Genetic diversity of endosymbiotic bacteria Wolbachia infecting two mosquito species of the genus Eretmapodites occurring in sympatry in the Comoros archipelago.

Introduction: The influence of Wolbachia on mosquito reproduction and vector competence has led to renewed interest in studying the genetic diversity of these bacteria and the phenotypes they induced in mosquito vectors. In this study, we focused on two species of Eretmapodites, namely Eretmapodites quinquevittatus and Eretmapodites subsimplicipes, from three islands in the Comoros archipelago (in the Southwestern Indian Ocean). Methods: Using the COI gene, we examined the mitochondrial genetic diversity of 879 Eretmapodites individuals from 54 sites. Additionally, we investigated the presence and genetic diversity of Wolbachia using the wsp marker and the diversity of five housekeeping genes commonly used for genotyping through Multiple Locus Sequence Typing (MLST). Results and discussion: Overall, Er. quinquevittatus was the most abundant species in the three surveyed islands and both mosquito species occurred in sympatry in most of the investigated sites. We detected a higher mitochondrial genetic diversity in Er. quinquevittatus with 35 reported haplotypes (N = 615 specimens, Hd = 0.481 and π = 0.002) while 13 haplotypes were found in Er. subsimplicipes (N = 205 specimens, Hd = 0.338 and π = 0.001), this difference is likely due to the bias in sampling size between the two species. We report for the first time the presence of Wolbachia in these two Eretmapodites species. The prevalence of Wolbachia infection varied significantly between species, with a low prevalence recorded in Er. quinquevittatus (0.8%, N = 5/627) while infection was close to fixation in Er. subsimplicipes (87.7%, N = 221/252). Both male and female individuals of the two mosquito species appeared to be infected. The analysis of MLST genes revealed the presence of two Wolbachia strains corresponding to two new strain types (STs) within the supergroups A and B, which have been named wEretA and wEretB. These strains were found as mono-infections and are closely related, phylogenetically, to Wolbachia strains previously reported in Drosophila species. Finally, we demonstrate that maternal transmission of Wolbachia is imperfect in Er. subsimplicipes, which could explain the presence of a minority of uninfected individuals in the field.

Combining transinfected Wolbachia and a genetic sexing strain to control Aedes albopictus in laboratory-controlled conditions.

The global expansion of Aedes albopictus has stimulated the development of environmentally friendly methods aiming to control disease transmission through the suppression of natural vector populations. Sterile male release programmes are currently being deployed worldwide, and are challenged by the availability of an efficient sex separation which can be achieved mechanically at the pupal stage and/or by artificial intelligence at the adult stage, or through genetic sexing, which allows separating males and females at an early development stage. In this study, we combined the genetic sexing strain previously established based on the linkage of dieldrin resistance to the male locus with a Wolbachia transinfected line. For this, we introduced either the wPip-I or the wPip-IV strain from Culex pipiens in an asymbiotic Wolbachia-free Ae. albopictus line. We then measured the penetrance of cytoplasmic incompatibility and life-history traits of both transinfected lines, selected the wPip-IV line and combined it with the genetic sexing strain. Population suppression experiments demonstrated a 90% reduction in population size and a 50% decrease in hatching rate. Presented results showed that such a combination has a high potential in terms of vector control but also highlighted associated fitness costs, which should be reduced before large-scale field assay.

Evidence of Eretmapodites subsimplicipes and Aedes albopictus as competent vectors for Rift Valley fever virus transmission in Mayotte.

Rift Valley fever (RVF) recently re-emerged in Mayotte. We described, for the first time, that the mosquito species Eretmapodites subsimplicipes, a highly abundant species in Mayotte, is a competent vector for the transmission of RVF virus using three distinct populations native to Mayotte. We also showed that Aedes albopictus specimens are able to transmit RVF virus (RVFV) as previously observed in mosquito populations of other countries emphasizing the need of the increase vigilance for this highly invasive species of global distribution. Altogether, these results underline the epidemiological importance of both species for RVFV transmission in Mayotte and contribute to better understand the RVF epidemiological cycle and help to implement efficient prevention measures.

Fitness costs associated with a GABA receptor mutation conferring dieldrin resistance in Aedes albopictus.

Understanding the dynamics of insecticide resistance genes in mosquito populations is pivotal for a sustainable use of insecticides. Dieldrin resistance in Aedes albopictus is conferred by the alanine to serine substitution (A302S or RdlR allele) in the γ-aminobutyric acid (GABA) receptor encoded by the Rdl gene. On Reunion Island, dieldrin resistance was initially reported in natural Ae. albopictus populations sampled in 2008 despite the ban of dieldrin since 1994. To monitor insecticide resistance in Ae. albopictus on the island and to identify its drivers, we measured (i) the frequency of resistance alleles in 19 distinct natural populations collected between 2016 and 2017, (ii) fitness costs associated with dieldrin resistance in laboratory-controlled experiments, and (iii) the resistance conferred by RdlR to fipronil, an insecticide widely used on the island and reported to cross-react with RdlR. The results show a persistence of RdlR in Ae. albopictus natural populations at low frequencies. Among the measured life history traits, mortality in pre-imaginal stages, adults' survival as well as the proportion of egg-laying females were significantly affected in resistant mosquitoes. Finally, bioassays revealed resistance of RdlR mosquitoes to fipronil, suggesting that the use of fipronil in natura could select for the RdlR allele. This study shows that dieldrin resistance is persistent in natural mosquito populations likely as a result of combined effects between fitness costs associated with RdlR and selection exerted by cross-reacting environmental insecticides such as fipronil.

Spatio-Temporal Dynamics of a Dieldrin Resistance Gene in Aedes albopictus and Culex quinquefasciatus Populations From Reunion Island.

The control of mosquito populations using insecticides is increasingly threatened by the spread of resistance mechanisms. Dieldrin resistance, conferred by point mutations in the Rdl gene encoding the γ-aminobutyric acid receptor, has been reported at high prevalence in mosquito populations in response to selective pressures. In this study, we monitored spatio-temporal dynamics of the resistance-conferring RdlR allele in Aedes (Stegomyia) albopictus (Skuse, 1895) and Culex (Culex) quinquefasciatus (Say, 1823) populations from Reunion Island. Specimens of both mosquito species were sampled over a 12-month period in three cities and in sites located at lower (<61 m) and higher (between 503 and 564 m) altitudes. Mosquitoes were genotyped using a molecular test detecting the alanine to serine substitution (A302S) in the Rdl gene. Overall, the RdlR frequencies were higher in Cx. quinquefasciatus than Ae. albopictus. For both mosquito species, the RdlR frequencies were significantly influenced by location and altitude with higher RdlR frequencies in the most urbanized areas and at lower altitudes. This study highlights environmental factors that influence the dynamics of insecticide resistance genes, which is critical for the management of insecticide resistance and the implementation of alternative and efficient vector control strategies.

Contrasted transmission efficiency of Zika virus strains by mosquito species Aedes aegypti, Aedes albopictus and Culex quinquefasciatus from Reunion Island.

BACKGROUND: Zika virus (ZIKV) is a mosquito-borne flavivirus that recently emerged in the South Pacific islands and Americas where unprecedented outbreaks were reported. Although Aedes aegypti is considered to be the main vector for ZIKV, other mosquito species have been shown to be potential vectors and differences in vector competence with respect to mosquito strain and ZIKV strain have been demonstrated. In this study we compared the vector competence of three mosquito species Aedes albopictus, Ae. aegypti and Culex quinquefasciatus from Reunion Island for three ZIKV strains. METHODS: Five mosquito strains (2 strains of Ae. albopictus, 1 of Ae. aegypti and 2 of Cx. quinquefasciatus) were exposed to three ZIKV strains: one African strain (Dak84) and two Asian strains (PaRi_2015 and MAS66). The vector competence parameters (infection rate, dissemination efficiency and transmission efficiency) and viral loads were examined at 14 and 21 days post-infection. RESULTS: The two Cx. quinquefasciatus strains did not become infected and were therefore unable to either disseminate or transmit any of the three ZIKV strains. Aedes albopictus and Ae. aegypti strains were poorly competent for the two Asian ZIKV strains, while both mosquito species displayed higher infection rates, dissemination and transmission efficiencies for the African ZIKV Dak84 strain. However, this African ZIKV strain was better transmitted by Ae. aegypti as compared to Ae. albopictus. CONCLUSIONS: Our results show that both Ae. albopictus and Ae. aegypti, from Reunion Island, are more likely to be competent for ZIKV in contrast to Cx. quinquefasciatus which appeared refractory to all tested ZIKV strains. This improves our understanding of the role of mosquito species in the risk of the ZIKV emergence on Reunion Island.

Aedes albopictus Adult Medium Mass Rearing for SIT Program Development.

For the production of several hundred thousands of Aedes albopictus sterile males for the implementation of a Sterile Insect Technique (SIT) program, no costly mass-rearing equipment is needed during the initial phases, as optimized rearing at laboratory scale can be sufficient for the first steps. The aim of this study was to maximize the egg production by optimizing adult rearing methods for Ae. albopictus. The effect of parameters such as male/female ratio, density of adults, membrane type for blood feeding, quantity of blood delivered, continuous or discontinuous blood feeding, and surface of substrates for egg laying on overall egg production was tested to find optimized conditions. Based on the number of eggs produced per cage in response to the parameters tested, the optimum cage set-up was seen to be 1500 adults in a 30 × 30 × 30 cm cage with a male/female sex ratio of 1:3, fed by fresh bovine blood for periods of 30 min using a cellulose membrane covering a 10 cm stainless steel plate heated by a Hemotek device, and the provision of five oviposition cups to collect eggs. With this set-up, production per cage can reach a maximum of 35,000 eggs per week.

Field evaluation of seasonal trends in relative population sizes and dispersal pattern of Aedes albopictus males in support of the design of a sterile male release strategy.

BACKGROUND: To develop an efficient sterile insect technique (SIT) programme, the number of sterile males to release, along with the spatial and temporal pattern of their release, has to be determined. Such parameters could be estimated from a reliable estimation of the wild population density (and its temporal variation) in the area to treat. Here, a series of mark-release-recapture experiments using laboratory-reared and field-derived Aedes albopictus males were carried out in Duparc, a selected pilot site for the future application of SIT in the north of La Reunion Island. METHODS: The dispersal, longevity of marked males and seasonal fluctuations in the population size of native mosquitoes were determined from the ratio of marked to unmarked males caught in mice-baited BG-Sentinel traps. The study was conducted during periods of declining population abundance (April), lowest abundance (September) and highest abundance (December). RESULTS: According to data collected in the first 4 days post-release, the Lincoln index estimated population size as quite variable, ranging from 5817 in April, to 639 in September and 5915 in December. Calculations of daily survival probability to 4 days after release for field and laboratory males were 0.91 and 0.98 in April, respectively, and 0.88 and 0.84 in September, respectively. The mean distance travelled (MDT) of released field males were 46 m, 67 m and 37 m for December, April and September experiments, respectively. For released laboratory males, the MDT was 65 m and 42 m in April and September, respectively. CONCLUSIONS: Theoretically, the most efficient release programme should be started in July/August when the mosquito population size is the lowest (c.600 wild males/ha relative to 5000 wild males estimated for December and April), with a weekly release of 6000 males/ha. The limited dispersal of Ae. albopictus males highlights the nessecity for the widespread release of sterile males over multiple sites and in a field setting to avoid topographical barriers and anthropogenic features that may block the migration of the released sterile male mosquitoes.

Construction of a genetic sexing strain for Aedes albopictus: a promising tool for the development of sterilizing insect control strategies targeting the tiger mosquito.

BACKGROUND: Aedes albopictus is an invasive mosquito species of global medical concern as its distribution has recently expanded to Africa, the Americas and Europe. In the absence of prophylaxis protecting human populations from emerging arboviruses transmitted by this mosquito species, the most straightforward control measures rely on the suppression or manipulation of vector natural populations. A number of environmental-friendly methods using mass releases of sterilizing males are currently under development. However, these strategies are still lacking an efficient sexing method required for production of males at an industrial scale. RESULTS: We present the first Genetic Sexing Strain (GSS) in Ae. albopictus, hereafter referred as Tikok, obtained by sex linkage of the rdl gene conferring dieldrin resistance. Hatch rate, larval survival and sex ratio were followed during twelve generations. The use of dieldrin at the third larval stage allowed selecting 98 % of males on average. CONCLUSION: A good production rate of Tikok males makes this GSS suitable for any control method based on mass production of Ae. albopictus males. Despite limitations resulting from reduced egg hatch as well as the nature of the used insecticide, the construction of this GSS paves the way for industrial sex separation of Ae. albopictus.

Laboratory evaluation of the effects of sterilizing doses of γ-rays from Caesium-137 source on the daily flight activity and flight performance of Aedes albopictus males.

The control of Aedes albopictus through Sterile Male Releases requires that the most competitive males be mass-reared and sterilized usually with gamma- or X-ray radiation prior to release. Developing an understanding of the impact of irradiation treatment on flight performance in sterile males is very important because any fitness cost may reduce the efficacy of SIT intervention in the field. Here, we examined the role of irradiation exposure and sugar-feeding on daily flight activity and performance of Ae. albopictus males sterilized during pupal stage with gamma-radiation at 35Gray from a Caesium 137 source. We used a previously developed automated video tracking system to monitor the flight activity of different groups of sterile and control non-sterile males over 24 hours in a flight arena. This monitoring took place under controlled laboratory conditions and we wished to quantify the daily flight activity and to highlight any changes due to radiation treatment and nutritional conditions (starved versus sugar fed). Our experimental evidence demonstrated a characteristic diurnal flight activity with a bimodal pattern regardless of the treatment. Precisely, both irradiated and non-irradiated males exhibited two distinct peaks in flight activity in the morning (6-8 a.m.) and late afternoon (4-6 p.m.). Under changing physiological conditions, irradiated males were generally more active over time and flew longer overall distances than control male populations. These results suggest some internal circadian control of the phase relation to the light-dark cycle, with evidence for modification of flight performance by nutritional status. The fact that daily activity patterns are alike in irradiated and control Ae. albopictus males, and that sterile males could display higher flight performance, is in contrast with the hypothesis that irradiation treatment appears to reduce the fitness of male mosquitoes. We discuss the implications of the present study in sterile-male release programs against Ae. albopictus.

Comparison of Efficiency of BG-Sentinel Traps Baited with Mice, Mouse-Litter, and CO2 Lures for Field Sampling of Male and Female Aedes albopictus Mosquitoes.

Determining the abundance and distribution of male mosquitoes in the wild and establishing species seasonality in candidate pilot sites is of particular interest with respect to the use of the sterile-male technique. With the knowledge that using mice as bait in BG-Sentinel traps effectively enhances Aedes albopictus male and female trapping success, the present study was designed to determine whether attractants derived from mouse odour blend could be a viable substitute for live mice to lure Ae. albopictus mosquitoes into traps. The effects of baiting BG-Sentinel traps with mice, carbon dioxide (CO2), and attractants derived from litter mouse odours (mouse litter (ML)) and a mouse odour blend (MOB) on the efficiency of trapping Ae. albopictus males and females were tested using a Latin square design. The BG-Sentinel trap baited with CO2 + ML caught a significantly larger number of mosquitoes compared to traps baited with mice only. The BG-Sentinel traps containing only CO2 or CO2 + MOB, however, did not catch significantly more mosquitoes compared to the other traps. The proportions of males caught in the BG-Sentinel traps did not differ significantly between the respective attractants. The results from this study confirm that CO2 bait is efficient to provide a reliable estimation method for Ae. albopictus adult male abundance in the wild, and suggest that mouse litter baits in combination with CO2 could be used to enhance Aedes trapping success in BG-Sentinel traps.

Cross-Mating Compatibility and Competitiveness among Aedes albopictus Strains from Distinct Geographic Origins - Implications for Future Application of SIT Programs in the South West Indian Ocean Islands.

The production of large numbers of males needed for a sustainable sterile insect technique (SIT) control program requires significant developmental and operational costs. This may constitute a significant economic barrier to the installation of large scale rearing facilities in countries that are undergoing a transition from being largely dependent on insecticide use to be in a position to integrate the SIT against Aedes albopictus. Alternative options available for those countries could be to rely on outsourcing of sterile males from a foreign supplier, or for one centralised facility to produce mosquitoes for several countries, thus increasing the efficiency of the mass-rearing effort. However, demonstration of strain compatibility is a prerequisite for the export of mosquitoes for transborder SIT applications. Here, we compared mating compatibility among Ae. albopictus populations originating from three islands of the South Western Indian Ocean, and assessed both insemination rates and egg fertility in all possible cross-mating combinations. Furthermore, competitiveness between irradiated and non-irradiated males from the three studied strains, and the subsequent effect on female fertility were also examined. Although morphometric analysis of wing shapes suggested phenoptypic differences between Ae. albopictus strains, perfect reproductive compatibility between them was observed. Furthermore, irradiated males from the different islands demonstrated similar levels of competitiveness and induced sterility when confronted with fertile males from any of the other island populations tested. In conclusion, despite the evidence of inter-strain differences based on male wing morphology, collectively, our results provide a new set of expectations for the use of a single candidate strain of mass-reared sterile males for area-wide scale application of SIT against Ae. albopictus populations in different islands across the South Western Indian Ocean. Cross-mating competitiveness tests such as those applied here are necessary to assess the quality of mass reared strains for the trans-border application of sterile male release programs.

Enhancement of the BG-sentinel trap with varying number of mice for field sampling of male and female Aedes albopictus mosquitoes.

BACKGROUND: Trapping male mosquitoes in the field is essential for the development of area-wide vector control programs with a sterile insect technique (SIT) component. To determine the optimal temporal and spatial release strategy, an estimation of the wild population density and its temporal dynamics is essential. Among the traps available for such data collection, the BG-Sentinel trap developed by the Biogents company uses a combination of visual cues, convection currents and olfactory signals. Although in numerous cases, this trap has shown high efficiency in sampling Aedes albopictus, in some cases low capture rates of Ae. albopictus males were recorded for the BG-sentinel mosquito trap baited with synthetic attractants. METHODS: The effects of modifying the BG-sentinel trap (by adding one mouse, two or three live mice to the trap) on the efficiency of trapping Ae. albopictus males and females was tested. The experiment was carried out in three distinct areas on La Réunion that have been selected for pilot field testing of the release of sterile male Ae. albopictus mosquitoes. The effect of four types of attractant (including the generic BG-Lure, one mouse or two to three mice) in baited BGS traps was tested with a Latin square design in order to control for the variability of different sampling positions and dates. RESULTS: At the three studied sites, the number of Ae. albopictus adults caught and the proportion of males per trap consistently increased with the number of mice present in the trap. CONCLUSION: The results from this study suggest that some new attractants derived from, or similar to, mouse odors could be developed and tested in combination with other existing attractive components, such as CO2 and heat, in order to provide a reliable estimation method for Ae. albopictus adult male abundance in the wild.

Comparison of Irradiation and Wolbachia Based Approaches for Sterile-Male Strategies Targeting Aedes albopictus.

The global expansion of Aedes albopictus together with the absence of vaccines for most of the arboviruses transmitted by this mosquito has stimulated the development of sterile-male strategies aiming at controlling disease transmission through the suppression of natural vector populations. In this context, two environmentally friendly control strategies, namely the Sterile Insect Technique (SIT) and the Wolbachia-based Incompatible Insect Technique (IIT) are currently being developed in several laboratories worldwide. So far however, there is a lack of comparative assessment of these strategies under the same controlled conditions. Here, we compared the mating capacities, i.e. insemination capacity, sterilization capacity and mating competitiveness of irradiated (35 Gy) and incompatible Ae. albopictus males at different ages and ratios under laboratory controlled conditions. Our data show that there was no significant difference in insemination capacity of irradiated and incompatible males, both male types showing lower capacities than untreated males at 1 day but recovering full capacity within 5 days following emergence. Regarding mating competitiveness trials, a global observed trend is that incompatible males tend to induce a lower hatching rate than irradiated males in cage controlled confrontations. More specifically, incompatible males were found more competitive than irradiated males in 5:1 ratio regardless of age, while irradiated males were only found more competitive than incompatible males in the 1:1 ratio at 10 days old. Overall, under the tested conditions, IIT seemed to be slightly more effective than SIT. However, considering that a single strategy will likely not be adapted to all environments, our data stimulates the need for comparative assessments of distinct strategies in up-scaled conditions in order to identify the most suitable and safe sterilizing technology to be implemented in a specific environmental setting and to identify the parameters requiring fine tuning in order to reach optimal release conditions.

Wolbachia-based population control strategy targeting Culex quinquefasciatus mosquitoes proves efficient under semi-field conditions.

In mosquitoes, the maternally inherited bacterial Wolbachia induce a form of embryonic lethality called cytoplasmic incompatibility (CI). This property can be used to reduce the density of mosquito field populations through inundative releases of incompatible males in order to sterilize females (Incompatible Insect Technique, or IIT, strategy). We have previously constructed the LR[wPip(Is)] line representing a good candidate for controlling field populations of the Culex quinquefasciatus mosquito in the islands of the south-western Indian Ocean. The main purpose of the present study was to fill the gap between laboratory experiments and field implementation, i.e. assessing mating competitiveness of these incompatible males under semi-field conditions. In a first set of experiments, we analyzed crossing relationships between LR[wPip(Is)] males and La Réunion field females collected as larvae in 19 distinct localities throughout the island. This investigation revealed total embryonic mortality, confirming the strong sterilizing capacity of LR[wPip(Is)] males. Subsequently, mating competitiveness of LR[wPip(Is)] males was assessed under semi-field conditions in the presence of field males and females from La Réunion. Confrontations were carried out in April and December using different ratios of LR[wPip(Is)] to field males. The results indicated that the LR[wPip(Is)] males successfully compete with field males in mating with field females, displaying even higher competitiveness than field males in April. Our results support the implementation of small-scale field tests in order to assess the feasibility of IIT against Cx. quinquefasciatus in the islands of southwestern Indian Ocean where this mosquito species is a proven competent vector for human pathogens.

Straightforward multi-object video tracking for quantification of mosquito flight activity.

Mosquito flight activity has been studied using a variety of different methodologies, and largely concentrates on female mosquito activity as vectors of disease. Video recording using standard commercially available hardware has limited accuracy for the measurement of flight activity due to the lack of depth-perception in two-dimensional images, but multi-camera observation for three dimensional trajectory reconstructions remain challenging and inaccessible to the majority of researchers. Here, in silico simulations were used to quantify the limitations of two-dimensional flight observation. We observed that, under the simulated conditions, two dimensional observation of flight was more than 90% accurate for the determination of population flight speeds and thus that two dimensional imaging can be used to provide accurate estimates of mosquito population flight speeds, and to measure flight activity over long periods of time. We optimized single camera video imaging to study male Aedes albopictus mosquitoes over a 30 h time period, and tested two different multi-object tracking algorithms for their efficiency in flight tracking. A. Albopictus males were observed to be most active at the start of the day period (06h00-08h00) with the longest period of activity in the evening (15h00-18h00) and that a single mosquito will fly more than 600 m over the course of 24 h. No activity was observed during the night period (18h00-06h00). Simplistic tracking methodologies, executable on standard computational hardware, are sufficient to produce reliable data when video imaging is optimized under laboratory conditions. As this methodology does not require overly-expensive equipment, complex calibration of equipment or extensive knowledge of computer programming, the technology should be accessible to the majority of computer-literate researchers.

Sugar-source preference, sugar intake and relative nutritional benefits in Anopheles arabiensis males.

Plant-derived sugar is the only source of dietary carbohydrate for males of most mosquito species. Male resource acquisition and utilization remain an under-researched area of behavior in vectors of human diseases. However, the renewed interest in the use of sterile males against disease vector mosquitoes reinforces the urgent need for studies on the behavioral and ecological processes that underpin male fitness and reproductive success. Here an attempt was made first to characterize the conditions and modes of resource acquisition (plant derived sugar meals) early in the life of An. arabiensis males, and second to test the hypothesis that the plants chosen for their sugar meals are those which maximize their fitness in terms of energy gains (i.e. amount of lipids, proteins, glycogen and glucose). Olfactometry assays demonstrated the ability of An. arabiensis males to discriminate among a sample of ten abundant flowering plants present in their natural habitats. In further experiments, we observed significant variations in the sugar intake rates that matched their olfactory preferences, with the most attractive plants eliciting significantly higher sugar intake rates. Consistent with our expectations, analyses of the whole-body free sugars, lipids and glycogen unequivocally showed that the energy reserve accumulated post-feeding is dependent on the diet of the adult males, with the preferred plants providing more energy reserves than the less preferred ones, despite mosquitoes actively feeding on both. Taken together, these results show that An. Arabiensis males are able to discern between food sources, preferentially feeding on those species of plant that provide the highest metabolic payoff. Ensuring or somehow heightening the ability to detect and obtain rewarding sugar meals by male mosquitoes reared for field release could enhance their competitive ability in the field.