Arenaria polytrichoides (Caryophyllaceae) is a common cushion plant occurring at high elevations in the Himalaya-Hengduan Mountains, SW China. It frequently has other non-cushion species growing within its canopy, forming a contrast with the surrounding areas because it creates patches of higher diversity and greater biomass. In this study, we examined the relationship between the cushions and associated non-cushion species along a gradient of cushion size. A total of 200 A. polytrichoides individuals were selected to fit four size classes. Field measurements were carried out to assess canopy structure, functional traits relevant to growth and reproduction, and soil quality below cushions along the size gradient. Furthermore, the size effect of cushions on the richness and abundance of species and biomass production was also examined. All the morphological variables examined exhibited a positive correlation with cushion size, as did the nutrients under cushions. Large and compact cushions were associated with higher soil nutrient contents compared with small and loose cushions. As a result of these biogenic environmental changes, there was a stronger facilitation effect performed by large cushions. Data pertaining to functional traits revealed that large cushions benefit from the enhanced resources within their compact structure and exhibit greater fitness and a higher reproductive output than small cushions. Our data indicated that interactions occur between cushion species and other plants depending on the size of the cushions, probably because of the greater heterogeneity of conditions beneath larger cushions. These findings provide a clear demonstration of the generally overlooked importance of the traits of nurse plants, such as size and age, in terms of their facilitative effects. Retama sphaerocarpa, increased with the shrub’s age and size. They considered that this was probably due to the greater heterogeneity under larger canopies. In the shrubland of the transition area between the Mediterranean and the semi-arid zone of Israel, Sarcopoterium spinosum resulted in a unimodal pattern of conspecific recruitment in relation to canopy density and age. In addition, many studies have focused on the variable effects of beneficiary species on nurse plants. For example, a common shrub in the Colorado Desert of California, USA, Ambrosia dumosa, exhibits an ontogenetic shift in response to adult neighbors: there is facilitation of seedlings and juveniles by adult neighbors, while there is competition between larger plants and their adult neighbors , which in turn influences the microenvironments created by cushions and, eventually, modulates the outcome of their interaction with other non-cushion species. As an adaptation to harsh and unpredictable environmental conditions, alpine cushion species often appear to have long life spans, during which their canopy structure and/or physiological status changes in response to ontogenetic processes. Based on the size–age relationship, some studies have used plant size represented by canopy diameter to estimate a cushion’s longevity . HMM has been identifies as a hotspot for temperate biodiversity  is located at an elevation of 4900 m at the Baima pass on the Baima Snow Mountains, Deqen County, Yunnan Province, SW China. This site was chosen because it supports a population of cushion species of a variety of sizes, and also because facilitation by s region , 2015.At high elevations in the HHM, the summer climate is monsoonal and characterized by cold rain or sleet, interrupted by short periods of intense solar radiation . Annual Air temperature (°C) and relative humidity  at 15 cm above the soil surface at the study site recorded every 30 min by using an integrated thermistor  indicated average temperature during the growing season from middle of June to the end of September 2015 was 4.87 ± 0.04°C , and average RH during daytime (0800–2000 h) of that period was 89.9 ± 0.24%. Absolute maximum and minimum temperatures were 17.80°C and -1.95°C recorded in the middle  and end  of the growing season 2015, respectively.Arenaria polytrichoides (Caryophyllaceae) is a long-lived and stress tolerant perennial herb that forms a hemispherical cushion; it is mainly found at high elevations in the HHM. Like most species with a cushion life form, A. polytrichoides cushions retain their dead leaves under the compact structure and these can be seen as litter and a humus trap. The dense canopy structure of A. polytrichoides cushions has been shown to result in soil below with a higher moisture and nutrient content than adjacent open areas and the cushions provide a more favorable microenvironment for many other non-cushion plants  of each species within each sample. All the diversity and biomass sampling was undertaken in the middle of August 2015 at the peak of the growing season.Cushion plants normally have a roughly elliptical shape. To obtain comparable sizes for examining species richness, abundance and biomass production in patches sampled in cushions and paired surrounding open areas  and volume (cm3) of each cushion were calculated using following equations:In addition, the height and the maximum  and minimum  canopy diameter of each cushion were measured. The canopy area (cma and b are mean maximum and minimum canopy diameter (cm), respectively.where a, b, and c are mean maximum and minimum canopy diameter (cm) and height of canopy (cm), respectively.where i, j) contained the abundance of the ith species in the jth sample. We then used this to determine the effect of cushion plants on species richness within and outside cushions in each of the four size classes. From the matrices, 500 samples were randomly drawn, with replacement, for each sample size (from one sample to the maximum number of samples); then the species richness of the 500 samples was calculated using Coleman’s algorithm (Arenaria cushions (ISR) was calculated as:To compare species richness of sampled patches of cushions and paired open areas in each size class, we generated a species × samples matrix containing each sampled cushion and open area of each size class, where each cell .where n curves . The estabundance and RIIbiomass) for each non-cushion species recorded in association with the canopy of cushions in the four size classes, separately. For each cushion size class, RIIabundance and RIIbiomass were calculated as follows:In order to examine the size effect of cushion on the abundance and biomass production of non-cushion species, we calculated the relative interaction index  was determined by weighing a densely packed volume of these dry soils. The remaining five soil samples from underneath cushions of each size class and in the open areas were sent to the Laboratory for Soil Analysis at the Agricultural Institute of Yunnan, where soil organic matter , available nitrogen , phosphorus (mg kg-1), and potassium (mg kg-1) were measured by using potassium dichromate heating oxidation-volumetric method (for SOM), alkali diffusion method (for available nitrogen), sodium bicarbonate extraction Mo-Sb Antispetrophotography method (for available phosphorus), neutral normal salt ammonium acetate extraction flame photometer method, and boiling nitric acid method (for available potassium), respectively. Because litter is an important source of N for cushion plants at high elevations (post hoc multiple comparisons (LSD/Tamhane). In addition, the differences between litter depths of cushions of different sizes were also examined with one-way ANOVA and post hoc multiple comparisons (LSD).Between 14 and 15 August 2015, 10 samples of topsoil (0–15 cm) beneath cushions of each size class and in open areas were collected. These soil samples were stored in sealed containers and fresh mass was determined immediately after sampling before being transported to the laboratory within 24 h. Gravimetric soil water content  of five samples of cushions of each size class and open areas were measured by mass loss after drying at 105°C for 72 h. Bulk density , i.e., the ratio between leaf dry mass and fresh leaf area. LMA exhibits inherent variations between different functional groups  and responds to environmental conditions , leaf position and leaf/plant age (reviewed by -1), i.e., the ratio between leaf dry mass and fully rehydrated fresh mass. LDMC is generally positively correlated with life span and negatively correlated with potential relative growth rate (RGR) and likelihood of physical damage. LMA and LDMC were measured in leaves of five cushions of each size class. (3) Foliar δ13C (‰) was determined using an isotope ratio mass spectrometer . Leaf δ13C values are commonly used for assessing plant photosynthetic activity and water use efficiency. Variations in leaf δ13C are caused by environmental factors  and morphology . These morphological factors explain most variations in leaf δ13C with respect to plant phenology, development and age .(1) Leaf mass per area (LMA) (g m-2) was calculated from the number of terminal branches counted in one randomly placed 2 cm × 2 cm quadrat per cushion; (4) leaf density (leaves cm-2) was calculated as the average number of leaves per terminal branch of 10 randomly selected branches multiplied by the number of branches cm-2. High values of branch and leaf density indicated high cushion compactness. In addition, these functional traits were considered to reflect the physiological status of the plants themselves  with Varimax rotation was used to reduce the number and multicollinearity of variables . The priF = 240.855, p < 0.001, also see Figure 1A). ISR increased with cushion size, ranging from ca. 6.5% with smallest cushions to 21.5% with largest cushions .There was significantly higher species richness recorded in sampled cushions than open areas for all the four size classes . Overall, both RIIabundance and RIIbiomass were positively related to cushion size , with the lowest mean values of both RIIabundance (0.04) and RIIbiomass (0.03) associated with the smallest cushions (class I), and the highest values of RIIabundance (0.38) and RIIbiomass (0.39) with the largest cushions in class IV . For the smallest cushion size class, 10 and 11 of 17 species  recorded exhibited positive RIIabundance and RIIbiomass values , but not for cushions in class I (p = 0.768), II (p = 0.324), and III (p = 0.183). For RIIbiomass, significant/marginally significant difference was found for cushions in the size classes III (p = 0.078) and IV (p = 0.017), but not in classes I (p = 0.768) and II (p = 0.324).Generally, a decreasing p = 0.584, values for SWC, bulk density, SOM, and the availability of P and K in soil under the smallest cushions in class I were significantly/marginally significantly higher than in open areas (Table 1). In contrast, all examined soil properties under the cushions of classes II, III, and IV had  significantly higher values than they did under the smallest cushions (Table 1). Cushions in class IV had marginally significantly higher soil potassium availability under them than soil under the cushions in class III . A significant difference in litter depth between open areas and cushions in different size classes was found . Pairwise tests indicated greater depth beneath large cushions than small cushions (Table 1).With the exception of nitrogen availability, for which Table 2). Other examined morphological traits  also increased with cushion size (Table 2).The selected cushions in the four size classes differed significantly in lateral spread, thickness, canopy area and volume , the post hoc test indicated a significantly higher LMA value for cushions in class IV (p = 0.042) than cushions in class I. Overall, a significant size effect on LDMC  was observed across the four size classes of Arenaria cushions, with increased LDMC with increasing cushion size (Table 3). In addition, pairwise testing revealed significantly lower LDMC values for cushions in class I than cushions in class II (p = 0.006), class III (p = 0.033), and class IV (p = 0.002). No significant difference in foliar δ13C  was found across the four size classes. A significant difference in flower number per area (FNA) was found between the cushion size classes , with a significantly higher FNA value for cushions in class IV than those in class I (p = 0.039) and class II (p = 0.007). The FNA value for cushions in class III was marginally significantly higher than for cushions in class II (p = 0.059).Although no significant difference in LMA was found between the four size classes (Table 2). Bigger cushions were larger, thicker and had higher branch and leaf density than smaller cushions (Table 2). The first three axes of the PCA based on the four morphological traits explained ca. 46.4, 28.9, and 23.2% of the variance, respectively. The first axis (PC1) represents cushion size  and thickness (PC1thickness = 0.924). Both the second (PC2) and third (PC3) axes represent cushion compactness .Overall, all functional traits used as proxies for cushion morphology varied with size class  and RIIbiomass (r2 = 0.919 p = 0.042) showed a significant/marginally significant positive relationship with PC1. ISR was significantly and marginally significantly positively related with PC3  and a model including PC2 and PC3 , respectively. The absolute difference in available N between cushions of different size classes and open areas exhibited a marginally significant positive correlation with PC1 . PC1 was also the best predictor of the changes in LMA . The model including PC2 and PC3 was marginally significantly related with the absolute difference in available K  between cushions of different size classes and open areas.Both RIIA. polytrichoides cushions on the composition of the plant community and the biomass production of focal plants increased as the cushions increased in size. Our data, thus, provide evidence of a mechanistic link between ontogenetic development of cushion species and the biogenic environmental changes in abiotic factors, with relevant consequences for plant–plant interactions.Previous studies indicated that the facilitative role of cushion plants at high elevations of the HHM relies on their ability to modify the surrounding environments, particularly soil nutrients . Here, wA. polytrichoides cushions on other plant species was previously mainly attributed to fertility islands produced under their dense canopy  and non-legume (Juniperus communis subsp. nana) species has been attributed to a higher decomposition rate due to greater microbial activity and/or particular chemical or physical characteristics of litter under their canopies (Androsace tapete) litter during the winter may be an important source of N for plant growth early in the growing season and probably explains subsequent facilitation of cushion species in cold environments  legume  facilitates the establishment and growth of other species in association with the ant size . Given tant size . In thisant size . It may R. sphaerocarpa and Juniperus communis was found to increase with shrub size/age, probably due to greater heterogeneity under the larger canopy (Arenaria polytrichoides vs. Potentilla articulata) in intensity of facilitation to distinct capacities for improving soil nutrients of two phylogenetically distant species but which shared similar cushion morphology  from a ‘young’ to an ‘old’ stage that shifted the species from facilitation to interference because of the extreme shade under the canopy of oldest plants  in the upper soil layer by cushions. A similar size–age related interaction has also been reported in a semi-arid environment, where plant diversity in the understory of r canopy . In addirphology . It has rphology . For Junl shrubs . In contt plants , A. polyht level . In contrichment . Establirichment . As the al Chile . These fse grows . Future The contribution of facilitation by cushion species to natural plant community properties is commonly discussed in the context of its importance along spatial stress gradients e.g., . Here atYY and J-GC conceived and designed the experiments. YY and J-GC performed experiments and analyzed the data. YY wrote the manuscript. CS and HS provided editorial advice on experiments and writing of 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.


The in vitro starch digestion yielded a high slowly digestible fraction (51%) compared to maltodextrin (9%). In the rooster digestibility model, the starch was highly digestible (94%). Consumption of slowly digestible starch (SDS), in an instant pudding or bar, yielded a significantly lower glycemic index compared to a control. At individual time points, the SDS bar and pudding yielded blood glucose levels with significantly lower values at 30–60 min and significantly higher values at 120–240 min, demonstrating a balanced energy release. This is the first study to comprehensively characterize the physiological responses to slowly digestible starch (tapioca and corn blend) in in vitro and in vivo studies.There is growing interest among consumers in foods for sustained energy management, and an increasing number of ingredients are emerging to address this demand. The SUSTRA™ 2434 slowly digestible carbohydrate is a blend of tapioca flour and corn starch, with the potential to provide balanced energy after a meal. The aim of the study was to characterize this starch’s digestion profile in vitro (modified Englyst assay) and in vivo (intact and cecectomized rooster study), and to determine its effects on available energy, by measuring post-prandial glycemia in healthy adults ( According to the World Health Organization (WHO), diabetes will be one of the leading causes of deaths by 2030 ,2. ElevaCarbohydrates are a commonly consumed staple food and represent 45–55% of daily energy intake in a Western diet . CarbohySome native starches have inherently high SDS content (A-type X-ray diffraction pattern granule), such as in waxy corn starch or RS (B-type X-ray diffraction pattern granule), such as in potato starch . During Starch digestibility is associated with GI. Slowly digestible carbohydrates, commonly abbreviated as SDC, are digested steadily, resulting in prolonged glucose release from the lumen of the small intestine into the blood stream, with blunted glycemia and therefore a lower insulin requirement . BecauseThe investigational material in the present study is a new, commercially available product, the SUSTRA™ 2434 slowly digestible carbohydrate. This product will be referred to as “slowly digestible starch” or SDS for the remainder of the report. This SDS is a blend of corn starch and tapioca flour, designed to deliver balanced energy and a reduced glycemic response when added to non-thermal applications. The aims of this study were to (1) characterize the in vitro digestibility profile of SDS; (2) confirm in vivo digestibility using a rooster model and (3) assess its glycemic response in a healthy population.® Plus 10 DE maltodextrin, Ingredion Incorporated, Bridgewater, NJ, USA), and SDS . A modified version of the Englyst method [Aspergillus niger, ≥260 U/mL, St. Louis, MO, USA) was added to centrifuged 12 g pancreatin in solution; (2) Invertase (40 mg)  was added, replacing 4 mL of the original liquid enzyme (3000 EU/mL and (3) Guar gum (Sigma G4129) was prehydrated in 0.05 M HCl solution (0.5% w/v) with 0.5% pepsin before being added to the digestion tubes. Released glucose was determined using glucose oxidase/peroxidase (GOPOD) reagent. Slowly available glucose (SAG) was calculated using the following equation: SAG = (slowly digestible starch/glucose amount release at 120 min) × 100. The total dietary fiber of test starch was measured using the AOAC 991.43 method [The in vitro digestion method compared maltodextrin (Globet method  was used3 method .n) content of a starch and maltodextrin, using both conventional and cecectomized roosters, as previously described [® N, LECO Corporation, St. Joseph, MI, USA; AOAC, 2006), and gross energy (GE) using a bomb calorimeter . The TMEn values, corrected for endogenous energy excretion based on previous data, were calculated using the following equation:fedEI equals the gross energy intake of the test substrate consumed; fedEE equals the energy excreted by the fed birds; 8.22 is the correction factor for uric acid; fedN equals the g nitrogen retained by the fed birds; fastedEE equals the energy excreted by the fasted birds; fastedN equals the g nitrogen retained by the fasted birds; and FI equals the grams of dry test substrate consumed. The database with conventional and cecectomized birds indicates that the N-corrected endogenous energy excretion by fasted birds was 9.25 kcal.The aforementioned SDS and maltodextrin were evaluated in roosters. Nutrient digestibility is determined by measuring nitrogen-corrected true metabolizable energy . All subjects provided written informed consent prior to starting the study. The clinical study was conducted at GI Labs .Inclusion criteria: Participants were healthy male or non-pregnant females, 18–75 years of age, with a body mass index (BMI) of ≥20 and ≤40 kg/m2. Participants were required to maintain their regular diet, supplement intake, physical activity and body weight throughout the study duration and refrain from smoking prior to each visit. On test days, subjects were not allowed to take any dietary supplements, until dismissal from the GI labs. Subjects were required to have normal fasting serum glucose (<7.0 mmol/L capillary corresponding to whole blood glucose <6.3 mmol/L), abstain from alcohol consumption and to avoid vigorous physical activity for 24 h prior to all test visits. Subjects had to have an understanding of the study procedures and be willing to provide informed consent to participate in the study and authorization to release relevant protected health information to the investigator.Exclusion criteria: Subjects were excluded if they failed to meet inclusion criteria, had a history of chronic disease, such Type 1 or 2 Diabetes, cardiovascular disease, cancer, gastrointestinal disorders; used medications within four weeks of the screening, had surgery within 3 months of screening, had an intolerance or allergy to test ingredients, had extreme dietary habits, had drastic body weight changes >3.5 kg within four weeks of screening duration, had the presence of any symptoms of an active infection during screening or study visits, had a history of alcohol or substance abuse, or had an unwillingness or inability to comply with the experimental procedures and to follow GI Labs safety guidelines.2). Eligible participants were studied on separate days over a period of 2 to 6 weeks. The interval between successive tests was no less than 48 h and no more than 2 weeks. Subjects completed six study visits in a random order, during which they consumed one of the following treatments: SDS bar, control bar, SDS pudding, control pudding, dextrose beverage 1 (50 g dextrose in 250 mL water) or dextrose beverage 2 (50 g dextrose in 250 mL water). The dextrose beverage was administered twice for glycemic index calculations. The study subject flow is shown in The study was a randomized, double-blinded, placebo controlled, cross-over design, with 14 healthy adults  ≥20.0 and <40.0 kg/mCold-pressed bar: The SDS bar and control bar were similar in appearance and were packaged in an opaque envelope with an alpha-numeric code for identification. Neither the study subjects nor the investigators knew the identity of the study foods. Both the SDS bar and control bar were matched for available carbohydrates (50 g). The SDS bar contained 24.9 g SDS  and 18.9 g corn syrup  while the control bar contained 22.1 g maltodextrin  and 21.8 g corn syrup . The portion sizes and nutrient composition of the study bars are shown in Pudding: The SDS pudding and control pudding were identical in appearance and were packaged in an opaque envelope with an alpha-numeric code for identification. Neither the study subjects nor the investigators knew the identity of the study foods. Both the SDS pudding and control pudding were matched for available carbohydrate (50 g). The test pudding contained 30.4 g SDS  and 10.9 g dextrose , and the control pudding contained 39.6 g dextrose . The remainder of available carbohydrate in both puddings was provided by whole (full-fat) milk. The puddings were prepared by mixing an entire 300 g package of powder with 840 g whole milk, whisking for 2 min and refrigerating overnight. The portion sizes and nutrient composition of the study puddings are shown in Participants were asked to maintain stable dietary and activity habits throughout the study. Prior to each study visit, participants refrained from drinking alcohol and from unusual levels of food intake or physical activity for 24 h. On each test occasion, subjects arrived at the clinical site after fasting for 10 to 12 h. Two fasting blood samples for glucose analysis  were obtained by fingerprick, 5 min apart and after the second sample, the subject started to consume a test meal. Each test meal was served with a drink of 1 or 2 cups of coffee or tea with 30 mL of 2% milk if desired, or water. At the first visit, each subject selected the type and volume of drink desired and the same type and volume of drink was consumed on subsequent visits. Subjects consumed the entire test meal within 10 min. At the first bite, a timer was started and additional blood samples for glucose analysis  were taken at 15, 30, 45, 60, 90, 120, 150, 180, 210 and 240 min after starting to eat. Blood samples were obtained from hands warmed with an electric heating pad for 3–5 min prior to each sample.After blood collection the tubes containing blood for glucose analysis were rotated to mix the blood with an anti-coagulant and then placed in a refrigerator until the last blood sample in the set had been collected. After all tubes were collected from one subject, the tubes were stored in a −20 °C freezer until analysis. Analysis was performed within 3 days of the study visit, using a YSI model 2300 STAT analyzer .n = 14 subjects provided 80% of the power required to detect a difference of 19%.The main effect of treatment was expected to have a SD of 23%/sqrt (2) because each subject tested each treatment in 2 types of food (bar and pudding). We wished to be able to detect a difference in glycemic response of 20%; The study was conducted in two blocks of three treatments; the two blocks contained either the bar or the pudding and within each block the tests consisted of one dextrose test and the control and test food. The six possible orders of three treatments were listed twice, one for each block. The six orders in each block were randomized using the Rand  function on an Excel spreadsheet. The order of the blocks was randomized in the same way and the process was repeated three times.The order of testing was assigned to the 14 subjects in the order they attended for the first visit; the extra orders were included in case of drop-outs which needed to be replaced.Glycemic index values were calculated based on previously published methods . The netp values < 0.05 were deemed statistically significant.Paired t-tests were conducted on blood glucose values at individual time points, net incremental area-under-curve (iAUC), and glycemic index using GraphPad Prism 7 . The digestibility profile of SDS and maltodextrin (control) was determined by measuring glucose release, as shown in Roosters provide a robust model to determine digestibility and/or fermentability of an ingredient. The true metabolizable energy (TMEn) for both SDS and maltodextrin (control) was high, indicating their complete digestibility . HoweverStudy demographics are shown in The inclusion of SDS in a cold-pressed bar and instant pudding yielded a significantly lower glycemic index compared to the control .SDS inclusion in the cold-pressed bar yielded a significantly different mean blood glucose concentration a and a 5Global health concerns, such as diabetes, can be addressed with lifestyle changes, including dietary modifications by replacing rapidly digestible carbohydrates with slowly digestible carbohydrates. Natural SDCs, such as whole grains and pulses, comprise slowly digestible starch and resistant starch/fiber fractions. Dietary fiber has profound health benefits, but can lead to gastrointestinal discomfort. Thus, there is a heightened interest in ingredients with high amounts of SDS and low RDS content, in order to deliver sustained glucose energy.Carbohydrate quality or prolonged digestion is an emerging area and studies to evaluate the effect of SDCs are currently relying on glycemic response and glycemic index measurements to define “sustained energy” . HoweverThis is the first study to characterize a SDS by adopting a translational approach, which involved in vitro screening of the ingredient and in vivo validation of slow but near complete digestibility and prolonged glucose release. In the in vitro assay, SDS had an over 50% slowly digestible fraction and showed a steady glucose release over 120 min of digestion which simulates digestion in the small intestine. SDS had low fiber and RDS fractions. Carbohydrate digestibility in humans is commonly determined in ileostomates . AlternaA recently published meta-analysis , with thAnother study with starches differing in hydrolysis rates, indicated a relation between starch in vitro digestibility and glycemic response in healthy men . SubjectGlycemic responses of other sugar-based SDCs, such as isomaltulose and sucromalt, were tested in low GI formulated meal replacement drinks in diabetics . Four enThe current study has few limitations. The modified in vitro method is a screening tool to characterize digestibility profile and is less dynamic in terms of measuring interactions with other nutrients and absorption uptake and does not completely reflect physiological responses. The human ileostomate model, which has been utilized to measure ingredient digestibility in some studies, was replaced with the cost-effective rooster model. Other limitations included lack of insulin response measurements, rate of glucose appearance and indicators of carbohydrate and fat oxidation.The SUSTRA™ 2434 slowly digestible carbohydrate is a blend of tapioca flour and corn starch, with the potential to provide balanced energy. The new SDS has been shown to be highly digestible in vivo and yielded a lower GI, compared to a rapidly digestible control. Incorporating SDS into a cold-pressed bar and instant pudding resulted in a lower glucose response in the first 60–90 min and a higher glucose response at two hours and beyond, suggesting steadily available energy. The reduced blood glucose response to SDS is due to the presence of a significant slowly digestible starch fraction and low rapidly digestible and resistant fractions, as documented in the in vitro study. The balanced energy from the SUSTRA™ 2434 slowly digestible carbohydrate is a solution to improve carbohydrate quality in non-thermal applications.


Nasutitermes ephratae gut-microbiome displaying the highest levels of wheat straw degradation, carboxylate production and enzymatic activity. Comparing the 16S rRNA gene diversity of the initial gut inocula to the bacterial communities in lignocellulose degradation bioreactors revealed important changes in community diversity. In particular, taxa such as Spirochaetes and Fibrobacteres that were highly abundant in the initial gut inocula were replaced by Firmicutes and Proteobacteria at the end of incubation in wheat straw bioreactors. Overall, this study demonstrates that termite-gut microbiomes constitute a reservoir of lignocellulose-degrading bacteria that can be harnessed in artificial conditions for biomass conversion processes that lead to the production of useful molecules.Termites are xylophages, being able to digest a wide variety of lignocellulosic biomass including wood with high lignin content. This ability to feed on recalcitrant plant material is the result of complex symbiotic relationships, which involve termite-specific gut microbiomes. Therefore, these represent a potential source of microorganisms for the bioconversion of lignocellulose in bioprocesses targeting the production of carboxylates. In this study, gut microbiomes of four termite species were studied for their capacity to degrade wheat straw and produce carboxylates in controlled bioreactors. All of the gut microbiomes successfully degraded lignocellulose and up to 45% w/w of wheat straw degradation was observed, with the  With more than 200 billion tons of non-food lignocellulosic biomass produced yearly, lignocellulose represents the most abundant source of renewable carbon on Earth  and/or harbor a mutualistic hindgut microbiome exclusively constituted by prokaryotes  in August 2011. After harvesting, the straw was milled to 2 mm and stored at room temperature (20–25°C).Microcerotermes parvus, Termes hospes, Nasutitermes ephratae, and one undescribed species closely related to N. lujae (herein after N. lujae) were selected as inocula for this study. The major selection criteria were based on the fact that higher termites harbor a predominantly bacterial gut microbiome and that the intestinal pH in each species is slightly acidic or near to neutrality  n = 500 guts per species per sampling campaign). Following cold anesthesia of termites, whole guts were removed by dissection on ice with sterile scissors and forceps. Once removed, the guts were immediately suspended in a physiological saline solution (PBS-buffer) and maintained on ice  at IRD. One thousand worker termites per species were randomly collected from their nest in two campaigns separated by a period of 3 months . Milled wheat straw (2 mm) was sterilized by autoclaving  and added to the medium (20 g.L−1) as the sole carbon source. Stirred bioreactors (400 rpm) were operated under strict anaerobic conditions, flushing the reactor with nitrogen after inoculation. The absence of dissolved oxygen was continuously monitored with a polarographic dissolved oxygen probe (AppliSens). The temperature was set to 35°C and pH was maintained at 6.15 by adding a 2 M NaOH solution. During the incubation, methane was monitored and, if necessary, inhibited by addition a solution of 2-bromoethanesulfonate (BES), a methanogenesis inhibitor, until a maximum concentration of 10 mM. A non-inoculated control bioreactor was operated under identical conditions. Samples were collected from all bioreactors every 2 days during the 20 days of incubation to characterize VFA and gas production. Samples from the initial and final times of incubation were used to determine LC degradation, enzymatic activities and microbial diversity.To assess the lignocellulose degradation capacity of the different gut microbiomes, two replicate anaerobic bioreactors (Applikon MiniBio 500) were conducted for each termite species. Following centrifugation  and elimination of the saline media, termite-gut homogenates (500 guts) were used to inoculate 400 mL of mineral media (MM), containing per liter of distilled water: KH−1. Injector, oven, and detector temperatures were 100, 60, and 140°C, respectively.Gas production was measured by monitoring pressure changes and gas composition was analyzed using a gas chromatograph (GC) HP 5890 equipped with a conductivity detector and a HAYSEP D column (molecular sieve of 5 Å). Argon was used as the carrier gas at a flow rate of 100 mL.minVolatile Fatty Acids (VFA) contained in the liquid phase of samples were analyzed using a Varian 3900 gas chromatograph equipped with a flame ionization detector and CP-Wax 58 (FFAP) CB column .Wheat straw concentration was determined at the beginning and at the end of the 20-day incubation by measuring the total (TS) and volatile (VS) solids. TS were determined using 10 mL samples that were first centrifuged , rinsed twice with distilled water and dried for 24 h at 105°C. The mineral fraction (MF) was estimated by mineralization of the samples at 500°C for 2 h, and VS were determined by subtracting MF from TS. Wheat straw degradation was reported as percentage of LC  related to the initial LC mass.Enzyme activity measurements were performed as previously described by Lazuka et al. . Briefly−1). DNA was purified using an AllPrep DNA/RNA Mini Kit (Qiagen) according to the manufacturer's instructions. DNA integrity and purity were checked using agarose gel (1%) electrophoresis. DNA concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo Scientific), measuring absorbance at 260 and 280 nm.16S rRNA gene copy number and bacterial diversity were analyzed on the initial termite gut samples and at the end of the bioreactor experiments. Samples (1.5 mL) were collected and centrifuged at 13,000 × g, for 5 min at 4°C. After removing the supernatant, the pellet was snap frozen in liquid nitrogen and stored at −80°C. Total DNA/RNA was extracted from these samples using a PowerMicrobiome RNA Isolation kit (MoBio Laboratories Inc. Carlsbad) following the manufacturer's instructions, but omitting the final DNAse steps. Cell lysis was carried out using a Fast Prep  . For each sample, assays were carried out in triplicate using 96-well real-time PCR plates (Eppendorf). The qPCR was performed in 25 μL containing 12.5 μL Master Mix , using primers BAC3388 and BAC805R (250 nM of each primer), the TaqMan probe BAC516F (100 nM) and DNA template ranging from 10–100 ng as previously described . The V3-V4 hypervariable region of the 16S rRNA gene was amplified from genomic DNA samples using the bacterial primers 343F (5′-CTT TCC CTA CAC GAC GCT CTT CCG ATC TAC GGR AGG CAG CAG-3′) and 784R (5′-GGA GTT CAG ACG TGT GCT CTT CCG ATC TTA CCA GGG TAT CTA ATC CT-3′) modified to add adaptors during the second PCR amplification. The first PCR amplification was performed in 50 μl reactions containing 1X PCR buffer, 2.5 U MTP Taq DNA Polymerase (Sigma), 0.2 mM of each dNTP, 0.5 mM of each primer and 2 ng of extracted DNA. After 30 amplification cycles , amplicons were purified using magnetic beads and quantified using a NanoDrop 1000 spectrophotometer. A second PCR amplification was performed at the GeT platform to add sequencing adapters and a unique index for each sample (details in Supplementary Data). The PCR products were purified using magnetic beads and their quality was ascertained using a High Sensivity DNA Analysis Kit (Agilent) and a BioAnalyzer 2100. DNA concentration was measured using a NanoDrop 1000 spectrophotometer. An equimolar pool was then prepared and loaded on a MiSeq Illumina cartridge, using reagent kit v3 which enabled paired 300-bp reads.Sequencing data were demultiplexed at the GeT platform and pair-ends reads were joined with Flash v1.2.6  package Phyloseq v1.6.1  of the National Center for Biotechnology Information (NCBI) under accession number N. ephratae, N. lujae, T. hospes and M. parvus) to degrade wheat straw as sole carbon source was assessed in controlled bioreactors, performing duplicates for each termite species (named r1 and r2). The quantity of DNA in each inoculum (500 guts) was estimated to be about 150 μg, implying that each inoculum contained a similar number of 16S rRNA gene copies (~1.5 × 1012). However, regarding T. hospes these values were lower , irrespective of the inoculum used. Small amounts of methane were detected in some of the bioreactors, but in this case its production was immediately inhibited by the addition of BES. During the initial phase of the culture, small amounts of H2 production were also detected, but this ceased at later stages (data not shown). VFA accumulation varied from 2.2 to 5.8 g.L−1 for the different termite gut inocula, with the N. ephratae gut microbiome yielding the highest concentration . It is also noteworthy that VFA production by N. lujae and M. parvus gut inocula displayed high variability between replicates , the one containing the T. hospes inoculum displayed lower xylanase activity (600 UA.mL−1). Regarding CMCase activity, a similar profile was observed in all bioreactors, but the measured activity was generally lower than xylanase activity . Finally, it was noted that for all termite gut inocula, more than 60% of the xylanase and cellulose activities were cell-bound.Monitoring various enzymatic activities on r2 duplicates at the end of the incubation  were close to saturation  at 97% sequence similarity threshold. Rare OTUs were filtered generating 671 final OTUs. Rarefaction curves, based on normalization by subsampling at 15,000 sequences per sample, showed that all samples . Indeed, only 8 minor OTUs were common to the four termite species.Abundant OTUs were defined herein as those displaying a minimum 2% abundance (min. 300 sequences) in at least one sample. From the 34 abundant OTUs detected across all gut samples, only one was common to three termite species M. parvus and the two species of Nasutitermes were dominated by Spirochaetes (>55%), followed by Fibrobacteres (except for N. lujae) and the candidate phylum Termite group 3 , Spirochaetes (21%), Bacteroidetes (13%) and Proteobacteria (12%). Overall, these results are consistent with previous descriptions of termite gut bacterial communities  and characterized by the absence of Bacteroidetes, while the second group was mainly composed of Bacteroidetes (20 to 50%) and Firmicutes (30 to 47%).A majority of sequences (64%) obtained at the end of the incubation period belonged to 59 OTUs that were common to all the samples. However, 273 OTUs representing 10% of sequences were sample-specific. The phylogenetic profiles of the bacterial communities present in the bioreactor at the end of the incubation were dominated by Firmicutes (particularly of the Clostridia class) and contained lower levels of Proteobacteria- and Bacteroidetes-related OTUs , even if carboxylate production was variable in replicate bioreactors. Regarding this variability, it is important to note that the different termite individuals were collected from their nests over a 3-month interval  and, in the specific case of N. lujae, the inoculum size of the two replicate experiments was different, with a two-fold difference in the number of 16S rRNA gene copies  and in both cases a rather consistent bacterial community composition was observed. Therefore, it appears that other factors, such as the specific physiological state of the gut microbiome at the time of the gut's withdrawal, could be responsible for experimental variability.Importantly, the extent of wheat straw degradation brought about by the termite gut microbiome was high compared to similar experiments performed using larger amounts of cow rumen inoculum  and those bound to the outer cell wall . In this respect, it is noteworthy that the bacterial communities in the bioreactors contained Clostridia, a class that contains cellulosome-producing members  were highly similar . Nevertheless, their relative abundance in the bioreactors was low, with maximal abundances of 3.6% Stenotrophomonas and 2.7% Acinetobacter in bioreactors inoculated with guts from M. parvus and N. ephratae, respectively. Higher abundance values were observed for Pseudomonas (12.3% in bioreactors inoculated with T. hospes guts), but facultative anaerobic metabolism has been reported for Pseudomonas, particularly, in relation to aromatics and lignoaromatics metabolism , particularly Clostridium termitidis OTU1, but also OTUs related to Lachnospiraceae  and Ruminococcaceae  were strongly enriched. C. termitidis was present in all bioreactors at an average of 16% of total reads. This species, previously identified in the N. lujae gut  genes than those of the Clostridiaceae family. This is remarkable, because many species belonging to the Clostridiaceae family are lignocellulolytic, being characterized by the fact that they bear cellulosomes and their genomes encode large numbers of xylanases  and Bacteroides (OTU18). Members of the Dysgonomonas genus are known for their lignocellulolytic potential  revealed that diversity was strongly decreased. For all termite gut inocula, the conditions prevailing in the bioreactors led to the selection of OTUs mainly related to Firmicutes, Bacteroidetes and Proteobacteria. This was particularly noticeable in the bioreactors inoculated with the  Imhoff, . Moreove Taylor, . In this2 requirements, altered host specific signaling and inter- taxa dependency relationships might explain the loss of certain phyla during our experiments.The final composition of the bacterial communities growing on wheat straw in the bioreactors was clearly distinct when compared to the parental termite gut communities Figure . DiversiNasutitermes gut microbiome (up to 45% w/w) are comparable with those mentioned above. Moreover, it is noteworthy that this level of degradation was achieved using a relatively intact substrate (simply milled to 2 mm) and in the absence of prior enrichment of microbial community. This suggests that it might be possible to achieve higher degradation levels using a pretreated substrate (Yan et al., Cubitermes (Brune and Kühl, Nasutitermes genus. Their hindguts are characterized by neutral or even slightly acidic pH values (Köhler et al., Nasutitermes microbiome performed best in our experimental conditions.It is difficult to compare the biomass-degrading potency of the termite gut microbiomes studied herein with those described in other studies, because the latter are often enriched microbial communities from compost, forest soils, or mangrove sediments grown on different substrates that were prepared in different ways (Feng et al., In conclusion, this study demonstrated that termite gut microflora can be grown in bioreactors, using lignocellulose as the sole carbon source to produce carboxylates. Further studies should focus on deeper characterization of the enriched microbial communities and identification of the key microbial and enzymatic drivers for lignocellulose bioconversion. Moreover, it will be worthwhile to investigate how modified experimental conditions can be used to further optimize the bioconversion process in order to maximize carboxylate production.LA, student on this project, did all bioreactors' experiments and data interpretation. He carried out the molecular diversity analysis and drafted the manuscript. AL, student in this project did the metabolite and enzymatic activity assays; she also trained LA to bioreactor utilization and monitoring. DS-D participated on termite rearing, selection and dissection; he trained LA to termite dissection. EM participated on termite selection for this project. DS-D and EM revised the manuscript critically. MO and GH-R participate on experimental design and project conception; they revised the manuscript critically for important intellectual content. GH-R is the main supervisor and MO is co-supervisor of LA and AL. GH-R is the project coordinator, conceived the study, and participated in its design and helped to draft the manuscript. All authors read and approved the final 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.


Acromyrmex echinatior and Atta cephalotes, which are both important functional herbivores in the New World tropics. Bacterial density remained similar across the developmental stages of Acromyrmex, but Atta brood had very low bacterial prevalences suggesting that bacterial gut symbionts are not actively maintained. We found that Wolbachia was the absolute dominant bacterial species across developmental stages in Acromyrmex and we confirmed that Atta lacks Wolbachia also in the immature stages, and had mostly Mollicutes bacteria in the adult worker guts. Wolbachia in Acromyrmex appeared to be transovarially transmitted similar to transmission in solitary insects. In contrast, Mollicutes were socially transmitted from old workers to newly emerged callows. We found that larval and pupal guts of both ant species contained Pseudomonas and Enterobacter bacteria that are also found in fungus gardens, but hardly or not in adult workers, suggesting they are beneficial only for larval growth and development. Our results reveal that transmission pathways for bacterial symbionts may be very different both between developmental stages and between sister genera and that identifying the mechanisms of bacterial acquisition and loss will be important to clarify their putative mutualistic functions.The social Hymenoptera have distinct larval and adult stages separated by metamorphosis, which implies striking remodeling of external and internal body structures during the pupal stage. This imposes challenges to gut symbionts as existing cultures are lost and may or may not need to be replaced. To elucidate the extent to which metamorphosis interrupts associations between bacteria and hosts, we analyzed changes in gut microbiota during development and traced the transmission routes of dominant symbionts from the egg to adult stage in the leaf-cutting ants  Obligate symbionts are strictly vertically transmitted, but facultative symbionts can be both vertically and horizontally transmitted  and three Atta cephalotes colonies  for sampling workers and brood immediately after collection in the field. Specimens belonged to three developmental stages: actively feeding larvae with clearly visible (under a stereoscope) guts, mature (brown) pupae close to hatching, and adult workers taken either from the top of the fungus garden or foraging around it. For Acromyrmex, we collected large workers and for Atta media workers of comparable size, of which individual guts were dissected in Gamboa and washed in sterile phosphate-buffered saline , transferred to 2 ml screw cap tubes, and stored at -80°C until DNA extraction. For subsequent experiments in the lab to confirm bacterial transmission patterns, we sampled similar specimens from three other A. echinatior  and four other A. cephalotes  colonies maintained in rearing rooms in Copenhagen (since 1996–2012) at ca. 25°C and 70% relative humidity and fed with bramble leaves, fruit fragments and dry rice. Before dissections, individuals of all developmental stages (including eggs) were always surface-sterilized by submerging them into 70% ethanol and then 2.5% bleach.Ant colonies were collected in Gamboa, Republic of Panama, in 2014. We used four A. echinatior colonies  and two of the A. cephalotes colonies (Ac2012-2 and Ac2012-3) to isolate brood and workers. The infection status of workers was repeatedly confirmed with PCR , Ac2012-2 pupae + Ac2012-3 workers and Ac2012-3 pupae + Ac2012-2 workers . Mature pupae were isolated from colonies and put in large Petri dishes together with fungus garden material and workers from paired colonies. Petri dishes were checked daily for the presence of newly hatched ants and such callows were marked with paint dots on their gasters. After eclosion, callows were together for 1 day with workers from the paired colony to improve the survival of hatched callow workers, which mostly died when they were kept in isolation. Then callows were transferred to a second Petri dish either alone or accompanied by three major and three minor workers from the other colony for 21 days. Ants of both groups were fed with aqueous 10% sucrose solution throughout the experiment and five guts per colony were dissected for pupae and callows both on the first and on the 21st day.To monitor transmission of gut bacteria in real time, we used three of the A. cephalotes), touchdown PCR was performed under the following conditions: 1 cycle at 95°C for 2 min, 20 cycles at 95°C for 20 s, the annealing temperature ranging from 60°C to 55°C (decreasing 0.3°C every cycle) for 15 s and 72°C for 5 min, then 20 cycles with the same conditions but at a constant 55°C annealing temperature, and finally 1 cycle at 72°C for 10 min. Data were deposited in GenBank (Bioproject PRJNA383138).DNA was extracted from frozen gut samples of field and lab colonies using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s instructions, with an extra step at which glass beads of 0.5 mm were added to lysis buffer and vortexed for 30 s. All samples were eluted using 100 μl of AE elution buffer, after which the extracted DNA from field colonies was sent to the Microbial Systems Laboratory at the University of Michigan for library preparation and 16S Miseq Illumina sequencing as described in EntAcro1 and EntAcro2 ] of Bray–Curtis dissimilarity indices, unweighted and weighted UniFrac distances and permutational multivariate analysis of variance  in PAST3. Bray–Curtis indices were also calculated in PAST3, while UniFrac distances were computed in mothur.To compare bacterial 16S rRNA gene copy densities across different samples, we used non-parametric tests in PAST3 because data were not normally distributed . P-valuepost hoc pairwise tests were run to determine significant differences across four sampling groups  in each experiment with combination of colonies . If there were many zero counts, zero-inflated, hurdle and negative binomial regression models were tested and the Akaike’s Information Criterion (AIC) was calculated for each of them to select the most appropriate model, which always turned out to be a negative binomial or hurdle model. To compensate for the complete separation problem in some colonies where concentrations of bacteria in pupae were zero for all individuals, we ran binomial-response generalized linear models using the bias-reduction method developed by To compare Mollicutes density in the transmission experiment, we ran a negative binomial regression model for the ddPCR counts in R , becausein situ hybridization (FISH), five ant workers, larvae and pupae from A. echinatior colony Ae507 and A. cephalotes colony 2012-2 were dissected in PBS and their guts placed in 4% paraformaldehyde overnight. For the permeabilization, deproteinization, and hybridization, we followed a previously described protocol , and guts were fixed in 2.5% glutaraldehyde (Sigma) in 0.1 M sodium cacodylate buffer (pH 7.4) for 2.5 h. This was followed by washings in the same buffer and postfixation in 1% OsO4 for 1 h, after which samples were placed in a 1% aqueous solution of uranyl acetate and left at 4°C overnight. Samples were then dehydrated in an ethanol series and acetone and embedded in Spurr low-viscosity resin (Ted Pella Inc.). Ultrathin sections of 60–70 nm thickness were stained with uranyl acetate and Reynolds lead citrate and examined with a transmission electron microscope .For transmission electron microscopy (TEM), larvae, pupae, and large workers of Figure 1A) were differentially distributed among the two leaf-cutting ant species . In Acromyrmex, there was no significant difference among the three developmental stages , but there was a significant difference among the same developmental stages in Atta . Gene copy numbers were similar in larvae and pupae of A. echinatior, with median densities of 1.79 and 1.43 copies per host cell, respectively, but less in Atta larvae (with a median of 0.14) and the lowest density in Atta pupae (0.067 copies per cell). The highest bacterial densities were observed in workers of both species  and without a significant difference between workers of these two species . However, bacterial cell numbers were always bimodally distributed across worker samples, with numbers ranging from 0.07 to 222 bacteria per host cell in A. cephalotes and from 0.82 to 114 in A. echinatior.Densities of bacterial 16S rRNA gene copies  and 32 bacterial classes and 374 OTUs were found across the gut communities of A. cephalotes , whereas Acromyrmex larvae harbored a higher bacterial diversity . Gut bacterial diversity in Atta appeared to be very different, with highly diverse pupal gut microbiomes  and moderately high diversity in both larvae  and adults  . Diversity measures were negatively correlated with overall bacterial abundance in A. cephalotes , but not in A. echinatior , suggesting the presence of dominant bacterial OTUs (when inverse Simpson index close to 1) leads to an increase of overall bacterial abundance in A. cephalotes, but not in A. echinatior . Overall, nMDS plots generated from a Bray–Curtis dissimilarity matrix revealed two non-overlapping clusters for A. echinatior and A. cephalotes  were identified across the gut communities of A. echinatior the most prevalent OTU was a Wolbachia, comprising an average of 76.0, 91.6, and 74.9% of the gut bacteria in larvae, pupae and workers, respectively, whereas the most abundant OTU in A. cephalotes belonged to the Mollicutes and reached average abundances of 17.2, 21.6, and 81.3% in the guts of larvae, pupae and workers, respectively. However, this only applied to two colonies because the third colony had low (<0.05%) infections in only two individuals and was thus considered to be Mollicutes-uninfected . We confirmed that this abundant OTU was identical to the one we identified as EntAcro1 across workers from six species of Panamanian Atta and Acromyrmex leaf-cutting ants (A. echinatior (Ae715) also had high gut infection levels with EntAcro1 .In A. echinatior, several OTUs in addition to the dominant Wolbachia OTU reached relative abundances of more than 10% in at least one individual  and other OTUs hovered between 3 and 10% in several individuals  were also observed in the fungus garden of Atta species  belong to the same families. This suggests that the fungus garden plays an important role in supporting the persistence of these bacteria within colonies, but further study will be required to fully understand their beneficial role for larvae or fungus gardens. The few Pseudonocardia bacteria that were present in larval guts of A. echinatior (with relative abundance ranging from 0.07 to 3.2%) could come from the cuticle of the workers feeding them or from the fungus garden assuming workers would inoculate these as well. Studies of bee species have shown that honey bee larvae harbor both bacteria belonging to the core gut microbiome of adults and other OTUs that potentially originate from nectar sources   . Some laanalyzed . In botho larvae , while wandibles . Althougy beetle . We thusEntAcro1 and EntAcro2) are beneficial for Acromyrmex and Atta leaf-cutting ants. In the adult workers, the Mollicutes were most abundant in the rectum, suggesting that this is the most favorable niche for them. Both Acromyrmex and Atta also maintain Rhizobiales bacteria, which have nitrogen fixing/preservation functions and form a biofilm adhering to the cuticle in the ileum and rectum of workers , which showed enormous variation in abundance both across and within colonies of A. echinatior and A. cephalotes. Although these bacteria are mostly known as mild pathogens  issued permits for collection and export of live and frozen ant material from Panama to Denmark (2014).MZ and JB carried out the field sample collection, MZ and PS performed the experiments and data analysis. MZ, PS, MS, and JB designed the study, drafted and wrote the manuscript. All authors read and approved the final 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.


Water is essential for the proper functioning of the body. Even though a recommendation exists for adequate water intake for Koreans, studies identifying actual water intake from all beverages and foods consumed daily in the Korean population are limited. Thus, we estimated total water intake from both beverages and foods and its association with energy intake and eating behaviors in Korean adults. We used a nationally representative sample of 25,122 Korean adults aged ≥19 years, from the Korean National Health and Nutrition Examination Survey 2008–2012. We performed multiple regression analyses, adjusting for sociodemographic and health-related variables to investigate the contribution of overall energy and dietary intakes and eating behaviors to total water intake. The mean total water intake excluding plain water was 1071 g (398 g from beverages and 673 g from foods) and the estimated plain water intake was 1.3 L. Among Korean adults, 82% consumed beverages (excluding plain water) and these beverages contributed to 10% of daily energy intake and 32% of total water intake from beverages and foods. For every 100 kcal/day in energy intake, water intake consumed through beverages and foods increased by 18 g and 31 g, respectively. Water intake from beverages and foods was positively associated with energy from fat and dietary calcium, but inversely associated with energy density and energy from carbohydrates. When there was a 5% increase in energy intake from snacks and eating outside the home, there was an increase in water intake from beverages of 13 g and 2 g, respectively. Increased daily energy intake, the number of eating episodes, and energy intake from snacks and eating outside the home predicted higher water intake from beverages and foods. Our results provide evidence suggesting that various factors, including sociodemographic status, dietary intakes, and eating behaviors, could be important contributors to the water intake of Korean adults. Findings from this cross-sectional analysis may provide insight into strategies for promoting adequate water intake among Koreans. Water is a crucial nutrient comprising 60% of human body weight ,2. It isMany longitudinal and cross-sectional studies have been conducted on water intake in relation to dietary intakes, weight status, and disease. It has been found that increasing one’s intake of non-caloric beverages such as plain water and coffee, including the replacement of sugar-sweetened beverages (SSBs) with plain water, is inversely associated with weight gain ,8,9 and Acknowledging the importance of water intake in maintaining health and preventing disease, many countries suggest recommendations for water intake to encourage the public to drink adequate amounts of water. In the United States (US), the Adequate Intake (AI) of total water was established by adding estimated water intake from foods and beverages obtained from nationally representative data . The EurThe beverage industry in Korea has grown and is now a global trend. According to recent statistics on the most frequently consumed foods in Korea, carbonated beverages are ranked third, blended beverages fourth, and fruit/vegetable beverages eighth . MoreoveGiven the limited number of studies attempting to quantify total water intake among Korean adults, investigations on the associations of water intake from foods and beverages with dietary intake and meal consumption attributes might help to lay the foundation for future work establishing better recommendations for water intake in Koreans. Therefore, the purpose of this study is (1) to describe water intake from beverages and foods in respect to beverage consumption; (2) to identify certain sociodemographic and health-related characteristics that can predict water intake; and (3) to investigate water intake in relation to energy intake and eating behaviors using a nationally representative sample of Korean adults from the Korea National Health and Nutrition Examination Survey (KNHANES) 2008–2012 data.The KNHANES conducted by the Korea Centers for Disease Control and Prevention (KCDC) and the Korean Ministry of Health and Welfare provide ongoing surveillance. The KNHANES data were collected to monitor the health and nutritional status among Koreans and to provide meaningful baseline data for health and nutrition policy design in Korea ,25. To en = 30,642). We excluded those with <500 or >5000 kcal/day of total energy intake (n = 469) and pregnant and lactating women (n = 612). We also excluded those who had incomplete information on socio-demographic characteristics (n = 2816), health-related behaviors (n = 1370), and reported implausible total water intake besides plain water intake (>3466 mL) (n = 253), for a final analytic sample of 25,122 .Data from the KNHANES 2008–2012 were used in this cross-sectional study to identify important contributors of water intake in Korea. All participants who were older than 19 years of age with a nutrition survey including a single 24-h dietary recall and health interview were eligible for inclusion in this study  water intake from beverages excluding plain water; (2) water intake from foods; (3) total water intake from beverages and foods other than plain water; and (4) usual plain water intake. As part of the 24-h dietary recall data, the water content of all beverages and foods consumed by participants in the KNHANES were publicly released . EstimatBased on the KNHANES coding scheme  and prevTo describe how each water variable varied according to dietary intakes and eating behaviors, we used several variables that may reflect energy intakes, dietary constituents, and meal consumption behaviors. We estimated total energy intake , % energy from macronutrients , dietary fiber, calcium, and sodium consumed . We alsoWe used several eating behavioral variables based on previous literature ,34 to as2)  [To determine an independent association between all water variables and dietary intakes and eating behavioral factors, potential confounders were included in our statistical model. Socio-demographic variables included: age ; income  ; educatise, ≥25) .p < 0.05 were considered statistically significant. We used SURVEY procedure with sample weight, stratum, and primary sampling unit recommended by the KNHANES analytical guidelines to account for unequal selection probabilities resulting from complex survey design and non-response [We used SAS  for statistical analyses in this study. All statistical tests were two-tailed and response ,26.We computed the adjusted means using multiple linear regressions to compare all types of water intake according to sociodemographic and health-related characteristics . Frequenp < 0.05). Compared to women, men consumed a higher amount of water from beverages and foods. Age was negatively correlated to water from beverage and total water intake, but positively correlated to water consumed through foods (p < 0.01). Income and education levels were positively associated with all water variables (p < 0.01). Like usual plain water intake, current smokers consumed higher amounts of water from beverages and total water, but lower amounts of water from foods than non-smokers (p < 0.01). Water intake from beverage and total water intake excluding plain water were higher on weekends than weekdays (p < 0.01). Those undertaking regular physical activity consumed more water from foods and total water compared with their counterparts (p < 0.01), but there was no association with water intake from beverages and usual plain water intake.The daily amount of total water intake from beverages and foods was 1071 g (1.1 L) and the mean usual plain water intake was 1.3 L among Korean adults . Usual pKorean adults consumed 451.0 ± 4.5 mL of beverages and 395.8 ± 4.0 g of water from beverages per capita each day . Beveragp < 0.01), but less likely to have alcohol compared to men (p < 0.01). Compared to younger adults (19–29 years of age), the consumption of unsweetened coffee/tea and alcohol was higher in other age groups, however, there was lower milk and 100% fruit juice consumption in adults aged over 30 years compared with younger adults (p < 0.01). The prevalence of consuming milk and unsweetened coffee/tea increased with level of income and education. Those with a higher education were more likely to consume milk, unsweetened coffee/tea, and even SSBs (p < 0.05). Current smokers and individuals reporting no physical activity were less likely to consume milk but more likely to consume SSBs.The prevalence of consuming different types of beverages according to sociodemographic and health-related characteristics was presented elsewhere see . Women wp < 0.01) (p < 0.01). Percent energy as protein was positively related to water intake from foods (p < 0.01), but negatively related to water from beverage and total water intake (p < 0.01). While percent energy as fat was positively correlated with all water variables (p < 0.01), percent energy as carbohydrates was negatively correlated (p < 0.01). Dietary fiber and sodium were significantly associated with higher water intake from foods and total water intake (p < 0.01), but they showed negative associations with water intake from beverages. Intake of usual plain water had no significant association with water intake from beverages and foods. However, water from foods and beverages were significantly related to each other and to total water intake excluding plain water after adjusting for covariates. All water intake from different types of beverages were positively associated with intake of water from beverages (p < 0.01), but such associations were observed in water from foods. The greatest contributors to water intake from beverages were water consumed as alcoholic beverages and 100% fruit juices. When there was a 100 g/day increase in the consumption of alcohols and 100% fruit juices, there was an increase in water consumed through beverages of 90.9 ± 0.8 g and 87.5 ± 4.4 g, respectively.Total daily energy intake was positively correlated to water intake from beverages and foods and total water intake excluding plain water ( < 0.01) . The 17.p < 0.01), but negatively associated with water from foods (p < 0.01). In case of the number of non-beverage foods consumed, the reverse association with water consumed from foods was found (p < 0.01). Particularly, the number of different types of beverages was the most important contributor of higher intake of water from beverages. When the number of unique beverages consumed increased, water consumed from beverages increased by 211.2 ± 2.7 g. The number of beverage-only episodes and eating duration were positively related to water consumed from beverages and total water intake besides plain water (p < 0.01), but negatively related to water content consumed through foods (p < 0.01). Reported total daily energy from main meals was negatively correlated with water intake from beverages (p < 0.01), but positively correlated with water consumed from foods and total water (p < 0.01). The number of total eating episodes and snack episodes were positively related to all water intakes (p < 0.01). A similar trend was observed for 24-h energy intake from snacks and reporting a snack in the 24-h dietary recall. Increasing the energy consumed outside the home had positive associations with intakes of water from beverages and foods and total water intake other than plain water (p < 0.01). For each 5% increase in energy from snacks and eating outside the home, the 30.2 ± 1.2 g and 12.6 ± 0.6 g, respectively, increases in water intake from beverages were found.Independent associations of eating behaviors with contribution to water intake are listed in This study described total water intake from beverages and foods consumed daily and explored the significant associations of water intake with energy and dietary intakes as well as eating behaviors using 25,122 weighed dietary information from Korean adult subjects in a nationally representative sample of the KNHANES 2008–2012. These findings might help to shed light on the important contributors to water intake and aid in understanding the current status of total water intake among Korean adults. On average, Korean adults consumed 1.1 L of total water, excluding plain water , based on the 24-h dietary recall data. Adding 1.3 L of usual plain water intake estimated from the FFQ-type question, the average amount of total water consumed daily by Koreans was 2.4 L, satisfying the AI of water intake for Koreans. This is somewhat lower than 3.1–3.2 L, the total water intake of Americans estimated from the National Health and Nutrition Examination Survey (NHANES) 2005–2006 data . In termWe found that those who were younger, had higher income and education, and exercised regularly showed higher intakes of total water excluding plain water. These sociodemographic and health-related characteristics were major determinants of not only water intake but also better dietary quality among Koreans . Higher It has been well-documented that vegetables and fruits are high-water-content foods, while oils, butter, and sugars contain the least water . In otheAnother interesting result was observed in relation to eating behaviors and water intake. Reported snacking at the time of recall and 24-h energy from snacks were positively correlated with water intake from beverage. It is well-known that higher consumption of beverages has a significant association with increased frequency of snacking . In a prThe present study has both limitations and strengths. Firstly, it used cross-sectional data which has limitations in identifying causal relationships between water intake and energy and dietary intakes and eating behaviors. Secondly, we adjusted for sociodemographic and health-related variables as covariates. However, we did not consider the influence of seasonal variations in water intake  since thIn conclusion, our study quantified total water intake and investigated the important nutritional and diet-related behavioral contributors of water intake among Korean adults. Water intake from beverages and foods was positively associated with total daily energy intake, but negatively associated with the energy density of non-beverage foods. The present study also found strong and independent associations between water intake and eating location (eating outside the home vs. at home), types of meals , and eating episodes. This additional information might help to provide more efficient strategies for nutritional education to prompt adequate water intake among people with various characteristics of dietary intakes and meal consumption behavior. Future studies using plain water intake measured by 24-h dietary recalls rather than FFQs or simple questionnaires are needed in order to answer the question “What are the best water intake guidelines for Koreans to ensure the proper role of water in the body?”


The patient was hospitalized for 3 days, required minimal oxygen and supportive care, and survived. Serology obtained on hospital admission was positive for hantavirus immunoglobulin M (IgM) and immunoglobulin G antibodies, with a positive result for Sin Nombre virus (SNV) (the primary etiologic agent of HPS in the United States) IgM at 1:6400, consistent with acute infection  in Colorado was notified of a confirmed case of hantavirus pulmonary syndrome (HPS). The patient was a previously healthy male migrant farm worker aged 25 years, living in farm quarters, and working in vegetable fields for 8 weeks before symptom onset. On July 20, he experienced sudden onset of fever, severe headache, myalgias, cough, and nosebleed. He was evaluated at an emergency department on July 23, where his temperature was 103.9°F (40.0°C), and his physical exam was notable for bronchial breath sounds and pulmonary crackles. Chest radiograph revealed bilateral interstitial infiltrates and small pleural effusions, and thrombocytopenia   on a ranch approximately 50 miles east of the farm described in this report. Environmental assessment of that patient’s farm home in 2014 revealed multiple rodent nests and excrement throughout the rural residential dwellings.This report highlights the importance of considering HPS in farm workers and in other occupations with risk for rodent exposure either at the workplace or in housing provided by the employer (


We conclude that vertebral CSA is negatively associated with LL, and that the greater degree of LL in females could, at least in part, be due to smaller vertebral cross-sectional dimensions. Studies are needed to examine the potential relations between vertebral CSA and spinal conditions known to be associated with increased LL, such as spondylolysis and spondylolisthesis.Lumbar lordosis (LL) is more prominent in women than in men, but the mechanisms responsible for this discrepancy are poorly defined. A recent study indicates that newborn girls have smaller vertebral cross-sectional area (CSA) when compared to boys—a difference that persists throughout life and is independent of body size. We determined the relations between vertebral cross-sectional area (CSA) and LL angle and whether sex differences in lumbar lordosis are related to sex differences in vertebral CSA. Using multi-planar magnetic resonance imaging (MRI), we measured vertebral cross-sectional area (CSA) and vertebral height of the spine of 40 healthy boys and 40 girls, ages 9–13 years. Measures of the CSA of the lumbar vertebrae significantly differed between sexes , while the degree of LL was significantly greater in girls than in boys . When all subjects were analyzed together, values for LL angle were negatively correlated to vertebral CSA ; this was also true when boys and girls were analyzed separately. Multivariate regression analysis indicated that vertebral CSA was independently associated with LL, even after accounting for sex, age, height or vertebral height, and weight. Similar negative relations were present when thoracic vertebrae were analyzed (Model P < 0.0001, R Lumbar lordosis (LL) refers to the anterior curvature of the lumbar spine, expressed in humans as a response to upright posture and bipedalism. Accumulating evidence indicates that LL is present in early childhood, increases during adolescence , and is Although variations in lumbar vertebral morphogenesis, such as vertebral wedging and orientation of lumbar facets, have been suggested to facilitate lumbar lordosis ,11,12, tSupport for the notion that the sexual dimorphism in vertebral size in humans could represent an evolutionary adaptation to bipedal obstetric load would come from demonstrating an association between vertebral CSA and LL angle. In the current study, we examined the degree of LL and vertebral cross-sectional dimensions in 40 boys and 40 girls, ages 9–13 years, and hypothesized that 1) adolescent girls have a greater degree of lumbar lordosis than boys, 2) the degree of lumbar lordosis is predicted by vertebral CSA, and 3) differences in lumbar lordosis between boys and girls are related to sex differences in vertebral CSA.The Institutional Review Board for clinical investigations at Children’s Hospital Los Angeles (CHLA) approved this study, which was compliant with the Health Insurance Portability and Accountability Act. Written informed assent and consent were obtained from all participants and their parents.For the purpose of this study, we chose boys and girls with ages corresponding to the adolescent growth spurt (9 to 13 years) and in early puberty when LL development markedly increases . ParticiAll MRI examinations were performed in the supine position with extended legs and without the use of general anesthesia or contrast enhancement. Subjects were examined using a research-dedicated 3.0 Tesla whole-body MRI scanner  with a standard 15-channel spine coil. Three dimensional T2-weighted turbo spin echo scans were taken with TE of 120 ms, TR of 3000 ms, a flip angle of 90°, and with a voxel size of 1.0 x 1.0 x 1.0 mm. For the purpose of this study, LL was measured as the angle between the superior endplate of L1 and the inferior endplate of L5  19], an, an19], The data were analyzed with the t-test for unpaired samples, and multivariate regression analysis was used to determine the association between vertebral CSA and LL. Collinearity among predictor variables was considered a tolerance level < 0.01 or variance inflation factor > 10. Confounding variables were considered a > 10% change in the CSA slope. Multivariate regression assumptions  were ascertained. Continuous variables are presented as mean ± SD unless otherwise indicated. Statistical significance was considered p < 0.05. Data was analyzed using SAS .The characteristics of study subjects are shown in As expected, measures of the CSA of the lumbar vertebrae significantly differed between sexes ; on averWhen all subjects were analyzed together, there were no associations between lordosis and age, weight, or standing or vertebral height . In contrast, values for LL angle were negatively correlated to vertebral CSA ; this was also true when boys and girls were analyzed separately  .2 increased from 0.14 to 0.37), but eliminated the contribution of sex .When lumbar vertebral CSA was replaced with the mean vertebral CSA of the low thoracic spine (T10–T12), the negative relationship persisted Click here for additional data file.