Cospeciation Between the Primary Endosymbionts of Mealybugs and Their Hosts

Mealybugs have an association with prokaryotic endosymbionts that are located in specialized cells called bacteriocytes. In order to compare the phylogeny of the host with that of the previously published phylogeny of the endosymbionts, 3.1 to 3.2 kilobase DNA fragments containing mitochondrial cytB (part), nd1,16S ribosomal DNA(rDNA), and 12S rDNA (part) were amplified and sequenced. A phylogenetic analysis of the data and a comparison with the trees obtained from endosymbiont genes and host 18S and 28S rDNA indicated that all the trees were similar. This result is consistent with an infection of a mealybug ancestor with a precursor of the endosymbiont followed by the vertical transmission of the endosymbiont to progeny. Comparison of the guanine + cytosine (G + C) contents of the mealybug mitochondrial genes with the same genes from other members of Sternorrhyncha and Arthropoda indicated that the mealybug genes had unusually low G + C contents in their DNAs (10.2 to 11.1 mol%).     

Secondary (γ-Proteobacteria) Endosymbionts Infect the Primary (β-Proteobacteria) Endosymbionts of Mealybugs Multiple Times and Coevolve with Their Hosts

Mealybugs (Hemiptera, Coccoidea, Pseudococcidae) are plant sap-sucking insects that have within their body cavities specialized cells containing prokaryotic primary endosymbionts (P-endosymbionts). The P-endosymbionts have the unusual property of containing within their cytoplasm prokaryotic secondary endosymbionts (S-endosymbionts) [C. D. von Dohlen, S. Kohler, S. T. Alsop, and W. R. McManus, Nature (London) 412:433-436, 2001]. Four-kilobase fragments containing 16S-23S ribosomal DNA (rDNA) were obtained from the P-endosymbionts of 22 mealybug species and the S-endosymbionts of 12 representative species. Phylogenetic analyses of the P-endosymbionts indicated that they have a monophyletic origin and are members of the β-subdivision of the Proteobacteria; these organisms were subdivided into five different clusters. The S-endosymbionts were members of the γ-subdivision of the Proteobacteria and were grouped into clusters similar to those observed with the P-endosymbionts. The S-endosymbiont clusters were distinct from each other and from other insect-associated bacteria. The similarity of the clusters formed by the P- and S-endosymbionts suggests that the P-endosymbionts of mealybugs were infected multiple times with different precursors of the S-endosymbionts and once the association was established, the P- and S-endosymbionts were transmitted together. The lineage consisting of the P-endosymbionts of mealybugs was given the designation “Candidatus Tremblaya” gen. nov., with a single species, “Candidatus Tremblaya princeps” sp. nov. The results of phylogenetic analyses of mitochondrial DNA fragments encoding cytochrome oxidase subunits I and II from four representative mealybug species were in agreement with the results of 16S-23S rDNA analyses, suggesting that relationships among strains of “Candidatus T. princeps” are useful in inferring the phylogeny of their mealybug hosts.     

Genotypes of Cryptosporidium Species Infecting Fur-Bearing Mammals Differ from Those of Species Infecting Humans

Of 471 specimens examined from foxes, raccoons, muskrats, otters, and beavers living in wetlands adjacent to the Chesapeake Bay, 36 were positive for five types of Cryptosporidium, including the C. canis dog and fox genotypes, Cryptosporidium muskrat genotypes I and II, and Cryptosporidium skunk genotype. Thus, fur-bearing mammals in watersheds excreted host-adapted Cryptosporidium oocysts that are not known to be of significant public health importance.     

How Humans Differ from Other Animals in Their Levels of Morphological Variation

Animal species come in many shapes and sizes, as do the individuals and populations that make up each species. To us, humans might seem to show particularly high levels of morphological variation, but perhaps this perception is simply based on enhanced recognition of individual conspecifics relative to individual heterospecifics. We here more objectively ask how humans compare to other animals in terms of body size variation. We quantitatively compare levels of variation in body length (height) and mass within and among 99 human populations and 848 animal populations (210 species). We find that humans show low levels of within-population body height variation in comparison to body length variation in other animals. Humans do not, however, show distinctive levels of within-population body mass variation, nor of among-population body height or mass variation. These results are consistent with the idea that natural and sexual selection have reduced human height variation within populations, while maintaining it among populations. We therefore hypothesize that humans have evolved on a rugged adaptive landscape with strong selection for body height optima that differ among locations.     

昆虫对植物的选择性以及植物化学成分对昆虫的影响

植物与昆虫之间的关系一直被人们作为重要的研究目标。昆虫依靠绿色植物生存,植物通过自身的化学物质影响昆虫的进化方向,两者形成了复杂的协同进化关系。本文阐述了昆虫在定居、产卵、取食过程中运用不同的嗅觉、味觉、触觉刺激标准来选择适宜的寄主或寄主位置的方法,以及植物体内的化学成分对昆虫的营养作用和通过毒杀、拒食、招引天敌寄生蜂等方式抵御昆虫的进攻。     

昆虫对植物次生性物质的适应策略

植物次生性物质是植食性昆虫在取食过程中遇到的主要障碍之一,也是天敌昆虫寻找寄主或猎物的主要信息来源。当今,昆虫学中的一些重要理论问题,如寄主植物的识别,食性的形成,植物求救信号的释放,天敌对寄主或猎物的识别和寻找机制等等,均与植物次生性物质有关。在长期的演化过程中,昆虫适应了植物次生性物质的种种不利作用,改变了这类物质对植物本身的防御作用,使其能充分地利用各分类阶元的植物次生性物质作为寻找寄主植物、昆虫寄主或猎物以及取食的信号。昆虫与植物次生性物质的这种关系是当今协同演化理论得以产生的主要依据之一。关于昆…     

Eicosanoid Signaling in Insects: from Discovery to Plant Protection

Prostaglandins (PGs) and related eicosanoids are signal moieties derived from arachidonic acid and two other C20 polyunsaturated fatty acids. They were discovered in the 1930s in the context of mammalian reproductive physiology; PGs were associated with the prostate gland, hence their name, and they stimulate uterine smooth muscle contraction. Determining PG chemical structures in the early 1960s and demonstrating that they mediate many human pathophysiological events in the 1970s stimulated intensive research over the following decades in universities, governments and the private sector. Interest in the biological significance of PGs in insects arose in the 1970s and 1980s, which opened a new research frontier. PGs act in reproduction, releasing egg-laying behaviors in some species and signaling egg-maturation events in the Drosophila and silk moth models. They act in insect immunity, mediating and coordinating cellular and humoral responses to wounds, infection and invasion. PGs act in ion transport physiology in insect Malpighian tubules and recta. These compounds also mediate physiological trade-offs between insect immunity and reproduction. Finally, they are central players in the molecular ecology of interactions between blood-feeding insects and their vertebrate hosts. Some PG functions are critical at specific, crucial moments in insect lives, moments we consider ‘emergencies,’ such as the immediate reactions to infection. Certain microbial species have keyed into insect PG signaling and they evolved mechanisms to disable insect immune reactions to infection by inhibiting key enzymes in PG biosynthesis. We provide proof-of-principle that RNA interference treatments designed to silence genes in PG signaling disrupts insect immunity. In this review we describe the history, chemistry and biology of PGs. We use this background to argue that because PGs and other eicosanoids act in emergency situations, they are visible targets for development and deployment of novel insect pest management technologies.     

Amazonian Anthrosols Support Similar Microbial Communities that Differ Distinctly from Those Extant in Adjacent, Unmodified Soils of the Same Mineralogy

We compared the microbial community composition in soils from the Brazilian Amazon with two contrasting histories; anthrosols and their adjacent non-anthrosol soils of the same mineralogy. The anthrosols, also known as the Amazonian Dark Earths or terra preta, were managed by the indigenous pre-Colombian Indians between 500 and 8,700 years before present and are characterized by unusually high cation exchange capacity, phosphorus (P), and calcium (Ca) contents, and soil carbon pools that contain a high proportion of incompletely combusted biomass as biochar or black carbon (BC). We sampled paired anthrosol and unmodified soils from four locations in the Manaus, Brazil, region that differed in their current land use and soil type. Community DNA was extracted from sampled soils and characterized by use of denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism. DNA bands of interest from Bacteria and Archaea DGGE gels were cloned and sequenced. In cluster analyses of the DNA fingerprints, microbial communities from the anthrosols grouped together regardless of current land use or soil type and were distinct from those in their respective, paired adjacent soils. For the Archaea, the anthrosol communities diverged from the adjacent soils by over 90%. A greater overall richness was observed for Bacteria sequences as compared with those of the Archaea. Most of the sequences obtained were novel and matched those in databases at less than 98% similarity. Several sequences obtained only from the anthrosols grouped at 93% similarity with the Verrucomicrobia, a genus commonly found in rice paddies in the tropics. Sequences closely related to Proteobacteria and Cyanobacteria sp. were recovered only from adjacent soil samples. Sequences related to Pseudomonas, Acidobacteria, and Flexibacter sp. were recovered from both anthrosols and adjacent soils. The strong similarities among the microbial communities present in the anthrosols for both the Bacteria and Archaea suggests that the microbial community composition in these soils is controlled more strongly by their historical soil management than by soil type or current land use. The anthrosols had consistently higher concentrations of incompletely combusted organic black carbon material (BC), higher soil pH, and higher concentrations of P and Ca compared to their respective adjacent soils. Such characteristics may help to explain the longevity and distinctiveness of the anthrosols in the Amazonian landscape and guide us in recreating soils with sustained high fertility in otherwise nutrient-poor soils in modern times.     

Posterior Midgut Epithelial Cells Differ in Their Organization of the Membrane Skeleton from Other Drosophila Epithelia

In epithelial cells, the various components of the membrane skeleton are segregated within specialized subregions of the plasma membrane, thus contributing to the development and stabilization of cell surface polarity. It has previously been shown that, in various Drosophila epithelia, the membrane skeleton components ankyrin and alphabeta-spectrin reside at the lateral surface, whereas alphabeta(H)-spectrin is restricted to the apical domain. By use of confocal immunofluorescence microscopy, the present study characterizes the membrane skeleton of epithelial cells in the posterior midgut, leading to a number of unexpected results. First, ankyrin and alphabeta-spectrin are not detected on the entire lateral surface but appear to be restricted to the apicolateral area, codistributing with fasciclin III at smooth septate junctions. The presumptive ankyrin-binding proteins neuroglian and Na(+),K(+)-ATPase, however, do not colocalize with ankyrin. Second, alphabeta(H)-spectrin is enriched at the apical domain but is also present in lower amounts on the entire lateral surface, colocalizing apicolaterally with ankyrin/alphabeta-spectrin. Finally, despite the absence of zonulae adherentes, F-actin, beta(H)-spectrin, and nonmuscle myosin-II are enriched in the midlateral region. Thus, the model established for the organization of the membrane skeleton in Drosophila epithelia does not hold for the posterior midgut, and there is quite some variability between the different epithelia with respect to the organization of the membrane skeleton.     

Drivers of Variation in Aboveground Net Primary Productivity and Plant Community Composition Differ Across a Broad Precipitation Gradient

Aboveground net primary production (ANPP) is a key integrator of C uptake and energy flow in many terrestrial ecosystems. As such, ecologists have long sought to understand the factors driving variation in this important ecosystem process. Although total annual precipitation has been shown to be a strong predictor of ANPP in grasslands across broad spatial scales, it is often a poor predictor at local scales. Here we examine the amount of variation in ANPP that can be explained by total annual precipitation versus precipitation during specific periods of the year (precipitation periods) and nutrient availability at three sites representing the major grassland types (shortgrass steppe, mixed-grass prairie, and tallgrass prairie) spanning the broad precipitation gradient of the U.S. Central Great Plains. Using observational data, we found that precipitation periods and nutrient availability were much stronger predictors of site-level ANPP than total annual precipitation. However, the specific nutrients and precipitation periods that best predicted ANPP differed among the three sites. These effects were mirrored experimentally at the shortgrass and tallgrass sites, with precipitation and nutrient availability co-limiting ANPP, but not at the mixed-grass site, where nutrient availability determined ANPP exclusive of precipitation effects. Dominant grasses drove the ANPP response to increased nutrient availability at all three sites. However, the relative responses of rare grasses and forbs were greater than those of the dominant grasses to experimental nutrient additions, thus potentially driving species turnover with chronic nutrient additions. This improved understanding of the factors driving variation in ANPP within ecosystems spanning the broad precipitation gradient of the Great Plains will aid predictions of alterations in ANPP under future global change scenarios.     

Properties of Desulfovibrio carbinolicus sp. nov. and Other Sulfate-Reducing Bacteria Isolated from an Anaerobic-Purification Plant

Several sulfate-reducing microorganisms were isolated from an anaerobic-purification plant. Four strains were classified as Desulfovibrio desulfuricans, Desulfovibrio sapovorans, Desulfobulbus propionicus, and Desulfovibrio sp. The D. sapovorans strain contained poly-beta-hydroxybutyrate granules and seemed to form extracellular vesicles. A fifth isolate, Desulfovibrio sp. strain EDK82, was a gram-negative, non-spore-forming, nonmotile, curved organism. It was able to oxidize several substrates, including methanol. Sulfate, sulfite, thiosulfate, and sulfur were utilized as electron acceptors. Pyruvate, fumarate, malate, and glycerol could be fermented. Because strain EDK82 could not be ascribed to any of the existing species, a new species, Desulfovibrio carbinolicus, is proposed. The doubling times of the isolates were determined on several substrates. Molecular hydrogen, lactate, propionate, and ethanol yielded the shortest doubling times (3.0 to 6.3 h). Due to the presence of support material in an anaerobic filter system, these species were able to convert sulfate to sulfide very effectively at a hydraulic retention time as short as 0.5 h.     

Genetic Subdivision of Chemosynthetic Endosymbionts of Solemya velum along the Southern New England Coast

Population-level genetic diversity in the obligate symbiosis between the bivalve Solemya velum and its thioautotrophic bacterial endosymbiont was examined. Distinct populations along the New England coast shared a single mitochondrial genotype but were fixed for unique symbiont genotypes, indicating high levels of symbiont genetic structuring and potential symbiont-host decoupling.Studies of endosymbioses between marine invertebrates and sulfur-oxidizing chemosynthetic bacteria have yielded tremendous insight into the biology of bacterium-eukaryote interactions. Though best described for deep-sea vents and cold seeps, these mutualisms, in which symbiont thioautotrophy supports the nutrition of both partners, are also ubiquitous in coastal sediments (17). Our understanding of these interactions stems largely from studies of symbioses involving protobranch bivalves in the family Solemyidae (16). Though solemyids and other species that form chemosynthetic symbioses occur globally, little is known about how symbionts and hosts are structured genetically across distinct populations. Characterizing these patterns is critical for understanding how symbiosis drives the coevolution of interacting species, as well as how environmental heterogeneity and dispersal affect local adaptation. This study examines the geographic structure of genetic variation in the symbiosis between chemosynthetic bacteria and the Atlantic protobranch Solemya velum.Solemya velum is ideal for studying the evolution of highly coadapted bacterium-eukaryote mutualisms. This small bivalve (∼1.5 to 3 cm) burrows in sulfide-rich coastal sediments, where it obtains most of its nutrition from thioautotrophic bacteria living within specialized gill cells (1, 10). Though observed from Florida to Canada (20), the distribution of S. velum is highly patchy, with seemingly suitable habitat often devoid of individuals (12). Consequently, molecular characterizations of this symbiosis have focused primarily on stable and locally abundant populations near Woods Hole, MA. Direct sequencing of the symbiont 16S rRNA gene from these individuals has revealed a single, unique phylotype clustering within the Gammaproteobacteria (5, 6, 9). DNA from this symbiont has been extracted from S. velum ovarian tissue, raising the hypothesis that symbionts are transmitted vertically from mother to offspring (11) and are therefore tightly coupled to the host''s life cycle and evolutionary history.If symbiont acquisition is strictly vertical in Solemya populations, the genealogies of the symbiont and the cotransmitted host mitochondrion should diverge in parallel (cospeciation) (8, 15, 18). However, lateral acquisition involving either symbiont uptake from the environment or horizontal transfer between co-occurring hosts has not been ruled out for Solemya populations and could decouple symbiont and host genealogies (18). Indeed, 16S phylogenies show that symbionts of diverse Solemya species are polyphyletic, a pattern inconsistent with the putative monophyly of the hosts (based on nonmolecular characters) and suggestive of multiple evolutionary origins (2, 9, 16). However, tests for symbiont-host codiversification below the species level in S. velum are lacking; sequence data from multiple populations will help resolve questions of cospeciation and symbiont transmission in this group.Here, distinct Solemya velum populations were genotyped to examine how symbiont diversity covaries with host diversity and geography. Individual bivalves (n = 12 to 22 per site) were collected from mudflats at four sites along the southern New England coast (Fig. ​(Fig.1A).1A). DNA was extracted from the symbiont-containing gills and used for PCR amplification of fragments of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the symbiont 16S gene and hypervariable internal transcribed spacer (16S-ITS) (Table ​(Table1;1; also see the supplemental material). Unambiguous contigs of 340 nucleotides (nt) for the COI locus and 716 nt for the 16S-ITS locus, including 241 nt of the 16S and 475 nt (∼95%) of the ITS, were generated via bidirectional direct sequencing of amplicons using BigDye chemistry. Symbiont identity was confirmed by blasting the 16S-ITS (Woods Hole [WH] phylotype) against an assembly of the S. velum symbiont genome from the same population (C. Cavanaugh, unpublished data). Blastn returned a single full-length hit with 100% identity across the locus. Genotype networks were then inferred via statistical parsimony in the program TCS (3).Open in a separate windowFIG. 1.(A) Locations of Solemya velum collection sites (stars) along the Atlantic Coast were Naushon Island, Woods Hole, MA (WH; 41.514°N, −70.712°W); Lake Tashmoo, Martha''s Vineyard, MA (MV; 41.465°N, −70.623°W); Judith Pond, RI (RI; 41.380°N, −71.502°W); and Shark River Island, NJ (NJ; 40.186°N, −74.030°W). (B) Parsimony networks of host COI and symbiont 16S-ITS genotypes. Open circle, single-nucleotide substitution in either the host COI (top; 340 nt) or symbiont 16S (241 nt); filled circle, single-nucleotide substitution in the ITS portion (475 nt) of the 16S-ITS sequence fragment (716 nt total); diagonal bar, single-nucleotide indel in the symbiont ITS; gen1 and gen2, genotypes 1 and 2. Values in parentheses show the number of S. velum individuals from which sequences were obtained at each site.

TABLE 1.

Symbiont and host primers used in PCR^a and direct sequencing

HIV-1 Vaccine-Induced T-Cell Reponses Cluster in Epitope Hotspots that Differ from Those Induced in Natural Infection with HIV-1

Several recent large clinical trials evaluated HIV vaccine candidates that were based on recombinant adenovirus serotype 5 (rAd-5) vectors expressing HIV-derived antigens. These vaccines primarily elicited T-cell responses, which are known to be critical for controlling HIV infection. In the current study, we present a meta-analysis of epitope mapping data from 177 participants in three clinical trials that tested two different HIV vaccines: MRKAd-5 HIV and VRC-HIVAD014-00VP. We characterized the population-level epitope responses in these trials by generating population-based epitope maps, and also designed such maps using a large cohort of 372 naturally infected individuals. We used these maps to address several questions: (1) Are vaccine-induced responses randomly distributed across vaccine inserts, or do they cluster into immunodominant epitope hotspots? (2) Are the immunodominance patterns observed for these two vaccines in three vaccine trials different from one another? (3) Do vaccine-induced hotspots overlap with epitope hotspots induced by chronic natural infection with HIV-1? (4) Do immunodominant hotspots target evolutionarily conserved regions of the HIV genome? (5) Can epitope prediction methods be used to identify these hotspots? We found that vaccine responses clustered into epitope hotspots in all three vaccine trials and some of these hotspots were not observed in chronic natural infection. We also found significant differences between the immunodominance patterns generated in each trial, even comparing two trials that tested the same vaccine in different populations. Some of the vaccine-induced immunodominant hotspots were located in highly variable regions of the HIV genome, and this was more evident for the MRKAd-5 HIV vaccine. Finally, we found that epitope prediction methods can partially predict the location of vaccine-induced epitope hotspots. Our findings have implications for vaccine design and suggest a framework by which different vaccine candidates can be compared in early phases of evaluation.     

Host-Parasite Interactions from the Inside: Plant Reproductive Ontogeny Drives Specialization in Parasitic Insects

Host plant interactions are likely key drivers of evolutionary processes involved in the diversification of phytophagous insects. Granivory has received substantial attention for its crucial role in shaping the interaction between plants and their seed parasites, but fine-scale mechanisms explaining the role of host plant reproductive biology on specialization of seed parasites remain poorly described. In a comparative approach using plant histological techniques, we tested the hypotheses that different seed parasite species synchronize their life cycles to specific stages in seed development, and that the stage they target depends on major differences in seed development programs. In a pinaceous system, seed storage products are initiated before ovule fertilization and the wasps target the ovule’s nucellus during megagametogenesis, a stage at which larvae may benefit from the by-products derived from both secreting cells and dying nucellar cells. In a cupressaceous system, oviposition activity peaks later, during embryogenesis, and the wasps target the ovule’s megagametophyte where larvae may benefit from cell disintegration during embryogenesis. Our cytohistological approach shows for the first time how, despite divergent oviposition targets, different parasite species share a common strategy that consists of first competing for nutrients with developing plant structures, and then consuming these developed structures to complete their development. Our results support the prediction that seed developmental program is an axis for specialization in seed parasites, and that it could be an important parameter in models of their ecological and taxonomic divergence. This study provides the basis for further investigating the possibility of the link between plant ontogeny and pre-dispersal seed parasitism.     

植物蛋白酶抑制剂在植物抗虫与抗病中的作用

综述了植物蛋白酶抑制剂抗虫与抗病作用的研究进展.蛋白酶抑制剂广泛存在于植物体内,与植物抗虫抗病密切相关.植物蛋白酶抑制剂能抑制昆虫肠道蛋白酶,使昆虫生长发育缓慢,甚至死亡.但取食蛋白酶抑制剂后,昆虫能迅速分泌对抑制剂不敏感的蛋白酶,而使蛋白酶抑制剂无效.食物蛋白的含量和质量也影响植物蛋白酶抑制剂的抗虫效果.病原菌的感染能诱导植物产生蛋白酶抑制剂,诱导产生的蛋白酶抑制剂能抑制病原菌的生长.     

Comparison of the Rates of Motility of Salmonella with Those of Other Enteric Bacteria

Comparison of the rates of motility through a semisolid medium of 16 common Salmonella sp., 14 Escherichia coli serotypes, 4 Arizona strains, 2 Escherichia freundii (Citrobacter) isolates, 2 Proteus sp., and 2 Pseudomonas sp. revealed the following. (i) Very closely related bacteria could demonstrate markedly different rates of progression. (ii) All of the salmonellae tested advanced faster than the Proteus and Pseudomonas test cultures but some Salmonella sp., notably S. choleraesuis and S. typhi, progressed relatively slowly compared to many other test cultures. (iii) The mean rate of motility for the fastest 14 Salmonella sp. (1.49 cm/hr) was not statistically greater than the mean value for the 14 E. coli serotypes (1.31 cm/hr) at the 1% level of significance. Selective motility procedures may not be a reliable means of isolating all Salmonella sp. from materials contaminated with other bacteria.     

水稻RubisCO的纯化及其与烟草RubisCO性质的比较

利用蔗糖密度梯度离心和DEAE-Sepharose fast flow柱层析等步骤从水稻叶片中纯化了RubisCO。此法不仅快速,而且酶的收得率高,酶的比活达1.15 μmol CO_2 min~(-1) mg~(-1)。 水稻RubisCO的热稳定性比烟草酶差,在活化时对Mg~(2 )较敏感。水稻和烟草RubisCO钝化态时总巯基数和表面巯基数相同,然而当酶活化后,水稻酶表面巯基数增加,而烟草酶则减少。当这些表面巯基被修饰后,水稻酶活性损失60％而烟草酶活力仅损失15％。水稻和烟草RubisCO的远紫外CD光谱有明显的区别,这显示了两者在二级结构、酶比活和性质上的重大差别。     

高羊茅和其他羊茅植株再生与遗传转化研究进展

高羊茅、紫羊茅和草地羊茅均为很重要的多年生冷季型牧草与草坪草,生物技术在其品种改良中具有很大的应用潜力。30年来,三种羊茅的组织培养、胚性培养物的长期保存以及遗传转化等研究取得了较大进展,已建立起多种植株再生体系和遗传转化技术,但作为单子叶植物,这些草种的组织培养和转基因遗传改良也还存在一些问题。本文就以上几方面的内容进行了综述。