Comparison of the diversity of the bacterial communities in the intestinal tract of adult Bactrocera dorsalis from three different populations

Aims: To (i) identify the bacterial communities in the gut of oriental fruit fly (Bactrocera dorsalis) adult and (ii) determine whether the different surroundings and diets influence the bacteria composition. Methods and Results: Polymerase chain reaction‐denaturing gradient gel electrophoresis (DGGE) fingerprinting was used to investigate bacterial diversity in the oriental fruit fly adult gut. The 16S rDNA cloned libraries from the intestinal tract of laboratory‐reared (LR), laboratory sterile sugar‐reared (LSSR) and field‐collected (FC) populations of oriental fruit fly were compared. Phylogenetic analysis of 16S rDNA revealed that Gammaproteobacteria were dominant in the all samples (73·0–98·3%). Actinobacteria and Firmicutes were judged to be major components of a given library as they constituted 10% or more of the total clones of such library. The Flavobacteria, Deltaproteobacteria, Bacteroidetes and Alphaproteobacteria were observed in small proportions in various libraries. Further phylogenetic analyses indicated common bacterial phylotypes for all three libraries, e.g. those related to Klebsiella, Citrobacter, Enterobacter, Pectobacterium and Serratia. libshuff analysis showed that the bacterial communities of B. dorsalis from the three populations were significantly different from each other (P < 0·0085). Conclusions: (i) The intestinal tract of B. dorsalis adult contains a diverse bacterial community, some of which are stable. (ii) Different environmental conditions and food supply could influence the diversity of the harboured bacterial communities and increase community variations. Significance and Impact of the Study: Comparison of the microbial compositions and common bacterial species found in this paper may be very important for the biocontrol of B. dorsalis.     

Allyl‐2,6‐dimethoxyphenol,a female‐biased compound,is robustly attractive to conspecific males of Bactrocera dorsalis at close range

Insect cuticular hydrocarbons (CHCs) play important roles in mate recognition and chemical communication. To explore the cues regulating courtship and mating behaviour in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), CHCs were extracted from mature virgin female and male oriental fruit flies using n‐hexane. Nine compounds – i.e., 4‐allyl‐2,6‐dimethoxyphenol (designated ‘compound 3’) and eight ester compounds – had significantly greater abundance in female samples than in males. Of these nine compounds, six (1, 2, 4, 5, 6, and 8) elicited electrophysiological responses from the antennae of male flies. Compound 3 did not elicit a detectable male antennal response, but in Y‐tube olfactometer bioassays, it exhibited robust attractiveness to conspecific males at close range. We speculate that compound 3 is a close‐range sex pheromone in B. dorsalis. A mating competition experiment revealed that compound 3 significantly increased the competitiveness of males, which implied that compound 3 might be used in control programs of B. dorsalis.     

Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) is not invasive through Asia: It's been there all along

Oriental fruit fly, Bactrocera dorsalis (Hendel), is a highly polyphagous fruit fly which, in the last 15 years, has invaded (with or without establishment) Africa, Europe and North America. As a direct result of these invasions, there is increasing research interest in the invasion history and spread patterns of this fly. A statement being repeatedly used in the B. dorsalis invasion literature is that the species was first identified from Taiwan in 1912 and that it subsequently spread through South‐East and South Asia during the 20th century. This assumption is incorrect and stems from: (a) an incomplete knowledge of B. dorsalis taxonomic history; and (b) a confounding of first taxonomic record with first invasion record. Rather than being first detected in Taiwan in 1912, the first record of oriental fruit fly was from “East India” (India orientali) under the synonymous name of Musca ferruginea by Fabricius in 1794, and in the 1910s, it was known not only from Taiwan, but widely across tropical Asia with records from India, Burma, Bengal, Sri Lanka (as Ceylon), Singapore and Indonesia (multiple islands). The taxonomic literature is very clear that oriental fruit fly has not invaded the rest of Asia from Taiwan since 1912, and this error should not continue to be repeated in the literature.     

Effects of radiation on the fertility of the Ethiopian fruit fly,Dacus ciliatus

The Ethiopian fruit fly, Dacus ciliatus (Loew) (Diptera: Tephritidae), is a significant pest of cucurbit crops in Asia and Africa and is currently controlled with insecticides. The sterilizing effect of gamma radiation on D. ciliatus adults was investigated to assess the suitability of sterile insect technique (SIT) for use as an alternative, non‐chemical strategy for the control of this pest. Late pupae (48 h before emergence) were irradiated with 60, 80, 100, 120, and 140 Gy of gamma rays emitted by a ⁶⁰Co source. Following emergence, the biological characteristics of the experimental cohorts (including all possible male‐female combinations of irradiated and untreated flies) were recorded. No significant negative effects of irradiation on pupal eclosion or the ability of newly emerged flies to fly were observed. Samples of eggs at reproductive fly‐ages (12‐, 15‐, and 17‐day‐old pairs) were collected and their hatch rates were assessed. At 60 Gy, females were completely sterilized, whereas complete sterilization of the males was observed only at 140 Gy (a small amount of fertility persisted even at 120 Gy). In addition to the above experiments, three fruit infestation trials were conducted with zucchini [Cucurbita pepo L. (Cucurbitaceae)] as the plant host and the pupae produced in those trials were collected and recorded. We observed significant (ca. 10%) infestation following treatment with up to 120 Gy and zero progeny only at 140 Gy, mirroring the egg‐hatch results. Our findings support the feasibility of SIT for the control of D. ciliatus.     

橘小实蝇神经肽sulfakinin的分子生物学特性及其表达模式

【目的】分析橘小实蝇sulfakinin的时空表达模式及其对饥饿的响应,初步明确sulfakinin在调节橘小实蝇行为和生理方面的功能。【方法】利用RT-PCR技术克隆橘小实蝇神经肽sulfakinin的c DNA序列,采用实时定量PCR技术分析该基因在橘小实蝇不同发育阶段、成虫不同组织、幼虫不同组织及其在饥饿胁迫下的表达模式,并运用半定量RT-PCR技术进一步验证。【结果】实时定量PCR分析结果表明,sulfakinin在橘小实蝇的幼虫期和成虫期显著性高表达,卵期和蛹期几乎不表达。其中早期成虫表达量最高,约为晚期成虫的2倍,幼虫期表达量位于两者之间。在橘小实蝇幼虫、成虫不同组织中,sulfakinin在中枢神经系统的表达量显著高于其他组织,在触角的表达量仅次于中枢神经系统。此外,橘小实蝇在经饥饿处理24 h的过程中,sulfakinin的表达量均出现下调,其中从饥饿处理结果可知在6 h下降幅度最大,12 h和24 h表达量下降幅度减少。半定量RT-PCR技术进一步检测结果与实时定量PCR技术检测结果一致。【结论】sulfakinin在橘小实蝇中枢神经系统可能调节橘小实蝇成虫对饥饿胁迫的响应。其在触角中的高表达可能与橘小实蝇嗅觉的敏感性相关。本研究为进一步研究橘小实蝇sulfakinin的生理功能、评估其药靶潜力奠定了理论基础。     

Maternal behaviours may explain riffle‐scale variations in some stream insect populations

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Microbiome changes through ontogeny of a tick pathogen vector

Blacklegged ticks (Ixodes scapularis) are one of the most important pathogen vectors in the United States, responsible for transmitting Lyme disease and other tick‐borne diseases. The structure of a host's microbial community has the potential to affect the ecology and evolution of the host. We employed high‐throughput sequencing of the 16S rRNA gene V3‐V4 hypervariable regions in the first study to investigate the tick microbiome across all developmental stages (larvae, nymphs, adults). In addition to field‐collected life stages, newly hatched laboratory‐reared larvae were studied to determine the baseline microbial community structure and to assess transovarial transmission. We also targeted midguts and salivary glands due to their importance in pathogen maintenance and transmission. Over 100 000 sequences were produced per life stage replicate. Rickettsia was the most abundant bacterial genus across all sample types matching mostly the Ixodes rickettsial endosymbionts, and its proportion decreased as developmental stage progressed, with the exception of adult females that harboured a mean relative abundance of 97.9%. Laboratory‐reared larvae displayed the lowest bacterial diversity, containing almost exclusively Rickettsia. Many of the remaining bacteria included genera associated with soil, water and plants, suggesting environmental acquisition while off‐host. Female organs exhibited significantly different β‐diversity than the whole tick from which they were derived. Our results demonstrate clear differences in both α‐ and β‐diversity among tick developmental stages and between tick organs and the tick as a whole. Furthermore, field‐acquired bacteria appear to be very important to the overall internal bacterial community of this tick species, with influence from the host bloodmeal appearing limited.     

Field evaluation of the relative attractiveness of enriched ginger root oil (EGO)lure and trimedlure for African Ceratitis species (Diptera: Tephritidae)

The males of some fruit flies (Diptera: Tephritidae) are known to be attracted to specific parapheromones. The trapping results between trimedlure (TML) and enriched ginger root oil (EGOlure) were compared at two experimental sites in Morogoro (Central Tanzania) for a period of 12 weeks co‐inciding with the main citrus season. Both attractants captured a comparable diversity of fruit flies, except that EGOlure also attracted fruit flies, such as Ceratitis cosyra, not normally found in TML‐baited traps. Both EGOlure and TML attracted mainly or exclusively male fruit flies, but the catches with EGOlure were equal or superior to those with TML. It is concluded that EGOlure should be considered as a suitable alternative for TML in detection, monitoring and control programs for African fruit flies of the genus Ceratitis. It has the added advantage that it combines the attractiveness with regard to species spectrum of both TML and terpinyl acetate.     

Potential for insecticide resistance in populations of Bactrocera dorsalis in Hawaii: spinosad susceptibility and molecular characterization of a gene associated with organophosphate resistance

The potential for populations to become resistant to a particular insecticide treatment regimen is a major issue for all insect pest species. In Hawaii, for example, organophosphate (OP)‐based cover sprays have been the chemical treatment most commonly applied against oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), populations since the 1950s. Moreover, bait spray treatments using spinosad were adopted as a major control tactic in the Hawaii area‐wide fruit fly pest management program beginning in the year 2000. To determine the current level of spinosad and OP tolerance of wild B. dorsalis populations, bioassays were conducted on flies collected from a range of geographic localities within the Hawaiian islands. Adult B. dorsalis flies were tested (1) for the level of susceptibility to spinosad using LC₅₀ diagnostic criteria, and (2) for the presence of alleles of the ace gene previously shown to be associated with OP resistance. Regarding spinosad tolerance, only flies from Puna, the one area lacking prior exposure to spinosad, showed any significant difference compared to controls, and here the difference was only in terms of non‐overlap of 95% fiducial limit values. With respect to OP tolerance, specific mutations in the ace gene associated with resistance to these insecticides were found in only two populations, and in both cases, these alleles occurred at relatively low frequencies. These results suggest that at the present time, populations of B. dorsalis in Hawaii show no evidence for having acquired resistance to the insecticides widely used in control programs.     

Population structure and colonization of Bactrocera dorsalis (Diptera: Tephritidae) in China,inferred from mtDNA COI sequences

The oriental fruit fly, Bactrocera dorsalis, is a serious pest of fruits and vegetables in South‐east Asia, and, because of quarantine restrictions, impedes international trade and economic development in the region. Revealing genetic variation in oriental fruit fly populations will provide a better understanding of the colonization process and facilitate the quarantine and management of this species. The genetic structure in 15 populations of oriental fruit fly from southern China, Laos and Myanmar in South‐east Asia was examined with a 640‐bp sequence of the mitochondrial cytochrome oxidase subunit I (COI) gene. The highest levels of genetic diversity were found in Laos and Myanmar. Low to medium levels of genetic differentiation (F_ST ≤ 0.134) were observed among populations. Pooled populations from mainland China differed from those in Laos and Myanmar (F_ST = 0.024). Genetic structure across the region did not follow the isolation‐by‐distance model. The high genetic diversity observed in Laos and Myanmar supports the South‐east Asian origin of B. dorsalis. High genetic diversity and significant differentiation between some populations within mainland China indicate B. dorsalis populations have been established in the region for an extended period of time. High levels of genetic diversity observed among the five populations from Hainan Island and similarity between the Island and Chinese mainland populations indicate that B. dorsalis was introduced to Hainan from the mainland and has been on the island for many years. High genetic diversity in the recently established population in Shanghai (Pudong) suggests multiple introductions or a larger number of founders.     

Comparison of male adult population densities of the oriental and artocarpus fruit flies,Dacus spp. (Diptera: Tephritidae), in two nearby villages in Penang,Malaysia

The populations of native male adult oriental fruit fly Dacus dorsalis (Hendel ) and artocarpus fruit fly D. umbrosus (F.) in two selected site (BU and SD) were estimated weekly by the capture-recapture technique using live traps baited with methyl eugenol. In BU where many varieties of fruit trees were grown, the estimated population densities of D. dorsalis were between 980 and 3100 male flies per ha between May and July, 1984. During the same period, in SD where there were fewer number and varieties of fruit trees, the estimated population densities were between 300 and 1000 flies per ha. The estimated population densities of D. umbrosus over the same period were between 570 and 1290 flies per ha in BU; and between 5 and 95 flies per ha in SD. Of a total 6828 marked D. dorsalis flies released only one fly (released 6 weeks earlier in BU) was caught in a different site.     

Onset and establishment of diazotrophs and other bacterial associates in the early life history stages of the coral Acropora millepora

Early establishment of coral–microbial symbioses is fundamental to the fitness of corals, but comparatively little is known about the onset and succession of bacterial communities in their early life history stages. In this study, bacterial associates of the coral Acropora millepora were characterized throughout the first year of life, from larvae and 1‐week‐old juveniles reared in laboratory conditions in the absence of the dinoflagellate endosymbiont Symbiodinium to field‐outplanted juveniles with established Symbiodinium symbioses, and sampled at 2 weeks and at 3, 6 and 12 months. Using an amplicon pyrosequencing approach, the diversity of both nitrogen‐fixing bacteria and of bacterial communities overall was assessed through analysis of nifH and 16S rRNA genes, respectively. The consistent presence of sequences affiliated with diazotrophs of the order Rhizobiales (23–58% of retrieved nifH sequences; 2–12% of 16S rRNA sequences), across all samples from larvae to 12‐month‐old coral juveniles, highlights the likely functional importance of this nitrogen‐fixing order to the coral holobiont. Dominance of Roseobacter‐affiliated sequences (>55% of retrieved 16S rRNA sequences) in larvae and 1‐week‐old juveniles, and the consistent presence of sequences related to Oceanospirillales and Altermonadales throughout all early life history stages, signifies their potential importance as coral associates. Increased diversity of bacterial communities once juveniles were transferred to the field, particularly of Cyanobacteria and Deltaproteobacteria, demonstrates horizontal (environmental) uptake of coral‐associated bacterial communities. Although overall bacterial communities were dynamic, bacteria with likely important functional roles remain stable throughout early life stages of Acropora millepora.     

Response of two parasitoid species (Hymenoptera: Braconidae,Figitidae) to tephritid host and host food substrate cues

The Neotropical‐native figitid Aganaspis pelleranoi (Brèthes) and the Asian braconid Diachasmimorpha longicaudata (Ashmead) are two parasitoids of Tephritidae fruit flies with long and recent, respectively, evolutionary histories in the Neotropics. Both species experienced a recent range of overlap. In Argentina, A. pelleranoi is a potential species in biological control programs against the pestiferous tephritid species, Anastrepha fraterculus (Wiedemann) and Ceratitis capitata (Wiedemann), whereas D. longicaudata is already used in open‐field releases against Medfly in central‐western Argentina. To characterize the host‐foraging strategies of A. pelleranoi and D. longicaudata, olfactometer experiments were conducted comparing responses to C. capitata and A. fraterculus larvae, in two kinds of food substrate: fruit and artificial larval medium. To control the possible influence of host larvae used for parasitoid rearing on olfactory response, two strains of both parasitoid species, reared on both tephrtid species, were studied. Volatiles directly emanating either from A. fraterculus or C. capitata larvae may be detected by both A. pelleranoi and D. longicaudata, although chemical stimuli originating from the combination of host larvae and the habitat of the host were preferred. However, olfactory cues associated with host larvae probably play a relevant role in host searching behaviour of A. pelleranoi, whereas for D. longicaudata, the host‐habitat olfactory stimuli would be highly essential in short‐range host location. The strain of the parasitoids did not affect host search ability on the two tephritid species evaluated. These evidences are relevant for mass production of both parasitoids and their impact following open‐field augmentative releases.     

Concordance of bacterial communities of two tick species and blood of their shared rodent host

High‐throughput sequencing is revealing that most macro‐organisms house diverse microbial communities. Of particular interest are disease vectors whose microbiome could potentially affect pathogen transmission and vector competence. We investigated bacterial community composition and diversity of the ticks Dermacentor variabilis (n = 68) and Ixodes scapularis (n = 15) and blood of their shared rodent host, Peromyscus leucopus (n = 45) to quantify bacterial diversity and concordance. The 16S rRNA gene was amplified from genomic DNA from field‐collected tick and rodent blood samples, and 454 pyrosequencing was used to elucidate their bacterial communities. After quality control, over 300 000 sequences were obtained and classified into 118 operational taxonomic units (OTUs, clustered at 97% similarity). Analysis of rarefied communities revealed that the most abundant OTUs were tick species‐specific endosymbionts, Francisella and Rickettsia, and the commonly flea‐associated bacterium Bartonella in rodent blood. An Arsenophonus and additional Francisella endosymbiont were also present in D. variabilis samples. Rickettsia was found in both tick species but not in rodent blood, suggesting that it is not transmitted during feeding. Bartonella was present in larvae and nymphs of both tick species, even those scored as unengorged. Relatively, few OTUs (e.g. Bartonella, Lactobacillus) were found in all sample types. Overall, bacterial communities from each sample type were significantly different and highly structured, independent of their dominant OTUs. Our results point to complex microbial assemblages inhabiting ticks and host blood including infectious agents, tick‐specific endosymbionts and environmental bacteria that could potentially affect arthropod‐vectored disease dynamics.     

Development of Asian parasitoids in larvae Of Drosophila Suzukii feeding on blueberry and artificial diet

In just a few years, the Asian fly Drosophila suzukii has invaded several continents and has become a very serious pest of many fruit crops worldwide. Current control methods rely on chemical insecticides or expensive and labour‐intensive cultural practices. Classical biological control through the introduction of Asian parasitoids that have co‐evolved with the pest may provide a sustainable solution on condition that they are sufficiently specific to avoid non‐target effects on local biodiversity. Here, we present the first study on the development of three larval parasitoids from China and Japan, the Braconidae Asobara japonica and the Figitidae Leptopilina japonica and Ganaspis sp., on D. suzukii. The Asian parasitoids were compared with Leptopilina heterotoma, a common parasitoid of several Drosophilidae worldwide. The three Asian species were successfully reared on D. suzukii larvae in both, blueberry and artificial diet, in contrast to L. heterotoma whose eggs and larvae were encapsulated by the host larvae. All parasitoids were able to oviposit one day after emergence. Asobara japonica laid as many eggs in larvae feeding in blueberry as in artificial diet, whereas L. heterotoma oviposited more in larvae on the artificial diet and the Asian Figitidae oviposited more in larvae feeding on blueberry. Ganaspis sp. laid very few eggs in larvae in the artificial diet, suggesting that it may be specialized in Drosophila species living in fresh fruits. These data will be used for the development of a host range testing to assess the suitability of Asian parasitoids as biological control agents in invaded regions.     

Larval competition within and between insecticide resistant and susceptible individuals in the oriental fruit fly,Bactrocera dorsalis

Insecticide resistance is a serious issue in agriculture. Ecological interactions may be used to manage the spread of resistance, but little information is currently available. In particular, virtually none is known about interactions between resistant and susceptible individuals. This study investigated competition between resistant and susceptible oriental fruit fly larvae (Bactrocera dorsalis). Competition in the larval stage was examined with an explicit consideration of individual identity (resistant or susceptible). Guava fruits were inoculated with eggs of susceptible and/or resistant flies, and their development and survival were monitored. Egg density influenced the time that larvae stayed in fruits as well as their survival. In addition, susceptible flies survived better when interacting with resistant flies than with other susceptible flies, indicating that susceptible flies are competitively superior to resistant flies. The results suggest that artificially creating environments that induce competition between susceptible and resistant flies can be useful for the management of insecticide resistance.     

Acceptability of Drosophila suzukii as prey for common predators occurring in cherries and berries

The invasive cherry vinegar fly, Drosophila suzukii, has been identified in Europe as a destructive fruit pest since its arrival in 2008. In the present laboratory study, three predatory insects (Orius majusculus, Chrysoperla carnea, and Forficula auricularia) naturally occurring on fruit crops in Europe were investigated for their ability to attack and feed on D. suzukii within and outside fruits. The predators were provided with various D. suzukii life stages (eggs, larvae, pupae or adults) exposed or within infested cherries. The anthocorid bug O. majusculus fed on eggs and larvae, but was not able to attack pupae. Larvae of the lacewing C. carnea preyed upon D. suzukii eggs, larvae and pupae and also captured adult flies. The European earwig F. auricularia was the most voracious predator of these three tested species. Although the earwigs were not able to catch adult flies, they readily preyed upon every other developmental stage. Adult O. majusculus or third instar larvae of C. carnea significantly reduced the offspring of D. suzukii from infested cherries, when these contained the egg stage of the pest. None of the predators were able to attack early larval stages inside the cherries. But pupae that protruded from the fruit epicarp or that had pupated outside the fruit were accessible to lacewing larvae and earwigs and significantly reduced by them. Orius bugs, lacewing larvae and earwigs were able, under laboratory conditions, to capture and prey upon various life stages of the invasive pest, if not completely concealed inside the fruit. Our findings suggest that these generalist predators may have some control capacity on infested fruit in cultivated fruit crops and also in non‐crop habitats.     

Effects of gamma irradiation on the life stages of yellow flower thrips, Frankliniella schultzei (Trybom) (Thysanoptera: Thripidae)

Irradiation at a minimum absorbed dose of 250 Grays (Gy) has been approved by the USDA as a quarantine treatment for certain fruits in Hawaii to control four species of tephritid fruit flies. Subsequent research must determine whether this dose is sufficient to control other quarantine pests, such as mealybugs, thrips, mites, beetles, moths, and scale insects, on other commodities with export potential that are approved for irradiation treatment for fruit flies. This study demonstrated that irradiation at 250 Gy caused non‐emergence of eggs and pupae, failure of larval development, and sterility of adults of yellow flower thrips, Frankliniella schultzei (Trybom). Adults were the most resistant stage tested, with 100% mortality at 57, 36 and 30 days post‐treatment for the 250, 350 and 400 Gy treatments, respectively. Untreated adults survived up to 66 days. After receiving an irradiation dose of 250 Gy, no one‐ to two‐day old eggs hatched successfully, while 3–4‐day old eggs hatched but did not develop beyond the larval stage. Of the controls, 96.0% of 1–2‐day old eggs and 75.9% of the 3–4‐day old eggs hatched and survived through pupation. No first or second instar larvae treated with a target dose of 250 Gy were able to pupate. When pupae were irradiated at 250 Gy, 37% emerged as adults and all were sterile compared to 88.3% emergence of controls.     

MicroRNA Let‐7 targets the ecdysone signaling pathway E75 gene to control larval–pupal development in Bactrocera dorsalis

MicroRNAs (miRNAs) regulate various biological processes during insect developme nt;however, their role in larval-pupal development in oriental fruit fly, Bactrocera dorsalis (Hendel) remains unknown. In the current study, we address the biological function of a conserved miRNA, Bdo-Let-7 in the regulation of BdE75 gene, which belongs to the ecdysone signaling pathway and participates in the larval-pupal development in B. dorsalis. Using dual luciferase reporter assay in HEK293T cells we show that Bdo-Let-7 miRNA interacts with the 3' untranslated region of BdE75 gene and suppresses its expression. The Bdo-Let?7 and BdE75 are also co-expressed in the larval-pupal stages and in different tissues of B. dorsalis .In in vivo experiments, the injectio n of Bdo-Let-7 agomir and antagomir in third instar larvae down- and up-regulated the expression of BdE75、 respectively. The 20-hydroxyecdysone (20E) injection assay shows that 20E up-regulated the expression of Bdo-Let-7 on the 5th day of the larvae. Moreover, abnormal pupation and eclosion were observed after larval Bdo-Let-7 antagomir injection. Based on these results, we show that Bdo-Let-7 regulates the ecdysone signaling pathway through the exact dose of BdE75 gene, and is indispensable for normal larval-pupal development in B. dorsalis.