Chrysomya Putoria Research Articles

A Preliminary Study of Carrion Insects and Their Succession in Luanda, Angola.

The study of the arthropod fauna involved in carcass decomposition is crucial for medicolegal forensic purposes. This knowledge is especially needed in countries with high levels of criminality and where the arthropod fauna is still under-recorded and not well known, such as Angola. We present data from the first survey on sarcosaprophagous flies and beetles in Angola, conducted in Luanda over a 17-d period, in the wet summer season. In total, 881 adult and immature specimens, belonging to 18 species, were collected from three pig carcasses that were sampled daily. Three species were not previously known from Angola. The dominant flies were Chrysomya albiceps (Wiedemann, 1819) (Diptera: Calliphoridae) and Chrysomya megacephala (Fabricius, 1794), but Chrysomya marginalis (Wiedemann, 1830), Chrysomya putoria (Wiedemann, 1830), and Liosarcophaga emmrichiana (Lehrer, 2002) (Diptera: Sarcophagidae) also bred on the carcasses. The dominant beetles were Dermestes maculatus De Geer, 1774 (Coleoptera: Dermestidae), Saprinus splendens (Paykull 1811) (Coleoptera: Histeridae), and Necrobia rufipes (De Geer, 1775) (Coleoptera: Cleridae). Because of the hot weather during the experimental period (~27°C), decomposition was extremely fast. Periods of insect activity on carcasses are presented and comparisons are made with other carrion communities reported from the Afrotropical region.

Bioactivity under laboratory conditions of Brevibacillus laterosporus towards larvae and adults of Chrysomya putoria (Diptera: Calliphoridae).

Brevibacillus laterosporus was tested for entomopathogenic activity towards larvae and adults of Chrysomya putoria (Diptera: Calliphoridae) under laboratory conditions. Sublethal effects related to feeding activity or development were observed, including reduction in larval weight gain, probably by inhibition of feeding, and variation in the duration of the developmental stages of the insect. Larval mortality was dose dependent following ingestion. The experiments were performed with newly emerged adults exposed to a sugar based diet containing spore suspensions. Concentrations of 1.13 × 109 CFU/ml caused 70.5% of mortality. The present study highlights the potential of B. laterosporus to control populations of C. putoria, a dipteran of medical-veterinary and sanitary importance, both in larval and adult stages.

Insecticidal effects of Ocimum sanctum var. cubensis essential oil on the diseases vector Chrysomya putoria

Context: The blowfly Chrysomya putoria is widely distributed throughout the Neotropical region and, besides transmitting pathogens; they could cause secondary myiasis. Botanical insecticides provide an alternative to synthetic pesticides because the excessive use of synthetic insecticides resulted in a progressive resistance of the pests to these chemicals, diminishing their effectiveness and generating consequences with negative environmental impact. The essential oil extracted from Ocimum sanctum (basil) has showed insecticidal activity against some insects but has no reported studies on the activity of this plant against flies. Aims: To evaluate the insecticidal effects of Ocimum sanctum var. cubensis Gomes essential oil on the post embryonic development of Chrysomya putoria. Methods: The colonies of Chrysomya putoria were established and maintained at the Laboratório de Entomologia Médica e Forense (FIOCRUZ), Rio de Janeiro, Brazil. The basil essential oil was tested in six concentrations (4.13, 8.25, 20.63, 41.25, 61.87 and 80,25 mg/mL). Mortality and changes in life cycle were recorded daily. Results: β-caryophyllene, β-selinene and eugenol, were the main constituents of the basil essential oil. The experiments demonstrated that in all concentrations tested, this essential oil shortening the duration of all post embryonic stages having a direct impact in the viability of this fly estimating the LC50 in 7.47 mg/mL of concentration. In addition, the essential oil caused morphological alterations in abdomen, wings and ptilinum at lower concentrations. Conclusions: This essential oil emerge as a good option for the control of the disease vector blowfly Chrysomya putoria.

Longevity, Fertility and Fecundity of Adult Blow Flies (Diptera: Calliphoridae) Held at Varying Densities: Implications for Use in Bioconversion of Waste.

Large numbers of flies are needed to produce the quantity of larvae required for insect bioconversion of waste. However, this 'mass-rearing' may negatively affect adult survival and reproductive output. This study assessed the suitability for mass-rearing of four blow fly species, Chrysomya chloropyga, Chrysomya chloropyga (Wiedemann), Chrysomya megacephala (F.), Chrysomya putoria (Wiedemann) and Lucilia sericata (Meigen). Flies were kept at densities of 20, 50, 100, 250, 500, and 1,000 flies per 30 × 30 × 30 cm cage with an even sex ratio. Time to 50% mortality (LT50) was recorded, and the effects of density, species, and sex on LT50, fecundity, and fertility were determined. Females survived longer than males across all species. There was evidence for a trade-off between survival and high fecundity in L. sericata and C. chloropyga at density 250. C. megacephala had low fecundity across all densities. At high densities, C. putoria had the lowest mortality and highest fecundity, making it the most suitable for mass-rearing.

Post‐embryonic development of Chrysomya putoria (Diptera: Calliphoridae) in swine and bovine tissues in South America: implications for forensic entomology

AbstractNecrophagous diptera are used in forensic entomology to measure the post‐mortem interval (PMI) of cadavers. PMI estimates based on fly larvae, however, can be inaccurate when larval development is retarded, or accelerated, by the nutritional composition of the cadaveric tissue. We compared the post‐embryonic development of Chrysomya putoria (Wiedemann, 1830) on different tissues of pigs and cows. Forty newly hatched fly larvae per treatment were placed on 60 g of the following swine tissues: liver (T1), swine lung (T2), swine heart (T3), swine muscle (T4), bovine heart (T5), bovine lung (T6) and bovine muscle (T7). There were four replicates/treatment. Experiments were conducted in controlled conditions (28°C day/26°C night, RH 70% ± 10% and 12 h of photophase). Fly larvae reared on swine liver took longer to develop (5.2 days) than flies reared on other substrates (from 3.5 to 4.5 days), and this difference was statistically significant. The total duration of development on swine liver (T1) was significantly slower (8.7 days) when compared to the other treatments. Larvae reared on swine lungs (T2) were significantly heavier in the third instar ( = 61.2 mg) than larvae reared on swine liver (T1), swine muscle (T4), bovine lung (T6) and bovine muscle (T7). Total larval survival was high (above 76%) in all treatments except for T1 (66%). There was no deviation from the expected adult sex ratio, and the percentage of abnormalities remained within the expected range. These results reinforce the idea that, to achieve greater accuracy in PMI estimates, the type of tissue on which the larvae develops needs to be taken into account.

Nocturnal oviposition behavior of blowflies (Diptera: Calliphoridae) in the southern hemisphere (South Africa and Australia) and its forensic implications

Published research has offered contradictory evidence of the occurrence of nocturnal oviposition by carrion-breeding blowflies, a behavior that can affect the interpretation of forensic estimates of a minimum post mortem interval (minPMI) by up to 12 hours, depending on latitude and season. The majority of published studies are from the northern hemisphere. Field experiments were conducted in South Africa and Australia that extend observations to species of the southern hemisphere. Various vertebrate carrion was exposed at night in summer under different lunar phases and/or artificial lighting, and in woodland and pasture areas. Three laboratory experiments were also conducted. No nocturnal oviposition occurred outdoors in Berry, Australia, but Lucilia cuprina, Lucilia sericata and Chrysomya megacephala laid eggs outdoors at night in Grahamstown and Durban, South Africa. In laboratory experiments L. sericata, L. cuprina, Chrysomya chloropyga and Chrysomya putoria laid eggs and Calliphora augur deposited larvae under nocturnal conditions. Chrysomya albiceps and C. chloropyga laid eggs in darkness with increasing likelihood as ambient temperature increased. This study shows that nocturnal ovi/larviposition by carrion-breeding blowflies is possible in both South Africa and Australia. The forensic issue is therefore not whether nocturnal oviposition occurs, but rather whether the conditions of a particular case are more or less conducive to it. Circadian rhythms and physiological thresholds (particularly temperature and humidity) appear to act individually and in conjunction to stimulate or inhibit nocturnal laying. The significance of carcass size, freezing and handling of carcasses and comprehensive quantification for experimental design is discussed, and recommendations are made for future laboratory and case scene experiments.

Checklist and distribution maps of the blow flies of Venezuela (Diptera, Calliphoridae, Mesembrinellidae).

A checklist of the 39 species of blow flies (Calliphoridae and Mesembrinellidae) so far known to occur in Venezuela is provided, based on a thorough literature review and the examination of ca. 500 specimens deposited in the main entomological collections of the country. Data from the literature and museum collections were used to generate distribution maps for 37 species. Three species are recorded from Venezuela for the first time: Chrysomya putoria (Wiedemann, 1830), Mesembrinella spicata Aldrich, 1925 and Mesembrinella umbrosa Aldrich, 1922.

Functional Response and Matrix Population Model of Podisus nigrispinus (Dallas, 1851) (Hemiptera: Pentatomidae) fed on Chrysomya putoria (Wiedemann, 1818) (Diptera: Calliphoridae) as Alternative Prey.

Among the predators with high potential for use in biological control, the species of the genus Podisus (Hemiptera: Pentatomidae) have received special attention for laboratory rearing, since they feed on different agricultural and forestry pest insects. However, the type of diet offered to insects in the laboratory may affect the viability of populations, expressed essentially by demographic parameters such as survival and fecundity. This study assessed demographic and development aspects in experimental populations of Podisus nigrispinus (Dallas, 1851) fed on larvae of Chrysomya putoria (Wiedemann, 1818) (Diptera: Calliphoridae) as an alternative prey. The demographic parameters fecundity and survival were investigated in life stages of P. nigrispinus with ecological modeling, by applying the Leslie matrix population model, producing histograms of life stages in successive time steps. The functional response of P. nigrispinus was also investigated on seven densities of C. putoria third-instar larvae at 24 and 48h. The survival of predators that reached adulthood was 65% and the development time from egg to adult was 23.15days. The predator showed a type III functional response for consumption of C. putoria at 24 and 48h. The Leslie-matrix simulation of the age structure provided perpetuation of the predator population over time steps and the prey proved to be feasible for use in rearing and maintenance of P. nigrispinus in the laboratory.

Assessment of the Abundance and Diversity of Calliphoridae and Sarcophagidae (Diptera) in Sites With Different Degrees of Human Impact in the Iberá Wetlands (Argentina).

The aim of the present study was to explore the diversity and describe the spatial distribution of Calliphoridae and Sarcophagidae along an urbanization gradient in the Iberá Wetlands. The flies were collected at 18 sampling points, three per site. In total, 3,318 flies were collected (3,077 blow flies and 241 flesh flies), distributed in 13 genera and 33 species. Calliphoridae was the most abundant, comprised 92.74% of all adult flies. Chrysomya albiceps (Wiedemann), Cochliomyia macellaria (F.), Chrysomya megacephala (F.), and Chrysomya putoria (Wiedemann) were the most abundant species, representing 82.21% of the total sample. The genus Oxysarcodexia Townsend was the most diverse with 10 species represented. The abundance of exotic species represents 62.85% of the total sample, showing a reduction toward less disturbed sites. The results of this study indicated that sites with intermediate impact had higher diversity than those where the disturbances are high or absent. Our findings provide the first assessment of Calliphoridae and Sarcophagidae diversity in the area of the Iberá Wetlands.

Post-embriononic development of Chrysomya putoria(Diptera: Calliphoridae) on a diet containing ampicillin in different concentrations.

Here we evaluate the effects of different concentrations of the antibiotic ampicillin on the growth and development of Chrysomya putoria. Third-generation, first instar larvae (L1) reared on 60 grams of homogenate+agar 65% were treated with ampicillin sodium. The experiment consisted of four replicates (40 larvae/replicate) of each antibiotic concentration tested (T1: 466µg/mL ; T2: 81.33 mg/mL and T3: 166.66mg/mL) and a T4: control. The body mass of the mature larvae, after they abandoned the diet, were recorded in batches of five. The variation between the mean body mass of larvae and the duration of larval and pupal stages, and overall duration of the development, viability and normal rates were analyzed by ANOVA. There were no significant differences between the four treatments in the following parameters: body mass of larvae that discontinued the diet as well as the duration of larval, pupal, and total development. The sex ratios found in the four treatments did not differ from those expected. Normality rates were 100% for all treatments. There were no significant differences between treatments for larval and overall viability, but pupal viability differed significantly between T1 and the control, T1 and T2, and between the control and T3. The antibiotic did not appear to significantly alter the development of C. putoria.

Intraguild predation influences oviposition behavior of blow flies (Diptera: Calliphoridae).

The objective of the present study was to determine whether blow flies (Diptera: Calliphoridae) are able to identify larvae of an intraguild predator species in the substrate and avoid laying eggs there. Blow flies oviposited in traps with different treatments: substrate only and substrate with larvae of Chrysomya albiceps (Wiedemann, 1819), Chrysomya megacephala (Fabricius, 1794), or Chrysomya putoria (Wiedemann, 1830). Ch. megacephala, Ch. putoria, and Lucilia eximia (Wiedemann, 1819) avoided laying eggs in the trap containing Ch. albiceps larvae. Cochliomyia macellaria (Fabricius, 1775) did not oviposit differently in each substrate but had overall low abundance. The prevalence of species on corpses may be influenced by the ability of the species to detect the presence of other species, mainly predators. In this sense, intraguild predation may result in misinterpretations of a crime scene and should be considered when assessing the minimum postmortem interval.

English

Cymbopogon citratus oil is used in folk medicine asrepellent and insecticide against insects. This study evaluated the insecticidal activity of C. citrates oil extracted from Brazil and Cuba and its main component citral as insecticides against Chrysomya megacephala, Chrysomya putoria and Lucilia cuprina. Variables monitored were:duration of post embryonic development, larval weight (mg), sex ratio, mortality index and percentage of morphological deformities. The essential oils were dissolved in dimethylsulfoxide (DMSO) and tested at concentrations of 5, 10, 25, 50, 75 and 100%, andcitral was diluted in DMSO yielding a concentration of 17.5 µg/µL.Substances were applied (1µL on the newly-hatched larvae. Results showed that both oils and citral had toxic effects on post-embryonic development of all tested blowflies. While the mortality for C. megacephala reaches 80% of the flies treated with essential oil, and values under 50% for the LC50; C. putoria has the highest ratio of deformities, once it becomes adult with values over 85%. The behavior for C. citrates essential oils and citral, changes from one variable to another, but in general sense, all are toxic to flies. Previous observations point out this essential oil as a potential alternative in those blowflies control. Key words: Arthropod, Calliphoridae, biopesticides, lemongrass, essential oil.

Induced Cannibalism in Experimental Populations of the Forensic Indicator Chrysomya putoria Wiedemann (Diptera: Calliphoridae).

We analyzed the behavior of third-instars of Chrysomya putoria as potential cannibals in experimental populations. Cannibalism rates were evaluated in three settings observed for 3, 6, 9, and 24 h, placing injured and uninjured larvae of C. putoria together. Our data heavily support that C. putoria larvae behave as cannibals when induced by a wound in another larva, and also after starving for 24 h. The probability of cannibalism increased as a function of time, both in no-choice and in choice experiments evidencing that time is a determining factor for cannibalism induction in C. putoria. However, the treatment combining injured with uninjured larvae showed the highest probability of cannibalism. These results suggest that C. putoria larvae may cannibalize under scarcity of food over long time or the presence of injured larvae. This study is useful to understand the behavior of C. putoria feeding on ephemeral substrates such as carrion or corpses and brings relevant and significant contribution to population ecology of blowflies and also forensic entomology.

Effect of age on cuticular hydrocarbon profiles in adult Chrysomya putoria (Diptera: Calliphoridae)

A species-specific complex mixture of highly stable cuticular hydrocarbons (CHCs) covers the external surface of all insects. Components can be readily analyzed by gas chromatography coupled to mass spectrometry (GC–MS) to obtain a cuticular hydrocarbon profile, which may be used as an additional tool for the taxonomic differentiation of insect species and also for the determination of the age and sex of adult and immature forms. We used GC–MS to identify and quantify the CHCs of female and male Chrysomya putoria (Wiedemann, 1818) (Diptera: Calliphoridae) from one to five days old. CHCs ranged from C21 to C35 for females and from C21 to C37 in males. Major compounds were the same for both sexes and were 2-MeC28, C29:1, n-C29, 15-,13-MeC29, 2-MeC30, C31:1, n-C31 and 15-,13-MeC31. The relative abundance of each component, however, varied with age. Cluster Analysis using Bray–Curtis measure for abundance showed that cuticular hydrocarbon profiles are a strong and useful tool for the determination of age in adult C. putoria.

Egg Developmental Time and Survival of Chrysomya megacephala and Chrysomya putoria (Diptera: Calliphoridae) Under Different Temperatures.

Chrysomya megacephala (F.) and Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae) are considered of forensic, medical, and veterinary importance in Brazil because of their necrophagous and synanthropic behaviour. The development of flies can be influenced by temperature, and species from the same genus usually have different responses to external variables. The egg development of blow fly can be a useful complementary technique to estimate the minimum postmortem interval. Thus, this study aimed to compare the egg developmental time and survival of C. megacephala and C. putoria at different temperatures to determine the optimal temperature for egg development and the linear regression for developmental time and temperature, thereby determining the minimum threshold (t) and thermal summation constant (K) for each species. Adults of both species were collected in the region of Campinas city, São Paulo state, Brazil. Eggs were incubated at eight constant temperatures between 05 ± 1°C and 35 ± 1°C and the egg developmental time and survival were evaluated. There was no egg survival at 5 and 10°C. The K for C. megacephala and C. putoria were 179.41 HD and 189.94 HD, respectively. The regression slopes and t (10°C) were similar for both species. The optimal temperature for egg survival was between 25 and 35°C, for C. megacephala and 20 and 30°C, for C. putoria. The present data were similar to most data available in the literature, but differences in the same species are a possibility.

Detection and preliminary physico-chemical properties of antimicrobial components in the native excretions/secretions of three species of Chrysomya (Diptera, Calliphoridae) in Brazil

Antibiotic-resistant bacteria in hospitals and communities increasingly threaten public health in Brazil and the rest of the World. There is an urgent need for additional antimicrobial drugs. Calliphorid blowfly larvae are a rich source of antimicrobial factors but the potential of Neotropical species has been neglected. This preliminary study evaluates the antimicrobial activity of the native excretions/secretions of larvae of three species of Brazilian calliphorids, Chrysomya megacephala, Chrysomya albiceps and Chrysomya putoria. Native excretions/secretions were collected from third instar larvae, sterile filtered and tested for antibacterial activity against Staphylococcus aureus 9518, Escherichia coli K12 4401 and Serratia marcescens 365. Turbidometric assays were made in micro-plates, using an ELISA reader, with readings taken up to 22h. Bacterial suspensions at the start and end of each experiment were also serially diluted, spread on nutrient agar plates and then colony forming units counted. The physico-chemical characteristics of the native excretions/secretions were also tested by freezing/thawing, boiling, and protease digestion. The native excretions/secretions of larvae from these three Chrysomya species significantly inhibited bacterial growth. Therefore, Brazilian calliphorid flies could potentially provide new classes of antibiotics.

Intrapuparial development of Chrysomya putoria (Diptera: Calliphoridae).

The intrapuparial development of 150 pupae of Chrysomya putoria (Wiedemann, 1830) was analyzed. The material was kept in a climate chamber at 27 degrees C (day) and 25 degrees C (night), 60 +/- 10% relative humidity (RH), and a photoperiod of 14:10 (L:D) h, which started at 06:00 a.m. Daily, 30 pupae were frozen at - 15 degrees C, for the sacrifice of the insects, and the immatures obtained were photomicrographed until the emergence of the adults. The following stages were observed: pupariation process: during this process, reduction of the larvae's first three body segments and the darkening of the cuticle occur; cryptocephalic phase after 18 h; phanerocephalic phase after 24 h; body division after 48 h. Between the third and the fourth days of the development, the stage ofpharate adult was reached, characterized by the following changes in the color of the compounds eyes: 1) orange eyes, after 66 h; 2) red eyes, after 72 h; 3) dark red eyes, after 90 h; 4) brown eyes, after 96 h. The time of intrapuparial development of C. putoria at 2 degrees C (day) and 25 degrees C (night), 60 +/- 10% RH, and a photoperiod of 14:10 (L:D) h was 5 d.

Rates of development of immatures of three species of Chrysomya (Diptera: Calliphoridae) reared in different types of animal tissues: implications for estimating the postmortem interval

Blowflies have major medical and sanitary importance because they can be vectors of viruses, bacteria, and helminths and are also causative agents of myiasis. Also, these flies, especially those belonging to the genus Chrysomya, are among the first insects to arrive at carcasses and are therefore valuable in providing data for the estimation of the minimum postmortem interval (PMImin). The PMImin can be calculated by assessing the weight, length, or development stage of blowfly larvae. Lack of information on the variables that might affect these parameters in different fly species can generate inaccuracies in estimating the PMImin. This study evaluated the effects of different types of bovine tissues (the liver, muscle, tongue, and stomach) and chicken heart on the development rates of larvae of Chrysomya albiceps Wiedemann, Chrysomya megacephala Fabricius, and Chrysomya putoria Wiedemann (Diptera: Calliphoridae). The efficiency of each rearing substrate was assessed by maggot weight gain (mg), larval development time (h), larval and pupal survival (%), and emergence interval (h). The development rates of larvae of all blowfly species studied here were directly influenced by the type of food substrate. Tissues that have high contents of protein and fat (muscle and heart) allowed the highest larval weight gain. For bovine liver, all Chrysomya species showed slower growth, by as much as 48h, compared to the other tissues. Different rates of development are probably associated with specific energy requirements of calliphorids and the nutritional composition of each type of food.

Biological activity of Pouteria sapota leaf extract on post-embryonic development of blowfly Chrysomya putoria (Wiedemann, 1818) (Calliphoridae)

Chemical insecticides have been the main way to control synanthropic flies of medical and veterinary importance; however, residuals of these products have become a factor impacting on the environment, as well as the potential toxicological that they may cause damage to humans and domestic animals. Phytochemical screening carried out with the aqueous crude extract of Pouteria sapota (Jacq.) H.E. Moore & Stearn, Sapotaceae, leaves showed that coumarins, reducing sugars, flavonoids and cyanogenic glycosides were its most abundant metabolites. This study evaluated the activity of the crude aqueous extract of this plant on the post-embryonic development of Chrysomya putoria. Larvae treated with 5, 10 and 25% extract showed a decrease in the pupal period and in the newly-hatched larvae to adult period when compared to the control groups. Larvae from the 25% extract group were the lightest (45.8mg) when compared with the control group (46.5mg). The larval and newly-hatched larvae to adult stages were more sensitive to the leaf extract from P. sapota (5%) and the treated flies showed the low viability (47.5 and 45.5% respectively). The results demonstrated that topic treatment with P. sapota could alter C. putoria post embryonic development.

Developmental rates of immatures of three Chrysomya species (Diptera: Calliphoridae) under the effect of methylphenidate hydrochloride, phenobarbital, and methylphenidate hydrochloride associated with phenobarbital

Entomotoxicology is focused on obtaining data on necrophagous entomofauna, for criminal investigations purposes. This study aimed to evaluate the effect of different concentrations of methylphenidate hydrochloride, phenobarbital, and their association on the developmental rate, larval and pupal survivorship, and the interval of emergence of adults of Chrysomya albiceps (Wiedemann), Chrysomya megacephala (Fabricius), and Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae). Considering the therapeutic dose (TD) of methylphenidate hydrochloride (0.29 mg/Kg), the concentrations tested were 10× TD, 50× TD, and 100× TD. For phenobarbital, the concentrations used were 1× TD (=150 mg/Kg), 3.3× TD, and 6.7× TD. For the association of the drugs, the combinations used were 10× TD-methylphenidate hydrochloride plus 1× TD-phenobarbital, 50× TD-methylphenidate hydrochloride plus 3.3× TD-phenobarbital, and 100× TD-methylphenidate hydrochloride plus 6.7× TD-phenobarbital. The control group, without addition of drug, was maintained under the same conditions of temperature (25 ± 1 °C), humidity (70 ± 10%), and photoperiod (12 h). Specimens of each group were weighed every 12 h until pupariation. The developmental rate of the three Chrysomya species immatures was monitored. For C. albiceps the developmental time was delayed in 24 h for methylphenidate hydrochloride group and in 12 h for the phenobarbital and the drugs association groups. The effect was observed only at specific ages for C. megacephala, without altering the developmental time. For C. putoria, the developmental time was delayed in 12 h for methylphenidate hydrochloride group and in 24 h for the phenobarbital and the drugs association groups. The emergence interval was similar among all experimental groups, but larval and pupal viabilities were affected in different ways.