A plant diterpene counteracts juvenile hormone-mediated gene regulation during Drosophila melanogaster larval development.

Many plant species possess compounds with juvenile hormone disruptor (JHD) activity. In some plant species, such activity has been attributed to diterpene secondary metabolites. Plant JHD diterpenes disrupt insect development by interfering with the juvenile hormone (JH)-mediated formation of JH receptor complexes. Here, we demonstrate that a plant extract and a diterpene from Lindera erythrocarpa (methyl lucidone) interfere with the formation of both methoprene-tolerant (Met)/Taiman and Germ cell-expressed (GCE)/Taiman heterodimer complexes in yeast two-hybrid assays in vitro. In addition to the in vitro JHD activity, the diterpene and the plant extract from L. erythrocarpa also disrupt the development of larvae and pupae in Drosophila melanogaster. Comparing the transcriptomes of juvenile hormone analog (JHA, methoprene)- and JHD (methyl lucidone)-fed wandering third-instar larvae revealed a large number of genes that were coregulated by JHA and JHD. Moreover, most (83%) of the genes that were repressed by methyl lucidone were significantly activated by methoprene, indicating that JHDs and JHAs have opposing effects on the transcriptional regulation of many JH-dependent genes. Gene ontology analysis also suggested that some of the genes activated-by-JHA/repressed-by-JHD play roles in spermatogenesis. Affymetrix microarray-based analysis indicated that the expression of genes activated-by-JHA/repressed-by-JHD was testis-specific. Together, these results suggest that JH is involved in testis-specific gene expression and that plant JHD diterpenes function as JH antagonists in such JHA-mediated gene regulation.

Species-Specific Interactions between Plant Metabolites and Insect Juvenile Hormone Receptors.

Because juvenile hormone (JH) controls insect development and its analogs are used as insecticides, juvenile hormone disruptors (JHDs) represent potential sources from which novel pesticides can be developed. Many plant species harbor JHD activity, which has previously been attributed plant secondary metabolites (i.e., diterpenes) that disrupt insect development by interfering with the JH-mediated heterodimer formation of insect juvenile receptor complexes. The results of the present study indicate that plant JHD activity is also concentrated in certain plant groups and families and that plant metabolites have insect group-specific activity. These findings suggest that reciprocal diversification has occurred between plants and insects through the evolution of the plant metabolites and JH receptors, respectively, and that plant metabolites could be developed into insect group-specific pesticides with limited effects on non-target species.

Conifer Diterpene Resin Acids Disrupt Juvenile Hormone-Mediated Endocrine Regulation in the Indian Meal Moth Plodia interpunctella.

Diterpene resin acids (DRAs) are important components of oleoresin and greatly contribute to the defense strategies of conifers against herbivorous insects. In the present study, we determined that DRAs function as insect juvenile hormone (JH) antagonists that interfere with the juvenile hormone-mediated binding of the JH receptor Methoprene-tolerant (Met) and steroid receptor coactivator (SRC). Using a yeast two-hybrid system transformed with Met and SRC from the Indian meal moth Plodia interpunctella, we tested the interfering activity of 3704 plant extracts against JH III-mediated Met-SRC binding. Plant extracts from conifers, especially members of the Pinaceae, exhibited strong interfering activity, and four active interfering DRAs (7α-dehydroabietic acid, 7-oxodehydroabietic acid, dehydroabietic acid, and sandaracopimaric acid) were isolated from roots of the Japanese pine Pinus densiflora. The four isolated DRAs, along with abietic acid, disrupted the juvenile hormone-mediated binding of P. interpunctella Met and SRC, although only 7-oxodehydroabietic acid disrupted larval development. These results demonstrate that DRAs may play a defensive role against herbivorous insects via insect endocrine-disrupting activity.

Identification of plant compounds that disrupt the insect juvenile hormone receptor complex.

Insects impact human health through vector-borne diseases and cause major economic losses by damaging crops and stored agricultural products. Insect-specific growth regulators represent attractive control agents because of their safety to the environment and humans. We identified plant compounds that serve as juvenile hormone antagonists (PJHANs). Using the yeast two-hybrid system transformed with the mosquito JH receptor as a reporter system, we demonstrate that PJHANs affect the JH receptor, methoprene-tolerant (Met), by disrupting its complex with CYCLE or FISC, formation of which is required for mediating JH action. We isolated five diterpene secondary metabolites with JH antagonist activity from two plants: Lindera erythrocarpa and Solidago serotina. They are effective in causing mortality of mosquito larvae at relatively low LD50 values. Topical application of two diterpenes caused reduction in the expression of Met target genes and retardation of follicle development in mosquito ovaries. Hence, the newly discovered PJHANs may lead to development of a new class of safe and effective pesticides.

Pedobacter luteus sp. nov., isolated from soil.

Two strains of Gram-staining-negative, rod-shaped bacteria that were motile by gliding, N7d-4(T) and B4a-b5, were isolated during a study of culturable bacteria in soil cultivated with potatoes. These isolates grew at 15-37 °C and at pH 6.5-7.0. The major cellular fatty acids were summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), anteiso-C15 : 0, iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 1ω9c. The major polar lipids were phosphatidyl-N-methylethanolamine and phosphatidylethanolamine. The strains contained d-18 : 0 and d-19 : 0 sphingosines. The DNA G+C contents of strains N7d-4(T) and B4a-b5 were 48.5 and 46.9 mol% (HPLC), respectively. A phylogenetic analysis based on 16S rRNA gene sequences showed that strains N7d-4(T) and B4a-b5 were affiliated with Pedobacter species in the family Sphingobacteriaceae. Strains N7d-4(T) and B4a-b5 shared 99.9 % sequence similarity, and the most closely related Pedobacter type strains were Pedobacter composti TR6-06(T) (96.5 and 96.7 % sequence similarity, respectively), P. oryzae N7(T) (95.4 and 95.6 %) and P. caeni LMG 22862(T) (94.0 and 94.4 %). Phenotypic data and phylogenetic inference clearly distinguished the two isolates from other Pedobacter species. Based on these data, the isolates are considered to represent a novel species of the genus Pedobacter, for which the name Pedobacter luteus sp. nov. is proposed. The type strain is N7d-4(T) ( = KCTC 22699(T)  = DSM 22385(T)).

Phycicoccus ochangensis sp. nov., isolated from soil of a potato cultivation field.

Two novel, Gram-positive, motile, coccal bacteria, strains L1b-b9(T) and B5a-b5, were isolated from a potato cultivation field in Ochang, Korea. These isolates grew at 10-45°C, pH 5.0-10.0, and in the presence of 8% (w/v) NaCl. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The major menaquinone was MK-8(H(4)) and the main cellular fatty acids were iso-C(14:0), iso-C(15:0), and anteiso-C(15:0). Polar lipids in strain L1b-b9(T) consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, and an unknown glyco-amino lipid. The G+C content of genomic DNA was 73.6 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed that strains L1b-b9(T) and B5a-b5 shared 99.36% similarity and formed a robust clade with the type species of the genus Phycicoccus. Strain L1b-b9(T) is related most closely to Phycicoccus cremeus V2M29(T) (97.52% 16S rRNA gene sequence similarity). On the basis of phylogenetic characteristics, the name Phycicoccus ochangensis sp. nov. is proposed for strain LIb-b9(T) (=KCTC 19695(T) [corrected] =JCM 17595(T)).

Potent anticariogenic activity of Aceriphyllum rossii and its components, aceriphyllic acid A and 3-oxoolean-12-en-27-oic acid.

Aceriphyllum rossii Engler (Saxifragaceae) have been used as a nutritious food in Korea. We found that the methanol extract of A. rossii root and its components, aceriphyllic acid A and 3-oxoolean-12-en-27-oic acid, potently inhibited the growth of the key cariogenic bacteria, Streptococcus mutans, with MIC of 2 to 4 microg/mL. They also showed antibacterial activity against other cariogenic bacteria such as S. oralis, S. sobrinus, and S. salivarius with the similar potency. In the time-kill study, aceriphyllic acid A reduced the viable counts of S. mutans by 90% in 1 min at 8 microg/mL, indicating that aceriphyllic acid A had the fast bacteriostatic activity. Severe damages of the cell surface of S. mutans by aceriphyllic acid A were observed by transmission electron microscopy, suggesting with its fast antibacterial activity that its mechanism of action might be membrane disruption. These results suggest that the methanol extract of A. rossii root and its components, aceriphyllic acid A and 3-oxoolean-12-en-27-oic acid, could have the great potential as natural agents for preventing dental caries.

Dokdonella ginsengisoli sp. nov., isolated from soil from a ginseng field, and emended description of the genus Dokdonella.

A Gram-negative, aerobic, rod-shaped, non-motile, non-spore-forming bacterial strain, designated Gsoil 191T, was isolated from a soil sample from a ginseng field in Pocheon Province, South Korea, and was characterized taxonomically by using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain Gsoil 191T belongs to the family Xanthomonadaceae and is related to Dokdonella fugitiva LMG 23001T (97.8% sequence similarity) and Dokdonella koreensis KCTC 12396T (96.9%). The G+C content of the genomic DNA was 68.7 mol%. The major respiratory quinone was Q-8 and the major fatty acids were iso-C17:1omega9c (30.6%), iso-C17:0 (21.6%) and iso-C15:0 (13.0%), supporting the affiliation of strain Gsoil 191T to the genus Dokdonella. DNA-DNA hybridization experiments showed that the DNA-DNA relatedness values between strain Gsoil 191T and its closest phylogenetic neighbours were below 40%. The results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain Gsoil 191T from recognized species of the genus Dokdonella. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain Gsoil 191T represents a novel species of the genus Dokdonella, for which the name Dokdonella ginsengisoli sp. nov. is proposed. The type strain is Gsoil 191T (=KCTC 12564T=DSM 17954T=CCUG 52462T).

Paenibacillus pueri sp. nov., isolated from Pu'er tea.

Pu'er tea is a fermented drink made from the leaves of the tea plant, Camellia sinensis. Two novel bacteria, designated strains b09i-3(T) and b13i-1, were isolated during the process of fermentation of this tea. These isolates were Gram-positive, endospore-forming, motile rods that grew at 25-42 degrees C and pH 5.5-10.4. The DNA G+C content was 56.6-58.4 mol%, the predominant isoprenoid quinone was MK-7 and the predominant cellular fatty acid was anteiso-C(15 : 0) (49.0-50 % of the total). Phylogenetic analysis based on 16S rRNA gene sequences showed that strains b09i-3(T) and b13i-1 shared 99.9 % similarity and were affiliated with a cluster within the family Paenibacillaceae. Strains b09i-3(T) and b13i-1 were related most closely to Paenibacillus ginsengihumi DCY16(T) (97 % 16S rRNA gene sequence similarity). Levels of DNA-DNA relatedness between the two novel isolates and P. ginsengihumi DCY16(T) were below 56 %. The phylogenetic and phenotypic characteristics of these novel isolates allowed them to be distinguished clearly from recognized species of the genus Paenibacillus. Based on these data, strains b09i-3(T) and b13i-1 are considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus pueri sp. nov. is proposed. The type species is b09i-3(T) (=KCTC 13223(T)=CECT 7360(T)).

Paenibacillus camelliae sp. nov., isolated from fermented leaves of Camellia sinensis.

A novel bacterium, strain blls-2(T) was isolated from Pu'er tea. The isolate was Gram-positive, endospore-forming motile rod that grew at 15 approximately 42 degrees C and pH 6.0 approximately 10.2. The DNA G+C content was 48.3 mol%, the predominant isoprenoid quinone was MK-7, and the predominant cellular fatty acid was anteiso-C15:0 (54.2%) followed by C16:0 (15.5%) and iso-C16:0 (8.2%). The polar lipid pattern of blls-2(T) was characterized by the presence of diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. Phy-logenetic analysis based on 16S rRNA gene sequence showed that the strain was affiliated within the Paenibacillaceae. The strain was most closely related to Paenibacillus granivorans A30(T), with a similarity of 97.1%. Based on the phylogenetic and phenotypic characteristics of strain blls-2(T), the isolate is thought to represent a novel taxon in the genus Paenibacillus. The name Paenibacillus camelliae sp. nov. is proposed for the fermented tea isolate; the type strain is blls-2(T) (= KCTC 13220(T)= CECT 7361(T)).

Sphingopyxis ginsengisoli sp. nov., isolated from soil of a ginseng field in South Korea.

A Gram-negative, aerobic, motile, rod-shaped bacterium (strain Gsoil 250(T)) was isolated from soil of a ginseng field in Pocheon province (South Korea) and was characterized using a polyphasic approach in order to determine its taxonomic position. Comparative analysis of 16S rRNA gene sequences showed that strain Gsoil 250(T) belonged to the family Sphingomonadaceae of the class Alphaproteobacteria, and was related to Sphingopyxis macrogoltabida (98.7 %), Sphingopyxis chilensis (98.2 %), Sphingopyxis alaskensis (97.9 %), Sphingopyxis taejonensis (97.9 %) and Sphingopyxis witflariensis (97.8 %). The phylogenetic distance from any other species with validly published names within the genus Sphingopyxis was greater than 3.8 %. The G+C content of the genomic DNA of strain Gsoil 250(T) was 69.2 mol%. Strain Gsoil 250(T) contained Q-10 as the predominant respiratory lipoquinone and C(18 : 1)omega7c and summed feature 4 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH; 34.6 %) as the major fatty acids. No 3-hydroxy fatty acids were detected. Major polar lipids consisted of sphingoglycolipid, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, diphosphatidylglycerol and two unknown glycolipids. Phenotypic and chemotaxonomic data supported the affiliation of strain Gsoil 250(T) to the genus Sphingopyxis. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain Gsoil 250(T) from the eight recognized Sphingopyxis species. Strain Gsoil 250(T) therefore represents a novel species, for which the name Sphingopyxis ginsengisoli sp. nov. is proposed, with the type strain Gsoil 250(T) (=KCTC 12582(T)=LMG 23390(T)).

Polyamine biosynthesis regulated by StARD expression plays an important role in potato wound periderm formation.

An acireductone dioxygenase (ARD) gene of potatoes was isolated from the expressed sequence tags (ESTs) of potato post-suberization cDNA libraries. The highest expression levels of the StARD gene and the protein appeared 36 h after suberization. An approximate 9-fold increase in ARD activity was detected at 36 h after wounding. Real-time reverse transcription-PCR (RT-PCR) analysis and immunolocalization studies revealed that StARD transcripts increase at the wound surface of potato tubers. The polyamine (PA) contents increased significantly after wounding at the wound surface. The increased PA content and ARD activity may play an important role in wound periderm formation.

Elevated H(2)O (2) production via overexpression of a chloroplastic Cu/ZnSOD gene of lily (Lilium oriental hybrid 'Marco Polo') triggers ethylene synthesis in transgenic potato.

Transgenic potato plants (SS2 and SS4) that overexpressed a chloroplastic copper/zinc superoxide dismutase lily gene were utilized as an H(2)O(2)-inducible system in order to study the role of H(2)O(2) as a signaling molecule in the biosynthesis of ethylene. SS2 and SS4 plants grown in vitro under sealed microenvironment (SME) conditions displayed anomalous phenotypes including reduction of stem elongation, radial stem growth, and promotion of root hair formation in the generated root, which were similar to ethylene-induced responses. In addition, SS4 plants showed severe vitrification in developing leaves and elevated ethylene production under SME conditions. After the ethylene action inhibitor AgNO(3), 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) inhibitor CoCl(2), and ACC synthase inhibitor L -aminoethoxyvinylglycine were added to the growth media, the anomalous phenotypes in SS4 plants reverted to their normal phenotype with a concurrent decrease in ethylene production. Northern blot analysis showed that ACO transcripts in SS4 plants were constantly at high levels under normal and SME conditions, indicating that a high level of H(2)O(2) in SS4 plants up-regulates ACO transcripts. Moreover, the direct treatment of H(2)O(2) in potato plants confirmed the elevated expression of the ACO gene. Taken together, these data suggest that the high concentration of H(2)O(2) in transgenic potato plants stimulates ethylene biosynthesis by activating ACO gene expression.

A culture-based study of the bacterial communities within the guts of nine longicorn beetle species and their exo-enzyme producing properties for degrading xylan and pectin.

In this study, bacterial communities within the guts of several longicorn beetles were investigated by a culture-dependent method. A total of 142 bacterial strains were isolated from nine species of longicorn beetle, including adults and larvae. A comparison of their partial 16S rRNA gene sequences showed that most of the bacteria constituting the gut communities can typically be found in soil, plants and the intestines of animals, and approximately 10% were proposed as unreported. Phylogenetic analysis demonstrated that the bacterial species comprised 7 phyla, and approximately half were Gammaproteobacteria. Actinobacteria were the second most populous group (19%), followed by Firmicutes (13%) and Alphaproteobacteria (11%). Betaproteobacteria, Flavobacteria, and Acidobacteria were minor constituents. The taxonomic compositions of the isolates were variable according to the species of longicorn beetle. Particularly, an abundance of Actinobacteria existed in Moechotypa diphysis and Mesosa hirsute, which eat broadleaf trees; however, no Actinobacteria were isolated from Corymbia rubra and Monochamus alternatus, which are needle-leaf eaters. Considerable proportions of xylanase and pectinase producing bacteria in the guts of the longicorn beetles implied that the bacteria may play an important role in the digestion of woody diets. Actinobacteria and Gammaproteobacteria were the dominant xylanase producers in the guts of the beetles.

Superoxide anion regulates plant growth and tuber development of potato.

A higher concentration of H2O2 was detected in the sense transgenic potato plant (SS4) with the lily chCu,ZnSOD sequence, whereas higher levels of O2(-) was detected in the antisense transgenic plant (SA1) than the WT plant. The elongation growth in SA1 was significantly inhibited by treatment with diphenyleneiodonium, an inhibitor of O2(-) generation, and promoted in the SS4 on treatment with herbicide methyl viologen, a generator of apoplastic O2(-) . Higher concentrations of GAs were detected during plant growth and the early stage of tuberization in SA1. Complete recovery of the above elongation growth and microtuberization pattern in transgenic plants following treatment of GA(3) or an inhibitor of gibberellin synthesis, paclobutrazol, indicate that these changes were mainly caused by active GA levels. In conclusion, a specific ROS (O2(-) ) acts as a signal transducer via GA biosynthetic pathways for the regulation of plant growth and tuber development of potato.

Brevibacillus ginsengisoli sp. nov., a denitrifying bacterium isolated from soil of a ginseng field.

A Gram-positive, rod-shaped, spore-forming bacterium, Gsoil 3088T, was isolated from soil from a ginseng field in Pocheon Province in South Korea and characterized in order to determine its taxonomic position. On the basis of 16S rRNA gene sequence similarity, strain Gsoil 3088T was shown to belong to the family Paenibacillaceae, being related to Brevibacillus centrosporus (96.6%), Brevibacillus borstelensis (96.3%), Brevibacillus parabrevis (96.1%), Brevibacillus formosus (96.1%), Brevibacillus brevis (96.1%) and Brevibacillus laterosporus (96.0%). The phylogenetic distances from other validly described species within the genus Brevibacillus were greater than 4.0% (i.e. there was less than 96.0% similarity). The G+C content of the genomic DNA was 52.1 mol%. Phenotypic and chemotaxonomic data (major menaquinone, MK-7; fatty acid profile, iso-C15:0, iso-C14:0 and anteiso-C15:0) supported the affiliation of strain Gsoil 3088T to the genus Brevibacillus. The results of physiological and biochemical tests allowed strain Gsoil 3088T to be distinguished genotypically and phenotypically from Brevibacillus species with validly published names. Strain Gsoil 3088T, therefore, represents a novel species of the genus Brevibacillus, for which the name Brevibacillus ginsengisoli sp. nov. is proposed. The type strain is Gsoil 3088T (=KCTC 13938T=LMG 23403T).

Paenibacillus panacisoli sp. nov., a xylanolytic bacterium isolated from soil in a ginseng field in South Korea.

A Gram-positive, facultatively anaerobic, motile, spore-forming bacterium, designated Gsoil 1411T, was isolated from soil of a ginseng field in Pocheon Province (South Korea) and was characterized using a polyphasic approach. Comparative analysis of 16S rRNA gene sequences revealed that strain Gsoil 1411T belongs to the family Paenibacillaceae, with closest sequence similarity to the type strains of Paenibacillus xylanilyticus (95.7%), Paenibacillus illinoisensis (95.2%) and Paenibacillus pabuli (94.8%). Strain Gsoil 1411T showed less than 94% sequence similarity to the type strains of other recognized members of the genus Paenibacillus. In addition, the presence of MK-7 as the major menaquinone, anteiso-C15:0 as a major fatty acid (44.8%) and the presence of PAEN513F and PAEN862F signature sequences suggest that it is affiliated to the genus Paenibacillus. The G+C content of the genomic DNA was 53.9 mol%. On the basis of its phenotypic characteristics and phylogenetic distinctiveness, strain Gsoil 1411T is suggested to represent a novel species within the genus Paenibacillus, for which the name Paenibacillus panacisoli sp. nov. is proposed. The type strain is Gsoil 1411T (=KCTC 13020T=LMG 23405T).

Antioxidant and anti-inflammatory activities of N-acetyldopamine dimers from Periostracum Cicadae.

A known N-acetyldopamine dimer, (2R,3S)-2-(3',4'-dihydroxyphenyl)-3-acetylamino-7-(N-acetyl-2''-aminoethyl)-1,4-benzodioxane (1) and a new N-acetyldopamine dimer, (2R,3S)-2-(3',4'-dihydroxyphenyl)-3-acetylamino-7-(N-acetyl-2''-aminoethylene)-1,4-benzodioxane (2) were isolated from the methanolic extracts of Periostracum Cicadae. Compounds 1 and 2 inhibited the Cu2+ -mediated, 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH)-mediated, and 3-morpholinosydnonimine (SIN)-1-mediated LDL oxidation in the thiobarbituric acid-reactive substances (TBARS) assay. The antioxidant activities of 1 and 2 were tested with respect to other parameters, such as lag time of conjugated diene formation, relative electrophoretic mobility (REM), and apoB-100 fragmentation on copper-mediated LDL-oxidation. Compounds 1 and 2 also showed 1,1-diphenyl-2-picrylhydrasyl (DPPH) radical scavenging activity. Compound 2 was more efficient than compound 1 at inhibiting the reactive oxygen species (ROS) generation, nitric oxide (NO) production, and nuclear factor-kappaB (NF-kappaB) activity as well as the expression of pro-inflammatory molecules such as inducible nitric oxide synthase (iNOS), interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and cyclooxygenase (COX)-2 in LPS-induced RAW264.7 cells.

Potent inhibition of the inductions of inducible nitric oxide synthase and cyclooxygenase-2 by taiwaniaflavone.

The improper productions of nitric oxide and prostaglandins following the inductions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are involved in the pathogenesis of chronic inflammation. Selaginella tamariscina is used as an oriental medicine for its anti-inflammatory effects. Here, we isolated taiwaniaflavone from S. tamariscina and investigated whether taiwaniaflavone affects the induction of iNOS and COX-2 in RAW264.7 macrophages stimulated with lipopolysaccharide. We found that taiwaniaflavone blocks the transactivations of iNOS and COX-2 genes by blocking the nuclear translocation of p65 and subsequent nuclear factor-kappaB inactivation. It is known that NF-kappaB activation is controlled by the phosphorylation and subsequent degradation of I-kappaBalpha, and in the present study, we found that the phosphorylation and degradation of I-kappaBalpha were also inhibited by taiwaniaflavone. Our findings indicate that taiwaniaflavone may provide a developmental basis for an agent against inflammatory diseases.