Caenorhabditis species disperse by traveling on invertebrate carriers. Here, we describe the morphology of a stress-resistant, dauer-like larval stage of the nematode Caenorhabditis inopinata, whose adults can grow to be nearly twice as long as its close relative, the model organism C. elegans.Body size is a fundamental organismal trait. However, as body size and ecological contexts change across developmental time, evolutionary divergence may cause unexpected patterns of body size diversity among developmental stages. This may be particularly evident in polyphenic developmental stages specialized for dispersal. The dauer larva is such a stage in nematodes, and C. inopinata is on average 13% shorter and 30% wider than the dauer larvae of C. elegans, despite its much longer adult stage. Additionally, many C. inopinata dauer-like larvae were ensheathed, a possible novelty in this lineage reminiscent of the infective juveniles of parasitic nematodes. Variation in dauer-like larva formation frequency among twenty-four wild isolates of C. inopinata was also observed, although frequencies were low across all isolates (< 2%), with many isolates unable to produce dauer-like larvae under conventional laboratory conditions.We find that a dauer-like, stress-resistant larval stage in two isolates of Caenorhabditis species thrive on rotting plants and disperse on snails, slugs, or isopods (among others) whereas C. inopinata is ecologically divergent and thrives in fresh Ficus septica figs and disperses on their pollinating wasps. While there is some unknown factor of the fig environment that promotes elongated body size in C. inopinata adults, the small size or unique life history of its fig wasp carrier may be driving the divergent morphology of its stress-resistant larval stages. Further characterization of the behavior, development, and morphology of this stage will refine connections to homologous developmental stages in other species and determine whether ecological divergence across multiple developmental stages can promote unexpected and opposing changes in body size dimensions within a single species.Most The online version contains supplementary material available at 10.1186/s40850-022-00131-y. One obvious fact of the life cycles of most organisms is that they typically get larger as they develop. Indeed, most animals span at least an order of magnitude in body size within a single generation through the course of development , 2. HoweThe ontogenetic niche of an organism is not limited by resource use. Organisms often have specialized developmental stages for specific life history strategies, and such partitioning is frequently used for dispersal. Locusts have a complex polyphenism in overcrowding conditions that transform solitary morphs into gregarious morphs that constitute famine-inducing swarms . Aphids Caenorhabditis elegans and C. inopinata live in substantially different habitats and differ substantially in size. In crowded, low food, high temperature, or otherwise stressful conditions, C. elegans is known to develop into a dispersal-specialized juvenile phase known as the dauer larva [C. elegans dauer larvae travel to new resources on invertebrate carriers such as snails, slugs, isopods, and myriapods [C. elegans dauer stage, the dauer stage from C. inopinata has never before been described. Do the substantial differences observed in the adult species of these close relatives persist throughout this developmental stage as well?As adults, the free-living bacterivorous nematodes er larva . The dauyriapods . The dauyriapods . Once ityriapods . As the yriapods . While aC. inopinata adults grow nearly twice as long as C. elegans, and they also develop nearly twice as slowly as C. elegans [C. inopinata lives in fresh Ficus septica figs and disperses on fig wasp pollinators [C. inopinata . Rather than echoing the shape differences observed in the adults, C. inopinata dauer-like larvae are shorter and fatter than C. elegans dauers. In addition, C. inopinata dauer-like larvae also exhibit a novelty in ensheathment that resembles the infective larvae of parasitic nematodes. This suggests that the evolution of body shape may not be in the same direction across all developmental stages, and stage-specific ecological contexts could potentially drive opposing directions of morphological change. elegans –17. Addilinators , 18. HerC. inopinata adults are much longer  than those of C. elegans . Reminiscent of the ensheathed infective larvae of parasitic nematodes [C. elegans dauers (n = 47 worms). Additionally, C. inopinata dauer-like larvae revealed pharyngeal constriction compared to conspecific L3 larvae . The C. inopinata dauer-like stage is then stress-resistant and maintains many of the hallmarks of diapause, dispersal nematodes.exposure , which kbriggsae . We alsoata Fig. B-D. The olt 46%, /46 wormsematodes , this walt 46%, 2/46 wormsC. inopinata dauer-like larvae were shorter in length than C. elegans dauers . This is surprising because all other developmental stages of C. inopinata were longer than comparable stages of C. elegans . However, this difference in tail length does not account for all of the length difference as C. inopinata dauer-like larvae were shorter than C. elegans dauer larvae even when tails were excluded . Conversely, C. inopinata dauer-like larvae were wider than C. elegans dauers . This increased width was observed across three independent series of measurements, albeit with different effect sizes . These width measures do not include the sheath , as this would inflate width estimates in ensheathed C. inopinata animals. Regardless, the increased width of the C. inopinata dauer-like larva is also unexpected because previous observations of non-dauer developmental stages reveal much smaller or negligible differences in width of reproductive stages . As C. inopinata dauer-like larvae are shorter and fatter than C. elegans dauers, they then appear to occupy different regions of morphological space relative to their respective non-dauer stages . Pairwise comparisons reveal C. inopinata dauer-like larvae to occupy different regions of morphospace compared to the dauer larvae of the other species  with the exception of the C. inopinata L2 larva (adjusted p = 0.26). k-means clustering likewise classifies C. inopinata dauers in clusters distinct from those of C. elegans, C. briggsae, and C. tropicalis dauers . In this framework, the C. inopinata dauer harbors distinct cluster classification from all other species-stage groups (chi-square adjusted p < 0.05).To further explore the hypothesis that s clade . However, the primary discriminant axis is largely driven by width . Indeed, although C. inopinata dauer-like larvae are shorter in length than C. elegans dauers , and C. briggsae dauers , these differences are small (6–15% shorter in length). Conversely, C. inopinata dauer-like larvae are much larger in width than the dauer larvae of all the other species . Thus, while C. inopinata dauer-like larvae occupy a distinct region of morphospace compared to the dauer larvae of other species, this is largely driven by increased width. This is in stark contrast to C. inopinata adults, who are dramatically elongated compared to their close relatives and harbor negligible differences in width . At 32 °C, C. inopinata was inviable . Additionally, in nature, dauer-like larva formation in C. inopinata is tied to fig and fig wasp developmental events [C. inopinata on them (fruit media prepared as in [C. inopinata did not proliferate on this fig media (N = 9 plates with complete sterility). We then continued isolating C. inopinata dauer-like larvae at 25 °C on NGM plates. As variation in dauer formation frequency has long been noted in C. elegans [C. inopinata wild strains from Okinawan Ficus septica figs . This is in contrast to other Caenorhabditis species, where many lines are able to produce abundant dauers in starvation conditions  [C. elegans. However, other nematode life stages have been shown to be particularly resistant to stress. For instance, unlike other reproductive stages, the L1 stage of C. elegans can be reared in the absence of food for weeks yet still be capable of reproduction [Ostertagia ostertagi and Haemonchus contortus harbor an L4 larval arrest state [C. inopinata biology diverge from its close relatives [C. inopinata. There are many hallmarks of the dauer stage that we do not explore here such as: nictation behavior [C. inopinata biology will be invaluable in definitively connecting this stress-resistant dauer-like larva to homologous developmental stages in other Caenorhabditis species.The nematode dauer larva is a stress-resistant, long-lived alternative developmental stage specialized for dispersal whose development is induced by adverse environments. Here, we described the broad morphological characteristics of a stress-resistant, dauer-like larval stage of r larvae , and thelug Fig. C and exhlug Fig. . These fhed Fig. D, a traispecies) . This adspecies) –30. Takeoduction . Additiost state , 32, 33.elatives –18, and behavior ; cuticulbehavior , 36; alabehavior ; metabolbehavior ; dauer ebehavior ; dauer pbehavior ; and thebehavior . Future C. inopinata dauer-like larvae were found to be shorter and fatter than the dauer larvae of other Caenorhabditis species  is also an appealing explanation considering C. inopinata dauer-like larvae have similar dimensions to C. inopinata L2 larvae ; when tC. inopinata , and otulations . RegardlC. inopinata has decreased length and increased width despite its elongated adult body size compared to C. elegans. This is possibly due to the divergent ecological contexts of these developmental stages that are specialized for dispersal or reproduction. Fig wasps are the dispersal vector of C. inopinata that are smaller and shorter-lived than those used by C. elegans, while figs have potentially released some selective constraint that allows longer adult body sizes compared to the rotting plant environments of C. elegans. Future characterization of this stage will reveal the extent of flexibility in the direction and magnitude of body shape evolution in a single species.Here, we showed that a stress-resistant, dauer-like larval stage in Escherichia coli strain OP50–1 for food as in [C. inopinata NKZ2 , C. briggsae , and C. tropicalis  were used. Fig media was prepared following [F. carica figs (Anna and Sarah™ dried black mission figs) were blended in a kitchen blender in distilled water to make 500 mL of fig slurry, which was centrifuged for 10 minutes at 8500 rpm. Supernatant was then removed and brought up to 800 mL with distilled water. The solution was then adjusted to pH 6–7. Agar was added to 3% and the media was then prepared and seeded with bacteria as above for nematode culture.Animals were grown on modified Nematode Growth Media with od as in . All anis NK74SC , 16), C.s PD1074 , C. brigollowing . BrieflyC. inopinata used for characterizing dauer formation variation. C. inopinata strains NKZ22 and NKZ27 were isolated from fresh F. septica figs from the island of Iriomote, Okinawa, Japan in May 2014  sequence and/or mating tests were performed in eleven of these lines, confirming their species identity  with high nematode density incubated at 25 °C  were retrieved from the data in [C. elegans N2 dauer; C. inopinata NKZ2 dauer-like larvae; C. briggsae HK104 dauer; C. tropicalis NIC122 dauer); “GCW 2019” ; “KAM 2022” . “EWH 2017” data were collected across 2 months and eleven imaging days. “GCW 2019” data were collected on one imaging day. “KAM 2022” data were collected across 2 months and eight imaging days. L3 larvae were isolated after bleach synchronization based on estimates of developmental rates at 25 °C in [Animals were mounted on agar pads and imaged on a dissecting “EWH 2017” data) or compound (“GCW 2019” and “KAM 2022” data) microscope. Animal length and width was measured using the ImageJ software ; curved  data in . All adu data in . For mea25 °C in . Sample 7” data oDauer formation frequency was determined as follows. Cultures on individual starved plates (as above) were washed into M9. Suspensions from single plates were split in half, and one was incubated in SDS as above while the other was incubated in M9 in parallel. All worms and carcasses were plated, and all live and dead worms were counted. Moving animals or animals with a normal, sinusoidal body posture were counted as alive ; unmoving animals with a stiff, straight posture were scored as dead. The dauer formation frequency for a given culture was estimated as the fraction surviving SDS times the fraction surviving in M9. This should account for non-dauer larvae death not induced by SDS.tidyverse [reshape2 [ape package [rstatix package [lsmeans package [vegan [pairwiseAdonis [K-means clustering was performed in base R with the kmeans function ; this was used to define clusters in length-width space. BIC was used to evaluate a range of values of k (k = 1–20), and BIC was minimized when k = 7. As this is also the number of developmental stages included in our data, this value was used for downstream analysis. The caret [MASS [ggplot2 [cowplot [lemon [ggforce [ggmap [ggtree [https://github.com/gcwoodruff/dauer_2020).All statistical analyses were performed in the R language . The pacidyverse  and reshreshape2  were use package  was used package  for visu package ; these w package  was usede [vegan  and pairseAdonis  packageshe caret  and MASSet [MASS  packages[ggplot2 , cowplot[cowplot , lemon [t [lemon , ggforce[ggforce , ggmap [e [ggmap , and ggt [ggtree  packagesAdditional file 1.Additional file 2.


Neoniphon sammara and Myripristis violacea) and an apogonid with a single rod bank (Ostorhinchus compressus), to determine the sensory advantage of multiple rod layers. Our results showed that fish with multibank retinas have both faster vision and enhanced responses to bright- and dim-light intensities. Faster vision was indicated by higher flicker fusion frequencies during temporal resolution electroretinography as well as faster retinal release rates estimated from their rhodopsin proteins. Enhanced sensitivity was demonstrated by broadened intensity-response curves derived from luminous sensitivity electroretinography. Overall, our findings provide the first functional evidence for enhanced dim-light sensitivity using a multibank retina while also suggesting novel roles for the adaptation in enhancing bright-light sensitivity and the speed of vision.Most vertebrates have one layer of the dim-light active rod photoreceptors. However, multiple rod layers, known as a multibank retina, can be found in over 100 species of fish, including several deep-sea species and one family of nocturnally active reef fish, the Holocentridae. Although seemingly associated with increased photon catch, the function of multibank retinas remained unknown. We used an integrative approach, combining histology, electrophysiology and amino acid sequence analysis, applied to three species of nocturnal reef fishes, two holocentrids with a multibank retina ( Due to the variability of light in their habitat, marine fishes possess the greatest diversity of visual adaptations among vertebrates –3. To carh1 ,7, high rh1 ,9 and thrh1 ,11. Althrh1 ,13.A retina containing multiple layers of rods is known as a multibank retina and can contain 2–28 layers of stacked rods ,14. MultThe family Holocentridae is composed of two subfamilies: squirrelfishes (Holocentrinae) and soldierfishes (Myripristinae). They mainly inhabit shallow depth ranges, however, a few species dwell as deep as 640 metres ,21. HoloNeoniphon sammara and Myripristis violacea, from the subfamilies Holocentrinae and Myripristinae, respectively), and a non-multibank control species, another nocturnally active reef fish, Ostorhinchus compressus (family Apogonidae). Firstly, we examined retinal structure using histology. Then, we studied the luminous sensitivity and temporal resolution of their vision by recording the electrophysiological response of the whole eye to different light stimuli, using electroretinography (ERG) [trans retinal, to be released from the opsin – a rate-limiting step in resetting of the phototransduction cascade) as a proxy for the speed of the regeneration of vision. Overall, our study sheds light on the unresolved function of an understudied visual adaptation in deep-sea and nocturnal coral reef fish as well as offering a broader insight into vision in vertebrates.In this study, the sensitivity hypothesis for the function of the multibank retina was tested by assessing the visual systems of two species from the family Holocentridae (hy (ERG) –31. Fina. 2 (a)https://www.cairnsmarine.com/), which also collects from the northern Great Barrier Reef. All collections and procedures were conducted under a Great Barrier Reef Marine Park Permit (G17/38160.1), a Queensland General Fisheries Permit (180731) and a University of Queensland's Animal Ethics Permit (QBI 304/16). Following euthanasia, all animals were photographed with a scale reference to quantify body length and eye diameter. Eyes were dissected and the eye cup preserved in RNAlater or paraformaldehyde  PFA in 0.01 M phosphate-buffered saline (PBS), pH 7.4) depending on the analyses.Details of all animals are given in electronic supplementary material, table S1. Adult fish were collected from the Great Barrier Reef around Lizard Island, Australia, or sourced from a supplier, Cairns Marine ( (b)O. compressus, N. sammara and M. violacea as described previously [2 of retina were estimated from sections using Fiji v1.53c [Five retinal regions  were dissected, processed and sectioned from one PFA-fixed eye each for eviously . The deni v1.53c  as descri v1.53c ). Densiti v1.53c  (figure  (c)in vivo on whole, intact eyes to assess visual function using methods similar to those described in [−1 gallamine triethiodide and ventilated with oxygenated seawater . After ≥40 min of dark adaptation, light stimuli were delivered to the eye using a custom-built, calibrated, broad-spectrum light source controlled via a PowerLab 4/26 DAQ module. Visual responses were detected through silver wire electrodes placed on the surface of the eye, amplified via a DP-103 amplifier and acquired in LabChart 8 v8.1.16. The system was grounded to the water of the recording chamber. Recordings were conducted at 28 ± 1°C at both day and night to control for any effects of temperature and circadian rhythm, respectively. Recordings were performed at the Lizard Island Research Station (LIRS) or the Queensland Brain Institute (QBI). Additional recordings were taken at both sites to compare results between the recording locations .Corneal electroretinography (ERG) recordings were conducted ribed in . Fish we (d)O. compressus, N. sammara and M. violacea. FFF is the frequency at which retinal responses no longer follow the frequency of evenly spaced light pulses. Dark-adapted FFF ERGs were recorded by increasing the frequency of white light stimuli of constant intensity from 5 Hz to 95 Hz at increments of 5 Hz. Light pulses were 10 ms in duration and were repeated 30 times. Recordings were conducted for bright (384 lux) and dim (4 lux) stimuli  ERGs on three or five individuals for day and night recordings, respectively, for  stimuli . The FFF (e)N. sammara , M. violacea (n = 4 for day and night) and O. compressus (n = 5 for day and night). The absolute (luminous) sensitivity of vision was determined using V/logI curves, which plot the normalized amplitude of the response, V , against the log of the intensity (I). These ERGs were recorded by increasing the intensity of a white light from 2.4 × 10−8 to 240 000 lux (i.e. −7.6 to 5.4 log10(lux)) in 0.3–0.6 log unit steps. Light stimuli were 100 ms pulses presented at 0.1–0.4 Hz  and were repeated 10 times for each intensity. The mean response amplitudes were normalized to the maximal response (Vmax) and plotted against stimulus intensity to obtain the V/logI curve [V/logI curves were used to calculate stimulus irradiances eliciting 5%, 50% (referred to as K50) and 95% of Vmax. K50 values are typically used to make interspecific comparisons of relative luminous sensitivity, while the dynamic range is a proxy for the breadth of visual functionality and was defined as the log10 irradiance range between 5% and 95% Vmax [max-normalized responses were also normalized to eye diameter  to obtain responses per unit of retina and analysed again as described above. To further understand how the visual response changed with intensity, representative ERG waveforms were analysed to obtain: (1) the time from stimulus presentation to the peak of the signal generated post-synaptic to the photoreceptors  and (2) the amplitude of the photoreceptor-derived peak . These values were obtained for dim (0.4 lux), moderate (125 lux) and bright (2165 lux for O. compressus and 5160 lux for N. sammara and M. violacea) stimuli, which matched the base, peak and decline of the V/logI curves, respectively.Absolute sensitivity ERGs were conducted for gI curve ,35. V/lo95% Vmax . All val (f)1/2t; min) compared with a wild-type reference (either zebrafish [N. sammara and F116S and A164G in M. violacea) and that any substitutions that also affected spectral sensitivity were excluded from these analyses  [O. compressus (MH979489.1), N. sammara (MW219675.1) and M. violacea (MW219672.1) [https://www.ncbi.nlm.nih.gov/genbank/) and translated into protein sequences. These were aligned and inspected for AA substitutions at each of the candidate sites in Geneious Prime v2021.1.1. Identified substitutions were used to estimate the cumulative change in retinal release  . Next, tessus MH99489.1, N release . The abs. 3 (a)O. compressus, N. sammara and M. violacea (n = 1). All three species had duplex retinas composed of both rods and cones. However, while O. compressus only had a single layer of rods , N. sammara and M. violacea had a maximum of 6 and 14 banks of rods, respectively . The densities of all cell types were heterogeneous across the retina in all species . The highest rod densities and summation of rods onto GC occurred in M. violacea  followed by N. sammara  and then O. compressus . An inverse pattern was observed for cone and GC densities in all regions, with O. compressus having the highest densities and M. violacea the lowest . Finally, INL cell densities were also highest in O. compressus and lowest in M. violacea for most regions  .Retinal architecture and cell densities were assessed in  (b)n = 3) and night (n = 5) . Under all conditions, N. sammara attained the greatest FFF ), followed by M. violacea  and then O. compressus  . However, the FFFs of O. compressus did not vary greatly with stimulus intensity. Finally, all species showed a trend towards lower FFFs at night compared with during the day, irrespective of stimulus intensity Temporal resolution ERGs were conducted to determine the FFF (the point at which responses to evenly spaced light pulses can no longer be distinguished as separate) in response to dim (4 lux) and bright (384 lux) stimuli at day ( (c)O. compressus (n = 5), N. sammara  and M. violacea (n = 4) during the day and night . Firstly, V/logI curves were normalized to either Vmax alone  or Vmax and eye size . In all species, V/logI curves produced non-monotonic functions, with the amplitude of the b-wave representing the response post-synaptic to the photoreceptor, generally increasing with stimulus intensity until the maximal amplitude (Vmax) was reached, before subsequently decreasing due to bleaching. Notably, a subtle decrease in gradient occurred in the curves from M. violacea between stimulus intensities of approximately 40 and 700 lux ), before continuing to increase until the response reached its peak. A closer examination of the ERG waveforms themselves revealed that, in all species, the speed of the visual response (i.e. time taken for the b-wave to reach its peak) became faster at higher intensities . Additionally, the photoreceptor-derived component of the waveform (i.e. a-wave amplitude) also increased at higher intensities, very minimally in O. compressus, more substantially in N. sammara and greatly in M. violacea .Absolute sensitivity ERGs were recorded for V/logI curves between diel period and species. The V/logI curves were bright-shifted during the day compared with the night for O. compressus and N. sammara, but not M. violacea. Furthermore, when considering the same diel period, the V/logI curves differed among the three species, with the nature of these differences quantified using 5% and 95% Vmax values, the dynamic range and K50 values. Interspecific trends in these values were similar irrespective of whether the data was normalized to Vmax alone  or Vmax and eye size O. compressus RH1 possessed four AA substitutions known to alter retinal release rate, while those in N. sammara and M. violacea had five and six AA substitutions, respectively (M. violacea (1/2t of 19.5 min), followed by N. sammara (20.8 min) and then O. compressus (21.8 min). Therefore, the rhodopsins of both holocentrids had faster estimated retinal release rates than O. compressus.The retinal release rates of each species' rhodopsin protein were estimated using AA substitutions. The ectively . These s. 4N. sammara and M. violacea had multiple rod banks across the entire retina. Similar to other nocturnal reef fishes [N. sammara and M. violacea showing greater adaptation for scotopic vision (i.e. higher rod densities and summation and multibank retinas) but inferior adaptation for photopic vision (i.e. lower cone densities) compared with O. compressus.Here, we investigated the retinal structure and visual function of nocturnal reef fishes with and without multibank retinas. Firstly, we confirmed that, at the morphological level, the three species investigated had visual systems that were well-adapted to their dim-light environments  was within the range for other nocturnal fishes for O. compressus (approx. 40 Hz) [N. sammara (70 Hz) and M. violacea (approx. 60 Hz) the range shown was characteristic of diurnal fishes [Secondly, this study examined temporal resolution (or speed) of vision in these fishes by determining the FFF (. 40 Hz) , for botl fishes ,30.O. compressus had the highest cone and lowest rod densities but not the highest peak FFF implies that more complex neuronal mechanisms are at play in the holocentrids compared with the cardinalfish, likely due to the structure of the multibank retina. Indeed, the higher speed of the multibank retina could be related to photoreceptor size. High sensitivity requires a large membrane surface area, and this is often achieved through large photoreceptors. However, large photoreceptors may have slower activation rates than small photoreceptors  [Lepidocybium flavobrunneum) which had a much lower FFF  . TheoretF  than thw waters ,50. Finaw waters .N. sammara and O. compressus were no exception, showing higher bright-light sensitivity during the day but higher dim-light sensitivity during the night. The sensitivity of M. violacea was, on the other hand, relatively constant. This indicates that M. violacea may only undergo a weak diel switch between photopic and scotopic systems. This is likely due to their lack of a well-developed photopic system to switch to, similar to some deep-sea fishes with pure rod retinas [Finally, we assessed luminous sensitivity  with increasing rod densities and rod banking. This supports the idea that the multibank retina enhances dim-light vision.Luminous sensitivity also varies with retinal structure and ecology. For example, diurnal fish (with higher cone densities) have better daytime bright-light vision, while nocturnal fish (higher rod densities) have greater night-time dim-light visual capabilities ,28. Nota0 values , was not species  or diffe species ,54. RegaVmax values) was also correlated with increasing rod densities and banking (and decreasing cone densities), but only during the night, when rods are the dominantly active photoreceptor type. Bright-light vision is usually cone mediated, however, in some species with rod-dominated retinas and few cones, e.g. mice, rods have been shown to function under bright-light conditions [O. compressus had higher daytime 95% Vmax values than M. violacea), our data also suggest that the rods have less involvement in photopic vision when cones can be used instead (e.g. when the retina has higher cone densities). However, the photopic visual capabilities of the holocentrids would likely still be sufficient to meet their daytime ecological demands, such as courtship and predator avoidance [Enhanced bright-light vision (i.e. bright-shifted 95% nditions ,56. Our voidance ,58. Overvoidance , holocenO. compressus despite their less well-developed photopic visual systems. Furthermore, work in mice has shown that rods can recover and respond to bright intensities and that this is facilitated by more efficient post-bleaching regeneration at higher light intensities compared with lower intensities [in vitro regeneration experiments to test the retinal release rates of holocentrid RH1 visual pigments may be used to explain how their rods continue to function at brighter intensities, similar to cones.Our study suggests that the rods in the holocentrid multibank retina can still function at brighter intensities. However, rhodopsin normally bleaches at high intensities. A key reason for this bleaching is the slower retinal release rate of rhodopsin compared with the cone opsins, and therefore, slower resetting of the phototransduction cascade ,60. Aminensities ,56. FutuM. violacea. The dual functionality of the skate retina was hypothesized to be due to the fact that they are dominantly but not exclusively nocturnal [Overall, our findings suggest a dual role for the holocentrid multibank retina, where at dim intensities it functions to enhance photon capture while at bright intensities, it functions to regenerate the visual response in a rod-dominated system, allowing the eye to function at both lower and higher intensities than a retina with a single rod bank. Interestingly, these dual rod- and cone-like physiological properties of the rods in the holocentrids are reminiscent of findings in skates ,62. Skatocturnal ,63, it mIrrespective of the underlying mechanism, enhanced visual functionality at both bright- and dim-light intensities aligns well with the ecology of holocentrids, since they are nocturnal foragers but are still somewhat active on the reef during the day . Overall


Recurrent respiratory tract infections (RRTIs) are one of the most common pediatric diseases. Although the pathogenesis of pediatric RRTIs remains unknown, ineffective B cell-dominated humoral immunity has been considered as the core mechanism. During the course of pediatric RRTIs, B cell-dominated humoral immunity has changed from “protector” of respiratory system to “bystander” of respiratory tract infections. Under physiological condition, Tfh cells are essential for B cell-dominated humoral immunity, including regulating GC formation, promoting memory B cell (MB)/plasma cell (PC) differentiation, inducting immunoglobulin (Ig) class switching, and selecting affinity-matured antibodies. However, in disease states, Tfh cells are dysfunctional, which can be reflected by phenotypes and cytokine production. Tfh cell dysfunctions can cause the disorders of B cell-dominated humoral immunity, such as promoting B cell presented apoptosis, abrogating total Ig production, reducing MB/PC populations, and delaying affinity maturation of antigens-specific antibodies. In this review, we focused on the functions of B and Tfh cells in the homeostasis of respiratory system, and specifically discussed the disorders of humoral immunity and aberrant Tfh cell responses in the disease process of pediatric RRTIs. We hoped to provide some clues for the prevention and treatment of pediatric RRTIs. The mosMechanistically, the core functions of humoral immunity in the respiratory system are against pathogens invasion, which B cell immune responses are indispensable . After p1.1It is generally thought that productive humoral immune responses require that naïve B cells and their differentiated progeny move among distinct micro-environments . The devIn general, in normal physiologic state, B cell distributions are significant differences according to the anatomic sites and age . In the 1.2It has been confirmed that ineffective humoral immune responses are associated with the occurrence of various pediatric diseases, and pediatric RRTIs is one of them ,26. Whilrespiratory syncytial virus (RSV), human rhinovirus, and influenza virus . Due to Tfh cells are mainly responsible for B cell programs, they have become an important breakthrough for rescuing B cell functions in pediatric RRTIs.Tfh cells are indispensable for humoral immune responses.In addition to Igs deficiency, the abnormal differentiation and activation of B cells are also the symbol of humoral immunity impairment in pediatric RRTIs. In recent years, some studies have confirmed that mature/naive B cell proportion in the peripheral circulation of children with RRTIs were significantly lower than that in HC . As discza virus . Despiteza virus . During za virus . Howeverza virus . Indeed,za virus . In addiza virus . Similarth RRTIs . Notable+T expressing CXCR5 in the secondary lymphoid organs [+T cell subsets [Bcl-6 are transcribed and translated into Bcl-6, which indicated the beginning of Tfh cell differentiation and maturation [+T cell sub sets, the differentiation and activation of Tfh cells also require cytokine signals, such as IL-6, IL-12,TGF-β, and IL-27 [+ Th cells, including CXCR5, ICOS, PD-1,CD40, and Bcl-6 [+CD4+T cells are currently named as circulating Tfh cells [Compared with Th1, Th2 and Th17 cells, Tfh cells were found fairly later. The disco very of Tfh cells was resulted from the detection of CD4d organs . With th subsets . The dif subsets . After Sturation . After mturation . Similarnd IL-27 . After snd Bcl-6 . Apparennd Bcl-6 . In the nd Bcl-6 . In the fh cells . Interesfh cells . Howeverfh cells .+T cells that specialized for helping B cells [−/−mice, which resulted in a significant reduction of GC B cells and isotype-switched antibody-secreting B cells and high-affinity antigen-specific IgG1 [. Classically, T cells migrate as amoeboid without requiring strong adhesion to extracellular matrix, while they rely on dynamic pseudopods to power their movement [As reported elsewhere, Tfh cells are the CD4 B cells . They pl B cells ,63. Tfh fic IgG1 ,62. Likefic IgG1 . As a cofic IgG1 ,66. Oncefic IgG1 . Classicmovement . ICOS camovement . Indeed,movement ,70. Unlimovement . Howevermovement . Likewismovement . Profounmovement . In humamovement .Table 1TExcept for cell-cell contacts, Tfh cells also can provide the helpful signals to B cells thought production of cytokines (paracrine action) . As ment1.3Streptococcus (GAS) [+T cells of children with RRTIs are skewed away from antigen-specific Tfh cell differentiation, which gave rise to the impairment of antigens-specific Igs production; Most importantly, Tfh cells from those patients acquire a killer identity, they can directly kill GC B cells by secreting granzyme B (GzmB) [+T cells and natural killer cells for killing of target cells [As described in the preceding sections, Tfh cells are essential in sustaining B cell dominated humoral immunity, which is necessary for respiratory pathogen clearance. Conversely, aberrant Tfh cell responses accounted for chronic lung diseases development, including asthma, human primary immune deficiencies (PICDs), sarcoidosis, pediatric RRTIs and their corresponding disease models . In the us (GAS) . Apparenus (GAS) . Similarus (GAS) . Noteworus (GAS) . In vivous (GAS) . At the B (GzmB) . In geneet cells . The expet cells . The excet cells . IL-21 det cells .Fig. 3Tf+T cells, PI3K signaling pathway can be activated by several surface receptors on them, such as TCR, CD28, ICOS, all of them are highly expressed on the Tfh cells [So far, the underlying mechanisms of aberrant Tfh cell responses are likely to remain unclear in pediatric RRTIs. The over-activation of PI3K signaling pathway is receiving much attention. In CD4fh cells . Therefofh cells ,86. Appafh cells ,88. Recefh cells . In thatfh cells . Interesfh cells .2This review summarized the current literatures to highlight the importance of Tfh and B cells in the homeostasis of respiratory system of children, and specifically discussed the abnormalities of their immune responses in pediatric RRTIs . In the JD wrote the manuscript. HC and HSL made substantial contributions to figures and tables production. WBC edited and proof read this manuscript. All authors read and approved the final manuscript. The final version of the manuscript was approved by all authors.All authors listed have significantly contributed to the development and the writing of this article.10.13039/501100001809National Natural Science Foundation of China (Grant no. 82305313).This work was supported by the No data was used for the research described in the article.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Geum longifolium (Maxim.) Smedmark 2006 belongs to the family Rosaceae, subfamily Rosoideae, tribe Colurieae. Geum longifolium is endemic to China and its whole herb is used in Chinese medicine. Here, the first complete chloroplast (cp) genome of G. longifolium was assembled and annotated based on genome skimming, and its phylogenetic position was investigated using phylogenomic evidence. The cp genome size of G. longifolium was 155,884 bp with the total GC content of 36.7%. Its cp genome presented a typical tetrad structure, composed of a large single copy (LSC) region , a small single copy (SSC) region , and a pair of inverted repeat (IR) regions . The cp genome encoded 129 genes, including 84 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Phylogenetic analysis indicated that G. longifolium was sister to G. elatum Wall. ex G.Don 1832 in current taxa sampling. This study can enrich the chloroplast genomic resource of Geum and lay the foundation for future phylogenetic studies on Geum. Geum longifolium (Maxim.) Smedmark Coluria longifolia Maxim. Geum longifolium is endemic to China and distributed in the alpine meadows of Gansu, Qinghai, Sichuan, Xizang, and Yunnan . The specimen was deposited at the herbarium of Inner Mongolia Normal University (NMTC)  under the voucher number Li QQ BDB1.Leaf sample of Geum macrophyllum Willd.1809  gene as the seed and 3,655,282-bp reads were mapped to G. macrophyllum cp genome. Sequencing depth and coverage map of G. longifolium was generated following the protocol of Ni et al.  as the reference. Chloroplast Genome Viewer (CPGView) was used to draw the circular cp genome map of G. longifolium and the structure of the genes that are difficult to annotate in the cp genome  method  as outgroups. The cp genome sequences of the above 17 accessions were aligned using MAFFT version 7.450  and the GTR + I + G model was selected as the best-fit model by PartitionFinder2 , and the total GC content was 36.7%. Its cp genome presented a typical tetrad structure, consisting of a large single copy (LSC) region , a small single copy (SSC) region , and a pair of inverted repeat (IR) regions . The GC content of LSC, SSC, and IR were 34.4%, 30.9%, and 42.6%, respectively. The cp genome contained 129 genes, including 84 protein-coding genes, 37 tRNA genes, and eight rRNA genes. In addition, the cp genome contained one pseudogene Ψycf1 located in the IRb/SSC junction. Ten unique genes  were cis-splicing and rps12 was trans-splicing .The cp genome of . The genus Geum was recovered as a monophyletic group in the phylogenetic tree . Within Geum, G. rupestre (Yü et Li) Smedmark G. urbanum L. 1753, G. japonicum var. chinense F.Bolle 1931, G. aleppicum Jacq. 1781, G. triflorum Pursh 1814, G. macrophyllum, G. longifolium and G. elatum Wall. ex G.Don 1832) in current taxa sampling.Tree topologies inferred by ML and BI analyses were identical, so the ML tree with bootstrap support values (BS) and Bayesian posterior probabilities (PP) was shown in Figure 3 G. longifolium was assembled and annotated based on genome skimming. The cp genome of G. longifolium has similar structure and gene size, and consistent gene composition and gene order to that of other Geum species  and the name was adopted in the Flora of China by Li et al. ( Geum based on phylogenetic studies of Colurieae (Smedmark and Eriksson Coluria longifolia were included in Geum as G. longifolium (Maxim.) Smedmark. Our phylogenetic analysis showed that G. longifolium was nested within the Geum species, which supported Smedmark’s taxonomic treatment to place this species within Geum (Smedmark . Phylogenetic analysis indicated that G. longifolium was sister to G. elatum in current taxa sampling. This study can enrich the chloroplast genomic resource of Geum and lay the foundation for future phylogenetic studies on Geum.In this study, using the related bioinformatics methods, the first complete cp genome ofi et al. . Smedmari et al.  made a rSmedmark . PhylogeGeum longifolium is not an endangered or protected plant.No ethical approval is required. Click here for additional data file.Click here for additional data file.Click here for additional data file.


Thoracotremata belong to the large group of “true” crabs (infraorder Brachyura), and they exhibit a wide range of physiological and morphological adaptations to living in terrestrial, freshwater and marine habitats. Moreover, the clade comprises various symbiotic taxa  that are specialised in living with invertebrate hosts, but the evolutionary history of these symbiotic crabs is still partially unresolved.Kroppcarcinus siderastreicola, Opecarcinus hypostegus and Troglocarcinus corallicola. A phylogenetic tree of the Thoracotremata was reconstructed using 13 protein-coding genes and two ribosomal RNA genes retrieved from three new gall crab mitogenomes and a further 72 available thoracotreme mitogenomes. Furthermore, we applied a comparative analysis to characterise mitochondrial gene order arrangement, and performed a selection analysis to test for selective pressure of the protein-coding genes in symbiotic Cryptochiridae, Pinnotheridae, and Varunidae (Asthenognathus inaequipes and Tritodynamia horvathi).Here we assembled and characterised the complete mitochondrial genomes (hereafter mitogenomes) of three gall crab species (Cryptochiridae): A. inaequipes and T. horvathi clustered together in a clade with free-living Varunidae species, highlighting that symbiosis in the Thoracotremata evolved independently on multiple occasions. Different gene orders were detected in symbionts and free-living species when compared with the ancestral brachyuran gene order. Lastly, the selective pressure analysis detected two positively selected sites in the nad6 gene of Cryptochiridae, but the evidence for positive selection in Pinnotheridae and A. inaequipes and T. horvathi was weak. Adaptive evolution of mitochondrial protein-coding genes is perhaps related to the presumably higher energetic demands of a symbiotic lifestyle.The results of the phylogenetic reconstruction confirm the monophyly of Cryptochiridae, which clustered separately from the Pinnotheridae. The latter clustered at the base of the tree with robust branch values. The symbiotic varunids  Sun et Symbiotic lifestyles in Thoracotremata likely evolved independently on multiple occasions. Aphanodactylidae and Pinnotheridae are not closely related, and Varunidae are retrieved in a distant phylogenetic position from Pinnotheridae and Cryptochiridae . The queThe mitogenomes of most metazoans have high nucleotide substitution rates, low extensive recombination rates and relatively conserved gene content, thus making it an informative molecular signal for phylogenetic reconstruction and adaptive evolution analysis . AdditioHapalocarcinus marsupialis Stimpson, 1859 s.l. , including three newly sequenced Cryptochiridae mitogenomes. Based on the inferred phylogeny reconstruction and comparative analysis, we aim to elucidate: (1) the monophyly and phylogenetic position of Cryptochiroidea; (2) the evolution of symbiosis in Thoracotremata; and (3) by means of a test for selective pressure, whether a symbiotic lifestyle results in positive selection for certain protein-coding genes (PCGs) in the mitogenome.Troglocarcinus corallicola Verrill, 1908, was sampled from Orbicella faveolata  in Anse à Jacques, Guadeloupe  on April 27, 2021. Kroppcarcinus siderastreicola Badaro, Neves, Castro & Johnsson, 2012, collected from Siderastrea siderea  on February 24, 2022, and Opecarcinus hypostegus , collected from Agaricia humilis Verrill, 1901 on March 14, 2022, were sampled in Piscadera Bay, Curaçao . The crabs were stored in 70% ethanol and transported to the University of Groningen, and from there the entire specimens were sent to the Beijing Genomics Institute (BGI) in Hong Kong for DNA extraction, and paired-end sequencing using the DNBSEQ-G400 platform.The three gall crab species used in this study were collected from two sites in the Caribbean. Sampling in Guadeloupe was authorised by the Direction de la Mer de Guadeloupe under Autorisation N°09/2021. Sampling in Curaçao was under collecting permits of the Curaçaoan Government provided to CARMABI (Government reference: 2012/48584).http://mitos.bioinf.uni-leipzig.de/index.py) (https://proksee.ca/) was used for the visualisation of the mitogenome maps. The assembled and annotated mitogenomes with gene features were uploaded to GenBank under accession numbers OQ308778 (K. siderastreicola), OQ308779 (O. hypostegus), and OQ308780 .The raw data were filtered in order to remove adapter, contaminated and low-quality sequences using fastp v.0.23.2  with defndex.py)  for annohttps://www.bioinformatics.org/sms2/codon_usage.html) with the invertebrate mitochondrial genetic code  barcode from the mitogenome by H. marsupialis HM.08 .After complete annotation of the mitogenome, the nucleotide composition for each species was calculated in MEGA X  and the tic code  and visu ggplot2  in R v.4 ggplot2 . The tra ggplot2 . Detailslis s.l.  were misSeventy-two thoracotreme and five heterotreme crabs (as outgroups) for which whole mitogenomes are available were retrieved from GenBank . With thThe 13 PCGs and two ribosomal RNA genes  were aligned separately using MAFFT v.7.407  and subs6 generations, sampling every 1,000 iterations. Consensus trees were constructed in MrBayes with a burnin of 25%. The average standard deviation of split frequencies was 0.03, surpassing the recommended threshold (<0.01) proposed by the software authors as a measure of convergence. The trees were visualised in FigTree v.1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/).Maximum Likelihood (ML) and Bayesian Inference (BI) approaches were used for the phylogenetic analyses. The selected partition schemes and best-fit substitution models are available in http://mitos.bioinf.uni-leipzig.de/index.py). The Mitochondrial Gene Order (MGO) of Xenograpsus testudinatus Ng, Huang & Ho, 2000, is based on Besides the five heterotreme crabs as outgroups, the mitogenomes of 75 thoracotreme crabs were annotated by MITOS1  to synonymous (dS) substitution rates (ω = dN/dS) was calculated using the codon-based maximum likelihood (CodeML) application in PAML v.4.7 . Codon aω ratios vary between branches in the phylogeny: this model allows an independent ω ratio for each branch on the phylogenetic tree. Furthermore, the results of ω ratios in the free-ratio model were classified into two groups: symbionts  and free-living crabs . The branch-site model  was applied in this study, which assumes that ω ratios vary among sites and across the branches of the phylogeny. The branches of three symbiotic lineages were labelled as foreground branches while the rest were set as background branches in the branch-site model in three different topologies only containing one of three symbiotic lineages.The branch model (free-ratio model) was used to test if the ω ratios differed significantly between grouped lineages  in the free-ratio model. The Likelihood-Ratio Test (LRT) was used to compare branch-site models determining the best fitting model to our data. The parameters setting for LRT followed ggplot2 v3.4.0  and H. marsupialis s.l. had the lowest value (69.54%).The nucleotide composition, AT-content and GC-content of the mitogenomes of the 75 thoracotreme crabs are summarised in T. corallicola), 11,149 (K. siderastreicola), 11,104 (O. hypostegus) and 11,068  codons. Nine PCGs  were located on the J-strand and four  on the N-strand. The start and stop codons of the four gall crab species are summarised in PCGs comprised 11,144  arm without the TΨC loop in both T. corallicola and K. siderastreicola, while the same scenario was found for tRNA-Asn and tRNA-Phe in T. corallicola, tRNA-Asp in O. hypostegus and tRNA-Gly in H. marsupialis s.l. , V. yui Hwang & Takeda, 1986 and Metaplax longipes Stimpson, 1858 formed a well-supported clade in BI , which differed from the pattern for the Indo-Pacific species H. marsupialis s.l. (pattern seven), where tRNA-His changed location and was transposed in between tRNA-Ser2 and tRNA-Phe , Pinnotheres pholadis De Haan, 1835 and Pinnaxodes major Ortmann, 1894 shared pattern one, while Pinnotheres excussus Dai in Dai, Feng, Song & Chen, 1980, Arcotheres sinensis , Arcotheres purpureus  and Arcotheres sp. shared pattern nine; Indopinnixa haematosticta  had pattern ten. Pattern three was only detected in A. inaequipes, whereas the other symbiotic varunid T. horvathi shared pattern two with the remaining Varunidae. As for the free-living crabs, five patterns, including the ancestral gene order of Brachyura, were found across the different families . Using the branch-site model, two amino acid sites in gall crabs were detected to be under positive selection in nad6 by BEB (posterior probability ≥ 95%), as well as five amino acid sites in atp8, one site in cob and two in nad5 with weak positive selection evidence. In pea crabs, genes atp8, cob, nad2, nad4, nad5 and nad6, as well as two genes atp8 and nad5 in Asthenognathus inaequipes and Tritodynamia horvathi, were inferred with weak positive selection evidence  within the Thoracotremata , Fig. S3H. marsupialis s.l. as closely related to an Ocypodoidea lineage , albeit without full branch support. Furthermore, a recent phylogenetic study conducted by e.g., Glyptograpsidae, Leptograpsodidae, Aphanodactylidae and Heloeciidae) and the insufficient number of representative species for several families  do not allow us to fully compare our results with their proposed new classification. In addition, insufficient representative species within individual families was also problematic in their analyses . Future studies working on the classification within Thoracotremata need to contain more species given the large number of undescribed species in some families . The switch from a free-living to a symbiotic lifestyle appears to be relatively common in marine invertebrates . tebrates .A. inaequipes and T. horvathi (Varunidae) in their dataset, but did not recognise the species as symbiotic. Astenognathus inaequipes is symbiotic with holothurians   . Tsang earunidae .Gene rearrangements occurred frequently in Thoracotremata . While mH. marsupialis s.l. has a unique pattern (pattern seven), and the Atlantic species K. siderastreicola, O. hypostegus and T. corallicola share the same MGO pattern  with the other Varunidae.Different MGO patterns were detected in symbiotic gall crabs, pea crabs and varunids. Tandem-duplication-random-loss took place in gall crabs, resulting in two patterns: the Indo-Pacific species A. inaequipes associates with holothurians, whereas T. horvathi associates with polychaetes , ychaetes . Sun et ychaetes . Moreove species . Both peFormica fusca Linnaeus, 1758 (Hymenoptera) sharing the same MGO with Ditrysia ; Ochrogaster lunifer Herrich-Schäffer, 1855), which is the largest clade of Lepidoptera. Here we detected MGO pattern four in 12 grapsoids , Orisarma sinense , Cristarma eulimene ). Pattern seven of H. marsupialis s.l. is shared by Scopimera intermedia Balss, 1934, a free-living crab in the Dotillidae , however, the number of species in this study is relatively small when compared to the overall number of extant species  in Thoracotremata. Pattern sharing has already been detected in crabs  could promote MGO rearrangements as mitogenomes are more likely to be influenced by evolutionary processes than nuclear genes  advantageous amino acid changes to facilitate adaptation to a symbiotic lifestyle. In a diverse range of taxa, adaptations to different lifestyles were studied, e.g., flying vs flightless in birds and insects, deep vs shallow habitats in fish, high vs low altitudes in grasshoppers and mud-dwelling vs terrestrial habitats in mud shrimps  factors, compared with free-living species, and in turn these factors could influence oxygen or energy usage. Cryptochiridae and Pinnotheridae have, for example, a very high reproductive investment when compared to their free-living counterparts e.g., . These sA. inaequipes and T. horvathi were retrieved in three separate lineages in our phylogenetic reconstruction, thus based on our results symbiosis independently evolved at least three times in the Thoracotremata. The recent recognition of two additional pea crab families in the Pinnotheroidea and the elevation of Aphanodactylidae to superfamily level highlights the need for more molecular data to further clarify their positions in the Thoracotremata. However, given the distant relationship between the Aphanodactyloidea and Pinnotheroidea, symbiosis likely evolved at least four times in the Thoracotremata (A. inaequipes and T. horvathi). Currently little is known about the exact role and function of these genes, although they are potentially linked to aerobic metabolism. Further research is needed to explore the possible link between mobility, reproduction, body size or other factors that are at play in these positive selection scenarios.Cryptochiridae, Pinnotheridae and the varunids otremata . Furtherotremata  and 3 sh10.7717/peerj.16217/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.16217/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.16217/supp-3Figure S1(A) Kroppcarcinus siderastreicola; (B) Opecarcinus hypostegus; (C) Troglocarcinus corallicola; and (D) Hapalocarcinus marsupialis s.l.Click here for additional data file.10.7717/peerj.16217/supp-4Figure S2AClick here for additional data file.10.7717/peerj.16217/supp-5Figure S2BClick here for additional data file.10.7717/peerj.16217/supp-6Figure S2CClick here for additional data file.10.7717/peerj.16217/supp-7Figure S2DClick here for additional data file.10.7717/peerj.16217/supp-8Figure S3Click here for additional data file.10.7717/peerj.16217/supp-9Supplemental Information 9Click here for additional data file.


Citrobacter species are Gram-negative opportunistic pathogens commonly reported in nosocomial-acquired infections. This study characterised four Citrobacter species that were isolated from surface water in the North West Province, South Africa.In silico taxonomic identification was conducted by using multi-locus sequence typing and average nucleotide identity. A pangenome was used to determine the phylogenomic landscape of the Citrobacter species by using 109 publicly available genomes. The strains S21 and S23 were identified as C. braakii, while strains S24 and S25 were C. murliniae and C. portucalensis, respectively. Comparative genomics and sequenced genomes of the ESBL-producing isolates consisted of n = 91; 83% Citrobacter species in which bla-CMY−101  and bla-CMY−59  were prevalent in C. braakii, and C. portucalensis strains, respectively. Macrolide  and aminoglycoside (acrD) efflux pumps genes were identified in the four sequenced Citrobacter spp. isolates. The quinolone resistance gene, qnrB13, was exclusive to the C. portucalensis S25 strain. In silico analysis detected plasmid replicon types IncHI1A, IncP, and Col(VCM04) in C. murliniae S24 and C. portucalensis S25, respectively. These potentially facilitate the T4SS secretion system in Citrobacter species. In this study, the C. braakii genomes could be distinguished from C. murliniae and C. portucalensis on the basis of gene encoding for cell surface localisation of the CPS (vexC) and identification of genes involved in capsule polymer synthesis (tviB and tviE). A cluster for the salmochelin siderophore system (iro-BCDEN) was found in C. murliniae S24. This is important when it comes to the pathogenicity pathway that confers an advantage in colonisation.Phenotypic antimicrobial susceptibility profiles of the isolates demonstrated their ability to produce the extended-spectrum β-lactamase (ESBL). Whole genomes were sequenced to profile antibiotic resistance and virulence genes, as well as mobile genetic elements. Citrobacter species are of significant concern due to their dissemination potential in freshwater systems. The presence of these ESBL and multidrug-resistant (MDR) pathogens in aquatic environments is of One Health importance, since they potentially impact the clinical domain, that is, in terms of human health and the agricultural domain, that is, in terms of animal health and food production as well as the environmental domain.The emerging and genomic landscapes of these ESBL-producing The online version contains supplementary material available at 10.1186/s12864-023-09867-4. Citrobacter species are characterised as Gram-negative, facultative anaerobe non-spore-forming rods that utilise citrate as the sole carbon source, and they belong to the family Enterobacteriaceae [Citrobacter species have also shown resistance to the common antimicrobials that are used to treat individuals who suffer from these infections [eriaceae . They areriaceae . Howevereriaceae . These Cfections .Citrobacter species can colonise the intestinal tract of humans and animals [Salmonella enterica subsp. enterica serovar Typhi str. In its turn, CT18  plays a role in pathogenicity. Citrobacter species can be identified in terms of different metabolic and virulence gene associations. These associations are evidenced by species such as C. korseri, where macromolecular secretion systems and pathogenic islands occur [iroBCDEN gene cluster) for colonisation can also be acquired by means of horizontal gene transfer in some members of the family of Enterobacteriaceae [Moreover,  animals , 6, as tds occur . The pateriaceae . These gCitrobacter has been classified into 11 species [Citrobacter species mimic Escherichia coli, so that they are readily misidentified when phenotypic microbiological tests are used [Enterobacteriaceae [Citrobacter species in the ecosystem [Based on microbiological phenotypic methods and DNA hybridisation technology, the genus  species . The gen species , 11. Mosare used , 12. Theeriaceae , 14. Howeriaceae –18. Thercosystem –22. Thiscosystem , 24.Citrobacter species that are multi-drug resistant, namely C. freundii, C. koseri, C. braakii and C. youngae, were recently identified and reported in the Democratic Republic of the Congo, Benin, Nigeria, Algeria, Tunisia and South Africa [Citrobacter spp. in river systems, possibly by means of faecal run-off [Enterobacteriaceae are aggravated by the rise of the antimicrobial-resistant bacteria phenomenon. This phenomenon has been identified as one of the most considerable health challenges globally [h Africa . In South Africa –28. The h Africa . Livesto run-off , 31. Theglobally –34.Enterobacteriaceae have been characterised as displaying the ability to produce ESBL [Citrobacter species strains can also be inferred by means of this approach. Environmental samples are often overlooked and could be the main drivers for AR that can be transmitted to animals and humans [Citrobacter species obtained from surface river water in the Mooi River, North West Province, by using antibiotic susceptibility testing, screening for ESBL production and whole-genome sequencing.Many uce ESBL . These Euce ESBL . A wholed humans –39. The E. coli species on selective differential SMAC-CT agar. All isolates were characterised as ESBL-producing species upon observing sub-culturing on CHROMagar ESBL media. The metallic blue colonies with reddish halo formation indicated the presence of Klebsiella, Enterobacter or Citrobacter species. Furthermore, the Enteropluri results showed similar biochemical reactions between isolates S21 and S24. S25 was the only isolate that was able to produce hydrogen sulphide. All four isolates were able to utilise citrate as a carbon source. On the basis of a disc diffusion assay (DDA), the antimicrobial patterns reflected in Table C. murliniae strain 24. All the isolates examined in the present project, apart from C. murliniae, were classified as multi-drug resistant (MDR), since they were resistant to beta-lactams, macrolides and aminoglycosides.Based on their inability to ferment sorbitol, the four isolates were presumptively identified as pathogenic Citrobacter genomes showed between 62 and 144 contigs . Strain S24 had a 99.26% ANI when the C. murliniae strain P080C CL was used as a reference. Strain S25 was identified by means of pubMLST as C. portucalensis and was found to share 90% of its identity with most of the C. freundii genomes. ANI classified this strain as C. portucalensis when C. portucalensis A60T was used as a reference.The scaffolds engendered by the sequenced C. braakii strains S21 and S23 have genomes that are 4.98 Mb and 4.80 Mb in size, respectively. Different plasmids replicon types were present in the C. murliniae strain S24 and C. portucalensis strain S25. These genomes were larger (> 5.2 Mb) than the C. braakii genomes sequenced in this study. This was confirmed by the high number of coding sequences (CDS) in these two strains. The GC content of Citrobacter genomes ranged between 50% and 52%. The GC content of sequenced C. braakii genomes was approximately 52%, which was the same as the reference strain C. braakii ATCC 8090T and C. braakii strain DY2019 of the present study. The GC of the C. murliniae strain S24 was 50.62%, which was the same as the C. murliniae strain PC080C CL as isolated from human faeces available in GenBank. However, the genome size of C. murliniae strain PC080C CL was 5.0 Mb, which was slightly lower than that of C. murliniae strain S24 as sequenced in the present study. The genome size of the C. portucalensis strain S25 was 51.49%, which was similar to the C. portucalensis A60T reference strain.The sequenced Citrobacter spp. was constructed to assess genetic diversity  in Rwanda.These two species, along with the unassigned Citrobacter genomes were deduced. Except for the Citrobacter spp. cluster, which could not be assigned with species identification, strains consistently grouped in accordance with their MLST profiles. MLST and pangenome analysis allocated the following five species to their respective clusters: C. braakii (n = 59), C. gillenii (n = 6), C. europaeus (n = 7), C. murliniae (n = 1), C. portucalensis (n = 31) and Citrobacter spp. (n = 4). The pangenome phylogenetic tree is represented by samples from animals (n = 21), humans (n = 44), the environment (n = 35) and plants (n = 11). Each Citrobacter species cluster consisted of human and animal isolates, except for the clusters of C. murliniae and C. gillenii.Through a de novo assembly-based pangenome study, the evolutionary relationships among 109 ESBL-Citrobacter species  were classified as ESBL-producing isolates comprising one of the 18 identified bla-CMY- genes: that is, bla-CMY-, bla-26CMY−, bla-CMY−39, bla-CMY−46, bla-CMY−59, bla-CMY−66, bla-CMY−70, bla-CMY−65, bla-CMY−71, bla-CMY−74, bla-CMY−77, bla-CMY−82, bla-CMY−83, bla-CMY−86, bla-CMY−93, bla-CMY−100, bla-CMY−101 and bla-CMY−104. No CMY genes were present in the C. europaeus and C. gillenii clustered strains. The bla-CMY−101  and bla-CMY−82  were prevalent in the genomes of C. braakii, whereas bla-CMY−59  and bla-CMY−77  occurred in the genomes of C. portucalensis.The vast majority of qnrB) were found in 42 of the 109 genomes of the Citrobacter species examined  of the C. europaeus strains that had been isolated from humans (n = 6), except for strain CB00169. The qnrB13 gene was present in the genome of the sequenced C. portucalensis strain S25 as isolated from the environment: however, this gene was also detected in human and animal samples. This strain clusters with the C. portucalensis genomes CB00079, PBIO1950 and PBIO1938 strains, which are qnrB17-positive. C. portucalensis appears to contain a variety of qnrB genes: eleven of these were determined in this cluster.The quinolone resistance genes  across the four Citrobacter species  genes in all four sequenced genomes. The pmrE gene, which affects the peptide antibiotics class by means of antibiotic target alteration, was the only AR gene found in C. braakii strain S21  differentiates this species from the other three genomes strains that were compared in this study.The phenotypic features that were identified by using DDA affirmed the presence of the extended beta-lactamase , and genes involved in capsule polysaccharide biosynthesis (tviBCDE) could be exclusively used to differentiate the C. braakii genomes from those of C. murliniae and C. portucalensis. The C. murliniae S24 strain exhibited a virulence profile that was distinct from the other genomes that had been compared. This genome consisted of the iroABCDEN gene cluster that encodes a heme receptor protein shuA for the iron-salmochelin siderophore system. Moreover, the iron uptake gene, chuY, was exclusive to this strain.All four isolates showed a similar extracellular enzyme production profile, except for isolate S24, which did not produce catalase and gelatinase. Overall, though, lecithinase, gelatinase, lipase, DNase, proteinase and catalase were produced. A heatmap was generated of the virulence gene profile across the four sequenced genomes so as to identify the main pathogenicity genes in sis Fig. . The presis Fig. . It was Citrobacter genomes of this study, excluding C. braakii S23, indicated the presence of mobile genetic elements such as insertion sequences (ISs) and transposons (Tns). The three genomes that had insertion sequences shared a commonly identified element, IS5075. Mobile genetic elements such as ISEsa1 and ISEsa2 were shared among C. murliniae and C. portucalensis genomes. The unique profile of IS elements in C. portucalensis strain S25 were found in IS903, ISSpu2 and ISSKox1. In contrast, ISEc33, ISKpn24 and ISKpn26 were present in C. murliniae strain S24. Except for the C. braakii strain S23 genome, only the transposase Tn5403 was found in the sequenced Citrobacter genomes in this study. Type IV coupling proteins (T4CPs) were present in all the sequenced Citrobacter spp. genomes, except in C. braakii strain S21.Table Citrobacter spp. consisted of a diverse category of translocation systems known as the bacterial Type IV secretion systems (T4SSs), except in the genome of C. braakii strain S23. Genome C. murliniae strain S24 presented a large number of T4SSs, which merits further investigation. The TraD, TraF, TraH and TraG Type IV secretion were found to be common among C. murliniae S23 and C. portucalensis strain S25. Except for C. braakii S23, all the other sequenced Citrobacter species carried plasmids, while C. murliniae consisted of different plasmids . The plasmid AA543 of C. murliniae strain S25 had a VirD4-TraABCDEFGH-TrbABCDEFGHIJKLV gene cluster, while plasmid AC978 had a TraX_F-TrbBCDEFGHIJLN gene cluster. Citrobacter portucalensis strain S25 carried four plasmids, and plasmid AB130 consisted of the TraABCEFGHIKLNVW gene cluster.Genomes of ds Table . PlasmidC. freundii (CP037735) (Supplementary Table C. murliniae strain S24 were classified as conjugative plasmids and were closely related to Klebsiella oxytoca strain CAV1374 plasmid pKPC (CP011635) and Escherichia coli JJ1886 (CP006788), respectively. None of the plasmids discovered in the reference strain P080C_CL and sequenced C. murliniae strain S24 were comparable. Sequence typing of the C. portucalensis strain S25 plasmid AB130 and AF578 were identified to be closely related to C. freundii (CP037735) and Salmonella enterica subsp. enterica serovar Cubana str. CFSAN002050 (NC_021819), respectively. Meanwhile, plasmids AF384 and AF578 of this strain were found to be closely related to Enterobacter roggenkampii (CP019840) and Pelobacter propionicus DSM 2379 (CP000484), respectively.Typing analysis of this plasmid AC978 demonstrated that it was related to the Citrobacter species that carried ESBL genes were identified during the present study, which assessed the characteristics of Enterobacteriaceae bacteria that circulate in the Mooi River system in the North West Province, South Africa. The prevalence of ESBL-producing Enterobacteriaceae is receiving increasing attention as a threat to global health [Citrobacter species were first assumed to be E. coli on the basis of conventional phenotypic microbiological tests. However, by utilising high-resolution taxonomic categorisation that included ANI and pangenome analysis, these isolates were verified as belonging to the Citrobacter species. The use of traditional methods for Citrobacter species identification is not recommended, because they are frequently flawed [Four l health . Environy flawed , 17.Citrobacter species among its various genetic clusters, which raises concerns about the spread of antibiotic-resistance genes in humans and animals as well as the acquisition of such genes. Additionally, AR genes, virulence genes and mobile genetic elements were investigated. In the case of river systems especially, the population genetic structure of Citrobacter genomes has not been fully documented. The first Citrobacter species genomes to be isolated from surface water in South Africa exhibit heterologous unique structures that are closely related to the human and animal-associated Citrobacter spp. genomes. These Citrobacter species were successfully identified in the present study as C. braakii (n = 2), C. murliniae (n = 1) and C. portucalensis (n = 1). Thus far, only one genome was sequenced and documented from a human feaces sample in Norwich, United Kingdom [C. murliniae: it is the only current genome that is available in GenBank. C. portucalensis has been well characterised by means of various biochemical tests [Citrobacter species such as C. braakii, C. freundii, and C. europaeus is lacking.The present project dispersed the environmental strains of the  Kingdom  for C. mal tests  However,Citrobacter species belong to the critically essential antibiotic classes [Enterobacteriaceae infections [Citrobacter spp. genomes of the present study displayed resistance against these critically and highly important antibiotic classes, the latter as described by the WHO [Antibiotic resistance remains a public health concern . Some of classes . Resistafections . The seq the WHO . The pheCitrobacter genomes was explored by means of WGS. We found that all species had genes that encoded diverse types of antibiotic efflux pumps, including resistance-nodulation-cell division (RND) types, major facilitator superfamily (MFS) types, ATP-binding cassette (ABC) types, all of which confer resistance to aminoglycoside antibiotics, fluoroquinolone and macrolides  were classified as ESBL-producing isolates consisting of one of the 18 identified bla-CMY− genes, including bla-CMY−26, bla-CMY−39, bla-CMY−46, bla-CMY−59, bla-CMY−66, bla-CMY−70, bla-CMY−65, bla-CMY−71, bla-CMY−74, bla-CMY−77, bla-CMY−82, bla-CMY−83, bla-CMY−86, bla-CMY−93, bla-CMY−100, bla-CMY−101 and bla-CMY−104. Most ESBL-producing isolates have been reported in Enterobacteriaceae [bla-CMY−101  and bla-CMY−82  were prevalent in the C. braakii genomes. The bla-CMY−101 and bla-CMY−82 genes have been reported from C. braakii strains that had been isolated from a river in China [Over the last decade, cyclines . The preeriaceae , 40. In bla-CMY−101 and bla-CMY−82, which contain isolates, are circulating in the river system, However, they are not limited to this environment, as some of bla-CMY−101 and bla-CMY−82 were isolated from plants and humans  [qnrB gene is prevalent in C. freundii strains isolated from human clinical specimens [Citrobacter is the origin of qnrB, as this gene is distributed in species such as C. freundii, C. braakii, C. youngae, and C. werkmanii, However, allele variation specific to each species does exist [A fluoroquinolone resistance indicator,  species , 47, whier spp.)  and the pecimens . Other ses exist , 49.C. braakii genomes can be distinguished from C. murliniae and C. portucalensis on the basis of gene encoding for cell surface localisation of the CPS (vexC) as well as genes involved in capsule polymer synthesis (tviB and tviE). Extant studies also report these genes in this kind of context [C. murliniae strain S24 could not produce catalase, which was confirmed by the absence of the katB gene. A gene cluster of the salmochelin siderophore system (iroBCDEN) was found in C. murliniae S24: it is important for the pathogenicity pathway that confers an advantage in colonisation. This gene cluster was first described in Salmonella enterica [E. coli strains, which suggests acquisition through horizontal gene transfer [iron-BCDEN gene clusters were identified in Citrobacter species, particularly in the C. murliniae genome.The present study demonstrated that the  context . The preenterica , and orttransfer . The preCitrobacter spp. strains were associated with iron uptake (chu and ent). Pathogenic bacteria need iron, which they must acquire during infection, if they are to thrive in a mammalian host [C. murliniae contained an additional iron update gene, namely chuF. This gene is an anearobilin reductase that exhibits kinetic cooperativity during the process of biochemical degradation in E. coli O157:H7 [Citrobacter., especially C. koseri, extant studies have found strains that consisted of this iron gene cluster [Other virulence factors identified in all ian host . However O157:H7 . Among m cluster . HoweverCitrobacter species [Citrobacter genomes strains, except in the case of C. braakii strain S23. This was most probably due to the fact that the plasmid was absent from the strain.The identified plasmids, as determined in the present study, were mostly associated with T4SSs. The latter is a broad distribution group that secretes macromolecules in Gram-negative and positive bacteria as well as other eukaryotic cells . They plCol) factors, such as plasmid Col(VCM04), determine the production of proteins called colicins, which can kill other bacteria. Given certain environmental conditions, plasmids can provide many different types of selective advantages, including antibiotic resistance, resistance to pollutants or UV and biofilm formation [We detected an incompatibility group of plasmids replicon genes, including IncP and IncH. The IncP plasmid replicon type plays important roles around the transfer of antibiotic resistance genes and other types of genetic information among bacteria . They alormation .C. Murliniae strain S24 consisted of three distinct types of T4SS gene clusters  that were encoded in the plasmid AA543: VirD4, TraABCDEFGH, and TrbABCDEFGHIJKLV. Meanwhile, the plasmid AC978 encoded the TraXF and Trb gene clusters TraXF and TrbBCDEFGHIJLN. It is well known that some T4SS components, such as the tra-operon involved in conjugation, exhibit significant sequence and structural homology and have identical protein activities that are associated with pathogenicity [Pseudomonas aeruginosa, Vibrio cholerae, enteroaggregative Escherichia coli, Burkholderia thailandensis, Serratia marcescens, Burkholderia malle, and Salmonella enterica, which are also involved in conjugative transfer and plasmid replication [The sequenced genicity , 60. Thelication .C. portucalensis, C. murliniae, and C. braakii are multi-drug resistant pathogens with intrinsic genes that encode forESBL. The use of whole-genome sequencing identified these species and the phylogenomic structure of the four Citrobacter species that circulate in surface water in the Mooi River of the North West Province, South Africa. The C. murliniae strain showed a unique profile with an iron-BCDEN gene cluster of salmochelin siderophore system. Fluoroquinolone resistance (qnrB13) was only detected in C. portucalensis, which was closely related to animal and human C. portucalensis.Our findings suggest that Citrobacter species, is that all four genomes carried the bla-CMY-gene, thus presenting a threat in its transmission potential for the environment and possibly other sectors, the latter as described in the One Health model.These genomes represent important reference points. They act as crucial pieces of detailed genetic information and phenotypic data that will be used as models for the characterisation of antibiotic resistance mechanisms and interactions. Of further importance is that the study, as centred on ESBL-producing Enterobacteriaceae and their potential antibiotic resistance within surface water in the Mooi River system. In summary of that study, water samples (100 ml) were processed by the Colilert-18  procedure. Selective media of Sorbitol MacConkey agar  infused with antibiotics  and 2.5 mg/L Potassium Tellurite  (SMAC-CT agar)) were further used to isolate presumptive pathogenic Enterobacteriaceae species incubated at 37 °C for 24 h.Samples were collected from a Mooi River site (26° 42’ 29.3” S 027° 06’ 20.6” E), as described in earlier part of the present project . HoweverE. coli ATCC 10,536, pathogenic E. coli O157:H7 ATCC 700,728 and ESBL-producing E. coli ATCC 35,218 . ESBL-producing Citrobacter isolates were confirmed phenotypically on CHROMagar™ ESBL . The agar differentiates between ESBL E. coli (red colony presentation) and ESBL Klebsiella, Enterobacter and Citrobacter spp. . The selection of these four isolates was based on their ability to produce the ESBL enzymes.The isolates were further subjected to Rainbow agar O157  infused with Novobiocin sodium salt (Sigma Aldrich), and were subsequently incubated at the same conditions as those of the SMAC-CT agar. The reference strains that were used in this study were non-pathogenic Enterobacteriaceae were analysed according to the manufacturer’s instructions on the Enteropluri-Test strip . A set of 13 biochemical reactions was detected, which included glucose fermentation, gas production, hydrogen sulphide production, lysine decarboxylation, ornithine decarboxylation, adonitol fermentation, lactose fermentation, arabinose fermentation, sorbitol fermentation, dulcitol fermentation, phenylalanine deamination, urea hydrolysis and citrate utilisation [These ESBL-producing lisation . Furtherlisation .Citrobacter isolates (n = 4) were sub-cultured onto Mueller-Hinton agar  by using the Kirby-Bauer disc diffusion method [https://clsi.org/standards/products/free-resources/access-our-free-resources/) and the clinical breakpoint for bacteria (v12.0) EUCAST guidelines .Pure cultures of n method  for a seCitrobacter isolates while following the manufacturer’s protocol. The concentration and quality of the DNA were determined by using the Qubit 2.0 fluorometer . The DNA integrity was monitored on a 2% agarose gel pre-stained with ethidium bromide and visualised on a UV transilluminator. Paired end libraries were generated by using the NEBNext kit  according to the NEBNext for Illumina protocol. Cluster generation and sequencing were performed by means of the MiSeq Reagent Kit V3 (2 × 300 bp) on the MiSeq 2000 .Overnight broth cultures of the bacterial isolates were centrifuged and pelleted for DNA extraction. The Chemagic kit  was used to extract the total genomic DNA of the Citrobacter spp. strains were assembled de novo by using the SPAdes v1.1.0 pipeline [Citrobacter species. The assembled contigs were annotated by means of the NCBI prokaryotic genome automatic annotation pipeline (PGAAP) [Quality of the sequenced reads were assessed by using FastQC software 0:10.1 . Trimmompipeline . The minpipeline  was addipipeline  was used (PGAAP) .Citrobacter spp. on the website https://pubmlst.org/ were used to determine the species identity. FastANI version 1.3 [Citrobacter species as references. Based on the ANI, a percentage identity of > 90% against the reference genome was considered to belong to the same species. For pangenome analysis, 109 Citrobacter publicly available genomes were retrieved from GenBank  [Citrobacter spp. By using the Virulence Factor Database  [The ABRicate pipeline, which was assessed on 25 January 2023, was employed to identify antibiotic resistance and virulence genes in the four genomes of the isolated sFinder)  with mindb vfdb) , 78 in tCitrobacter by means of Mobile Element Finder v1.0.3 (2023-01-20) on the Center for Genomic Epidemiology platform [Mobile genetic elements were investigated on the sequenced genomes of the platform . Plasmidplatform . The MOBplatform , and Oriplatform  was usedBelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5


Staphylococcus pseudintermedius is an important cause of clinical infections in small-animal-veterinary medicine. Evolutionary changes of strains using multilocus sequence typing (MLST) have been observed among S. pseudintermedius in European countries and the United States. However, there are limited or no studies on the detection of methicillin resistant Staphylococcus pseudintermedius (MRSP) and predominating MLST strains in South Africa. Therefore, this study aimed to determine the molecular epidemiology of S. pseudintermedius in South Africa. Twenty-six, non-duplicate, clinical isolates from dogs were obtained as convenience samples from four provinces in South Africa. The Kirby Bauer disk diffusion test was used to determine antimicrobial susceptibility. We used Resfinder and the Comprehensive Antibiotic Resistance Database (CARD) to detect antimicrobial resistance genes. Virulence genes were identified using the virulence factor database and Basic Local Alignment Search Tool (BLASTN) on Geneious prime. geoBURST analysis was used to study relationships between MLST. Finally, the maximum likelihood phylogeny was determined using Randomized Axelerated Maximum Likelihood (RAxML). Twenty-three isolates were confirmed as S. pseudintermedius of which 14 were MRSP. In addition to β-lactam antimicrobials, MRSP isolates were resistant to tetracycline (85.7%), doxycycline (92.8%), kanamycin (92.8%), and gentamicin (85.7%). The isolates harbored antimicrobial resistance genes -Ie-aph(2”)-Ia, ant(6)-Ia, and aph(3’)-III) and virulence genes . MLST analysis showed that ST2228, ST2229, ST2230, ST2231, ST2232, ST2318, ST2326 and ST2327 are unique sequence types in South Africa. Whereas, previously reported major STs including ST45, ST71, ST181, ST551 and ST496 were also detected. The geoBURST and phylogenetic analysis suggests that the isolates in South Africa are likely genetically related to isolates identified in other countries. Highly resistant MRSP strains  were reported that could present challenges in the treatment of canine infections in South Africa. Hence, we have gained a better understanding of the epidemiology of MRSP in the African continent, the genes involved in resistance and virulence factors associated with these organisms. Staphylococcus pseudintermedius is a commensal bacterium that affects the skin and mucosa and can transiently colonize human beings [S. pseudintermedius is attributed primarily to the mecA gene harbored within a mobile genetic element called SCCmec which encodes penicillin binding protein 2a (PBP2a) [S. pseudintermedius is the most common staphylococcal species isolated from dogs in South Africa [et al. revealed a high occurrence (83.89%) of S. pseudintermedius and 85.9% prevalence of mecA positive carriage among clinical S. pseudintermedius isolates from five geographical dispersed laboratories in South Africa [S. pseudintermedius isolated from dogs has not been investigated in this country.In dogs, n beings –4. Theren beings ,5–7. Met (PBP2a) –10. The  (PBP2a) ,12. S. ph Africa . Recentlh Africa  However,S. pseudintermedius worldwide. As a result, CC71/ CC258 in Europe, CC68 in the United States, and CC45/CC112 in Asia have been established as dominant clonal complexes in the past [et al. reported ST39, ST200, ST54, ST204, ST18 among S. pseudintermedius isolates from a veterinary hospital in Zambia [S. pseudintermedius isolated from dogs are lacking.Multi-locus sequence typing is a well-established method used to identify dominant MRSP lineages and geographical dissemination of n Zambia . In Botsn Zambia . In Soutmec type Vt whereas ST71 in Europe is associated with SCCmec type III [nanB gene for sialidase, while ST496 in France carries the genes for surface adhesion virulent spsI and spsF [et al. reported multidrug resistance and increasing levels of resistance to fluoroquinolones and sulfonamides among S. pseudintermedius isolated from dogs at a veterinary teaching hospital [et al. reported high penicillin and tetracycline resistance among S. pseudintermedius isolates implicating tetM and blaZ genes [There are significant regional differences in patterns of antimicrobial resistance and virulence factors associated with distinct clonal lineages. For example, a low percentage of tetracycline and chloramphenicol resistance was reported from isolates belonging to the CC71 lineage in North America compared to isolates belonging to the CC71 lineage in Europe . In Frantype III ,17. Addiand spsF ,17. In Shospital . In ZambaZ genes . HoweverS. pseudintermedius as a result of the acquisition of SCCmec and the international movement of animals [S. pseudintermedius has changed over the years in South Africa. Therefore, the aims of this study were to determine the phenotypic resistance profile, to identify resistant genes, and virulence genes, to determine MLST profiles as well as geoBURST analysis of MRSP clones circulating in South Africa.Recent studies in various countries show a changing population structure among  animals ,14,19. AS. pseudintermedius isolates were inoculated onto Columbia blood agar plates containing 5% sheep blood (Remel) and incubated overnight at 37°C in 5% CO2. The isolates were identified as S. pseudintermedius using the matrix-assisted laser desorption/ionization-time of flight mass spectrometry . S. pseudintermedius species assignment was made when the log(score) values were ≥ 2 [A total of 26, non-duplicate, clinical samples collected in 2021 from dogs were received as convenience samples from a veterinary diagnostic laboratory in South Africa, Gauteng. The samples were obtained from Johannesburg, Middleburg, Cape Town, Port Elizabeth, Pietermaritzburg, and Randburg and submitted for diagnostics analysis at a veterinary diagnostic laboratory. Clinical isolates were taken from post-operative infections, cystitis, otitis, wound, abdominal fluids, left front limb, pyoderma, and hock hygroma. Isolates were then shipped to the University of Tennessee Veterinary College, bacteriology lab in transport media for further analysis. S. pseudintermedius isolates. The Kirby-Bauer disk diffusion method was used and interpreted as recommended by the most current Clinical and Laboratory Standards Institute (CLSI) veterinary guidelines available at the time of the study [S. aureus by Rezaeifar et al. [Antimicrobial susceptibility tests were performed on all confirmed he study .Mueller he study . Interprr et al. . Additior et al. . The CLSr et al. . Whereasr et al. . Erythrohttp://bioinf.spbau.ru/spades). Annotation and analysis of the genomes was performed using the PATRIC software [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA892647.Three or four bacterial colonies were suspended in 3 ml of sterile tryptic soy broth, incubated at 37 C° overnight, and a commercial kit  was used for DNA extraction. The concentration was measured using a nanodrop and Qubit 4 fluorometer . Sequencing libraries were constructed using a Nextera DNA sample prep kit  according to the manufacturer’s instructions. The genomes were sequenced using a MiSeq platform  with a single end read length of 150 bp at the University of Tennessee Immunology Laboratory. Whole genome sequences were assembled using SPAdes (software . The genhttps://card.mcmaster.ca/) databases on PATRIC were used to identify resistance genes. Virulence genes were detected using the Virulence Factor Database (VFDB), additionally the Geneious prime was used for BLASTN analysis of virulence genes [ResFinder (genomic epidemiology.org), CARD  was used to determine the sequence type (ST) of each isolate as we described previously [S. pseudintermedius database, we ascertained the clonal relationships of the sequence types obtained in this study through goeBURST clustering analysis on PHYLOViZ [Multilocus sequence typing (MLST) of seven housekeeping genes  in PATRIC were used to identify 655 protein families from genes that were present as a single copy per genome . For eacAll proportions and 95% confidence intervals in this study were performed using SAS ® 9.4 .S. pseudintermedius and distributed geographically as shown in S. pseudintermedius alleles were unique and were assigned STs in this study . Five of the MRSP isolates belonged to ST496, ST1308, ST45, ST181 and ST1111. Whereas seven of the MSSP isolates were previously reported on the pubMLST database .Based on MALDI-TOF, twenty-three isolates were confirmed as All isolates were resistant to at least one antibiotic whereas 87% of the isolates were multidrug resistant (MDR)  . Most ofblaZ gene which encodes for narrow spectrum beta-lactamases was detected in all but one isolate (S. pseudintermedius isolates harbored tetM resistance genes whereas one isolate harbored tetK. The dfrG gene conferring the trimethoprim-sulfamethoxazole resistance phenotype was detected in 14 isolates. Most (11/23) of the isolates in this study harbored macrolide resistance, ermB genes. Chloramphenicol acetyltransferase genes (catpC221) were detected in six isolates. Isolates harbored aac6-aph2 (14/23), ant6-Ia (9/23) and aph3-III (9/23) that confer resistance to acetyltransferase, nucleotidyl transferase and phosphotransferase, respectively.The  isolate . SixteenadsA), exfoliative toxin (speta) and intracellular adhesion gene (icaA and icaD)  . WhereasThe isolates representing the assigned STs  were singletons and were not related to the STs in the database . These SS. pseudintermedius genomes and the 23 isolates in this study were generated  observed among dogs with pyoderma and otitis in South Africa . High leospitals . Whereas31, 2017 . Therefoet al. reported a significant increase in the proportions of S. pseudintermedius resistant isolates to trimethoprim-sulfamethoxazole, clindamycin and orbifloxacin between 2007 and 2012 in South Africa (13). Canine antibiotic resistance in South Africa may be influenced by changes in veterinary prescription practices. Therefore, more studies elucidating the implications of antibiotic use and MRSP in South Africa should be a priority for future studies.Resistance to antimicrobials was higher among MRSP compared to MSSP isolates. In addition to β-lactam resistance, MRSP isolates in this study were resistant to tetracyclines, aminoglycosides, folate inhibitors, macrolide and lincosamides. This is similar to results reported among MRSP isolates in Argentina and Finland ,35. In ablaZ, tetM, ermB, and dfrG) were found to be the predominant genes encoding resistance in S. pseudintermedius. Resistance to aminoglycosides was associated with the presence of adenyl nucleotidyl transferase gene (ant(6)-Ia), phosphotransferase gene (aph(3’)-III), and the acetyltransferase/phosphotransferase gene (aac(6’)-Ie-aph(2”)-Ia) in all isolates except five that only harbored the aac(6’)-Ie-aph(2”)-Ia gene. Other studies have reported a strong correlation between the resistant phenotypes and resistance genes detected among S. pseudintermedius isolates [Similar to other studies, the genes responsible for penicillin, tetracyclines, erythromycin and trimethoprim-sulfamethoxazole , nanB  and spsM . The nanB gene contributes to colonization by offering a carbon source for growth, forming biofilms, or increasing adherence if receptors on the host are exposed [spsM cell wall protein and sdrE have been associated with mediating binding, invasion, and degradation of epithelial cells [LukS-I and LukF-I) that are responsible for destruction of canine polymorphonuclear leukocytes (PMNs) by pore formation and cell lysis [S. pseudintermedius lineages has previously been associated with variation in the surface proteins. Therefore, more studies analyzing the functional expression of virulence genes among canine S. pseudintermedius isolates could inform on the successful lineages in South Africa.The prevalence of virulence genes among  studies ,38,39. N exposed . The spsal cells ,41. Wherll lysis . The sucS. pseudintermedius population structure isolated from dogs in South Africa could be non-clonal. In comparison, Dos Santos et al. concluded that the population of S. pseudintermedius was weakly clonal due to the presence of recombination among STs worldwide [spsI and spsF associated with adhesion to the extracellular matrix [tetK which encodes tetracycline resistance through efflux pumps. This is a unique feature that has been identified for ST551 among MRSP isolated from dogs in Poland and Slovenia [S. pseudintermedius strains in South Africa could help in successful treatment of canine infections.geoBURST analysis could not identify a main clonal founder and clonal structure of MSSP and MRSP isolates in South Africa. In previous studies, MRSP isolates were associated with fewer clones than MSSP isolates, which had relatively less clonal expansion ,5,19,36.orldwide ,19,43. Torldwide . Of concr matrix . The isoSlovenia . Hence, S. pseudintermedius isolates submitted to a veterinary diagnostic laboratory in South Africa. The results from this study suggest the presence of a highly diverse clonal population structure of S. pseudintermedius. Additionally, highly resistant clonal lineages such as ST71, ST45, ST551 and ST496 were detected that pose a threat to the successful treatment of MRSP infections in South Africa. The collection of S. pseudintermedius and MRSP in South Africa is underrepresented compared to worldwide continental reports. However, information on antimicrobial resistance and molecular epidemiology of MRSP in South Africa is vital to obtain a universal understanding of this organism. Therefore, future studies should investigate the occurrence and patterns of multidrug resistant MRSP isolated from canine clinical samples representing a larger population of dogs in South Africa.This is the first report addressing the phenotypic and genotypic characterization of canine S1 Table(XLSX)Click here for additional data file.S2 Tablehttps://pubmlst.org/organisms/staphylococcus-pseudintermedius.The alleles were assigned using (TXT)Click here for additional data file.