships (Southwood 1961). There is some evidence, j90 however, that young oak leaves may be more nu- tritious for larvae than leaves of other trees at- tacked by the same insect species. For example, winter moth (Operophtera brumata L., Geome- 50 tridae) larvae develop into heavier pupae when E 60 fed on young oak leaves than when reared on z ( L.) or blackthorn ( spi- 0 nosa L.), two species commonly used as host plants in the field (G. R. Gradwell, personal com- munication). Young oak leaves are also the most satisfactory plant food for larvae of the gypsy Apr May Jun Jul Aug Sep Oct 1 Received July 21, 1969; accepted December 29, 1969. MONTH 2 Present address: Department of Entomology and Sec- FIG. 1. Number of Lepidoptera species feeding as lar- tion of Ecology and Systematics, Cornell University, vae on oak leaves in Britain from April to October. Ithaca, N.Y. 14850. (Data from Feeny 1966, p. 150-160.) 566 PAUL FEENY Ecology, Vol. 51, No. 4 are relatively scarce on oak leaves after mid-June, Older larvae tend to spin down on their silk very high densities are frequently reached in May, threads prematurely from defoliated trees, and when the average annual population density of many fail to complete their growth on low vegeta- the commonest species, the winter moth, is ap- tion. Though mass starvation of larvae in Wy- proximately 100/M2 of oak canopy area (ground tham is rare, competition for food probably man- projection) in WAythamWood, near Oxford, and ifests itself as reduced fecundity of the resulting the annual density has been known to reach adults (Varley and Gradwell 1958a). Pathogens 1,200/m2 of oak canopy area on a single tree (Var- are known to contribute substantially to insect ley and Gradwell 1958a, and personal communica- mortality during defoliation years. Golosova tion). The infestations are occasionally sufficient (1965) reports the almost total annihilation of high to cause complete defoliation of oak trees over a densities of Phigalia pilosaria Schiff. ( pedaria wide area in late May, as occurred in southern Fab.) (Geometridae) and Apocheima (- Biston) England in 1965 (Fig. 2). The trees respond to hispidaria Schiff. (Geometridae) on oak in west- the insect attack by producing "lammas" shoots, ern Russia within 10 days by nuclear polyhedrosis and the photosynthetic area is largely restored by virus. This virus was detected among a high late June. density of winter moth larvae in Wistman's Wood, Years of greatest oak defoliation tend to be fol- Devon, in 1965, though its effect on the cater- lowed by years in which densities of many early- pillar population there is unknown (P. Feeny, feeding Lepidoptera species are relatively low and unpublished results). In Wytham Wood, micro- vice versa (Varley and Gradwell 1958a). Wind- sporidian infection regularly leads to some mortal- borne dispersal of the young larvae of many spe- ity (Canning 1960, Varley and Gradwell 1962a). cies, including the winter moth, results in high Finally, a proportion of the larvae suffer preda- mortality through failure to find alternative food. tion and parasitism, and pupae are subject to density-dependent predation in the soil beneath the trees (Varley and Gradwell 1963a). In view of the evidence for increased mortality and reduced natality among high densities of lar- vae feeding on oak leaves early in the year, it is curious that very little insect damage occurs on gs oak leaves after mid-June. There is evidence that selection for early feeding may be very strong, at least in some species. Varley and Gradwell (1960) found that the heaviest mortality of winter moth ..r..i;. :. - ; occurs between the time in November and Decem- ber when the females lay their eggs on the twigs and the following April and May, when the first instar larvae are feeding on the young oak leaves. This mortality is largely responsible for the an- nual variations in population density and is prob- ably related to weather, which influences the rel- ative timing of bud burst on the trees and insect egg hatch. If the majority of the trees open their buds later than the peak egg hatch, a high mor- tality of caterpillars results, since the young lar- vae are unable to penetrate the closed buds. In any one year, in a given area may vary by as much as 2 weeks in the date of bud burst, so that early trees tend to carry higher insect pop- ulations than late-opening trees, which may escape defoliation even in years of very high caterpillar density. The average annual mortality of winter FIG. 2. Oak tree almost totally defoliated by Lepidop- moth due to this winter disappearance is about tera larvae, Wytham Wood, June 1965. In foreground, 90%o (G. R. Gradwell, personal communication), leaves of a sycamore, Acer pseudo-platanus L. (largely largely made up of larvae hatching too soon to untouched in years of low or moderate insect density on oak), reveal damage from Lepidoptera larvae which had enter the oak buds. This high mortality of first dispersed in their early instars from neighboring oaks. instar larvae, together with subsequent competi- Summer 1970 OAK LEAF TANNINS AND WINTER MOTH FEEDING 567 tion for food, seems a high biological "penalty" food (Perrins 1965). While perhaps a contribut- to pay to ensure that the remaining larvae com- ing factor, predation can probably be discounted mence feeding on the leaves at the earliest possible as a major selective force favoring early larval chance, and presumably it must be offset by some feeding. There is even less evidence to suggest considerable selective advantage for early feeding. that parasitism might act in this way. More prob- It is difficult otherwise to understand why selec- ably, the suspected selection pressure forcing early tion has not favored a later egg hatch and exten- larval feeding is due in large part to changes in sion of feeding periods to enable larvae to utilize the leaves of the host plant which render older the apparent abundance of relatively untouched leaves less suitable as larval food. The study food after June. described here, suggested by Professor G. C. Var- The population dynamics of oak-feeding Lepi- ley, provides evidence in favor of this hypothesis. doptera in Wytham Wood have been studied for several years by Varley and Gradwell (1958a, ExPERIMENTAL MATERIALS 1963b). They conclude that the fluctuations in The study area density from year to year, largely synchronous for The trees selected for study stand among mixed all early-feeding species, are the determined by deciduous woodland in Wytham Great Wood, action density-independent of weather, but that the Berkshire, England, approximately 3 miles west relative population levels of the different species of the Oxford City boundary. One mature oak are largely governed by differing efficiencies of tree (tree M, 0.8 m dbh, ca. 17 m high) was the specific parasites, acting in a delayed density- studied intensively; comparative analyses were dependent manner (Varley 1947). While para- made on leaves from the nearby trees 1, 5, 8, 9, sitism may be sufficient to level control at low the and 15 within the area studied by Varley and less common Lepidoptera species, which seem to Gradwell (whose numbering is retained) and oc- suffer from more efficient parasites, parasitism of casionally on other oak trees in Wytham Wood. the more abundant species is unable to prevent the On May 6, 1966, the longest leaf in each of 20 host density from occasionally reaching the food terminal leaf clusters taken at random from the limit. lower shade branches of each of the six main Predation by birds appears to be unimportant study trees was measured to estimate relative ad- in regulating numbers of Lepidoptera feeding on vancement of leaf growth. These measurements oak leaves. Spring caterpillars form an important showed that tree M (5.5 + 0.2 cm) is the earliest item of food for populations of the chief predators, and tree 15 (2.2 + 0.1 cm) the latest of the six the great tit (Parus major L., Paridae) and the trees, an order which is likely to vary little from blue tit (Parus caeruleus L., Paridae), in Wytham year to year (Varley and Gradwell 1958b). Wood (Perrins 1965). However, a detailed study of the food taken by the parents to great tit nest- Insect samples from tree M, 1964 in lings Wytham showed that oak-feeding larvae On the first four sampling dates Lepidoptera were taken in quantity only for a limited period larvae of each species were counted from 25 leaf (Rovama 1970). A study of the predation by clusters collected at random from the upper in tits during the outbreak of winter moth Wy- branches of the tree and 25 leaf clusters from the tham in 1948 revealed that only 2-5%o of the lower branches. This method was inadequate after caterpillars were taken (Betts 1955, Varley and June, due to the scarcity of larvae, and the three Gradwell 1962a). There is substantial evidence summer samples were therefore taken by exten- in the literature for the inability of bird predators sive beating of the lower branches over a beating to prevent insect outbreaks (e.g., Morris 1958, tray. 1963, Clark 1964). To estimate the number of larvae dropping from It follows from the work of Perrins (1965) that the tree to pupate, two metal trays, each 0.5 m2 newly fledged great tit nestlings would probably in area, were placed beneath the tree canopy take a higher proportion of oak-feeding caterpil- within 10 ft of the trunk and the number of larvae lars if the annual peak of larval abundance oc- falling into the trays counted at 3- to 5-day inter- curred a week or more later in the season. Even vals. The trays contained sufficient water to so, mortality due to tit predation would be most drown the larvae falling into them and were pro- unlikely to approach that currently suffered by tected from bird predation by wire netting. the larvae as a direct consequence of the early- feeding habit (see above). Moreover, the popu- Selection of larvae for laboratory experiments lation density of breeding tits in Wytham is limited The winter moth was selected for laboratory to a considerable extent by the supply of winter study since it is the species for which the apparent 568 PAUL FEENY Ecology, Vol. 51, No. 4 selection for early feeding has been most clearly TABLE 1. Number of Lepidoptera larvae collected from demonstrated. It is also the most abundant spe- the leaves of tree M on seven sampling dates in 1964a cies on oak; its life history is well known, and May May May June July July Sept. females can conveniently be collected to provide Species 12 18 25 1 1 24 30 a supply of eggs. Orthosia crude 1 The winter moth has a single generation each Cosmia trapezirna 2 1 year. After emergence in November and Decem- prasinana 2 Eupithecia abbreviata 1 1 ber and pairing on the oak trunks at night, the Oporinia dilutata I wingless females climb to lay their eggs (about Operophterabrumata 23 19 5 Erannis le tcophaearia 2 2 2 150 per female) in crevices in the bark of twigs Erannis aurantiaria 1 and branches in the crowns of the trees. After Erannis marginaria 1 2 egg hatch the following April at about the time of Erannis defoliaria 1 Phigalia pilosaria 1 bud burst, the caterpillars feed on the young Cacoecia sorbiana 1 1 leaves and pass through five instars before they Cacoecia lecheana 4 4 Pandemic corylana 1 spin down to the ground on silk threads as fully cerasana 1 1 fed larvae in late May. On reaching the ground, Tortriz loeflingiana 4 3 1 1 a short soil sur- Tortrix viridana 11 11 6 1 they pupate distance below the Eucoernacorticana 4 9 13 5 face, where they remain until the adults emerge Ypsolophus spp. 6 2 7 2 to repeat the cycle the following winter. 59 54 39 13 0 2 0 Winter moth eggs and larvae were usually col- Total lected from Wytham Wood, though on June 17- aData for the first four dates are samplesfrom 50 leaf clusters(25 fromupper leaves and 25 fromlower leaves); data for the last threedates representresults of 18, 1965, when larvae in Wytham had already extensivebeating of lowerbranches, using a beating tray. pupated, a collection was made from Wistman's Wood, Devon (1,500 ft above mean sea level), LARVAL FEEDING PERIODS where oak bud burst and larval egg hatch occur The results of a census of the Lepidoptera lar- annually about 2 weeks later than in Wytham. vae feeding on the leaves of tree M, taken during Gradwell, Collection of leaf samples for analysis 1964, agreed with the finding of Varley, and others that larval feeding on oak leaves is Buds and leaves were collected as intact leaf concentrated in the spring (Table 1). Almost clusters in polyethylene bags and immediately no larvae were detected by extensive beating after placed in a portable refrigeration box containing mid-June, even though this method samples many crushed ice. Representative leaf samples for anal- more leaves than the 50 leaf clusters examined on ysis were ensured by making a collection of leaf each spring sampling date. clusters from several branches in the appropriate The peak fall of fully fed larvae (prior to pupa- part of the tree. Most analyses were carried out tion in the soil) occurred in late May for the win- on leaves or buds from the south side of the tree ter moth, followed a few days later by Eucosma crown ("upper sun leaves") and from the north- corticana Hubn. (Eucosmidae) (Table 2). (Lar- ern branches at the base of the tree ("lower shade vae of Tortrix viridana pupate on the leaves and leaves"), since a comparison of these two regions are thus not detected by tray samples.) The tray is most likely to reveal any chemical variations data reveal that the density of winter moth larvae among the leaves of a single tree. on tree M was about 200/M2 canopy area (ver- Leaves were always collected between 1400 hr tical ground projection). The highest density was and 1630 hr B.S.T. to reduce variation in results 310/M2 on tree 5, the lowest 20/M2 on tree 15 due to time of day. Samples for sugar analysis (G. R. Gradwell, personal communication). Thus were collected as far as possible on days of high tree M held a high spring caterpillar population, sunshine hours, to reduce any variation resulting consistent with its relatively early date of bud from differing rates of photosynthesis in different burst. weather conditions. Leaves were cut from the clusters and immediately processed during the GROWTH OF WINTER MOTH LARVAE ON OAK evening following collection. Simultaneously with LEAVES OF DIFFERENT AGES each analysis, leaves from each randomized col- An experiment was conducted to determine lection of leaf clusters were dried at 80'C to con- whether or not winter moth larvae are able to stant weight in an oven to determine water con- grow satisfactorily on oak leaves somewhat older tent. The procedures used to determine sugar than those they feed on naturally. Lower shade and protein content of leaf samples and to estimate oak leaves from tree M were collected on May 16, changes in leaf "toughness" are described later. 1964, and transferred to a freezer at -20?C. A Summer 1970 OAK LEAF TANNINS AND WINTER MOTH FEEDING 569 TABLE 2. Number of Lepidopteralarvae per square meter of canopy area dropping to pupate beneath tree M on 50 T eight sampling dates in 1964

May May May June June June June June Species 22 25 28 1 5 8 11 16 0 Young leaves E 40-40 /tL O (May 16th) Orthosiacruda 1 1 Conistrs vaccinia 1 Cosmiatrapezina 1 2 2 Eupitheciaabbreviata 2 2 1 1 I-~~~~~~~~~~ Operophterabrumata 63 90 21 1 Erannisleucophaearia 2 1 2 Erannisaurantiaria 1 1 5 1 Eranniamarginaria 1 Erannisdefoliaria 2 1 > 20 / T More mature leaves Phyditaspissicella 1 20 so 0 (May 28th onwards) Cacoeciaxylosteana 1 Z Pandemis corylana 1 1 Tortrixloeflingiana 1 3 Eucsma corticana 1 15 39 39 6 6 2 Carcinaquercana 1 LithocolleWisapp. 3 3 Ypselephusapp. 2 6 2 1 2 0 June Total 67 102 53 54 44 8 9 5 ay 29 ~~1 14 1'8 22 2 6 3'0 further leaf collection from the same branches on DATE (1964) May 28 was stored in the freezer for 1 night to FIG. 3. Mean larval and pupal weights of groups of 25 fourth-instar winter moth larvae reared on young and equalize any effects of deep-freeze storage between more mature oak leaves. Initial rise in fresh weight, the two collections. On May 29, 50 fourth-instar representing fourth- and fifth-instar larval feeding phase, winter moth larvae, reared from an egg hatch is followed by decline during pre-pupal phase until pupa- delayed by cooling the eggs at 4?C, were placed tion is complete. Five per cent confidence intervals for individually on thawed leaves (25 on May 16 pupal weight means are shown. leaves and 25 on May 28 leaves) in small plastic containers at room temperature and humidity and LEAF TOUGHNESS AND GROWTH OF their growth followed by weighing at intervals WINTER MOTH LARVAE of a few days. At each weighing new leaves were The most obvious change in young oak leaves, substituted, the early leaves from the original apart from their rapidly darkening color during deep-frozen sample and the more mature leaves May, is their increase in toughness. The thin from lower shade samples from tree M, collected and fragile leaves of early May rapidly become the previous day and deep-frozen overnight. thicker and more difficult to tear apart. The in- Both larval peak weights and pupal weights of creasing toughness of the leaves is due largely to the winter moth larvae were markedly lower on the deposition of cellulose, hemicelluloses, pectins, the diet of mature oak leaves (May 28 onwards) and other materials which consolidate the cell walls. than on the diet of early leaves (May 16) (P < It is probably also connected with the decreasing 0.001 for the difference in mean pupal weight). water content of the leaves, rendering them in- The early leaf diet of May 16 corresponded to the creasingly less succulent. The water content of natural food of the fourth-instar larvae in Wytham both upper and lower leaves of tree M was found Wood. Moreover, of the 21 pupae from the early to drop sharply during late May and early June leaf sample, 10 hatched into normal adults, where- (Fig. 7a), confirming the observations of Edel'man as of the 10 pupae from the more mature leaf diet (1963) on oak (Q. robur) trees in the Soviet no adults emerged. The curves of mean indi- Union. vidual weight (Fig. 3) followed the usual larval Thus increase in leaf toughness may have a pattern of a rapid growth phase to peak weight, significant effect on the ability of insect larvae to followed by a decline due to loss of water and gut feed satisfactorily, and such an effect may account contents before pupation and, finally, a leveling- for the results obtained in the previous experiment. out as pupation is completed. Tanton (1962) has shown that mustard beetles It is concluded that some change or combination (Phaedon cochleariae Fab.), reared on the leaves of changes occurs in oak leaves over a period of of kale, turnip, and other vegetables, suffer greater only 2-3 weeks in late May which has a markedly larval and pupal mortality as the toughness of the adverse effect on the larval growth rates, pupal leaves is increased. Leaf toughness also has a weights, and adult emergence of the winter moth. considerable influence on the choice of foodplant 570 PAUL FEENY Ecology, Vol. 51, No. 4 by young locusts and grasshoppers (Williams mated by the force required to detach the leaf disc. 1954). This method provides a better simulation of the biting action of a caterpillar than does the pierc- Comparison of toughness of May and ing action of a needle. Jine oak leaves The complete device, 12 cm in length, and its Williams (1954) described a "penetrometer" component parts are shown in Fig. 4. A leaf is which he used to estimate leaf texture in connec- placed between two aluminum blocks, fastened tion with experiments on the feeding habits of together by two slip screws. Through a cylindri- Acrididae. His method, with minor modifications, cal hole in the blocks, lined with a brass sleeve, has since been used by Tanton (1962). The prin- runs a steel punching rod ( 5 mm diam), to the ciple of the method is simply to measure the force upper end of which is attached a circular metal (estimated as weight of sand) required to cause plate, supporting a beaker. This is gradually filled a sharp needle to pierce the tightly stretched leaf. with sand at a constant rate from a wide bore After consultation with members of the Depart- chromatography column. As soon as the lower ment of Engineering, Oxford University, a device end of the punching rod punches a disc of leaf tis- was constructed to punch out leaf discs with a sue from the leaf, the rod drops suddenly into the metal rod, entirely flat on its lower surface (Fig. hole in the lower block. Flow of sand is imme- 4). The lower surface of the rod was slightly diately halted and the beaker and contents weighed. rounded along its circumference to avoid imme- Leaves were placed between the blocks in such a diate tearing of the leaf by a sharp metal edge. way that the disc punched out was well clear of The action of the rod was therefore to break the the major leaf ribs and the device was thoroughly leaf tissue initially by a shearing action, after which cleaned between successive determinations. the disc of leaf tissue would be detached from the The change in toughness of lower sun leaves leaf by a combination of shearing and tearing from a single oak tree (tree 9) was measured over forces. The texture of the leaves was then esti- the period in late May and early June (1966) when most of the caterpillars pupate. On each occasion a large number of leaf clusters was col- lected and transferred to the laboratory in a re- _ ~~ ~ ~ ~: ~S ...... I . i f i -...... - frigeration box. The clusters were thoroughly _~~~~~~~~~~~~~~~~~~~~~~...... mixed and individual clusters withdrawn at ran- dom. Toughness estimations were made only on the longest leaf in each of the clusters withdrawn. In all, 20 leaves from each sample were measured and the toughness values plotted graphically against leaf growth (Fig. 5). Although toughness values vary considerably within each sample, the differences between the two samples are substan- tial and the values do not overlap. The force re- quired to punch out a leaf disc in the manner described is approximately seven times greater on June 10 than on May 19, a difference of only 22 days. The rapid increase in the toughness of oak leaves during late May and early June is apparent.

Growth of winter moth larvae on leaf powder diets The effect on winter moth growth of removing leaf toughness was studied by the method of Moreau (1965). Lower sun oak leaves were col- lected from tree M on May 13 and June 1, 1965. They were transferred rapidly from the refrigera- tion container (0-50C) to an oven at 1000C. After drying, the leaves were ground to a fine powder in a ball mill and incorporated into agar FIG. Device for of leaves. 4. estimating"toughness" 22 ml Above: Fully assembled. Below: Componentparts. (See diets, containing the following ingredients: text for description.) distilled water , 14 g leaf powder, 0.03 g aureomy- Summer 1970 OAK LEAF TANNINS AND WINTER MOTH FEEDING 571 TABLE 3. Peak weights and growth increments (peak E less initial weight) of winter moth larvae reared on 9g 900 agar diets containing leaf powders from oak leaves of two different ages 0 0 0 o0 0 Diet from May Diet from June 0 Item 13 leaves 1 leaves ~~700 ~~0 0 Mean initial weight (mg) 24.8?2.3 23.7 ?3.6 -' Mean peak weight (mg) 37.1?2.5 35.3 ?2.0 Z 0 ~~~~0 ? JUNE 10th Mean growth increment (mg) 12.3?2.0 11.6?3.0 z 0 Z500- 0 a. parable age difference (Fig. 3), which suggests 0 that the inability of larvae to grow well on June oak leaves may be partly attributable to the rapid ' 300 increase in toughness of the leaves. larvae to feed on mature oak a * * MAY 19th The inability of leaves because of leaf toughness is not a completely satisfactory explanation for the lack of extensive insect attack on mature oak leaves in the field. 100 F X - Many insect larvae, even in their early instars, have mandibles which would be amply strong "'0 5 6 7 8 9 10 11 12 enough to cut through mature oak leaves. More- over, several species of Lepidoptera feed as larvae (cm) LEAF LENGTH on mature oak leaves in the normal course of their FIG. 5. Toughness values of May 19 and June 10 oak cases only in their early in- leaves. life histories, in some stars (Feeny 1966). In view of the high mor- tality of young winter moth larvae discussed ear- cin (antibiotic), 0.15 g methyl p-hydroxybenzoate lier, it seems remarkable that selection has not (antibiotic), 1.0 ml vitamin solution (McMorran favored the development of stronger mandibular 1965), and 2.5 g agar (plain Japanese) dissolved muscles by more species, enabling them to extend in 62 ml distilled water. The vitamin solution their feeding periods into the summer. That this was included to compensate for the probable de- has not occurred in the majority of species sug- struction of leaf vitamins during drying. The gests that other factors make oak leaves less suit- first five ingredients were thoroughly mixed in a able for larval growth as the season progresses homogenizer, after which the agar solution was and that the inability of larvae to ingest tough added at 700C and the whole mixture blended until leaves may be a consequence rather than a cause homogeneous. The diets were allowed to set to of early feeding. a rigid gel in autoclaved glass jars. Twenty fifth-instar winter moth larvae, collected SEASONAL VARIATION IN CARBOHYDRATE AND the previous morning in Wistman's Wood, were PROTEIN CONTENT OF OAK LEAVES placed on each of the two leaf diets. Each larva Variation of nutritional value seemed the most was reared at 200C in an individual plastic con- obvious property of oak leaves which might con- tainer, 4 cm in diameter and 2.5 cm deep, and tribute to selection for early larval feeding. As- at intervals a few Unfortunately, weighed of days. suming that caterpillars feed as rapidly as possible, many of the larvae died before pupation due to growth rate could be determined by the concen- nuclear polyhedrosis virus, so that pupal weights tration in the ingested leaf material of the essen- were not available for comparison. However, the tial ingredients in shortest supply relative to the initial period of growth up to peak larval weight insect's requirements. In this study attention was appeared normal by comparison with previous ex- restricted to the major leaf nutrients, carbohydrate periments with powder diets, and the mean and protein, which are likely to be important in growth increments (differences between peak determining maximum insect growth rates owing weight and initial weight) were therefore used for to rapid formation of new tissues and correspond- a comparison of the larval growth on the two diets. ing requirements for energy. The difference between the mean growth in- crements on the two diets was not significant Polysaccharides (Table 3) and is considerably smaller than that Few insects have been shown to digest cellulose, found for larval growth on entire leaves of com- the chief structural carbohydrate of leaves, and 572 PAUL FEENY Ecology, Vol. 51, No. 4 the few examples known, such as the termites, min and then immediately homogenized in the achieve this breakdown with the aid of a spe- same solvent with a blender. The homogenates cialized microbial gut fauna. Digestion of cellu- were filtered under suction and the residues washed lose has never been reported in phytophagous several times with fresh hot solvent. The com- Lepidoptera larvae (Wigglesworth 1953), and it bined filtrates from any one sample were distilled seems, therefore, that this source of carbohydrate to dryness under vacuum to remove ethanol and is ignored, which suggests that carbohydrate is the residue shaken into solution or suspension in not generally limiting for phytophagous insects. distilled water by means of an ultrasonorator. If this was not the case, one would have expected Polyphenols and other contaminants of high molec- strong selection for the production of a cellulose ular weight were then removed by "clearing with enzyme, as has occurred in the snails, or in the lead" (Horwitz 1955). Saturated aqueous lead development of a symbiotic cellulase-containing acetate solution was mixed with the extract, fol- gut fauna, as in the termites. lowed by just enough saturated aqueous sodium Ability to digest starch, the other chief poly- oxalate solution to precipitate all the lead as its meric carbohydrate source in leaves, is widespread insoluble oxalate. The precipitate was filtered off among insects, including phytophagous larvae and the filtrate, now colorless, clear and free from (Wigglesworth 1953). However, attempts to de- lead, made up to 1 liter. After "clearing with lead" tect the presence of an amylase in the salivary the reducing materials in the extract consist al- glands or the mid-gut of winter moth larvae were most entirely of sugars (Horwitz 1955), which entirely unsuccessful. Evidently this enzyme is control experiments showed to be unaffected by either completely absent in the winter moth, as the procedure. The cleared extracts were accord- has been found for some other phytophagous Lepi- ingly analyzed for sugar content by a colorimetric doptera (Wigglesworth 1953), or present in such adaptation of the ferricyanide method of Hagedorn small amounts that its activity is very restricted. and Jensen (1923), which determines reducing Since the winter moth does not appear to uti- materials. The procedure used (Folin 1928, lize leaf polysaccharides, it must be assumed that Fuller 1964, K. W. Fuller, personal communica- its needs for energy are adequately met by the tion) was as follows. To 1 ml of diluted extract free sugars, and, perhaps, nitrogenous compounds was added 1 ml of 0.004 M potassium ferricyanide present in leaves. The sugar content of oak leaves in 0.1 N sodium hydroxide solution. After heating was therefore studied at various times during the for 10 min in a boiling water bath, the solution year to determine whether there was any corre- was cooled and mixed with 1 ml of a solution ob- lation between sugar content and larval feeding tained by dissolving 1 g ferric sulfate, 2 g sodium period. Most, if not all, larvae of phytophagous dodecyl sulfate, and 50 ml phosphoric acid Lepidoptera so far studied secrete the enzyme in- (AnalaR 88%o) in 2.5 liters of distilled water. vertase (Wigglesworth 1953), so that both sucrose The optical density of the Prussian blue color at and monosaccharides are likely to be important 660 nm was then read in a Unicam manual spec- items of diet. trophotometer and converted to monosaccharide concentration (expressed in terms of D-glucose) Sugars by reference to a standard graph of optical density against glucose concentration, prepared from con- Ethanolic extracts of freshly collected July oak trols. leaves were evaporated to dryness under vacuum; Sucrose was determined by hydrolysis of ex- the residues were taken up in distilled water and tract samples for 45 min at 20'C with invertase shaken for 1 hr with a mixed-bed ion exchange (British Drug Houses preparation, diluted 2,000 resin ("Amberlite IRC 50": "Amberlite IRA times in 0.1 M sodium acetate/acetic acid buffer 401," 1: 2; British Drug Houses, Poole, Dorset). at pH 4.5) to glucose and fructose, followed by Paper chromatograms of the de-ionized solutions determination of the reducing sugars. Subtrac- were run in ethyl acetate/pyridine/water (12: 5: tion of the original reducing sugar determinations 4) solvent alongside samples of known sugars. from the readings obtained after hydrolysis of Aniline/diphenylamine and silver nitrate sprays sucrose gave the sucrose concentrations in terms (Smith 1960) revealed only D-glucose, D-fructose, of glucose. All analyses were carried out in du- and sucrose in the oak leaf extracts. plicate. The quantitative sugar content of upper sun The reducing sugar content of oak leaves (Fig. and lower shade leaves from tree M was estimated 6c) was found to follow no obvious seasonal trend, at intervals throughout the 1964 growing season. in spite of considerable fluctuation during May Weighed samples (50 g) of fresh leaves were and June, and remained at 3.0-7.5% dry wt transferred to boiling ethanol (96%o v/v) for 20 throughout the season in both upper and lower Summer 1970 OAK LEAF TANNINS AND WINTER MOTH FEEDING 573 60 a rently uncertain whether or not nitrogen is a limit-

Number of ing factor for defoliating insects (Painter 1953, Larvae per 40 Stark 1965, Johnson 1968). Larval growth of 50 Leaf Clusters* 20 many species of Lepidoptera is better on leaves of plants which have been fertilized with nitrogen (e.g., Rodriguez 1960), yet there is evidence that 100 may have a deleterious effect Number of n b such fertilization Larvae per (see review by Stark 1965). m2 Canopy nitrogen of plants (usually Area 50 Most of the 80-90% in leaves) is present in the form of protein. The total nitrogen content of the leaves is then a rough a C measure of its total amino acid and protein content % Dry Wt phytophagous larvae so far 6 .~o% oO \ 0 (Long 1961). All as D-Glucose ; , 0 0 to digest leaf protein 4 *Z^ studied are apparently able -0 1953), though only about 50% 2 (Wigglesworth is assimilated (Fraenkel 1953). Determination 0- of total nitrogen is probably a good estimate of a O.* d RIME %DryWt 0 d the maximum amount of nitrogen available for as D-Glucose A * 0 to 0 insect growth. Leaf protein content is frequently 0 0 / multiplied by 6.25 24 =8 determined as per cent nitrogen in 0' (Long 1961), and this procedure was followed Apr' May Jun I Jul I Ausg Sep | the present study. FIG. 6. Seasonal variation of (a) number of lepidop- On five dates during the 1965 season, buds or terous caterpillars feeding on oak leaves (data from leaves from tree M were collected for analysis to Table 1) (*last three samples obtained with beating discover whether the total nitrogen content varied tray) ; (b) number of lepidopterous caterpillars falling to affect insect larvae. Leaf to pupate (data from Table 2); (c) reducing sugar (glu- in a manner likely cose and fructose) content of oak leaves; and (d) sucrose material was dried to constant weight, ground to content of oak leaves. * = upper sun leaves; o = lower a fine powder in a ball mill, and analyzed in trip- shade leaves. licate for nitrogen by the Kjeldahl method (Brad- street 1965, W. R. C. Handley, personal commw- leaves. Sucrose content, however, showed a nication). The protein content of lower shade or marked increase in early June (Fig. 6d), reaching upper sun leaves differed little on any one date, about 8% dry wt in the upper leaves and about but protein content of both upper and lower leaves 5% dry wt in the lower leaves, levels which were decreased markedly during the early part of the maintained until late September. It has been season from about 309 (5%o dry wt nitrogen) in known for some time that sucrose content in leaves April buds to about half this value in late May tends to remain low during rapid growth and to and thereafter (Fig. 7b). These values were con- increase as the leaves approach maturity (e.g., firmed by analysis of the leaves of six trees in 1966 Barnell 1936). (Fig. 7b), and they agree closely with those found It is clear that the majority of oak-feeding for oak leaves in Russia by Shvetsova (Edel'man caterpillars feed during the period when sugar 1963). A high nitrogen content in young growing content of the leaves is at its lowest and not, as leaf tissue is, of course, expected and has been would be expected if sugars were nutritionally shown for many plants (e.g., Long 1961). Its limiting, when sugar content is at or near its max- coincidence in oak leaves with the main period of imum seasonal value. This tends to confirm the larval feeding is striking and supports the view suspicion that carbohydrate is not limiting the that nitrogen content may be one of the most im- growth of these insects and is not likely to be a portant factors governing early feeding. significant factor in determining early larval feed- The leaves of trees 8 and M contained notice- ing periods. ably less nitrogen on May 6 than the four other were the fur- Protein trees (Fig. 7b). These two trees thest advanced on May 6 and their leaf nitrogen The quantitative nutritional requirements of content had presumably started to drop before phytophagous insects, especially requirements for that of the other four trees. dietary nitrogen, are as yet poorly known (Painter 1953, Friend 1958). Though populations of suck- Micronutrients and water ing insects are in general favored by an increase Fraenkel (1953) concludes that leaves are ex- of soluble nitrogen in their host plant, it is cur- cellent sources of the micronutrients which most 574 PAUL FEENY Ecology, Vol. 51, No. 4 75 tain increasing amounts of tannin as the growing season progresses. A study was therefore under- % Fresh Wt taken to determine the seasonal variation of the qualitative and quantitative composition of oak leaf phenolic compounds and to determine whether 35 _=: o- - these compounds might influence the growth pe- 5 b0 riods of insect larvae. % Wt Dry 25 9 / 1 = 11 15 ___ Seasonal variation in the tannin content

5 M of oak leaves 5 0 Changes in the phenolic content of leaves from 5 ~~~~~~~~~~C * tree M were studied by two-way paper chroma- % Dry Wt tography throughout the growing season (Feeny 3 and Bostock 1968). Although more than 20 phenolic compounds were detected, the most ob-

0' vious components were the condensed and hydro- 40 lyzable tannins. Although the level of hydrolyz- d 30 able tannin remained approximately constant from 20 April throughout the season, condensed tannin

10 I did not appear in the leaves until late May (Fig. 8). Total tannin content of upper sun leaves was Apr May Jun Jul Aug Sep found to increase during the 1965 season from FIG. 7. Seasonal variation of (a) water content, (b) 0.66% dry wt in April to 5.50% dry wt in Sep- protein content, (c) tannin content, and (d) protein: tember (Fig. 7c). The intensities of chromat- tannin ratio of oak leaves. * = upper sun leaves; o = ogram spots indicated that the increase was due lower shade leaves; A = 1966 data for lower shade leaves a conclusion sup- of trees 1, 5, 8, 9, 15, and M. largely to the condensed tannin, ported by the much higher rate of incorporation of insects require for growth (Friend 1958, House carbon-14 label into condensed rather than hydro- 1961, 1962). However, seasonal deficiencies in lyzable tannin in July leaves (see below). Hydro- micronutrients may be limiting for some insects. lyzable and condensed tannins were separated from Ellis, Carlisle, and Osborne (1965) found that the crude tannin mixture by column chromatog- sexual maturation in the desert locust (S. gregaria raphy. An investigation of their chemical and Forsk.) is delayed when the insects are fed on chromatographic properties is described elsewhere senescent leaves of Brassica spp., due to a shortage (Feeny and Bostock 1968). of gibberellins, which are present in the young of growth of winter moth larvae leaves. It is possible, also, that decreasing water Inhibition by oak leaf tannins content of oak leaves (Fig. 7a) may contribute to selection for early feeding (V. B. Wigglesworth, An artificial diet, containing casein as protein personal communication). The effects of seasonal source, was developed for rearing winter moth changes in the content of water and micronutrients larvae in the laboratory. The presence in the diet were not studied in the present work, but the feed- of as little as 1% fresh wt of oak leaf tannin, ex- ing of some Lepidoptera larvae on oak leaves late tracted from September oak leaves, caused a sig- in the season suggests that sufficient water and nificant reduction in larval growth rate and pupal micronutrients are available for insect growth. weight, which is likely also to reduce fecundity (Feeny 1969). OAK LEAF TANNINS AND PROTEIN DIGESTION Tannins are widely recognized to form com- BY WINTER MOTH LARVAE plexes with proteins and to inhibit the action of There is considerable evidence that leaf poly- enzymes (e.g., Pridham 1963, Goldstein and Swain phenols, including the tannins (Haslam 1966), can 1965). Oak leaf tannins form complexes with have adverse effects on the growth and survival of both casein and nettle leaf protein at pH 5.0 (the a wide range of organisms (see below). Oak pH of macerated July oak leaves), the degree of leaves, bark, and galls have been known for cen- complex formation depending on the ratio of the turies to develop high concentrations of tannins, protein to tannin concentrations and on the time which have been used in the manufacture of leather of contact between protein and tannin (Feeny and inks. Some evidence (Brown, Love, and 1969). When complexed with oak leaf tannin, Handley 1962) indicates that Q. robur leaves con- casein is completely protected from hydrolysis by Summer 1970 OAK LEAF TANNINS AND WINTER MOTH FEEDING 575

1.0 1.0

APRIL JUNE 0.8 0.6

LU- 40.6 (D) 0.6 (+)-GALLOCATECHIN

~~~GALLICD1f~~~~~(Hj Aj) GALLIC ACID Z 0.4 k~~~~~~~~~~~hJ~ ~ ~ ~~ ~ACID 0. v' and lt@id 'PS O vow0 ~(+)-CATECHIN 4U 0. URCTN K ELLAGIC ACID 0.2 ELLAGIC ACID S | % / @ QGUERCETIN | ^ @ G / @ QUERCETIN 0 iSu 0 N S aT 0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 Rf VALUES IN BUTANOL:ACETIC ACID : WATER(60 :15 : 25) FIG. 8. Representations of two-way paper chromatogramsof aqueous acetone extracts of descaled April oak buds and June oak leaves from tree M. All components shown are phenolic, as revealed by ferric chlo- ride/potassium ferricyanide reagent. Identity of componentsis indicated where known. Vanillin-positive phe- nolic components (including condensed tannin C) are indicated by black shading. Streaks D1, D2, and H represent hydrolysable tannins. (After Feeny and Bostock 1968).

mammalian trypsin at pH 7.6, but at pH 9.2 (the material, the protease level in the insect gut, and pH of the mid-gut of the winter moth) some pro- the period available for digestion. Furthermore, tein digestion occurs, presumably owing to partial winter moth larvae were observed by the author dissociation of protein-tannin complexes under to feed by biting off pieces of leaf from around the alkaline conditions. At pH 9.2, enzymic hydrol- edge of previously damaged tissue. During ex- ysis of complexes between condensed or hydro- posure to the air between rupture of the cells and lyzable oak leaf tannins and either casein or nettle ingestion of the tissue by the larvae, it is likely leaf protein is increasingly inhibited as the pro- that the tannins would undergo oxidation which portion of tannin in the original complex is raised, is almost certain to increase their inhibitory inter- the inhibition being more marked with condensed action with proteins (Byrde, Fielding, and Wil- than with hydrolyzable tannin. Inhibition of liams 1960). However, a rough estimate of the growth of winter moth larvae by oak leaf tannins possible effect of the tannins can be obtained if is likely to be due to the formation of relatively in- the behavior of casein is taken to represent that of digestible complexes with the available protein, oak leaf protein in the field. At pH 9.2, the thus reducing the rate of assimilation of dietary amount of amino acid nitrogen released from nitrogen (Feeny 1969). casein after enzymic digestion for 3 hr is reduced If it is assumed that the tannins used in the by about half if the casein is previously complexed laboratory experiments are chemically identical to at pH 5.0 with a third of its weight of condensed the tannins originally present in oak leaves and, tannin (Feeny 1969). In September oak leaves again, if the conditions obtained in the laboratory the ratio of tannin to protein is approximately experiment can be considered to parallel the field 1: 3, so that the amount of available protein would situation, then oak leaf tannins, and especially be of the order of 7 g/100 g of leaf dry wt, com- condensed tannin, are likely to reduce the avail- pared with approximately 30 g/100 g leaf dry wt ability of protein nitrogen to insects feeding on in very young leaves, in which no condensed tan- the leaves. At present the extent of this reduction nin is present. If a caterpillar must ingest, let us cannot be estimated precisely since so many fac- say, about 10 g (dry wt equivalent) of leaf mate- tors are unknown. The effect of the tannins would rial to complete its growth during April and May, depend, for example, on the concentrations of tan- and if growth rate is limited by nitrogen supply, nins in the leaves, the nature of the proteins pres- then at least 40 g would have to be ingested to ent, the time available for complex formation after achieve similar growth on September leaves. ingestion of the leaves, the extent to which some An estimate of the availability of the protein in of the tannin may form complexes with non-protein oak leaves may be obtained from the protein: 576 PAUL FEENY Ecology, Vol. 51, No. 4 tannin ratio. The variation of this ratio, plotted for the upper sun leaves of tree M during 1965 (Fig. 7d), clearly demonstrates the sharp fall in protein availability during the early summer, re- sulting from the simultaneous decline in leaf pro- tein content (Fig. 7b) and increase in tannin con- tent (Fig. 7c).

The fate of tannins ingested by winter moth larvae The generalized nature of the interaction be- tween tannins and proteins, involving extensive formation of hydrogen and perhaps covalent bonds (Haslam 1966), would probably render difficult the development by insects of specific detoxication mechanisms, such as are known for alkaloids and other poisonous principles in plants (e.g., Hodgson, Self, and Gutherie 1965). Hollande ( 1923) ON claimed to have discovered tannin "crystals" in cells of the mid-gut of T. viridana larvae, reared on oak leaves, and these may have represented an 4..". ._ adaptation to feeding on tannin-containing plants. However, condensed tannins are chemically amor- phous, often with a wide range of molecular weights resulting from different degrees of poly- merization, and their manifestation as crystals seems most unlikely. In the present study the fate of tannins ingested by winter moth larvae was investigated by histological and autoradio- FIG. 9. Part of a branch of an oak tree (Q. robur) enclosed in a polyethylen bag for preparation of 14C_ graphical methods. labeled tannins. Tube containing labeled barium carbo- Radioactively labeled tannins were first pre- nate projects from lower right corner of bag. pared as follows. A south-facing branch of an isolated oak tree, Q. robur, in Wytham Wood was Condensed tannin (water insol) 95.7 mg enclosed securely in a large transparent polyethy- 344.5 millimicrocuries lene bag (capacity 130 liters) at 0700 hr B.S.T. Hydrolyzable tannin 40.7 mg on June 30, 1966 (Fig. 9). At 0900 hr B.S.T., 6.9 millimicrocuries 14CO2 (about 1 mc) was released into the bag by injecting 30%o perchloric acid through a rubber The total incorporation of activity into the tannin septum into a glass tube which was sealed half represented 0.044% of the activity present in the way along its length through the wall of the bag original barium carbonate. and which contained 14C-labeled barium carbo- Three fourth-instar winter moth larvae were nate. The gas released amounted to 0.02% of reared individually for 7 days on 0.7 g samples of the hag capacity. After 12 hr (including 7 hr of artificial diet (Feeny 1968) incorporating either bright sunshine), the bag was removed and a week '4C-labeled condensed tannin (water sol) (7 mg) or 14C-labeledhydrolyzable tannin (3.3 mg), which later the leaves (360 g, fresh wt) were harvested had previously been complexed with the dietary and extracted with 70% (v/v) acetone/water. casein. The larvae, which had consumed all their Crude tannin was isolated from the (0.475 g) diet and were now in mid-fifth (last) instar, were extract by an ether-precipitation technique (Feeny dissected and their guts fixed in formol/saline and Bostock 1968) and the condensed and hydro- fixative for 20 hr. The guts were embedded in lyzable tannins separated by chromatography on paraffin and serial sections cut at 6-15 p.. These Sephadex columns (Somers 1966) and thick were stained in Haematal 8, counterstained with paper. Yields and activities of the tannin frac- Biebrich Scarlet (Baker 1962), and then coated tions, measured by a Nuclear Chicago series 724 with Kodak AR 10 stripping film (Kukita and liquid scintillation system, were as follows: Fitzpatrick 1955) and placed in a sealed container Condensed tannin (water sol) 22.0 mg at 40C. 86.9 millimicrocuries Slides were withdrawn after 4 days, 4 weeks, Summer 1970 OAK LEAF TANNINS AND WINTER MOTH FEEDING 577 and 4 months, developed (Brunet and Small 1959), TABLE 4. Comparison of larval feeding habits of early- and examined under oil immersion (X500). No feeding (June or earlier) and late-feeding (July or later) Lepidoptera species on oak leaves (Data from incorporation of the 14Clabel in the mid-gut tissue Feeny (1966, p. 150-160), compiled from Scorer (1913), was detected, though considerable incorporation Stovin (1944), Allan (1949), Ford (1949), South was found in mid-gut tissue of control larvae which (1961), H. B. D. Kettlewell and G. R. Gradwell, per- had been reared on a diet containing denatured sonal communication.) 14C-labeled Chlorella protein (Radiochemical Cen- tre, Amersham, England). Percentage of Percentage of early-feeding late-feeding Several winter moth larvae, reared on oak Feeding habit (not species species leaves or on artificial diets containing tannin, were mutually exclusive) (total 111) (total 90) dissected during the 1965 and 1966 seasons. Their 1) Larva completes growth on guts were fixed in formol/saline, Zenker, or mer- oak leaves in one sesaon curic/acetic fixatives. Sections from these prep- (excluding leaf miners) 92 42 arations, embedded in paraffin, were examined 2) Larva completes growth on after staining with Masson tricolor, haemalum/ low herbage, after initial feeding on oak leaves 3 11 light green, or ferric chloride, but no trace could be found of tannin "crystals," such as were re- 3) Larva overwinters and com- pletes growth in following ported by Hollande (1923) in larvae of T. yiri- season 4 38 dana. 4) Larva bores into parenchyma The mid-gut walls of three winter moth larvae, (leaf miner) 3 26 reared on tannin-containing diets, were removed from the peritrophic membranes, containing the gut contents. After staining with 5% (w/v) (Table 4). Although larvae of the lunar double- ferric chloride solution to locate phenolic mate- stripe moth (Minucia lunaris Schiff., Noctuidae) rials (Nierenstein 1934), the peritrophic mem- feed on oak leaves in July and August, they will brane and its contents in each case turned deep only survive on fresh tender oak leaves put out blue, but no trace of color could be seen on the from the stumps of recently felled or fallen trees gut lining. ("stool oak") and the larvae cannot survive when These results suggest that tannins are largely, fed on mature oak leaves (H.B.D. Kettlewell, if not entirely, retained inside the peritrophic mem- personal communication). Life histories of spe- brane of the winter moth mid-gut. cies with slow larval growth on mature oak leaves could represent adaptations to a reduced avail- of in Adaptations of oak-feeding Lepidoptera species to ability nitrogen the diet, due partly to the increase in leaf tannin content. the presence of tannins in their food Edel'man (1953) suggests that the nutrient requirements of larval A comparison of the life histories, as far as they Lepidoptera on oak leaves in Russia are adapted are known, of the early-feeding and late-feeding to the available nutrient levels in the leaves. Late- Lepidoptera species on oak suggests that some feeding larvae are adapted to a diet rich in carbo- ecological adaptations may have been evolved, hydrate and poor in protein. whereby the effects of tannins are minimized. A relatively high proportion (26%) of late- Though the most intense feeding pressure by larval feeding larval species are leaf miners (Table 4), Lepidoptera on oak leaves occurs in the spring, some of which grow fairly rapidly, even in autumn. nevertheless many species feed on the leaves later One function of this feeding habit may be the in the year (Feeny 1966, p. 150-160). Although avoidance of tannins by feeding on the spongy few detailed life history studies are available, the parenchymatous tissue while ignoring the palisade records of naturalists suggest that early-feeding cells, in which the tannins are believed to be con- larvae tend to complete their growth rapidly (2-3 centrated (see below; and G. R. Gradwell, per- weeks), whereas late-feeding species, such as Chi- sonal communication). The high pH in the mid- mabache (= ) fagella (Oecophoridae) gut of many phytophagous larvae (Wigglesworth grow slowly and may take several months to com- 1953), including the winter moth, may be an plete their growth. Late-feeding species may even adaptation to feeding on polyphenol-containing fail to complete their growth in a single season and leaves, since leaf protein-tannin complexes are many overwinter as larvae before completing their increasingly dissociated at high alkalinity, thus growth the following spring or drop from the trees releasing a higher proportion of protein for diges- to complete their growth on ground herbage tion (Feeny 1969). 578 PAUL FEENY Ecology, Vol. 51, No. 4 DEFENSIVE FUNCTION OF TANNINS IN PLANTS absence of attack by caterpillars. Acorn produc- Tannins occur widely in the plant kingdom, tion is likely to suffer especially severely from especially in the Angiospermae (Nierenstein 1934, high caterpillar density since developing fruits are Bate-Smith and Metcalfe 1957, Brown, Love, and eaten by the larvae (Varley and Gradwell 1962b). Handley 1962). Concentrations may reach 40% Young (1965) reports that insect larvae are ap- dry weight in the bark of some species of Quercus parently able to kill within a short period oak trees and 15% dry weight in tea leaves (Meyer, Ander- which have previously been weakened by drought. son, and Bbhning 1960). Tannins are known It is clear, therefore, that oak trees are appreciably from almost all types of tissue (Esau 1965), but affected by insect attack, so that the evolution of in leaves they tend to be concentrated in the pal- some kind of defensive mechanism might be ex- isade cells of the parenchyma (Haberlandt 1914, pected. The work described in the present paper McNair 1965). The tannins are almost invari- suggests that one function of the leaf tannins may ably present in vacuoles, separated from the pro- be to inhibit attack by insects by reducing the nu- toplasm by a membrane (Nierenstein 1934), and tritive value of the leaves and perhaps also by disruption of this membrane may cause precipita- influencing palatability (see below). The pos- tion of protoplasmic proteins by the tannins (Doby sible action of tannins as repellents determining 1965). the suitability of food plants for insects has been Until recently the functions of tannins and many mentioned previously (Fraenkel 1953, Wise and other secondary chemicals in plants were largely Jahn 1952, Lipke and Fraenkel 1956), though unexplained by plant physiologists (Lipke and with little or no experimental evidence. Fraenkel 1956, Fraenkel 1959). Among the phys- Considerable evidence now suggests that plant iological functions ascribed to tannins (mostly with tannins have a general protective function. Hand- little experimental evidence) have been protec- ley (1961) has found that tannins reduce the tion against desiccation, storage of reserve mate- ability of microbial organisms to break down leaf rials somehow related to carbohydrate metabolism, protein, owing to the formation of leaf protein- regulation of cellular oxidation, protection of cell tannin complexes. Tannins are known to inhibit turgor, or merely accumulation of metabolic waste the growth of fungi (e.g., Wise and Jahn 1952, products (Lutz 1928, Nierenstein 1934, Esau Pridham 1960, Swain and Bate-Smith 1962, Wil- 1965, McNair 1965). It was early recognized liams 1963) and the transmission of viruses that tannins might have some protective function (Bawden and Kleczkowski 1945, Cadman 1960), against other organisms (Haberlandt 1914, Nier- probably by tanning the fungal pectolytic enzymes enstein 1934), but much experimental proof was (Byrde, Fielding, and Williams 1960, Williams lacking (Byrde, Fielding, and Williams 1960). 1963) and the virus nucleoprotein (White 1956, It is a precept of modern chemical ecology, how- Cadman 1960). Vukavic and Maksimovic (1956) ever, that energy is not likely to be "wasted" in suggest that increasing polyphenol content of oak the production of secondary chemicals, which fre- leaves may account for the decrease in digesti- quently involve specialized biosynthetic pathways, bility of crude protein in oak leaf fodder by sheep unless there is some compensating adaptive advan- as the season progresses. Simple phenols, such tage to the organisms in question (e.g., Janzen as chlorogenic acid and catechins, may be biolog- 1969, Whittaker and Feeny, unpublished data,3 ically active in a similar way, though their effect Sondheimer and Simenone 1970). The biosynthe- may be dependent on prior oxidation to quinones sis of tannins, initially from active acetate by way (Johnson and Schaal 1952, Byrde, Fielding, and of the acetate and shikimic acid pathways (Haslam Williams 1960). 1966), involves considerable quantities of sugar The astringent taste of leaf polyphenols may and energy and one would expect, therefore, to influence leaf palatability, regardless of their effect find some substantial compensating advantage to on the digestibility of protein. The rate of break- tannin-containing plants. down of fallen oak (Q. robur) and (Fagus Varley and Gradwell (1962b) found a signifi- sylvatica, Fagaceae) leaves by the soil fauna (and cant inverse correlation between the summer especially by earthworms) is inversely propor- growth of timber in oak trees and the caterpillar tional to the polyphenol content of the leaves. Ex- density during the spring of the same year. They traction of leaf polyphenols with 50% methanol estimate that the mean annual summer growth rendered them more palatable to earthworms of the trees might increase by as much as 60% (Heath and King 1964, Heath and Arnold 1966, and the total timber by as much as 40% in the King and Heath 1967). Tannins have been shown 3Whittaker, R. H., and P. P. Feeny. MS. Allelochem- to render the herbage of Lespedeza cuneata (Le- ics: chemical interactions between species. guminosae) less palatable to sheep (Wilkins et Summer 1970 OAK LEAF TANNINS AND WINTER MOTH FEEDING 579 al. 1953), Shpan-Gabrielith (1965) found that season progresses. The ability of tannins to form tannin-containing plants are repellent to locusts complexes with proteins and thus reduce the avail- (Schistocerca gregaria Forsk.), and Stahl, long ability of nitrogen to at least some herbivores is ago, found that tannins rendered leaves unpala- believed to contribute to their defensive function table to snails (Haberlandt 1914). in plants. In some cases tannins are actually toxic to an- From the observation that plants are largely imals. For example, oak leaves of various Quer- intact and rarely depleted by herbivores, Hairston, cus species (including Q. robur) are toxic to cat- Smith, and Slobodkin (1960) deduce that herbi- tle, sheep, and goats, and oak poisoning is a vore populations generally are not limited by their severe economic problem on the ranges of the food supply. The present work lends support to American southwest and elsewhere (Kingsbury the critics of such a deduction (notably Murdoch 1964). Dollahite, Pigeon, and Camp (1962) 1966, Ehrlich and 1967). In the case of found that symptoms of natural oak poisoning oak leaves, at least, although there is "apparently" could be produced in rabbits and cattle by repeated an excess of food available for herbivores through- doses of tannin. The effect was thought to be out the summer and fall and although this food due partly to combination of tanning materials may have a high carbohydrate and calorific con- with protein in the gastro-intestinal tract, result- tent, it is not necessarily available as suitable food ing in lower nitrogen assimilation. Tannins are for phytophagous insects or other herbivores. The toxic to chicks and depress growth by lowering widespread occurrence of tannins in plants sug- the rate of food intake and also by reducing the gests that the phenomenon may be more general. uptake of nitrogen from ingested food (Vohra, Kratzer, and Joslyn 1966). Growth depression ACKNOWLEDGMENTS in rats, due to ingestion of tannic acid, was found This research was carried out during the tenure of an to be due partly to decrease in food consumption. Agricultural Research Council Studentship and a Domus Senior Scholarship at Merton College, Oxford. The However, the proportion of dietary nitrogen ex- author is especially grateful to P. C. J. Brunet, K. W. creted was increased sixfold relative to controls Fuller, G. R. Gradwell, W. R. C. Handley, H. B. D. when 8% tannic acid was incorporated in the diet Kettlewell, D. J. Osborne, G. C. Varley, and V. B. (Glick and Joslyn 1966). In much of the work Wigglesworth for helpful advice and discussion and to tannins mammals G. R. Gradwell, N. E. Johnson, G. C. Varley, and R. H. on toxicity of to and birds, hy- Whittaker for kindly criticizing the manuscript. drolyzable tannins are implicated as the most im- Figs. 7c and 8 are reproducedby permission of Phyto- portant toxic agents (see preceding four refer- chemistry. ences). There seems little doubt, then, that tannins can LITERATURE CITED act to defend plants from attack by other orga- Allan, P. B. M. 1949. Larval food plants. Watkins nisms. They appear to act as a "broad spectrum" and Doncaster, London. 126 p. Baker, J. R. 1962. Experiments on the action of mor- defensive mechanism against herbivores and path- dants 2. Aluminium-haematein. Quart. J. Microscop. ogens and may exert their effect by three different Sci. 103: 493-517. means: as repellents affecting palatability, as Barnell, H. R. 1936. Seasonal changes in the carbo- growth inhibitors affecting protein availability, and hydrates of the wheat plant. New Phytol. 35: 229- 266. as direct toxic agents. Such a wide defensive Bate-Smith, E. C., and C. R. Metcalfe. 1957. Leuco- capability may not be the original or only function anthocyanins 3. The nature and systematic distri- of plant tannins, but it may account, at least in part, bution of tannins in dicotyledonous plants. J. Linn. for their widespread occurrence in the plant king- Soc. London 55: 669-705. dom. Bawden, F. C., and A. Kleczkowski. 1945. Protein precipitation and virus inactivation by extracts of CONCLUSIONS strawberry plants. J. Pomol. Hort. Sci. 21: 1-70. Betts, M. M. 1955. The food of titmice in oak wood- Increasing leaf toughness..is probably the chief land. J. Anim. Ecol. 24: 282-323. proximate factor preventing winter moth larvae Bradstreet, R. B. 1965. The Kjeldahl method for or- from feeding normally on mature oak leaves. It ganic nitrogen. Academic Press, London and New is suggested that of several possible ultimate fac- York. 239 p. tors for Brown, B. R., C. W. Love, and W. R. C. Handley. responsible the evolution of spring feeding 1962. Protein-fixing constituents of plants: Part III. by larvae of the winter moth, and possibly of Rep. on Forest Res. (1962): 90-93, H.M.S.O., London. other oak-feeding species of Lepidoptera, one of Brunet, P. C. J., and P. L. Small. 1959. An improved the most important is the seasonal decline in the radioautographic method for demonstrating tyrosine availability of nitrogen. This decline is due to uptake and tyrosinase activity in melanocytes. Quart. J. Microscop. 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