Coliphages as indicators of primary wastewater treatment efficiency by constructed wetlands.

Constructed wetlands are an efficient and cost-effective system for the treatment of wastewater that can be reused for diverse purposes, including irrigation; however, few studies have determined the efficiency of microbial removal by constructed wetlands in tropical regions. Therefore, the present study aimed to determine the microbial quality of the influent and effluent of a constructed wetland in Puerto Rico, using traditional bacterial indicators (i.e., thermotolerant coliforms and enterococci), as well as somatic and male-specific (F+) coliphages. Results showed that over 99.9 and 97.7% of thermotolerant coliforms and enterococci were removed after treatment by constructed wetlands, respectively. Notably, approximately 84.0% of male-specific (F+) coliphages were removed, while somatic and total coliphages exhibited differing removal percentages at different steps during treatment by constructed wetlands. The potential risk of the presence of enteric viruses in treated wastewater by constructed wetlands may increase when considering traditional bacterial indicators exclusively. The present study may aid in the efforts to determine public health concerns associated with the exposure of bioaerosols resulting from wastewater treatment by constructed wetlands.

Dietary fat composition shapes bile acid metabolism and severity of liver injury in a pig model of pediatric NAFLD.

The objective of this study was to investigate the effect of dietary fatty acid (FA) composition on bile acid (BA) metabolism in a pig model of NAFLD, by using a multiomics approach combined with histology and serum biochemistry. Thirty 20-day-old Iberian pigs pair-housed in pens were randomly assigned to receive 1 of 3 hypercaloric diets for 10 wk: 1) lard-enriched (LAR; n = 5 pens), 2) olive oil-enriched (OLI; n = 5), and 3) coconut oil-enriched (COC; n = 5). Animals were euthanized on week 10 after blood sampling, and liver, colon, and distal ileum (DI) were collected for histology, metabolomics, and transcriptomics. Data were analyzed by multivariate and univariate statistics. Compared with OLI and LAR, COC increased primary and secondary BAs in liver, plasma, and colon. In addition, both COC and OLI reduced circulating fibroblast growth factor 19, increased hepatic necrosis, composite lesion score, and liver enzymes in serum, and upregulated genes involved in hepatocyte proliferation and DNA repair. The severity of liver disease in COC and OLI pigs was associated with increased levels of phosphatidylcholines, medium-chain triacylglycerides, trimethylamine-N-oxide, and long-chain acylcarnitines in the liver, and the expression of profibrotic markers in DI, but not with changes in the composition or size of BA pool. In conclusion, our results indicate a role of dietary FAs in the regulation of BA metabolism and progression of NAFLD. Interventions that aim to modify the composition of dietary FAs, rather than to regulate BA metabolism or signaling, may be more effective in the treatment of NAFLD.NEW & NOTEWORTHY Bile acid homeostasis and signaling is disrupted in NAFLD and may play a central role in the development of the disease. However, there are no studies addressing the impact of diet on bile acid metabolism in patients with NAFLD. In juvenile Iberian pigs, we show that fatty acid composition in high-fat high-fructose diets affects BA levels in liver, plasma, and colon but these changes were not associated with the severity of the disease.

MULTI-OMICS as Invaluable Tools for the Elucidation of Host-Microbe-Microbiota Interactions.

"Omics" is becoming an increasingly recognizable term, even to the general public, as it is used more and more often in everyday scientific research [...].

Interaction of bacterial metagenome and virome in patients with cirrhosis and hepatic encephalopathy.

OBJECTIVE: Altered bacterial composition is associated with disease progression in cirrhosis but the role of virome, especially phages, is unclear. DESIGN: Cross-sectional and pre/post rifaximin cohorts were enrolled. Cross-sectional: controls and cirrhotic outpatients (compensated, on lactulose (Cirr-L), on rifaximin (Cirr-LR)) were included and followed for 90-day hospitalisations. Pre/post: compensated cirrhotics underwent stool collection pre/post 8 weeks of rifaximin. Stool metagenomics for bacteria and phages and their correlation networks were analysed in controls versus cirrhosis, within cirrhotics, hospitalised/not and pre/post rifaximin. RESULTS: Cross-sectional: 40 controls and 163 cirrhotics (63 compensated, 43 Cirr-L, 57 Cirr-LR) were enrolled. Cirr-L/LR groups were similar on model for end-stage liver disease (MELD) score but Cirr-L developed greater hospitalisations versus Cirr-LR (56% vs 30%, p=0.008). Bacterial alpha/beta diversity worsened from controls through Cirr-LR. While phage alpha diversity was similar, beta diversity was different between groups. Autochthonous bacteria linked negatively, pathobionts linked positively with MELD but only modest phage-MELD correlations were seen. Phage-bacterial correlation network complexity was highest in controls, lowest in Cirr-L and increased in Cirr-LR. Microviridae and Faecalibacterium phages were linked with autochthonous bacteria in Cirr-LR, but not Cirr-L hospitalised patients had greater pathobionts, lower commensal bacteria and phages focused on Streptococcus, Lactococcus and Myoviridae. Pre/post: No changes in alpha/beta diversity of phages or bacteria were seen postrifaximin. Phage-bacterial linkages centred around urease-producing Streptococcus species collapsed postrifaximin. CONCLUSION: Unlike bacteria, faecal phages are sparsely linked with cirrhosis characteristics and 90-day outcomes. Phage and bacterial linkages centred on urease-producing, ammonia-generating Streptococcus species were affected by disease progression and rifaximin therapy and were altered in patients who experienced 90-day hospitalisations.

Consumption of High-Fructose Corn Syrup Compared with Sucrose Promotes Adiposity and Increased Triglyceridemia but Comparable NAFLD Severity in Juvenile Iberian Pigs.

BACKGROUND: Fructose consumption has been linked to nonalcoholic fatty liver disease (NAFLD) in children. However, the effect of high-fructose corn syrup (HFCS) compared with sucrose in pediatric NAFLD has not been investigated. OBJECTIVES: We tested whether the isocaloric substitution of dietary sucrose by HFCS would increase the severity of NAFLD in juvenile pigs, and whether this effect would be associated with changes in gut histology, SCFA production, and microbial diversity. METHODS: Iberian pigs, 53-d-old and pair-housed in pens balanced for weight and sex, were randomly assigned to receive a mash diet top-dressed with increasing amounts of sucrose (SUC; n = 3 pens; 281.6-486.8 g/kg diet) or HFCS (n = 4; 444.3-724.8 g/kg diet) during 16 wk. Diets exceeded the animal's energy requirements by providing sugars in excess, but met the requirements for all other nutrients. Animals were killed at 165 d of age after blood sampling, and liver, muscle, and gut were collected for histology, metabolome, and microbiome analyses. Data were analyzed by multivariate and univariate statistics. RESULTS: Compared with SUC, HFCS increased subcutaneous fat, triacylglycerides in plasma, and butyrate in colon (P ≤ 0.05). In addition, HFCS decreased UMP and short-chain acyl carnitines in liver, and urea nitrogen and creatinine in serum (P ≤ 0.05). Microbiome analysis showed a 24.8% average dissimilarity between HFCS and SUC associated with changes in SCFA-producing bacteria. Body weight gain, intramuscular fat, histological and serum markers of liver injury, and circulating hormones, glucose, and proinflammatory cytokines did not differ between diets. CONCLUSIONS: Fructose consumption derived from HFCS promoted butyrate synthesis, triglyceridemia, and subcutaneous lipid deposition in juvenile Iberian pigs, but did not increase serum and histological markers of NAFLD compared with a sucrose-enriched diet. Longer studies could be needed to observe differences in liver injury among sugar types.

Multiomics and Health: A Holistic Approach to Better Understand the Role of the Microbiome.

The present Special Issue focuses on the latest approaches to health and public health microbiology using multiomics [...].

Dysregulated FXR-FGF19 signaling and choline metabolism are associated with gut dysbiosis and hyperplasia in a novel pig model of pediatric NASH.

To investigate the role of bile acids (BAs) in the pathogenesis of diet-induced nonalcoholic steatohepatitis (NASH), we fed a "Western-style diet" [high fructose, high fat (HFF)] enriched with fructose, cholesterol, and saturated fat for 10 wk to juvenile Iberian pigs. We also supplemented probiotics with in vitro BA deconjugating activity to evaluate their potential therapeutic effect in NASH. Liver lipid and function, cytokines, and hormones were analyzed using commercially available kits. Metabolites, BAs, and fatty acids were measured by liquid chromatography-mass spectrometry. Histology and gene and protein expression analyses were performed using standard protocols. HFF-fed pigs developed NASH, cholestasis, and impaired enterohepatic Farnesoid-X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling in the absence of obesity and insulin resistance. Choline depletion in HFF livers was associated with decreased lipoprotein and cholesterol in serum and an increase of choline-containing phospholipids in colon contents and trimethylamine-N-oxide in the liver. Additionally, gut dysbiosis and hyperplasia increased with the severity of NASH, and were correlated with increased colonic levels of choline metabolites and secondary BAs. Supplementation of probiotics in the HFF diet enhanced NASH, inhibited hepatic autophagy, increased excretion of taurine and choline, and decreased gut microbial diversity. In conclusion, dysregulation of BA homeostasis was associated with injury and choline depletion in the liver, as well as increased biliary secretion, gut metabolism and excretion of choline-based phospholipids. Choline depletion limited lipoprotein synthesis, resulting in hepatic steatosis, whereas secondary BAs and choline-containing phospholipids in colon may have promoted dysbiosis, hyperplasia, and trimethylamine synthesis, causing further damage to the liver.NEW & NOTEWORTHY Impaired Farnesoid-X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling and cholestasis has been described in nonalcoholic fatty liver disease (NAFLD) patients. However, therapeutic interventions with FXR agonists have produced contradictory results. In a swine model of pediatric nonalcoholic steatohepatitis (NASH), we show that the uncoupling of intestinal FXR-FGF19 signaling and a decrease in FGF19 levels are associated with a choline-deficient phenotype of NASH and increased choline excretion in the gut, with the subsequent dysbiosis, colonic hyperplasia, and accumulation of trimethylamine-N-oxide in the liver.

Potential Applications of Human Viral Metagenomics and Reference Materials: Considerations for Current and Future Viruses.

Viruses are ubiquitous particles comprising genetic material that can infect bacteria, archaea, and fungi, as well as human and other animal cells. Given that determining virus composition and function in association with states of human health and disease is of increasing interest, we anticipate that the field of viral metagenomics will continue to expand and be applied in a variety of areas ranging from surveillance to discovery and will rely heavily upon the continued development of reference materials and databases. Information regarding viral composition and function readily translates into biological and clinical applications, including the rapid sequence identification of pathogenic viruses in various sample types. However, viral metagenomic approaches often lack appropriate standards and reference materials to enable cross-study comparisons and assess potential biases which can be introduced at the various stages of collection, storage, processing, and sequence analysis. In addition, implementation of appropriate viral reference materials can aid in the benchmarking of current and development of novel assays for virus identification, discovery, and surveillance. As the field of viral metagenomics expands and standardizes, results will continue to translate into diverse applications.

General Anesthesia Alters the Diversity and Composition of the Intestinal Microbiota in Mice.

Dysbiosis of the intestinal microbiota has been shown to result in altered immune responses and increased susceptibility to infection; as such, the state of the intestinal microbiome may have profound implications in the perioperative setting. In this first-in-class study, we used 16s ribosomal RNA sequencing and analysis in a mouse model of general anesthesia to investigate the effects of volatile anesthetics on the diversity and composition of the intestinal microbiome. After 4-hour exposure to isoflurane, we observed a decrease in bacterial diversity. Taxonomic alterations included depletion of several commensal bacteria including Clostridiales. These data identify volatile anesthetics as potential contributors to microbial dysbiosis in the postoperative patient.

Assessing the microbial quality of a tropical watershed with an urbanization gradient using traditional and alternate fecal indicators.

Urbanization affects the microbial loading into tropical streams, but its impact on water quality varies across watersheds. Rainfall in tropical environments also complicates microbial dynamics due to high seasonal and annual variations. Understanding the dynamics of fecal contamination in tropical surface waters may be further hindered by limitations from the utilization of traditional microbial indicators. We measured traditional (Enterococcus spp. and Escherichia coli), as well as alternate (enterophages and coliphages) indicators of fecal contamination in a tropical watershed in Puerto Rico during a 1-year period, and examined their relationship with rainfall events across an urbanization gradient. Enterococcus spp. and E. coli concentrations were 4 to 5 logs higher in non-urbanized or pristine sites when compared to enterophages and coliphages, suggesting that traditional fecal indicator bacteria may be natural inhabitants of pristine tropical waters. All of the tested indicators were positively correlated with rainfall and urbanization, except in the most urbanized sites, where rainfall may have had a dilution effect. The present study indicates that utilizing novel indicators of microbial water quality may improve the assessment of fecal contamination and pathogen risk for tropical watersheds.

Transcriptome analysis of bacteriophage communities in periodontal health and disease.

BACKGROUND: The role of viruses as members of the human microbiome has gained broader attention with the discovery that human body surfaces are inhabited by sizeable viral communities. The majority of the viruses identified in these communities have been bacteriophages that predate upon cellular microbiota rather than the human host. Phages have the capacity to lyse their hosts or provide them with selective advantages through lysogenic conversion, which could help determine the structure of co-existing bacterial communities. Because conditions such as periodontitis are associated with altered bacterial biota, phage mediated perturbations of bacterial communities have been hypothesized to play a role in promoting periodontal disease. Oral phage communities also differ significantly between periodontal health and disease, but the gene expression of oral phage communities has not been previously examined. RESULTS: Here, we provide the first report of gene expression profiles from the oral bacteriophage community using RNA sequencing, and find that oral phages are more highly expressed in subjects with relative periodontal health. While lysins were highly expressed, the high proportion of integrases expressed suggests that prophages may account for a considerable proportion of oral phage gene expression. Many of the transcriptome reads matched phages found in the oral cavities of the subjects studied, indicating that phages may account for a substantial proportion of oral gene expression. Reads homologous to siphoviruses that infect Firmicutes were amongst the most prevalent transcriptome reads identified in both periodontal health and disease. Some genes from the phage lytic module were significantly more highly expressed in subjects with periodontal disease, suggesting that periodontitis may favor the expression of some lytic phages. CONCLUSIONS: As we explore the contributions of viruses to the human microbiome, the data presented here suggest varying expression of bacteriophage communities in oral health and disease.

Global transcription of CRISPR loci in the human oral cavity.

BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are active in acquired resistance against bacteriophage and plasmids in a number of environments. In the human mouth, CRISPR loci evolve to counteract oral phage, but the expression of these CRISPR loci has not previously been investigated. We sequenced cDNA from CRISPR loci found in numerous different oral bacteria and compared with oral phage communities to determine whether the transcription of CRISPR loci is specifically targeted towards highly abundant phage present in the oral environment. RESULTS: We found that of the 529,027 CRISPR spacer groups studied, 88 % could be identified in transcripts, indicating that the vast majority of CRISPR loci in the oral cavity were transcribed. There were no strong associations between CRISPR spacer repertoires and oral health status or nucleic acid type. We also compared CRISPR repertoires with oral bacteriophage communities, and found that there was no significant association between CRISPR transcripts and oral phage, regardless of the CRISPR type being evaluated. We characterized highly expressed CRISPR spacers and found that they were no more likely than other spacers to match oral phage. By reassembling the CRISPR-bearing reads into longer CRISPR loci, we found that the majority of the loci did not have spacers matching viruses found in the oral cavities of the subjects studied. For some CRISPR types, loci containing spacers matching oral phage were significantly more likely to have multiple spacers rather than a single spacer matching oral phage. CONCLUSIONS: These data suggest that the transcription of oral CRISPR loci is relatively ubiquitous and that highly expressed CRISPR spacers do not necessarily target the most abundant oral phage.

Prospective epidemiological pilot study on the morbidity of bathers exposed to tropical recreational waters and sand.

A prospective cohort epidemiological pilot study was performed at three tropical beaches with point- and non-point-sources of fecal pollution to characterize the risk of illness among swimmers and non-swimmers. There was an increased risk of illness in swimmers as compared to non-swimmers, even when waters met current microbial standards for recreational water quality. Illnesses included gastrointestinal (GI), skin and respiratory symptoms, earache and fever. Odds ratios (ORs) ranged from 0.32 to 42.35 (GI illness), 0.69 to 3.12 (skin infections), 0.71 to 3.21 (respiratory symptoms), 0.52 to 15.32 (earache) and 0.80 to 1.68 (fever), depending on the beach sampled. The indicators that better predicted the risks of symptoms (respiratory) in tropical recreational waters were total (somatic and male-specific) coliphages (OR = 1.56, p < 0.10, R(2) = 3.79%) and Escherichia coli (OR = 1.38, p < 0.10, R(2) = 1.97%). The present study supports the potential of coliphages as good predictors of risks of respiratory illness in tropical recreational waters. This is the first study that has determined risks of illness after exposure to tropical recreational waters with point- and non-point sources of fecal contamination. The results give an opportunity to perform epidemiological studies in tropical recreational waters in Puerto Rico which can include more participants and other indicators and detection techniques.

Antibiotic-resistance and virulence genes in Enterococcus isolated from tropical recreational waters.

The prevalence of enterococci harboring tetracycline- and vancomycin-resistance genes, as well as the enterococcal surface protein (esp) has mostly been determined in clinical settings, but their prevalence in tropical recreational waters remains largely unknown. The present study determined the prevalence of tetM (tetracycline-resistance), vanA and vanB (vancomycin-resistance) in the bacterial and viral fractions, enterococci and their induced phages isolated from tropical recreational marine and fresh waters, dry and wet sands. Since lysogenic phages can act as vectors for antibiotic-resistance and virulence factors, the prevalence of the mentioned genes, as well as that of an integrase-encoding gene (int) specific for Enterococcus faecalis phages was determined. Up to 60 and 54% of the bacterial fractions and enterococci, respectively, harbored at least one of the tested genes suggesting that bacteria in tropical environments may be reservoirs of antibiotic-resistance and virulence genes. int was detected in the viral fractions and in one Enterococcus isolate after induction. This study presents the opportunity to determine if the presence of bacteria harboring antibiotic-resistance and virulence genes in tropical recreational waters represents a threat to public health.

Evaluation of Enterococcus-infecting phages as indices of fecal pollution.

No microbial source tracking tool satisfies all the characteristics of an ideal indicator of human fecal pollution. For this reason, the potential of Enterococcus faecalis phages (enterophages) as markers of this type of contamination was tested by using eight Enterococcus type strains as the possible hosts. The prevalence of enterophages in animal feces and domestic sewage were determined, as were the inactivation rates in raw sewage at 4 °C and surface and tap waters at 22 °C. Enterophages were exclusively detected in raw sewage (up to 66.0 plaque forming units (PFU)/100 mL), suggesting a strictly human origin; and exhibited inactivation rates of approximately 0.002 to 0.05, 0.3 to 0.5 and 0.4 to 1.4 log day(-1) in raw sewage and surface and tap waters, respectively, similar to those of previous reports on human enteric viruses under similar conditions. Interestingly, phages infecting other Enterococcus type strains were detected in both animal feces and domestic sewage in concentrations of up to 335.8 PFU/g and 96.0 PFU/100 mL, and certain phage isolates infected several of the strains tested. This clearly indicates the possible promiscuous nature of some Enterococcus phages and thus opens up the opportunity to further characterize these as indices of specific fecal sources.

Viral Metagenomics as a Tool to Track Sources of Fecal Contamination: A One Health Approach.

The One Health framework recognizes that human, animal, and environmental health are linked and highly interdependent. Fecal contamination of water, soil, foodstuff, and air may impact many aspects of One Health, and culture, PCR-based, and sequencing methods are utilized in the detection of fecal contamination to determine source, load, and risk to inform targeted mitigation strategies. Viruses, particularly, have been considered as fecal contamination indicators given the narrow host range many exhibit and their association with other biological contaminants. Culture- and molecular-based methods are considered the gold-standards for virus detection and for determining specific sources of fecal contamination via viral indicators. However, viral metagenomics is also being considered as a tool for tracking sources of fecal contamination. In the present review, studies tracking potential sources of fecal contamination in freshwaters, marine waters, foodstuff, soil, and air using viral metagenomics are discussed to highlight the potential of viral metagenomics for optimizing fecal source tracking. Limitations of the use of viral metagenomics to track fecal contamination sources, including sample processing, nucleic acid recovery, sequencing depth, and bioinformatics are also discussed. Finally, the present review discusses the potential of viral metagenomics as part of the toolbox of methods in a One Health approach.

Deciphering Diets and Lifestyles of Prehistoric Humans through Paleoparasitology: A Review.

Parasites have affected and coevolved with humans and animals throughout history. Evidence of ancient parasitic infections, particularly, reside in archeological remains originating from different sources dating to various periods of times. The study of ancient parasites preserved in archaeological remains is known as paleoparasitology, and it initially intended to interpret migration, evolution, and dispersion patterns of ancient parasites, along with their hosts. Recently, paleoparasitology has been used to better understand dietary habits and lifestyles of ancient human societies. Paleoparasitology is increasingly being recognized as an interdisciplinary field within paleopathology that integrates areas such as palynology, archaeobotany, and zooarchaeology. Paleoparasitology also incorporates techniques such as microscopy, immunoassays, PCR, targeted sequencing, and more recently, high-throughput sequencing or shotgun metagenomics to understand ancient parasitic infections and thus interpret migration and evolution patterns, as well as dietary habits and lifestyles. The present review covers the original theories developed in the field of paleoparasitology, as well as the biology of some parasites identified in pre-Columbian cultures. Conclusions, as well as assumptions made during the discovery of the parasites in ancient samples, and how their identification may aid in better understanding part of human history, ancient diet, and lifestyles are discussed.

The Skin Microbiome: Current Techniques, Challenges, and Future Directions.

Skin acts as a barrier that promotes the colonization of bacteria, fungi, archaea, and viruses whose membership and function may differ depending on the various specialized niches or micro-environments of the skin. The group of microorganisms inhabiting the skin, also known as the skin microbiome, offers protection against pathogens while actively interacting with the host's immune system. Some members of the skin microbiome can also act as opportunistic pathogens. The skin microbiome is influenced by factors such as skin site, birth mode, genetics, environment, skin products, and skin conditions. The association(s) of the skin microbiome with health and disease has (have) been identified and characterized via culture-dependent and culture-independent methods. Culture-independent methods (such as high-throughput sequencing), in particular, have expanded our understanding of the skin microbiome's role in maintaining health or promoting disease. However, the intrinsic challenges associated with the low microbial biomass and high host content of skin microbiome samples have hindered advancements in the field. In addition, the limitations of current collection and extraction methods and biases derived from sample preparation and analysis have significantly influenced the results and conclusions of many skin microbiome studies. Therefore, the present review discusses the technical challenges associated with the collection and processing of skin microbiome samples, the advantages and disadvantages of current sequencing approaches, and potential future areas of focus for the field.

Edible flora in pre-Columbian Caribbean coprolites: Expected and unexpected data.

Coprolites, or mummified feces, are valuable sources of information on ancient cultures as they contain ancient DNA (aDNA). In this study, we analyzed ancient plant DNA isolated from coprolites belonging to two pre-Columbian cultures (Huecoid and Saladoid) from Vieques, Puerto Rico, using shotgun metagenomic sequencing to reconstruct diet and lifestyles. We also analyzed DNA sequences of putative phytopathogenic fungi, likely ingested during food consumption, to further support dietary habits. Our findings show that pre-Columbian Caribbean cultures had a diverse diet consisting of maize (Zea mays), sweet potato (Ipomoea batatas), chili peppers (Capsicum annuum), peanuts (Arachis spp.), papaya (Carica papaya), tomato (Solanum lycopersicum) and, very surprisingly cotton (Gossypium barbadense) and tobacco (Nicotiana sylvestris). Modelling of putative phytopathogenic fungi and plant interactions confirmed the potential consumption of these plants as well as edible fungi, particularly Ustilago spp., which suggest the consumption of maize and huitlacoche. These findings suggest that a variety of dietary, medicinal, and hallucinogenic plants likely played an important role in ancient human subsistence and societal customs. We compared our results with coprolites found in Mexico and the United States, as well as present-day faeces from Mexico, Peru, and the United States. The results suggest that the diet of pre-Columbian cultures resembled that of present-day hunter-gatherers, while agriculturalists exhibited a transitional state in dietary lifestyles between the pre-Columbian cultures and larger scale farmers and United States individuals. Our study highlights differences in dietary patterns related to human lifestyles and provides insight into the flora present in the pre-Columbian Caribbean area. Importantly, data from ancient fecal specimens demonstrate the importance of ancient DNA studies to better understand pre-Columbian populations.