Flavobacterium are widely distributed and produce various polysaccharide-degrading enzymes. Many species in the genus have been isolated and characterized. However, few studies have focused on marine isolates or fish pathogens, and in-depth genomic analyses, particularly comparative analyses of isolates from different habitat types, are lacking. Here, we isolated 20 strains of the genus from various environments in South Korea and sequenced their full-length genomes. Combined with published sequence data, we examined genomic traits, evolution, environmental adaptation, and putative metabolic functions in total 187 genomes of isolated species in Flavobacterium categorized as marine, host-associated, and terrestrial including freshwater. A pangenome analysis revealed a correlation between genome size and coding or noncoding density. Flavobacterium spp. had high levels of diversity, allowing for novel gene repertories via recombination events. Defense-related genes only accounted for approximately 3% of predicted genes in all Flavobacterium genomes. While genes involved in metabolic pathways did not differ with respect to isolation source, there was substantial variation in genomic traits; in particular, the abundances of tRNAs and rRNAs were higher in the host-associdated group than in other groups. One genome in the host-associated group contained a Microviridae prophage closely related to an enterobacteria phage. The proteorhodopsin gene was only identified in four terrestrial strains isolated for this study. Furthermore, recombination events clearly influenced genomic diversity and may contribute to the response to environmental stress. These findings shed light on the high genetic variation in Flavobacterium and functional roles in diverse ecosystems as a result of their metabolic versatility.Members of the genus Flavobacterium is a diverse group of bacteria that are found in a variety of environments. While most species of this genus are harmless and utilize organic substrates such as proteins and polysaccharides, some members may play a significant role in the cycling for organic substances within their environments. Nevertheless, little is known about the genomic dynamics and/or metabolic capacity of Flavobacterium. Here, we found that Flavobacterium species may have an open pangenome, containing a variety of diverse and novel gene repertoires. Intriguingly, we discovered that one genome (classified into host-associated group) contained a Microviridae prophage closely related to that of enterobacteria. Proteorhodopsin may be expressed under conditions of light or oxygen pressure in some strains isolated for this study. Our findings significantly contribute to the understanding of the members of the genus Flavobacterium diversity exploration and will provide a framework for the way for future ecological characterizations.The genus  Flavobacterium in the phylum Bacteroidota (formerly Bacteroidetes)  isolated from various environments, including the rhizosphere, freshwater, sediment, and sea ice  concentrations or temperatures , spoilage foods, or clinical species  in their habitats. Some Flavobacterium spp. have a critical role in the cycling of organic matter  in diverse environments, with implications for bioremediation (The genus oidetes)  consistseratures 4. The geratures . Studies species –12. Neediation –15. Soediation –18.Primary research on the genus has largely focused on the identification and characterization of novel species from marine or terrestrial environments despite their putative ecological importance in diverse habitats. Habitat diversity can result from genomic integration of advantageous genes for adaptation to heterogenous ecological niches by horizontal gene transfer (HGT) . In addiFlavobacterium. However, an evolutionary, metabolic versatility or genomic dynamic investigation of the genus is lacking using pangenome analysis. Therefore, in this study, we isolated Flavobacterium strains from various samples in South Korea and fully sequenced their genomes. We then evaluated genomics traits with the publicly available Flavobacterium genomes by a pangenome approach. We grouped genomes based on the isolation sources  involved in the ethylmalonyl-CoA (EMC) pathway were found in some genomes analyzed in this study. The EMC pathway contributes to central carbon in numerous Alphaproteobacteria and Actinobacteria  over competitors in simple sugar-limited environments  in the glyoxylate cycle (an anabolic pathway) and an anaplerotic variant of the TCA cycle were found in some genomes . In addibacteria . Althougronments , 42 or hronments  than wasClostridium, Acidaminococcus, and Megasphaera of the phylum Bacillota (formerly Firmicutes) (Flavobacterium and suggests that members of this genus have high metabolic versatility.We identified genes for butyryl-CoA dehydrogenase (Bcd) and electron transfer flavoprotein (EtfAB) in all genomes. The Bcd/Eft complex for energy conservation is found in a few bacterial strains in the genera micutes) –46. ThFlavobacterium spp. have niche adaptation via plant glycan metabolism, including genes for rhamnogalacturonan utilization  provided by corals and algae. These results suggest that marine-associated members of the genus Flavobacterium could contribute to organic carbon recycling in their ecological niche . In particular, the significant differences were estimated between terrestrial Flavobacterium genomes (avg. 55.78) and marine genomes (avg. 45.64)  and host-associated genomes (avg. 46.34) . However, we did not detect significant differences between genomes belonging to the marine and host-associated genomes . In addition, we evaluated the number of CAZymes commonly predicted by three methods , revealing significant variation across (isolated) habitats , with no significant differences between each site. We then compared only the number of CAZymes predicted by at least one method relative to the total number of predicted genes, showing lower values for terrestrial Flavobacterium (avg. 17.17) than for host-associated and marine groups . However, we observed a statistically significant difference , particularly, between terrestrial and marine (post-hoc Dunn’s test P = 0.0198) and host-associated and terrestrial Flavobacterium genomes (post-hoc Dunn’s test P = 0.0239) . These findings were supported by the estimation statistics  of P = 0.0160 and P = 0.0028, respectively . Taken together, these results suggest that genes encoding CAZymes in genomes of terrestrial Flavobacterium might be densely distributed even though the number of the CAZymes was slightly lower than those in the other clades.To evaluate differences in carbohydrate degradation and/or transformation ability among isolation sites, the CAZy repertoire was evaluated based on the number of CAZymes  per Mbp (sequence length) . We deteendo-fucosidase for fucan hydrolysis in brown algae)  metabolism via GH78 and GH106 families , pectin (GH28), agar GH50), and fucoidan  gene families in all genomes , and fuc, 53. No n algae)  was idenn algae)  was founfamilies  47)..endo-fucFlavobacterium genomes than in genomes of other clades (Two GH families (GH29 [fucosidase] and GH50 [beta-agarase]) were slightly more abundant in host-associated r clades . In factr clades .Flavobacterium lutivivi isolated from activated sludge (https://flusurver.bii.a-star.edu.sg/). In addition, we found the other neuramidase sequence  in the genome of Siccirubricoccus deserti isolated from a desert sand sample  in the genome (GCF_014642275) of d sludge . Howeverd sample . Based od sample . In thisd sample . To the 8 family .Flavobacterium spp. might harbor a great number of genes involved in phage defense. The number of defense system-related genes in all genomes ranged from 61 to 182 (avg. 103.12) (Flavobacterium spp. genome (R2 = 0.741). The average percentages of total predicted genes classified as defense-related for the terrestrial, host-associated, and marine groups were 2.94%, 3.13%, and 3.06%, respectively. The proportion of defense-related genes differed slightly between the terrestrial and host-associated groups . These values were comparable to those for pathogenic or intracellular parasitic genera, such as Rickettsia, Francisella, Neisseria, and Yersinia   and was Yersinia . In addiFlavobacterium have been isolated and characterized in nonmarine environments  (Flavobacterium foetidum (GCF_004634245). The relative abundance of tRNA and rRNA genes was highest in the host-associated group . In addition, there were no significant differences in the proportion of rRNA and tRNA genes among total genes among groups . However, based on the number of the tRNA and rRNA genes, significant difference was only found in the tRNA gene count with respect to isolation sources . In particular, the tRNA gene count differed significantly between the host-associated and terrestrial groups  and supported estimation statistics  . While host-associated Flavobacterium spp. exhibited a higher abundance of tRNAs and rRNAs, it is difficult to assert that they possess superior colonization and/or stress adaptation capabilities in comparison to other groups. Nonetheless, marine taxa may have a higher capacity to exploit nutrients in resource-limited or low-temperature environments by a K-strategy (Most members of the genus shwater) . Speciesshwater) –65 or shwater) –68 in shwater) . No rRNAstrategy , 69.n = 87) of the 139 genes were identified as resistance genes for benzylkonium  chloride  and mercury . In addition, two genes accounted for more resistance genes in the terrestrial (68.48%) and marine (68.75%) groups than in the host-associated group (41.94%). Even though residual BAC levels are regulated in some countries, the potential environmental crisis is expected to increase. BAC is widely used as a disinfectant in industrial, healthcare, or animal culture industries  isolated from an eroded salmon fin  was identified as a complete lysogenic phage using Vibrant and was closely related to the Escherichia phage phiX174 (NC_001422), as determined using ViPTree and VIRIDIC . These results were supported by a phylogenetic analysis based on major capsid protein sequences  and Gokushovirinae, with circular single-stranded DNA genomes (Enterobacter) and obligate intracellular parasitic bacteria , respectively. To our knowledge, no members of the genus Microvirus have been identified in Flavobacterium spp. Krupovic and Forterre  of F. succinicans LMG 10402 in salmon.Bacteriophages, which are abundant viruses, have critical ecological functions, including roles in the control of microbial abundance and community structure as well as effects on genome plasticity in prokaryotes –76. We studies , 78 of d studies . We detelmon fin . F. succequences . phiX174 genomes , 81. MemForterre  reportedacterium –85. ItFlavobacterium isolated from coastal waters  in the class Flavobacteria contain functionally diverse rhodopsins. In fact, in nutrient-poor marine environments, marine Flavobacterium with PR genes may have an ecological benefit with respect to light-stimulating growth or osmotic stress elimination  was detected in the genomes and was adjacent to the PR gene. However, the genomes of four strains lacked genes (crtEIY) involved in retinal biosynthesis, except for crtB (phytoene synthase). Of note, only 16 strains isolated in this study had a gene encoding 4,4′-diapophytoene desaturase (CrtN). CrtN is a component of the C30 carotenoid  biosynthesis pathway (crtMNOPQ)  .Flavobacterium spp. under anoxic and oligotrophic conditions  were included in the HGT analysis. Only 279 HGT events were detected in the genus and species . The values for amino acid identity ranged from 74.46% to 100% (avg. 84.08%).As mentioned above, volution , 21. TheFlavobacterium genomes. We obtained 1–68 predicted recombination events per genome, except for two genomes [Flavobacterium plurextorum CCUG 60112 (GCF_002217395) isolated from farmed rainbow trout (Oncorhynchus mykiss) and strain N2469 isolated from the gut of Korean rockfish (Sebastes schlegelii)]. The average number of recombination events in host-associated genomes (28.53) was slightly higher than those in terrestrial (23.65) and marine (23.00) genomes. However, there were no significant differences in combination events with respect to habitat type . Three key model parameters  were estimated: R/θ = 1.016 (0.996–1.042), δ = 51.89 bp (51.12–52.59 bp), and ν = 0.065 (0.064–0.065) . In addition, the mutation rate was approximately 0.985 (θ/R). Each recombination event involved approximately 3.361 substitutions. The ratio of recombination to mutation was 3.413 (r/m = R/θ × δ × ν) (Flavobacterium genomes (0.821) than in other groups . However, the ratios of recombination to mutations for host-associated genomes (4.995) were higher than those of other groups  and all Flavobacterium genomes. This indicates that homologous recombination might contribute more substantially to host-associated Flavobacterium genomes than to genomes in other groups  or Marine Agar 2216 (Difco), and incubated at 30°C for 1 week under dark conditions. Single colonies were isolated through several subcultures (at least five) via transfer to new R2A or MA plates. To select bacterial strains belonging to the genus  studies , 105. Fiated see .For complete genome sequencing, the genomic DNA of each strain was extracted using a Monarch Genomic DNA Purification Kit  according to the manufacturer’s instructions. Then, a gDNA library was prepared using the SMRTbell  Kit  following the protocol for the PacBio RSII platform at DNA Link  and Theragen Bio . All sequence reads from each strain were completely assembled using Flye (v2.8.3)  or HGAP3n = 187 including 20 strains isolated in this study) were predicted and annotated using prokka (v 1.14.5) with default parameters. For KEGG Orthology (KO numbers) and COGs database analyses, BlastKOALA (Flavobacterium genomes (For the (pan)genome analysis, publicly available complete or draft genomes were downloaded from Genome Taxonomy Database  . One hunastKOALA  and RPS-astKOALA  were use genomes . Ribosom genomes . To anal genomes . Prophag genomes . For add genomes , VIRIDIC genomes , and VIC genomes  for prot genomes .https://www.r-project.org/). Phylogenomic analysis of the genomes, including those for closely related genera , was performed based on single-copy marker genes as described previously , the GFF3 file of all genomes was downloaded from GenBank (NCBI) or produced by Prokka (see above) with default parameters . The Roaeviously , 119. Breviously , and a meviously  with theeviously . The releviously  and ultreviously . ClonalFeviously , 125. Aveviously .Flavobacterium genomes, including genomes of new isolates in this study. Related bacteriorhodopsin sequences were obtained from publicly available data  or in the dark  with shaking at 180 rpm. For oligotrophic cultivation , bacterial cells were grown in artificial freshwater medium supplemented with 0.5% (vol/vol) peptone and 1× vitamin mixture (600). All culture experiments were performed in triplicate.Four  mixture . The pH Flavobacterium were compared by nonparametric analysis of variance , followed by Dunnett’s multiple comparison tests using GraphPad Prism 9 . Sequentially, if a significant difference was expected, additional Welch’s t-tests as estimation statistics  were performed for comparisons between two groups  in the genus o groups . Correla


Borrelia spielmanii, which manifested as a large erythema chronicum migrans rash. The patient completely recovered after a 15-day course of amoxicillin. Absence of pathognomonic signs prevented distinguishing B. spielmanii from other etiologies as cause in this case-patient. We describe a rare case of early Lyme borreliosis in France caused by  Borrelia garinii, B. afzelii, and, more rarely, B. burgdorferi sensu stricto. Lyme borreliosis is endemic in France except the southern region. Mean annual incidence of Lyme borreliosis in France may be as high as 84 cases/100,000 persons  in the Netherlands , I. ricinus ticks in the Crimea peninsula , which revealed presence of IgG and absence of IgM for B. burgdorferi sensu lato. Western blotting using LymeCheck Optima IgG & IgM  revealed presence of Borrelia spp.–specific IgG for p100, VlsE, p58, and p41 antigens and a faint band for B. spielmanii–specific ospC antigen  of sequenced amplicons showed 100% identity of the biopsy sequences with strain A14S of B. spielmanii for both the 910 bp–long portion of 16S rRNA gene (Genbank accession no. AF102056) and 260 bps-long amplicon of ospA (Genbank accession no. CP001469). We deposited our sequences into Genbank (accession nos. OR192893 and OR234396). The phylogenic tree (B. spielmanii strains.We amplified portions of 16S rRNA (nic tree  showed tB. azfelii, B. garinii, and B. burgdoferi sensu stricto had been identified from patients in France (B. spielmanii. Previously, only B. spielmanii infection and found only 2 published case reports. A 69-year-old woman from Slovenia showed skin manifestations described as redness, mild local itching, burning, and pain on the left knee and later a 24 × 20 cm ring-like lesion on the left thigh (B. spielmanii was detected in isolates from 4/242 patients with ECM from Germany and Slovenia (B. spielmanii lived in Munich where a higher proportion of ticks were positive for that pathogen. This finding argues for sporadic occurrences of the infection in other locations. We reviewed available literature on clinical descriptions of human cases of Borrelia spp. as potential causes of Lyme disease, prompting need for broader surveillance. However, additional well-documented reports on ECM as a manifestation of B. spielmanii are needed to provide new information about the epidemiology of this Borrelia in Europe. In summary, our study has contributed more data on Borrelia spielmanii in France. Additional information on study on Lyme disease caused by


COI) and cytochrome b (Cytb) genes and D-loop sequences were analyzed for 37 clariid catfish species. The highest intraspecific nearest neighbor distances were 85.47%, 98.03%, and 89.10% for COI, Cytb, and D-loop sequences, respectively. This suggests that the Cytb gene is the most appropriate for identifying clariid catfish and can serve as a standard region for DNA barcoding. A positive barcoding gap between interspecific and intraspecific sequence divergence was observed in the Cytb dataset but not in the COI and D-loop datasets. Intraspecific variation was typically less than 4.4%, whereas interspecific variation was generally more than 66.9%. However, a species complex was detected in walking catfish and significant intraspecific sequence divergence was observed in North African catfish. These findings suggest the need to focus on developing a DNA barcoding system for classifying clariid catfish properly and to validate its efficacy for a wider range of clariid catfish. With an enriched database of multiple sequences from a target species and its genus, species identification can be more accurate and biodiversity assessment of the species can be facilitated.DNA barcoding without assessing reliability and validity causes taxonomic errors of species identification, which is responsible for disruptions of their conservation and aquaculture industry. Although DNA barcoding facilitates molecular identification and phylogenetic analysis of species, its availability in clariid catfish lineage remains uncertain. In this study, DNA barcoding was developed and validated for clariid catfish. 2,970 barcode sequences from mitochondrial cytochrome c oxidase I ( Clarias) reached 94,277 tons in 2022 in Thailand [Clarias batrachus), bighead catfish , and whitespotted clarias (C. fuscus), are common in the global aquaculture market [C. gariepinus) through various trade channels have significantly been decreasing the populations of walking and bighead catfish. Introduction of North African catfish for improving production, such as hybridization by producing hybrids with other species, negatively affects native fish fauna via predation and hybridization [Over 3,000 species, 478 genera, and 36 families of catfish have been identified; these fish represent an important global protein source. In particular, the yield of clariid catfish (Thailand . Clariidhttp://www.fishbase.org) is limited by the impact of living conditions or processed products, rendering classification difficult or even impossible. New fish species have been described using molecular approaches that overcome the challenges associated with morphology-based identification [Diverse sources of clariid catfish and misidentification of certain species, such as whitespotted clarias, make it difficult to classify individual species precisely. Morphological character-based classification methods are insufficient and remain controversial due to morphologies's similarity among clariid catfish ,9,10. Idfication ,12. DNA fication ,13,14. Dfication . The accCOI) and cytochrome b (Cytb) genes and D-loop sequences, for which large sequence accessions have been deposited in the public repository of clariid catfish. Species identities were validated using nucleotide barcodes and species delimitation analyses, which revealed high accuracy.A quality check of available nucleotide sequences of clariid catfish in public repositories is necessary to establish an international standard for database management ,16. HereCOI, Cytb, and D-loop sequences, which are frequently used for identification of clariid catfish species and their phylogenetic analysis [COI and Cytb coding sequences were translated into the amino acid sequences using Molecular Evolutionary Genetics Analysis version X (MEGA-X) [Partial mitochondrial nucleotide sequences of clariid catfish were acquired from public repositories, with focus on the analysis ,15,17-20analysis . The sel(MEGA-X)  and alig(MEGA-X) .COI, Cytb, and D-loop sequence datasets. Substitution saturation was evaluated by plotting the number of transitions (s) and transversions (v) against Kimura 2-parameter (K2P) sequence divergences and comparing the information entropy-based index (Iss) with critical values (Iss.c) [ss is significantly lower than Iss.c, then substitution saturation does not occur in the sequences [COI, Cytb, and D-loop sequence datasets [The alignment length, variable positions, and GC content were compared among the  (Iss.c) -26, as i (Iss.c) . If Iss equences . Nucleotequences  was applequences -32. To iequences . The perequences . Barcodidatasets . To assedatasets . MarkersCOI, Cytb, and D-loop sequence datasets using a tree-based approach [Clarotes laticeps) was used as the outgroup for the COI and Cytb datasets  methods in MrBayes version 3.2.6 to examine the approach . Wide-he for COI ; and GenOI [Cytb ), whereaAB097696 ). Four cAB097696 . The treAB097696 ,29,31.http://species.h-its.org) using the default settings. An ultrametric tree was constructed using the "chronos" function in the R program and used as input for the GMYC analyses. The GMYC delimitation method with a single threshold model was performed using the "gmyc" function in the R package "splits" .The delimitation of clariid catfish species was assessed using two approaches: the Bayesian Poisson tree process (bPTP) method  and the https://datadryad.org/stash/share/JTJgvoR1X35SeML9B7A2H0BFaHRKPTz0JorqYW8BJyw).The full dataset and metadata from this study are available from the Dryad Digital Repository  by the Animal Experiment Committee of Kasetsart University, and conducted in accordance with the Regulations on Animal Experiments at Kasetsart University.COI, Cytb, and D-loop region of clariid catfish were compiled to test whether DNA barcoding could identify erroneous sequences in public repositories. Their sequence lengths were varied, and a tradeoff was observed between maximum of alignment length and taxonomic coverage. To address intraspecific variability, most species were represented by multiple specimens, with an average of 75 specimens per species. However, the data of only one specimen were available for 14 species, including 6 in the COI dataset , 9 in the Cytb dataset , and 2 in the D-loop sequence dataset . The nucleotide sequence database contained 2,970 barcoding sequences from 3,026 accession number sequences of clariid catfish, including 670 COI sequences (710 bp), 782 Cytb sequences (510 bp), and 1,518 D-loop sequences (593 bp). No stop codons or frameshift mutations were found in any of the sequences, indicating that they were all partial COI and Cytb genes fragments. The dataset ultimately included with varying base pairs and 346, 138, and 420 bp of parsimonious informative sites, respectively.The nucleotide sequences of mitochondrial COI dataset, the minimum intraspecific K2P sequence divergence was 0% for C. dumerilii, Valenciennes clariid (C. dussumieri), C. jaensis, mudfish (C. laeviceps), C. magur, and slender walking catfish (C. nieuhofii). The maximum intraspecific K2P sequence divergence was 133.09% ± 23.40% in North African catfish (C. gariepinus). The minimum interspecific K2P sequence divergence was 0% in Angolian walking catfish (C. angolensis), C. dumerilii, whitespotted clarias (C. fuscus), C. jaensis, smoothhead catfish , and C. magur. The maximum interspecific K2P sequence divergence was 7.90% ± 0.00% in mudfish (C. laeviceps). For the Cytb dataset, the minimum intraspecific K2P sequence divergence was 0% in Alluaud catfish, whitespotted clarias (C. fuscus), smoothhead catfish , C. maurus, C. pseudonieuhofii, and Werner's catfish (C. werneri). The maximum intraspecific K2P sequence divergence was 14.97% ± 0.00% in C. gabonensis. The minimum interspecific K2P sequence divergence was 0.26% ± 0.60% in C. punctatus. The maximum interspecific K2P sequence divergence was 11.31% ± 0.00% in Werner’s catfish (C. werneri) (C. fuscus). The maximum intraspecific K2P sequence divergence was 469.46% ± 578.35% in North African catfish (C. gariepinus). The minimum interspecific K2P sequence divergence between North African catfish and other clariid catfish was 110.63% ± 19.58%. The maximum interspecific K2P sequence divergence was 124.28% ± 19.58% for North African catfish. The maximum and minimum interspecific sequence divergences were used to establish a barcoding gap for species identification . According to the pairwise comparison, the Cytb barcoding gap was significantly different from those of COI  and D-loop gaps . By contrast, no significant differences were observed between the COI and D-loop sequence datasets . The COI, Cytb, and D-loop sequences had 85.47%, 98.03%, and 89.10% of intraspecific nearest neighbor distances, respectively  . For thefication . The intergences . The threctively .COI, Cytb, and D-loop sequence datasets strongly supported that 296 specimens belonged to a single clariid catfish species with a high posterior probability. The highest percentage of monophyletic groups (42%) was observed in the phylogenetic tree of the COI sequence dataset, whereas lower percentages of monophyletic groups (30% and 28%) were observed in the phylogenetic trees of the Cytb and D-loop sequence datasets, respectively  could not be used to differentiate between highly similar species because they showed conflicting results in the database, and they were classified as “group 2,” which exhibited higher-level similarity to multiple species. Moreover, intraspecific sequences divergences displayed interspecific sequence divergence, including Clarias sp., C. batrachus, C. camerunensis, C. angolensis, C. fuscus, and C. liocephalus, thus indicating a discrepancy between the nomenclature and DNA barcodes. This was evident in both public data repositories GenBank (https://www.ncbi.nlm.nih.gov/) and BOLD (https://www.boldsystems.org/). “Group 3” consisted of 11 unique COI sequences and 1 unique Cytb sequence that showed no similarity with most sequences under the same species name in public data repositories, such as Clarias sp., C. batrachus, C. fuscus, C. camerunensis, C. angolensis, and C. liocephalus. This suggests that the barcode reference data available for these species, which are obtained from public repositories, are insufficient. For validation of species identifications based on public repositories and BOLD, a reference database with at least three barcoded specimens for each species and a conspecific distance match of less than 1% are required, which is not applicable for the current clariid catfish barcodes in the COI, Cytb, and D-loop sequence dataset [Clarias sp., Angolian walking catfish (C. angolensis), bighead catfish , North African catfish (C. gariepinus), smoothhead catfish , Valenciennes clariid (C. dussumieri), walking catfish (C. batrachus), whitespotted clarias (C. fuscus), C. buthupogon, C. camerunensis, C. pachynema, C. gabonensis, and C. magur for COI and Clarias sp., bighead catfish , blackskin catfish (C. meladerma), blunttooth catfish (C. ngamensis), North African catfish (C. gariepinus), slender walking catfish (C. nieuhofii), walking catfish (C. batrachus), whitespotted clarias (C. fuscus), C. anguillaris, C. gabonensis, C. kapuasensis, forest walking catfish (C. leiacanthus), C. maurus, C. pseudonieuhofii, and C. punctatus for Cytb, suggesting that species identifications in clariid catfish using these markers remain uncertain. However, most of species whose D-loop sequences were examined were categorized into classes 2 and 3, indicating that D-loop sequences are not applicable for identification of clariid catfish species, which is consistent with the findings for D-loop sequences in other vertebrates [As of April 2023, public repositories have amassed 59,304 nucleotide sequences from clariid catfish, and they include 3,748 mitochondrial and 55,556 nuclear sequences. Most of the nuclear sequences were obtained from functional gene analyses -47. Over catfish ,15,17-20tebrates . The suctebrates . In Falatebrates , 98%–100COI gene for more than 1,000 teleost species indicated that the probability of intraspecific sequence divergence was 3% or less [Amphilius platychir) and Amphilius rheophilus, have shown a cutoff score at the species level that may be influenced by the rate of mutation or the existence of cryptic species [Cytb dataset showed no saturation in the saturation analyses, which is consistent with the positive barcoding gap, whereas the COI and D-loop sequence datasets showed saturation. The transition to transversion ratios of mitochondrial DNA in clariid catfish are similar to those in many teleosts with a larger excess of transitions compared to transversions [Cytb gene may be more informative in the clariid catfish lineage at the species level than the D-loop sequences that are appropriate for population studies, whereas the COI gene may not be fit for systematic studies of specific lineages [Cytb dataset that was –0.15 to 0.11 for the species identification of clariid catfish, while the gaps of the COI and D-loop sequence datasets were –1.64 to 0.08 and –1.12 to –0.98, respectively. These values are consistent with the average barcoding gap of 83.6% reported in studies in other fish [COI gene [C. magur, may have been mistaken for walking catfish because of differences in head shape and pectoral spine serration from those found in Southeast Asia [The analysis of nucleotide sequence divergence of the  or less . However species . Low seq species . In the versions . The Cytlineages . Intrasplineages . To imprlineages . In the her fish ,20,54. TCOI gene ,15. Mislast Asia . Karyotyast Asia . The preLarge intraspecific sequence divergences were observed in North African catfish, which were more than expected in the present study. This may be caused by the genetic difference between domesticated strains of the same species. Nonetheless, all specimens were classified as belonging to the same species, using the bPTP and GMYC methods. The ZZ/ZW sex determination system is believed to be the same as the ancestral system for North African catfish, which is supported by most of the reported sex chromosome systems from Africa and Israel. However, some studies of different populations from Israel, Hungary, and China suggest an XX/XY system or the possible coexistence of both sex chromosome systems or polygenic sex determination in North African catfish -62. UnliCOI and D-loop sequence datasets had lower power than the Cytb dataset because they had fewer informative sites. The overlap in the distribution of intra- and interspecific sequence divergence resulted in unclear cutoff values for species identification. The paradox of deep intraspecific and shallow interspecific sequence divergence indicates the need for further verification of specimen collection accuracy. However, species identification based on the Cytb dataset was unambiguous for Alluaud's catfish , whitespotted clarias (C. fuscus), North African catfish (C. gariepinus), C. intermedius, smoothhead catfish , bighead catfish , blunttooth catfish (C. ngamensis), slender walking catfish (C. nieuhofii), C. olivaceus, C. pseudonieuhofii, and Werner's catfish (C. werneri). The optimal quality and traceability of the data associated with reference barcodes must be ensured by developing and adhering to best practices. To build a robust reference sequence library in public repositories, specimens from various geographical locations are required improvement of the Cytb barcoding gap value will provide more reliable representative data. However, cryptic species complexes in clariid catfish lineages may cause an overlap intraspecific and interspecific sequence divergences.The Pangasius macronema) and striped catfish , resulting from geographical isolation and substantial habitat reorganization [Pseudotanganikallabes prognatha (new genus and species of clariid catfish) from Clarias might be larger than that detected by morphology. This raises the question of whether Pseudotanganikallabes and Clarias belong to the same genus with high intraspecific genetic variability or multiple species. Such ambiguously identified species often form species complexes when close examination is carried out for specimen collected from broad geographicareas [C. gabonensis in our dataset is required to determine the presence of new species.DNA barcoding and species delimitation methods can reveal the existence of cryptic species among known species, thus providing an objective means of testing evolutionary independence. The integration of these methods is crucial for accurately determining regional biodiversity, and the consistent results obtained through all means have confirmed the boundary of species. Compared the results of sequence divergence with species delimitation in North African catfish, one individual was clustered away from walking catfish and whitespotted clarias individuals, with divergence in the interspecific range (group 3 and class 3) and clear cutoff intervals, using the two specific delimitation methods (the bPTP and GMYC methods). Whether this sequence belongs to a different cryptic species or represents a distant gene pool within the same species remains unclear. This individual may indicate a different ecotype with a diverse geographical location or may have resulted from a species identification error by observation. Such high intraspecific sequence divergence has also been reported previously in haiwels (nization . Intraspnization . Our resnization , which ihicareas . A thoroThe BOLD identification system and a public data repository for BLASTN comparison of nucleotide sequences were created for different purposes and operated differently ,67. BOLDCytb gene barcoding is highly effective for identifying clariid catfish precisely because it is reliable and informative with a large dataset of nucleotide sequences. Thus, guidelines for international standards and digital infrastructure to manage genetic resources of clariid catfish have been introduced, which brings out the full potential of biological resource stored in public nucleotide repositories. Future studies should prioritize barcoding for specific clariid catfish groups, such as walking catfish or North African catfish, which have high utility value for aquaculture production, and validate the efficacy of the marker as a barcoding region for a broader selection of species. A comprehensive DNA barcode library can also aid in identifying new species and improving our understanding of endemic clariid catfish resources.The lack of accurate taxonomic and genetic information has caused misidentification of clariid catfish, resulting in hindering the monitoring of habitat changes caused by habitat loss, overfishing, and non-native species introductions, which is prerequisite for planning their conservation and aquaculture. The limitations of BOLD and present public repositories for clariid catfish, where sequences from potentially misidentified specimens were contained, were revealed in this study. Thus, taxonomic uncertainties in challenging morphological groups and similar situations can be resolved. Therefore, the validated DNA barcode sequence library of clariid catfish obtained in this study can serve as a reference for examining species boundaries among closely related taxa, which is required for planning conservation strategies and increasing aquaculture productivity. Mitochondrial


Hycleus cichorii and Hycleus phaleratus are the two most commercially significant species due to their capacity to produce CTD in males. In this investigation, we conducted a gene expression profiling analysis of male and female individuals of these two species, utilizing the Illumina Hiseq4000 platform. We identified 7,983 expressed genes, including 2,823 differentially expressed genes (DEGs) shared by both male and female blister beetles. Nineteen genes related to CTD biosynthesis in the terpenoid backbone biosynthesis pathway were identified, including hydroxymethylglutaryl-CoA reductase , which demonstrated a significant correlation with CTD content. Furthermore, hydroxymethylglutaryl-CoA synthase  and isopentenyl-diphosphate Delta-isomerase  were also found to be significantly up-regulated in males. Comparative analysis revealed that NADP+-dependent farnesol dehydrogenase  and farnesyl diphosphate synthase  had the highest copy number in these beetles, significantly higher than the copy number of the other four non-Meloidae insects. The analysis of the protein-protein interaction network of genes related to CTD biosynthesis revealed that the acetyl-CoA C-acetyltransferase  gene was the central gene, exhibiting greater expression in male blister beetles than in females. This study offers novel insights into the mechanisms of CTD biosynthesis in blister beetles and enhances our comprehensions of the association between particular genes and CTD content.Cantharidin (CTD) is a well-established defensive toxin synthesized by blister beetles, displaying both therapeutic potential and toxicity. Among these beetles,  Jiang, Lü et al. found that 3-hydroxy-3-methylglutary-CoA reductase (HMGR) and cytochrome P450 gene (CYP4BM1) also affect CTD production in Epicauta chinensis [E. chinensis after mating, and multiple tissues may be involved in the process.Cantharidin C10H12O, CTD, a globally –8. At thglobally . Two patglobally –14. Seveed genes . Anothers of CTD . Additiohinensis . FurtherCTD is a sesquiterpenoid biosynthesized primarily by adult male blister beetles and transferred to adult females as a defensive substance to safeguard their offspring , 12. MorHycleus cichorii Linnaeus and Hycleus phaleratus Pallas are listed in the Pharmacopoeia of the People’s Republic of China and commonly used in artificial culture [H. cichorii and H. phaleratus are currently available in public databases. This observation, along with the availability of correlational studies in these organisms [H. cichorii and H. phaleratus as models for investigating CTD biosynthesis genes. We aimed to explore sex-based differences in gene expression levels between H. cichorii and H. phaleratus using the Illumina Hiseq4000 platform, by selecting samples from both females and males of H. cichorii and H. phaleratus obtained after 30 days of segregation breeding. In addition, we investigated CTD biosynthesis genes in the "terpenoid backbone biosynthesis" pathway and compared them with four non- Meloidae insects. We constructed an interaction network of CTD biosynthesis genes firstly. Our findings provide valuable insights into sex-related expression, as well as CTD biosynthesis in Hycleus.RNA sequencing (RNA-seq) is a highly effective method for measuring gene expression levels and provides a strong foundation for future research. Gene networks play an important role in various organisms and systems, effectively helping to reveal the essential rules of a large number of biological processes and reactions in organisms . The dry culture , 23–25.  culture , 26, 27,rganisms , 23–25, Hycleus cichorii and Hycleus phaleratus adults were collected from soybean fields  in Luodian, Guizhou Province, China. After morphological species and gender identification, the insects were immediately stored in liquid nitrogen for RNA extraction. We sequenced female and male samples of these two beetles using the Illumina 4000 platform. Total RNA (~10 μg) was extracted from whole body using the TRIzol Reagent . Potential genomic DNA was removed by using DNase I. Poly (A) + RNA was extracted using poly-T oligo-coated magnetic beads. After purification, first-strand cDNA was synthesized using the Superscript II reverse transcriptase  and random hexamer primers. The cDNA was further converted into double-stranded DNA. The library was constructed using the TruSeq® RNA Sample Prep Kit (Illumina). After library quality control, pair-end sequencing was performed using Illumina HiSeq™ 4000 platform at BGITech .Newly emerged https://deweylab.github.io/RSEM/rsem-calculate-expression.html) and Zhao, X., et al [Raw reads were filtered by SOAPnuke  to obtai., et al , the bowp-value less than or equal to 0.001.Estimated mapped read count matrix was used to calculate fragments per kilobase per million mapped fragments (FPKM) by using RSEM  package.p-value was less than 0.05.To understand the functions of the DEGs, ClusterProfiler  was carrTribolium castaneum (TCAS), Dendroctonus ponderosae (DPON), Bombyx mori (BMOR), and Drosophila melanogaster (DMEL), were downloaded from Ensembl database (ftp://ftp.ensemblgenomes.org/pub/metazoa/release-49). Subsequently, the proteins were searched again the KEGG database with diamond program . The best hits were selected to obtain the CTD-related genes. Protein-protein interaction (PPI) network among the CTD-related genes was analyzed using the Search Tool for the Retrieval of Interacting Gene (STRING) [The key enzymes in the pathway of ‘terpenoid backbone biosynthesis’ (map00900) were annotated using the diamond program  with par(STRING)  database®FAST Universal qPCR Kit  on an optical 96-well plate with a Step One PlusTM Real Time PCR System  in accordance with the manufacturer’s instructions. Thermal cycling conditions involved 40 cycles at 95°C for 5 s and 60°C for 30 s. After thermal cycling, we used the ABI Prism 7500 SDS software to analyze and adjust the results automatically. The data were exported to EXCEL for a 2-ΔΔCt analysis. We performed four independent biological replicates for each treatment.To validate the expression of three key CTD-related genes between sexes, we conducted quantitative real-time PCR (qRT-PCR) analysis using the same source of insects described above. These insects were collected in Luodian, Guizhou, China. For each sample, we extracted total RNA from approximately 5 individuals and used the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit  to synthesize cDNA from 1 μg of total RNA following the manufacturer’s instructions. To normalize cDNA templates, we used UBE3A (ubiquitin-protein ligase E3A) and RPL22e  as internal references, as suggested by Vandesompele et al. . We confH. cichorii was more than that in H. phaleratus, and the number of genes expressed in female was more than that in male. The hierarchical clustering showed that the samples were generally divided by sex rather than breed  to provide insights regarding CTD biosynthesis. In total, 19 CTD-related genes were detected, all of which are only involved in the MVA pathway , HMGS, and IDI , HMGR, ACAT, FDPS, mevalonate kinase , diphosphomevalonate decarboxylase , HMGS and IDI , LOC662066, HMGS, IDI, LOC664130, LOC656773, geranylgeranyl diphosphate synthase, type III (GGPS1), LOC659113 and FDPS  was determined to be the core gene of the interaction network. Both RNAseq and Quantitative Real-Time PCR demonstrated that the expression level of ACAT  in males was higher than that in females . Most blister beetles are sexually dimorphic with respect to CTD production, requiring a deeper understanding of the sex expression and CTD biosynthesis. Many studies have employed Illumina sequencing platform as it provides high-quality sequences for gene expression analysis. In this research, we identified 3,955 DEGs in H. cichorii and 6,122 DEGs in H. phaleratus with RNA-seq reads from both females and males of H. cichorii and H. phaleratus obtained after 30 days of segregation breeding. As breeding conditions mature, biological replicates should be included in the future study because they can improve the quality and reliability of analysis results [M. cichorii, these genes are only involved in MVA pathways. FOHSDR is the gene with the highest number of copies, and it has significantly more copies in the two blister beetles than in the other four non- Meloidae insects. The recent comparative genomic analysis of M. aulica suggested that none of the known genes related to CTD biosynthesis were included in expanded significantly gene families under GO and KEGG enrichment analyses [FOHSDR gene family is expanded in two blister beetles. Two previously uncharacterized CTD related genes were found in M. aulica with a draft genome [HMGR was reported to exhibit a positive correlation in the fat body of male E. chinensis [HMGR is higher in male H. phaleratus than in female H. phaleratus. Besides, two CTD-related genes, namely HMGS and IDI, have caught our attention because they were significantly up-regulated in male of two blister beetles. These findings were also confirmed by PCR experiments. Interaction networks provide insights for inferring the function of genes or proteins [ACAT , the first enzyme that catalyzes the conversion of acetyl-CoA into acetoacetyl-CoA in the MVA pathway [ACAT also displayed higher expression in males compared to females. This is the first study to characterize the CTD-related interaction network with STRING database [CYP15A1) and JH esterase (JHE) [CTD is a secondary metabolite derived from blister beetles and CTD biosynthesis is becoming a research hotspot. As reported, CTD has the potential to be a major compound used in resistance management . More im results . The GO analyses . Furthert genome . We belihinensis , and we proteins . We founproteins , 43. ACA pathway , was foudatabase . The divH. cichorii and H. phaleratus were investigated by using Illumina deep-sequencing technology, and CTD biosynthesis-related genes were explored. A total of 2,823 DEGs were identified by comparing female and male individuals of the two blister beetle species. Additionally, nineteen CTD biosynthesis-related genes involved in the ‘terpenoid backbone biosynthesis’ pathway (map00900) were obtained. HMGS and IDI, the two key genes in the main MVA pathway, were up-regulated in male beetles from both species. Comparative analysis revealed that FOHSDR and FDPS are the top two highest copy number genes, and the copy number of these two genes is much higher than that of the other four non- Meloidae insects. A CTD biosynthesis-related gene network was firstly constructed using the STRING database, which revealed 584 pairs of interactions. ACAT, the core gene in the network, was up-regulated in male beetles. These results provide a broader view of CTD biosynthesis mechanism as well as provide valuable insights for genomics study in Hycleus.In summary, sex expression profiles of S1 FigH. cichorii, HCI_M refers to male H. cichorii, HPH_F refers to female H. phaleratus, and HPH_M refers to male H. phaleratus.HCI_F refers to female (DOCX)Click here for additional data file.S2 FigH. cichorii, HCI_M refers to male H. cichorii, HPH_F refers to female H. phaleratus, and HPH_M refers to male H. phaleratus.HCI_F refers to female (DOCX)Click here for additional data file.S3 FigHycleus.The highlighted red boxes represent the genes that are present in the (DOCX)Click here for additional data file.S4 FigH. cichorii (HC-F), male of H. cichorii (HC-M), female of H. phaleratus (HP-F), male of H. phaleratus (HP-M).The relative quantities indicate the levels of putative transcripts normalized to the internal standard UBE3A (ubiquitin-protein ligase E3A) and RPL22e . The bars indicate the standard deviation of four repeats. Note: female of (DOCX)Click here for additional data file.S1 Table(XLS)Click here for additional data file.


Enterococcus faecium (E. faecium) is a commensal bacterium found in the gastrointestinal tract. In immunocompromised patients, such as hematopoietic stem cell transplant (HSCT) recipients, it is a frequent course of extraintestinal infections. Identifying HSCT patients who are predisposed to developing such infections could enable the development of targeted strategies to reduce their risk. We aimed to develop a machine learning (ML) model to predict E. faecium infection in HSCT patients based on gut metagenomic data.E. faecium infection. We used 80% of the data to train the model to predict clinical E. faecium infection. The model was evaluated on the remaining 20% of the data  using the area under the curve (AUC) and confusion matrix.In a cohort of HSCT recipients, fecal samples were collected around the time of transplantation. The samples generated metagenomic data derived from high-throughput DNA sequencing. The patients were followed for clinical infections up to 30 days post-sampling. As an interpretable ML model, we utilized the Q-Lattice, implementing symbolic regression to identify interactions among variables. We chose 20 bacterial species and 37 antibiotic resistance genes (ARGs) within the gut, associated with clinical E. faecium infections were observed within 30 days after the sampling of 656 gut material retrieved from 276 HSCT recipients. We found three bacterial species  and one ARG (patA) that contributed to predicting the E. faecium infection . All features in this final model, except for patA, were negatively associated with E. faecium infection (Spearman’s |rho| < 0.2). The ML model demonstrated 100% sensitivity and negative predictive value in predicting the infection in both the development and validation sets .Twenty-nine clinical an overview of the prediction modelPrediction model for E. faecium Infection as a block diagram (on top) where the predictors Bacteroides dorei, Blautia wexlerae, Fusicatenibacter saccharivorans, and patA in the gut were selected by the model and the model representation as a closed-form equation (bottom). The confusion matrices display model performance on the development set (top) and validation set (bottom) where true denotes positive (infected) and false denotes negative (not infected). Also “expected” here means “truth” or “actual” class. Software package, Feyn in Python, was used for the model development and visualizations.E. Faecium infection.The ML approach identified complex relationships between multiple microbial agents in predicting the risk of the infection. The predictive results were internally validated but requires external validation. These findings suggest that ML analyses of the gut microbiome is useful in predicting clinical Roger Paredes, M.D, Ph.D., Atea: Advisor/Consultant|Gilead: Advisor/Consultant|Lilly: Advisor/Consultant|MSD: Advisor/Consultant|MSD: Grant/Research Support|Pfizer: Advisor/Consultant|Roche: Advisor/Consultant|Theratechnologies: Advisor/Consultant|ViiV: Advisor/Consultant|ViiV: Grant/Research Support


Borrelia genospecies, and host complement-mediated killing is a major determinant in these interactions. In North America, only a subset of Borrelia burgdorferi lineages cause the majority of disseminated infections in humans. We hypothesize that differential resistance to human complement-mediated killing may be a major phenotypic determinant of whether a lineage can establish systemic infection. As a corollary, we hypothesize that borreliacidal action may differ among human subjects. To test these hypotheses, we isolated primary B. burgdorferi clones from field-collected ticks and determined whether the killing effects of human serum differed among those clones in vitro and/or whether these effects were consistent among human sera. Clones associated with human invasiveness did not show higher survival in human serum compared to noninvasive clones. These results indicate that differential complement-mediated killing of B. burgdorferi lineages is not a determinant of invasiveness in humans. Only one significant difference in the survivorship of individual clones incubated in different human sera was detected, suggesting that complement-mediated killing of B. burgdorferi is usually similar among humans. Mechanisms other than differential human complement-mediated killing of B. burgdorferi lineages likely explain why only certain lineages cause the majority of disseminated human infections. Reservoir host associations have been observed among and within  Borrelia burgdorferi sensu lato complex [Borrelia genospecies [B. afzelii and B. garinii are considered rodent-associated and avian-associated, respectively [Borrelia genospecies can survive in the presence of serum from the taxonomic class of hosts they typically infect but not others [Borrelia species may also be due to differential serum (complement)-mediated killing [Host associations exist between and within genospecies of the  complex ,2,3,4,5.ospecies ,2,4. Theectively ,7. Partit others ,2,4,8. Tt others . The comt others ,11. We a killing .Borrelia burgdorferi sensu stricto (hereafter B. burgdorferi) is a tick-borne human pathogen with observed host associations among its diverse lineages [B. burgdorferi lineages in the United States which can be distinguished by the nucleotide sequences encoding the outer surface protein C (OspC) [B. burgdorferi lineages have a higher fitness in experimentally infected animals [B. burgdorferi lineages [B. burgdorferi lineages [B. burgdorferi lineages characterized by ospC genotypes A, B, I, K, and N are considered human-invasive, and the other lineages rarely disseminate in humans [lineages ,13. TherC (OspC) ,14,15. E animals ,17,18. Flineages ,20,21. Hlineages ,23,24,25n humans .B. burgdorferi lineages may be mediated by differential resistance to host complement. This idea is supported by recent experiments demonstrating that differential resistance to killing by host serum is a factor contributing to the fitness of different strains within B. burgdorferi in laboratory animals [B. burgdorferi has a multifunctional and redundant set of anti-complement outer surface proteins [B. burgdorferi strains and confer strain-specific survival differences in host serum [B. burgdorferi lineages that efficiently infect a particular host will survive better in serum collected from that host compared to other B. burgdorferi lineages. The observed host preferences by different  animals ,27. B. bproteins ,9. The ast serum ,12,27,28B. burgdorferi lineages. We determined whether human-invasive clones have higher survival probabilities compared to human noninvasive clones and if these effects are consistent among human sera. B. burgdorferi is considered to be resistant to the borreliacidal activity of human serum [B. burgdorferi have typically been used in similar experiments, and survival in human serum has been found to vary widely among strains [B. burgdorferi clones characterized by their ospC sequence. The B. burgdorferi cultures used in these experiments better represent the spirochetes potentially transmitted to humans by feeding ticks.In this study, we focused on the observed associations between humans and an serum ,2,8,9,29 strains ,33,34,35B. burgdorferi clones were isolated directly from field-collected Ixodes scapularis ticks by limiting dilution and were frozen at −80 °C [ospC) sequence using Luminex technology , as previously described [ospC sequences were detected by either method. The clones used in this study represent two invasive B. burgdorferi lineages (ospC genotypes A and I) and three noninvasive lineages . Hereafter, they are referred to as clones-A, -I, -G, -H, or -M. The actual invasiveness to humans of the isolated clones cannot be verified, but their predicted invasiveness is based on previous research [t −80 °C ,37 .Human blood was collected from two males and one female, hereafter referred to as PP, CR, and LJ, respectively. This study did not meet the definition of human subject research, and IRB approval was not required. The blood was collected in serum separator tubes, allowed to clot at room temperature for up to 45 min and then centrifuged at 1500 RCF for 10 min to separate serum. Sera from multiple tubes from each donor were pooled, filtered through a 0.22 um filter, and stored in 0.5 mL aliquots at −80 °C. Aliquots were tested for a functional alternative complement pathway using the Total Complement Functional Screen ELISA kit  to ensure complement activity was retained in the collected serum. One aliquot of human serum was heat-inactivated at 56 °C for 30 min before testing. Complement activity was detected in serum collected from each donor, while complement activity was completely abolished in the heat-inactivated serum aliquot. All blood donors were negative for anti-B. burgdorferi clones to human serum was measured as previously described [6 cells/mL in BSK-H media without rabbit serum . Then, 400 μL of human serum was added to 400 μL of culture (yielding 50% human serum concentration), and 200 μL of this mixture was aliquoted into triplicate wells on a 96-well plate and incubated at 34 °C for 4 h. BSK-H incomplete was substituted for 50% human serum as a positive control. Viable cells were counted by darkfield microscopy at 0 and 4 h. Viable cells were defined as those that were visibly motile and retained a characteristic spirochete shape [n = 6 total replicates for positive control). The sensitivity of the escribed . The clote shape ,40. The Odocoileus virginianus) as a control to observe and validate the serum-mediated killing of spirochetes [B. burgdorferi growth [The same assay was performed using the clones mixed with 50% white-tailed deer serum . The five clones survived in human serum  but wereB. burgdorferi clones was observed, but it did not conform to expectations based on the clones’ human-invasive or -noninvasive designation. It was expected that invasive clones-A and -I would have a significantly higher survivorship compared to the noninvasive clones -G, -H, and -M. However, clone-A had the lowest percent survival values, while clone-I had a survivorship similar to clones-G, -H, and-M when incubated in human serum (n = 18) included clone-A. None of these pairwise comparisons yielded a significantly higher survivorship of an invasive clone compared to a noninvasive clone. Differential survival among the an serum . Severalan serum . The oveThe survivorship of individual clones incubated in human sera  was generally homogenous . The perB. burgdorferi lineages. Reservoir host associations exist among Borrelia genospecies, and these associations are maintained by differential host-serum-mediated killing [Borrelia species to strains within a single species, B. burgdorferi [B. burgdorferi lineages [B. burgdorferi lineages associated with human-invasive and -noninvasive infections had different survival rates in media supplemented with human serum.We hypothesized that differential resistance to human complement may be a determinant of differential human infectivity among  killing ,2,4. Thegdorferi ,27,41. Plineages ,23,24,25B. burgdorferi lineages are more resistant to human-complement-mediated killing compared to the lineages that rarely disseminate in humans. Previous research has demonstrated that B. burgdorferi is resistant to human-serum-mediated killing but that variation can occur among strains within B. burgdorferi [B. burgdorferi clone survived, and significant differences in survivorship were detected among the clones. The observed borreliacidal activity was likely complement-mediated, but we cannot exclude the possibility of other heat-sensitive factors. The survival differences, however, did not correspond to whether the clones are considered invasive or noninvasive in humans as defined by their ospC genotype. We used a single clone to represent a B. burgdorferi lineage, but spirochetes within a B. burgdorferi lineage can have different serum-survival phenotypes and invasiveness levels in hosts [ospC genotype may help resolve this potential concern. These data do not support the hypothesis that the invasive gdorferi ,29,31,34in hosts ,21,22,27We speculated that the survival of a clone in human serum may be impacted by the specific individual from whom the serum was collected. Previous studies with similar assays have typically used pooled human serum from many individuals, serum collected from one donor, or do not clearly match serum-survival results with specific individuals if there are multiple donors ,33,34,42B. burgdorferi lineages and their predicted invasiveness in humans. Recent studies have reported B. burgdorferi strain-specific survival in different host sera [B. burgdorferi strains is a major factor affecting fitness in laboratory animals [B. burgdorferi strains does not always match their infectivity in hosts and is most likely due to complement-independent mechanisms [B. burgdorferi strain cN40 had a high survivorship in white-footed mouse (Peromyscus leucopus) serum but a low fitness in mice [B. burgdorferi strains and their capacity to cause disseminated infections in C3H/HeJ (Mus musculus) mice, although M. musculus complement is unstable in vitro, which may have affected these results [It is unclear why our data do not show a connection between the serum resistance of ost sera ,27,41. T animals ,27. Howechanisms ,27. For  in mice  also fou results ,43. B. burgdorferi lineages to infect humans. OspC may play a direct role, since it is polymorphic and interacts with several host molecules [ospC gene can be used for defining B. burgdorferi lineages, other linked genetic elements likely have a greater impact on the invasiveness of lineages in humans [B. burgdorferi lineages do exhibit differential resistance to human-complement-mediated killing which corresponds to invasiveness, but these differences were not detected in our in vitro assay. Other factors besides human-complement-mediated killing are likely facilitating the differential propensity of certain olecules ,46,47,48olecules . It is an humans . AnotherB. burgdorferi clones representing canonical human-invasive lineages are not more resistant to human-complement-mediated killing. These results also show that human-complement-mediated borreliacidal activity does not usually differ among individuals. Additional research is necessary to understand the determinants of invasiveness in humans by different B. burgdorferi lineages. A more detailed understanding of the factors shaping associations between humans and B. burgdorferi lineages may lead to better Lyme disease management strategies. We have demonstrated that