Fungal endophytes which invade insect galls: insect pathogens,benign saprophytes,or fungal inquilines?

Fungi are frequently found within insect galls. However, the origin of these fungi, whether they are acting as pathogens, saprophytes invading already dead galls, or fungal inquilines which invade the gall but kill the gall maker by indirect means, is rarely investigated. A pathogenic role for these fungi is usually inferred but never tested. I chose the following leaf-galling-insect/host-plant pairs (1) a cynipid which forms two-chambered galls on the veins of Oregon white oak, (2) a cynipid which forms single-chambered galls on California coast live oak, and (3) an aphid which forms galls on narrowleaf cottonwood leaves. All pairs were reported to have fungi associated with dead insects inside the gall. These fungi were cultured and identified. For the two cynipids, all fungi found inside the galls were also present in the leaves as fungal endophytes. The cottonwood leaves examined did not harbor fungal endophytes. For the cynipid on Oregon white oak, the fungal endophyte grows from the leaf into the gall and infects all gall tissue but does not directly kill the gall maker. The insect dies as a result of the gall tissue dying from fungal infection. Therefore, the fungus acts as an inquiline. Approximately 12.5% of these galls die as a result of invasion by the fungal endophyte.     

Endophytic yeasts in leaf galls

Yeast abundance and species diversity of endophytic complexes in galls (cecidia) formed on the leaves of Salix fragilis, Salix caprea, Quercus robur, Tilia cordata, and Ulmus laevis and the epiphytic yeast communities of undamaged leaves of these plants were studied. Dynamics of yeast abundance in the galls was significantly different from that of the epiphytic yeast communities. Maximum numbers of endophytic yeast cells in the galls (up to 10⁴ CFU/g) were comparable to abundance of epiphytic yeasts. A total of 14 species of endophytic yeasts were isolated from galls of different plants. Ascomycetous yeasts were found to predominate in the insect galls on willows and oak, while basidiomycetous yeasts dominated in mite galls on linden and elm, as well as on plant leaves. These results indicate that gall formation may be considered not only as a bidirectional pathological process of the interaction between plants and invertebrates, but also as a process in which the endophytic microbial population of the galls plays an important role.     

The structure of cynipid oak galls: patterns in the evolution of an extended phenotype

Galls are highly specialized plant tissues whose development is induced by another organism. The most complex and diverse galls are those induced on oak trees by gallwasps (Hymenoptera: Cynipidae: Cynipini), each species inducing a characteristic gall structure. Debate continues over the possible adaptive significance of gall structural traits; some protect the gall inducer from attack by natural enemies, although the adaptive significance of others remains undemonstrated. Several gall traits are shared by groups of oak gallwasp species. It remains unknown whether shared traits represent (i) limited divergence from a shared ancestral gall form, or (ii) multiple cases of independent evolution. Here we map gall character states onto a molecular phylogeny of the oak cynipid genus Andricus, and demonstrate three features of the evolution of gall structure: (i) closely related species generally induce galls of similar structure; (ii) despite this general pattern, closely related species can induce markedly different galls; and (iii) several gall traits (the presence of many larval chambers in a single gall structure, surface resins, surface spines and internal air spaces) of demonstrated or suggested adaptive value to the gallwasp have evolved repeatedly. We discuss these results in the light of existing hypotheses on the adaptive significance of gall structure.     

Polyamine anabolic/catabolic regulation along the woody grapevine plant axis

The distribution of the endogenous PA fractions throughout the entire perennial woody grapevine (Vitis vinifera L.) plant was studied, along with the expression profiles of the PA anabolic and catabolic genes and their substrates and secondary metabolites. Putrescine fractions increased with increasing leaf age, although the expression of its biosynthetic enzymes Arg and Orn decarboxylases decreased. Orn transport from young organs dramatically enhanced putrescine biosynthesis in older tissues, via the Orn decarboxylase pathway. S-adenosylmethionine decarboxylase and spermidine synthase genes were down-regulated during development in a tissue/organ-specific manner, as were spermidine and spermine levels. In contrast, amine oxidases, peroxidases and phenolics increased from the youngest to the fully developed vascular tissues; they also increased from the peripheral regions of leaves to the petioles. Hydrogen peroxide generated by amine oxidases accumulated for the covalent linkage of proteins via peroxidases during lignification. These results could be valuable for addressing further questions on the role of PAs in plant development.     

Herbivorous insects alter the chlorophyll metabolism of galls on host plants

Five types of insect-induced galls derived from three host plant leaves were analyzed for their carotenoid (Car), chlorophyll (Chl), and Chl biosynthesis porphyrins such as protoporphyrinogen IX (PPIX), magnesium protoporphyrin (MGPP) and protochlorophyllide (Pchlide), and Chl degradation intermediates including chlorophyllide (Chlide), pheophytin (Phe), pheophorbide (Pho), and phytylated and dephytylated pigments, and compared to ungalled portions of the same leaf. Galls contain significantly lower levels of Chl-related compounds (CRCs) than ungalled portions of host leaves. The mole percent of porphyrin and the ratios of Chlide/Phe and phytylated/dephytylated pigments are both very different between galls and host leaves. We, therefore, conclude that leaf-derived gall is a kind of non-leaf green tissue, that herbivorous insects alter gall Chl biosynthesis and degradation pathways, that Mg-chelatase, Mg-dechelatase, and chlorophyllase may be the major non-lethal enzymes in galls, and that while ungalled host leaves take Chl → Phe → Pho and Chl → Chlide → Pho as the major and minor degradation routes, respectively, all galls are in contrast with the host leaves.     

Developmental plasticity and reduced susceptibility to natural enemies following host plant defoliation in a specialized herbivore

Host-specific phytophagous insects that are short lived and reliant on ephemeral plant tissues provide an excellent system in which to investigate the consequences of disruption in the timing of resource availability on consumer populations and their subsequent interactions with higher tropic levels. The specialist herbivore, Belonocnema treatae (Hymenoptera: Cynipidae) induces galls on only newly flushed leaves of live oak, Quercus fusiformis. In central Texas (USA) episodic defoliation of the host creates variation in the timing of resource availability and results in heterogeneous populations of B. treatae that initiate development at different times. We manipulated the timing of leaf flush in live oak via artificial defoliation to test the hypothesis that a 6- to 8-week delay in the availability of resources alters the timing of this gall former’s life cycle events, performance and survivorship on its host, and susceptibility to natural enemies. B. treatae exhibits plasticity in development time, as the interval from egg to emergence was significantly reduced when gallers oviposited into the delayed leaf flush. As a consequence, the phenologies of gall maturation and adult emergence remain synchronized in spite of variation in the timing of resource availability. Per capita gall production and gall-former performance are not significantly affected by the timing of resource availability. The timing of resource availability and natural enemies interact, however, to produce strong effects on survivorship: when exposed to natural enemies, B. treatae developing in galls initiated by delayed oviposition exhibited an order-of-magnitude increase in survivorship. Developmental plasticity allows this gall former to circumvent disruptions in resource availability, maintain synchrony of life cycle events, and results in reduced vulnerability to natural enemies following defoliation of the host plant.     

Impact of whole-canopy and systemic insecticidal treatments on Callirhytis cornigera (Hymenoptera: Cynipidae) and associated parasitoids on pin oak

The gall wasp Callirhytis cornigera (Osten Sacken) is a cynipid with alternating generations that produce large, woody stem galls and tiny blister-like leaf galls on pin oak, Quercus palustris Muenchhausen, in the United States. We tested 3 approaches to control the leaf-galling generation, and determined their impact on associated parasitoids and effectiveness in reducing numbers of new stem galls. First, trees were sprayed with bifenthrin or chlorpyrifos in late March to kill females emerging from stem galls before they oviposited into buds. Second, concentrated solutions of abamectin, imidacloprid, or bidrin were injected from pressurized containers into tree sapwood to control larvae developing in young leaf galls. Finally, systemic insecticides (acephate, abamectin, dimethoate, or imidacloprid) were sprayed at early leaf expansion (2 May) or to young, expanded leaves (17 May) to target larvae in leaf galls. Parasitoids, mostly eulophids, accounted for approximately 70% mortality of leaf-galling C. cornigera larvae on untreated trees. Whole-canopy sprays during C. cornigera emergence from stem galls reduced overall numbers of galled leaves and leaf galls. Trunk injections of bidrin or abamectin resulted in significant mortality of gall inhabitants, including parasitoids. However, neither of the aforementioned approaches significantly reduced numbers of new stem galls. Sprays of abamectin, dimethoate, or imidacloprid applied on 2 May caused high mortality of all gall inhabitants. There was no net benefit, however, because parasitism caused a similar reduction in C. cornigera survival on unsprayed shoots. Sprays applied later in leaf expansion had little impact on gall inhabitants. Of the treatments tested, bifenthrin sprays at bud break provided the greatest reduction in new leaf galls, whereas bidrin injections provided the greatest reduction in gall wasps emerging from galled leaves. This study suggests that gall wasp outbreaks are unlikely to be controlled by a single treatment, regardless of application method.     

Why do the galls induced by Calophya duvauae Scott on Schinus polygamus (Cav.) Cabrera (Anacardiaceae) change colors?

One of the galling herbivores associated to the superhost Schinus polygamus (Cav.) Cabrera (Anacardiaceae) is Calophya duvauae Scott (Hemiptera: Calophyidae). The galls are located on the adaxial surface of leaves and vary from red to green. The levels of their pigments were herein investigated in relation to age. Samples were collected between June 2008 and March 2009, in a population of S. polygamus at Canguçu municipality, Rio Grande do Sul, Brazil. Galls were separated by color, measured, dissected, and the instar of the inducer was determined. The levels of photosynthetic (chlorophyll a and b, total chlorophylls, and carotenoids) and protective pigments (anthocyanins) were also evaluated. Red galls were more numerous than the green ones, and the induction should occur preferentially on young leaves, but may also occur on mature leaves. Immature stages of C. duvauae were observed in all samples throughout the year, characterizing its life cycle as multivoltine. There was a significant correlation between the instar of the inducer and the size of the gall (r = 0.675, p < 0.001), with larger galls corresponding to more advanced instars. The ratio between red and green galls varied over the samples, with the highest (96%) and lowest (38%) frequency of red galls observed in September, and December 2008, respectively. The average amounts of chlorophyll b and total chlorophylls were 70% lower in the gall tissues when compared to non-galled portions of galled leaves. There is a notable reduction in the contents of all pigments in the galls when compared to non-galled leaves, especially for total anthocyanins in green galls. The red galls presumably had the constant stimuli of healthy gall inducers, whereas in green galls they died prematurely, due to the interference of parasitoids and inquilines. The alterations in gall structure and color were related to the gradual decreasing in galling stimuli.     

Histocytological aspects of four types of ambrosia galls on Machilus zuihoensis Hayata (Lauraceae)

Four types of prosoplasmatic galls induced by Daphnephila midges are found on leaves of Machilus zuihoensis, a species endemic to Taiwan: urn- and small urn-shaped, obovate, and hairy oblong galls. In addition to containing nutritive tissues, these galls are lined with fungal hyphae. The objective of this study was to describe and compare the structural organization of the various gall morphologies and to examine the ultrastructure of the nutritive and fungal cells lining the gall chambers. The morphology and ultrastructure of mature-stage galls were examined by light, scanning electron, and transmission electron microscopy. Diverse epidermal cell shapes and wax textures were observed in the leaves and galls of M. zuihoensis. In small urn-shaped, obovate, and hairy oblong galls vascular bundles extend from the gall base to near the centre of the gall top. In contrast, vascular bundles in urn-shaped galls are distributed in the gall wall and extend to close to the outer gall top. Trichomes were present only abaxially on leaves and on hairy oblong gall surfaces. Starch granules, tannins, and mucilage were distributed differently among the four gall types. Further, fungal mycelia spread in the interior gall wall and partially passed through the intercellular spaces of nutritive cells and reached the sclerenchyma. Histological analyses revealed that the surface structure of galls differs from that of the leaf and that the epidermal organization differs among the four gall types. Different types of leaf galls on the same plant have different patterns of tissue stratification and contain different ergastic substances. The results of this study will contribute to the understanding of tritrophic relationships and the complex interactions among parasitic gall-inducing insects, mutualistic fungi, and host plants.     

Characterisation of the effects of leaf galls of Clusiamyia nitida (Cecidomyiidae) on Clusia lanceolata Cambess. (Clusiaceae): Anatomical aspects and chemical analysis of essential oil

Galls develop in different plant organs and are induced by the activity of various organisms. Some studies have investigated the ecological interactions between species of Clusia and gall-inducing insects. The goal of our study is to characterise changes in leaf anatomy caused by the activity of gall insects in Clusia lanceolata. Additionally, we also investigated the chemical composition of volatile compounds of normal leaves and those with galls to detect possible effects on the host plants. For anatomical studies, we used botanical material fixed in FAA₅₀. Transversal sections of the leaf blade were obtained from samples of leaves located on the third and fourth nodes from both male and female individuals. Material was studied from both sexes both with unaffected leaves and leaves containing galls. Fresh leaves of C. lanceolata were used for the extraction of volatile compounds, which were submitted to stem distillation using a modified Clevenger apparatus determining the oil yields subsequently (w/w). The unaffected leaves of female and male individuals of C. lanceolata exhibit similar anatomical structures. However, galls on leaves of both sexes show anatomical differences. The activity of the gall insect Clusiamyia nitida induces several changes in the foliar anatomy and the distribution of metabolic compounds in new tissues during gall development. However, the larvae are not able to induce significant changes in the volatile compounds of inflected leaves from male and female individuals.     

Variation in the Degree of Pectin Methylesterification during the Development of Baccharis dracunculifolia Kidney-Shaped Gall

Insect galls may be study models to test the distribution of pectins and arabinogalactan-proteins (AGPs) and their related functions during plant cell cycles. These molecules are herein histochemically and immunocitochemically investigated in the kidney-shaped gall induced by Baccharopelma dracunculifoliae (Psyllidae) on leaves of Baccharis dracunculifolia DC. (Asteraceae) on developmental basis. The homogalacturonans (HGAs) (labeled by JIM5) and the arabinans (labeled by LM6) were detected either in non-galled leaves or in young galls, and indicated stiffening of epidermal cell walls, which is an important step for cell redifferentiation. The labeling of HGAs by JIM7 changed from young to senescent stage, with an increase in the rigidity of cell walls, which is important for the acquaintance of the final gall shape and for the mechanical opening of the gall. The variation on the degree of HGAs during gall development indicated differential PMEs activity during gall development. The epitopes recognized by LM2 (AGP glycan) and LM5 (1–4-β-D-galactans) had poor alterations from non-galled leaves towards gall maturation and senescence. Moreover, the dynamics of pectin and AGPs on two comparable mature kidney-shaped galls on B. dracunculifolia and on B. reticularia revealed specific peculiarities. Our results indicate that similar gall morphotypes in cogeneric host species may present distinct cell responses in the subcelular level, and also corroborate the functions proposed in literature for HGAs.     

The chemical composition of plant galls: are levels of nutrients and secondary compounds controlled by the gall-former?

The chemical composition of galled and ungalled plant tissue was compared in a series of experiments. Gall and adjacent plant tissue was analysed for 20 species of gall-former on 11 different plant species. There were clear differences between galled and ungalled tissue in levels of nutrients and secondary compounds. Gall tissue generally contained lower levels of nitrogen and higher levels of phenolic compounds than ungalled plant tissue. The gall tissue produced by the same plant in response to different species of gall-former differed in chemical composition, as did the gall-tissue from young and mature galls of the same species. The chemical differences between gall and plant tissues were studied in more detail in two field manipulations. Firstly, the seasonal changes in phenolic biosynthesis in Pontania proxima and P. pedunculi (Hymenoptera: Tenthredinidae) gall tissue were compared to those of their host plants, Salix alba and S. caprea. In both types of gall tissue, phenolic levels declined as the season progressed, but levels in the surrounding plant tissue increased. When the gall insects were killed with insecticide, phenolic levels in the galled tissue dropped to the same level as those in adjacent plant tissue. Secondly, the density of Cynips divisa (Hymenoptera: Cynipidae) galls on Quercus robur leaves was reduced by removing half the galls present, either those from the central region of the leaf or those from the edge. Decreasing gall density increased the size of the remaining galls and the weight of the insects, but these effects were most marked when the galls remaining were growing centrally on the leaf, i.e. when the galls from the edge had been removed. Decreasing gall density increased the nitrogen content of the remaining galls, again to a greater extent in galls growing centrally on the leaf. The results of these studies suggest that the levels of nutrients and secondary compounds in gall tissue are usually markedly different to those of surrounding plant tissue, and that gall-formers may produce species-specific and temporally variable changes in the chemical composition of gall tissue. Received: 7 July 1997 / Accepted: 29 September 1997     

Structure,biochemistry and ecology of entomogenous galls in Selaginella Pal. Beauv. (Selaginellaceae) from India

Abstract

We studied the structure of newly found entomogenous galls in three Indian species of Selaginella, and the biochemical changes during gall development as well as their seasonal abundances in 10 squares of 1.5 m² under two differing habitat conditions, open and covered. Adult wasps (Cynipidae) initiate two types of gall formation by oviposition, spherical galls on vegetative shoots and elongated, club-shaped strobilar galls on reproductive shoots. Galls are anatomically and biochemically different from the unaffected shoots. Vegetative shoot apices bear more galls (84.6%) than reproductive shoot apices (15.4%). Gall frequency is significantly higher in covered (94.8%) than in open habitat (5.2%), and its seasonal peak occurs earlier in the former (43.1% during the rainy season) than in latter habitat (52.8% in autumn). We discuss the relationship between relative species density and gall seasonality as well as the possible role of certain environmental factors that make covered habitats more favorable to the gall inducer.     

Fungal diversity in galls of baldcypress trees

The baldcypress midge (Taxodiomyia cupressi and Taxodiomyia cupressiananassa) forms a gall that originates from leaf tissue. Female insects may inoculate galls with fungi during oviposition, or endophytes from the leaf tissue may grow into the gall interior. We investigated fungal diversity inside of baldcypress galls, comparing the gall communities to leaves and comparing fungal communities in galls that had successful emergence versus no emergence of midges or parasitoids. Galls of midges that successfully emerged were associated with diverse gall fungal communities, some of which were the same as the fungi found in surrounding leaves. Galls with no insect emergence were characterized by relatively low fungal diversity.     

Distribution of metabolites in galled and non-galled foliar tissues of Tibouchina pulchra

This paper reports the contents of foliar metabolites of Tibouchina pulchra (Melastomataceae) in (a) galls induced by a lepidopteran, (b) remaining parts of the galled leaf after gall removal, (c) leaves opposite to the galled leaf, and (d) leaves of non-infested stem branches (control). The parameters assayed were soluble phenols, flavonoids, tannins, lignins, fibers, soluble carbohydrates, lipids and organic nitrogen. Differences in the parameters assayed were evaluated using Principle Components Analysis. Compared to other tissues, galls showed significantly higher contents of soluble phenols, tannins, lignins, fibers, soluble carbohydrates and lipids, and significantly lower contents of flavonoids and organic nitrogen. Apart from gall tissues, in most cases no significant differences were detected in the quantitative analyses among the leaf tissues assayed. Flavonols and flavones were not detected in galls. Other tissues revealed a similar flavonoid pattern, characterized by 3-O-monoglycosides of kaempferol, myricetin and quercetin. A luteolin glycoside was obtained exclusively from control leaves. Carbohydrate amounts are lower in the foliar tissues closer to the galls than in non-galled tissues. Palmitic acid was essentially the sole fatty acid found in all tissues analysed. The high lipid content of the galls suggests that such substances represent the main energy source for the insect, and suggests that the studied galls could be classified as cynipid galls. The observed metabolic changes taking place in the galls strengthen the hypotheses that galls behave as new organs, operating a metabolic machinery of their own.     

The number of cecidomyiid insect galls affects the photosynthesis of Machilus thunbergii host leaves

Previous studies of the impacts of galls on host leaf photosynthesis do not suggest any general trends, with a reported range of effects from negative to positive. In this study, photosynthetic characteristics such as chlorophyll fluorescence (Fv/Fm), photosynthetic capacity, and stomata conductance were determined in two types of fruit-like galls (red ovoid and green obovate galls) induced by Daphnephila taiwanensis and Daphnephila sueyenae, respectively, in order to investigate whether the number of galls affects the photosynthesis of galled leaves of Machilus thunbergii. In 2008, chlorophyll fluorescence and photosynthetic capacity were negatively correlated with gall numbers, non-significantly and significantly, respectively, whereas stomata conductance was positively but non-significantly correlated with gall numbers. In 2009, photosynthesis capacity and stomata conductance were negatively, but non-significantly, correlated with gall numbers. Results imply that photosynthesis in M. thunbergii leaves is slightly affected by the number of cecidomyiid insect galls, and that the higher the gall number, the greater the negative effect that galls have on host leaf photosynthesis and subsequent infection.     

The protein content of tissues in cynipid galls (Hymenoptera: Cynipidae): Similarities between cynipid galls and seeds

Cynipid galls are examples of induced plant development, where the gall inducer is in control of cell differentiation and morphogenesis of a new plant organ. This study concentrates on the tissues of the larval chamber common to all cynipid galls. The protein content of the inner gall tissue was compared to that of non‐gall plant tissues. We investigated three oak and two rose galls and their respective host plants. Total protein signatures of inner gall tissues were different from those of non‐gall plant tissues, and among the five galls. N‐terminal sequences were obtained for two abundant proteins from the inner gall tissues of D. spinosa and A. quercuscalicis, which were common to all galls, at 62 and 43 kDa. Database queries suggest the 62 kDa protein to be homologous to a protein disulphide isomerase (PDI), and the 43 kDa protein to be homologous to NAD‐dependent formate dehydrogenase (FDH). A naturally biotinylated protein was detected at 33 kDa during Western analyses with streptavidin. Western analyses revealed the presence of the biotinylated protein and PDI in the inner gall tissues of all five galls, while FDH was only detected in A. quercuscalicis and A. fecundator. PDI was also common to all non‐gall tissues, while FDH was not detected in non‐gall tissues, and the biotinylated protein was only detected in seeds. The proteins identified in the inner gall tissue suggest that (a) inner gall tissues in some galls are under respiratory stress, and (b) cynipid gall formation might involve the ectopic expression of seed‐specific proteins.     

Partitioning of herbivore hosts across time and food plants promotes diversification in the Megastigmus dorsalis oak gall parasitoid complex

Communities of insect herbivores and their natural enemies are rich and ecologically crucial components of terrestrial biodiversity. Understanding the processes that promote their origin and maintenance is thus of considerable interest. One major proposed mechanism is ecological speciation through host‐associated differentiation (HAD), the divergence of a polyphagous species first into ecological host races and eventually into more specialized daughter species. The rich chalcid parasitoid communities attacking cynipid oak gall wasp hosts are structured by multiple host traits, including food plant taxon, host gall phenology, and gall structure. Here, we ask whether the same traits structure genetic diversity within supposedly generalist parasitoid morphospecies. We use mitochondrial DNA sequences and microsatellite genotypes to quantify HAD for Megastigmus (Bootanomyia) dorsalis, a complex of two apparently generalist cryptic parasitoid species attacking oak galls. Ancient Balkan refugial populations showed phenological separation between the cryptic species, one primarily attacking spring galls, and the other mainly attacking autumn galls. The spring species also contained host races specializing on galls developing on different host‐plant lineages (sections Cerris vs. Quercus) within the oak genus Quercus. These results indicate more significant host‐associated structuring within oak gall parasitoid communities than previously thought and support ecological theory predicting the evolution of specialist lineages within generalist parasitoids. In contrast, UK populations of the autumn cryptic species associated with both native and recently invading oak gall wasps showed no evidence of population differentiation, implying rapid recruitment of native parasitoid populations onto invading hosts, and hence potential for natural biological control. This is of significance given recent rapid range expansion of the economically damaging chestnut gall wasp, Dryocosmus kuriphilus, in Europe.     

Polyamines and the Cell Cycle of Catharanthus roseus Cells in Culture

Investigation was made on the effect of partial depletion of polyamines (PAs), induced by treatment with inhibitors of the biosynthesis of PAs, on the distribution of cells at each phase of the cell cycle in Catharanthus roseus (L.) G. Don. cells in suspension cultures, using flow cytometry. More cells treated with inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were accumulated in the G₁ phase than those in the control, while the treatment with an inhibitor of spermidine (SPD) synthase showed no effect on the distribution of cells. The endogenous levels of the PAs, putrescine (PUT), SPD, and spermine (SPM), were determined during the cell cycle in synchronous cultures of C. roseus. Two peaks of endogenous level of PAs, in particular, of PUT and SPD, were observed during the cell cycle. Levels of PAs increased markedly prior to synthesis of DNA in the S phase and prior to cytokinesis. Activities of ADC and ODC were also assayed during the cell cycle. Activities of ADC was much higher than that of ODC throughout the cell cycle, but both activities of ODC and ADC changed in concert with changes in levels of PAs. Therefore, it is suggested that these enzymes may regulate PA levels during the cell cycle. These results indicate that inhibitors of PUT biosynthesis caused the suppression of cell proliferation by prevention of the progression of the cell cycle, probably from the G₁ to the S phase, and PUT may play more important roles in the progression of the cell cycle than other PAs.