Dioscorea hispida tuber plant to explore their potential as renewable materials. The extraction of the Dioscorea hispida starch and Dioscorea hispida fibres was carried out and the chemical composition, physical, thermal, morphological properties, and crystallinity were studied. The chemical composition investigations revealed that the Dioscorea hispida starch (DHS) has a low moisture t (9.45%) and starch content (37.62%) compared to cassava, corn, sugar palm, and arrowroot starches. Meanwhile, the Dioscorea hispida fibres (DHF) are significantly low in hemicellulose (4.36%), cellulose (5.63%), and lignin (2.79%) compared to cassava, corn hull and sugar palm. In this investigation the chemical, physical, morphological and thermal properties of the Dioscorea hispida fibre and Dioscorea hispida starch were examined by chemical composition investigation, scanning electron microscopy (SEM), particle size distribution, thermogravimetric analysis (TGA), X-ray powder diffraction (XRD), and Fourier transform infrared (FTIR), respectively. It was found that Dioscorea hispida waste is promising alternative biomass and sustainable material with excellent potential as a renewable filler material for food packaging applications.This study was driven by the stringent environmental legislation concerning the consumption and utilization of eco-friendly materials. Within this context, this paper aimed to examine the characteristics of starch and fibres from the  Non-renewable materials have caused critical environmental issues in many countries in the world regarding waste disposal ,2,3. ThiRecent environmental regulation has forced production industries to seek for other potential materials for the development of their product that are more environmentally friendly than synthetic materials  ,16. In rBiocomposites are changing the research activities carried out in the scope of material engineering because the number of beneficial properties proven, such as biodegradability, lightweight, low energy consumption, usability, and eco-friendly ,31,32,33Currently, biocomposites are used in various applications, e.g., automotive, aircraft, building materials, sports equipment, and food packaging . From thDioscorea hispida plants, also known as Ubi Gadong in Malaysia is a poisonous tuber plant that contains the alkaloid of dioscorine [Dioscorea sp. discovered in numerous parts around the globe, specifically in the subtropical and tropical regions [Dioscorea hispida tubers is edible after the removal of the dioscorine compound [Dioscorea hispida starch is largely based upon empirical studies that investigated its use as a crude drug for inflammation [Dioscorea hispida starch demonstrated the hydrogel created from the starch functioned as an antibacterial agent to prevent microorganism activity [oscorine . Recent  regions . Studies regions . The stacompound . It requcompound . The knoammation . Previouactivity . Additioactivity . This isactivity ,46. Geneactivity . These gactivity ,61.Dioscorea hispida plants. Tuber parts have been particularly identified and some of the recent research works published are tabulated in Dioscorea hispida plants can also generate large quantities of sustainable lignocellulosic materials every year, which can be used for the production of starch, bioplastic, and potential biocomposites to minimize environmental impact and renewability.Up to now, several studies have been carried out on various parts of Dioscorea hispida species as filler materials. In this study, the chemical, physical, morphological, and thermal properties of the Dioscorea hispida fibre and Dioscorea hispida starch were examined by Chemical Composition Investigation, Scanning Electron Microscopy (SEM), Particle Size Distribution, Thermogravimetric analysis (TGA), X-ray Powder Diffraction (XRD), and Fourier Transform Infrared (FTIR). To the best of our knowledge, no previous study had proposed Dioscorea hispida as a promising material with strong potential in the development of biodegradable films. In comparison with other natural fibres and starch, the objective of this work was to characterize novel starch and fibre from Dioscorea hispida tuber as renewable materials for food packaging applications. It must be noted that the Dioscorea hispida tubers used in this study were chemically treated to remove the toxin.According to the literature, limited studies have been conducted on the investigation of the native starch and fibre solid wastes from Dioscorea hispida was collected from a local farm M Abd Halim Enterprise in Kuala Terengganu, Malaysia. Dioscorea hispida starch and fibres were extracted from the fresh tubers. All samples were characterized in powder form.The native Dioscorea hispida starch and fibre from the Dioscorea hispida tuber was conducted with a number of processes  at a temperature of 60 °C. The fibres were shredded, sieved through a 300 µm mesh sieve  and characterized prior to application as filler in the Dioscorea hispida starch biocomposites.The extraction of rocesses . InitialDioscorea hispida starch. The procedures used to study acid detergent fibre (ADF), neutral detergent fibre (NDF), ash, lignin (LIG), hemicellulose, and cellulose of Dioscorea hispida fibres were adapted from [Dioscorea hispida fibres. The most practised procedure was used for evaluating the amount of fibre constituents, such as hemicellulose, cellulose, and lignin. The amount of hemicellulose and cellulose were determined by using Equations (1) and (2), respectively.The analysis was conducted to investigate the moisture, ash, starch, crude protein, energy, and carbohydrate contents of ted from . The ADFm = mass (g), V = volume (cm3)The density was determined using an AccuPyc II 1340 pycnometer gas  intrusion using helium gas flow. The samples underwent oven drying at a temperature of 105 °C for 24 h to eliminate the moisture content within the fibres and starch. Then, the samples were placed inside a desiccator to remove water traces from the dried sample before putting inside the pycnometer. Samples’ densities were calculated from measurements at a temperature of 27 °C using Equation (3).i and after, Wf the heating to evaluate the moisture content using Equation (4)The moisture content investigation was performed with fibres and starch samples. The samples were heated in an oven at 105 °C for 24 h. The samples were weighed before, WThe instrument Mastersizer 2000 E Ver. 5.52  was used to identify the particle size distribution for all samples through a built-in Q-space powder feeder. Prior to distribution analysis, the particle size for the tested samples was investigated with a 1000 μm sieve.The instrument scanning electron microscope  was used to determine the samples’ surface morphology. The samples were coated with a layer of gold and 20 kV voltage was passed through at high vacuum condition to produce an electron beam. The electrons were connected with the sample atoms and produced signals giving a report on the surface topography, by generating images of high resolution.The thermogravimetric analyser  was applied to analyse the thermal behaviour of the samples. The samples with the range of mass 5–10 mg were placed in platinum crucibles and the temperature was raised to 600 °C from room temperature under a heating rate of 10 °C/min in a nitrogen atmosphere with flow rate, 50 mL/min.−1 to 400 cm−1 with 4 cm−1 of spectral resolution. The sample preparation was conducted using the potassium bromide (KBr) disc method, and the scan per specimen used was 16 scans.The infrared spectrometer  was used to obtain the FTIR spectrum for samples at frequencies over a wide spectral range, 4000 cmθ) at scattering speed of 0.02 (θ) s−1. The operating current and voltage were set to 35 mA and 40 kV, respectively.XRD analysis was conducted using a 2500 X-ray diffractometer  with an angular range from 5° to 60°  and starch had low moisture contents of 9.45% and 37.62%, respectively compared to cassava, corn, sugar palm, and arrowroot starches. Meanwhile, the Dioscorea hispida fibres (DHF) were significantly low in hemicellulose (4.36%), cellulose (5.63%), and lignin (2.79%) compared to cassava, corn hull, and sugar palm. The composition of these components could affect the characteristics of thermoplastic starch-based film composites, including the water barrier properties [f plants . Howeveroperties .Dioscorea hispida starch was relatively high due to the presence of phosphate groups. In terms of metals, the ash of native starches consists mainly of potassium, calcium, sodium, and magnesium [Dioscorea hispida tuber composition, even though they differed according to genetic, ecological, and agronomic factors [In addition, the ash content in the agnesium . Carbohy factors ,67. This factors . Since tDioscorea hispida starch showed slightly higher density, 1.74 g/cm3 compared to other natural starches shown in The most important factor in the materials selection process is the molecular weight of the materials since it could affect the performance of the products . MoreoveDioscorea hispida fibre is the amount of cellulose, hemicellulose, and lignin in the fibre. The results of this study were compared to other studies’ findings, as tabulated in Dioscorea hispida fibre were considerably low compared to other fibres. Furthermore, the lignin content (2.79%) in the Dioscorea hispida fibre was comparatively lower than the other samples’ lignin contents, as shown in Dioscorea hispida tubers. Apart from that, Dioscorea hispida fibre density (1.47 g/cm3) values were smaller compared to sugar palm fibres but higher than cassava and corn hull fibres [The critical finding for l fibres ,70.Dioscorea hispida fibres was relatively low compared to the other samples. The current study found that the relative amounts of different compounds discovered in natural fibres varied with each plant, including different parts of the same plant [Ibrahim et al.  reportedme plant . Generalme plant . Previoume plant . Similarme plant ,47.Fibres are assumed to be natural source composites mainly consisting of holocellulose  and lignin, with low sugar, starch, protein, extractives, and ash content . The incDioscorea hispida starch and Dioscorea hispida fibre samples. It was apparent that considerable weight loss occurred up to ~100 °C in all of the samples as shown in Dioscorea hispida starch and Dioscorea hispida fibre. For example, Dioscorea hispida starch sample’s onset thermal degradation temperature was about ~41.3 °C, while for samples of Dioscorea hispida fibre was ~40.7 °C. It might be due to the amount of moisture content of the samples  than Dioscorea hispida fibre, as presented in Dioscorea hispida starch and Dioscorea hispida fibre. The migration of volatile materials occurred as a result of the movement of water from the lower interior part of the starch and fibres to a better water potential region, at the surfaces of Dioscorea hispida starch and Dioscorea hispida fibre, as the water molecules on the surface of samples were evaporated. Hence, this movement of water indirectly moved the volatile materials, leaving them on the Dioscorea hispida starch and Dioscorea hispida fibre surfaces [Thermal stability is frequently assessed by identifying the thermal decomposition’s onset temperature.  samples . The evasurfaces .Dioscorea hispida starch and 203.9 °C for Dioscorea hispida fibre. The results showed that the initial heat tolerance of Dioscorea hispida starch was considerably greater than Dioscorea hispida fibre. It was revealed that when temperatures reached 140 °C, the inorganic materials and lignin of Dioscorea hispida fibre were degraded after the thermal decomposition of hemicellulose, cellulose, and volatiles [Dioscorea hispida starch began with the decay of the water-soluble amylopectin at the onset temperature and continued until achieving a weight loss of approximately 66.1%. The highest rate of thermal degradation of Dioscorea hispida starch occurred at about 310 °C and left 20.6% residue of ash. These results were in good agreement with the findings in previous works [Nevertheless, the temperatures of the first thermal decomposition were about 260.4 °C for onset temperature of olatiles . The firus works ,70.Dioscorea hispida fibre was consistent with the degradation of hemicelluloses. The remarkable weight loss was due to the degradation of significant elements of lignocellulose, including hemicellulose, cellulose, and finally, lignin, which normally degrades at the temperature of higher than 140 °C [Dioscorea hispida fibre, similar to other lignocellulosic material decompositions [Dioscorea hispida starch and Dioscorea hispida fibre samples were found to be in the range of 20% to 25% at a higher temperature (550 °C)  . This waperature . Hemicelbranches . These rDioscorea hispida starch and Dioscorea hispida fibre samples are displayed in Dioscorea hispida starch appeared as a polyhedral shape obtained from the raw starch isolated from the Dioscorea hispida tubers with a range of size from 1.8 µm to 3.5 µm. The shape was different from other Dioscorea sp. due to the dissimilarity in biological sources and the environments of plant growth [Dioscorea sp. starch and found that Dioscorea alata had three different shapes; triangular, ellipsoid, and polyhedral [Dioscorea opposita starch such as cake-shaped, oval, and ground granules. Jiang et al. [Dioscorea hispida fibre sample showed the presence of a polyhedral shape and exhibited individualized irregular granules with a clear smooth surface and no evidence of any fissure. The fibres were much bigger than Dioscorea hispida starch granules in this study with a range of size from 3.1 µm to 5.1 µm.SEM images of t growth . Previoulyhedral . In addig et al.  publisheg et al. . The DioDioscorea hispida samples showed a single mode distribution profile for their sizes. The graph was analyzed to approximate the gradation size range of the Dioscorea hispida fibre and Dioscorea hispida starch particles. According to the refractive guideline for the sample taken from the Malvern Instrument Mastersizer 2000, the refractive index value was denoted as 1.334. Thus, the particle size distribution of Dioscorea hispida fibre and Dioscorea hispida starch are presented in Dioscorea hispida fibre particles had dimensions of less than 18 µm and the majority sizes of the particles were 333 µm. Meanwhile, 10% of Dioscorea hispida starch particles had less than 3 µm and 877 µm for the majority sizes of the particles. However, the findings of the current study were unparalleled with the previous research of corn starch [The particle size distribution of n starch ,61. ThesThe results indicated that the mean particle size of starch powder for the volume-weighted mean was 167 µm. The mean particle diameter demonstrated crystallinity pattern and degree of hardness inside the starch granules . Recent Dioscorea hispida fibre sample presented a larger particle size compared to Dioscorea hispida starch due to the morphological structure related to the samples. Nonetheless, the findings of this study strengthened the idea that it can convey an excellent dispersion behaviour that is significant in reinforcing composites.Moreover, the particle size distribution of the fibres indicated that the volume-weighted mean was 344 µm. The Dioscorea hispida starch and fibre waste are portrayed in −1, the second stage was between 800 cm−1 to 1500 cm−1, followed by the third stage within the C–H stretch area (2800 cm−1 to 3000 cm−1). The final stage showed intense peaks indicating the O–H groups’ presence in every filler part associated with the hydroxyl group discovered in hemicellulose, cellulose, and lignin [Dioscorea hispida starch and Dioscorea hispida fibre showed behaviour patterns below 800 cm−1 in the region related to the vibrations of the glucose pyranose unit [−1 in the FTIR spectrum of Dioscorea hispida fibre was mainly ascribed to the C=O stretching vibration of the ester linkage of the carboxylic group of ferulic and p-coumaric acids of hemicellulose and lignin [−1 were shown as aromatic skeletal vibrations of the operative lignin group in DHF, while the band observed at 1635 cm−1 corresponded to lignin composition. These findings were supported by determining the chemical composition of Dioscorea hispida fibres as shown in −1 indicated the structural polymer stretching of the aromatic groups existing in the lignin form. From −1 reflected the stretching of the C–H and O–H groups, correspondingly. Peaks found in the 3300–3000 cm−1 region were attributed to the adsorbed water. The absorbance bands around 856, 930, 996, 1077, 1149, and 1242 cm−1 were related to the C–O stretching, C–H rocking vibrations, and C–O–C asymmetric valence vibration, respectively, and these peaks indicated cellulose in the carbohydrates [Dioscorea hispida starch appeared to have complex spectra in the second region with outstanding overlapping originating from the monomer glucose units C–O vibrational stretching. This result might explain that the presence of the C–O–H bending mode was due to the band appearance at 1078 cm−1, while the coupling modes of C–C and C–O stretching exhibited the peak at 1148 cm−1. However, the peak at 1339 cm−1 of the infrared wavenumbers indicated CH2 bending modes [−1 was related to the water fragment vibrations in the amorphous region of the starch granules, while another possible explanation for this was the emergence of broad infrared bands. The intense peaks at 3262 cm−1 meant the presence of O–H groups in Dioscorea hispida starch. A possible explanation for this might be the Dioscorea hispida starch that was very sensitive to water molecules due to the presence of hydroxyl groups. It can be noted that the fourth vibration in the region of the O–H stretch hydroxyl groups resulted in the formation of the last band [The FTIR spectra for raw d lignin . Thus, tose unit . The pead lignin . It can hydrates . The Diong modes . In addiDioscorea hispida starch and Dioscorea hispida fibre samples. The fibres characterization was performed via the XRD diffractograms. Dioscorea hispida fibres are primarily composed of cellulose, hemicellulose, and lignin, while the lignin is amorphous having a benzene ring with cellulose existing in a crystalline form in nature [Dioscorea hispida starch possessed 27.5% crystallinity index, which was lower than Dioscorea hispida fibres of 39.0%. This was probably due to the difference in the samples’ contents, which was referred to the investigations of the chemical composition. The crystallinity index of Dioscorea hispida starch and Dioscorea hispida fibre supported the agreement of Ibrahim et al. [Dioscorea hispida fibres and Dioscorea hispida starch samples. This might be attributed to the contents of lignin, cellulose, hemicellulose, moisture, and amylose of the fibres [Dioscorea sp. which agreed on the crystallinity of the starches of Dioscorea hispida starch [XRD Analysis is frequently used to investigate the percentage of crystallinity and the structure as for the samples in this study. n nature . Based om et al.  which ree fibres ,74. The a starch . Therefoa starch . Accordia starch , the modDioscorea hispida starch and Dioscorea hispida fibres were successfully extracted from Dioscorea hispida tubers after removing the dioscorine compound in the tubers. The chemical composition investigations, particle size distribution, scanning electron microscopy (SEM), X-ray powder diffraction (XRD), thermogravimetric analysis (TGA), and Fourier transform infrared (FT-IR) of DHS and DHF samples were explored, and their chemical composition, thermal properties, and morphologies were compared. The chemical composition investigations revealed that Dioscorea hispida starch has low moisture (9.45%) and starch content (37.62%) compared to cassava, corn, sugar palm, and arrowroot starches. Meanwhile, the Dioscorea hispida fibres (DHF) are significantly low in hemicellulose (4.36%), cellulose (5.63%), and lignin (2.79%) compared to cassava, corn hull, and sugar palm Thermogravimetric analysis (TGA) showed that the Dioscorea hispida starch maximum decomposition temperature was 309.7 °C, and for Dioscorea hispida fibres, it was 315.4 °C. The particle size determination and SEM analysis results also contributed to the excellent thermal stability of Dioscorea hispida starch and Dioscorea hispida fibres. The crystallinity index of Dioscorea hispida starch was 27.5%, which was lower than that of Dioscorea hispida fibres of 39.0%. This experiment revealed that Dioscorea hispida waste could be promising alternative biomass and sustainable material with excellent potential as a renewable filler material for food packaging applications. Tests with other geographical origins of Dioscorea hispida tubers should be conducted in a future study to determine the best characteristics for the production of biodegradable packaging films.


Did late Roman traditions persist, did new customs emerge, and did both amalgamate into new cultural expressions? A prime area to investigate these population and settlement historical changes is the Carpathian Basin (Hungary). Particularly, we studied archaeological and anthropological evidence, as well as radiogenic and stable isotope ratios of strontium, carbon, and nitrogen of human remains from 96 graves at the cemetery of Mözs-Icsei dűlő. Integrated data analysis suggests that most members of the founder generation at the site exhibited burial practises of late Antique traditions, even though they were heterogeneous regarding their places of origin and dietary habits. Furthermore, the isotope data disclosed a nonlocal group of people with similar dietary habits. According to the archaeological evidence, they joined the community a few decades after the founder generation and followed mainly foreign traditions with artificial skull modification as their most prominent characteristic. Moreover, individuals with modified skulls and late Antique grave attributes attest to deliberate cultural amalgamation, whereas burials of largely different isotope ratios underline the recipient habitus of the community. The integration of archaeological and bioarchaeological information at the individual level discloses the complex coalescence of people and traditions during the 5th century.The decline of the Roman rule caused significant political instability and led to the emergence of various ‘Barbarian’ powers. While the names of the involved groups appeared in written sources, it is largely unknown how these changes affected the daily lives of the people during the 5 Archaeoaditions . The strge diets . The δ15n period –91. If w13C (-16.6 to -15.9 ‰) and δ15N (9.8 and 11.2 ‰) values , and similar δues Figs . The isoues Figs . The cluues Figs  as well grave 43 , an archgrave 43  and excemunities .th century, the practise flourished especially during the middle third of the 5th century, when it occurred frequently among both males and females [Especially the high number of children with artificially deformed skulls and strontium isotope ratios in the centre of the local range suggests that the arriving group became culturally and socially dominant at the cemetery. They retained the custom of shaping the bodies of their offspring irreversibly, and artificial skull deformation became an integral part of the community’s identity , 94. Whi females . The wid females , 96.pars pro toto, these could be understood as a symbolic brick structure, a later, simplified version of the construction and illustrate the hybridization of different strands of traditions. Grave 86, the burial of a juvenile female, shows that local artefact types, in this case late Roman earrings, could also appear together with artificial skull deformation. Reversely, there is no example of foreign artefact types in brick-lined graves, which usually contained very few or no grave goods and disappeared shortly after the arrival of the foreign group.The co-habitation of people with different cultural backgrounds also facilitated an amalgamation of local and foreign traditions in the same burials. The brick-lined structure of Grave 56 represents a late Antique tradition, while the artificially deformed skull of the locally born child buried in it is characteristic for the newly arriving group. A possible interpretation is that the members of the founding generation already practised artificial skull deformation. However as this would be the only example of an earlier dating of this tradition, the explanation of cultural amalgamation is more plausible. While the ‘fashion’ of artificial skull deformation became popular in an instant, burial customs may have changed slower and retained their local character during the transition. Moreover, the age (7–8 years old) of the individual buried in Grave 56 presents a chronological problem: subsequent generations could have the same archaeological dating, because children  may have died before their parents. Another example is grave 71, an adult male with a deformed skull and few bricks placed in his grave. As 87Sr/86Sr ratios above or in the upper section of the local range as well as children with isotope ratios in the restricted central part of the local range  in the Carpathian Basin [4 plant, which first started to play a role in the every-day diet in the area during the Late Bronze Age and Early Iron Age [3 cereals, such as barley, wheat, and rye [3 cereals exceeded millet by far [th century. However, written sources suggest that the upheavals at the end of the Roman Empire also triggered an increase of millet cultivation in association with a nomadic lifestyle of incoming ‘Barbarian’ people [The δan Basin , 98. ThiIron Age , 98. Anc and rye , 100. Tht by far . There i’ people . Alternath century AD, and the burials from Mözs revealed the highest average δ13C values among five roughly contemporary sites [13C values of the burials at Mözs may indeed be ascribed to higher shares of millet in their diet. The data distribution within the burial community suggests that individuals with non-modified skulls and those who were buried in brick-lined graves consumed less millet than those with artificial skull deformations, the supposedly incoming group with non-local traditions th centuraditions . This obth century being a highly dynamic time in the Carpathian Basin. Residential changes were widely common and provided the basis for cultural hybridization at the site and even personal level. Placed into the historical narrative, this could be understood as the emergence of a Roman-‘Barbarian’ Mischkultur (mixed culture) [Archaeological, anthropological, and stable isotope analyses of almost 100 individuals from the cemetery of Mözs demonstrated the coalescence of people and their cultural traditions. The site represents the transition period after the decline of the Roman Empire through the rise and fall of the Hunnic power and ends before the formation of ‘Barbarian’ kingdoms in the Carpathian Basin (430/440-470/480 AD). The community had a very recipient habitus. It accepted and integrated men, women, and children of different geographical and cultural backgrounds during the two to three generations of its existence. The isotope data indicate that residential changes played a remarkable role and occurred not only on an individual basis, but also in groups of a shared cultural background and lifestyle. The founder generation of the cemetery predominantly comprised people, who represented late Antique traditions, grew up in different locations and consumed a diet with variable shares of millet. Little later, a group from a shared area of origin and with similar dietary habits that included millet consumption mixed in. They exhibited foreign, probably non-Roman traditions, among which artificial skull deformation was the most remarkable. While the cemetery grew, both local and these non-local traditions even amalgamated in the same burials. At Mözs, archaeological and bioarchaeological data attest to the 5culture) , in whicS1 TableS1A Table. Anthropological, archaeological and isotope data of the burials from the cemetery of Mözs as well as their classification into Sr isotope data ranges and interpreted group affiliations. S1B Table. Results of carbon, nitrogen and strontium isotope analysis of animal bones and teeth from Mözs. S1C Table. Strontium isotope data of human teeth and bones as well as animal teeth of Neolithic sites in up to 20 km distance from Mözs. S1D Table. Carbon and nitrogen isotope ratios of the quality control standards analysed along with the collagen samples from Mözs. S1E Table. Summary counts and percentages of individuals with 87Sr/86Sr ratios in the different local and non-local isotope ranges according to age and sex, burial groups, and skull deformations. S1F Table. Descriptive statistics of C and N isotope data of various groups of individuals at the cemetery of Mözs, results of pairwise comparisons among these groups and of Mözs with other datasets of the 5th century AD.(XLSX)Click here for additional data file.S1 FigThe skulls from grave 34 and grave 43 represent examples of an adult female and an infans II child with very similar deformations in an upward-backward direction and recessions resulting from binding (deformation variant II). The skull of grave 85 (infans I) combines an artificial deformation of variant I (circular binding and strong upward deformation) with post-mortem deformation.(PDF)Click here for additional data file.S2 FigThe sites are grouped according to landscapes as mapped in (PDF)Click here for additional data file.S3 Fighttps://map.mbfsz.gov.hu/arcgis/services/fdt100/fdt_100/MapServer/WMSServer.For the legend of the geological map, please refer to: ttps://map.mbfsz.gov.hu/fdt_alapszelvenyek/. WMS server of geological map: (PDF)Click here for additional data file.S4 FigThe individuals are grouped according to burial groups A, B and C, and within these groups by subadult individuals as well as adult females and males. Within each group, the data are sorted from lower to higher values. See text for definition of the local strontium isotope ranges.(PDF)Click here for additional data file.S5 Figrd quarter of the 5th century (°), and individuals with similar C, N and Sr isotope data (‘) are labelled.The data are grouped according to sex and the presence or absence of artificial skull modifications. Representatives of the supposed founder generation (*), indication of inhumation in the 3(PDF)Click here for additional data file.S6 Fig13C values.The grey ellipse highlights a tight cluster of individuals with highly similar Sr and N isotope ratios. Bold burial numbers indicate individuals who exhibit also very similar δ(PDF)Click here for additional data file.S7 Fig13C values.While there are no significant differences among the animal data, the human bones from Mözs yielded the highest average δ(PDF)Click here for additional data file.


Trialeurodes vaporariorum. We tested the trapping efficacy of the LED water pan trap, assessed the most attractive LED light and analyzed its efficacy under different weather conditions. The results showed that the LED water pan trap was too inefficient to be useful. Green LEDs were more attractive than yellow LEDs, UV LEDs and green-UV combinations. Regardless of sunny or cloudy conditions, the green LED trap caught more than twice the number of whiteflies than the yellow sticky card alone under summer shading conditions. Our study suggests that LED traps have a significant field application value in whitefly mass trapping and may also be efficient for other diurnal insects. The design of LED traps specific for diurnal insects is discussed.Population control of small sucking insects has been challenging, and alternative control methods are constantly being sought. Visual traps have long been used to monitor and control pests. Colored sticky cards are widely used for diurnal pests, but their effects are influenced by environmental light conditions. Artificial light traps are mostly used for nocturnal pests. Here, we explored and evaluated light-emitting diode (LED) traps for the monitoring and control of small diurnal sucking insects using greenhouse tests targeting the greenhouse whitefly  Due to their small size and their difficulty to detect, in addition to their short life cycle, high fecundity, and outbreak propensity, some small sucking insects, e.g., whiteflies, aphids, and thrips, have become major pests on many agricultural crops worldwide, causing serious economic losses each year. These pests, not only cause direct damage to plant tissues, but also transmit plant viruses and facilitate infections through bacteria and fungi. Furthermore, they excrete sticky honeydew, which contaminates the plant surface and serves as a growth substrate for pathogens . ChemicaSome olfactory and visual cues are attractive to insects. Plant volatile chemicals may facilitate short-distance orientation to host plants. Sex pheromones and aggregation pheromones are responsible for conspecific chemical communication and lead to calling behavior and conspecific aggregation, respectively. Visual cues play important roles in host plant location and migration navigation. All these attractive elements could be used alone, or combined with insect traps, and serve as physical control tactics.Insects display phototactic behaviors. They can be attracted to specific light sources depending on wavelength and intensity ,5. BasedTrialeurodes vaporariorum (Westwood), an important pest worldwide, as a target insect to explore and evaluate the application potential of LED lights for the mass trapping of small diurnal insects in a greenhouse field test. The results of this study will provide valuable information for the development of a commercial highly efficient LED trap for the control of the greenhouse whitefly and will also be referential for the application of LED traps for other small diurnal insects.To our knowledge, no efficient artificial light traps targeting these small diurnal sucking pests have been commercialized yet. Light-emitting diodes (LEDs), a recently developed semiconductor light source, could be a promising light source for insect traps. LEDs have many outstanding advantages, e.g., high luminous efficiency, low electricity consumption, small size, light weight, long lifespan, low heat emission and environmental friendliness . FurtherA killing device and an attractive light source constitute the two major parts of a light trap. Regarding killing devices, the water pan trap is the simplest, least expensive and most commonly used among ordinary farmers. Water pan traps have been used to forecast outbreaks or mass trap aphids, thrips, etc. ,20. In oRegarding attractive light sources, many aphids and greenhouse whiteflies are attracted to targets that reflect or transmit light in the green-yellow range of the spectrum 520–610 nm), with yellow being the most attractive ,21,22. M20–610 nmFinally, considering the most important environmental factor, solar radiation, in color/light trapping efficacy, we determined how much the trapping efficacy would be improved by LEDs under sunny and cloudy weather conditions.T. vaporariorum to determine whether this inexpensive trap could be used to mass trap the greenhouse whitefly. Then, we compared the attractiveness of green, yellow, and UV LED lights and their combinations to T. vaporariorum to identify the most attractive LED light for trapping. Finally, we analyzed the potentiation effect of green LEDs under different weather conditions. This study will provide valuable information about the field application potential of LED insect traps for small diurnal pests. Further suggestions for the design and development of LED traps, specific for small diurnal insects, are discussed.In summary, in this study, we tested the trapping efficacy of the LED water pan trap for The current study was carried out between 14 May and 29 August 2019 at Harmony Farm in the city of Erdos, Inner Mongolia Autonomous Region, China .The LED traps used in this study were CD-IL400-8KIG1 traps, with a rated power of 8 W and an output voltage of 6–11 V (DC). The LED lamps were arranged in a corncob shape consisting of 6 light beams. The wavelengths of the LED light used in this study were as follows: Green light, 525 ± 5 nm; yellow light, 585 ± 5 nm; and UV light, 365 ± 5 nm. Both LED traps and LED lamps were provided by Changchun Cedar Electronics Technology Co., Ltd., Changchun, China A. The LEThe experiment was carried out in a 100 × 10 m greenhouse containing tomato from 14 May to 17 June 2019; temperatures ranged from 9–30 °C and the relative humidity ranged from 40%–90%. LED water pan traps A were huThe experiment was carried out in an intelligent greenhouse containing tomato planting of 20 × 50 m, with a temperature of 13–28 °C and relative humidity of 50%–90%. To directly compare the trapping efficacy of the water pan and yellow sticky card for greenhouse whiteflies, 4 green LED water pan traps equipped with a yellow sticky card B perpendThese experiments were carried out in the intelligent greenhouse. Starting in July, the shelf of the greenhouse was shaded from 10 am to 5 pm on sunny days. The temperature ranged from 15–28 °C, and the relative humidity ranged from 50%–90% during the experiment. The tomatoes (20 × 50 m) were planted in a high photosynthetic efficiency pattern in rows (50 m long) in the direction of magnetic south by west 15°. All yellow sticky cards were hung perpendicular to the rows and at the top of the tomato plants. In order to determine the different catch efficacies of the sunward sides (southward side) and dark sides (northward side) of the yellow sticky cards, 4 yellow sticky cards were evenly distributed in the middle row, with a 10 m distance between two traps. Considering the low whitefly population in the greenhouse at the time, the greenhouse whiteflies, trapped on the two sides of the yellow sticky cards, were checked every 3 days for 18 days.Then, we determined whether the relative position of the LED lamp in relation to the yellow sticky card would affect the trapping efficacy of the LED-lit yellow sticky card traps. Green LED lamps were set at either the sunward side or the dark side of the yellow sticky card refer to C, each cThis experiment was conducted in a different 100×10 m greenhouse. Before the experiment, we randomly picked 68 leaves from the top 3-7 branches of 15 tomato plants to estimate the greenhouse whitefly population level. Considering both larvae and adults, 3.7 ± 6.5 (mean ± SD) greenhouse whiteflies were present on each leaf. During the experiment, the greenhouse was shaded overhead from 10 am to 5 pm on sunny days; the temperature ranged from 15–32 °C, and the relative humidity ranged from 40%–90%. The whole planting area was divided into 3 blocks, and 3 LED-lit yellow sticky card traps refer to C,D with We collected all the data, including counts from the yellow sticky card alone and the green LED-lit yellow sticky card trap, at the same time from the shaded greenhouse, as described above. Each trap had 30 replicates. The data were further divided into two groups depending on the weather conditions: sunny or cloudy. The effects of weather conditions on the trapping efficacy of each type of trap were analyzed.t tests.Statistical analysis was performed using Statistical Product and Service Solutions software version 19.0 . Paired sample t tests were conducted to compare the number of greenhouse whiteflies captured between the water pan and the yellow sticky card and on the two sides of the yellow sticky cards. Analysis of variance (ANOVA) followed by the least significant difference (LSD) test for means comparisons were performed on the most attractive LED light trap data. Data were log-transformed to homogenize the variance when necessary but are presented as original data in the graphs. Other data were analyzed by independent samples Data visualization was conducted with GraphPad Prism 7 .T. vaporariorum population was very low in the greenhouse and only fewer than 3 whiteflies were found occasionally on some yellow sticky cards. One month later, the average number of total whiteflies captured by each yellow sticky card reached 46.9 ± 13.9 . However, no whitefly was caught by the LED water pan trap or by plant checking . Further analysis (by the LSD test) indicated that the yellow sticky card supplemented with a green LED light caught significantly more greenhouse whiteflies than the other 3 traps. The yellow LED light showed no significant effect on the trapping efficacy of the yellow sticky card, and the number of greenhouse whiteflies caught by the yellow sticky card supplemented with the UV LED light was the least efficient and caught significantly less whiteflies than the other 3 traps . Further analysis (by the LSD test) indicated that the presence of a UV LED light had no significant effect on the trapping efficacy of the green LED light. All 3 yellow sticky card traps equipped with a green LED light caught significantly more greenhouse whiteflies than the yellow sticky card alone . On the other hand, both traps caught significantly more greenhouse whiteflies on sunny days than on cloudy days . Furthermore, the green LED-lit yellow sticky card trapped even more whiteflies on cloudy days than the yellow sticky card alone on sunny days , the green LEDs increased the number of captures by more than one-fold compared with the yellow sticky card alone during the summer shading operation. Therefore, we suggest that both colored card and LED light should be included in the design of an LED insect trap to achieve the greatest attraction. Our study suggests that the LED trap has significant field application value in the mass trapping of greenhouse whiteflies and may also be efficient for the trapping of other diurnal insects if equipped with a species-specific attractive color card and LED light.The purpose of this study was to explore and evaluate the field application of LED insect traps for the monitoring and control of small diurnal sucking pests by using greenhouse whiteflies, Based on this study, we developed the patent “A LED insect trap for small diurnal insects” (Application number: China 2019221589118).


Mesocyclops aspericornis) on a vector species (Aedes aegypti) by laboratory experiments under different temperatures, nutrition and prey-density conditions. (3) We found that copepod predation reduced intraspecific competition among Aedes larvae and then shortened the survivor’s aquatic life and increased their pupal weight. In addition, the predatory efficiency of copepods was reduced at high temperatures. Furthermore, performance of copepod offspring fell when the density of mosquito larvae was high, probably because mosquito larvae had adverse effects on copepod growth through competition for food resources. (4) These results suggest that the increase in mosquitoes will not be suppressed solely by the application of copepods if the density of mosquito larvae is high or ambient temperature is high. We need to consider additional control methods in order to maintain the efficiency of copepods to suppress mosquito increase.(1) Dengue is the most spread mosquito-borne viral disease in the world, and vector control is the only available means to suppress its prevalence, since no effective treatment or vaccine has been developed. A biological control program using copepods that feed on mosquito larvae has been practiced in Vietnam and some other countries, but the application of copepods was not always successful. (2) To understand why the utility of copepods varies, we evaluated the predation efficiency of a copepod species ( Aedes mosquitoes transmit dengue virus that cause dengue fever and dengue haemorrhagic fever [Aedes (Stegomyia) aegypti (Linnaeus) and Aedes (Stegomyia) albopictus (Skuse). A noticeable means for Aedes control is the use of copepod predators. Since the discovery of copepod predation on mosquito larvae, they have been used to control Aedes larvae in temperate, subtropical and tropical regions [ic fever . Because regions ,6,7,8,9. regions ,3,4,5,10 regions . In a re regions . However regions .Aedes, increase more rapidly at a higher temperature if it does not exceed harmful upper limits and food is not limited [Copepods predate on younger (mainly first instar) mosquito larvae more efficiently in comparison with later-stage larvae ,14. Ther limited . HoweverMesocyclops aspericornis (Daday) as an agent to suppress the increase in Ae. aegypti populations by laboratory experiments in which rearing conditions, i.e., temperature, the amount of foods for mosquitoes and copepods, and the initial density of mosquito larvae, are manipulated. We used Ae. aegypti but not Ae. albopictus as a prey species because of the following two reasons. First, the former species is more sensitive to environmental changes in population growth than the latter [Ae. ageypti eggs immediately hatch, but Ae. albopictus eggs vary in hatching time. Therefore, it is difficult to prepare a large number of first instar larvae for experimental use in the latter.This study aims to understand the effects of environmental conditions on the effectiveness of a copepod species e latter . Second,M. aspericornis were established with females collected from water containers placed in a domestic area of Ho Chi Minh City (10˚46’10”N and 106˚40’55”E), Vietnam in 2003. They were maintained in 15-L plastic boxes (28 × 38 × 14 cm) according to the methods described by Suárez et al. (1992). Copepods were fed with Chilomonas paramecium infusion that was cultured with wheat seeds (50seeds/L) in 1-L plastic jars at 27 °C for seven days or longer. The chemical oxygen demand (COD) of infusion always exceeded 100 mg/L according to our measurements using commercial kits .Mass cultures of Ae. aegypti females collected from Ho Chi Minh City, Vietnam, in 2005. Adult mosquitoes that emerged were maintained at 27 ± 1 °C and 70% relative humidity under 14L/10D (14 h light - 10 h dark) photoperiod conditions. They were fed with a 3% sucrose solution and allowed to suck blood from an anesthetized mouse once a week. Newly hatched Ae. aegypti first instar larvae (within 2 hours after hatching) were used in the experiments.A laboratory colony was established with Ae. aegypti was evaluated by larval mortality, the duration of the first-instar and whole-larval periods, and pupal dry weight [M. aspericornis was evaluated by the number of offspring produced and the development time of offspring from egg to adulthood. A treatment comprises of 10 or 50 newly hatched Ae. aegypti larvae with or without a primiparous M. aspericornis female in a 200-mL plastic cup  containing 100 mL of “nutrient rich” or “nutrient poor” water. To prepare Ae. aegypti larvae, dried eggs were soaked in water for 2 hours, and then newly hatched larvae were collected. The nutrition medium was Chilomonas paramecium infusion. The COD level of “nutrient rich” water was adjusted to 20 mg/L by diluting the infusion, and 5 mL of food medium was added daily while the COD level of “nutrient poor” water was adjusted to 4 mg/L and one mL of food medium added daily. Thirty replicates were prepared per treatment.Experiments were carried out under eight conditions  at three temperatures  under 14L/10 D photoperiod in incubators . We measured water temperature in experimental cup without animals and foods placed in the incubators using StowAway Tidbit data loggers , and we confirmed that water temperature fluctuated within the range of ± 1 °C. The population performance of y weight . The perAedes larvae occurred was approximately 20 mg/L as a mode . All treatments were checked daily to record the number of surviving mosquito larvae and growth of copepods. When mosquito pupae were observed, they were collected and dipped in 60 °C hot water to kill. Then, they were placed on paper using a pipette to absorb moisture, transferred to a 96-well plastic plate to dry at 32 °C for 48 hours, and measured for dry weight using a digital balance . Observations were terminated when all mosquitoes had pupated or died.Temperatures of 20, 27 and 32 °C were chosen to cover the range of the annual minimum and maximum temperatures of Ho Chi Minh, where the mosquito strain was collected. The COD of water in experimental cups was determined with reference to COD in mosquito habitats in Ho Chi Minh; i.e., COD in field containers in which The effects of temperature, nutrition conditions, initial larval density and the copepod presence on mosquito performance was analyzed by generalized linear model (GLM). The analysis on mortality was performed with the logit link function and the binomial distribution error, and the analysis on the larval development time and mosquito pupal weight was with the identity link function and the normal distribution error. The effects of temperature, nutrition, initial larval density on copepod performance was also analyzed using GLM with the identity link function and the normal distribution error. For explanatory variables, temperature, nutrition, initial larval density and the copepod presence and interaction terms of these parameters were used in the analysis of mosquito performance, while the same parameters, except for the copepod presence, were applied in the analysis of copepod performance. All statistical analyses were performed using JMP version 11.2.1 .Ae. aegypti larvae and copepod presence) significantly affected larval mortality and developmental time and pupal body weight of Ae. aegypti (p < 0.001). The mortality of first instars was most influenced by the presence of copepod  was generally higher at a lower temperature, under nutrition-poor conditions and at a low initial density of Ae. aegypti larvae  significantly affected the production and developmental time of copepod offspring  was generally reduced at higher temperatures, under nutrition-rich conditions and at a high initial density of Ae. aegypti larvae. This seems to be related to the prey-size selection of copepods and to the difference in their temperature response. Copepods preferentially feed on small mosquito larvae [Ae. aegypti larvae increase body size more rapidly and could escape from copepod attack. This result may explain why the application of copepods was not successful in a hot location . It may also explain the results of copepod application in Laos where Ae. aegypti was eliminated from wells but not from water containers [Bacillus thuringiensis israelensis (Bti), permethrin, methoprene, or pyriproxyfen may be effective, because these kill mosquito larvae but do not harm copepods [Anopheles and Aedes populations under copepod-based control programs [Under the presence of o larvae ,14, possntainers ,25; i.e.copepods ,26. Furtprograms . As a new method of controlling mosquitoes—a mass release of genetically modified mosquitoes—is becoming applicable in addition to the conventional means. Yakob et al. performed a mathematical simulation of three control methods, a mass release of infertile males, Wolbachia-infected mosquitoes and genetically modified mosquitoes carrying lethal genes, and pointed out that the former two may enhance the survival of adult mosquitoes by reducing intraspecific competition of mosquitoes . MoreoveAedes larvae and result in a faster development and larger pupal weight of survivors. In addition, high temperature, nutrition-rich conditions and a high larval density of Aedes reduced the predatory efficiency of M. aspericornis, probably because Aedes larvae increased body size more rapidly and were able to escape from copepod attack. The effect of a high density of mosquito larvae is to hamper copepod growth through interspecific competition for food. These results suggest that the utility of copepods as a mosquito-controlling agent could be reduced under higher temperature, organic rich water, and higher densities of mosquitoes.Our study demonstrates that copepod predation could reduce intraspecific competition among


Zea mays L.). We grew maize plants in an agricultural loamy soil and sampled rhizosphere soil at distinct locations along maize roots. We applied high-throughput sequencing, followed by diversity and network analyses in order to track changes in relative abundances, diversity and co-occurrence of rhizosphere microbiota along the root axis. Apart from a reduction of operational taxonomic unit (OTU) richness and a strong shift in community composition between bulk soil and root tips, patterns of microbial community assembly along maize-roots were more complex than expected. High variation in beta diversity at root tips and the root hair zone indicated substantial randomness of community assembly. Root hair zone communities were characterized by massive co-occurrence of microbial taxa, likely fueled by abundant resource supply from rhizodeposition. Further up the root where lateral roots emerged processes of community assembly appeared to be more deterministic . This shift toward significance of deterministic processes was revealed by low variability of beta diversity, changes in network topology, and the appearance of regular phylogenetic co-occurrence patterns in bipartite networks between prokaryotes and their potential protistan predators. Such patterns were strongest in regions with fully developed laterals, suggesting that a consistent rhizosphere microbiome finally assembled. For the targeted improvement of microbiome function, such knowledge on the processes of microbiome assembly on roots and its temporal and spatial variability is crucially important.It is by now well proven that different plant species within their specific root systems select for distinct subsets of microbiota from bulk soil – their individual rhizosphere microbiomes. In maize, root growth advances several centimeters each day, with the locations, quality and quantity of rhizodeposition changing. We investigated the assembly of communities of prokaryotes (archaea and bacteria) and their protistan predators  along the longitudinal root axis of maize ( The predictable assembly of specific subsets of the soil microbiota in the rhizosphere of plants has led to the characterization of plant species-specific “microbiomes” . As the Differences in resource supply (bottom–up processes) are thought to be the main driver of microbiome assembly . The vas–1 but up to 7 cm day–1  or manipulation of rhizodeposition (niche modification) along the growing root axis . RandomnAs root growth progresses, the initially random community assembly at the root tip is expected to be replaced at some point by increasingly deterministic processes that lead to the typical plant-associated microbiomes. Deterministic assembly of microbial communities can arise through two mechanisms. The first is competition for resources, a mechanism that is likely to increase when rhizodeposits subside . The secTo gain insights into the self-organization of the rhizosphere microbiome, we investigated the assembly of rhizosphere specific microbial communities of prokaryotes and their protistan predators along the longitudinal root axis of maize plants at clearly defined root regions. We investigated the early assembly at root tips to communities of subsequently older root regions until first order lateral roots dominated root architecture. We hypothesized an immediate reduction of alpha diversity of prokaryotes in rhizosphere as compared to bulk soil due to a competitive advantage of the fast-growing, copiotrophic taxa from rhizodeposition. Differences in beta diversity, however, should be maximized on root tips, due to an increased likelihood of random priority effects in the early stages of community assembly. Further on, we expected that root regions with highest resource availability  would favor many taxa indiscriminately, becoming visible by increased evenness and in networks by high positive co-occurrence of taxa. Finally, we expected the strongest selective forces when rhizodeposits subside, both by microbial competition and predation, which eventually leads to the assembly of the microbiome. Since the “microbiome” is predicated on deterministic processes, it would be characterized by the strongest shift in community structure (beta diversity) compared to bulk soil, together with a strongly reduced variation of beta diversity . At the same time, co-occurrence networks would show a more structured topology, on the one hand due to competitive exclusion and on the other hand by taxa which coevolved during the assembly process and can mutually coexist in the rhizosphere. The existence of top-down processes by protists through selective grazing should be revealed in potential trophic networks between prokaryotes and protists by clear phylogenetic patterns of both negative co-occurrences  as well as positive co-occurrences .Zea mays seeds (inbred line B73) were surface sterilized with 10% H2O2 for 10 min and germinated on wet filter paper under sterile conditions at 18°C in the dark. After 3 days, 40 seedlings of similar length were selected and separately planted into cylindrical perspex microcosms  filled with 885 cm3 of 1 mm sieved sandy loam soil at a bulk density of 1.46 ± 0.1 g dry wt cm–3, corresponding to 1300 ± 80 g dry wt microcosm–1. The bottom of each microcosm was closed with a nylon gauze (30 μm mesh size) for watering. Soil water content was kept constant at 22%VWC by daily weighing and replacement of water. Tubes were wrapped in aluminum foil to protect soil and roots from light. Plants were grown in a climate chamber with a day–night regime of 12/12 h (350M PAR) at 24°C/18°C and 65% humidity.g, the supernatant was discarded, and the pellet was used for DNA extraction. DNA-extraction and purification were done using the FastDNA® SPIN Kit for soil and the GENECLEAN® SPIN Kit , following the manufacturer’s instructions.Microcosms were sampled 9 days after planting. Using sterile forceps and scissors, 1 cm root samples with adhering soil from (i) the root tip, (ii) the root hair zones, (iii) the region where the first lateral root primordia emerged as earliest lateral roots, and (iv) from a subsequent region with fully developed lateral roots  were traA two-step PCR yielding a c. 350 bp long fragment of the V4 region of the SSU/18S gene was conducted using primers  targetinA 1 μl of purified DNA from soil samples served as the template for the first PCR, resulting in amplicons of which 1 μl was used for the second semi-nested PCR. Both PCR-rounds were conducted with reagents in the following final concentrations: Dream Taq polymerase  0.01 units, Thermo Scientific Dream Taq Green Buffer, dNTPs 0.2 mM and primers 1 μM. All PCRs were conducted in duplicates to reduce the possible artificial dominance of few amplicons by PCR competition and then pooled. The thermal program for the reaction started with a 2 min denaturation step at 95°C, followed by 95°C for 2 min,  repeated 24 times and a final elongation step at 72°C for 5 min. After checking for successful amplification and potential contamination by gel electrophoresis, 24 μl of the PCR-product of each sample were purified and normalized using SequalPrep Normalization Plate Kit  to obtain a concentration of 1–2 ng/μl per sample. Purified amplicons were pooled, concentrated and sequenced on an Illumina MiSeq platform  at the Cologne Center for Genomics . With the MiSeq v3 Reagent kit, 2 × 300 cycles were performed, producing 300 bp long paired-end reads.Reads were processed using the customized MOTHUR pipeline v.39.5 .e–50 and the PR2 database  using VSEARCH  accordindatabase , keepingdatabase , allowindatabase , resultiTM (TaKaRa Bio Group) and 10 μM of forward and reverse primer each. The thermal program for the reaction started with a 5 min denaturation step at 95°C, followed by  repeated 30 times and a final elongation step at 72°C for 5 min. The sequencing was done on the Illumina HiSeq platform  by Magigene Technology Co., Ltd. . With a HiSeq v2 Reagent kit, 2 × 250 cycles were performed, producing 250 bp long paired-end reads.The forward primer 515F (5′-GTGCCAGCMGCCGCGGTAA-3′)  and the Quality trimming and adapter clipping of the raw reads was performed using Trimmomatic . Paired-post hoc test. After the removal of one outlier from the prokaryote and two from the cercozoan dataset permutational multivariate analysis of variance (PERMANOVA) using Bray–Curtis dissimilarity, permuted 999 times, was employed to test for differences in prokaryote and cercozoan community structure across samples from different root regions.Statistical analyses and data visualization were performed in R version 3.5.1 . With ou1  which measures the complexity of a network, average clustering coefficient, average path distance and modularity were calculated. Based on detected modules among-module connectivity and inter-module connectivity were calculated, and nodes were assigned to one of four possible network roles: network hubs, module hubs, connectors or periphers.Co-occurrence networks incorporating communities containing bacteria, archaea, and Cercozoa were based on single OTUs and generated to assess co-occurrence or potential interactions between species. To assess the complexity and specificity of community structures along roots, network analyses were conducted for communities at the four different sampled root regions and from bulk soil. For network construction and analysis of topological features the molecular ecological network analysis pipeline (MENAP)1  was used2, including connectance which is the proportion of possible links that are established, links per OTU, number of compartments, and modularity for the cercozoan community. The network graphs were visualized in Cytoscape 3.7.2 pe 3.7.2 .F4,186 = 14.12, p < 0.001). Bulk soil and root tips showed highest prokaryote alpha diversity  and evenness  compared to older root regions  and protists . Beta diversity of prokaryote communities was lowest in bulk soil, highest at root tips and the root hair zone, and decreased again toward lateral root primordia and lateral roots, respectively , indicative of a scale-free network structure with a topology of many nodes with few connections and some highly connected nodes within modules . BS, YC, and RS provided sequences of prokaryota. MB and LR wrote the manuscript with assistance of MW. PY and FH contributed significantly with their expertise on maize root development and exudation. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Adiponectin and Resistin correlate with insulin sensitivity and cardiovascular risk, respectively. This study aimed to identify lifestyle factors that modulate changes in Adiponectin and Resistin levels after gastric banding positioning (LapGB).2). After surgery, a dietitian checked the adherence of S-Ob to an Apulian hypocaloric diet (aphypoD)/physical activity (phA) and, according to their high or low compliance to aphypoD/phA, S-Ob were included in group 1 (n = 14) or 2 (n = 13) respectively. Serum Adiponectin and Resistin were also measured in 10 healthy controls.Before (T0), 3 months (T3), 6 months (T6), and 12 months (T12) after LapGB, serum Adiponectin and Resistin levels were evaluated in a single-centre prospective study including a cohort of 27 non-diabetic obese subjects , significantly decreased Resistin levels at T12 and increased Adiponectin levels at both T6 and T12 as compared with baseline. Group 2 showed 24.6 %EWL at T12, decreased Adiponectin levels at T6 and T12 as compared with baseline, but unaltered Resistin levels. After surgery, group 1 followed aphypoD/phA, while group 2 did not.LapGB fails to improve cardiovascular risk markers (Resistin) in S-Ob not improving lifestyle. Future studies might investigate these findings in a larger cohort and display whether aphypoD is more effective than other dietary intervention on cardiovascular risk in subjects undergoing LapGB or other Bariatric procedures. •Gastric banding (LapGB) plus a 12-month lifestyle program was followed by a % excess weight loss >40.•Resistin significantly declined in subjects adhering to a 1-year LapGB/lifestyle intervention.•In subjects not following any lifestyle program after surgery, Adiponectin unexpectedly decreased.•LapGB fails to change cardiovascular risk or insulin sensitivity without a healthy lifestyle. After closure of the ring, the stomach takes on the appearance of an asymmetrical egg-timer, with a small upper gastric pouch  and the remaining stomach below the band. This surgical technique is considered as “gastric restrictive” because it produces a significant reduction of the stomach volume and is associated with the feeling of early satiety ,2.Adiponectin and Resistin are cytokines playing a role in chronic low-grade inflammation. It is known that inflammation and endothelial dysfunction are involved in atherosclerotic plaque formation, and that dysregulation of adipokines in human obesity may contribute to this process ,4. ResisIn this study we analyzed lifestyle factors that can modulate the changes in Resistin circulating levels and some surrogate markers of insulin sensitivity  in a cohort of severely obese subjects (S-Ob) consecutively undergoing the positioning of laparoscopic adjustable gastric banding (LapGB). We found that, following bariatric surgery, subjects showing a good compliance to healthy lifestyle changes experienced a significant weight loss and reduction in Resistin.22. The exclusion criteria were current smoking, pregnancy, Type 2 and Type 1 diabetes mellitus, gestational diabetes, or other specific types of diabetes related to chronic pancreatitis, oral steroid therapy.This was a prospective study including 27 patients  consecutively followed at the Outpatient Obesity Clinic of the Unit of Endocrinology of the University Hospital “Consorziale Policlinico” in Bari, Italy. The inclusion criteria were age between 20 and 55 years and BMI 35–65 kg/mThe study was conducted in compliance with the Declaration of Helsinki and European Guidelines on Good Clinical Practice. The Research Registry Unique Identifying Number for the study is Researchregistry5052.Before and after surgery, physical activity levels were recorded using the short form of International Physical Activity Questionnaire (IPAQ). IPAQ is a good instrument to facilitate surveillance of physical activity based on a global standard . The sho3t-Test for normally distributed data. For non-normally distributed data, Wilcoxon signed-ranks test and Mann–Whitney U test were used and p-values <0.05 were considered statistically significant. Repeated Measures Anova tested comparisons between variables in different groups at different time-points.NCSS Statistical software  was used for statistical analysis. For normally distributed data, the results are presented as mean ± standard deviation (SD) unless stated otherwise. Comparisons between independent groups were performed with the Student's www.randomization.com.The sample size was calculated assuming a 35% difference in response between the two groups (high/low compliance). We estimated that 20 patients would have been the number required for the study to have 80% power and an α error of 5%. A per-protocol analysis was applied to the trial. Instead of 20 subjects we recruited 27 subjects (7 more subjects) because we were expected to have some dropouts. The randomization list was generated using the online resource available at 42  (range 36.77–57.86). Comorbidities included arterial hypertension , joint disease , dyslipidemia , gallbladder stones (1 patient in group 1), anxiety-depression (3 patients in group 1), cardio-respiratory diseases , and vascular disease . Baseline anthropometric variables, arterial blood pressure levels, heart rate, lipid pattern, liver function tests, HOMA-IR and Matsuda index . Before Bariatric surgery, each patient included in the study gave a written informed consent to allow the bariatric medical team belonging to Department of Emergency and Organ Transplantation of the University of Bari “Aldo Moro” to use data of the study for research purposes only. The members of the bariatric medical team were formally accepted by the Director of the University Hospital of Bari.University of Bari “Aldo Moro” .Funds to Gabriella Garruti from the Gabriella Garruti designed the study, was involved in the follow up of patients before and after surgery and wrote the paper.Michele De Fazio was involved in the surgical procedures and in the discussion of results.Palma Capuano was involved in patient selection and surgical procedures and in the discussion of results.Gennaro Martinez was involved in the surgical procedures.Maria T. Rotelli was coordinating the measurements of Adiponectin and Resistin and all the other laboratory tests reported in the paper.Francesco Puglisi designed the study, was involved in patient selection and surgical procedures and in the discussion of results.Nicola Palasciano was involved in the surgical procedures and in the discussion of results.Francesco Giorgino was involved in the discussion of results.http://www.researchregistry.com1. Name of the registry: 2. Unique Identifying number or registration ID: Researchregistry50523. Hyperlink to the registration (must be publicly accessible):Gabriella Garruti is fully responsible for the work and the conduct of the study, had access to the data, and controlled the decision to publish on the behalf of all the Authors involved in the study.Before Bariatric surgery each patient included in the study gave a written informed consent to allow the bariatric medical team belonging to Department of Emergency and Organ Transplantation of the University of Bari Aldo Moro to use data for research pourposes only.Not commissioned, externally peer reviewed.Authors declare that they have no competing financial interests in relation to the work described.


Contrarily, quinoa exhibited a Na+ includer character with the highest accumulation detected in the shoots. Expression levels of main genes involved in Na+ homeostasis  showed different patterns between amaranth and quinoa, with a marked higher basal expression in amaranth roots. These results highlight the important differences in the physiological and molecular responses of amaranth and quinoa when confronted with salinity.Yield losses due to cultivation in saline soils is a common problem all over the world as most crop plants are glycophytes and, hence, susceptible to salt stress. The use of halophytic crops could be an interesting alternative to cope with this issue. The Amaranthaceae family comprises by far the highest proportion of salt-tolerant halophytic species. Amaranth and quinoa belong to this family, and their seeds used as pseudo-cereal grains have received much attention in recent years because of their exceptional nutritional value. While advances in the knowledge of salt tolerance mechanisms of quinoa have been remarkable in recent years, much less attention was received by amaranth, despite evidences pointing to amaranth as a promising species to be grown under salinity. In order to advance in the understanding of strategies used by amaranth to confront salt stress, we studied the comparative responses of amaranth and quinoa to salinity (100 mM NaCl) at the physiological, anatomical, and molecular levels. Amaranth was able to exhibit salt tolerance throughout its life cycle, since grain production was not affected by the saline conditions applied. The high salt tolerance of amaranth is associated with a low basal stomatal conductance due to a low number of stomata  and degree of stomata aperture  of leaves, which contributes to avoid leaf water loss under salt stress in a more efficient way than in quinoa. With respect to Na Amaranthus spp.) has greatly increased in recent years. Some species are consumed as vegetables and others as grain-producing crops, being the most important species of these last group Amaranthus cruentus, Amaranthus caudatus, and Amaranthus hypochondriacus. These species are recognized by their high protein content and have been proven to be non-allergenic food with seeds of remarkable nutraceutical properties  is one of the very few halophyte seed crops where numerous experiments have been carried out under conditions of salinity (Amaranthaceae). It is an exciting perspective to advance in the knowledge of the salt tolerance mechanisms in species such as amaranth and quinoa, since the high genetic variability of both species constitutes an attractive feature for their adaptation to most of the arable regions from tropical to temperate climates, under different environmental conditions , involved in Na+ retrieval from the xylem under salt stress, as well as NHX1 (Na+/H+ exchanger 1), involved in the vacuolar Na+ compartmentation . Thus, quinoa CqHAK5-like transporter drives K+ influx into EBCs of the leaf to contribute to osmotic balance of cytosol against osmotic pressure due to the salt-containing vacuoles (+ Outward-Rectifying) is also important as it is involved in the long-distance K+ transport from root to shoot, toward the xylem vessels  and with short times of salt exposure, conditions that are not agronomically realistic. Therefore, if we are interested in identifying salt tolerance mechanisms and selecting useful traits with the goal of upholding grain production, lower salt levels should be applied, and salt tolerance evaluated through the whole plant life cycle. This study has been carried out to investigate the comparative mechanisms used by amaranth and quinoa when a not so high salt level is applied (100 mM of NaCl), which warrants seed production, a key agronomic trait. Here we show the different anatomical, physiologic, and molecular responses in amaranth and quinoa when confronted to such salinity level, as well as the expression levels of the main genes involved in NaAmaranthus caudatus) , and quinoa (Chenopodium quinoa) , it was found that Kwicha Perú (K1) was the most tolerant amaranth variety to salinity, being able to maintain vegetative development when exposed to extremely high levels of salinity (300 mM of NaCl for 11 days)  –3. Simil11 days) . Therefo–2s–1 at the plant level provided by fluorescent tubes , and 18–25°C and 50–60% of temperature and RH, respectively.Seeds of the selected varieties of amaranth (K1) and quinoa (QQ) were germinated in darkness, in an 8:3 (v/v) mixture of peat and perlite, respectively, at 28°C temperature and 90% of relative humidity (RH). After emergence, plants were transferred to a pot (14 cm diameter) and grown in a controlled-condition growth chamber with 16 h light and 8 h darkness photoperiod, with the light of a photosynthetic photon flux (400–700 nm) of 350 μmol mTo unravel the strategies displayed by amaranth to confront salt stress compared to quinoa in terms of maintenance of vegetative development, a short-term salt treatment assay was carried out in a controlled-condition culture chamber. After 35 days of germination, nine plants of each species were irrigated with half-strength Hoagland solution  as contr–2s–1 of mean natural light irradiance ; and in summer, 50/22°C day/night temperatures, 40% relative humidity, and 1,200 mmol m–2s–1 of mean natural light irradiance (PAR). At the fifth fully developed leaf stage, 20 plants of each species were transplanted to the greenhouse and grown in containers with coconut peat, using a drip irrigation system and a fertigation solution as described by –1). After 30 days of growing in these conditions, 10 plants per species were subjected to salt treatment with fertigation solution where 100 mM NaCl was added , maintaining the other 10 plants of each species growing in fertigation solution without NaCl (control treatment). When plants reached their physiological maturity, 125 and 110 days after germination for amaranth and quinoa, respectively, irrigation was suppressed, and 15 days later, when plants were fully dried (seed humidity <12%), seeds of primary, secondary, and tertiary (the latter only appeared in quinoa) panicles were harvested, weighted, and finally fully dried in an oven at 60°C.To evaluate salt tolerance of amaranth and quinoa in terms of grain yield, a long-term salt treatment assay was carried out in a polyethylene greenhouse with natural conditions located in Santomera municipality  in an area of semiarid conditions typical of South-east Spain. The conditions of the greenhouse were the following: in spring, 40/15°C day/night temperatures, 48% relative humidity, and 600 mmol m–1) were calculated as [(FW - DW)/DW], where FW and DW are fresh weight and dry weight, respectively. Root/shoot water content ratio was calculated by dividing root water content (mg g DW–1) by shoot water content (mg g DW–1) of each individual plant. Shoot water content is the sum of the stems’ and the adult leaves’ water contents.Vegetative samples  of plants from the short-term salt treatment assay were oven-dried at 60°C for 72 h, and water contents (mg g DW–1) were converted to osmotic potential (1 mOsm = -2.408 kPa). Crude leaf sap extracts were obtained by centrifugation of previously frozen material in liquid nitrogen.Osmotic potential was determined by measuring osmolality in the sap extracts of the leaves by the freezing point depression method using an automatic osmometer . Osmolalities (mOsm kggs (mmol H2O m–2s–1), which indicates the degree of stomatal opening or closing by the rate of CO2 entry and H2O exit through the stomata, and the foliar transpiration rate, E (mmol H2O m–2s–1) were analyzed. Working conditions of the CIRAS-2 equipment were as follows: 1.7 cm2 of measurement leaf surface, 480 ppm reference CO2 level, 25.7 to 26.8°C temperature of the leaf in the measurement chamber, 195 ml min–1 gas flow rate in the measurement chamber, ambient pressure (1.010 mb) and 500 μmol m–2s–1 photosynthetic photon flux density flow (PAR).To analyze stomatal conductance and foliar transpiration, a portable photosynthesis measurement system  was used in intact upper and fully expanded leaves after 20 days of treatment  of adult plants (55 days after germination). The stomatal conductance, 2 were processed as described by –2) and stomatal aperture . These parameters were determined using the free ImageJ software . Finally, for leaf tissue anatomical measurements, toluidine blue-stained sections were observed under light microscope , and digital images were obtained, 18 randomly selected images, from nine plants per species and treatment, were analyzed using ImageJ software. Mesophyll and bundle sheath cell size (μm2) of amaranth, and palisade and spongy parenchyma cell size (μm2) of quinoa were measured. Adaxial and abaxial epidermis cell size (μm2), leaf thickness (μm), and cell density (cell mm–2) were also measured for both species.For microscopy analyses, sections of 1 mm3:HClO4 (2:1 v/v) solution and analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES) in an ICAP 6500 DUO/IRIS Intrepid II XLD equipment . Measurements were carried out at the Ionomics Platform of CEBAS-CSIC .Dried tissues used for water content determination were milled to powder, digested for 8 h in a concentrated HNO+ homeostasis, AhSOS1 (AH006272-RA), AhHKT1;1 (AHYPO_003592-RA), AhNHX1 (AHYPO_008765-RA), and in K+ homeostasis AhHAK5 (AHYPO_013402-RA) and AhSKOR (AHYPO_001358-RA), were obtained by blasting the mRNA sequences of CqSOS1, CqHKT1;1, CqHKT1;2, and CqHAK5  genome  was obtained by blasting the mRNA sequence AtSKOR of A. thaliana against the quinoa (C. quinoa) genome   genome , using P) genome , using t sativa) . Amino asoftware –10. All n = 9). Fresh roots, stems, and the first fully developed leaf were immediately frozen in liquid nitrogen and pulverized in a mortar. Then, 120 mg of the frozen powder was used for total RNA extraction with the E.Z.N.A.® Plant RNA Kit  according to the manufacturer’s instructions. Contaminant DNA was removed with RNase-free DNase Set I . Total RNA was quantified in a Nanodrop 1 ND-2000 spectrophotometer , and 1 μg was used for cDNA synthesis with the iTaq® iScript cDNA synthesis kit (Bio-Rad). One microlitre of the cDNA sample was used for gene amplification using the iTaq® Universal SYBR Green Supermix (Bio-Rad). Amplification reactions were carried out in a Rotor Gene 3000 (Corbett Research). All primers used for quantitative RT-qPCR are listed in Elongation Factor 1a as housekeeping gene of each species, AhEF1a (AH015912-RA) for amaranth and CqEF1a for quinoa  after 20 days of treatment. For these analyses, three biological replicates were considered, each one consisting of three plants  in 20% TCA. The mixture was incubated at 90°C in a shaking water bath for 30 min, and the reaction was stopped by placing the reaction tubes in an ice bath. The samples were centrifuged at 14,000 × g for 10 min, and the absorbance of the supernatant was read at 532 nm in a spectrophotometer . The value for non-specific absorption at 600 nm was subtracted. The amount of the MDA–TBA complex (red pigment) was calculated using the extinction coefficient 155 mM–1cm–1. Results were expressed as nmol MDA produced per gram of fresh weight per hour (nmol MDA g–1h–1).Malondialdehyde (MDA) was quantified in adult leaves, as indicator of cell membrane lipid peroxidation, by means of the thiobarbituric acid reactive substrates (TBARS) assay, using the protocol described by 2–stock solution in 100 ml of phosphate buffer . Absorbance at 734 nm was measured to check ABTS•− formation. Fresh leaf, 0.2 g, was grounded in 10 ml in the mentioned above buffer and centrifuged for 10 min at 9,500 rpm at 4°C. Of the sample, 40 μl was mixed with 1.960 μl of the ABTS•− radical solution to measure the antioxidant activity at 734 nm for a period of 6 min. The decrease in absorbance at 734 nm observed after the addition of each compound was used to calculate the Trolox equivalent antioxidant capacity (TEAC). A calibration curve was prepared with increasing concentrations of Trolox . The TEAC activity was calculated according to –1 FW, which has the same antioxidant capacity as the analyzed sample .In order to evaluate the total antioxidant activity of the plant tissues, the Trolox Equivalent Antioxidant Capacity (TEAC) assay was performed as described by Seeds (grain) were manually separated using sieves and filters of different pore size to remove the rest of the dry panicle. Total yield (g) was calculated weighting all harvested seeds by panicle . The average seed weight (mg) was calculated weighting 100 seeds per plant (mg 100 seeds/100). The number of seeds per panicle was calculated by dividing the total seed yield (g) between average seed weight.p ≤ 0.05) to determinate in each combination of factors where differences were significant, denoted by different letters.Experimental data are presented as mean ± standard error (SE) of 18 plants per species (nine plants per each treatment). Statistical analysis was performed using SPSS 19.0 software package by a two-way analysis of variance (ANOVA) to determinate the interaction between different factors , carrying out a factorial design and a one-way analysis of variance (ANOVA), followed by Tukey’s test , only Kwicha Peru (K1) did not reduce the germination percentage when increasing the salinity levels, whereas the rest of the varieties were differently affected. Thus, a sharp inhibition was found in Blanco (B) and Burganda (Bur) varieties starting from 100 mM NaCl, and Oscar Blanco (OB) and Kwicha Granada (K2) varieties at 150 mM NaCl  at the eighth developed leaf stage (adult plants) for 20 days, we did not find significant changes in shoot vegetative biomass between the control condition and salt stress. Contrarily, root growth was significantly reduced with salinity in both species, although the reduction was greater in quinoa (50%) than in amaranth (25%) . The maiRegarding traits related to osmotic homeostasis, first, water contents were analyzed in the root and leaf at the end of the experiment . These ss reduction induced by salinity was significantly greater in quinoa (59%) than in amaranth (37%) (gs) and transpiration ratio (E), values of both traits in control condition were significantly higher in quinoa than in amaranth  .The leaf Ψth (37%) . Regardictively) . Under sIn amaranth, stomatal density (SD) was lower in the adaxial than in the abaxial surface under control condition, and it was in the abaxial surface where the SD was significantly reduced (27.3%) by salinity, as it is illustrated in the micrographs . In quin4 plants, with the bundle sheath (BS) cells containing centripetally located chloroplasts, and a layer of mesophyll cells surrounding the BS. These cells were those altered by salinity, as mesophyll cells significantly reduced their size with salinity and increased their cell density, while BS cells remain unmodified  as a marker of oxidative stress of plants, as well as the total antioxidant capacity measured as TEAC in leaves of both species . In amar+ distribution within the plant , but only one had homology with quinoa CqHKT1;1 and CqHKT1;2, the protein AHYPO_003592-RA, which was named AhHKT1;1 since it is the isoform showing higher homology to the quinoa one (HKT1 family (AH 014429-RA) in amaranth had higher homology with HKT1;3 of Beta vulgaris and HKT1;1 of Suaeda salsa and Salicornia bigelovii. However, its expression was practically zero in amaranth in the control condition as well as in salt stress, and therefore, it was not included in the gene expression analyses.In order to analyze the transcript levels of the main genes involved in uptake and long-distance Naectively . In the inoa one . The othSOS1, HKT1;1, and NHX1 genes were all much higher in the roots of amaranth than in those of quinoa , both showed a similar response as their shoot vegetative biomasses were not affected by salinity . HoweverSalinity tolerance is achieved through different mechanisms operating simultaneously, but these were differently activated in amaranth and quinoa. The first effect induced by salinity is reduction of water uptake by the root due to osmotic stress. However, the water contents in roots of amaranth and quinoa increased after 20 days of 100 mM NaCl treatment compared to control plants, which indicates that root ability to uptake water under salinity conditions was similar in both species, independent of the different root development of each. However, the differences between amaranth and quinoa came out in the leaves, as only amaranth increased its leaf water content with salinity but not quinoa . The hydThe other mechanism contributing to the high salinity tolerance of halophytes is reduced water loss through the leaves , and thiAn interesting question is to elucidate whether there were differences between both species in the oxidative stress caused by salinity. Several studies have linked a high antioxidant capacity with a high osmotic stress tolerance in abiotic stress . Salinit+ homeostasis under salinity are very different. Amaranth exhibited higher Na+ accumulation in the root followed by the stem and a much lower accumulation in the leaf, with this pattern similar enough to that shown by glycophytic or Na+ excluder species , and either the HKT1-type gene in each different shoot organ analyzed: CqHKT1;1 was induced by salinity in the stem and CqHKT1;2 in the leaf  was analyzed, we observed a marked decrease under saline conditions in roots in both species, as it occurred in Arabidopsis with AtHAK5 in salt stress  to confront salt stress.One trait correlated with salt tolerance is the Na species  but also species . In our the leaf . When tht stress  (Supplemt stress . These rot level . Salinit vessels . Howevern quinoa . Taking + ions in a non-toxic way in the roots. Finally, the high constitutive expression levels of genes involved in Na+ and K+ homeostasis could be a key point in the salt tolerance displayed by amaranth. These results constitute an important advance in the knowledge of the tolerance mechanisms operating in a promising species such as amaranth, able to maintain seed yield under salinity, and they point out the importance of considering simultaneously the anatomical, physiological, and molecular changes caused by salinity in order to develop efficient strategies to increase salt tolerance.In conclusion, the main strategy of amaranth to maintain osmotic homeostasis is based on its high ability to reduce water loss through the leaves under salt stress. On the other hand, amaranth displayed a higher root/leaf water content ratio than quinoa, both at the control and under salt stress, which suggests that amaranth presents a higher ability than quinoa to harbor NaThe original contributions generated for this study are included in the article/YE, AF-O, BM, FF, and IE performed the experiments. MB and IE contributed in the conceptualization of the study and design of the experiments. IE, MB, and JE-F conceived the project and research, and supervised the experiments. YE, FF, MB, and IE contributed to the analysis and interpretation of the results. YE, MB, and IE wrote the manuscript with contributions from all the authors. All authors have critically reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mucormycosis is a rare, opportunistic fungal infection, which can progress acutely or subacutely in immunosuppressed patients. It is a devastating disease in patients with poorly controlled diabetes mellitus and immunocompromised conditions. It causes decaying and extensive lesions of the soft tissue caused by fungi belonging to the order of Mucorales and is often fatal. Due to its aggressive nature, early detection and prompt management are of great importance for a good prognosis. We report a case of mucormycosis post-dental extraction in a patient with uncontrolled diabetes mellitus. Mucormycosis is an opportunistic deep fungal infection, which ranks third after candidiasis and aspergillosis . It is cA 55-year-old male presented to the Department of Oral and Maxillofacial Surgery at Saveetha Dental College and Hospital complaining of pain and sinusitis of his left alveolus following maxillary tooth extraction three weeks ago. Examination revealed facial cellulitis involving his left buccal, maxillary and infraorbital areas with difficulty in opening the mouth due to pain. Intra-orally foul-smelling, black discoloration of the mucosa of the left half of the palate with necrotic bone was observed. He was vitally stable and afebrile with a blood pressure 130/76 mmHg and a pulse rate of 82 beats/min; he was fully conscious with a Glasgow Coma Scale (GCS) 15/15, and gave a history of uncontrolled non-insulin-dependent diabetes mellitus. The patient was admitted in our department and an incisional biopsy taken from the left maxillary alveolar ridge revealed the presence of nonvital bone, necrotic fibrous connective tissue, foreign material, and flattened, broad, wide-angle hyphae with thrombosis suggestive of mucormycosis. Laboratory investigations revealed hemoglobin 12.5 g/dL , WBC count of 12,700 cells/cu.mm , differential count of 78% neutrophil , 19% lymphocytes , 2% eosinophil , 1% monocyte , platelet count of 3 lakh cells/cu.mm , serum urea of 53 mg/dL , serum creatinine of 1.2 mg/dL , serum glutamic-oxaloacetic transaminase (SGOT) of 12 IU/L , and HBA1C of 14.4% . Facial and brain MRI was performed which revealed mucolytic and destructive foci involving the left maxilla, posterior aspect of the left nasal bone, left pterygoids, greater wing of the sphenoid and left temporal bone and also large foci diffusing peripherally involving the left parapharyngeal space extending to the base of the skull and a peripheral ring-enhancing foci in the anterior temporal lobe with adjacent perifocal edema suggestive of possible intracranial extension. Facial CT showed obliteration of the left maxillary sinus and swelling on the left side of face  under general anesthesia was carried out for sequestrectomy of maxilla, total curettage of the maxillary antrum and complete debridement was done. The patient was administered IV amphotericin B as an infusion at 1 mg/kg/day in 100 mL of 5% dextrose over one to two hours for a period of 14 days with daily monitoring of kidney functions and electrolyte levels. The patient was discharged from the hospital after 14 days with an improved general condition. Healthy granulation tissue was seen and the wound healed uneventfully in 14 days Figure .Resolution of facial swelling and granulation of extraoral sinus was observed at the end of 14 days Figure .  The CT of facial bones at three months follow up revealed osteolysis of left maxilla, hard palate, posterior aspect of the nasal bone, left zygoma, left orbit, left sphenoid and bilateral pterygoid process and clivus with mild to moderate thickening in all paranasal sinuses and nasal cavities with occlusion of left ostiomeatal unit and bilateral turbinate hypertrophy Figure . Given the serious and potentially fatal complication of tooth extraction, we reported a case of nonhealing extraction socket in a diabetic patient leading to mucormycosis. In approximately 50% of mucormycosis infections, diabetes mellitus was the predisposing factor. This could be due to the greater availability of glucose, lower pH, persistent reduction of blood flow, and subsequent decreased serum inhibitory activity against the pathogen and increased expression of some host receptors that mediate invasion of human epithelial cells causing abnormal phagocytosis and cell-mediated immune abnormalities . Oral muWe infer that quick diagnosis is crucial in the treatment planning and anti-fungal treatment enables speedy recovery. Diabetes mellitus is the most common risk factor with other risk factors being immunocompromised patients with pre-existing malignancies and recipients of bone marrow transplants. Most patients with diabetes suffer from mucormycosis involving the paranasal sinuses in the form of a rhinocerebral mucormycosis involving both the sinus and orbit and communicating to the brain. In the past, the mortality rate of the rhinocerebral type was 88%, but recently the survival rate of rhinocerebral mucormycosis averages 21%-73% depending on the circumstances. Liposomal amphotericin B is the drug of choice. The outcome is more favorable in 70% of the patients when surgical therapy was combined with anti-fungal therapy and balancing the diabetic levels. Though the incidence of mucormycosis following a tooth extraction is extremely low, nevertheless when it occurs, may cause significant morbidity and mortality.