Importance of long-term research in classical biological control: an analytical review of a release against the cabbage seedpod weevil in North America

Abstract:  Cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham) (Col., Curculionidae), is an invasive alien pest that is spreading in North America. To aid with planning for introductions of European parasitoids in North America, we examined the status of the only classical biological control release against this pest in North America, which in 1949 introduced Mesopolobus morys , Stenomalina gracilis and Trichomalus perfectus (Hym., Pteromalidae). Weevils and parasitoids were reared in 2005 from mass collections of seedpods of Brassica napus , Brassica rapa and Raphanus raphanistrum (Brassicaceae) from 18 sites in the Fraser Valley, near Vancouver, British Columbia, Canada. Of the three European parasitoid species that were originally released, only S. gracilis was found. The predominant hymenopterous parasitoid species were Trichomalus lucidus , S. gracilis , Mesopolobus moryoides (Pteromalidae), Necremnus tidius (Eulophidae) and Eupelmus vesicularis (Eupelmidae). These constituted over 97% of the parasitoids reared, although overall parasitism was low. Only M. moryoides is clearly North American in distribution; other than S. gracilis , the remaining species were either accidentally introduced or are Holarctic in distribution. Based on these results, re-releases of M. morys and T. perfectus in North America should be considered as part of a classical biological control programme. However, redistribution of S. gracilis is not recommended at present because of potential conflicts with biological control programmes against weeds. Ongoing re-examination of classical biological control programmes can further our understanding of failure of release programmes, particularly when re-examination can be made in the light of improved taxonomy and systematics of the target and agent species.     

Avoiding conflicts between insect and weed biological control: selection of non-target species to assess host specificity of cabbage seedpod weevil parasitoids

Abstract:  Classical biological control of insect pests and weeds may lead to potential conflicts, where insect pests are closely related to weed biological control agents. Such a conflict may occur in the classical biological control of the cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham) in North America, which belongs to the same subfamily, Ceutorhynchinae, as a number of agents introduced or proposed for introduction against non-indigenous invasive weed species. We propose a step-by-step procedure to select non-target species and thereby to develop a non-target species test list for screening candidate entomophagous biological control agents of a herbivore pest insect in a way that would simultaneously evaluate non-target potential on weed biological control agents and other non-target species. Using these recommendations, we developed a non-target test list for host specificity evaluations in the area of origin (Europe) and the area of introduction (North America) for cabbage seedpod weevil parasitoids. Scientifically based predictions on expected host–parasitoid interactions and ecological information about the ecological host range in the area of origin can help avoid conflicts, while still allowing the introduction of safe and effective agents against both insect pests and weeds.     

Flight behaviour of the cabbage seedpod weevil

The influence of host odour, windspeed, position of the sun, and temperature on flight behaviour of the cabbage seedpod weevil (Ceutorhynchus assimilis Paykull) were studied. This weevil showed a positive anemotaxis (upwind flight) inside the odour plume of a host crop (Brassica napus L.). Outside the odour plume the weevil showed a pronounced phototaxis at windspeeds below 1.5 m/s. At higher windspeeds, the seedpod weevils flew downwind. The cabbage seedpod weevil flies most readily at low windspeeds (less than 0.5 m/s) and at temperatures above 22 °C.     

Determining the host specificity of the biological control agent Trichomalus perfectus (Hymenoptera: Pteromalidae): the importance of ecological host range

We determined the host range of the parasitoid Trichomalus perfectus (Walker), a candidate for classical biological control of cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham), an important pest of canola in Canada. Studies were conducted in Europe and in North America. In laboratory experiments, the levels of parasitism (acceptance) of Ceutorhynchus turbatus Schultze, C. cardariae Korotyaev, C. omissus Fall and C. querceti (Gyllenhal) by T. perfectus were not significantly different than of the target host C. obstrictus. Although C. typhae (Herbst), C. pallidactylus (Marsham), C. americanus Buchanan, C. neglectus Blatchely and Ceutorhynchus sp. nr. nodipennis were parasitised by T. perfectus, the levels of parasitism were significantly lower on these species than on C. obstrictus. Ceutorhynchus peyerimhoffi Hustache, C. erysimi (Fabricius), C. alliariae H. Brisout, C. roberti Gyllenhal, Mogulones borraginis (Fabricius), Mononychus vulpeculus (Fabricius) and the leaf-mining fly Scaptomyza flava (Fallén) were not attacked. Ecological host range surveys in Europe corroborated the prediction that T. perfectus would attack C. cardariae at similar rates to C. obstrictus. In North America, the recent discovery of T. perfectus in a C. omissus population suggests that laboratory findings predicting that C. omissus is a preferred host may be the case in the field. We found that T. perfectus attacks larvae of some Ceutorhynchus spp. feeding on Brassicaceae and does not attack species outside of that host range. Thus, the parasitoid can be defined as narrowly oligophagous. These results demonstrate the value of ecological host range studies in the area of origin to validate hypotheses generated through laboratory host range experiments.     

A test using Wolbachia bacteria to identify Eurasian source populations of cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham), in North America

Previous research using mitochondrial haplotypes indicates that North American populations of cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham), originated from at least two separate introductions from source populations in Eurasia. We tested this hypothesis by comparing the genetic variation of symbiotic Wolbachia bacteria in C. obstrictus among seven North American and four European populations. Because Wolbachia are maternally inherited, infections acquired by a host species at one geographic location theoretically may be present in derivative populations that have established in new regions. Use of the conserved MLST Wolbachia genes gatB, coxA, hcpA, fbpA, and ftsZ identified one strain present in all beetles. Use of the variable wsp gene identified three distinct isolates of this strain that appear to co-occur in all populations and potentially in all individuals. Use of the variable wspB gene provided independent support for the presence of these isolates and evidence of a wspB pseudogene. The lack of genetic variation for these Wolbachia genes among host populations prevents their use to clarify the origins of C. obstrictus in North America. However, the results are an interesting example illustrating disjunction in genetic variation between mitochondria and a maternally-inherited symbiont.     

Mortality factors affecting the cabbage seedpod weevil,Ceutorhynchus obstrictus (Marsham), in its area of origin: A life table analysis

The cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham) (Coleoptera: Curculionidae), is a widely distributed invasive pest of cruciferous crops in North America. Control measures rely mostly on the application of insecticides but alternative control strategies such as classical biological control are under evaluation. To investigate the impact of parasitoids and other mortality factors on C. obstrictus populations, life table studies were conducted between 2005 and 2007 in 13 winter oilseed rape fields in Switzerland, part of the native range. Under field conditions females only realized approximately 50% of their potential lifetime fecundity, varying between 96 and 631 eggs per individual. Total generational mortality was higher than 99.6% in each year. Overwintering mortality of adults was the major population limiting factor, contributing approximately 50% to the overall generational mortality of C. obstrictus, whereas factors acting on the immature stages were responsible for the remaining 50%. Among the mortality factors of the immature stages, egg, larval and pupal mortality contributed 9–12%, 25–28% and 4–6% to the generational mortality, respectively. Larval ecto-parasitism on its own accounted for 7–15% of the generational mortality. Comparison of life table data presented here with that from North America will be invaluable for elucidating the mortality factors that regulate C. obstrictus populations in Europe, the region of origin.     

Incorporation of Novel <Emphasis Type="Italic">Ceutorhynchus obstrictus</Emphasis>-resistant Canola Genotypes Into Mixed Cropping Strategies: Evidence for Associational Resistance

Canola genotypes resistant to the cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham) (Coleoptera: Curculionidae), have recently been developed through introgression of Sinapis alba L. to Brassica napus L. Several lines express antixenosis and antibiosis resistance and have been shown to be less attractive to weevils in visual and olfactory behavioral bioassays. This paper details a small-plot study that assessed the effects on distribution dynamics of weevil adults and larvae of interspersing susceptible among resistant genotypes relative to monocultures over two growing seasons. Results indicate that mixes reduced weevil numbers and oviposition in pods of susceptible genotypes. These results are consistent with associational resistance.     

Bottom-up effects of Brassica napus nutrition on the oviposition preference and larval performance of Ceutorhynchus obstrictus (Coleoptera: Curculionidae)

The cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham) (Coleoptera: Curculionidae), is a serious pest of canola, Brassica napus L. and Brassica rapa L., both in Europe and North America. Nitrogen and sulfur levels can be readily manipulated by growers and could potentially be exploited to improve current pest management strategies. In an effort to better understand the relationship between host plant nutrition and C. obstrictus oviposition preference and its larval developmental biology, we exposed gravid females to host plants grown under differing regimes of nitrogen and sulfur. Results indicated that plants grown with a higher supply of nitrogen were preferred as hosts. Plants with a higher sulfur supply were also preferred but only in plants grown at lower nitrogen levels. In contrast, larval development time increased with increasing nitrogen levels although larval dry weights were unaffected. The results were inconsistent with the preference-performance hypothesis. The inconsistencies between oviposition choice and larval growth and development may be due to a conflict between maternal and larval fitness. Observed changes in larval development time may not represent a significant fitness cost for larval C. obstrictus, as no corresponding change in larval weight was observed. Possible causal mechanisms for the increase in development time include differences in oil, protein and glucosinolate contents of the seed. Fertilizer management regimes currently recommended were considered to be optimal for management of C. obstrictus as the yield benefits from higher rates of nitrogen fertilization would more than compensate for increased level of infestation.     

Use of life tables to predict the impact of introducing exotic parasitoids,against the cabbage seedpod weevil in North America

ABSTRACT

Cabbage seedpod weevil, Ceutorhynchus obstrictus (Coleoptera: Curculionidae), is an invasive alien species that impacts seed production of canola, Brassica napus and B. rapa (Brassicaceae), one of Canada’s major agricultural crops. To determine the need for and potential impact of introduction of one or more natural enemies for biological control it is essential to understand the mortality factors associated with C. obstrictus both in the native range and in the introduced range. Life tables were constructed for Ontario and British Columbia populations to determine the mortality factors affecting C. obstrictus. These were compared with life tables generated in Switzerland, the area of origin of the weevil. Results indicated that introduction of an exotic specialist parasitoid into canola growing regions of Canada would substantially reduce C. obstrictus populations. This study is the first to compare life tables of an invasive pest in the areas of origin and introduction.     

Biology and host specificity of Ceutorhynchus scrobicollis (Curculionidae; Coleoptera), a root-crown mining weevil proposed as biological control agent against Alliaria petiolata in North America

Ceutorhynchus scrobicollis is a root-crown mining weevil proposed for release as biological control agent of Alliaria petiolata (Brassicaceae, Thlaspideae), a European biennial herb, currently invading temperate North America. Using a combination of laboratory, common garden and fieldwork we studied biology, ecology and host range of C. scrobicollis, a univoltine species that oviposits and develops in A. petiolata rosettes in fall and spring. Individual C. scrobicollis can be long-lived (>2 years) and females show a second oviposition period. Weevils did not attack any of 31 test plant species outside the Brassicaceae. Within the Brassicaceae, five species allowed complete larval development under no-choice conditions. In subsequent choice tests, three of these five species (Nasturtium officinale, Peltaria alliacea and Thlaspi arvense; which are of European origin) were attacked. North American Rorippa sinuata was the only native species to be attacked by C. scrobicollis and only under no-choice conditions. Results of subsequent impact experiments showed that C. scrobicollis attack changed plant architecture but had no effect on overall plant vigour and reproductive output of R. sinuata, suggesting lack of impact on demography or population dynamics. A petition for field release of C. scrobicollis in North America has been submitted.     

Plant nutrients and the spatiotemporal distribution dynamics of Ceutorhynchus obstrictus (Coleoptera: Curculionidae) and its parasitoids

The cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham), is an alien invasive pest of canola, Brassica napus L., in North America. Field populations of both adults and larvae are known to be aggregated in their distributions, but the causal mechanism for this clustering is not understood. We investigated the hypothesis that spatial distributions of C. obstrictus adults, larvae, and their parasitoids were directly or indirectly related to host plant quality as indicated by leaf tissue nutrient contents. During the years of 2005 and 2007, these insect populations were sampled in a grid pattern in three commercial fields of B. napus located near Lethbridge, Alberta, Canada. Leaf samples were collected from plants in each grid plot and analyzed for nutrient contents. Spatial patterns and associations among the collected variables were analyzed using Spatial Analysis by Distance IndicEs software. We observed significant spatial associations among C. obstrictus adults, larvae, and parasitoids. The distributions of C. obstrictus adults and larvae were correlated with several plant nutrients, most notably nitrogen and sulfur. Evidence was found for a possible nitrogen-sulfur interaction in which ovipositioning C. obstrictus females appeared to prefer plants with high levels of sulfur and low levels of nitrogen. Spatial tracking of C. obstrictus larvae by its parasitoids was evident in only one field, and this appeared related to comparatively low parasitoid populations in two of the three fields. The associations between C. obstrictus and plant nutrients may have possible applications in fertility management, trap cropping, and precision insecticide application technology.     

Flight activity and dispersal of the cabbage seedpod weevil (Coleoptera: Curculionidae) are related to atmospheric conditions

The cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham) (Coleoptera: Curculionidae), is an invasive pest of canola (Brassica napus L. and Brassica rapa L.) in western Canada. Under current climatic conditions, C. obstrictus is spreading from established populations in southwestern Alberta at ≈ 55 km/yr. We studied the influence of climatic conditions on C. obstrictus flight behavior in 2007 and 2008 and eastward dispersal from the western border of Saskatchewan from 2002 to 2007. Positive linear relationships between increases in mean temperature and flight height and between greater mean maximum temperature and expanded dispersal distances were significant. Increases in relative humidity were associated with reduced flight heights and dispersal distances. We developed models that predict the relationships of temperature and relative humidity with flight height and with dispersal distance. We also discuss implications for C. obstrictus dispersal under current climatic conditions and in the context of predicted climate change.     

Host range of Ceutorhynchus assimilis (Coleoptera: Curculionidae), a candidate for biological control of Lepidium draba (Brassicaceae) in the USA

Ceutorhynchus assimilis has been selected as a potential biological control agent of Lepidium draba, which is a Eurasian invasive weed in North America. Preliminary studies indicated specificity of this weevil collected in southern France on L. draba. This result was in discord with the pest status of C. assimilis found in the literature. Host-specificity tests based both on field and laboratory experiments showed heterogeneity in the host spectrum of the weevils reared from different host-plants as determined by larval development. However, no distinguishable morphological differences could be visually detected between the populations feeding on different host-plants. All sampled populations of weevils were polyphagous as adults. Weevils reared from L. draba were specific to this plant for their complete larval development. Conversely, populations living on other wild and cultivated Brassicaceae species were not able to use L. draba as a host plant. Such differentiation is further highlighted by other biological aspects such as plant infestation rates, sex-ratio, duration of larval development, and differences in the timing of their life cycles. These results demonstrate that C. assimilis, an insect species formerly considered as a pest of Brassicaceae, is characterized by its host-range variability, with one population being potentially useful in the biological control of L. draba. Moreover, this example points to the need to test multiple populations of biological control agents in assessing risk.     

Baseline susceptibility and insecticide resistance monitoring in European populations of Meligethes aeneus and Ceutorhynchus assimilis collected in winter oilseed rape

Pollen beetle, Meligethes aeneus (Fabricius) (Coleoptera: Nitidulidae), and cabbage seed weevil, Ceutorhynchus assimilis (Paykull) (Coleoptera: Curculionidae), are important pests in the production of European winter oilseed rape, Brassica napus L. (Brassicaceae), which is grown on several million hectares in Europe. Insecticide treatments are common to control both pests once they exceed economic damage thresholds; however, not many chemical classes are available for their control in European oilseed rape. Particularly pollen beetles recently developed high levels of pyrethroid resistance impairing field control at recommended rates in many countries, whereas no resistance is yet reported to another important insecticide, thiacloprid. The major objective of this study was to investigate the spatio‐temporal susceptibility status of pollen beetle against the recently introduced insecticide thiacloprid. From 2009 to 2012 more than 630 populations of pollen beetle collected in 13 countries were monitored for resistance to thiacloprid by using an adult vial test. No shifting to lower susceptibility of pollen beetle to thiacloprid has been observed between 2009 and 2012. Furthermore, we were able to show that pollen beetle larvae are extremely susceptible to thiacloprid, whereas within strains larvae are significantly more resistant than adults to pyrethroids such as lambda‐cyhalothrin. Dose‐response data for thiacloprid against cabbage seed weevil populations collected in 2011 in Germany, Sweden, and Ukraine showed a 10‐fold higher intrinsic sensitivity compared to pollen beetle, and showed only a low variation in response. In addition, we also tested 17 cabbage seed weevil populations collected in five countries against lambda‐cyhalothrin with low variation in response (three‐fold), suggesting full baseline susceptibility and no resistance to pyrethroids. The implications of the data presented for resistance management in coleopteran pests in winter oilseed rape are discussed.     

Biology and host specificity of Aulacobaris fallax (Coleoptera: Curculionidae), a potential biological control agent for dyer’s woad,Isatis tinctoria (Brassicaceae) in North America

Dyer’s woad, Isatis tinctoria, a plant of Eurasian origin is a problematic weed in western North America against which a classical biological weed control programme was initiated in 2004. Three European insect species were selected as candidate agents to control this invasive species, including the root‐mining weevil Aulacobaris fallax. To determine its suitability as an agent, the biology and host specificity of A. fallax were studied in outdoor plots and in the field between 2004 and 2006 in its native European range. Aulacobaris fallax is a univoltine species that lays its eggs from March to August into leaf stalks and roots of dyer’s woad. Larvae mine and pupate in the roots and adults emerge from August to October. Up to 62% of the dyer’s woad plants at the field sites investigated were attacked by this weevil. In no‐choice host‐specificity tests, A. fallax attacked 16 out of 39 species and varieties within the Family Brassicaceae. Twelve of these are native to North America. In subsequent multiple‐choice tests, seven species, all native to North America, suffered a similar level of attack as dyer’s woad, while none of the European species were attacked. Our results demonstrate the importance of including test plant species that have not co‐evolved with the respective candidate agent. In sum, we conclude that the risk of non‐target effects is too high for A. fallax to be considered as a biological control agent for dyer’s woad in the United States.     

Identities and provenance of the gelechioid moths originally described by Francis Walker from 'unknown countries'

Abstract. The taxonomic placement and possible type-localities of twelve species and one genus of Gelechioidea described by Francis Walker from 'unknown countries' are discussed. Opisina arenosella Walker is the senior synonym of Nephantis serinopa Meyrick and Opisina Walker is the senior synonym of Nephantis serinopa Meyrick and Opisina Walker the senior synonym of Nephantis Meyrick; Aroga compositella (Walker), new combination, is the senior synonym of A.coloradensis (Busck); Cryptolechia metaphaeella Walker is a junior synonym of Antaeotricha schlaegeri (Zeller); A.albulella (Walker), new combination, is the senior synonym of A.vestalis (Zeller), and A.spurcatella (Walker), new combination, is the senior synonym of A.chloromis (Meyrick). A lectotype for C.metaphaeella Walker is designated.     

Cold hardiness and overwintering survival of the cabbage seedpod weevil,Ceutorhynchus obstrictus

The cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham) (Coleoptera: Curculionidae), is a serious pest of brassicaceous crops in temperate regions and a chronic member of the pest complex that attacks canola in Canada. We conducted several laboratory and field experiments to quantify winter survival and its role in the population dynamics of this insect. We estimated the supercooling point of the weevil at ?7 °C and its survival over 8 weeks decreased significantly at ?5 °C relative to 5 °C, but extending the overwintering period at 5 °C to 18.5 weeks had no effect on mortality. Cumulative sub‐freezing degrees estimated from air temperature, and especially from soil temperature, were highly correlated with weevil survival. Our linear regression model predicted poor survival of the weevils in typical winters in northern Alberta. Our results indicate that if milder winters prevail, as predicted by global warming, there is potential for the weevils to establish and become a serious pest in northern canola‐growing regions of Canada.     

Biology and field host range of Ceutorhynchus cardariae,a potential biological control agent for Lepidium draba

Lepidium draba (Brassicaceae) is a clonal herb, originating from Eurasia, which is invasive in North America. A classical biological control project was initiated in 2001, and the gall‐forming weevil Ceutorhynchus cardariae was prioritized as a candidate agent. We studied its biology and field host range between 2003 and 2014 in the laboratory and a common garden in Switzerland and in the field in Romania. Ceutorhynchus cardariae is a univoltine to bivoltine species. In Switzerland, oviposition usually started at the beginning of March and can occur at temperatures as low as 2.5°C. Galls are formed on stems, leaf stalks and midribs of L. draba rosettes and bolting plants. Gall size increased with an increasing number of larvae per gall. The three larval instars feed inside the galls and leave the plant to pupate in the soil once mature. In Switzerland, development from egg to adult took about 12 weeks in spring. Adults emerged from May to July. After a brief feeding period, adults aestivate. From late summer, feeding recommenced and females may oviposit, forming a partial second generation. Eggs and all larval instars can be found in galls throughout winter. The rate of larval ectoparasitism reached 78%, while endoparasitism was low with a maximum of 2.3%. Lepidium draba populations differed in their suitability for development (number of C. cardariae produced), indicating that effectiveness of C. cardariae – in case released – may be variable. In the field, we observed that gall formation by C. cardariae can severely stunt or even kill shoots. Investigations on the field host range of C. cardariae indicated that only the closely related Lepidium campestre may act as an alternative host for the weevil in Europe. Host‐specificity tests are underway to determine its environmental safety before field release in North America is being considered.     

The responses of the cabbage seed weevil (Ceutorhynchus assimilis) to the odour of oilseed rape (Brassica napus) and to some volatile isothiocyanates

The responses of the cabbage seed weevil,Ceutorhynchus assimilis Payk. (Coleoptera: Curculionidae) to volatiles from oilseed rape (Brassica napus L.) were tested using a linear track olfactometer. Weevils were attracted towards the odour of rape during a short period before diapause and for most of their postdiapause life. Odours from both the flowering and green parts of the plant were attractive, but the odour of a non-host plant (Bellis perennis L.) was not. An entrainment extract of flowering rape volatiles in pentane was attractive, but significantly less so than the odour of flowering rape itself. Attraction was found to 3-butenyl and 4-pentenyl isothiocyanate (NCS), but not to 2-phenylethyl isothiocyanate. A mixture of the three NCSs was more attractive than the individual NCSs. However, the concentration required to produce a response was still relatively high. Isothiocyanates, along with other volatiles from rape, probably play a role in host plant recognition by the seed weevil.     

The spatio-temporal distribution dynamics of the cabbage seedpod weevil, Ceutorhynchus obstrictus (Coleoptera: Curculionidae), and its larval parasitoids in canola in western Canada

Distribution patterns of the cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham) (Coleoptera: Curculionidae), and its larval parasitoids were investigated in commercial fields of spring canola (Brassica rapa L. and Brassica napus L.) in southern Alberta, Canada, from 2002 to 2004 in relation to developmental stages of its host plants. Adult weevils invaded fields along one or more fronts when crops were in bud to early flower. Significant clustering of adults along field edges in early stages of invasion was followed by more homogeneous distributions as canola reached the mid to late flowering and pod enlargement stages. Larval weevil distributions, as indicated by exit holes in siliques at the end of the season, were often aligned spatially with adult distributions, but they did not coincide in all regions of the fields. The primary ectoparasitoid species attacking weevil larvae comprised Necremnus tidius (Walker) (Hymenoptera: Eulophidae), and Trichomalus lucidus (Walker), Chlorocytus sp., and Pteromalus sp. (Hymenoptera: Pteromalidae). Parasitism rates increased from 0.1 to 5.0% over the three years of study. Parasitoid distributions were often, but not consistently, spatially associated with high densities of C. obstrictus larvae. Lack of close spatial alignment of parasitoids and their hosts probably reflects low parasitoid numbers in comparison with an abundant resource of weevil larvae, and a lack of co-evolutionary history between host and parasitoids. Some parasitoids invaded fields early in host plant development, at the same time that weevils invaded. Unfortunately the synchronous invasions of host and parasitoids indicate that insecticidal applications to reduce adult weevil infestations may be detrimental to these beneficial species.