Wolbachia在玉米螟赤眼蜂内的三重感染

Wolbachia是一类广泛存在于节肢动物体内的共生菌。玉米螟赤眼蜂Trichogramma ostriniae是我国玉米田间的优势赤眼蜂种, 据报道, 赤眼蜂种内有Wolbachia感染。本文利用Wolbachia的16s rDNA和wsp基因引物通过PCR方法对玉米螟赤眼蜂的野生种群进行了调查, 发现以wsp基因为鉴定依据, 检测的所有个体都感染了3种Wolbachia ［wOstGDAa (GenBank accession no. EU157103), wOstGDAb (GenBank accession no. EU157104) 和 wOstGDB (GenBank accession no. EU157105)］。本文首次报道了野生赤眼蜂种群内Wolbachia的三重感染率几乎为100%。根据本研究的结果, 可以推测当不同种赤眼蜂寄生同一寄主时, Wolbachia可能会在不同赤眼蜂种间进行横向传播。     

Quantification of Wolbachia copy number in Trichogramma eggs (Hymenoptera: Trichogrammatidae): Lysozyme treatment significantly improves total gene yield from the Gram-negative bacterium

The Wolbachia bacterium is one of the most prevalent endosymbionts of arthropods. The bacterium is known to induce four distinct reproductive manipulations in its various hosts; namely, cytoplasmic incompatibility, feminization, male killing and parthenogenesis induction. The bacterial titer has been thought to have an important role in expression of these phenotypes. Although host eggs of the bacterium are the place where all of the manipulations take place, in most cases, they are not big enough to be subjected to mechanical disruption to extract genetic materials from them. Furthermore, DNA extraction protocols generally involve washing steps in which part of the DNA is lost. With the simple three step digestion protocol we developed, we were able to preserve the whole genome and measure the titer of Wolbachia in individual Trichogramma eggs by real-time quantitative polymerase chain reaction (PCR), which was comparable to the titer by visual observation.     

孤雌产雌生殖品系松毛虫赤眼蜂产卵强度对Wolbachia诱导的其生殖表型的影响

[目的]松毛虫赤眼蜂Trichogramma dendrolimi是一种用于鳞翅目害虫生物防治的重要卵期寄生蜂.本研究旨在明确孤雌产雌生殖品系松毛虫赤眼蜂的产卵强度对其体内Wolbachia滴度及Wolbachia诱导的孤雌产雌生殖表型的影响.[方法]在室内调查了3个处理组松毛虫赤眼蜂雌蜂不同产卵强度(每日仅1 h供寄...     

温度通过影响Wolbachia滴度调控赤眼蜂生殖方式

【目的】揭示温度对宿主体内Wolbachia滴度及其调控宿主生殖作用的影响。【方法】以感染Wolbachia且营孤雌产雌生殖的食胚赤眼蜂Trichogramma embryophagum为对象,在22,25,28和31℃4个梯度温度下连续培养5代,观察生殖方式、性比等生物学特性;采用实时荧光定量PCR以Wolbachia特异基因——外膜蛋白基因(wsp)、二磷酸果糖醛缩酶基因(fbp A)和酰胺转移酶基因(gat B)为靶标对内生菌进行定量分析。【结果】22和25℃条件下连续培养5代,食胚赤眼蜂生殖方式均未发生改变(无雄蜂出现),且5代赤眼蜂群体Wolbachia滴度没有明显差异;28℃下,食胚赤眼蜂在F3代开始有雄蜂出现,至F5代雄蜂比例明显增多,且体内Wolbachia滴度在F2代开始下降,至F5代显著下降;31℃下,F2代即有雄蜂出现,至F5代已经恢复成产雄孤雌生殖,体内Wolbachia滴度也在F2代开始下降,F3代开始显著降低,到F5代Wolbachia滴度极小甚至检测不到。【结论】高温可改变营孤雌产雌生殖赤眼蜂的生殖方式及体内Wolbachia滴度,且随着处理代数的增加以及温度的升高作用显著,即高温对营孤雌产雌生殖赤眼蜂生殖方式改变程度与其体内Wolbachia滴度呈负相关。     

Wolbachia感染对拟澳洲赤眼蜂寿命、生殖力和嗅觉反应的影响

研究了通过共享同一寄主卵,短管赤眼蜂Trichogramma pretiosum体内共生的Wolbachia被水平传递到拟澳洲赤眼蜂T. confusum体内后,Wolbachia对新宿主拟澳洲赤眼蜂的影响。结果表明： Wolbachia的侵染能使拟澳洲赤眼蜂进行不完全的产雌孤雌生殖,增加拟澳洲赤眼蜂子代雌性比例,但却导致了雌蜂寿命缩短和繁殖力降低的生理损失。Wolbachia感染的当代处女蜂及其建立的种群连续5代(F₁～F₅),其子代雌蜂百分率分别为79.17%、76.60%、68.66%、72.58%、68.15%和64.06%,基本上呈现出逐代降低的趋势,并越来越接近对照的63.85%。处女蜂及F₁～F₅代雌蜂的平均寿命分别为4.33、5.50、5.60、6.68、7.32和7.50天,而未感染交配雌蜂寿命为7.59 天;处女蜂及F₁～F₅代雌蜂平均产卵量分别为11.33、70.00、86.41、93.90、102.92和124.38粒/雌,除F₅代外,均显著低于未感染交配雌蜂的产卵量134.81粒/雌。用四臂嗅觉仪测定了Wolbachia新宿主拟澳洲赤眼蜂对寄主小菜蛾的嗅觉反应,结果表明Wolbachia的侵染具轻微干扰拟澳洲赤眼蜂嗅觉反应的负面影响。未感染Wolbachia的拟澳洲赤眼蜂及Wolbachia供体短管赤眼蜂对寄主小菜蛾具较强的嗅觉反应,其雌蜂在小菜蛾腹部鳞片正己烷提取液和小菜蛾卵表正己烷提取液处理区内滞留时间显著或极显著长于对照区。而感染Wolbachia的拟澳洲赤眼蜂F₂代和F₃代雌蜂,尽管在小菜蛾腹部鳞片正己烷提取液处理区内滞留时间比对照区长,但没有达到显著水平;其F₂代雌蜂在小菜蛾卵表正己烷提取液处理区内滞留时间与对照区相比,也没有达到显著水平。随Wolbachia在拟澳洲赤眼蜂种群中垂直感染代数的增加,拟澳洲赤眼蜂对寄主小菜蛾的嗅觉反应恢复正常,在Wolbachia感染后的 F₄～F₆代,雌蜂在两种提取液处理区内的滞留时间均显著或极显著长于对照区。     

安荔赤眼蜂在中国野外首次发现及其体内Wolbachia的检测

【目的】为丰富赤眼蜂Trichogramma的种类资源,明确野外新采集的一种赤眼蜂的种类,探明该赤眼蜂所感染Wolbachia的类型。【方法】采用挂米蛾Corcyra cephalonica卵卡法在华南农业大学树木园诱集到两批赤眼蜂,通过形态鉴定和PCR扩增ITS2序列并测序分析的分子鉴定手段对野外采集的赤眼蜂材料进行种类鉴定;通过PCR扩增Wolbachia的外膜蛋白基因(wsp)序列,检测赤眼蜂体内Wolbachia的感染情况;通过PCR扩增wsp序列和多位点序列分型(multilocus sequence typing, MLST)对检测到的赤眼蜂体内的Wolbachia进行同源性分析。【结果】所诱集到的两批赤眼蜂均被鉴定为安荔赤眼蜂Trichogramma oleae Voegelé & Pointel,体内Wolbachia的感染率达100%。该Wolbachia株系与安荔赤眼蜂(前南斯拉夫品系)、短管赤眼蜂T. pretiosum(乌拉圭品系)以及T. deion(荷兰品系)体内Wolbachia亲缘关系较近,属于B超组Sib亚组,对应MLST序列型为ST486。【结论】安荔赤眼蜂T. oleae为中国野外首次发现,是完全感染Wolbachia的产雌孤雌生殖品系。本研究为害虫生物防治提供了一种新的天敌种类资源,并为进一步探明Wolbachia与赤眼蜂的互作提供了研究材料。     

Effect of cold storage of adult Trichogramma brassicae,T. cacoeciae and T. evanescens (Hym.: Trichogrammatidae)

Abstract

Trichogramma cacoeciae Marchall, T. brassicae Bezdenko and T. evanescens Westwood could be useful in biological control programmes of agricultural insect pest. The possibility of storing adult Trighogramma species at low temperatures, and the effect of such storage on the quality of the parasitoids and its fecundity were studied. Trighogramma cacoeciae, T. brassicae and T. evanescens adults were stored for 1, 2, 3, 4 and 5 days at 4 ± 1°C in a refrigerator, 60 – 70%RH and full darkness. Storage at 4 ± 1°C reduced fecundity and longevity of female parasitoids.     

四环素对孤雌产雌松毛虫赤眼蜂生殖表型及适合度的影响

共生菌Wolbachia能够诱导许多节肢动物发生产雌孤雌生殖现象。由于该菌无法在宿主细胞外离体培养,人们常采用抗生素处理获得Wolbachia去除或滴度降低的宿主种群,用以研究Wolbachia对宿主的调控作用,但抗生素可能会对宿主产生附加效应。前人研究主要集中在抗生素处理是否可以恢复感染寄生蜂的两性生殖,较少关注抗生素对宿主适合度的影响。本研究利用不同浓度四环素(0、5 mg/mL、15 mg/mL和25 mg/mL)连续饲喂5代来明确四环素对产雌孤雌生殖的松毛虫赤眼蜂Trichogramma dendrolimi适合度的影响。研究结果如下:(1)随各浓度四环素处理世代的增加,雌蜂的繁殖力先降低后升高。在F_1代、F_2代和F_5代中,雌蜂繁殖力随四环素浓度的增加而显著下降,但在F_3代和F_4代中,抗生素浓度对雌蜂繁殖力无显著差异;(2)在四环素不同浓度处理下,F_1代和F_3代的羽化率与对照无显著差异,在F_2代、F_4代和F_5代中,四环素对羽化率存在负面影响;(3)经四环素处理的松毛虫赤眼蜂子代雄性比显著高于对照组。在5 mg/mL的四环素处理条件下,松毛虫赤眼蜂子代雄性比随处理世代的增加而升高,但在25 mg/mL的四环素处理条件下随世代的增加先升高后下降。结果表明:四环素能够使孤雌产雌松毛虫赤眼蜂恢复两性生殖,但对松毛虫赤眼蜂的适合度存在不利影响。此外,随着四环素处理世代的增加,松毛虫赤眼蜂对抗生素产生了一定的适应能力。     

Biological studies on two neotropical egg parasitoid species: Trichogramma nerudai and Trichogramma sp. (Hymenoptera: Trichogrammatidae)

The main biological attributes of two Neotropical egg parasitoids, the arrhenotokous Trichogramma nerudai and the thelytokous Trichogramma sp., were assessed under controlled laboratory conditions. Developmental time from egg to adult, and parasitoid survival, fecundity and fertility were studied using life tables. Results showed that T. nerudai had a faster developmental time than Trichogramma sp. (13.014±0.4019 and 13.595±0.4931 days, respectively). Both species showed similar life table statistics, r_m was 0.222 and 0.225 for T. nerudai and Trichogramma sp., respectively. Parasitoid survival averaged 95% for both species. The results obtained are discussed in the context of selecting one of these natural enemies as a potential biological control agent for the European pine shoot moth Rhyacionia buoliana (Lepidoptera: Tortricidae) in pine forests and the codling moth Cydia pomonella (Lepidoptera: Tortricidae) in apple orchards in Argentina.     

米蛾体内Wolbachia的wsp基因序列测定与系统发育分析

Wolbachia是广泛分布于节肢动物体内的一类共生菌, 它们参与多种调控寄主的生殖活动机制。通过对wsp基因的特异性扩增和测序,发现了Wolbachia在米蛾Corcyra cephalonica (Stainton)体内的感染。利用所测序列和其他已发表的序列建立系统树,结果表明米蛾体内Wolbachia属于B大组的Pip类群,与其寄生物茧蜂及赤眼蜂中的Wolbachia各株系遗传距离相差较远。据此推测米蛾体内感染的Wolbachia不是由寄生物（茧蜂、赤眼蜂）水平传播所致。     

Comparison Between the Control Efficacy of Trichogramma evanescens Westwood (Hymenoptera: Trichogrammatidae) and Two Trichogramma cacoeciae Marchal Strains Against Grapevine Moth ( Lobesia botrana Den. & Schiff.), Depending on Their Release Density

In the first generation of the grape moth Lobesia botrana, Trichogramma evanescens spread to a greater extent than Trichogramma cacoeciae and parasitized most of the grape moth eggs. Reduction of damage to grapes was improved by doubling the number of T. evanescens release points in the first and second generations. In the second generation of the grape moth, T. cacoeciae was more efficient than T. evanescens . The highest proportion of individuals belonging to the reared and natural T. cacoeciae strains were found in the plot in which they were initially released. The total number of individuals emerging from parasitized eggs eggs was the same for each strain, which would suggest that neither predominated. Whichever T. cacoeciae strain considered, a density of 800 release points ha -1 did not induce greater parasitization than a density of 400 release points ha -1 .     

不同温度下松毛虫赤眼蜂孤雌产雌品系和两性生殖品系对米蛾卵的寄生功能反应

赤眼蜂部分蜂种或品系受Wolbachia侵染营孤雌产雌生殖。 通过室内试验分析了在4个恒温（20, 25, 30和35℃）下松毛虫赤眼蜂Trichogramma dendrolimi两性生殖品系和孤雌产雌品系对米蛾Corcyra cephalonica卵的寄生功能反应, 旨在比较不同温度两品系的寄生功能反应差异, 评价孤雌产雌品系在生物防治中的应用潜力。 结果表明： 松毛虫赤眼蜂两个品系对米蛾卵寄生作用均随寄主密度的增加而增大;随温度的升高松毛虫赤眼蜂两品系的功能反应类型由Ⅲ型改变为Ⅱ型。孤雌产雌品系以30℃的处置时间最短(0.0207 d), 最大日寄生量为48.31粒卵, 其次是25℃, 35℃最小;两性生殖品系以25℃的处置时间最短(0.0188 d), 最大日寄生量为53.08粒卵, 其次是30℃, 20℃最小; 松毛虫赤眼蜂两品系的寄生功能反应存在显著差异, 30℃下孤雌产雌品系为Ⅱ型功能反应而两性生殖品系为Ⅲ型。 从处置时间来看, 20℃时两品系无显著性差异(P≥0.05), 在25℃和35℃孤雌产雌品系寄生米蛾卵时花费的时间显著长于两性生殖品系(P<0.05), 而30℃却相反。 可见, 寄主密度、 温度和Wolbachia影响松毛虫赤眼蜂功能反应。     

Effectiveness of Trichogramma ostriniae and T. nubilale for controlling the European corn borer Ostrinia nubilalis in sweet corn

Seven field releases of Trichogramma ostriniae and T. nubilale (Hymenoptera: Trichogrammatidae) were made separately and in combination in a sweet corn field to compare the level of parasitism in sentinel eggs of the European corn borer, Ostrinia nubilalis (Lepidoptera: Pyralidae). The results indicate that the levels of egg parasitism among different release dates differed mainly because of changes in weather and plant architecture within the season. The level of egg parasitism by releasing T. ostriniae alone was found to be 15% higher than that by releasing T. nubilale alone, and 20% higher than by releasing the combination of the two species. Further analyses using the logistic regression model for independent and correlated data indicated T. ostriniae to be more efficient at discovering host egg masses and to have higher levels of egg parasitism than T. nubilale. Mutual interference between T. ostriniae and T. nubilale was the main factor for the lower level of egg parasitism when T. ostriniae and T. nubilale were released together. The results suggest that T. ostriniae is the better candidate for augmentative releases for control of the European corn borer, and the two species should not be released into a corn field at the same time.     

Effect of host egg viability on reproduction and development of Trichogramma cacoeciae and T. principium (Hymenoptera: Trichogrammatidae)

A combination of a sterile insect technique, resulting in infertile eggs, and of an egg parasitoid, should provide better control of codling moth than either alone. Non-choice laboratory experiments were conducted with infertile and fertile codling moth eggs to evaluate the potential parasitism and reproduction of Trichogramma cacoeciae Marchal and T. principium Sug. et Sor. The tendency of T. cacoeciae females to attack infertile eggs was similar to that for fertile eggs, whereas T. principium showed a greater preference for infertile eggs than fertile eggs. The fertility status of the host did not affect the number of eggs that were parasitized but fewer F₁ progeny emerged from infertile eggs when parasitized than from fertile eggs. When T. cacoeciae and T. principium parasitized infertile host eggs their mean developmental time was prolonged but the viability and quality of parasitoid progeny was not influenced by the fertility status of the host eggs. The study demonstrates the compatibility of use of T. cacoeciae and T. principium in an integrated program employing the sterile insect technique for codling moth management.     

两种赤眼蜂对小菜蛾卵的寄生能力和种间竞争

在实验室内研究了本地天敌拟澳洲赤眼蜂和外来天敌短管赤眼蜂在不同卵龄和不同空间条件下对小菜蛾卵的寄生和种间竞争.结果表明,在0～62 h的小菜蛾卵上,拟澳洲赤眼蜂和短管赤眼蜂单独接蜂时对小菜蛾卵的寄生率、子代羽化率、子代雌蜂百分率随卵龄下降,而后代发育死亡率则随卵龄增加.短管赤眼蜂对各龄小菜蛾卵的寄生率、子代羽化率、子代雌蜂百分率均较拟澳洲赤眼蜂高,而子代蜂的发育死亡率则较拟澳洲赤眼蜂低.两种蜂对小菜蛾卵龄的要求都不太严格,在小于48 h的小菜蛾卵上都能产卵寄生,且寄生率高于50%,但短管赤眼蜂寄生对小菜蛾卵龄的要求更宽松.在两种蜂混合接蜂时,后代中短管赤眼蜂的比例在各处理中都高于50%,且随寄主卵龄增加,当小菜蛾卵龄大于48 h后,短管赤眼蜂的比例高达100%,说明短管赤眼蜂对小菜蛾有更强的寄生能力和竞争能力.拟澳洲赤眼蜂和短管赤眼蜂单独接蜂时,在4～22cm^3空间范围内,各处理间对小菜蛾卵的寄生率没有显著差异,而短管赤眼蜂的寄生率(76.4%～86%)略低于拟澳洲赤眼蜂(88.7%～92.3%).当空间大于53 cm^3时,寄生率显著下降,在102cm^3空间时显著降低到50%.混合接蜂时各处理间寄生率差异不显著.两种接蜂方式对后代羽化率和雌蜂百分率没有太大影响,蜂的后代发育死亡率在两种蜂单独接蜂时随空间而增加.在4～102cm^3空间范围内,混合接蜂后代雄蜂中短管赤眼蜂从80%以上降低到20%以下,说明短管赤眼蜂的竞争能力随接蜂空间的加大而降低.     

wFlu: Characterization and Evaluation of a Native Wolbachia from the Mosquito Aedes fluviatilis as a Potential Vector Control Agent

There is currently considerable interest and practical progress in using the endosymbiotic bacteria Wolbachia as a vector control agent for human vector-borne diseases. Such vector control strategies may require the introduction of multiple, different Wolbachia strains into target vector populations, necessitating the identification and characterization of appropriate endosymbiont variants. Here, we report preliminary characterization of wFlu, a native Wolbachia from the neotropical mosquito Aedes fluviatilis, and evaluate its potential as a vector control agent by confirming its ability to cause cytoplasmic incompatibility, and measuring its effect on three parameters determining host fitness (survival, fecundity and fertility), as well as vector competence (susceptibility) for pathogen infection. Using an aposymbiotic strain of Ae. fluviatilis cured of its native Wolbachia by antibiotic treatment, we show that in its natural host wFlu causes incomplete, but high levels of, unidirectional cytoplasmic incompatibility, has high rates of maternal transmission, and no detectable fitness costs, indicating a high capacity to rapidly spread through host populations. However, wFlu does not inhibit, and even enhances, oocyst infection with the avian malaria parasite Plasmodium gallinaceum. The stage- and sex-specific density of wFlu was relatively low, and with limited tissue distribution, consistent with the lack of virulence and pathogen interference/symbiont-mediated protection observed. Unexpectedly, the density of wFlu was also shown to be specifically-reduced in the ovaries after bloodfeeding Ae. fluviatilis. Overall, our observations indicate that the Wolbachia strain wFlu has the potential to be used as a vector control agent, and suggests that appreciable mutualistic coevolution has occurred between this endosymbiont and its natural host. Future work will be needed to determine whether wFlu has virulent host effects and/or exhibits pathogen interference when artificially-transfected to the novel mosquito hosts that are the vectors of human pathogens.     

Assessing the Potential of Entomopathogenic Nematodes to Control the Grape Root Borer Vitacea polistiformis (Lepidoptera: Sesiidae) Through Laboratory and Greenhouse Bioassays

Seventeen entomopathogenic nematode species and strains were evaluated for virulence to the grape root borer, Vitacea polistiformis (Harris) in laboratory and greenhouse bioassays. Heterohabditis bacteriophora strain GPS11 and H. zealandica strain X1 produced a larval mortality rate of over 85% of larvae embedded in the root cambium in laboratory bioassays. The nematode species H. marelata and H. bacteriophora strain Oswego produced mortality rates of over 75%. Of the Steinernema species tested, S. carpocapsae strain 'All' performed the best with a mortality rate of 69%. All other nematode species and strains tested, with the exception of S. bicornutum , produced some degree of larval mortality. In the greenhouse bioassays, 93% control was achieved with H. zealandica strain X1 applied at 4 ×109 infective juveniles (IJs) acre1 -1 (9.88 ×10 9 IJs ha -1 ). H. bacteriophora strain GPS11 successfully reproduced in grape root borer larvae. The numbers of IJs produced within infected larvae were related to larval size. The survival rate of neonate larvae on grape root sections was 61%, which thus provides a means to rear the neonate larvae for bioassays.     

Review of Pasteuria penetrans: Biology,Ecology, and Biological Control Potential

Pasteuria penetrans is a mycelial, endospore-forming, bacterial parasite that has shown great potential as a biological control agent of root-knot nematodes. Considerable progress has been made during the last 10 years in understanding its biology and importance as an agent capable of effectively suppressing root-knot nematodes in field soil. The objective of this review is to summarize the current knowledge of the biology, ecology, and biological control potential of P. penetrans and other Pasteuria members. Pasteuria spp. are distributed worldwide and have been reported from 323 nematode species belonging to 116 genera of free-living, predatory, plant-parasitic, and entomopathogenic nematodes. Artificial cultivation of P. penetrans has met with limited success; large-scale production of endospores depends on in vivo cultivation. Temperature affects endospore attachment, germination, pathogenesis, and completion of the life cycle in the nematode pseudocoelom. The biological control potential of Pasteuria spp. have been demonstrated on 20 crops; host nematodes include Belonolaimus longicaudatus, Heterodera spp., Meloidogyne spp., and Xiphinema diversicaudatum. Pasteuria penetrans plays an important role in some suppressive soils. The efficacy of the bacterium as a biological control agent has been examined. Approximately 100,000 endospores/g of soil provided immediate control of the peanut root-knot nematode, whereas 1,000 and 5,000 endospores/g of soil each amplified in the host nematode and became suppressive after 3 years.     

Pattern of Egg Management by Trichogramma cacoeciae and T. dendrolimi (Hymenoptera: Trichogrammatidae)

The effect of temporary host deprivation on parasitization rates of Trichogramma cacoeciae Marchal and T. dendrolimi Matsumura was investigated. The study was conducted with females that we allowed to engage in 3 days of oviposition after various periods of host deprivation. It seems that the production and management of eggs by the two species is completely different. During the first day of oviposition, parasitization by T. cacoeciae was almost unaffected after 1 to 5 days of host deprivation. As deprivation time increased, however, the number of parasitized hosts decreased from an average of 28.6 ±2.0 hosts provided at emergence to an average of 12.5 ±2.3 hosts when the waiting time was 10 days. The number of hosts parasitized on the first day of parasitization by T. dendrolimi were not affected whatever the waiting test period. During the second or third days of oviposition, the lack of suitable hosts for T. cacoeciae did not depress egg-laying potentiality, whereas a strong reduction in parasitization rates by T. dendrolimi occurred in the next 2 days of oviposition whatever was the waiting period. This leads to ca. 50% reduction in total activity of 3 days of oviposition. Only in T. cacoeciae was it possible to distinguish between ageing and host deprivation. The data suggest that T. dendrolimi is a typical proovigenic species, while T. cacoeciae is neither definitely proovigenic nor synovigenic. A slight decrease in rate of emergence of offspring of T. cacoeciae females that had waited 8 to 10 days for their hosts was observed. As far as biological control is concerned, the efficiency of T. dendrolimi females may be more sensitive to host deprivation than T. cacoeciae.