A network-based approach to deciphering a dynamic microbiome's response to a subtle perturbation.

Over the past decades, one main issue that has emerged in ecological and environmental research is how losses in biodiversity influence ecosystem dynamics and functioning, and consequently human society. Although biodiversity is a common indicator of ecosystem functioning, it is difficult to measure biodiversity in microbial communities exposed to subtle or chronic environmental perturbations. Consequently, there is a need for alternative bioindicators to detect, measure, and monitor gradual changes in microbial communities against these slight, chronic, and continuous perturbations. In this study, microbial networks before and after subtle perturbations by adding S. acidaminiphila showed diverse topological niches and 4-node motifs in which microbes with co-occurrence patterns played the central roles in regulating and adjusting the intertwined relationships among microorganisms in response to the subtle environmental changes. This study demonstrates that microbial networks are a good bioindicator for chronic perturbation and should be applied in a variety of ecological investigations.

A network approach to investigating the key microbes and stability of gut microbial communities in a mouse neuropathic pain model.

BACKGROUND: Neuropathic pain is an abnormally increased sensitivity to pain, especially from mechanical or thermal stimuli. To date, the current pharmacological treatments for neuropathic pain are still unsatisfactory. The gut microbiota reportedly plays important roles in inducing neuropathic pain, so probiotics have also been used to treat it. However, the underlying questions around the interactions in and stability of the gut microbiota in a spared nerve injury-induced neuropathic pain model and the key microbes (i.e., the microbes that play critical roles) involved have not been answered. We collected 66 fecal samples over 2 weeks (three mice and 11 time points in spared nerve injury-induced neuropathic pain and Sham groups). The 16S rRNA gene was polymerase chain reaction amplified, sequenced on a MiSeq platform, and analyzed using a MOTHUR- UPARSE pipeline. RESULTS: Here we show that spared nerve injury-induced neuropathic pain alters gut microbial diversity in mice. We successfully constructed reliable microbial interaction networks using the Metagenomic Microbial Interaction Simulator (MetaMIS) and analyzed these networks based on 177,147 simulations. Interestingly, at a higher resolution, our results showed that spared nerve injury-induced neuropathic pain altered both the stability of the microbial community and the key microbes in a gut micro-ecosystem. Oscillospira, which was classified as a low-abundance and core microbe, was identified as the key microbe in the Sham group, whereas Staphylococcus, classified as a rare and non-core microbe, was identified as the key microbe in the spared nerve injury-induced neuropathic pain group. CONCLUSIONS: In summary, our results provide novel experimental evidence that spared nerve injury-induced neuropathic pain reshapes gut microbial diversity, and alters the stability and key microbes in the gut.

Maternal gut microbes shape the early-life assembly of gut microbiota in passerine chicks via nests.

BACKGROUND: Knowledge is growing on how gut microbiota are established, but the effects of maternal symbiotic microbes throughout early microbial successions in birds remain elusive. In this study, we examined the contributions and transmission modes of maternal microbes into the neonatal microbiota of a passerine, the zebra finch (Taeniopygia guttata), based on fostering experiments. RESULTS: Using 16S rRNA amplicon sequencing, we found that zebra finch chicks raised by their biological or foster parents (the society finch Lonchura striata domestica) had gut microbial communities converging with those of the parents that reared them. Moreover, source-tracking models revealed high contribution of zebra finches' oral cavity/crop microbiota to their chicks' early gut microbiota, which were largely replaced by the parental gut microbiota at later stages. The results suggest that oral feeding only affects the early stage of hatchling gut microbial development. CONCLUSIONS: Our study indicates that passerine chicks mainly acquire symbionts through indirect maternal transmission-passive environmental uptake from nests that were smeared with the intestinal and cloacal microbes of parents that raised them. Gut microbial diversity was low in hand-reared chicks, emphasizing the importance of parental care in shaping the gut microbiota. In addition, several probiotics were found in chicks fostered by society finches, which are excellent foster parents for other finches in bird farms and hosts of brood parasitism by zebra finches in aviaries; this finding implies that avian species that can transfer probiotics to chicks may become selectively preferred hosts of brood parasitism in nature. Video Abstract.

Characterization of benzimidazole resistance in Haemonchus contortus: integration of phenotypic, genotypic and proteomic approaches.

The field strain of Haemonchus contortus has a long history of anthelmintic resistance. To understand this phenomenon, the benzimidazole resistance profile was characterized from the Malaysian field-resistant strain by integrating phenotypic, genotypic and proteomic approaches. The faecal egg count reduction test (FECRT) demonstrated that benzimidazole resistance was at a critical level in the studied strain. The primary resistance mechanism was attributed to F200Y mutation in the isotype 1 ß-tubulin gene as revealed by AS-PCR and direct sequencing. Furthermore, the protein response of the resistant strain towards benzimidazole (i.e., albendazole) treatment was investigated via two-dimensional difference gel electrophoresis (2D-DIGE) and tandem liquid chromatography-mass spectrometry (LC-MS/MS). These investigations illustrated an up-regulation of antioxidant (i.e., ATP-binding region and heat-shock protein 90, superoxide dismutase) and metabolic (i.e., glutamate dehydrogenase) enzymes and down-regulation of glutathione S-transferase, malate dehydrogenase, and other structural and cytoskeletal proteins (i.e., actin, troponin T). Findings from this study are pivotal in updating the current knowledge on anthelmintic resistance and providing new insights into the defence mechanisms of resistant nematodes towards drug treatment.

Structure and membership of gut microbial communities in multiple fish cryptic species under potential migratory effects.

The animal gut microbiota evolves quickly towards a complex community and plays crucial roles in its host's health and development. Factors such as host genetics and environmental changes are regarded as important for controlling the dynamics of animal gut microbiota. Migratory animals are an important group for studying how these factors influence gut microbiota because they experience strong environmental perturbations during migration. The commercially important grey mullet, Mugil cephalus, is a cosmopolitan species complex that display reproductive migration behaviour. There are three cryptic species of M. cephalus fish distributed across the Northwest Pacific, and their spawning sites overlap in the Taiwan Strait. This extraordinary natural occurrence makes the grey mullet an ideal model organism for exploring the nature of wild animal-gut microbiota relationships and interactions. This study investigates the diversity and structure of the gut microbial community in three cryptic M. cephalus species using 16S rRNA amplicon sequencing. Gut microbial compositions from adult and juvenile fish samples were analysed. Our results indicate that gut microbial communities within the grey mullet share a core microbiome dominated by Proteobacteria, Firmicutes and Actinobacteria. However, the structures of gut microbial communities were more distinct between adult mullet groups than they were between juvenile ones. Intriguingly, we found that adult fish that migrate to different geographical tracts harbour gut microbiota similar to historical records of seawater microflora, along their respective migration routes. This observation provides new insights into the interaction between aquatic animal gut microbial communities and the environments along their hosts' migratory routes, and thus warrants future study.

A systematic approach re-analyzing the effects of temperature disturbance on the microbial community of mesophilic anaerobic digestion.

Microbial communities are key drivers of ecosystem processes, but their behavior in disturbed environments is difficult to measure. How microbial community composition and function respond disturbances is a common challenge in biomedical, environmental, agricultural, and bioenergy research. A novel way to solve this problem is to use a systems-level perspective and describe microbial communities as networks. Based on a mesophilic anaerobic digestion system of swine manure as a tool, we propose a simple framework to investigate changes in microbial communities via compositions, metabolic pathways, genomic properties and interspecies relationships in response to a long-term temperature disturbance. After temperature disturbance, microbial communities tend towards a competitive interaction network with higher GC content and larger genome size. Based on microbial interaction networks, communities responded to the disturbance by showing a transition from acetotrophic (Methanotrichaceae and Methanosarcinaceae) to methylotrophic methanogens (Methanomassiliicoccaceae and Methanobacteriaceae) and a fluctuation in rare biosphere taxa. To conclude, this study may be important for exploring the dynamic relationships between disturbance and microbial communities as a whole, as well as for providing researchers with a better understanding of how changes in microbial communities relate to ecological processes.

Occurrence of zoonotic Cryptosporidium and Giardia duodenalis species/genotypes in urban rodents.

This report describes the detection of zoonotic Cryptosporidium muris, C. parvum subgenotype IIa and Giardia duodenalis genotype B in urban rodents in Malaysia. A rare occurrence of C. meleagridis was also reported suggesting a role of rodents in mechanical transmission of this pathogen. Utilization of DNA sequencing and subtyping analysis confirmed the presence of zoonotic C. parvum subtypes IIaA17G2R1 and IIaA16G3R1 for the first time in rodents.

A novel molecular and chromosomal lineage of the anthropophilic Simulium (Simulium) rufibasis subgroup (Diptera: Simuliidae) in Taiwan.

The Simulium rufibasis subgroup is one of three subgroups of the Simulium (Simulium) tuberosum species-group; it is characterized by a pair of clustered stout hairs on the ventral surface of female abdominal segment 7. A member of the S. rufibasis subgroup in Taiwan was investigated morphologically and genetically using the universal cytochrome c oxidase subunit I (COI) barcoding gene and polytene chromosomal banding pattern. The Taiwanese material is morphologically similar to S. rosliramlii Takaoka & Chen from Vietnam and represents the second species of the S. rufibasis subgroup known from Taiwan. It also represents a novel molecular lineage that is distinct from three other primary lineages identified as S. doipuiense, S. doipuiense/S. rufibasis, and S. weji previously reported from Thailand. The mitochondrial evidence for a distinct lineage in Taiwan is supported by chromosomal analysis, which revealed unique sex chromosomes. For nomenclatural stability, we associate the name S. arisanum Shiraki with the Taiwanese entity. Originally described from females from Taiwan, S. arisanum until now has remained an enigmatic species.

Habitat and indigenous gut microbes contribute to the plasticity of gut microbiome in oriental river prawn during rapid environmental change.

Growing evidence points out that the capacity of organisms to acclimate or adapt to new habitat conditions basically depends on their phenomic plasticity attributes, of which their gut commensal microbiota might be an essential impact factor. Especially in aquatic organisms, which are in direct and continual contact with the aquatic environment, the complex and dynamic microbiota have significant effects on health and development. However, an understanding of the relative contribution of internal sorting (host genetic) and colonization (environmental) processes is still unclear. To understand how microbial communities differ in response to rapid environmental change, we surveyed and studied the environmental and gut microbiota of native and habitat-exchanged shrimp (Macrobrachium nipponense) using 16S rRNA amplicon sequencing on the Illumina MiSeq platform. Corresponding with microbial diversity of their living water areas, the divergence in gut microbes of lake-to-river shrimp (CK) increased, while that of river-to-lake shrimp (KC) decreased. Importantly, among the candidate environment specific gut microbes in habitat-exchanged shrimp, over half of reads were associated with the indigenous bacteria in native shrimp gut, yet more candidates presented in CK may reflect the complexity of new environment. Our results suggest that shrimp gut microbiota has high plasticity when its host faces environmental changes, even over short timescales. Further, the changes in external environment might influence the gut microbiome not just by providing environment-associated microbes directly, but also by interfering with the composition of indigenous gut bacteria indirectly.

Inferring microbial interactions in thermophilic and mesophilic anaerobic digestion of hog waste.

Anaerobic digestion (AnD) is a microbiological process that converts organic waste materials into biogas. Because of its high methane content, biogas is a combustible energy source and serves as an important environmental technology commonly used in the management of animal waste generated on large animal farms. Much work has been done on hardware design and process engineering for the generation of biogas. However, little is known about the complexity of the microbiology in this process. In particular, how microbes interact in the digester and eventually breakdown and convert organic matter into biogas is still regarded as a "black box." We used 16S rRNA sequencing as a tool to study the microbial community in laboratory hog waste digesters under tightly controlled conditions, and systematically unraveled the distinct interaction networks of two microbial communities from mesophilic (MAnD) and thermophilic anaerobic digestion (TAnD). Under thermophilic conditions, the well-known association between hydrogen-producing bacteria, e.g., Ruminococcaceae and Prevotellaceae, and hydrotrophic methanogens, Methanomicrobiaceae, was reverse engineered by their interactive topological niches. The inferred interaction network provides a sketch enabling the determination of microbial interactive relationships that conventional strategy of finding differential taxa was hard to achieve. This research is still in its infancy, but it can help to depict the dynamics of microbial ecosystems and to lay the groundwork for understanding how microorganisms cohabit in the anaerobic digester.

Inferring Microbial Interactions in the Gut of the Hong Kong Whipping Frog (Polypedates megacephalus) and a Validation Using Probiotics.

The concerted activity of intestinal microbes is crucial to the health and development of their host organisms. Investigation of microbial interactions in the gut should deepen our understanding of how these micro-ecosystems function. Due to advances in Next Generation Sequencing (NGS) technologies, various bioinformatic strategies have been proposed to investigate these microbial interactions. However, due to the complexity of the intestinal microbial community and difficulties in monitoring their interactions, at present there is a gap between the theory and biological application. In order to construct and validate microbial relationships, we first induce a community shift from simple to complex by manipulating artificial hibernation (AH) in the treefrog Polypedates megacephalus. To monitor community growth and microbial interactions, we further performed a time-course screen using a 16S rRNA amplicon approach and a Lotka-Volterra model. Lotka-Volterra models, also known as predator-prey equations, predict the dynamics of microbial communities and how communities are structured and sustained. An interaction network of gut microbiota at the genus level in the treefrog was constructed using Metagenomic Microbial Interaction Simulator (MetaMIS) package. The interaction network obtained had 1,568 commensal, 1,737 amensal, 3,777 mutual, and 3,232 competitive relationships, e.g., Lactococcus garvieae has a commensal relationship with Corynebacterium variabile. To validate the interacting relationships, the gut microbe composition was analyzed after probiotic trials using single strain (L. garvieae, C. variabile, and Bacillus coagulans, respectively) and a combination of L. garvieae, C. variabile, and B. coagulans, because of the cooperative relationship among their respective genera identified in the interaction network. After a 2 week trial, we found via 16S rRNA amplicon analysis that the combination of cooperative microbes yielded significantly higher probiotic concentrations than single strains, and the immune response (interleukin-10 expression) also significantly changed in a manner consistent with improved probiotic effects. By taking advantage of microbial community shift from simple to complex, we thus constructed a reliable microbial interaction network, and validated it using probiotic strains as a test system.

MetaMIS: a metagenomic microbial interaction simulator based on microbial community profiles.

BACKGROUND: The complexity and dynamics of microbial communities are major factors in the ecology of a system. With the NGS technique, metagenomics data provides a new way to explore microbial interactions. Lotka-Volterra models, which have been widely used to infer animal interactions in dynamic systems, have recently been applied to the analysis of metagenomic data. RESULTS: In this paper, we present the Lotka-Volterra model based tool, the Metagenomic Microbial Interacticon Simulator (MetaMIS), which is designed to analyze the time series data of microbial community profiles. MetaMIS first infers underlying microbial interactions from abundance tables for operational taxonomic units (OTUs) and then interprets interaction networks using the Lotka-Volterra model. We also embed a Bray-Curtis dissimilarity method in MetaMIS in order to evaluate the similarity to biological reality. MetaMIS is designed to tolerate a high level of missing data, and can estimate interaction information without the influence of rare microbes. For each interaction network, MetaMIS systematically examines interaction patterns (such as mutualism or competition) and refines the biotic role within microbes. As a case study, we collect a human male fecal microbiome and show that Micrococcaceae, a relatively low abundance OTU, is highly connected with 13 dominant OTUs and seems to play a critical role. MetaMIS is able to organize multiple interaction networks into a consensus network for comparative studies; thus we as a case study have also identified a consensus interaction network between female and male fecal microbiomes. CONCLUSIONS: MetaMIS provides an efficient and user-friendly platform that may reveal new insights into metagenomics data. MetaMIS is freely available at: https://sourceforge.net/projects/metamis/ .

Functional analysis for gut microbes of the brown tree frog (Polypedates megacephalus) in artificial hibernation.

BACKGROUND: Annual hibernation is an adaptation that helps many animals conserve energy during food shortage in winter. This natural cycle is also accompanied by a remodeling of the intestinal immune system, which is an aspect of host biology that is both influenced by, and can itself influence, the microbiota. In amphibians, the bacteria in the intestinal tract show a drop in bacterial counts. The proportion of pathogenic bacteria is greater in hibernating frogs than that found in nonhibernating frogs. This suggests that some intestinal gut microbes in amphibians can be maintained and may contribute to the functions in this closed ecosystem during hibernation. However, these results were derived from culture-based approaches that only covered a small portion of bacteria in the intestinal tract. METHODS: In this study, we use a more comprehensive analysis, including bacterial appearance and functional prediction, to reveal the global changes in gut microbiota during artificial hibernation via high-throughput sequencing technology. RESULTS: Our results suggest that artificial hibernation in the brown tree frog (Polypedates megacephalus) could reduce microbial diversity, and artificially hibernating frogs tend to harbor core operational taxonomic units that are rarely distributed among nonhibernating frogs. In addition, artificial hibernation increased significantly the relative abundance of the red-leg syndrome-related pathogenic genus Citrobacter. Furthermore, functional predictions via PICRUSt and Tax4Fun suggested that artificial hibernation has effects on metabolism, disease, signal transduction, bacterial infection, and primary immunodeficiency. CONCLUSIONS: We infer that artificial hibernation may impose potential effects on primary immunodeficiency and increase the risk of bacterial infections in the brown tree frog.

Population Structure and Historical Demography of the Oriental River Prawn (Macrobrachium nipponense) in Taiwan.

The oriental river prawn (Macrobrachium nipponense) is a non-obligatory amphidromous prawn, and it has a wide distribution covering almost the entire Taiwan. Mitochondrial DNA fragment sequences of the cytochrome oxidase subunit I (COI) and 16S rRNA were combined and used to elucidate the population structure and historical demography of oriental river prawn in Taiwan. A total of 202 individuals from six reservoirs and three estuaries were separately collected. Nucleotide diversity (π) of all populations was 0.01217, with values ranging from 0.00188 (Shihmen Reservoir, SMR, northern Taiwan) to 0.01425 (Mingte Reservoir, MTR, west-central Taiwan). All 76 haplotypes were divided into 2 lineages: lineage A included individuals from all sampling areas except SMR, and lineage B included specimens from all sampling locations except Chengching Lake Reservoir (CLR) and Liyu Lake Reservoir (LLR). All FST values among nine populations were significantly different except the one between Jhonggang River Estuary (JGE, west-central Taiwan) and Kaoping River Estuary (KPE, southern Taiwan). UPGMA tree of nine populations showed two main groups: the first group included the SMR and Tamsui River Estuary (TSE) (both located northern Taiwan), and the second one included the other seven populations (west-central, southern and eastern Taiwan). Demographic analyses implied a population expansion occurred during the recent history of the species. The dispersal route of this species might be from China to west-central and west-southern Taiwan, and then the part individuals belonging to lineage A and B dispersed southerly and northerly, respectively. And then part individuals in west-central Taiwan fell back to and stay at estuaries as the sea level rose about 18,000 years ago.

Inferring microbial interaction network from microbiome data using RMN algorithm.

BACKGROUND: Microbial interactions are ubiquitous in nature. Recently, many similarity-based approaches have been developed to study the interaction in microbial ecosystems. These approaches can only explain the non-directional interactions yet a more complete view on how microbes regulate each other remains elusive. In addition, the strength of microbial interactions is difficult to be quantified by only using correlation analysis. RESULTS: In this study, a rule-based microbial network (RMN) algorithm, which integrates regulatory OTU-triplet model with parametric weighting function, is being developed to construct microbial regulatory networks. The RMN algorithm not only can extrapolate the cooperative and competitive relationships between microbes, but also can infer the direction of such interactions. In addition, RMN algorithm can theoretically characterize the regulatory relationship composed of microbial pairs with low correlation coefficient in microbial networks. Our results suggested that Bifidobacterium, Streptococcus, Clostridium XI, and Bacteroides are essential for causing abundance changes of Veillonella in gut microbiome. Furthermore, we inferred some possible microbial interactions, including the competitive relationship between Veillonella and Bacteroides, and the cooperative relationship between Veillonella and Clostridium XI. CONCLUSIONS: The RMN algorithm provides the reconstruction of gut microbe networks, and can shed light on the dynamical interactions of microbes in the infant intestinal tract.

Effects of Host Phylogeny and Habitats on Gut Microbiomes of Oriental River Prawn (Macrobrachium nipponense).

The gut microbial community is one of the richest and most complex ecosystems on earth, and the intestinal microbes play an important role in host development and health. Next generation sequencing approaches, which rapidly produce millions of short reads that enable the investigation on a culture independent basis, are now popular for exploring microbial community. Currently, the gut microbiome in fresh water shrimp is unexplored. To explore gut microbiomes of the oriental river prawn (Macrobrachium nipponense) and investigate the effects of host genetics and habitats on the microbial composition, 454 pyrosequencing based on the 16S rRNA gene were performed. We collected six groups of samples, including M. nipponense shrimp from two populations, rivers and lakes, and one sister species (M. asperulum) as an out group. We found that Proteobacteria is the major phylum in oriental river prawn, followed by Firmicutes and Actinobacteria. Compositional analysis showed microbial divergence between the two shrimp species is higher than that between the two populations of one shrimp species collected from river and lake. Hierarchical clustering also showed that host genetics had a greater impact on the divergence of gut microbiome than host habitats. This finding was also congruent with the functional prediction from the metagenomic data implying that the two shrimp species still shared the same type of biological functions, reflecting a similar metabolic profile in their gut environments. In conclusion, this study provides the first investigation of the gut microbiome of fresh water shrimp, and supports the hypothesis of host species-specific signatures of bacterial community composition.

Examining the condition-specific antisense transcription in S. cerevisiae and S. paradoxus.

BACKGROUND: Recent studies have demonstrated that antisense transcription is pervasive in budding yeasts and is conserved between Saccharomyces cerevisiae and S. paradoxus. While studies have examined antisense transcripts of S. cerevisiae for inverse expression in stationary phase and stress conditions, there is a lack of comprehensive analysis of the conditional specific evolutionary characteristics of antisense transcription between yeasts. Here we attempt to decipher the evolutionary relationship of antisense transcription of S. cerevisiae and S. paradoxus cultured in mid log, early stationary phase, and heat shock conditions. RESULTS: Massively parallel sequencing of sequence strand-specific cDNA library was performed from RNA isolated from S. cerevisiae and S. paradoxus cells at mid log, stationary phase and heat shock conditions. We performed this analysis using a stringent set of sense ORF transcripts and non-coding antisense transcripts that were expressed in all the three conditions, as well as in both species. We found the divergence of the condition-specific anti-sense transcription levels is higher than that in condition-specific sense transcription levels, suggesting that antisense transcription played a potential role in adapting to different conditions. Furthermore, 43% of sense-antisense pairs demonstrated inverse expression in either stationary phase or heat shock conditions relative to the mid log conditions. In addition, a large part of sense-antisense pairs (67%), which demonstrated inverse expression, were highly conserved between the two species. Our results were also concordant with known functional analyses from previous studies and with the evidence from mechanistic experiments of role of individual genes. CONCLUSIONS: By performing a genome-scale computational analysis, we have tried to evaluate the role of antisense transcription in mediating sense transcription under different environmental conditions across and in two related yeast species. Our findings suggest that antisense regulation could control expression of the corresponding sense transcript via inverse expression under a range of different circumstances.

Role of antisense RNAs in evolution of yeast regulatory complexity.

Antisense RNAs (asRNAs) are known to regulate gene expression. However, a genome-wide mechanism of asRNA regulation is unclear, and there is no good explanation why partial asRNAs are not functional. To explore its regulatory role, we investigated asRNAs using an evolutionary approach, as genome-wide experimental data are limited. We found that the percentage of genes coupling with asRNAs in Saccharomyces cerevisiae is negatively associated with regulatory complexity and evolutionary age. Nevertheless, asRNAs evolve more slowly when their sense genes are under more complex regulation. Older genes coupling with asRNAs are more likely to demonstrate inverse expression, reflecting the role of these asRNAs as repressors. Our analyses provide novel evidence, suggesting a minor contribution of asRNAs in developing regulatory complexity. Although our results support the leaky hypothesis for asRNA transcription, our evidence also suggests that partial asRNAs may have evolved as repressors. Our study deepens the understanding of asRNA regulatory evolution.

Evolution of cis-regulatory elements in yeast de novo and duplicated new genes.

BACKGROUND: New genes that originate from non-coding DNA rather than being duplicated from parent genes are called de novo genes. Their short evolution time and lack of parent genes provide a chance to study the evolution of cis-regulatory elements in the initial stage of gene emergence. Although a few reports have discussed cis-regulatory elements in new genes, knowledge of the characteristics of these elements in de novo genes is lacking. Here, we conducted a comprehensive investigation to depict the emergence and establishment of cis-regulatory elements in de novo yeast genes. RESULTS: In a genome-wide investigation, we found that the number of transcription factor binding sites (TFBSs) in de novo genes of S. cerevisiae increased rapidly and quickly became comparable to the number of TFBSs in established genes. This phenomenon might have resulted from certain characteristics of de novo genes; namely, a relatively frequent gain of TFBSs, an unexpectedly high number of preexisting TFBSs, or lower selection pressure in the promoter regions of the de novo genes. Furthermore, we identified differences in the promoter architecture between de novo genes and duplicated new genes, suggesting that distinct regulatory strategies might be employed by genes of different origin. Finally, our functional analyses of the yeast de novo genes revealed that they might be related to reproduction. CONCLUSIONS: Our observations showed that de novo genes and duplicated new genes possess mutually distinct regulatory characteristics, implying that these two types of genes might have different roles in evolution.