Feeding behaviour of underyearling hybrids of the grass carp, Ctenopharyngodon idella♀ and the bighead, Hypophthalmicthys nobilis♂ on selected species of aquatic plants

Underyearling hybrids of the cross Ctenopharyngodon idella ♀ (grass carp) and Hypophthalmichthys nobilis ♂ (bighead), averaging 104.8 mm total length were stocked for two weeks in cement tanks holding 605 1 of water with various combinations of submersed or floating aquatic plants. The submersed species most preferred were Ceratophyllum demersum, Chara sp. and Najas guadalupensis. Hydrilla verticillata, Hygrophila polysperma, Myriophyllum pinnatum and Utricularia foliosa were fed on from a minor to moderate extent. No feeding damage was observed on Egeria densa and Potamogeton illinoensis. Azolla caroliniana, Lemna sp. and Wolffia columbiana were readily consumed while Salvinia rotundifolia was not preferred when in combination with these floating plant species. Feeding damage to the submersed roots of Eichhornia crassipes and Pistia stratiotes was minor during a 30 day period. Hybrid fingerlings readily fed on mosquito larvae and small leeches when offered to them in an aquarium on one occasion.     

Intestinal microbiota of gibel carp (Carassius auratus gibelio) and its origin as revealed by 454 pyrosequencing

The intestinal microbiota has received increasing attention, as it influences growth, feed conversion, epithelial development, immunity as well as the intrusion of pathogenic microorganisms in the intestinal tract. In this study, pyrosequencing was used to explore the bacterial community of the intestine in gibel carp (Carassius auratus gibelio), and the origin of these microorganisms. The results disclosed great bacterial diversities in the carp intestines and cultured environments. The gibel carp harbored characteristic intestinal microbiota, where Proteobacteria were predominant, followed by Firmicutes. The analysis on the 10 most abundant bacterial operational taxonomic units (OTUs) revealed a majority of Firmicutes in the intestinal content (by decreasing order: Veilonella sp., Lachnospiraceae, Lactobacillales, Streptococcus sp., and Lactobacillus sp.). The second most abundant OTU was Rothia sp. (Actinobacteria). The most likely potential probiotics (Lactobacillus sp., and Bacillus sp.) and opportunists (Aeromonas sp., and Acinetobacter sp.) were not much abundant. Bacterial community comparisons showed that the intestinal community was closely related to that of the sediment, indicating the importance of sediment as source of gut bacteria in gibel carp. However, 37.95 % of the OTUs detected in feed were retrieved in the intestine, suggesting that food may influence markedly the microbiota of gibel carp, and therefore may be exploited for oral administration of probiotics.     

Genetic diversity among and within cultured cyanobionts of diverse species of <Emphasis Type="Italic">Azolla</Emphasis>

The cyanobionts isolated from 10 Azolla accessions belonging to 6 species (Azolla mexicana, A. microphylla, A. rubra, A. caroliniana, A. filiculoides, A. pinnata) were cultured under laboratory conditions and analyzed on the basis of whole cell protein profiles and molecular marker dataset generated using repeat sequence primers (STRR(mod) and HipTG). The biochemical and molecular marker profiles of the cyanobionts were compared with those of the free-living cyanobacteria and symbiotic Nostoc strains from Anthoceros sp., Cycas sp. and Gunnera monoika. Cluster analysis revealed the genetic diversity among the selected strains, and identified 3 distinct clusters. Group 1 included cyanobionts from all the 10 accessions of Azolla, group 2 comprised all the symbiotic Nostoc strains, while group 3 included the free-living cyanobacteria belonging to the genera Nostoc and Anabaena. The interrelationships among the Azolla cyanobionts were further revealed by principal component analysis. Cyanobionts from A. caroliniana-A. microphylla grouped together while cyanobionts associated with A. mexicana-A. filiculoides along with A. pinnata formed another group. A. rubra cyanobionts had intermediate relationship with both the subgroups. This is the first study analyzing the diversity existing among the cultured cyanobionts of diverse Azolla species through the use of biochemical and molecular profiles and also the genetic distinctness of these free-living cyanobionts as compared to cyanobacterial strains of the genera Anabaena and Nostoc.     

Inactivation by gene disruption of 2‐cysteine‐peroxiredoxin in Synechocystis sp. PCC 6803 leads to increased stress sensitivity

2‐Cysteine‐peroxiredoxins (2‐CP) constitute a ubiquitous group of enzymes which reduce toxic alkyl hydroperoxides. In higher plants it was shown that the nuclear encoded 2‐CPs are posttranslationally imported into the chloroplasts, the site of most active oxidative metabolism in plants (Baier and Dietz 1997, Plant J. 12; 179‐190). The genome of the bluegreen alga Synechocystis (EMBL acc. # D64000) encodes a 2‐CP which shares 60% homology to higher plant 2‐CPs on the gene level and about 70% on the level of the mature protein. In order to elucidate the physiological significance of 2‐CPs for photosynthetic organisms, the 2‐CP gene was mutated in Synechocystis sp. PCC 6803 by insertion of a kanamycin gene cartridge. Following complete segregation mutant lines were analyzed for growth and photosynthetic parameters. The mutants revealed decreased growth rates as compared to the wild type. Growth inhibition was relieved after lowering the concentration of Fe or trace elements in the growth medium. Chlorophyll a fluorescence transients as induced by saturating light pulses were used as indicator for the state of photosynthesis. The effective quantum yield decreased at lower light intensities in the mutants as compared to the wild type Synechocystis . Simultaneously, electron transport rates saturated at lower light intensities in the mutants. These data provide the first evidence that 2‐CPs play a pivotal protective role in photosynthesis.     

Feeding behaviour of yearling and older hybrid grass carp

Hybrid grass carp resulting from the cross of a female grass carp, Ctenopharyngodon idella (Val.), and a male bighead carp, Hypophthalmichthys ( Aristichthys ) nobilis Rich., 12–18 months old ( c . 300 mm T.L.) were studied in a two-part experiment to determine feeding preference and total daily consumption fish^-1 on selected species of aquatic plants. Fish were maintained in circular pools with 6840·8 1 of water inside a temperature-controlled greenhouse. Preference tests were conducted at three temperature ranges; 25–28° C, 17–20° C and 12–15° C. Based on the time to complete consumption or the relative quantity consumed, the most preferred plant was Lemna gibba when in combination with six other species. Chara sp., Najas guadalupensis and Potamogeton peciinatus were readily consumed and considered to be of about equal preference. Ceratophyllum demersum and Myriophyllum brasiliense were least preferred. Hybrid grass carp generally consumed as much plant material species^-1 and in the same order of preference at the 12–15°C range as they did at 25–28° C. In the second part, mean daily consumption (g) fish^-1 at 25·7–31·0° C for five plant species tested separately was as follows: Chara sp. 369·8; Lemna gibba 178·2; Najas guadalupensis 172·6; Hydrilla verticillata 106·4 and Ceratophyllum demersum 8·8.     

Introduction of the Exocelina ekari-group with descriptions of 22 new species from New Guinea (Coleoptera,Dytiscidae, Copelatinae)

The Exocelina ekari-group is here introduced and defined mainly on the basis of a discontinuous outline of the median lobe of the aedeagus. The group is known only from New Guinea (Indonesia and Papua New Guinea). It contained four species to date: Exocelina astrophallus (Balke, 1998), Exocelina atowaso (Shaverdo, Sagata & Balke, 2005), Exocelina munaso (Shaverdo, Sagata & Balke, 2005), and Exocelina polita (Sharp, 1882). Twenty two new species are described herein: Exocelina alexanderi sp. n., Exocelina anggiensis sp. n., Exocelina arfakensis sp. n., Exocelina bifida sp. n., Exocelina brahminensis sp. n., Exocelina bundiensis sp. n., Exocelina edeltraudae sp. n., Exocelina ekari sp. n., Exocelina eme sp. n., Exocelina evelyncheesmanae sp. n., Exocelina hansferyi sp. n., Exocelina irianensis sp. n., Exocelina kakapupu sp. n., Exocelina knoepfchen sp. n., Exocelina oceai sp. n., Exocelina pseudosoppi sp. n., Exocelina soppi sp. n., Exocelina unipo sp. n., Exocelina utowaensis sp. n., Exocelina waigeoensis sp. n., Exocelina weylandensis sp. n., and Exocelina wondiwoiensis sp. n. The lectotype of Copelatus politus Sharp, 1882 is designated. A checklist and identification key to all species of the group are provided and important diagnostic characters (habitus, color, male antennae and protarsomeres 4–5, median lobes and parameres) are illustrated. Data on the distribution and habitat requirements are given. Representatives of the Exocelina ekari-group are so far mostly known from lowland to lower montane habitats of the northern and central parts of New Guinea, the group is less diverse in higher altitudes.     

鲢对藻类摄食效应的室内模拟研究

为研究鲢(Hypophthalmichthys molitrix)对藻类的摄食作用, 采用小环藻(Cyclotella)、小球藻(Chlorella)、微囊藻(Microcystis)和东湖原水开展了小规格鱼种的养殖试验, 对鲢摄食和排泄物进行了研究。结果显示: 在3 种纯培养藻种投喂下鲢存活率从高到低依次是小环藻组 小球藻组 微囊藻组;相应地, 鲢对藻的平均摄食率排序也是小环藻组 小球藻组 微囊藻组。在显微镜下观察粪便发现大部分小环藻为空壳, 一部分小球藻外部形态结构不完整, 外部形态完整的小球藻则色泽暗淡、内部结构紊乱, 微囊藻没有明显变化;3 种藻类细胞的受损率分别为20.04%、7.13%和1.97%。叶绿素荧光活性显示, 粪便中小环藻基本失去光合活性, 小球藻活性极显著降低(P0.01), 微囊藻活性明显降低(P0.05), 东湖原水处理组光合活性较对照极显著降低(P0.01)。对东湖水饲喂试验组排泄物的检测发现, 浮鞘丝藻Planktolyngya、鱼腥藻Anabeana(有破损)、假鱼腥藻Pseudoanabaena 和颤藻Oscillatoria 细胞受损严重;空星藻Coelastrum 和盘星藻Pediastrum 细胞部分破损;针杆藻Synedra、小环藻、桥弯藻Cymbella 和舟形藻Navicula 多为空壳。鱼粪中平裂藻Merismopedia、微囊藻、浮鞘丝藻、鱼腥藻、颤藻、卵囊藻Oocystis、栅藻Scenedesmus 和衣藻Chlamydomonas 在重新培养后恢复了生长。本研究显示硅藻尤其是小环藻、针杆藻和舟形藻对鲢摄食最敏感;栅藻、卵囊藻和平裂藻对摄食压力不敏感。研究表明鲢捕食对藻类群落结构有明显的改变效应, 利用鲢的滤食进行藻类群落调节和水华防控具有可行性。       

Zooplankton abundance and diversity in Central Florida grass carp ponds

The effect of the Asian grass carp (Ctenopharyngodon idella Val.) upon the zooplankton in three adjacent experimental ponds (0.139 ha each) was studied for one year. The ponds contained nine species of aquatic macrophytes. Grass carp were stocked into Pond 1 (65 per ha) and Pond 2 (611 per ha) three months after the study was started. At the time of stocking, physichochemical and biological parameters were similar among the ponds.Grass carp did not affect water quality and had little direct or indirect effect upon the zooplankton in the ponds. The abundance and species diversity of zooplankton (number of species, Shannon Index, and Simpson Index) were not significantly different (P < 0.05) between ponds prior to grass carp stocking. After stocking with grass carp, the number of species and species diversity were found to be significantly different (P < 0.05) between Pond 1 (low grass carp stocking rate) and Pond 2 (high grass carp stocking rate). Additionally, significant differences (P < 0.05) in populations between ponds were found for Lecane luna, Monostyla bulla, Lepadella ehrenbergii, Polyarthra sp., and Synchaeta sp. Temporal variation rather than grass carp was probably responsible for those differences. The number of zooplankton per group (Rotifera, Cladocera, and Copepoda) and species diversity were not significantly different (P<0.05) between ponds containing different stocking rates of grass carp.During the study, zooplankton were collected with a shallow-water sampler. No significant differences (P < 0.05) between collections were found for the sampler and a No. 20 mesh nylon zooplankton net.     

The role of tubificid oligochaetes in the transmission of Goussia carpelli

Tubifex tubifex and Limnodrilus hoffmeisteri, fed intestinal tissue of carp containing oocysts of Goussia carpelli, produced infections in laboratory-reared carp. Sporocysts ingested by the tubificids released the sporozoites that were found to be motile in the intestinal contents and incorporated in intestinal cells of the oligochaetes. Tubificids remained infective for carp at least up to 57 days postexposure (PE). Tubificids were also able to transmit G. carpelli to carp that had recovered from a previous infection, whereas attempts of direct transmission of G. carpelli among these carp failed. Direct transmission to uninfected carp by fecal contamination was possible. Organisms from the pond plankton, benthic Limnadia sp., and chironomid larvae did not transmit G. carpelli to carp.     

Effect of L-3,4-dihydroxyphenylalanine, ornithine and γγ-aminobutyric acid on winter wheat resistance to grain aphid

The association between the concentrations of L-3,4-dihydroxyphenylalanine (L-DOPA), ornithine, and γ-aminobutyric acid (GABA) in winter wheat cultivars with their antibiotic resistance to the grain aphid Sitobion avenae (F.) (Hom., Aphididae) was studied. The antibiosis was measured under field conditions using the intrinsic rate of natural increase ( r _m ). The content of the studied non-protein amino acids was estimated in flag leaf at the beginning of anthesis growth stage. Highly significant negative correlations were found between the r _m values and concentrations of L-DOPA (r = −0.937) and ornithine (r = −0.907), but not with GABA (r = −0.363). The contribution of these allelochemicals to the resistance of the plants to insects is discussed.     

Native β-glucuronidase activity in sugarbeet (Beta vulgaris)

β-Glucuronidase (EC 3.2.1.31) activity, initially thought absent from plants, has been found in a number of plant families. During an analysis of Agrobacterium -mediated transformation of sugarbeet ( Beta vulgaris L.), significant glucuronidase activity was observed in control (non-transformed) tissues when the fluorogenic substrates 4-methylumbelliferyl-β- d -glucuronic acid, resorufin glucuronic acid and 3-carboxyum-belliferyl-β- d -glucuronic acid were used to quantify β-glucuronidase activity under standard protocol conditions. Similarly, the colorigenic substrate p -nitrophenyl-β- d -glucuronide was hydrolyzed by this sugarbeet-derived glucuronidase. Biochemical and immunological data are presented to indicate significant differences between sugarbeet-derived glucuronidase and that from Escherichia coli (EC 3.2.1.31) encoded by gusA . These differences provide means of distinguishing between the two activities in extracts that contain a mixture of both. Use of X-glue, the substrate utilized in histochemical localizations of glucuronidase activity, gave no reaction product (i.e., indigo precipitate) at pH 7.0. However, at pH 3.0, 4.0 and 5.0 formation of the indigo precipitate was evident within 1 h at 37°C in sugarbeet callus and by 4 h in leaves and petioles. The specific activity of sugarbeet glucuronidase was observed to be strongly pH dependent, with an optimum near pH 4.0. The use of various β-glucuronidase assay techniques as applied to transformation of sugarbeet is discussed.     

Aeromycoflora in the Central Milk Dairy of Calcutta, India

Intramural aeromycological survey was performed at the Central Milk Dairy, Calcutta, covering eight locations within the Dairyusing Burkard personal volumetric air sampler. The locations were butter cold storage (−2 °C), cold store (8 °C), packaging section (23 °C), milk processing section (24 °C), reconstituent of skimmed milk (24 °C), quality control lab (25 °C), raw milk reception (28 °C) and loading dock (26 °C). A number of fungal spores, conidia and mycelia were recorded in different rooms: the highest spore quantity was recorded in the packaging section (23 °C) and the minimum at the butter cold store (−2 °C). The dominant spores consisted of Aspergillus niger, A flavus,Cladosporium sp., Fusarium sp., Curvularia sp.,Alternaria sp., Torula sp., Myrotheciumsp., Helminthosporium sp., Periconia sp.,Nigrospora sp. and Pithomyces sp. This revised version was published online in June 2006 with corrections to the Cover Date.     

Role of β-amylase in starch metabolism during soybean seed development and germination

Differences in starch metabolism during seed development and germination of two soybean [ Glycine max (L.) Merrill] genotypes with normal seed β-amylase activity ['Williams' ( Sp 1^b and 'Altona' ( Sp 1^b)] and two soybean genotypes with undetectable seed β-amylase activity ['Chestnut' ( Sp 1^au) and 'Altona' ( Sp 1)] were investigated. Starch and soluble sugar profiles were essentially the same during seed development and germination. Total amylase activity of Williams and Altona ( Sp 1^b) peaked just prior to seed maturity and then dropped off slowly; whereas, the total amylase activity of Chestnut and Altona ( sp 1) was very low throughout seed development and germination. The differences in amylase activity between Altona ( Sp 1 ^b) and Altona ( sp 1) was also seen in leaves. α-Amylase activity was similar in the four genotypes when β-amylase was inhibited with Hg²⁺ but was higher in the two genotypes with normal β-amylase activity when β-amylase was inhibited with heat plus Ca²⁺. Low levels of starch phosphorylase activity were detected throughout seed development and germination, and the activity was similar in three of the genotypes and higher in Altona ( sp 1).
The protein, oil and oligosaccharide contents of mature seeds of the four genotypes were similar. Altona ( sp 1 ^b) and ( sp 1), which appear to be near isogenic lines, were not different in any morphological character or yield.
Altona ( Sp 1 ^b) showed greater hydrolysis of soybean seed starch than Altona ( sp 1), but the evidence indicates that the mutation resulting in greatly reduced or missing β-amylase activity has no effect on starch metabolism of developing and germinating soybean seeds.     

DNA homology of α-globin gene among five races of carp, Cyprinus carpio

Common DNA fragments of the α-globin gene were observed in five races of adult common carp: mirror, scaly-German, Yamato-Suwa, Yamato-Saku, and Yamato-hybrid carp. There were different hybridization patterns between the Japanese carp and the German carp. Three to four DNA fragments hybridized with α-globin and indicated polymorphysim in the globin gene of carp.

Zusammenfassung

DNA-Homologie des α-Globin-Genes bei fünf Karpfenrassen, Cyprinus carpio
Identische Fragmente des α-Globin-Genes wurden bei fünf Rassen des adulten gemeinen Karpfens (Spiegeikarpfen, deutscher Schuppenkarpfen, Yamato-Suwa-, Yamato-Saku-und Yamato-Hybrid-karpfen) gefunden. Es gab verschiedene Hybridisationsmuster zwischen dem japanischen und dem deutschen Karpfen. Drei bis vier DNA-Fragmente hybridisierten mit α-Globin und wiesen auf einen Polymorphismus des Globingenes des Karpfens hin.

Résumé

Homologie de DNA dans le gène d'α-globine chez cinq races de carpe, Cyprinus carpio
Des fragments communs de DNA dans des gènes d'α-globine ont été observés chez cinq races de carpe commune adulte: carpe miroir, allemande écailleuse, Yamato-Suwa, Yamato-Saku et Yamato-hybride. Il existait différentes combinaisons d'hybridation entre la carpe allemande et la carpe japonaise. Trois à quatre fragments de DNA hybridisaient avec l'α-globine et indiquaient un polymorphisme dans les gènes de globine de carpe.     

太湖梅梁湾大型控藻围栏对浮游甲壳动物群落结构的影响

2005年对太湖梅梁湾大型鲢、鳙控藻围栏内外浮游甲壳动物群落结构的季节变化进行了监测.结果表明:围栏内外的环境因子、浮游植物生物量、浮游甲壳动物种类组成无显著差异.但鲢、鳙放养对浮游甲壳动物的生物量产生了较大的影响,围栏内浮游甲壳动物的总生物量和枝角类的生物量显著低于围栏外.总体上,枝角类各种类的生物量受鲢、鳙放养的影响程度大于桡足类的种类.太湖梅梁湾浮游甲壳动物的季节演替明显,大部分种类只是季节性出现.冬季和春季以溞(Daphnia sp.)和近邻剑水蚤(Cyclops vicnus)等大型种类为主,夏季和秋季以象鼻溞(Bosmina sp.)、角突网纹溞(Ceriodaphnia cornuta)和中华窄腹剑水蚤(Limnoithona sinensis)等小型种类为主.典范对应分析表明,透明度、温度和浮游植物的生物量是影响浮游甲壳动物季节变化的主要因素.     

Changes in the population of seed bacteria of transgenerationally Cd‐exposed Arabidopsis thaliana

Plant‐associated bacteria can have beneficial effects on the growth and health of their host. Nevertheless, the role of endophytic bacteria present in seeds has not been investigated in depth. In this study, the cultivable endophytic population of seeds from Arabidopsis thaliana exposed to 2 μm cadmium for several generations (Cd seeds) was compared with a population isolated from seeds of plants that were never exposed to Cd (control seeds). We observed obvious differences between the two types of seed concerning genera present and phenotypic characteristics of the different isolates. Sinorhizobium sp. and Micrococcus sp. were only found in control seeds, while Pseudomonas sp., Bosea sp. and Paenibacillus sp. were only found in Cd seeds. Sphingomonas sp., Rhizobium sp., Acidovorax sp., Variovorax sp., Methylobacterium sp., Bacillus sp. and Staphylococcus sp. occurred in varying numbers in both types of seed. Metal tolerance and 1‐aminocyclopropane‐1‐carboxylate deaminase activity were predominantly found in strains isolated from Cd seeds, while the production of siderophores, indole‐3‐acetic acid and organic acids was more prevalent in endophytes isolated from control seeds. These data support the hypothesis that certain endophytes are selected for transfer to the next generation and that their presence might be important for subsequent germination and early seedling development.     

Vitamins A, C, and E and β-Carotene Content in Seeds of Seven Species of Vicia L.

Abstract: To determine the vitamins A, C, and E and β-carotene content of Vicia species that can be used in animal feed, a high performance liquid chromatography (HPLC) method was used to investigate the vitamin and β-carotene content in mature and immature seeds of seven Vicia species ( Vicia anatolica Turrill., V. ervilia (L.) Willd., V. michauxii Sprengel, V. mollis Boiss. et Hausskn. ex Boiss., V. noeana Reuter ex Boiss., V. peregrina L., and V. sericocarpa Fenzl.), which are useful plants in animal feed in the eastern Anatolia region in Turkey. The vitamin content was found to differ between mature and immature seeds. The levels of vitamins A, C, and E and β-carotene were higher in mature seeds than in immature seeds ( P < 0.01).
(Managing editor: Wei WANG)     

Nitrogen-fixing chemo-organotrophic bacteria isolated from cyanobacteria-deprived lichens and their ability to solubilize phosphate and to release amino acids and phytohormones

AIMS: Cyanobacteria-deprived lichens of the species Canoparmelia caroliniana, Canoparmelia crozalsiana, Canoparmelia texana, Parmotrema sancti-angeli and Parmotrema tinctorum were screened for the presence of chemo-organotrophic nitrogen-fixing bacteria. METHODS AND RESULTS: Fifty-three lichen samples subjected to enrichment selection using a nitrogen-free minimal medium were positive for acetylene reduction. Seventeen isolates, able to fix nitrogen, belonged to Gamma-proteobacteria group and were identified as: Acinetobacter sp., Pantoea sp., Pseudomonas sp., Pseudomonas stutzeri, Serratia marcescens and Stenotrophomonas maltophilia, according to 16S rRNA gene sequences and biochemical tests. The excretion of amino acid and phytohormone and the ability of mineral phosphate solubilization were determined in 14 isolates. All isolates were able to release amino acids and 3-indoleacetic acid. About 64% of the isolates solubilized phosphates and 30% released ethylene. CONCLUSIONS: These data confirm sparse evidence from the literature on the occurrence of chemo-organotrophic nitrogen-fixing bacteria in cyanobacteria-deprived lichens; the isolates presented physiologic features which might benefit the host if they are expressed when the bacteria are harboured by lichens. SIGNIFICANCE AND IMPACT OF THE STUDY: Chemo-organotrophic nitrogen-fixing bacteria were isolated from a high percentage (72.6%) of cyanobacteria-deprived lichens. All isolates presented important physiological characteristics, some of which are being described here for the first time.     

Heterodera raskii n. sp. (Heteroderidae: Tylenchina), a Cyst Nematode on Grass, from Hyderabad,India

Heterodera raskii n. sp. is described and illustrated from specimens collected from roots of bulb grass, Cyperus bulbosus, in Hyderabad, India. The new species belongs to the ''goettingiana'' group and differs from closely related H. cyperi by the elongate ovoid shaped cysts and females, greater fenestral length, width, vulval slit, and absence of egg sac. The stylet knob shape was round in second-stage juveniles and posteriorly sloping in females and males of H. raskii n. sp., while it was anteriorly directed in second-stage juveniles and spherical in females and males of H. cyperi.