Bracoviruses recruit host integrases for their integration into caterpillar’s genome

Some DNA viruses infect host animals usually by integrating their DNAs into the host genome. However, the mechanisms for integration remain largely unknown. Here, we find that Cotesia vestalis bracovirus (CvBV), a polydnavirus of the parasitic wasp C. vestalis (Haliday), integrates its DNA circles into host Plutella xylostella (L.) genome by two distinct strategies, conservatively and randomly, through high-throughput sequencing analysis. We confirmed that the conservatively integrating circles contain an essential “8+5” nucleotides motif which is required for integration. Then we find CvBV circles are integrated into the caterpillar’s genome in three temporal patterns, the early, mid and late stage-integration. We further identify that three CvBV-encoded integrases are responsible for some, but not all of the virus circle integrations, indeed they mainly participate in the processes of early stage-integration. Strikingly, we find two P. xylostella retroviral integrases (PxIN1 and PxIN2) are highly induced upon wasp parasitism, and PxIN1 is crucial for integration of some other early-integrated CvBV circles, such as CvBV_04, CvBV_12 and CvBV_24, while PxIN2 is important for integration of a late-integrated CvBV circle, CvBV_21. Our data uncover a novel mechanism in which CvBV integrates into the infected host genome, not only by utilizing its own integrases, but also by recruiting host enzymes. These findings will strongly deepen our understanding of how bracoviruses regulate and integrate into their hosts.     

CLP gene family,a new gene family of Cotesia vestalis bracovirus inhibits melanization of Plutella xylostella hemolymph

Polydnaviruses (PDVs) are obligatory symbionts of parasitoid wasps and play an important role in suppressing host immune defenses. Although PDV genes that inhibit host melanization are known in Microplitis bracovirus, the functional homologs in Cotesia bracoviruses remain unknown. Here, we find that Cotesia vestalis bracovirus (CvBV) can inhibit hemolymph melanization of its host, Plutella xylostella larvae, during the early stages of parasitization, and that overexpression of highly expressed CvBV genes reduced host phenoloxidase activity. Furthermore, CvBV-7-1 in particular reduced host phenoloxidase activity within 12 h, and the injection of anti-CvBV-7-1 antibody increased the melanization of parasitized host larvae. Further analyses showed that CvBV-7-1 and three homologs from other Cotesia bracoviruses possessed a C-terminal leucine/isoleucine-rich region and had a similar function in inhibiting melanization. Therefore, a new family of bracovirus genes was proposed and named as C -terminal L eucine/isoleucine-rich P rotein (CLP). Ectopic expression of CvBV-7-1 in Drosophila hemocytes increased susceptibility to bacterial repression of melanization and reduced the melanotic encapsulation of parasitized D. melanogaster by the parasitoid Leptopilina boulardi. The formation rate of wasp pupae and the eclosion rate of C. vestalis were affected when the function of CvBV-7-1 was blocked. Our findings suggest that CLP genes from Cotesia bracoviruses encoded proteins that contain a C-terminal leucine/isoleucine-rich region and function as melanization inhibitors during the early stage of parasitization, which is important for successful parasitization.     

Transient expression of specific Cotesia plutellae bracoviral segments induces prolonged larval development of the diamondback moth, Plutella xylostella

A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses a segmented and dispersed genome that is located on chromosome(s) of its symbiotic endoparasitic wasp, C. plutellae. When the host wasp parasitizes larvae of the diamondback moth, Plutella xylostella, at least 27 viral genome segments are delivered to the parasitized host along with the wasp egg. The parasitized P. xylostella exhibits significant immunosuppression and a prolonged larval development. Parasitized larvae take about 2 days longer than nonparasitized larvae to develop until the wandering stage of the final larval instar, and die after egress of the full grown wasp larvae. Developmental analysis using juvenile hormone and ecdysteroid analogs suggests that altering endocrine signals could induce the retardation of larval developmental rate in P. xylostella. In this study we used a transient expression technique to micro-inject individual CpBV genome segments, and tested their ability to induce delayed larval development of P. xylostella. We demonstrated that a CpBV segment was able to express its own encoded genes when it was injected into nonparasitized larvae, in which the expression patterns of the segment genes were similar to those in the larvae parasitized by C. plutellae. Twenty three CpBV genome segments were individually cloned and injected into the second instar larvae of P. xylostella and their effects assessed by measuring the time taken for host development to the cocooning stage. Three CpBV genome segments markedly interfered with the host larval development. When the putative genes of these segments were analyzed, it was found that they did not share any common genes. Among these segments able to delay host development, segment S27 was predicted to encode seven protein tyrosine phosphatases (CpBV-PTPs), some of which were mutated by insertional inactivation with transposons, while other encoded gene expressions were unaffected. The mutant segments were unable to induce prolonged larval development of P. xylostella. These results suggest that CpBV can induce prolonged larval development of P. xylostella, and that at least some CpBV-PTPs may contribute to the parasitic role probably by altering titers of developmental hormones.     

IkB genes encoded in Cotesia plutellae bracovirus suppress an antiviral response and enhance baculovirus pathogenicity against the diamondback moth, Plutella xylostella

An endoparasitoid wasp, Cotesia plutellae, parasitizes larvae of the diamondback moth, Plutella xylostella, with its symbiotic polydnavirus, C. plutellae bracovirus (CpBV). This study analyzed the role of Inhibitor-kB (IkB)-like genes encoded in CpBV in suppressing host antiviral response. Identified eight CpBV-IkBs are scattered on different viral genome segments and showed high homologies with other bracoviral IkBs in their amino acid sequences. Compared to an insect ortholog (e.g., Cactus of Drosophila melanogaster), they possessed a shorter ankyrin repeat domain without any regulatory domains. The eight CpBV-IkBs are, however, different in their promoter components and expression patterns in the parasitized host. To test their inhibitory activity on host antiviral response, a midgut response of P. xylostella against baculovirus infection was used as a model reaction. When the larvae were orally fed the virus, they exhibited melanotic responses of midgut epithelium, which increased with baculovirus dose and incubation time. Parasitized larvae exhibited a significant reduction in the midgut melanotic response, compared to nonparasitized larvae. Micro-injection of each of the four CpBV genome segments containing CpBV-IkBs into the hemocoel of nonparasitized larvae showed the gene expressions of the encoded IkBs and suppressed the midgut melanotic response in response to the baculovirus treatment. When nonparasitized larvae were orally administered with a recombinant baculovirus containing CpBV-IkB, they showed a significant reduction in midgut melanotic response and an enhanced susceptibility to the baculovirus infectivity.     

A copy of cystatin from the diamondback moth Plutella xylostella is encoded in the polydnavirus Cotesia plutellae bracovirus

Cystatins (CSTs) are reversible and competitive inhibitors of cysteine proteases. Some polydnaviruses encode viral CSTs that have been speculated to play a crucial role in viral pathology. Four CSTs have been reported in the episomal genome of a polydnavirus, Cotesia plutellae (synonymous with C. vestalis) bracovirus (CpBV). These 4 CSTs share high sequence homologies with other bracoviral CSTs. Further sequence analysis showed that 2 of the CpBV-CSTs are identical. The remaining 3 CSTs have been designated CpBV-CST1, CpBV-CST2, and CpBV-CST3. Expression analysis indicated that CpBV-CST2 was not expressed in any stage of Plutella xylostella, either parasitized or non-parasitized by C. plutellae. However, both CpBV-CST1 and CpBV-CST3 were expressed in all stages of P. xylostella. Interestingly, these 2 genes were also expressed in non-parasitized P. xylostella in all developmental stages. A CST sequence from the non-parasitized larva was 100% identical with that of CpBV-CST1 for the entire open reading frame (ORF). To understand the role of CpBV-CST1 in viral pathology, the ORF was cloned into a eukaryotic expression vector and transiently expressed in non-parasitized larvae. The in vivo transient expression lasted for at least 4 days. Under this condition, the treated larvae suffered significant suppression in immune responses and in development. These results suggest that CpBV-CSTs play a crucial role in parasitism, altering host immune and developmental processes by interrupting normal interactions between CSTs and cysteine proteases in P. xylostella.     

Transient transcription of a putative RNase containing BEN domain encoded in Cotesia plutellae bracovirus induces an immunosuppression of the diamondback moth, Plutella xylostella

A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses segmented genome located on chromosome(s) of an endoparasitoid wasp, C. plutellae. An episomal viral segment (CpBV-S3) consists of 11,017 bp and encodes two putative open reading frames (ORFs). ORF301 shows amino acid sequence homologies (28-50%) with RNase T2s of various organisms. It also contains BEN domain in C-terminal region. ORF302 is a hypothetical gene, which is also found in other bracoviruses. Both genes were expressed in larvae of Plutella xylostella parasitized by C. plutellae. Their expressions were detected in all tested tissues including hemocyte, fat body, gut, and epidermis. To analyze effects of these genes on the parasitism, the segment of CpBV-S3 was injected to nonparasitized larvae of P. xylostella, in which the two genes were expressed at least for 4 days post-injection. The larvae injected with CpBV-S3 exhibited significant immunosuppression, such as reduction in total hemocyte population and impairment in nodule formation behavior of hemocytes in response to bacterial challenge. Each gene expression in the treated larvae was inhibited by co-injecting respective double strand RNA (dsRNA) specific to each ORF. Injection of dsRNA of ORF301 could rescue the immunosuppression of the viral segment-treated larvae, while dsRNA specific to ORF302 did not. These results suggest that a putative RNase fused with a BEN domain encoded in CpBV-S3 plays a parasitic role in inducing host immunosuppression in the parasitism.     

Plant Resistance Affects the Olfactory Response and Parasitism Success of Cotesia vestalis

Understanding the factors influencing host-selection behavior of parasitoids is essential in studies on host-parasitoid ecology and evolution, and in combining sustainable strategies of pest management, such as host-plant resistance and biological control. The effects of host-plant resistance on the olfactory response and parasitism success by Cotesia vestalis, a parasitoid of diamondback moth (Plutella xylostella) larvae were examined. Here, it was demonstrated that host-plant resistance can strongly influence foraging behavior and parasitism success of the parasitoid. In olfactometer experiments, C. vestalis did not differentiate between crucifer plant types with similar levels of susceptibility or resistance to P. xylostella but showed a strong preference for susceptible compared with partially-resistant host plants. The influence of previous oviposition activity varied with the host-plant type experienced by the parasitoid. In cage experiments, C. vestalis preferred to parasitize P. xylostella larvae on a susceptible plant compared with larvae on a partially resistant host plant when exposed to hosts for 24 h. However, this preference appeared to be transitory, and was not found after 96 h exposure. The present study suggests that combining partial host-plant resistance with biological control by C. vestalis for the control of P. xylostella may in some circumstances be antagonistic and negatively affect parasitism success.     

Movement of transgenic plant-expressed Bt Cry1Ac proteins through high trophic levels

The movement of Bacillus thuringiensis (Berliner) (Bt) Cry1Ac endotoxin through high trophic levels was assessed to help elucidate the effects of Bt toxin on non‐target insects. The diamondback moth (Plutella xylostella L., Lepidoptera: Plutellidae), the parasitic wasp (Cotesia vestalis Haliday, Hymenoptera: Braconidae) and the predatory green lacewing Chrysoperla carnea (Stephen) (Neuroptera: Chrysopidae) were used as a model system in this laboratory study. Bt‐resistant P. xylostella larvae fed Cry1Ac‐expressing transgenic oilseed rape (OSR, Brassica napus L., Cruciferae), before and after parasitization by C. vestalis, consumed Cry1Ac with the ingested plant material but only a proportion of Cry1Ac consumed was recovered from the bodies and faeces of P. xylostella larvae. Cry1Ac was not detected in newly emerged parasitoid larvae. In contrast, Cry1Ac was detected in C. carnea larvae fed on resistant P. xylostella larvae reared on Bt OSR. However, no Cry1Ac could be detected in C. carnea larvae when the lacewings were transferred to P. xylostella larvae reared on conventional OSR and tested 24–48 h. The metabolizing ability of Cry1Ac is discussed for the larvae of P. xylostella and C. carnea.     

Identification and characterization of defensin genes from the endoparasitoid wasp Cotesia vestalis (Hymenoptera: Braconidae)

Defensins are members of a large and diverse family of antimicrobial peptides (AMPs) containing three or four intramolecular disulfide bonds. They are widely distributed from vertebrates to invertebrates, and serve as critical defense molecules protecting the host from the invasion of pathogens or protozoan parasites. Cotesia vestalis is a small endoparasitoid wasp that lays eggs in larvae of Plutella xylostella, a cosmopolitan pest of cruciferous crops. We identified and characterized three full-length cDNAs encoding putative defensin-like peptides from C. vestalis, named CvDef1, CvDef2 and CvDef3. Phylogenetic analyses of these sequences showed that they are present in two clades, CITDs and PITDs, indicating a diversity of defensins in C. vestalis. We analyzed their expression patterns in larvae, pupae and adults by semi-quantitative RT-PCR. The results showed that CvDef1 mRNA was expressed from the end stage of the second instar larva, CvDef3 mRNA from the early stage of the second instar larva, and CvDef2 mRNA was expressed in all developmental stages of C. vestalis. Furthermore, CvDef1 showed antimicrobial activity against gram-positive and gram-negative bacteria. Growth kinetics of Staphylococcus aureus indicated that CvDef1 had much better antimicrobial ability than ampicillin, making it a potential candidate for practical use. Transmission electron microscopic (TEM) examination of CvDef1-treated S. aureus cells showed extensive damage to the cell membranes. Our results revealed the basic properties of three defensins in C. vestalis for the first time, which may pave the way for further study of the functions of defensins in parasitism and innate immunity of C. vestalis.     

An Evidence for Host Translation Inhibitory Factor Encoded in a Polydnavirus,Cotesia glomerata Bracovirus,Genome and Its Expression in Parasitized Cabbage White Butterfly,Pieris rapae

Polydnavirus is a DNA virus symbiotic to some endoparasitic wasps and plays a critical role in accomplishing successful parasitic life cycle of host wasps. Host translation inhibitory factor (HTIF) has been found in some polydnaviral genomes and performs parasitic functions leading to host immunosuppression and redirecting host nutrient usage to wasp development. The cabbage white butterfly, Pieris rapae, parasitized by a gregarious endoparasitoid, Cotesia glomerata, undergoes several physiological alterations including immune malfunctioning and failure of pupal metamorphosis. C. glomerata possesses its own symbiotic polydnavirus, C. glomerata bracovirus (CgBV). Its genome consisted of at least 12 segments in unequal amounts. Parasitized P. rapae hemolymph contained HTIF-like protein, which was determined through an immunoblotting assay using HTIF antibody of C. plutellae bracovirus (CpBV). RT-PCR using HTIF primers of CpBV produced an HTIF-like gene in P. rapae larvae parasitized by C. glomerata. Also, this HTIF-like gene was encoded in CgBV genome and its partial sequence of CgBV showed highly homology (98.5%) to amino acid sequence of an HTIF of CpBV, called CpBV15a. These results suggest that a common HTIF-like moiety may be shared among Cotesia-associated bracovirus.     

Genome size estimation of an endoparasitoid wasp,Cotesia plutellae,using quantitative real-time polymerase chain reaction

An endoparasitoid wasp, Cotesia plutellae, is a natural enemy against the diamondback moth, Plutella xylostella, which is the most destructive insect pest of cruciferous crop plants. The wasp genome contains genetic information of several parasitic factors, such as its symbiotic virus (C. plutellae bracovirus), venom, teratocyte as well as the parasitoid itself. These parasitic factors interfere with physiological processes of the immature stages of P. xylostella and need to be analyzed concerning their genetic components. A full genome sequence of C. plutellae would be highly informative to determine functional genes associated with these parasitic factors. Before a full genome sequence analysis of C. plutellae can be undertaken, an estimate of genome size is needed. In this study, we used a strategy using a quantitative real-time polymerase chain reaction (qPCR) to measure the wasp genome size. qPCR determined the number of a single copy gene hexokinase in a total mass of genomic DNA. The resulting molecular weight of the DNA sample was used to calculate the genome size in base pair (bp). This estimation approach indicated a genome size of C. plutellae of 186.06 ± 1.21 Mb.     

菜蛾盘绒茧蜂卵携带的免疫抑制因子

抑制寄主昆虫的免疫反应是内寄生蜂存活的关键。菜蛾盘绒茧蜂Cotesia vestalis（Haliday）寄生小菜蛾Plutella xylostella （L.）幼虫后,蜂卵如何逃避和抑制寄主的免疫攻击,尚未得到全面揭示。本文采用电镜技术系统观察了菜蛾盘绒茧蜂卵表面的超微结构。结果显示：蜂卵表面覆盖有纤维层和絮状的类病毒样纤丝（VLFs）,同时携带了含多分DNA病毒粒子（PDV）的萼液。在寄生初期,包裹在蜂卵表面的纤维层和VLFs首先起到保护蜂卵不被小菜蛾血细胞包囊的被动防御作用。随后,PDV发挥主动的免疫抑制作用。通过假寄生手段,证明了菜蛾盘绒茧蜂PDV (CvBV) 具有较持久的克服寄主免疫攻击的能力,是主要的免疫抑制因子。在假寄生后连续8 d的观察时间内,菜蛾盘绒茧蜂的蜂卵均未被包囊。结果提示,在菜蛾盘绒茧蜂-小菜蛾寄生体系中,菜蛾盘绒茧蜂采取被动防御和主动攻击两种方式应对寄主小菜蛾的免疫攻击。     

Relative influences of plant type and parasitoid initial density on host–parasitoid relationships in a tritrophic system

To explore the effects of bottom-up and top-down forces on the relationships between a host, Plutella xylostella (L.) (Lepidoptera, Plutellidae), and its parasitoid, Cotesia vestalis (Haliday) (Hymenoptera, Braconidae), a short-term field experiment was established as a factorial experiment using three different host plants (Brassica pekinensis cv. Yuki F1, Brassica oleracea var. capitata cv. Midorimaru F1 and B. oleracea var. botrytis cv. Snow Crown) in the presence of C. vestalis at two different levels (low and high initial release). The tritrophic interactions were monitored by census counts of live adults 20?days after parasitoid release. The mean numbers of P. xylostella and C. vestalis adults were compared using log-linear analysis of deviance. Also, differences in the levels of parasitism were analysed using logistic analysis of deviance. There was a significant effect of host plant type on the abundance of P. xylostella, the abundance of C. vestalis and the percentage parasitism of P. xylostella by C. vestalis. The mean number of P. xylostella adults per cage on common cabbage or cauliflower was significantly greater than that on Chinese cabbage. The mean number of C. vestalis adults and the proportion of hosts attacked by C. vestalis per cage were significantly greater on Chinese cabbage compared with common cabbage or cauliflower. Indeed, initial parasitoid release did not significantly affect the abundance of P. xylostella but there was a significant influence of initial parasitoid release on the abundance of C. vestalis and the levels of parasitism of P. xylostella by C. vestalis. The mean number of C. vestalis adults and the proportion of P. xylostella parasitised by C. vestalis per cage were greater in high level of parasitoid release compared with low level of parasitoid release. However, there were no significant interacting effect of the factors (plant type?×?parasitoid initial abundance) on the abundance of P. xylostella, the population size of C. vestalis and parasitism of P. xylostella by C. vestalis.     

Parasitism of Cotesia spp. Enhances Susceptibility of Plutella xylostella to Other Pathogens

Two endoparasitoids, Cotesia plutellae and C. glomerata, parasitize the diamondback moth, Plutella xylostella, and induce significant host immunosuppression. This study analyzed the susceptibility changes of the parasitized P. xylostella against other pathogens using an entomopathogenic bacterium, Xenorhabdus nematophila (Xn), and a viral pathogen, Autographa californica nucleopolyhedrosis virus (AcNPV). The P. xylostella parasitized by either C. plutellae or C. glomerata exhibited higher susceptibilities to both microbial pathogens than the nonpara-sitized. To determine the parasitism factors inducing the enhanced susceptibility, three polydnaviral genes so far successfully cloned were selected from C. plutellae bracovirus (CpBV). CpBV-lectin and CpBV15 α/β were inserted into AcNPV under a CpBV promote and analyzed in their pathogenicities against P. xylostella larvae. Two AcNPVs recombined with CpBV15α/β were more potent than the control AcNPV recombined with an enhanced green fluorescent protein gene or the AcNPV recombined with CpBV-lectin. These results suggest that the wasp parasitization enhances other pathogen susceptibilities by inducing host immunosuppression, in which the symbiotic polydnavirus can play significant role in the enhanced susceptibility.     

Transient expression of a polydnaviral gene, CpBV15beta, induces immune and developmental alterations of the diamondback moth, Plutella xylostella

The diamondback moth, Plutella xylostella, parasitized by its endoparasitoid wasp, Cotesia plutellae, undergoes various physiological alterations which include immunosuppression and an extended larval development. Its symbiotic virus, C. plutellae bracovirus (CpBV), is essential for their successful parasitization with more than 136 putative genes encoded in the viral genome. CpBV15β, a CpBV gene, has been known to play significant role in altering host physiological processes including hemocyte-spreading behavior through inhibition of protein synthesis under in vitro conditions. In the current study, we investigated its specific involvement in physiological processes of the host by transient expression and RNA interference techniques. The open reading frame of CpBV15β was cloned into a eukaryotic expression vector and this recombinant CpBV15β was transfected into nonparasitized 3rd instar P. xylostella by microinjection. CpBV15β was expressed as early as 24 h and was consistent up to 72 h. Due to the expression of this gene, plasma protein levels were significantly reduced and the ability of the hemocytes to adhere and spread on extracellular matrix was inhibited, wherein CpBV15β was detectable in the cytoplasm of hemocytes based on an indirect immunofluorescence assay. To confirm the role of CpBV15β, its double stranded RNA could efficiently recover the hemocyte-spreading behavior and synthesis of plasma proteins suppressed by the transient expression of CpBV15β. In addition, the larvae transfected with CpBV15β significantly suffered poor adult development probably due to lack of storage proteins. Thus these results demonstrate the role of CpBV15β in altering the host physiological processes involving cellular immune response and metamorphic development, which are usually induced by wasp parasitization.     

Starvation and herbivore-induced plant volatiles affect the color preferences of parasitic wasps

Using light-emitting diode spotlights, we examined the responses of Cotesia vestalis, a parasitoid of diamondback moth (DBM), Plutella xylostella larvae, with different hunger level to different chromatic cues. Naïve satiated female wasps showed no significant preference for either green, yellow, orange, or red spotlighted areas over a control area with background fluorescent light. When starved for 2 h, female wasps preferred yellow and green light over the control area, but not orange or red light. We also tested the effects of DBM-larvae-induced cabbage-plant volatiles, which attract female wasps, on wasp responses to green versus yellow light. In control experiments with no plant volatiles, starved wasps showed no color preference. However, when synthetic volatiles were present, the wasps preferred green over yellow light. We concluded that both hunger level and herbivore-induced plant volatiles were important factors affecting the response of parasitic wasps to light of different color.     

A viral lectin encoded in Cotesia plutellae bracovirus and its immunosuppressive effect on host hemocytes

An endoparasitoid wasp, Cotesia plutellae, induces immunosuppression of the host diamondback moth, Plutella xylostella. To identify an immunosuppressive factor, the parasitized hemolymph of P. xylostella was separated into plasma and hemocyte fractions. When nonparasitized hemocytes were overlaid with parasitized plasma, they showed significant reduction in bacterial binding efficacy. Here, we considered a viral lectin previously known in other Cotesia species as a humoral immunosuppressive candidate in C. plutellae parasitization. Based on consensus regions of the viral lectins, the corresponding lectin gene was cloned from P. xylostella parasitized by C. plutellae. Its cDNA is 674 bp long and encodes 157 amino acid residues containing a signal peptide (15 residues) and one carbohydrate recognition domain. Open reading frame is divided by one intron (156 bp) in its genomic DNA. Amino acid sequence shares 80% homology with that of C. ruficrus bracovirus lectin and is classified into C-type lectin. Southern hybridization analysis indicated that the cloned lectin gene was located at C. plutellae bracovirus (CpBV) genome. Both real-time quantitative RT-PCR and immunoblotting assays indicated that CpBV-lectin showed early expression during the parasitization. A recombinant CpBV-lectin was expressed in a bacterial system and the purified protein significantly inhibited the association between bacteria and hemocytes of nonparasitized P. xylostella. In the parasitized P. xylostella, CpBV-lectin was detected on the surface of parasitoid eggs after 24 h parasitization by its specific immunostaining. The 24 h old eggs were not encapsulated in vitro by hemocytes of P. xylostella, compared to newly laid parasitoid eggs showing no CpBV-lectin detectable and easily encapsulated. These results support an existence of a polydnaviral lectin family among Cotesia-associated bracovirus and propose its immunosuppressive function.