BP. Some sites contemporary to the Pre-Pottery Neolithic B still provide evidence for the usage of wild grasses, which possibly reflects the utilization of edible arable weeds and continuous gathering of wild grasses by more mobile groups.The present study investigates the occurrence of wild grasses at Epipalaeolithic and aceramic Neolithic sites in the Near East in order to assess their role in subsistence economies alongside the emergence of cereal cultivation. We use Chogha Golan in the foothills of the central Zagros Mountains  as a case study, where the archaeobotanical data suggest the frequent exploitation of a complex of wild grasses for almost 2,000 years. Domesticated emmer replaced these wild grasses as the major food resources towards the end of occupation at the site . We discuss possible implications of this development and conclude that the traditional concept of pre-domestication cultivation seems unsuited for explaining the patterns from Chogha Golan. These data are in good accordance with the overall picture in the Zagros Mountains, where wild grasses were routinely gathered throughout the early Holocene. In contrast, wild grasses were gradually replaced by wild cereals in the Levantine corridor since the end of the Pleistocene. However, several sites located in this region provide evidence for a continuous exploitation of wild grasses alongside emerging cereal cultivation and most of these taxa were part of the earliest segetal floras that evolved with the appearance of domestic cereals throughout the 11 The aceramic Neolithic in the Near East is characterized by the emergence of sedentary farming communities and marks a major change in human subsistence economies. Although still disputed –4, many Pistacia, Ficus, Vitis, Capparis or Amygdalus as gathered foods [Today we owe this intensive archaeobotanical research a much clearer picture of emerging cultivation than during the 1990s and substantial methodological advances for the analysis of Neolithic plant assemblages. However, such a heavy focus on emerging cereal cultivation and domestication automatically tends to neglect the exploitation of wild plant resources by early farming communities. Many authors acknowledged the presence of a high diversity of potentially usable wild plants in aceramic Neolithic assemblages, but predominantly interpreted fruits and nuts from ed foods ,35,39–41ed foods –44.Based on finds from Ohalo II, Hayonim cave and Kebara cave we know that wild plant foods, including grasses and legumes, have been exploited by Middle Palaeolithic and Epipalaeolithic hunter-gatherers –47. PartIn this paper we present additional evidence for the routine exploitation of wild grasses alongside wild cereals from the aceramic Neolithic site of Chogha Golan in the foothills of the central Zagros Mountains. Based on this case study we discuss how the emergence of agriculture on the site relates to the gathering of wild grains that directly “competed” with domesticated cereals in the subsistence economy. In this regard, our main goals are to investigate how the roles of wild grasses and wild and domestic cereals change throughout the site’s occupation and whether the traditional pre-domestication cultivation hypothesis is suited to explain this development.By using Chogha Golan as a starting point, we systematically reviewed the occurrence of wild grasses at Epipalaeolithic and aceramic Neolithic sites in the entire Fertile Crescent. Our main goal is to trace the development of wild grass exploitation in relation to emerging cereal cultivation and domestication. For investigating this, we conducted a spatial and temporal analysis on the occurrence of wild grasses and cereals. In addition to proportions and ubiquity values, we considered the interpretation of the wild grasses by the individual authors and their occurrence in specific contexts. The major aims of these analyses are to evaluate how the role of wild grasses changed through time and whether we see differences in plant exploitation strategies among the sub-regions of the Fertile Crescent. This is of major significance for understanding the Neolithic transition, because the harvests of wild grasses directly compete with cultivated grains and may shed light on the reasons why initial cereal cultivation appeared with a high temporal variability throughout the Near East.A further goal of this paper is to identify constraints in the currently available information on Near Eastern wild grass use and to evaluate how future research should address this issue to proceed in reconstructing the origin of charred grains in anthropogenic deposits.The aceramic Neolithic site of Chogha Golan is located in the foothills of the Central Zagros Mountains in Iran . MembersPistacia-Amygdalus woodland steppe and the riparian vegetation were exploited for wood resources. The faunal material is dominated by ungulates and contains a high abundance of fish remains, but the preliminary results and the small sample size do not allow drawing final conclusions on possible management practices or morphological domestication [Aegilops sp. remains [Riehl et al. presented first results on the palaeoecology of Chogha Golan . Accorditication . The avatication ,44. A cotication  and we d remains ,27,44.For the present study the first author analyzed 23 additional flotation samples from AH VII to III of the deep sounding in order to increase the sample size for the occupation period that precedes the beginnings of agriculture at Chogha Golan and is contemporary to the early and middle PPNB of the Levantine sequence. A radiocarbon date from AH VIII dates to about 10,600 cal. BP and predates the sediments studied in this paper, for which radiocarbon dates accumulate between ca. 10,000 (AH VI-IV) and 9,800 (AH III-II) cal. BP . WhetherThe analyzed flotation samples derive from two main sediment types. Collapsed and decayed building debris intermixed with occupation deposits formed AHs II, III, V and VII, whereas AHs IV and VI represent midden deposits mostly consisting of ash, charcoal, bone fragments and stone artifacts . SedimenArchaeobotanists traditionally divide Poaceae taxa into groups according to the dimensions of their caryopses. As an alternative approach we decided to use grain weight, based on the average 1000 seed weight given in the Seed Information Database of the Royal Botanic Gardens Kew . Due to Avena sterilis or Aegilops spp. as cereals will be discussed below. In this regard it is important to note that the functional interpretations assigned to taxa within a group are not mandatory and single taxa can still be interpreted differently. Combining different criteria based on physiological characteristics of grains and possible usage currently provides the most plausible, knowledge-based classification for our analyses.The “small-seeded wild grasses” comprise genera with an average grain weight of less than 3g. Many species in this group are commonly interpreted as representative of dung-burning or fuel collection activities. From this group we separate all genera with an average grain weight of 3g or more. These taxa are commonly regarded as potential food plants or arable weeds and most of them have a reduced probability of being preserved in herbivore dung due to their larger grain size ,68,69. WCorrespondence analysis is a multivariate statistical method routinely used by archaeologists to examine the complex relationships between variables in a contingency table . In our The original dataset included 93 taxa in 38 samples. To avoid the bias of rare species and a poor preservation, we omitted all taxa with a ubiquity lower than 10% and all samples containing less than 100 identified specimens from the analysis. Because this procedure would have substantially influenced the original dataset, we combined rare taxa with a similar taxonomic rank or ecological implication in order to reach a ubiquity of 10%. Following this procedure we could maintain the majority of the original data and generated a table comprising 47 taxa in 36 samples.Multivariate statistics can be used to disentangle cultivated species, arable weeds, gathered resources and dung-derived plant remains, given a large dataset deriving from a multitude of contexts is available (see e.g. ). As theWe systematically reviewed the published archaeobotanical literature for examining wild grass use throughout the Epipalaeolithic and aceramic Neolithic. After screening all published reports, we defined standard criteria for subsequent analyses, which resulted in differing sets of sites for the conducted analyses and allowed to consider a maximum number of datasets for reviewing wild grass use . A totalPistacia shell fragments, which resulted in a strong proportional decrease of all other plant groups [For our analyses we used the proportions of the different Poaceae taxa and categories together with their ubiquity values. Taphonomic processes and diverging quantification methods can result in a heavy overrepresentation of single botanical taxa in charred assemblages. For instance, several PPNA sites in the southern Levant yielded extremely high counts for fig nutlets and t groups ,35,72. TUbiquity values were used to plot the occurrence and frequency of selected genera over time. We defined a threshold of ten flotation samples to include sites into this analysis. Different occupation phases have occasionally been combined in order to reach this minimum number of samples. For Chogha Golan we combined the samples from the deep sounding with those from excavation area A  to obtaiPhalaris sp. and Phleum type, together with small indeterminate grains, contribute the major portion to the grass remains. Large to medium-seeded taxa such as Avena sp., Bromus sp., Hordeum sp., Eremopyrum sp., Taeniatherum caput-medusae and Triticoid type form a second abundant group. They are mostly represented by grains and yielded only few identifiable chaff remains. Along with fragmented spikelet bases of T. caput-medusae, spikelets of an indeterminate type have been frequently found. Among the cereal species, Hordeum spontaneum is well represented by both grains and rachis segments. The domestic-type rachises reach their highest proportion in AH VII with 3.6%, still indicative of a population with shattering ears. Species of Triticum only yielded glume bases and spikelet forks, but virtually no identifiable grains. The major portion of the identifiable rachis and glume base fragments could be attributed to emmer wheat, whereas einkorn constitutes a minor component of the wheat chaff. The emmer chaff from AH II was identified as phenotypically domesticated [Aegilops sp. grains and chaff yielded proportions comparable to these typical cereal species. We therefore regard Aegilops sp. as economically equally important as H. spontaneum and the Triticum species and include it into the cereal category at Chogha Golan  to AH IV (23%) and again decrease towards AH II (15%). In their entirety, these large to medium-sized grains constitute higher proportions to the analyzed material than cereal grains. However, the lower grain volumes of the medium-seeded grasses have to be taken into account while assessing the relative importance of these taxa compared the cereals . The spikelets of the indeterminate type and Taeniatherum caput-medusae fluctuate throughout the sequence (10–34%) and only reach proportions comparable to the cereal species in AH V.Triticum spp. only yielded chaff, the temporal development of the different grasses becomes apparent by plotting all these data together  abundantly occur in the analyzed samples. Particularly Atriplex sp. and Alyssum sp. seeds reach high counts in the samples from the midden deposits of AH VI. Nutshell fragments of Pistacia represent the only remains of a gathered tree-fruit. They constantly occur in all samples, but are significantly less abundant in the middens of AH VI and IV ” [Avena sterilis associated with Hordeum spontaneum in a storage context at Gilgal clearly indicate that some large to medium-seeded wild grasses did represent important foods in the Levantine PPNA [Although the assemblages from the southern Levant show a less marked dominance of wild cereal grains, pre-domestic cultivation has been proposed for all sites included in this analysis ,35,40,72 cereals ,40. Howerum (…)” . Followiine PPNA . Weiss eine PPNA , which wPuccinellia species, which Fairbairn et al. interpreted as a possible fuel source [Outside the Levantine corridor, evidence for an increased focus on the wild progenitor species during the PPNA does not exist. For central Anatolia we are currently left with only one site, Pinarbaşi, where wild cereals are virtually absent. The only abundant grass remains are charred culm nodes and the small grains of a l source . In contl source ,51,60,64l source ,60,64. HThe overall pattern that was characteristic for the PPNA continues into the early and middle PPNB, where domestic cereals provide unequivocal evidence for the beginnings of farming ,25. SiteSuch a clear focus on a cereal-based subsistence strategy continues to be rare outside the Levantine corridor in the early and middle PPNB. Aşikli Höyük represents the only site in central Anatolia where domestic cereals were cultivated ,82. ReceEremopyrum-type and Stipa sp. grains positively and significantly correlated with cereal grains, supporting their interpretation as common field weeds. Interestingly, grains of Taeniatherum caput-medusae did not show a significant correlation, but were found in storage contexts together with Eremopyrum-type grains [Taeniatherum and Eremopyrum as arable weeds at the site [Full agricultural societies are established in all regions of the Fertile Crescent with the late PPNB . The arce grains . The aute grains  and corrthe site . Grassesthe site .Lolium sp. represents the most common weed at these sites, which is accompanied by Aegilops sp. at Sabi Abyad II and by Bromus sp. at El Kowm.A remarkable development in the Levantine corridor, particularly the upper Euphrates area, is the increase in non-cereal taxa towards the late PPNB. Whereas de Moulins did not strictly rule out continuing gathering activities at Abu Hureyra , this paPhalaris, 42%) and ZAD 2 . Colledge regarded the Phalaris grains as possible food sources or arable weeds [Plotting the proportions of small caryopses through time did not reveal a temporal trend towards increasing or decreasing percentages . Insteadle weeds , whereasle weeds ,72.Sporobolus, Aeluropus or Eragrostis were abundantly brought into the site. Besides Çatalhöyük, such an approach to disentangle arable weeds, crops and dung-derived seeds was only applied to Sheikh-e Abad, where Whitlam also argued for dung burning as the most likely source for the high abundance of the Poa-type caryopses [The highest proportions of small-seeded grasses with more than 60% of all grains occur, with increasing percentages, at Ganj Dareh, Çatalhöyük East (VI-PXII), Chogha Golan (AH VII-II), Körtik Tepe (Younger Dryas occupation), Pinarbaşi and Sheikh-e Abad (Trench 3). Most authors explained this phenomenon with the collection of herbivore dung or the grasses itself for fuel ,39,57,90aryopses . Riehl aaryopses .A total of 47 sites/phases could be included in the analysis of wild grass ubiquity over time. Most identified Poaceae taxa in Epipalaeolithic and aceramic Neolithic assemblages occur infrequently among these sites and their identification rarely has major implications for reconstructing gathering or cultivation activities. In contrast, some genera are very frequent among the analyzed datasets, which possibly indicates a role in the prehistoric subsistence economies. Among these frequently identified non-cereal taxa, two different developments through time can be observed.Aegilops, Avena, Eremopyrum, Hordeum, Stipa and the monotypic genus Taeniatherum. Many authors consider them as gathered resources at Epipalaeolithic and PPNA sites, with food as the most frequently mentioned usage. Particularly remains of Aegilops spp., Avena spp., Hordeum spp., Stipa spp. and Taeniatherum caput-medusae seem to be uniformly interpreted in this way, given they occur with a certain abundance in the analyzed assemblages [The first set of taxa abundantly occurs at Epipalaeolithic to PPNA sites as well as during the PPNB . They doemblages ,73,74,88emblages ,32,35,76Eremopyrum species are relatively rare on most PPNA sites except for the early phases of Mureybet, where grains occur in many samples but always with low numbers. Van Zeist and Bakker-Heeres discussed a possible interpretation of Eremopyrum as an arable weed [Eremopyrum grains and other potential weeds decreases alongside the increase of einkorn towards phase III. This seems to speak against an interpretation as crop-processing by-products and they leave the interpretation of these taxa undetermined.ble weed . HoweverEremopyrum, Hordeum and Stipa grains have very high ubiquities, de Moulins and Colledge did not rule out deliberate gathering activities [With the appearance of domestic cereals during the early and middle PPNB, the abundance of these taxa does not substantially change. All above mentioned genera comprise species which occur as arable weeds in the Near East today ,93. Basetivities ,42. Thestivities .Taeniatherum caput-medusae grain storages represent stored weeds outsorted from the crop harvests [The role of the considered grass taxa as arable weeds at late PPNB sites is mostly unquestioned ,89,95,96harvests ,53. De Mharvests .Echinaria capitata and Lolium spp. grains substantially increase in abundance towards the PPNB, where they are present at much more sites than during the Epipalaeolitic and PPNA. Bromus spp. grains occur on most sites of all periods, but similarly show higher ubiquity values in the PPNB. The increase in abundance of Phalaris species can be seen in their frequency among the sites. Phalaris spp. grains are present at around 23% of the Epipalaeolitic to PPNA sites and at 52% of the PPNB sites.Bromus pseudobrachystachys/tigridis as gathered foods at Ohalo II, where the grains occur with high numbers in a plant-food preparation area in a brush hut [Lolium sp. was presumably gathered at Sheikh-e Abad [Phalaris may have been exploited at Pinarbaşi, Netiv Hagdud, el-Hemmeh and Chogha Golan . During the PPNB, all these taxa apparently became common arable weeds. Lolium species developed into the most successful segetals, whose grains are present in 94% to 100% of all samples from late PPNB Ghoraifé and Ramad [Phalaris and Bromus are abundant prior to the emergence of domestic emmer and may represent gathered resources.Evidence for the exploitation of these taxa prior to the PPNB is limited. Weiss and his colleagues interpreted rush hut ,73,74. Lh-e Abad , but nownd Ramad . A regioWe conducted a correspondence analysis to test, whether the analyzed flotation samples from occupation and midden deposits differ in their taxonomic composition. Interestingly, the samples followed a parabolic distribution in the scatter plot. Such a pattern is the result of a factor within the data table, which orders the samples along a specific gradient. This means that the samples at both ends of the ordered data table show the largest difference and all other samples are gradually more similar to one another. Such a pattern can be problematic, e.g. for ecologists, because the distances between the data points in a parabolic plot do not reflect the “real” distances between the observed cases. However, this issue is of limited importance in an archaeological application . The casAtriplex sp. and Alyssum sp. and bulbils of Poa bulbosa accumulated here.At Chogha Golan the cases represent flotation samples characterized by their taxonomic composition, which derive from the deep sounding of a tell excavation. The parabolic distribution of the samples therefore means that their composition follows a strong temporal trend throughout the sequence, which allows us to interpret the compositional differences between the samples as a temporal development. The type of deposit is only a secondary factor influencing the formation of the charred assemblages. As an exception to this overall pattern, only three samples from the midden deposits of AH VI are clearly separated from the other samples along axis one, because possibly gathered seeds of Hordeum spontaneum, Triticum spp. and Aegilops sp., which also applies to the older horizons XI to VIII [Avena, Bromus, Eremopyrum and Hordeum species, the indeterminate Triticoid type and Taeniatherum caput-medusae raise the question of whether they were exploited as food resources and how their state in the subsistence economy relates to the wild cereals.In the first archaeobotanical reports about Chogha Golan the presence of cereals, their status as pre-domestic or domestic cultivars and the high proportions of small-seeded grasses between AH V and III were discussed in detail ,44,56,64 to VIII ,64. ThesH. spontaneum and possibly even Aegilops sp. were cultivated at Chogha Golan [H. spontaneum and the presence of many wild grasses representing potential segetals. However, in light of accumulating archaeobotanical data for Chogha Golan and other aceramic Neolithic sites in the Zagros Mountains, we became increasingly cautious in interpreting the observed patterns using this explanatory framework. Although the patterns from Chogha Golan basically fulfill the criteria commonly used to apply the pre-domestication cultivation hypothesis, we see considerable differences between this dataset and those from the Levantine corridor.We previously argued that ha Golan ,44,64. Sha Golan ,35,37,99Central for the proposition of pre-domestication cultivation, particularly for sites in the upper Euphrates area, was the interpretation of several potential weeds as representative of an early segetal flora, but also the gradual and clear decrease of gathered resources contemporaneous to a marked increase in wild cereals ,36. ThisH. spontaneum and Aegilops sp. presumably represent staple foods and that they were routinely exploited throughout the entire occupation period. Large to medium-seeded grasses exhibit the same patterns and, following the general picture for wild grass exploitation in the Zagros Mountains, they presumably represent routinely gathered foods as well. However, we are still confronted with the exploitation of diverse wild grasses throughout an occupation period of about 2,000 years. During the major part of this period, but at least 1,000 years, the inhabitants constructed permanent architecture indicative of a sedentary community. This leads us to the question if humans could continuously rely on these resources without any form of resource management? A look at the taxonomic composition of the identified wild grasses with regard to their present day ecology might give an additional clue for reconstructing subsistence practices.Applying the traditional concept of pre-domestication cultivation to Chogha Golan would ignore all these patterns, which we identified during our ongoing analyses. In light of this conclusion we have to ask how we will now characterize this subsistence economy? We are left with the finding that Aegilops spp., often dominate the herbaceous vegetation and grow together with e.g. Avena sterilis, Bromus spp., Hordeum bulbosum or Taeniatherum caput-medusae  . In addiWhen we assume taphonomic processes as the main factors causing this variability, we are confronted with a geographical rather than a temporal pattern. As concluded above, the Taurus-Zagros Mountains and central Anatolia display considerably different developments in subsistence practices as opposed to sites in the Levantine corridor. All sites with small-seeded grasses making up more than 60% of the Poaceae grains are located here, which suggests local subsistence practices being a major factor that induces this pattern. In the Zagros Mountains, subsistence strategies are characterized by the exploitation of wild grasses for food and the small-seeded taxa possibly represent deliberately collected resources as well. Riehl and her colleagues favor this explanation for Chogha Golan and Körtik Tepe, whereas the collection of grasses or herbivore dung as fuel has been suggested by most other authors ,39,57,90Bolboschoenus nutlets as being dung-derived, because their ecology made sedges the most plausible taxa being grazed by livestock and not harvested with the grown crops [Bolboschoenus nutlets as well as the small grains from Aeluropus sp., Crypsis sp., Eragrostis sp. and Sporobolus sp. are indeed dung-derived [Alopecurus grains from the early levels remains unclear. Çatalhöyük is therefore the only early Neolithic site where most small-seeded grasses could reliably be linked to dung-burning. The comprehensive application of multivariate statistics, in combination with a good situation for contextualized sampling and the application of established criteria to recognize dung-derived plant materials, represents the basis for these very informative archaeobotanical studies. Such a comprehensive approach was/could not be applied the other Anatolian or Zagrosian sites and we are therefore left with a quite unsatisfactory picture, which does not allow us to draw robust conclusions on the high proportions of small-seeded grasses at the sites under discussion. We should not take abundant small-seeded grasses per se as indicators for dung-burning, because different taphonomic factors also resulted in the incorporation of large to medium-sized grains in charred assemblages and include gathering and crop-processing. Whitlam eventually noted that herbivore dung as a major source for large amounts of small-seeded grasses in charred assemblages must remain hypothetical unless dung pellets including identifiable grains are found [Fairbairn et al. reported dung-pellets for the early phases at Çatalhöyük and considered particularly wn crops . Further-derived ,87,91, wre found  and we fHordeum spontaneum formed a major component of the consumed grasses and was harvested for almost 2,000 years without inducing the establishment of non-shattering ears. Then, as soon as domesticated emmer wheat dominates the cereal remains by ca. 9,800 cal. BP, all wild grasses decrease in relative abundance. This complex of wild grasses was presumably harvested as staple foods and was subsequently outcompeted by a domestic crop. Whether the wild cereals were ever cultivated at Chogha Golan is therefore not clear and we consider the ability of the exploited wild grasses to invade anthropogenically disturbed habitats around the sedentary village as an important factor that possibly facilitated their sustainable long-term exploitation. To which degree management practices were applied to maintain the wild grass stands is unclear, but this scenario significantly deviates from the traditional concept of pre-domestication cultivation and must be seen as a successful and resilient long-term subsistence strategy that did not heavily focus on wild progenitor species.Chogha Golan provides an exceptional case for analyzing the development of early Neolithic subsistence strategies and for evaluating the importance of gathered resources in relation to emerging crop cultivation. The inhabitants of the Neolithic village routinely exploited a complex of wild grasses comprising wild cereals, large to medium-seeded and possibly also small-seeded taxa. th and 11th millennium cal. BP. This is in sharp contrast to the development in the Levantine corridor, where wild grasses formed important components of Epipalaeolithic subsistence strategies and were gradually replaced by wild cereals as major food sources since the end of the Younger Dryas.The archaeobotanical results from Chogha Golan are in good accordance with the overall patterns of the aceramic Neolithic in the Zagros Mountains. Wild grasses were exploited as major food sources at many sites in the uplands and in the foothills bordering the Mesopotamian plain throughout the 12For evaluating wild grass gathering in relation to the emergence of cereal cultivation, we analyzed the occurrence of wild grasses throughout almost 15,000 years considering the entire Fertile Crescent. We were able to visualize spatial patterns and long-term developments, which we regard as crucial for interpreting the role of wild grasses throughout the aceramic Neolithic. We provide additional evidence for a considerable diversity among early Neolithic subsistence strategies on the inter-regional level and conclude that many wild grasses were gathered for food, fuel or other purposes from the Epipalaeolithic onwards and demonstrably into the PPNA period. Many of these gathered grasses were part of the earliest segetal communities that emerged with the beginnings of farming, emphasizing the potential dietary value of these weed species. This pattern is of major importance for evaluating the status of weeds in early farming communities. The archaeobotanical datasets from e.g. Çatalhöyük, Abu Hureyra or el-Hemmeh suggest the utilization of arable weed species, which is plausible in light of this result. A high diversity of wild grasses also co-occurs with domestic cereals at some sites in the “marginal zones” of the Levant, such as Jilat 7, which presumably indicates the utilization of these species by groups that maintained a more mobile lifestyle.Finally we want to emphasize the great potential multivariate approaches have to reconstruct prehistoric subsistence practices. Our review unmistakably shows that we will not proceed in disentangling arable weeds, deliberately collected food resources and dung burning activities if we do not implement the full spectrum of analytical techniques available to date. Among these, multivariate statistical methods such as correspondence analysis, coupled with contextualized sampling from relatively extensive excavations, represent the most promising approach that has been successfully applied to a small number of sites. It is clear that some assemblages are not suited for such analyses due to a poor preservation of the plant remains or small excavation areas, but many published assemblages fulfill these criteria and can be investigated in future analyses to more reliably interpret the role of wild resources in relation to emerging cultivation and domestication.S1 TableBased on the Seed Information Database of the Royal Botanic Gardens Kew .(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Fig(A) The landscape around Chogha Golan; (B) outline of the site with the location of the deep sounding and excavation Area A in the center of the tell; (C) the south profile of the deep sounding showing Archaeological Horizons I to XI and the related z-values. Figures and photos by M. Zeidi.(TIF)Click here for additional data file.S2 FigCalculations are based on the average 1000 seed weight given in the Seed Information Database (SID) of the Royal Botanic Gardens Kew . Numbers(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 FigH. spontaneum and Aegilops sp. together.Note that the strong temporal trend throughout the sequence is also visible in the Poaceae remains. Whereas the older samples from AH VII and VI have low percentages of small grains (right end of axis 1), samples from the middle part of the analyzed sequence are dominated by these taxa. Emmer wheat chaff remains characterize samples from AH III and II, where small grains are still very abundant (left end of axis 1). Large to medium seeded wild grasses are in all samples more abundant than grains of (TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.


Metal ions aside from Mg2+ or Mn2+ can act as cofactors for some DNA pols but not for others. Here we report on the ability of several divalent metal ions to substitute for Mg2+ or Mn2+ with BST DNA polymerase (BST pol), an A family DNA pol. We selected the metal ions based on whether they had previously been shown to be effective with other DNA pols. We found that Co2+ and Cd2+ were the only cations tested that could replace Mg2+ or Mn2+. When Co2+ was substituted for Mg2+, the incorporation efficiency for correct dNTPs increased 6-fold but for incorrect dNTPs there was a decrease which depended on the incoming dNTP. With Mn2+, base selectivity was impaired compared to Co2+ and Cd2+. In addition, Co2+ and Mn2+ helped BST pol to catalyze primer-extension past a mismatch. Finally both Co2+ and Mn2+ enhanced ground-state binding of both correct and incorrect dNTPs to BST pol: Dideoxy terminated primer-template complexes.Although Mg Even though DNA pols utilize the physiologically relevant Mg2+, Mg2+ can substitute for Mg2+ in the nucleotidyl transfer reaction but, when this occurs, it tends to lower the base selectivity dramatically . We have also looked at the ability of different divalent metal ions to support extension past a mismatched primer/template terminus. We have attempted to explain why BST pol can utilize only selected divalent cations as cofactors based on the properties of these metal ions and we have compared our results with other DNA pols.2, CaCl2, CdSO4, CoCl2, CuSO4, CrKSO4, MgCl2, MnCl2, NiCl2, SrCl2, FeCl2, and Zn(CH3COO)2) were from Fluka and were >99% pure.All chemicals were of the highest grade available and were used as purchased. dNTPs were from Roche , [γ-32P]ATP was from MP Biomedicals , and T4 polynucleotide kinase was from New England Biolabs . The metal ion salts . All primer strands were gel purified using PAGE (19:1% (w/v) acrylamide: Disacrylamide gels containing 8 M urea). The primer was labeled with [γ-32P]-ATP using T4 polynucleotide kinase and was annealed to unlabeled templates (in a 1:1.1 ratio) by heating to 95 °C, followed by slowly cooling to 25 °C . For equ13A), 40 nM BST pol enzyme and 10 mM MgCl2 in 66 mM Tris-HCl (pH 7.3). The mixture was preincubated at 23 °C for 10 min and the reaction initiated by adding 500 μM dTTP. 5 μL aliquots were withdrawn at various times  and quenched with 0.5 M EDTA. The assay was repeated by replacing MgCl2 with BaCl2, CdSO4, CuSO4, CrKSO4, FeCl2, MnCl2, NiCl2, SrCl2, and Zn(CH3COO)  were employed for each kpol and Kd,app determination.Single turnover experiments were carried out as described above with the exception that DNAPdd)  and the metal ion. Peak emission intensities at 365 nm were plotted as a function of [dTTP] (or [dCTP]) and fit to a hyperbolic equation to obtain the ground-state equilibrium dissociation constant (Kdg) for incoming dTTP (or dCTP).The emission spectra of dideoxy-primer/templates (ddP/T) containing 2AP at the templating position were recorded at 23 °C with a Photon Technology International Alphascan scanning spectrofluorometer. The assay contained 200 nM P/T (DNAPdd) , 1 μM BSThe amount of product formed at each [dNTP] at different times was fit by non-linear regression to obsk is the observed rate constant at a particular [dNTP], and A is the observed amplitude of product formation and T is time. To determine the pre-steady-state kinetic parameters kpol and Kd,app, kobs was plotted against the [dNTP] and data were fitted to Where kobs is the observed rate at a given [dNTP]. In cases where saturation was not achieved with the incoming dNTP, data were fit to Where kon is defined as the ratio of kpol to the Kd,app value for the incoming dNTP. Equilibrium fluorescence titration data were fit to the hyperbolic equation.Where f is the observed fluorescence intensity and fmax is the maximum decrease in fluorescence intensity at saturating [dTTP] and Kd,g is the ground-state equilibrium dissociation constant for the incoming dNTP.Where 2+  was ~12-fold greater than that of the primer-template [P/T]. The concentration of product DNA obtained at different [dTTP] were plotted against reaction time and fitted to kobs at each [dTTP]. The kobs values were then plotted against [dTTP] which yielded a kpol of 20 ± 2 s−1 and Kd,app of 26 ± 11 μM with Mg2+. The kpol values obtained using Co2+, Mn2+ and Cd+2 were 123 ± 12, 166 ± 14, and 15 ± 1 s−1 respectively. Surprisingly, Kd,app values for the correct nucleotide incorporation were very similar with all metal ions varying between 15–28 μM. 2+, Mg2+, Co2+, and Cd2+ respectively. The results, summarized in 13A) with Co2+, Mn2+, and Cd2+ were increased by ~6-, ~10-, and 1.3-fold respectively compared to Mg2+.Even though Mgom human , viral  in the presence of different metal ions. Kd,g) and apparent binding affinity  for an incoming dNTP. In the presence of Mg2+, the Kd,g value for dTTP binding opposite 2AP was determined to be 1 μM  respectively. 2+ and Co2+.We next tested whether BST pol is able to similarly discriminate against an incorrect incoming dNTP in the presence of these metal ions. The change in 2AP fluorescence was measured as a function of [dCTP] to determine the 2+, Mn2+, Co2+ and Cd2+. We chose a DNA oligo containing a dA/dC mismatch (DNAACMM) as this represents a purine-pyrimidine pair containing a nascent wobble mispair having the correct size but with distorted geometry. As compared to extension past a matched base-pair at the primer terminus, where the kpol was 20 ± 2 s−1 and the Kd,app was 26 ± 11 μM, the extension past a duplex DNA containing a mismatch at the P/T terminus in the presence of Mg2+ resulted in a kpol of 0.13 ± 0.01 s−1 and a Kd,app value of 618 ± 185 μM. This sharp decrease in the kpol value (200-fold) accompanied by a substantial increase in the Kd,app value (24-fold) results in ~4800-fold decrease in the incorporation efficiency of dNMP incorporation when BST pol encounters a dA/dC mismatch at the primer terminus in the presence of Mg2+.We next determined how efficiently BST pol could extend a duplex DNA containing a mismatch at the P/T terminus in the presence of Mg2+, the Kd,app was 1100 μM, higher than that obtained in the presence of Mg2+, but the kpol was 10-fold higher, showing that the incorporation of correct dNMP past the mismatched DNA was more readily accomodated compared to Mg2+. In the presence of Mn2+, the incorporation efficiency of correct dNMPs past the dA/dC mismatch was 100-fold higher than with Mg2+, demonstrating that the correct dNMP was much more readily incorporated past an dA/dC mismatch compared to Mg2+. This enhanced incorporation efficiency with Mn2+ directly resulted from a 20-fold increase in the kpol and a ~5-fold decrease in the Kd,app values. When Mg2+ was replaced with Cd2+ in the assay, saturation with the correct incoming dNTP was not achieved so we were unable to determine the actual kpol and Kd,app values. Consequently we had to plot the rates observed versus [dNTP] and determined the kpol/Kd,app value from the slope  in the presence of Cd2+ suggesting that, unlike Co2+ and Mn2+, Cd2+ does not promote extension past a dA/dC mismatch. The kinetic parameters for extension past a dA/dC mismatch are summarized in With Cohe slope . Compare13A and all four dNTPs, quenching the reaction at various times. 2+, Mn2+, Co2+, and Cd2+. The results show that all four metal ions were able to support processive DNA synthesis with BST pol. As expected with Mg2+, a fully duplexed DNA product was obtained. Intermediate products were observed from 0.05–1 s while majority of these intermediate length products were converted to full length product at the longer time points (5–30 s)  an 18mer product was also found  , Panel Ahe assay , Panel Bso found , Panel Cith Cd2+ , Panel D2+, Mn2+, and Ni2+ have been characterized as “mutagenic”, since they reduce the fidelity of DNA synthesis 62+, Mg[H2O]62+) there is a greater energy penalty with Mg2+ compared to Mn2+, when the inner sphere coordination number is changed from 6→5→4, indicating more rigid coordination requirements for Mg2+ complexes compared to Mn2+. This in turn allows dNTPs (correct and incorrect) to be accessible to the nucleotide binding pocket with Mn2+ [2+, could explain the lower base selectivity in the presence of Mn2+ [Apart from these factors, the preference of different divalent cations for a given coordination geometry plays a critical role in the nucleotidyl transfer reaction. Studies by Xia et al.  showed tomplexes . The hig of Mn2+ . Also, iith Mn2+ . In addi2+ is the universal metal ion cofactor for all DNA polymerases. Even though Mg2+ is preferred by pols, Mn2+ and Co2+ can also be used in place of Mg2+ but this usually results in a decrease in replication fidelity [2+, and Zn2+ were able to act as cofactors of pol β [2+. On the other hand, both these divalent cations were inactive with RB69pol [2+ has been shown to support catalysis for all DNA polymerases albeit with greatly reduced activity except for human pol α [2+ is also not able to activate BST pol. Interestingly, Fe2+ exhibits properties conducive to supporting catalysis with pols but fails to catalyze nucleotidyl transfer with BST pol, RB69pol [Mgfidelity –11,15. Cof pol β  while on RB69pol . Ni2+ haan pol α , human pan pol α , and Dpo RB69pol ,31 and H RB69pol ,33 but t2+ is the best divalent cation cofactor for pols in terms of the rate of nucleotidyl transfer reaction, base selectivity, exonuclease activity, and the efficiency of extension past mismatched DNA. Other divalent cations can substitute for Mg2+ but they affect the various fidelity checkpoints in the minimal kinetic scheme (In summary, Mgc scheme  and thei


Through their thorough investigation of the Hadza, a nonindustrialized language community in Tanzania, Lindsey and colleagues (2015) developed a new approach to understand the evolution of color terms. In the present commentary, I discuss the possibility that some of their results might be explained by the lacking control of saturation of their color stimuli. The saturation of colors plays an important yet widely neglected role in color naming. The additional analyses presented here suggest that the results on Hadzane color naming could be due to variations in saturation in the stimulus set rather than being evidence for universal constraints on color term evolution. The origin of color categories has preoccupied many scientific disciplines because it is the prime example for studying the biological, ecological, and cultural factors that relate language to perception . Yet, thThe theoretical and mathematical approach of this study is truly intriguing. However, with respect to the observed cross-cultural motifs, it is important to note that this study measured color naming with a set of color stimuli that varied strongly in chroma and saturation. Saturation, chroma, and colorfulness (referred to hereafter as “saturation”) generally refer to the difference of a color from achromatic colors, such as black, white, and gray. Until recently, saturation had been neglected in studies on color naming. Seminal studies e.g.,  used a sIn fact, the variation of saturation across the classical set of Munsell chips predicts the cross-cultural regularities in color naming observed in those seminal studies . It coulIn their recent study, consensus), and the frequency of observer pairs that produced any color term rather than answering “Don’t know”  = 0.68 and r(21) = 0.74, both p < .001). With a toolbox for robust correlation analyses (p < .001).Results of the correlational analyses are illustrated in analyses , I shower = 0.39 and r = 0.41, both p = .03 in a one-tailed t test). Nameability of English observers was at ceiling .Lindsey and colleagues also report data for Somali and English observers as control groups. Somali observers (These correlations suggest that the motifs Lindsey and colleagues observed in the Hadzane color lexicon are possibly due to a peculiarity of the stimulus set rather than reflecting universal constraints on color term evolution. This finding highlights the importance of controlling perceptual parameters of stimuli when investigating cognitive phenomena, such as language. This is particularly true for saturation, which has been widely neglected as a dimension of color stimuli in studies on color naming. With respect to the origin of color categories, the question about the evolution of color terms is still open until regularities across languages can be revealed with unbiased stimuli. To exclude the possibility that observed cross-cultural patterns in color categorization are merely due to a peculiarity of the stimulus sample, it is important to investigate these patterns with sets of color stimuli in which the prototypes of English color terms are not particularly saturated and salient.


Excessive drinking accounted for approximately 4,300 deaths each year among persons aged <21 years during 2006–2010,The national YRBS is a cross-sectional, biennial school-based survey of 9th–12th grade students in U.S. public and private schools that monitors the prevalence of health risk behaviors. During 1991–2015, a three-stage cluster sample design was used to select nationally representative samples of students; sample sizes ranged from 10,904 to 16,410. During each cycle, students completed an anonymous, self-administered questionnaire. Response rates ranged from 60% to 71%. Data were weighted to account for oversampling of non-Hispanic black and Hispanic students and nonresponse, and to produce national estimates of health risk behaviors among U.S. high school students who attend public or private schools. Details of the YRBS methodology have been published previously.Current drinking was defined as consuming one or more alcoholic drink on ≥1 days during the past 30 days. Binge drinking was defined as consuming five or more alcoholic drinks in a row on ≥1 days during the past 30 days. The usual source of alcohol and binge drinking intensity also were assessed. The prevalence of current drinking was calculated among students overall. The prevalence of binge drinking was calculated among students overall and current drinkers. The usual source of alcohol and binge drinking intensity were calculated among current drinkers only.Data from 1991–2015 were used to examine trends in current drinking, binge drinking, and binge drinking among current drinkers, adjusted for sex, race/ethnicity, and grade. Trends were analyzed using logistic regression models and interaction terms. Time was modeled as a continuous variableThe overall prevalence of current drinking among U.S. high school students declined significantly from 50.8% in 1991 to 44.7% in 2007, then further declined to 32.8% in 2015 . Trend aThe prevalence of binge drinking among current drinkers increased significantly from 62.2% in 1991 to 66.6% in 1997, then declined significantly to 57.8% in 2015 . Among mIn 2015, the prevalence of current drinking increased significantly with school grade from 23.4% among 9th grade students to 42.4% among 12th grade students, as did the prevalence of binge drinking, which was 10.4% among 9th grade students and 24.6% among 12th grade students . SimilarIn 2015, 36.4% of binge drinkers and 55.7% of current drinkers who did not binge drink usually obtained alcohol from someone who gave it to them. Binge drinkers were more than three times more likely than current drinkers who did not binge drink to give someone money to purchase alcohol (30.7% compared with 8.8%) and to purchase alcohol themselves (8.8% compared with 2.8%). Among binge drinkers, 43.8% consumed eight or more drinks in a row. Among binge drinkers, the prevalence of consuming eight or more drinks in a row was significantly higher among male (50.5%) than female (35.3%) students.Overall, current and binge drinking declined significantly among U.S. high school students from 1991 to 2015.Other national surveys have also reported declines in current and binge drinking among high school–aged students since the 1990s, although specific prevalence estimates vary  might also help reduce underage drinking  were also binge drinkers, and more than two in five binge drinkers consumed eight or more drinks in a row.Despite progress, current and binge drinking remain common among high school students, and many students who binge drink do so at high intensity . Widespread use of evidence-based prevention strategies for excessive drinking  could help reduce underage drinking and related harms.


Artemisia cina's main constituent. However, due to its toxicity to humans, it is no longer in use. Kazakhstan is looking to introduce this plant as an anthelmintic drug for veterinary purposes, despite the known toxic properties of the santonin. The objective of this study was to develop a fast and specific method for the identification of santonin and its precise quantitation using HPLC-UV in order to avoid unnecessary intoxication, which is paramount for the development of veterinary medicines. The results obtained showed that santonin appears at around 5.7 minutes in this very reliable HPLC method. The validation of the method was performed by the investigation of parameters such as precision, accuracy, reproducibility and recovery. The method was used to identify and quantify santonin in leaves of A. scoparia, A. foetida, A. gmelinni, A. schrenkiana, A. frigida, A. sublesingiana, A terra-albae, and A. absinthium from Kazakhstan as well as in three different extracts of leaves of A. cina. This study has provided a faster and simpler method for the identification and quantification of this compound in other species of Artemisia of economic importance.Santonin, a powerful anthelmintic drug that was formely used to treat worms, is  Artemisia cina is a very important plant as it has high economic significance in medicines, fodder, food, ornamentals or is also used as soil fertilizers in habitats that have been disturbed. It is an endemic plant of South Kazakhstan. It is a grey-green shrub with numerous woody stems at the base, with thrice pinnately dissected leaves and small erect flowers from the tubular yellow flowers, which are usually collected when in bloom +; 1H-NMR  δH: 1.01 . Samples were re-suspended in 10 ml of methanol prior to be analysed by HPLC.The leaves of the seven other About 1mg of santonin was accurately weighed out in a glass vial and dissolved in 1ml of acetonitrile.About 10mg of the supercritical CO2 extract was weighed out in a glass vial and dissolved in 5ml of chloroform and 5ml of acetonitrile. The vial was then placed in a water bath at 60oC to ensure that everything was dissolved to avoid particles present to block the column and cause the machine to clog. To ensure that there were no particles suspended in the solution, each sample was filtrated using a 0.45mm filter (Nylon66) before placement into the HPLC machine.Ten microlitres of the samples and standard solutions were injected in quadruplicates and eluted using the following gradient at a flow of 1.0ml/min with water (A) and acetonitrile (B): 0min, 35:65 (A:B); 5min, 35:65; 10min, 45:55; 15min, 55:45; 20min, 65:45. According to , the mact-test was used to investigate the intercept and the regression coefficient. Both the calibration line and the residuals were clearly examined and assessed.Reference solutions were prepared by diluting santonin with acetonitrile in the following concentrations: 1250μg/ml; 625μg/ml; 312.5μg/ml; 156.25μg/ml; 78.13μg/ml; 39.06μg/ml; 19.53μg/ml; 9.77μg/ml; 4.88μg/ml. These solutions were analysed in quadruplicates (n = 4) using the conditions already described. These results allowed for a calibration curve to be constructed. The Student’s Eleven replicate samples were made fresh for each day using the methods mentioned above and these were analysed on three separate days on the same equipment, by the same analyst to ensure that the technique could be replicated and also to reduce the chances of errors made if the experiment was carried out by multiple people.The recovery experiment was carried out to investigate the accuracy of this method for santonin. The samples were prepared using concentrations of santonin at 1250μg/ml, 625μg/ml and 312.5μg/ml and each of these were mixed with 1mg/ml of the CO2 extract solution. The ratio of the samples prepared in was 1:1 santonin and CO2 extract, respectively. These solutions were injected into the HPLC machine and the recovery was calculated by comparing what the theoretical value of santonin should be and what the value actually was from the experiment, expressed in percentage.Each sample of the different species was investigated against santonin using the same method devised in this study.A HPLC method for the analysis of santonin was devised using parameters interpreted from previous publications , 14. TheA calibration curve was made  using thCalibration curves for santonin at 9 different concentrations  in replicates of four (n = 4) were run in four different days. In all graphics, the results were very similar and the R2 value obtained was always above 0.99 indicating that the results were significant. The calibrations curves were used for the quantification of santonin later in the studied samples.Figs Upon investigating the quantity of santonin at approximately 5.7 minutes in the HPLC of these extracts,there were significant differences in the quantity of santonin extracted using the two different techniques with the santonin quantity in the supercritical CO2 being 3 times higher than that of the santonin from the chloroform extract . The conArtemisia genus or from any other plants that could contain santonin. The development of this HPLC method was based on previous papers and studies on santonin and sesquiterpene lactones as there has been little research carried out with A. cina. Miraldi et al. (1998) devised a new method to extract and quantify santonin using High Performance Liquid Chromatography in Artemisia caerulescens ssp. cretacea (Fiori) Br.-Catt. & Gubell.As it is known that the major compound santonin has potential toxicity , 6, 7, 8et al. made modifications to previous methods in an attempt to devise a faster, easier and more accurate extraction and HPLC analytical procedure. The mobile phase consisted of an isocratic elution with acetonitrile: water (4: 6) for the duration of 10 minutes (a flow rate from 0.5 mL/min to 0.8 mL/min). This was followed by a 5 minute linear gradient of acetonitrile: water from 4:6 to 6:4 with a flow rate from 0.8 mL/min to 1.0 mL/min. and then back to an isocratic elution with acetonitrile: water (6:4) for an additional 15 minutes (flow rate 1.0 mL/min). The UV absorbance was monitored at 236nm. The injection volume was 20 μL in all the experiments and all their analyses were conducted at room temperature. Their calibration curve was made by using the injections of five different concentrations of santonin varying from 0.00625g to 0.1 g. At each concentration, the values obtained were acceptable .In their experiment Miraldi et al. (1998).Their results showed santonin to have a retention time of approximately 14 minutes. By using the method devised in this study, the retention time for santonin fell to less than 6 minutes, more than 8 minutes faster than the method devised by Miraldi Upon investigation the accuracy and precision of the method devised in this paper, the recovery of santonin from the supercritical CO2 extract was over 95% while its recovery from the Chloroform extract was over 90%. This confirms the validity of this method, which can also be used to examine santonin presence in other species.Artemisia species were quantified for santonin using this method. The calibration curve previously obtained shows a valid regression line y = 25705x - 1540 (R2 = 0.99) which was used for the quantification of the plant species listed in To finalize our validation, seven other Artemisia cina possess the most santonin (1.96g/100g leaves) and several other species show no santonin present. Most of the other species with close to zero or zero santonin content were traditionally used as oral medications in some countries and are still in practise now. A. gmelinii leaves were used in south and south-east Asia to treat inflammatory liver conditions [A. absinthium was used by the indigenous as an antispasmodic, for the restoration of declining mental function, febrifuge, anthelmintic and inflammation of the liver, and to improve memory [A. scoparia is well known in Pakistan to treat pain, inflammation and febrile conditions [A. frigida is commonly used as in folk medicine to treat joint swelling, sore carbuncle, and abnormal menstruation [It is clearly shown in nditions . A. absie memory . A. scopnditions . In Mongtruation . It is i2 extractions are more expensive than those using solvents such as chloroform or ethanol. In this case, as seen in 2 was 250.40μg/ml. These results report a clear difference in the quantity of isolated santonin using the different methods of extraction. They also provide useful information for selecting extraction techniques based on the quantity of santonin allowed in the extract for medicinal purposes (veterinary medicine).It was also of interest to see if there were any differences in the quantity of santonin isolated using different extraction techniques. This is because techniques such as supercritical and subcritical COHowever many difficulties are present when attempting to quantify compounds in a plant because their contents are affected by many variables. For example, it is common to observe diurnal variations in plant secondary chemical components  as well A. cina to be reintroduced in Kazakhstan as a veterinary medicine is dependent on the quantity of santonin in the plant to have effect but with minimum toxic activity. This registration of veterinary medicines does not require as much specialized expertise and examinations compared to human medicines. Although the toxicity for santonin at the doses of 60mg for children and 200mg for adults is well documented, we also know that the LD50 for mammals is in the range of 900mg/kg [A. cina.The potential for 900mg/kg , which c


Escherichia coli (DEC) and non-typhoidal Salmonella (NTS) are major intestinal pathogens in developing countries, and the indiscriminate use of antibiotics has greatly contributed to resistant strains. Hence, the aim of the present study is to identify the antimicrobial resistance patterns and the molecular characteristics of DEC and NTS in southwest, China.Bacterial diarrhea is one of the most common causes for medical consultations, mortality and morbidity in the world. Diarrheagenic 1121 diarrheal patients and 319 non-diarrheal subjects across all age groups were recruited from four sentinel hospitals from June 2014 to July 2015 in Kunming City, Yunnan Province. Each stool specimen was collected to detect DEC and NTS with standard microbiological and molecular methods. Antimicrobial resistance testing was performed with the Kirby-Bauer disk diffusion method, and the standards for antimicrobial susceptibility testing complied with the Clinical and Laboratory Standards Institute (CLSI). Molecular characterization of strains was carried out using pulsed-field gel electrophoresis (PFGE). A structured questionnaire was used to record basic epidemiological data . Data were analyzed using Chi-square or Fisher’s exact test.χ2 = 20.69, P < 0.001, OR = 4.36, 95% CI: 2.19–8.65), and the prevalence of NTS isolated from diarrhea cases was higher than that of non-diarrheal cases across all age groups . The rates of resistance to ten antibiotics of DEC and NTS showed significant differences . The rates of resistance to Amoxicillin and Clavulafiate (AMC), Cephalothin (CEP), Gentamicin (GEN) and Sulfamethoxazole-Trimethoprim (SXT) of DEC isolated from diarrhea cases were higher than those of NTS isolated from diarrhea patients . Ciprofloxacin (CIP) was the most sensitive antibiotic for DEC and NTS strains isolated from diarrhea cases. Resistance rates of DEC isolates from cases and controls to more than three kinds antimicrobials  showed no significant differences . Pulsotype patterns of DEC strains were highly diverse; however, the pulsotype pattern of NTS strains was closely related to the serotype. The pattern of S. enteritidis was highly similar, but the S. enterica Typhimurium strain was discrete.DEC was detected in 127 (11.33%) diarrhea cases and 9 (2.82%) non-diarrheal cases  contains supplementary material, which is available to authorized users. Please see Additional file Acute diarrheal illness is still a major public health problems resulting in medical consultations, mortality and morbidity worldwide, especially in low- and middle-income countries –3. In adEscherichia coli (DEC), non-typhoidal Salmonella (NTS), Shigella spp. and Vibrio cholerae, among others. In China, annually, 70 million cases of infectious diarrhea are reported , 1 μl reverse primer (10 μmol), 1 μl DNA template and 7 μl H2O. The cycling conditions for each subtype DEC were 95 °C for 5 min, 40 cycles of 95 °C for 5 s, 60 °C for 30 s. Fluorescence was recorded at the annealing stage. In addition, these ten putative DEC colonies were also inoculated in nutrient agar media to culture a single strain at 37 °C for 18 h and to obtain single colonies. If the qPCR of the putative DEC colony was positive, a colony from the single strains was chosen and DNA extracted. Single primes were selected to detect the subtype of DEC [Salmonella-Shigella agar  to detect NTS. Systematic biochemical identification of NTS was performed using the VITEK® 2 Compact instrument . When NTS was determined to be positive, further serological testing was used to identify the subtype. Detailed detection procedures can be found in the references [The DEC strain was divided into five subtypes by their virulence genes as follows: enteroaggregative (10 μmol), 22, 1 μ(10 μmol), 22, 1 μferences , 25.E. coli ATCC 25922 was selected as the control strain.The Kirby-Bauer disk diffusion method was used to detect antibiotic susceptibility of enteric bacterial pathogens. These antimicrobials were ampicillin , amoxicillin/clavulanic acid AMC, 20/10, 10 μg), cefotaxime (30 μg), cephalothin , gentamicin , nalidixic acid , tetracycline , ciprofloxacin , rifampicin  and Sulfamethoxazole-Trimethoprim . DEC and NTS were cultured at 37 °C for 18–24 h, and the ring size was measured to judge the antibiotic resistance according to the Clinical and Laboratory Standards Institute  of the United States . The digestion condition of each plug was 45 U XbaI at 37 °C for 2 h. The CHEF - Mapper  was used for electrophoresis, and electrophoresis conditions for DEC and NTS were 6.76 s–35.38 s and 2.16 s–63.80 s for 19 h, respectively. A Bio-Rad Gel Doc XR system  was used to observe and record the gel results. Detailed detection procedures are found in previous studies [PFGE was conducted to assess clonal-relatedness in accordance with the PulseNet protocol procedure for NTS and  studies .OR) and 95% confidence intervals (CIs) of categorical variables were calculated using two-tailed Chi-square or Fisher’s exact tests. Significant differences were taken as the level of P < 0.05 for two-tailed tests. The PFGE patterns of DEC and NTS were analyzed with BioNumerics 5.10 software . A dendrogram was constructed using the Dice similarity coefficient with 1.00% optimization and a tolerance coefficient and un-weighted pair group methods with the arithmetic mean algorithm (UPGMA).Data analysis was performed by Statistical Product and Service Solutions (SPSS v24.0) software . Odds ratio , and the prevalence of NTS isolated from cases was higher than that of the controls across all age groups . In diarrhea cases, EPEC  was the most common subtype of DEC, followed by EAEC , EIEC  and ETEC . In addition, S. enteritidis  was the most common subtype of NTS in cases, followed by S. enterica Typhimurium .In total, 1121 diarrhea cases and 319 non-diarrheal cases were recruited into this study from June 2014 to July 2015. DEC was detected in 127 (11.33%) diarrhea cases and 9 (2.82%) controls . The resistance of DEC to AMP, NAL, TCY, REP and SXT was highly prevalent . Resistance to AMP, NAL, TCY, REP and SXT was also highly prevalent , but the resistance rates of the subtypes of DEC to AMP, AMC, CEP, CTX, GEN, CIP, TCY, REP and SXT were not significantly different.Many of the enteric bacterial pathogens isolated from diarrhea cases were widely resistant to antibiotics in Kunming City Table . The ratχ2 = 191.16, P < 0.001). Resistance to AMP, NAL, TCY and REP was also highly prevalent . The resistance rates of S. enteritidis and S. enterica Typhimurium to AMP, NAL, TCY and REP was very serious , but the resistance rates of the subtypes of NTS to AMC, CEP, CTX, GEN, CIP, TCY, REP and SXT were not significantly different.The rates of resistance to these ten antibiotics of NTS were significantly different , CEP , GEN , TCY  and SXT  were significantly different. The resistance rate of DEC to AMC, CEP, GEN, TCY and SXT were more serious than the resistance rates of NTS to those antibiotics, but the resistance rate of DEC and NTS to AMP, CTX, NAL, CIP and REP was not significantly different. In diarrheal cases in patients less than 5 years of age, the resistance rate of DEC and NTS strains to AMC , CEP , GEN , TCY  and SXT  were significantly different, but there were no significant differences among the resistance rates of DEC and NTS to AMP, CTX, NAL, CIP and REP. In diarrheal patients over 5 years of age, the resistance rate of DEC and NTS to those ten antibiotics were not significantly different.In diarrheal patients, the resistance rate of DEC and NTS strains to AMC , CEP  and CTX  were higher than those of the DEC strain isolated from patients over 5 years of age. The resistance rate of DEC isolated from cases under 5 years of age and over 5 years of age to AMC, GEN, NAL, CIP, TCY, REP and SXT were not significantly different. The resistance rate of NTS strains isolated from patients less than 5 years of age and over 5 years of age to these ten antibiotics were not significantly difference.In diarrhea patients, the resistance rate of DEC isolated from cases under 5 years of age to AMP . For the other nine antibiotics, the resistance rate of DEC strains isolated from diarrheal cases and subjects were not significantly different. For subjects under 5 years, the resistance rate of DEC strains from diarrheal patients were not significantly different from those resistance rates of DEC strains isolated from controls. In subjects over 5 years, the resistance rate for CIP of DEC isolated from diarrhea patients was lower than that of controls , and the resistance rates of DEC strains isolated from cases and controls for the other nine antibiotics were not significantly different.As shown in Table n = 22, 17.32%) or six  of the most common antimicrobials, followed by only seven types of antimicrobials  and only four types of antimicrobials . No DEC strains from cases were resistant to ten types of antimicrobials. In addition, DEC strains isolated from controls showed relatively high resistance to only six types of antimicrobials , followed by only nine types of antimicrobials . Resistance to only one, two, seven or eight types of antimicrobials was not found. The resistance rate of DEC isolated from diarrhea cases to only nine types of antimicrobials was lower than that of DEC isolates from controls , and the resistance rates to only one, two, three, four, five, six, seven, eight or ten types of antimicrobials were not significantly different for DEC isolated from diarrhea cases and controls. Resistance rates of DEC isolates from cases and controls to more than three types of antimicrobials were not significantly different .DEC isolates from diarrhea patients were resistant to only five (P = 0.047), and the resistance rate of DEC strains to only three antibiotics or four antibiotics were lower than those of NTS to only three antibiotics or four antibiotics . For diarrhea cases under 5 years of age, the resistance rate of DEC strains to only one antibiotic or four antibiotics were all lower than those of NTS to only one antibiotic or four antibiotics . The MDR resistance rate of NTS among diarrhea cases under 5 years of age and over 5 years of age were not significantly different.In diarrhea patients across all age groups, the resistance rate of DEC to only one type of antibiotic was lower than that of NTS to only one type of antibiotic  and NTS (9.52%) to CTX is concerning, and should not be ignored. It may be that this resistance strain to third-generation cephalosporin produces ESBL, an enzyme that confers resistance to cephalosporin antibiotics and oxyimino-β-lactam synthetic drugs, as well as to other penicillin drugs. ESBL can be inhibited with clavulanic acid . With inThe resistance rates of DEC and NTS in this study were similar to those in a previous study in China , but werIn both diarrheal and non-diarrheal subjects, the MDR rates of DEC and NTS were over 75.00%, a finding similar to other studies , 46. It S. enteritidis strains showed almost identical characteristics, but there was no connection between any two diarrhea cases based on epidemiological investigation. It can be concluded that S. enteritidis formed a dominant strain in local populations in recent years. Patterns of S. enterica Typhimurium strains showed discrete characteristics suggesting that the preponderant strain was not local, as seen in other studies [The molecular pattern of DEC were highly polymorphic, as found in another study . This ma studies , 49. TheSeveral limitations of this study can be noted. First, antibiotic resistance genes (ARGs) have important associations with strains of phenotypic resistance, but ARGs were not detected in this study. Hence, the relationship between ARGs and phenotypic resistance was not revealed. Second, the strains producing carbapenemases and ESBL were not detected, which was obstructing treatment to some extent. Third, the serotypes of the DEC strains were not detected, and thus the correlation between DEC serotypes and DEC pulsotype patterns were not clear. Therefore, further research including ARGs, phenotypic resistance and ESBL should be conducted in the future.Antibiotic resistance of bacterial strains was very significant, and multidrug-resistant strains were widely prevalent in diarrheal children. Hence, it is urgent to regulate antibiotic use. Combining the study of the molecular characteristics of enteric bacterial pathogens with systematic epidemiological investigation can provide accurate evidence to track the source of infections.Additional file 1:Multilingual abstract in the five official working languages of the United Nations. (PDF 838 kb)Additional file 2:Raw data on antimicrobial microbial resistances used in this work. (SAV 35 kb)


Concern is growing regarding the prospects of achieving the global elimination of lymphatic filariasis (LF) by 2020. Apart from operational difficulties, evidence is emerging which points to unique challenges that could confound achieving LF elimination as extinction targets draw near. Diethylcarbamazine (DEC)-medicated salt may overcome these complex challenges posed by the endgame phase of parasite elimination. We calibrated LF transmission models using Bayesian data-model assimilation techniques to baseline and follow-up infection data from 11 communities that underwent DEC salt medication. The fitted models were used to assess the utility of DEC salt treatment for achieving LF elimination, in comparison with other current and proposed drug regimens, during the endgame phase. DEC-medicated salt consistently reduced microfilaria (mf) prevalence from 1% mf to site-specific elimination thresholds more quickly than the other investigated treatments. The application of DEC salt generally required less than one year to achieve site-specific LF elimination, while annual and biannual MDA options required significantly longer durations to achieve the same task. The use of DEC-medicated salt also lowered between-site variance in extinction timelines, especially when combined with vector control. These results indicate that the implementation of DEC-medicated salt, where feasible, can overcome endgame challenges facing LF elimination programs. Despite the impressive expansion of the program since its inception in 2000, with 63 LF endemic countries currently under MDA2, delays in program implementation, issues with compliance, and contraindications of the two major drugs used in annual MDA, viz. ivermectin (IVM) and diethylcarbamazine (DEC), in some areas of Sub-Saharan Africa, suggest that the global elimination of LF is unlikely to be achieved by 2020 without rapid scale-up and/or via the discovery and application of more effective intervention technologies5.In 1997, the World Health Assembly (WHA) adopted resolution WHA 50.29 calling for the elimination of the mosquito-borne macroparasitic disease, lymphatic filariasis (LF), as a global public health problem. This lead to the establishment of the Global Program to Eliminate Lymphatic Filariasis (GPELF) by the World Health Organization (WHO) in 2000, which advocated the meeting of this goal by 2020 by all endemic countries through the application of annual mass drug administration (MDA) maintained over at least 4–6 years6, and cases in southern India, Nigeria, and Tanzania report that 8–10 years of annual MDA treatments have not halted transmission9. These slowed down but persistent low-level LF transmission regimes near elimination points could reflect the emergent effects of a range of factors, such as (1) the operation of diverse density dependent feedback loops that can maintain transmission resilience in the face of perturbations10, (2) the impacts of diversity and modularity in the transmission process by involving two hosts and heterogeneous mosquito larval habitats that can contain the effects of perturbations locally, and (3) the inter-connectedness of host and vector populations over space and time that can sustain regional transmission even when local parasite extinction in individual sites is achieved14. In addition to these ecological shifts, operational challenges affecting control programs can also be amplified during the late stages of elimination. For example, achieving effective coverage in hard-to-reach communities, managing costs of surveillance at low infection levels, and preserving the commitment of international and local organizations become increasingly critical16. With less than three years remaining for the GPELF to meet its 2020 target, these findings indicate that it is crucial not only to scale up programs and focus on developing new tools to accelerate the meeting of elimination targets, such as is being investigated with triple drug therapy17, but also to update program strategies to appropriately manage these complex, emergent challenges that are uniquely posed by the endgame phase of parasite elimination15.Evidence, in the meantime, is also emerging in countries that have already received multiple annual MDAs, which points to the unique challenges that could confound achieving LF elimination as parasite extinction targets near. First, post-MDA surveillance studies reporting persistent microfilarial (mf) infections after many years of treatment efforts draw attention to the shifting dynamics of LF near transmission thresholds. For instance, three decades of treatment on the remote island of French Polynesia was not able to eradicate LF25. Perhaps most famously, DEC-medicated salt was used in the vast majority of endemic regions of China starting in the 1980s after the first pilot trials in Tengxian County, Shandong Province, yielded impressive results. Following three years of treating positive cases with DEC tablets, elimination of LF was expedited by adopting community-based DEC-medicated salt treatment for six months. Community mf prevalence was reduced from 8.89% to 0.63% during the salt treatment and no new infections emerged during the next 10 years27. Several additional trials followed subsequently in other provinces with similar results. Eventually comprising 73.9% of total treatments, DEC-medicated salt contributed considerably to the elimination of LF  from all endemic provinces in China26.DEC-medicated salt has played a major role in the elimination of LF in a limited number of settings in Africa, Central America, and Asia28. While generally medicated salt with 0.3% concentration DEC was administered for a period of time based on the mf rate and density reflected in surveys, the duration of treatment ranged from 3–6 months and the dosage ranged from 4.5 to over 9 grams DEC per person. Furthermore, in some areas, several intermittent 3-month courses of DEC-medicated salt were required to reduce infection. While salt was strictly controlled by health authorities in some regions, it was not feasible in others and was instead added to food products such as soy26. Finally, vector control and sanitation efforts were implemented in addition to chemotherapy in some regions28. This high variation between treatment regimens has made it difficult to extract a clear-cut strategy framework with respect to DEC-medicated salt from the success of the salt programs in China.However, because provinces in China adapted their strategies differently in response to evolving research, the DEC-medicated salt regimen used to control LF was not uniform29. A Bayesian data-model assimilation approach was used to fit the model to longitudinal infection data observed in 11 study communities which underwent DEC salt medication, and the resulting calibrated models were used to assess the utility of using salt treatment for achieving LF elimination once infection was reduced to low levels. The results were also used to assess the impact of DEC medication as a strategy for addressing the challenge of heterogeneous LF transmission dynamics between sites during the elimination phase4. The performance of DEC-medicated salt as an endgame strategy in comparison with other drug regimens was also systematically evaluated in relation to variations in drug coverage and intensity of supplementary vector control (VC).Here, we undertook a modelling study to systematically evaluate the utility of using DEC-medicated salt as an endgame strategy for achieving LF elimination by comparing its impact, relative to other current and proposed drug regimens, on the dynamics of parasite extinction during the endgame stage of elimination. The analysis was based on extending our recently developed dynamical LF transmission model to allow for the continuous application of DEC in the form of medicated salt30. The results show that the Bayesian Melding (BM)-based data assimilation fitting procedure which relied on the pass/fail binary fitting criterion was able to reproduce the baseline mf infection data for all 11 sites when compared to either the constructed or observed age prevalences  identified for each study site based on their fits to baseline age-stratified mf prevalence data, either directly observed (squares) or constructed (circles and crosses), are shown in Fig. ω, and mf kill rate, ε, pertaining to DEC-medicated salt were estimated from their posterior parameter distributions derived from the parameter vectors that passed through both the baseline (observed/constructed) and follow-up overall mf prevalence data for each site. While there have been studies inferring the rate of mf clearance due to DEC-medicated salt33, there have been limited corresponding studies inferring the kill rate of adult worms and they report discrepant results33. The posterior distributions of ε and ω for two of the study sites, Kwemwale/Nkumba, Tanzania, and Miton, Haiti, are shown in Fig. p-values < 2.2e–16), suggesting the existence of significant between-site heterogeneity in applied salt drug efficacies. The median values of estimated monthly worm and mf kill rates are tabulated for all villages in Supplementary Table For each of the 11 sites, the monthly worm kill rate, 29. The estimated between-site breakpoint distributions at both the ABR and at TBR statistically differed . The 50%, 75%, and 95% elimination probability (EP) thresholds as derived via the application of an inverse cumulative density-based exceedance function to these site-specific breakpoint distributions are given in Supplementary Table 34. Typically, mf breakpoint values were lower at ABR (0.014% to 0.098% across sites) than at TBR (0.047% to 1.388%), and declined progressively with increasing EP probabilities (to as low as 0.014% at the 95% EP at ABR). In this study, the 95% EP threshold was considered the target endpoint when evaluating the effect of intervention strategies. Values of this threshold at ABR were used as targets for assessing parasite transmission elimination in the case of drug interventions, whereas values at TBR were used when the added effects of VC were modelled29.Distributions of model-generated mf breakpoints at the baseline annual biting rate (ABR) and at the threshold biting rate (TBR) were calculated for each site in this study using the numerical stability analysis method described previouslyIn this exercise, we evaluated and compared the impact of DEC salt as an endgame intervention strategy in relation to the performance of the other currently used or proposed LF drug interventions. An endgame strategy here refers to the use of a treatment regimen and/or VC only after annual MDA has decreased the community-level mf prevalence below the WHO-mandated threshold of 1% mf. Figure p < 0.0001). A post-hoc Tukey test confirmed the impression from Table p < 0.0001, Supplementary Table p < 0.0001, Supplementary Table p < 0.001, Supplementary Table Figure 35. DEC-medicated salt was consistently shown in this modelling study, where a Bayesian statistical approach was used to calibrate and learn LF process-based mechanistic models based on data from diverse sites ) or via the introduction of DEC-fortified salt into a competitive market as is being done in the case of Guyana48 and Haiti50. This suggests that regional or country-specific research into the necessary regulatory environment, partnerships with salt producers and importers, social marketing, learning from national salt iodization programs, and expansion of the skills of public health managers to manage fortified salt distribution will be required if the strategy is to be effectively deployed48. The second major barrier is the use of the salt intervention in areas of Sub-Saharan Africa co-endemic with onchocerciasis51. The primary issue here is the contraindicated use of DEC in these areas due to risk of inducing Mazzotti reactions and adverse ocular events in persons positive for Onchocerca volvulus51. While this may present a major issue, it is pertinent to note that the low dosage of DEC salt administered through fortified salt coupled with low prevalence of onchocerciasis (as is expected in many current co-endemic regions in Africa due to long-term ivermectin MDA) could lessen this problem enough to support its deployment. This rationale, for example, underlies the growing interest in conducting randomized control trials for assessing the potential value of using triple drug therapy with IDA in these areas51. These considerations, along with the modelling results presented here, firstly indicate a need for policy makers to re-appraise the use of DEC-medicated salt in LF control programs during the endgame phase, and secondly support conduction of urgent field and operations research into its possible deployment in settings where it is deemed to be programmatically feasible.In summary, we have presented a simulation study rooted in field data which indicates that using DEC-medicated salt even at moderate coverage may offer a potent intervention option to disrupt LF transmission efficiently and rapidly during the endgame phase compared to currently used or proposed MDA-based drug administration approaches. Including VC will further enhance this differential effect of community salt treatment. Our results are robust to data limitations and to the transmission heterogeneities that may govern LF extinction dynamics in the field. Although these results indicate a high prognosis for the use of DEC salt as an effective endgame intervention for eliminating LF transmission, major barriers to its programmatic use exist and need to be sufficiently resolved if its potential is to be realized. As highlighted by Lammie 25. The data comprised pre-treatment baseline infection prevalence in each community determined using blood surveys for mf prevalence prior to the introduction of DEC-medicated salt , microfilarial intensity (M), and a measure of acquired immunity (I) to the parasite moderated by the total worm load (P + W = WT) are formulated as partial differential equations over time (t) and host age (a). In the mosquito host, the burden of infective L3 stage larvae (L) is described by an ordinary differential equation. The model is genus-specific with respect to the uptake of microfilariae by the mosquito vector. In this analysis, the functional form for mosquitoes of the Culex genus was applied. The model parameters and functions are fully described in the Supplementary Information (SI)  algorithm in order to select N  parameter vectors, θ, or models applicable to a site based on their likelihood for describing the observed local data34. However, as noted by Raftery and Bao, SIR may not perform well in all cases53. In the current application, for example, the SIR algorithm selected very few unique parameter vectors because there were a handful of such vectors with high likelihoods given the data. These parameter vectors were consequently sampled many times resulting in a sparsely populated posterior parameter space. Bayesian techniques which bypass the evaluation of likelihoods, such as the use of a binary criterion of fit or model selection via Approximate Bayesian Computation (ABC), offer alternative methods to judge a model simulation based on its ability to mimic observed features of data54. For this work, the binary criterion of fit (pass/fail) proposed by Spear and Hubbard54 was implemented to address this problem using the algorithm below, wherein model generated mf prevalences, M¸ were judged against the observed age-stratified mf prevalence data, D, and N parameter vectors were selected only when their predicted age-prevalences fell within the 95% confidence intervals of the age-stratified data for j-2 data points :We used a data-model assimilation technique based on the Bayesian Melding (BM) algorithm to calibrate and identify localized LF transmission models based on the mf prevalence data observed in each of our study sites. This was done by “melding” the observed baseline mf data from each site with model-generated outputs in order to learn models describing the localized parasite transmission dynamics per site47. The derived age-prevalence infection data were then used in the model fitting procedures described above, effectively allowing the integration of partially observed data into the model. See the SI for details  the national demographic profile applicable to the site in question, and 2) by conversion of the community-level mf prevalence to reflect either a plateau or concave age-infection profile known typically to occur in LF endemic regions34. Drug efficacy parameters quantified in this study thus reflected the monthly pre-patent and patent worm kill rate, ω, mf kill rate, ε, and a period of months, p, for which the newly produced mf continue to be killed after the ith administration of the drug at time TMDAi. These rates are applied in such a way that the total population burden of worms and mf are adjusted according to the observed drug coverage, C29, where s is the worm sex ratio, α is the mf production rate, φ is the worm mating probability, and γ is the mf death rate  primarily in that a small dose of DEC is consumed daily as opposed to a larger dose applied once or twice annually. In the case of annual or biannual MDA, the use of DEC is modelled by assuming an instantaneous killing effect on mf and worms at the time of ingestion. However, in the case of daily consumption, there is a continuous input of the drug at low concentration on a daily basis, suggesting a more frequent pulsing or spacing of discrete treatments than in the case of annual or biannual MDA. While it is possible to model DEC salt application using continuous functions, here, we approximated the daily drug intake instead as a monthly pulse where impacts on mf and adult worms are aggregated to reflect clearance rates per month. This was done to facilitate consistency with the monthly scale of the other parameters employed in the modelω and ε were randomly selected from a range of plausible prior values using latin hypercube sampling and assigned to each of the model fits achieved using the BM procedure described above. This was repeated 500 times to derive a total set of 500 × 500 parameter vectors, each vector now containing its own quantified drug efficacy parameters. Second, simulations of the effects of monthly DEC salt treatment were run for each vector to obtain 500 mf prevalence trajectories forward in time per vector. Finally, all models whose mf trajectories arising from the monthly DEC salt intervention that passed through the 95% binomial confidence intervals of all but one follow-up survey data points in a site were selected, and their drug parameter values used to generate the corresponding site-specific distributions of values for ω and ε.Site-specific monthly worm and mf kill rates as a result of DEC salt medication were estimated as follows. First, values of viz. 1) DEC+ALB, and 2) IDA, were modelled in this study to provide a comparison with the effects of DEC salt medication. The use of these drugs in MDA programs are modelled as described above with TMDAi occurring annually or biannually with regimen-specific efficacy values40. To evaluate the added effect of VC as a supplement to drug-based interventions, the impact of long-lasting insecticidal bed nets with three main actions against mosquito biting was modelled: 1) deterring the insects from entering the home, 2) inhibiting their ability to feed on humans, and 3) killing them56. The model formulation is described elsewhere29.Two alternative combination MDA regimens; 35, which relies on a bisection root-finding method for identifying transmission and worm breakpoint thresholds34.Site-specific transmission endpoints were calculated in this study as before using a numerical stability analysis approachCustom code used to generate the results of this paper can be made available by the authors upon request. The authors declare that the data used in support of this study and its findings are available within the paper and its Supplementary Information files.Supplementary Information