Immunological mechanisms and therapy approaches in psychotic syndromes were recently supported by the discovery of autoantibody-associated limbic and non-limbic encephalitis. However, how clinical diagnostic procedures in psychiatry should be adapted to these new insights is still unclear. In this study, we analyzed the cerebrospinal fluid (CSF) and neuroimmunological alterations and their association with cerebral MRI (cMRI) and electroencephalographic (EEG) findings. From 2006 to 2013, we acquired 180 CSF samples from psychotic patients. Between 2006 and 2009, CSF examinations were only performed in cases in which organic brain disease was suspected. Since then, this procedure has been integrated into our routine diagnostic workup. CSF basic diagnostics were supplemented by measuring antineuronal antibodies against intracellular synaptic antigens, antibodies against intracellular onconeural antigens, antibodies against neuronal cell surface antigens and thyroid antibodies. In addition, cMRIs and EEGs were conducted. We found white cell counts elevated in 3.4% of the cases, albumin quotient elevated in 21.8%, and protein concentration elevated in 42.2%. Evidence of intrathecal immunoglobulin synthesis was found in 7.2% of the cases. Antibodies measured against neuronal cell surface antigens were positive in 3.2%. Reactivity on antibodies against intracellular onconeural antigens were detected in 3.5%. Serum thyroid antibodies were elevated in 24.7%. Abnormalities were found in 39.5% of cMRIs and in 34.3% of EEGs. The main finding of our study was the high prevalence of CSF and autoantibody abnormalities in 54.4% of psychotic patients. In combination with cMRIs and EEGs, 75.6% showed abnormal findings. Our results are discussed with regard to the concept of immunological encephalopathy. Future studies should analyze the efficacy of immunomodulatory therapies. Psychosis was identified as a possible immunological disease as early as 1930 in the work of Lehmann-Facius among others. He described a highly specific method called “brain–lipoid reaction” to detect immunological signals in psychotic patients. This in turn led to the autoimmune hypothesis of schizophrenia Roeder, . Immunolhttp://www.nice.org.uk; German S3-Praxisleitlinie, www.dgppn.de), CSF examinations are not recommended in the diagnostic routine work up of psychotic or affective disorders. However, there have been several recent reports pointing to good response to immune therapy in patients with classical psychiatric manifestations of psychotic or affective disorder without organic symptoms or signs  investigation is the most precise method of detecting and characterizing central nervous system inflammatory processes, even when cerebral magnetic resonance imaging (cMRI) or electroencephalographic (EEG) results are normal. In most guidelines  We wanted to investigate the frequency of CSF and neuroimmunological alterations and their association with cMRI and EEG findings in psychotic syndromes. We hypothesized to find increased prevalences of CSF-basic laboratory abnormalities and autoantibody rates in psychotic patients as well as an association between CSF and cMRI/EEG abnormalities. (2) We wanted to analyze how a change in diagnostic approach might affect the respective immunological findings. Could it be that our new diagnostic approach to offer CSF studies to all psychotic subjects is excessive and produces lots of negative findings? (3) We wanted to analyze on a case by case level, whether the findings of immunological signals have therapeutic implications for our patients.The study received approval from the local ethics committee of the University of Freiburg (EK-Fr 609/14). All patients included gave written informed consent for CSF studies during the clinical diagnostic work up.From 2006 to October 2013, we acquired 180 CSF samples from psychotic patients at the Clinic of Psychiatry and Psychotherapy of Freiburg University Hospital. Lumbar punctures (LPs) were generally performed during the initial manifestation of psychotic symptoms. Three clinically defined subgroups were analyzed: (1) patients with a schizophreniform syndrome, (2) patients with a schizoaffective syndrome, and (3) patients with psychotic syndromes in the context of other disorders .In all cases, paired CSF and serum samples were taken at the same time. CSF diagnostics were performed in the CSF laboratory of the Department of Neurology. CSF white blood count (WBC) and cytological differentiation of CSF sediment were established with manual microscopy  using a Fuchs-Rosenthal counting chamber . Basic quantitative protein diagnostics included total CSF protein concentration, albumin, and Ig G, M, and A concentrations in CSF and serum, respectively . For detection of a blood–brain barrier (BBB) dysfunction, we calculated the age-related albumin quotient since this marker is accepted as the “gold standard” for the estimation of the integrity of the BBB . Mainly, descriptive statistics were calculated for the psychotic patient group. For group comparison (respective age) between patients with and without clear-cut antibody abnormalities, we used a Mann–Whitney Of the 180 examined psychotic patients, 101 were female and 79 were male; the mean age was 34.67 (standard deviation ±14.7) years.r = 0.185, p = 0.013, N = 180) and the albumin quotient . There was no significant correlation between age and WBC  or age and IgG-index . Table Table Table n = 9) were younger on average compared with antibody negative patients (n = 171), however, these differences were not significant .Patients with clear-cut antibody findings . Of the available 175 EEG datasets, 60 showed pathological abnormalities (34.3%). Intermittent generalized slow activity was found most frequently  showed abnormalities in 98 of the 180 cases (54.4%). cMRI or EEG abnormalities were found in 91 of the 180 psychotic patients (50.6%). Total abnormalities in CSF analysis and cMRI/EEG analysis were detected in 136 of the 180 cases (75.6%).The main finding of our study was the high percentage of CSF and autoantibody abnormalities in 54.4% of all psychotic patients and overall organic abnormalities (including cMRI and EEG) in 75.6%. Before discussing the possible relevance of these findings, we have to stress the shortcomings of our open study.The entire study is open and followed clinical practice. Therefore, while the ecological validity might be high, the sample was generated in a very unsystematic way. The total collective includes a group of patients with suspected organic brain disease identified between 2006 and 2009 and a more systematic screening group since 2009. This includes all psychotic patients who gave their written consent for LP. While the majority of patients agreed to all proposed diagnostic procedures could not clarify retrospectively how many patients rejected CSF studies. Therefore, this sample cannot be regarded as representative. Still, since this issue is rather new to psychiatry, it is difficult to obtain data from more systematic samples. To our knowledge, this is the first study published in the literature looking at a large clinical sample of unselected consecutively diagnosed psychotic patients who were comprehensively investigated using CSF analysis, the measurement of different neuroimmunological markers (autoantibodies), EEG and cMRI. The major shortcoming of this study is the fact that we did not analyze a control group for comparison. However, this is an open clinical observational study, and for ethical reasons, we would not have obtained approval for doing LPs in healthy controls from our ethics committee, due to possible complications. The high rates of organic pathologies might also be influenced by medication effects, comorbidities, or patient history . In spite of these limitations, we feel that this study is of clinical significance because we were able to collect a large sample of psychotic patients, who were diagnosed by experienced senior psychiatrists in a tertiary care hospital on a strictly clinical basis. Presently CSF studies do not belong to the recommended diagnostic procedures. However, given the good therapeutic response to immunomodulatory therapies in some patients with immunological encephalopathy the issue of whether these recommendations should be modified and adapted to the latest findings in neuroimmunology is of obvious clinical relevance.One might have hypothesized that the change in diagnostic protocol, essentially introducing CSF examination as a routine diagnostic measure, would result in a majority of negative findings. This, however, was not the case. While the prevalence of elevated CSF WBC count went down from 9.1 to 2.1%, the same was not true for all other global CSF basic signals. In particular, we found strong evidence pointing to disturbed BBB function in psychotic patients.Several studies analyzing CSF in schizophrenia have reported differences, especially in BBB function. Most previous studies are older, and therefore, were assessed under different environmental and technical frameworks . Anti-VGKC complex encephalitis often leads to limbic encephalopathy and symptoms of seizures, memory loss, and affective symptoms  should be considered. For screening, thyroid hormones and particularly thyroid peroxidase and thyroglobulin antibodies should be investigated (Castillo et al., The issue of limbic encephalitis and related immunological encephalopathies as a possible cause for schizophreniform or also affective syndromes is still new in psychiatry. Some authors summarize all antibody associated autoimmune encephalopathies within the umbrella concept of immunological encephalopathies and propose a standardized symptomatic immunomodulatory therapeutic approach for all of these diseases including anti-VGKC- and anti-NMDAR-encephalitis as well as SREAT (Friese and Magnus, The issue of adapting clinical diagnostic and therapeutic standards to the advent of concepts of immunological encephalopathy and limbic encephalitis will most likely be very controversial. In many—if not in the majority—of psychiatric clinics, there won't be diagnostic competence for doing routine CSF studies. Additionally, many psychotic patients will not comply with doing CSF studies and in this constellation, currently, we don't do such investigations neither. The question of cost-effectiveness also arises. Does it make sense to diagnose immunological encephalopathy in a patient with psychological symptoms when it is unclear how this patient should then be treated? While such considerations question the rational of offering CSF studies to all patients with acute psychosis, there are also many arguments in favor of such a procedure. The vast majority of our psychotic patients did comply with these investigations and in fact were actively pursuing this road. One likely reason is that the disease model of “psychosis as an inflammatory brain process” was easier to accept for these patients and their families than the concept of schizophrenia. In quite a few of our immunological encephalopathy patients, the response to immune therapy was very good and in fact much better than the response to the classical variant of symptomatic psychopharmacology. These patients were happy to receive a more causal therapy than, for example, only taking symptomatic antipsychotic drugs.Clinicians must balance the potential benefits of a diagnostic procedure against its medical risks and costs. With the medical risk and financial costs of doing a CSF study being very low, and based on our data one has to judge whether it is worthwhile doing CSF studies in 100 patients in order to detect three with increased WBC counts, about 40 with some evidence of BBB disturbance, seven with OCBs, and seven with autoantibodies against some neuronal cell surface or intracellular antibodies. Given the potentially devastating course of schizophreniform syndromes, the authors of this paper feel that our data strongly point in favor of more comprehensive CSF investigations as routine procedures in psychotic patients. There is still a very large demand for research not only in the diagnostic but also in the linked therapeutic dimension. In case CSF findings like ours were clearly linked to beneficial responses to immunomodulatory treatments, this obviously would change the diagnostic and therapeutic reality in psychiatry in a dramatic way.Annette Baumgartner: Consulting and lecture fees, grant and research support from Bayer Vital GmbH, Biogen Idec, Merck Serono, Novartis, Sanofi-Aventis and Teva. Oliver Stich: Consulting and lecture fees, grant and research support from Bayer Vital GmbH, Biogen Idec, Genzyme, Merck Serono, Novartis, Sanofi-Aventis and Teva. Ludger Tebartz van Elst: Advisory boards, lectures, or travel grants within the last three years: Eli Lilly, Janssen-Cilag, Novartis, Shire, UCB, GSK, Servier, Janssen, and Cyberonics. Dominique Endres, Evgeniy Perlov, Tilman Hottenrott, and Rick Dersch declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The striatum has traditionally mainly been associated with playing a key role in the modulation of motor functions. Indeed, lesion studies in animals and studies of some neurological conditions in humans have brought further evidence to this idea. However, better methods of investigation have raised concerns about this notion, and it was proposed that the striatum could also be involved in different types of functions including cognitive ones. Although the notion was originally a matter of debate, it is now well-accepted that the caudate nucleus contributes to cognition, while the putamen could be involved in motor functions, and to some extent in cognitive functions as well. With the arrival of modern neuroimaging techniques in the early 1990, knowledge supporting the cognitive aspect of the striatum has greatly increased, and a substantial number of scientific papers were published studying the role of the striatum in healthy individuals. For the first time, it was possible to assess the contribution of specific areas of the brain during the execution of a cognitive task. Neuroanatomical studies have described functional loops involving the striatum and the prefrontal cortex suggesting a specific interaction between these two structures. This review examines the data up to date and provides strong evidence for a specific contribution of the fronto-striatal regions in different cognitive processes, such as set-shifting, self-initiated responses, rule learning, action-contingency, and planning. Finally, a new two-level functional model involving the prefrontal cortex and the dorsal striatum is proposed suggesting an essential role of the dorsal striatum in selecting between competing potential responses or actions, and in resolving a high level of ambiguity. The basal ganglia have been a topic of research for more than 100 years. This array of subcortical nuclei located at the base of the brain has intrigued researchers to investigate their contribution to behavior. The putamen and caudate nucleus, nuclei from the basal ganglia collectively referred to as the striatum, have received a lot of interest. Before the early 1990s, the majority of studies were performed on animals following a brain lesion to the area of interest. The main goal of these studies was to assess the specific deficits after carefully producing brain lesions leading to characterization of striatal functions via an animal model. In humans, most studies, that have reported effects of striatal lesions, correlated deficits with the location of the brain lesion either postmortem, or following a traumatic brain injury. However, during that period, investigative methods were not sensitive enough to assess the extent of subcortical injuries with accuracy due to their location in the brain as hidden structures.in vivo observation of the striatum became possible and this development opened up the field to study healthy human individuals. Traditionally, relying on data mainly derived from animal research, the striatum was believed to play a role in motor functions only. The striatum was described to be involved in the execution of sequences  and magnetic resonance imaging (MRI), Several models have been proposed in order to explain the role of the striatum in relation to the cortex. Graybiel  has propOne can distinguish between two major types of models with respect to the involvement of the prefrontal cortex and the dorsal striatum in cognitive function. The first type stresses the importance of the striatum in learning, such as in implicit learning , insisting on the rule or set generation required to choose an action. We also provide evidence that this role is independent of the type of stimuli being processed and may not contradict the role of the striatum in implicit and sequence learning.The anatomy of the basal ganglia has been largely described in the past few years Nolte, . The basWith respect to the general organization, the striatum and subthalamic nucleus receive excitatory glutamatergic inputs from the cerebral cortex. The striatum projects to the GPi via monosynaptic inhibitory gamma-aminobutyric acid (γ-GABA) connections. After receiving the signal the GPi sends inhibitory γ-GABAergic projections to the thalamus, which then transfers excitatory projections to the cerebral cortex closing the loop. This is referred to as the “direct pathway” which will favor a behavior. Additionally, there is an “indirect pathway” by which the striatum sends inhibitory projections toward the external segment of the globus pallidus (GPe), which in turn will send γ-GABAergic projections to the subthalamic nucleus. The subthalamic nucleus then sends excitatory projections to the GPi, which inhibit the thalamus and reduce cortical excitation. This “indirect pathway” has an overall inhibitory effect on behavior. Lastly, a third “hyperdirect pathway” has been characterized: the cerebral cortex sends excitatory projections directly to the subthalamic nucleus bypassing the striatum and stimulating the GPi by inhibiting the thalamus, leading to movement suppression. It has been proposed that this “hyperdirect pathway” acts as a regulatory mechanism to inhibit unwanted movements.Evidence supporting the existence of fronto-striatal loops, as previously proposed by Alexander et al. , has beeIn humans, several studies have reported cases in which alterations of the striatum led to prefrontal cortex dysfunction. This is the case in Parkinson's disease, a neurodegenerative disease characterized by alteration of the dopaminergic neurons in the substantia nigra pars compacta , cingulate motor areas, and also from the motor nuclei of the thalamus  and one that requires 4–5 moves . In this context, the difficulty resides in the planning of the movements of the beads before the actual execution. Using PET, studies have shown the involvement of fronto-striatal regions, involving the caudate nucleus, during the execution of the difficult planning compared to the simpler planning condition  as opposed to the native language (i.e., L1; Klein et al., As discussed above, studies performed in the last two decades, have accumulated a substantial amount of data supporting the “action value” model suggesting a strong contribution of the striatum in stimulus-response associations (Hassani et al., As pointed out by Lieberman , the dorWe propose that the PFC and striatal regions interact in order to plan and/or to execute new actions. In both cases, the idea of self-initiated involvement is predominant. The first level concerns the organization of information in working memory depending on the task at hand. The mid-DLPFC, which occupies area 9/46 and 46, was proposed to play a key role in the monitoring process, which consists of keeping track of the occurrences and non-occurrences of events within working memory. On the other hand, area 47/12 of the VLPFC contributes to the active retrieval process, which allows active comparison between different stimuli held within working memory, and promotes active selection based on specified characteristics. Finally, the posterior region of the PFC is more involved in conditional association between a stimulus and a response. The second level of the model involving the striatum only contributes if a novel action, which involves uncertainty and/or complex manipulation of information, is required. The connection from the DLPFC to the caudate nucleus is more specifically involved in the planning of action, while the VLPFC and the caudate nucleus is more likely linked to its selection, whereas the posterior PFC and the closely related premotor cortex together with the putamen are involved in its execution (Figure This model was largely inspired by results obtained in our laboratory, as we have shown significant involvement of the caudate nucleus and the DLPFC in self-initiated monitoring (Provost et al., Another possible interpretation that needs to be discussed is the possibility that the caudate nucleus might be driven by task difficulty. Indeed, the two concepts are hard to differentiate in the context of executive functioning, especially considering that greater ambiguity can be driven by increasing the number of possible choices. Task difficulty is indeed inherent to more complex cognitive processing, which is relative to the level of novelty and ambiguity. However, it depends on how “task difficulty” is defined and measured. We cannot rule out the effect of task difficulty as being part of the level of ambiguity between stimuli. In consideration of that fact, we argue in favor of the novelty aspect of a task and the ambiguity related to it requiring more cognitive control resulting in an increase of the task difficulty in some instances. As the task difficulty increases, more time and brain resources are usually required to execute a task (Just et al., The goal of this review was to investigate the relationship between the dorsal striatum and the prefrontal cortex with respect to executive processes by exposing a functional model. As thoroughly discussed earlier, the dorsal striatum participates in various tasks involving specific processes linked to executive functioning. Our review highlights the possibility that the functional role of the dorsal striatum may be similar across seemingly different processes, such as planning, set-shifting, bilingualism, and finger movements. In all cases, competition between two or more elements is implied, and only a self-generated action from the individual could resolve the conflicting situation. The notion of competition between stimuli and the involvement of the individual into sorting out the possibilities to execute the proper goal-directed behavior seems crucial for the functional contribution of the striatum. Implicitly associated with the planning of an action, the notion of novelty is provided as suggested with the proposed model of fronto-striatal interaction. This proposed model underlines the essential contribution of the striatum in specific situations involving prefrontal activity. As this model was derived from observations in healthy individuals, it would be interesting to examine pathological situations like Huntington's disease or Parkinson's disease. Indeed, neuroimaging data from Parkinson's disease patients showed impairments in set-shifting and self-initiated actions (Monchi et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Major depressive disorder (MDD) presumably includes heterogeneous subgroups with differing pathologies. To obtain a marker reflecting such a subgroup, we analyzed the cerebrospinal fluid (CSF) levels of fibrinogen, which has been reported to be elevated in the plasma of patients with MDD. Three fibrinogen-related proteins were measured using aptamer-based analyses and CSF samples of 30 patients with MDD and 30 controls. The numbers of patients with an excessively high level (>99 percentile of the controls) was significantly increased (17 to 23%). Measurement reproducibility of these results was confirmed by an ELISA for fibrinogen (Pearson’s r = 0.77). In an independent sample set from 36 patients and 30 controls, using the ELISA, results were similar (22%). When these two sample sets were combined, the number of patients with a high fibrinogen level was significantly increased . By using diffusion tensor imaging, we found white matter tracts abnormalities in patients with a high fibrinogen level but not those patients with a normal fibrinogen level, compared with controls. Plasma fibrinogen levels were similar among the diagnostic groups. Our results point to a subgroup of MDD represented by increased CSF fibrinogen and white matter tract abnormalities. Major depressive disorder (MDD) is supposed to include heterogeneous subgroups with different biological mechanisms contributing to their disease346Cerebrospinal fluid (CSF) is derived mainly from capillaries in the brain tissue, rather than the choroid plexus, and is in continuity with the brain interstitial fluidFibrinogen is crucial for blood clotting, inflammation, and angiogenesis. Previous studies showed that plasma fibrinogen levels were elevated in patients with MDD121314152 = 0.06). Five patients had an abnormally high level of SL000424 . Five patients had an abnormally high level of SL003341 .We evaluated the levels of three fibrinogen-related molecules in SOMAscan analyses: SL000022, SL000424, and SL003341 which were supposed to represent the fibrin D-dimer, fibrinogen, and fibrinogen γ-chain, respectively, in the first sample. Seven patients with MDD had an abnormally high level of SL000022  . Two of −5; SL000022 and SL003341, r = 0.66, p=9.3 × 10−9; SL000424 and SL003341, r = 0.93, p = 2.2 × 10−27; The expression pattern of the three fibrin-related proteins correlated with each other. In particular, SL000424 and SL003341 showed a nearly perfect correlation . To specify which molecules the three SOMAscan aptamers had measured, we examined the correlation between SOMAscan and ELISA measurements using each ELISA kit and the samples analyzed by SOMAscan (n = 32). There was a strong correlation between fibrinogen (ELISA) and SL000022 .We measured CSF fibrinogen using ELISA in the second sample. Here again the cutoff value of fibrinogen was defined as 99 percentile of the levels in the controls. Eight of 36 patients showed an abnormally high fibrinogen level .When the data  from the two sample sets were combined, the numbers of patients with a high fibrinogen level was significantly increased compared with controls . When data from the two samples sets were combined after z score transformation, the mean fibrinogen level in patients was significantly higher than in controls  = 7.42, p=0.007, partial η2 = 0.168, 2 = 0.05, −8, partial η2 = 0.42).We analyzed clinical features of patients with high fibrinogen levels in both the first and second samples. Compared with patients with a normal fibrinogen level, patients with a high fibrinogen level had a significantly higher mean age  = 12.5, p = 0.0008, partial η2 = 0.037). None of the four subjects (3 patients and 1 control) with a high plasma fibrinogen level had an abnormally high CSF fibrinogen level.The fibrinogen level in plasma was ~1000 times higher than that in CSF. There was no significant correlation between plasma and CSF fibrinogen levels , Japanese version36CSF samples were obtained by lumbar puncture between 1000 h and 1600 h. After neurologic examinations, each participant received local anesthesia followed by a lumbar puncture at L3–4 or L4–5 using an atraumatic pencil point needle . The initial 2 mL of CSF was used for laboratory tests, including number of cells, total protein, and glucose. Then 8 mL of CSF was collected in a low protein adsorption tube  and immediately chilled on ice. The CSF was centrifuged  and the supernatant was dispensed into 0.5 mL aliquots in low-protein adsorption tubes  and stored in a deep freezer (−80 °C) until use. Simultaneously, blood samples were collected by venipuncture and plasma samples were obtained by centrifugation.Workers at SomaLogic Inc.  who were blind to the diagnosis, performed the proteomic assessments as described elsewhere41Fibrinogen Human ELISA Kits (ab108841) were purchased from Abcam . Samples were diluted to 1:50 for the CSF and 1:10000 for the plasma using the diluent attached to the kit. An ELISA kit for Fibrin D-dimer (#RK023A) was purchased from Hyphen BioMed , and CSF samples were diluted to 1:4 using the sample diluent attached to the kit. An ELISA kit for fibrinogen gamma (SEC477Hu) was purchased from Uscn Life Science Inc.  and CSF samples were diluted to 1:10 using phosphate buffered saline.MRI was performed on a Magnetom Symphony 1.5-tesla . High spatial resolution, 3-dimensional (3D) T1-weighted images, diffusion tensor imaging (DTI), conventional T2-weighted images, and fluid attenuation inversion recovery images of the brain were obtained for the morphometric study. Detailed information on MRI parameters are described elsewhereThe SOMAscan results for 3 fibrinogen-related proteins were first analyzed using Microsoft Excel . The cut off value for each protein was determined as the 99 percentile of 30 control subjects using the “PERCENTILE” function. Further analyses were performed with the SAS program . Analysis of covariance  was used to identify the effects of diagnosis on quantitative values (such as fibrinogen levels) or the effect of fibrinogen levels  on quantitative values (such as HAM-D scores). The relationships among protein levels (measured by SOMAscan or ELISA) were analyzed by Pearson’s correlation test. The increases in the number of patients with high fibrinogen levels were assessed by one-sided Fisher’s exact test. Odds ratio (OR) and 95% confidence intervals (CI) were also obtained by the SAS program. Z scores were calculated using the mean and standard deviation of the control group in each sample set using the following formula; /(standard deviation).We prepared three MDD and age-, sex- matched control sample sets. SOMAscan was performed on a “first sample”, consisting of 30 MDD patients and 30 controls . CSF fibHow to cite this article: Hattori, K. et al. Increased cerebrospinal fluid fibrinogen in major depressive disorder. Sci. Rep.5, 11412; doi: 10.1038/srep11412 (2015).


Scenedesmus is known for its phenotypic plasticity in response to grazing risk. However, the benefits of colony formation induced by infochemicals from zooplankton should come with costs. That is, a tradeoff in benefit-to-cost ratios is likely under complex environmental conditions. In this study, we hypothesized that the coexistence of Scenedesmus and its competitors decreases the formation of anti-grazer colonies in Scenedesmus. Results demonstrated that the presence of a competitor Microcystis aeruginosa inhibited inducible defensive colony formation of Scenedesmus obliquus, and the established defensive colonies negatively affected the competitive ability of S. obliquus. The proportion of induced defensive colonies in cultures was dependent on the relative abundance of competitors. Under low competition intensity, large amount of eight-celled colonies were formed but at the cost of decreased competitive inhibition on M. aeruginosa. By contrast, defensive colony formation of S. obliquus slacked in the presence of high competition intensity to maintain a high displacement rate (competitive ability). In conclusion, S. obliquus exhibited different responses to potential grazing pressure under different intensities of competition, i.e., Scenedesmus morphological response to grazing infochemicals was affected by competition against Microcystis.The green alga  Mytilus edulis are induced by predation cues to increase shell thickness, enhance abductor muscle strength, and produce more byssal threadsDaphnia pulex produces neck teeth on the dorsal anterior margin of the head in the presence of the predator Chaoborus americanusThe production of different phenotypes from a single genotype in response to environmental conditions is termed “phenotypic plasticity”, which is common in organisms124Scenedesmus spp. is a typical case. Upon detection of infochemicals released by herbivores such as Daphnia, Scenedesmus populations become dominated by four- and eight-celled colonies instead of unicellsScenedesmus populations that are dominated by colonies can effectively deter feeding by small grazers such as Daphnia cucullata; therefore, induced colony formation is an efficient strategy for Scenedesmus to escape grazing by predators with a diet limited by the prey sizeCompetition and predation are considered the major selective pressures that shape the composition of pelagic photoautotrophic communities11Scenedesmus colonies experience a higher risk of sedimentation than unicells1720Scenedesmus.However, large Scenedesmus. It has been recognized that organisms face trade-offs to allocate the limited resources to grow or to counteract stress2224The disadvantages of forming colonies, including accelerated sinking rate and decreased nutrition and light absorption, may affect the competitive ability of Scenedesmus in response to infochemicals released by grazers, we hypothesized that defense and competition operate at the expense of each other and Scenedesmus may make trade-offs under pressure from both predators and competitors. Accordingly, this study aimed to examine the costs and the trade-off of forming grazer-induced defensive colonies in Scenedesmus under different levels of inter-species competition. Microcystis aeruginosa, one of the most common bloom-forming cyanobacteria, was used as the competitor of S. obliquus. Both algae are found in freshwaters world-wide and often coexist in the same area26M. aeruginosa has been reported to have significant allelopathic influence on co-existing phytoplankton282930M. aeruginosa can also form huge thousand-celled colonies, but the M. aeruginosa strain selected in this study was unicellular with no colony formation when exposed to Daphnia grazing infochemicalsDaphnia magna to simulate S. obliquus colony formation in the presence of competitors.Considering the trade-off mechanism and the high morphological plasticity of S. obliquus in the control groups were dominated by unicells over the 7 d period, with less than 12% large eight-celled coenobia   . Unicell< 0.001) . The coms ceased . In the S. obliquus decreased significantly in the Daphnia filtrate-treated co-cultures with 1-fold M. aeruginosa when compared to the control monoculture group (p = 0.001). Growth rates of S. obliquus in other four group cultures showed no significant differences compared with the control monoculture group. Two-way ANOVA was conducted to analyze the influence of Daphnia filtrate and the density of competitors on the growth rates of S. obliquus and M. aeruginosa  versus t were positive in all cultures, indicating that S. obliquus could displace M. aeruginosa in both experiments. In co-cultures with the same initial cell densities of both algae, the slope decreased in the Daphnia filtrate-treated group compared with the control, but the decrease was not statistically significant . The displacement rates during days 3 to 7 were further analyzed, because colony formation induced by Daphnia infochemicals peaked on day 3. Since multicellular colonies have lower surface area-to-volume ratios than unicells, nutrition uptake of S. obliquus may be affected under competition by M. aeruginosa and the potential costs may emerge after day 3. Significant decrease was detected in the Daphnia filtrate-treated group . For co-cultures with 5-fold M. aeruginosa, the slopes of the control and Daphnia filtrate-treated groups were similar . Coexistence with M. aeruginosa decreased the formation of inducible colonies by S. obliquus for potential reallocation of resources in response to competition.Separate linear regressions were fitted for all co-cultures to compare the displacement of d groups . The sloScenedesmus is considered to benefit from the ability of forming plastic morphs in response to biotic and abiotic environmental factors, particularly herbivory3525Scenedesmus, the benefits of being plastic have been well documented15ScenedesmusDesmodesums subspicatus preinduced and prolonged by Daphnia infochemicals had suppressed competitive ability compared to unicellular ones. Thus, it is possible that colonial morph interferes with the rapid growth of S. obliquus for the reduction of resource uptake owing to its decreased surface area-to-volume ratio. Therefore, we hypothesized that Scenedesmus formed different morphs as a trade-off between defense and competition.S. obliquus between defense and growth in the presence of competitors. Competitive displacement rates indicated that colony formation exerted adverse effects on the growth and competitive ability of S. obliquus , which lowers the amount of infochemicals detected by S. obliquus cells and thus lowers their morphological response.Besides the possible trade-off of M. aeruginosa inhibited inducible defensive colony formation of S. obliquus. The proportion of induced defensive colonies in cultures was dependent on the relative abundance of competitors. Under low competition intensity, large amount of eight-celled colonies were formed but at the cost of decreased competitive inhibition on M. aeruginosa. By contrast, defensive colony formation of S. obliquus slacked in the presence of high competition intensity to maintain a high competitive ability. S. obliquus exhibited different responses to potential grazing pressure under different intensities of competition. These results demonstrate that phenotypic plasticity allows organisms to undergo controllable phenotypic changes to reduce fitness costs by optimizing the benefit-to-cost ratios when responding to different environmental challenges.In summary, the presence of a competitor S. obliquus (Chlorophyta) FACHB-416 and M. aeruginosa (Cyanobacteria) FACHB-927 were obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology China (FACHB-collection) and maintained in our laboratory in a modified BG-11 medium4Cl as nitrogen source. Before the experiment, stock algae were regularly transferred to fresh media at a ratio of 0.3 d−1 for 10 d to maintain the logarithmic growth phase. The grazer Daphnia magna used to produce infochemicals in the experiment was a laboratory clone. The only food source of D. magna during the cultivation was S. obliquus grown in continuous cultures. The stock cultures of S. obliquus, M. aeruginosa, and D. magna were all stored in a climate-controlled chamber at 25 °C under fluorescent light at 40 μmol quanta·m−2·s−1 in a 14:10 h light-dark cycle. All experiments were conducted under these conditions.Scenedesmus were demonstrated to be associated with the grazing progress and linked to alga-grazer interaction11Daphnia feeding Scenedesmus” to obtaining the infochemicals. Test water containing infochemicals from D. magna was obtained as previously described15D. magna was washed to remove surface contamination and allowed to evacuate their guts in sterile distilled water for at least 12 h and then incubated at a density of 300 ind·L−1 with sufficient S. obliquus as food. After 24 h, D. magna was removed, and the cultures were filtered through a 0.10 μm membrane filter . The filtrates served as the treatment media containing infochemicals released by Daphnia. Cultures of Scenedesmus without Daphnia were filtered as well and used as the control media. NH4Cl was used as nitrogen source to easily adjusting the nitrogen levels of filtrates since the nitrogenous metabolites of the crustacean is ammonia. Appropriate ammonium and phosphate were added to the control filtrate according to the measured concentrations of nitrogen and phosphorus in the Daphnia filtrate, and thus the nitrogen and phosphorus concentrations in the treatment and control media were adjusted to the same levels.Since the infochemicals that evoke anti-grazing colonies of S. obliquus and M. aeruginosa, we executed the experiment using two initial density proportions (Scenedesmus: Microcystis = 1:1 and 1:5) with 10% filtered S. obliquus culture medium or Daphnia filtrate. The initial algal density of S. obliquus in each culture was approximately 1.0 × 105 cells·mL−1, and the initial algal densities of Microcystis were approximately 1.0 × 105 and 5.0 × 105 cells·mL−1, respectively. In the experiments, algal suspensions were transferred to 250 mL cellulose-plug stoppered Erlenmeyer flasks containing 150 mL of medium. Six treatments were carried out, and each treatment was run in triplicate. To avoid sedimentation, the algal cultures were manually shaken twice a day.A flow diagram of experimental design was shown in S. obliquus and M. aeruginosa were determined as the slope of logarithm abundance versus timeScenedesmus under competitors, the competitive displacement rate was adapted52Y(t) = ln[NScenedesmus(t)/NMicrocystis(t)], was calculated and regressed against time (d). The displacement rates for Scenedesmus were the slopes of the linear regressionsSamples (1 mL) were collected daily and preserved in Lugol’s fixative (2%). Algal densities and different-celled colony distributions were counted under a microscope  using a hemocytometer. Different algal particles  were counted, and the mean proportions of cells in different morphs were calculated. The numbers of cells per particle were calculated by dividing the total cell number by the number of particles. Growth rates of Daphnia filtrate as the two factors. Significant differences between treatments were detected by t-test. Statistical analyses were performed using SigmaPlot 11.0. The differences between slopes of the linear regressions of Y(t) versus t were analyzed by ANCOVA in SPSS 10.0.Data values are expressed as means of 3 replicates ± SE. Colony formation, proportions of different celled populations, and growth rates were compared by two-way ANOVA, with the presence/absence of the competitor and How to cite this article: Zhu, X. et al. Grazer-induced morphological defense in Scenedesmus obliquus is affected by competition against Microcystis aeruginosa. Sci. Rep. 5, 12743; doi: 10.1038/srep12743 (2015).


Epididymal anomalies and patent processus vaginalis are frequently found in boys with cryptorchidism or hydrocele. We conducted this study to evaluate the association between epididymal anomalies and testicular location or patent processus vaginalis in boys with undescended testis or hydrocele.Children undergoing surgery with undescended testis  or communicating hydrocele  were included. Testicular locations and epididymal anomalies were investigated prospectively. An anomalous epididymis was defined as anomalies of epididymal fusion that consisted of loss of continuity between the testis, the epididymis, and the long looping epididymis. The epididymis was considered normal when a normal, firm attachment between the testis, the caput, and the cauda epididymis was present.P < 0.001). The incidence of epididymal anomalies in boys with undescended testis was significantly different according to testis location. Epididymal anomalies were observed in 100 %, 91.4 %, and 39.3 % of cases when the testis was located in the abdomen, inguinal canal, and distal to the external inguinal ring, respectively (P < 0.001).The mean ages of groups A and B were 24.6 ± 19.7  and 31.4 ± 20.6 months . The incidence of epididymal anomalies was significantly higher in group A than that in group B (65.4 % vs. 13.5 %, We conclude that epididymal anomalies were more frequent in boys with undescended testis than in boys with hydrocele, and that these anomalies were more frequent when undescended testis was at a higher level. These results suggest that testicular location is associated with epididymal anomalies rather than patent processus vaginalis. Epididymal and vasal anomalies are associated with undescended testis. These anomalies occur in association with undescended testis at varying degrees of 32–79 % according to various epididymal anomaly diagnostic criteria –7. TheseThe surgeon must be aware of associated epididymal anomalies when operating to correct undescended testes or hydrocele and not to dissect an elongated epididymis, a cranially located testis with a descended long or dissociated epididymis, or a cranially located testis . AlthougSome reports have suggested that epididymal anomalies associated with more cranially located testis are more severe than those associated with more caudally located testis among boys with undescended testis . HoweverWe included children undergoing an inguinal exploration for undescended testis or a communicating hydrocele in the urologic department of our hospital between January 2011 and July 2013. A communicating hydrocele was defined by the size change history and a sac extending to the groin on ultrasound sonography. We performed a cross-sectional study to determine the incidence of epididymal anomalies in these children. Boys with any of the following were excluded from the study: associated disorders, such as hypospadias, exstrophy, imperforated anus, sex development disorders, ectopic testes, or vanishing testis. The spermatic cord was isolated after incising the cremasteric fascia, and the hernia sac was high-ligated when present. Orchiopexy was performed by placing the testis into the subdartos pouch. Hydrocelectomy was performed using a typical inguinal approach, and the testis was delivered to the groin so the epididymal anomaly could be examined. Written informed consent for data collection and participation in the study was obtained from a parent or guardian. This study received approval from the local ethics committee . The study procedures complied with the guidelines provided by the Declaration of Helsinki.Testicular locations and epididymal anomalies were investigated carefully. According to morphological classification by Barthold and Redman , an anomP-values < 0.05 were deemed significant.SPSS ver. 17 for Windows software  was used for statistical analyses. Data were analyzed with the chi-square test. A total of 229 boys (258 testes) with undescended testis (group A: 136 boys and 162 testes) or with hydrocele (group B: 93 boys and 96 testes) were included in this study. The mean ages in groups A and B were 24.6 ± 19.7  and 31.4 ± 20.6 months , respectively. In total, 110 patients  in group A had unilateral lesions and 26 patients had bilateral lesions. A total of 90 boys in group B had unilateral lesions  and three had bilateral lesions. The patient demographic data are shown in Table P < 0.001) (Table P < 0.001)  than in boys with hydrocele (13.5 %).The estimated overall incidence of epididymal anomalies in boys with undescended testis and hydrocele was 46.1 %, suggesting that testicular location contribute more to epididymal anomalies than patent processus vaginalis. Epididymal anomalies were found more frequently in boys with a higher testicular location. All boys with undescended testis located in intra-abdominal area had epididymal anomalies, 91.4 % when the undescended testis was in the inguinal canal, and 39.3 % when it was distal to external inguinal ring.Controversies exist regarding the role of the epididymis in testicular descent, as epididymal anomalies have been found in 36–79 % of boys with undescended testis. Most studies that have reported anatomical abnormalities of epididymis in boys with undescended testis have been sporadic and used different methods, and no recent data were found –7. MollaFactors that have been suggested as important for normal testicular descent include normal gubernaculum, epididymis, and intra-abdominal pressure, and innervation by the genitofemoral nerve of the gubernaculums , 11. HowKnowledge of normal testicular anatomy is necessary to understand epididymal anomalies. The normal epididymis curves along the testis but the epididymal body may be slightly detached from the testis body. The epididymal head remains in close proximity with the cranial pole of the testis, while the cauda epididymis disappears within the gubernaculum . The tesEpididymal anomalies are associated with maldescent of the testis and may account for impaired future fertility , 4. A clIn the present study, epididymal anomalies were detected in all boys when the affected testis was found in the intra-abdominal area, whereas 91.4 % showed an abnormal condition when the testis was in the inguinal canal and 39.3 % when it was found distal to the external inguinal ring. More severe epididymal anomalies and vassal anomalies are encountered in boys with testis located in the intra-abdominal or high inguinal position in a small case series [We conclude that epididymal anomalies were more frequent in boys with undescended testis than in boys with hydrocele, and that these anomalies were more frequent when the undescended testis was at a higher level. This result suggests that testicular location, rather than patent processus vaginalis, is associated with epididymal anomalies.


Several published surveys have shown that chiropractors are generally split in their opinions regarding the right to prescribe drugs in chiropractic practice. Many of these studies have been limited by low response rates, leaving the generalizability of their findings open to question. The aim of the current study was to ascertain the general attitudes of chiropractors in Ontario, Canada toward the inclusion of drug prescription rights in their scope of practice. Relationships between these attitudes and the number of years in practice including differences in philosophical orientation were also explored.A 14-item questionnaire was developed and invitations sent via e-mail to all eligible 2,677 chiropractors in active practice registered electronically with the College of Chiropractors of Ontario in February 2015. Data were collected and analyzed using descriptive and inferential statistics.960 questionnaires were completed for a 36 % response rate. The majority of respondents agreed that chiropractors should be permitted to prescribe musculoskeletal medications such as over-the-counter and prescription-based analgesics, anti-inflammatories, and muscle relaxants. Over two-thirds also felt that with limited prescriptive authority chiropractors could help reduce patients’ reliance on these types of drugs. Over three-quarters were opposed however to chiropractors having full prescribing rights. The majority indicated they recommend over-the-counter medications to acute and chronic patients to some extent in clinical practice. Nearly two-thirds perceived their knowledge of musculoskeletal medications as high or very high, while a similar proportion perceived their knowledge of drugs for non-musculoskeletal conditions to be low or very low. A majority of respondents felt that further education in pharmacology would be necessary for those in the profession wishing to prescribe medications. More recent graduates and those who espoused a broad scope of chiropractic practice were most in favour of limited prescribing rights for the profession.A majority of responding Ontario chiropractors expressed interest in expanding their scopes of practice to include limited drug prescription. These results together with those of other recent surveys could indicate a shift in chiropractors’ attitudes toward drug prescription rights within the profession. Further surveys and/or qualitative studies of chiropractors in other jurisdictions are still needed.The online version of this article (doi:10.1186/s12998-015-0066-7) contains supplementary material, which is available to authorized users. In some jurisdictions in the world chiropractors can gain licensure to prescribe medications from a limited formulary of over-the-counter (OTC) and/or prescription-based medications for common musculoskeletal conditions, such as non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, and muscle relaxants , 2. SomeTo date several published surveys –9 have sYet despite being divided over prescribing rights in general, there is evidence to suggest that many chiropractors often recommend OTC medications to patients in practice. For example, while just over half of respondent chiropractors from surveys in Australia  and OklaContention also exists over the scope of prescription-based drug use in chiropractic practice. In New Mexico, USA, for example, chiropractors can gain licensure to prescribe from a limited formulary of musculoskeletal medications . HoweverQuestions also remain as to why the chiropractic profession is split toward prescribing rights in the first place. Some evidence suggests that this division in attitudes may be reflective of differences in philosophical orientation, with so-called ‘mixer’ chiropractors being in favour and ‘straight’ chiropractors being opposed . HoweverThe aim of this study was therefore to ascertain the general attitude (s) of chiropractors from Ontario, Canada toward the inclusion of drug prescription rights in their scope of practice. In doing so, three main areas were investigated: (i) Ontario chiropractors’ attitudes and opinions to drug prescription rights, (ii) the frequency of OTC drug recommendation by Ontario chiropractors, and (iii) Ontario chiropractors’ current knowledge of drug prescription. This study also sought to determine if there was a relationship between Ontario chiropractors’ attitudes toward drug prescription rights and (a) the number of years in chiropractic practice or employment, and (b) philosophical orientation/preferred style of practice.A survey of all 2,900 chiropractors in active chiropractic practice registered through the 2014–2015 electronic directory of the College of Chiropractors of Ontario (CCO) .All qualified participants in this study were contacted via e-mail messages, at one-week intervals, up to six times over the course of six weeks. The first e-mail was a pre-notification message containing an introduction to the survey and its purpose, as well as a link to a review article on the topic of prescribing rights in chiropractic . The nexThe questionnaire was divided into four sections. Section 1 consisted of four questions recorded on a 5-point Likert scale ranging from ‘strongly agree’ to ‘strongly disagree’ that focused on chiropractors’ attitudes to drug prescription rights. Section 2 consisted of two questions regarding OTC drug recommendations in chiropractic practice. Responses to both questions were recorded on a 5-point scale ranging from ‘never’ to ‘routinely.’ Section 3 contained three questions asking about the chiropractors’ current knowledge of drug prescription. Responses to the first two questions were recorded on a 5-point Likert scale ranging from ‘very high’ to ‘very low.’ Responses to the third question were recorded on a 3-point ‘yes,’ ‘no,’ or ‘don’t know’ verbal scale. Section 4 asked demographic questions including: (i) age, (ii) gender, (iii) chiropractic college of graduation, (iv) number of years in chiropractic practice or employment, and (v) chiropractic philosophical orientation/scope of practice. For this last item, respondents were asked to choose between one of three categories, as defined by McDonald et al. , which bAn assessment of the questionnaire’s face validity , 17 was a priori, that chiropractors with a higher number of years in practice and/or who preferred a focused (or ‘straight’) chiropractic scope of practice would hold more negative views toward drug prescription rights. Relationships between these two grouping variables and the various attitudinal response variables from section 1 of the questionnaire were explored using the chi-square test of independence for nominal/categorical data [p < 0.05, and all data analysis was carried out using SPSS .Responses to all questions were analyzed using descriptive statistics. Central tendencies were measured as means and standard deviations for continuous data, while medians were used for ordinal data . Categorcal data . In ordePrior to data collection, ethics approval (E67/05/15) was obtained through the Anglo-European College of Chiropractic Research Ethics Sub-Committee. The Research Ethics Board Secretariat for Health Canada was also contacted and further ethics review in Canada was deemed not necessary due to the nature of the research being undertaken in this study. All data collected for this study was recorded anonymously and stored securely in a password protected electronic database.n = 77) or those previously having opted out of receiving SurveyMonkey® surveys (n = 93). Of the remaining 2,677 questionnaires that were sent, completed questionnaires were received from 960 respondents (35.9 % response rate), representing the views of nearly one-quarter of the profession in Ontario at the time.After removing duplicate and invalid e-mail addresses from the 2014–2015 CCO directory, the questionnaire was sent to 2,847 chiropractors in Ontario, representing more than two-thirds (68.0 %) of all chiropractors in active practice registered with the CCO at the time of the survey . One hundred and seventy questionnaires were automatically returned as undeliverable due to change of recipient e-mail addresses , increasing majorities (55 % and 61 %) of respondents respectively were in favour of chiropractors prescribing anti-inflammatory and/or analgesic medications. An even greater majority favouring limited prescribing rights in the present study suggests that there may be a possible shift in chiropractors’ attitudes toward drug prescription rights occurring within the profession in Ontario.In Switzerland, where chiropractors already have limited prescribing rights, the profession is more united regarding drug prescription in chiropractic , 10 and Concerning the issue of pharmacology education, the current study found that Ontario chiropractors were quite confident regarding their perceived knowledge towards prescribing musculoskeletal medications. In fact, nearly two-thirds of respondents indicated that their current knowledge of these drugs was ‘high’ or ‘very high.’ Interestingly nearly equal numbers perceived their current knowledge of drugs for non-musculoskeletal conditions as ‘low’ or ‘very low.’ The first finding is surprising given that the basic chiropractic educational curriculum contains only 12 h of coursework in pharmacology . A possiDespite evidence to suggest that chiropractors in Ontario and elsewhere are interested in gaining limited prescriptive privileges, a large majority of respondents in the current study did not favour the idea of chiropractors having full prescribing rights. More than three-quarters disagreed that chiropractors should be able to gain an expanded scope to allow for the prescription of any and all medications, including controlled substances. This finding is consistent with those of previous surveys of chiropractors from Australia , the UniAnother finding of the current study was that a large number of Ontario chiropractors in this survey tend to recommend OTC drugs to their patients. For instance, when asked how often they suggested non-prescription analgesic and NSAID medications to acute and chronic patients in clinical practice, 81 % and 67 % of respondents indicated that they did so to some extent respectively. These non-prescription drug utilization rates are comparable to those of other published studies of practising chiropractors , 22, andWhen exploring possible reasons for why some chiropractors have differing views toward drug prescription, an association was found in this study between respondents’ opinions and the number of years in practice. For instance, chiropractors who had practised for 15 years or less were significantly more in favour of musculoskeletal drug prescription rights versus those with greater than 15 years’ experience. This difference in opinion between the two groups could possibly reflect slightly differing views toward evidence-based practice. For example, several clinical guidelines endorse the use of mild analgesics and/or anti-inflammatories in the management of various musculoskeletal conditions –31. Yet As for philosophical orientation, this study showed that there was a strong relationship between this variable and Ontario chiropractors’ attitudes to drug prescription rights. For instance, almost all of the ‘broad scope’ respondents in the survey were in favour of Ontario chiropractors gaining prescriptive rights for treating musculoskeletal conditions, whereas very few of the ‘focused scope’ group felt the same way. These findings are consistent with those from the study by McDonald et al.  where moThere may be a middle ground concerning chiropractic prescribing rights where some level of agreement within the profession could be reached. For instance, evidence from the literature including results from the current study suggest that among chiropractors who hold favourable views toward drug prescription, prescription privileges limited to within a musculoskeletal scope of practice would be preferred , 6, 8. An = 960) obtained in this study was higher than those of other published surveys on chiropractic prescribing rights [This study has some limitations. First, the overall response to the survey was relatively low (36 %) thus raising the likelihood of non-response/exclusion bias in the results . Howeverg rights , 5–10, aA second limitation of this study is that it excluded retired chiropractors and those not on the electronic 2014–2015 directory of the CCO. As these groups represented the minority (32 %) of all licensed chiropractors in Ontario at the time of the survey, this was felt to be less of an issue. Nonetheless, there is a risk that retired chiropractors and/or those who did not have an e-mail address listed with the CCO may have held systematically different views toward drug prescription rights compared to chiropractors listed on the electronic CCO directory.Thirdly, chiropractors’ attitudes to drug prescription rights were measured in this study using a closed-answer format only. Although good for aggregating data from large study populations, the disadvantage to using this survey method is that it does not allow participants to expand upon responses or offer alternative viewpoints , and thiThis study revealed that a majority of Ontario chiropractors were in favour of incorporating limited drug prescription rights into their scope of practice, were generally confident regarding their knowledge of musculoskeletal medications, and tended to recommend OTC drugs such as mild analgesics and/or anti-inflammatories to patients to some extent in clinical practice. However, respondents did not favour the idea of chiropractors having full prescribing rights, were not confident in their knowledge of drugs for non-musculoskeletal conditions, and felt that further education and training in pharmacology should be necessary for those in the profession wishing to prescribe medications. Those who had been in practice for less than 15 years favoured musculoskeletal prescribing rights more so than chiropractors with more than 15 years’ experience; however the overall majority in both groups still favoured limited prescribing rights for the profession. As for philosophical orientation, the majority of broad and middle scope respondents in this study also favoured limited chiropractic prescribing rights, whereas those in the focused scope group did not. Further surveys and/or qualitative studies of chiropractors in other jurisdictions are needed in order to validate these findings.


Soluble fragments of the amyloid precursor protein (APP) generated by α- and β-secretases, sAPPα and sAPPβ, have been postulated as promising new cerebrospinal fluid (CSF) biomarkers for the clinical diagnosis of Alzheimer’s disease (AD). However, the capacity of these soluble proteins to assemble has not been explored and could be relevant. Our aim is to characterize possible sAPP oligomers that could contribute to the quantification of sAPPα and sAPPβ in CSF by ELISA, as well as to characterize the possible presence of soluble full-length APP (sAPPf).We employed co-immunoprecipitation, native polyacrylamide gel electrophoresis and ultracentrifugation in sucrose density gradients to characterize sAPP oligomers in CSF. We have characterized the presence of sAPPf in CSF from NDC and AD subjects and demonstrated that all forms, including sAPPα and sAPPβ, are capable of assembling into heteromers, which differ from brain APP membrane-dimers. We measured sAPPf, sAPPα and sAPPβ by ELISA in CSF samples from AD (n = 13) and non-disease subjects  before and after immunoprecipitation with antibodies against the C-terminal APP or against sAPPβ. We demonstrated that these sAPP heteromers participate in the quantification of sAPPα and sAPPβ by ELISA. Immunoprecipitation with a C-terminal antibody to remove sAPPf reduced by ~30% the determinations of sAPPα and sAPPβ by ELISA, whereas immunoprecipitation with an APPβ antibody reduced by ~80% the determination of sAPPf and sAPPα.The presence of sAPPf and sAPP heteromers should be taken into consideration when exploring the levels of sAPPα and sAPPβ as potential CSF biomarkers.The online version of this article (doi:10.1186/1750-1326-10-2) contains supplementary material, which is available to authorized users. Abnormal accumulation of β-amyloid peptide (Aβ) in the brain is characteristic of Alzheimer’s disease (AD). The Aβ peptide is generated by processing a large type I transmembrane spanning glycoprotein, the amyloid precursor protein (APP), through the successive action of proteolytic enzymes called secretases. Sequential processing of APP begins with either the action of α-secretase, or alternatively by β-secretase, followed by γ-secretase cleavage ; see alsA. When AInterestingly, it has been demonstrated that full-length APP containing an intact cytoplasmic domain also exists as a soluble form (sAPPf) , 12. TheIn this study, we investigate the presence of sAPPf in AD CSF, as well as the oligomerization of sAPP species, to determine how this may affect the measurement of sAPPα/sAPPβ levels by currently available ELISA immunoassays.To determine the presence of sAPPf in human CSF, we first examined samples by Western blotting using different anti-APP antibodies Figure B. A simiTo further characterize sAPPf in human CSF, we performed immunoprecipitation/Western blotting assays Figure C. CSF saNext, we characterized sAPP oligomers from brain membranes and CSF samples by blue native-PAGE Figure A. Brain Previous studies have employed gradient centrifugation to characterize APP complexes . Sucrosep < 0.001). After immunoprecipitation with Sigma-Ct . This corroborates the existence of heteromers containing sAPPf and sAPPβ, but also of sAPPf and sAPPα.The presence of sAPP heteromers in CSF is expected to affect the determination of sAPPα and sAPPβ by specific ELISAs. To check this possibility, we analyzed CSF samples immunoprecipitated with different antibodies to pull-down potential heteromers. CSF samples from 13 probable AD subjects, characterized for T-tau, P-tau and Aβ42 levels determined by ELISA kits . In this ELISA kit, the detector antibody recognizes an epitope located between the β-secretase and α-secretase cleavage sites, and thus, similarly to the 6E10 antibody, detects sAPPα and sAPPf, but not sAPPβ. Samples (n = 10 in both the AD and NDC samples) were analyzed before and after immunoprecipitation with the IBL-β antibody. Levels of sAPP (sAPPf and sAPPα) decreased greatly after immunoprecipitation with IBL-β in both the AD and NDC groups . Interestingly, the levels of sAPPf correlated with Aβ42 in the AD group , but not in the NDC cases. There was no association of sAPPf levels with age or gender.Finally, to assess whether sAPPf levels are altered in AD, we analyzed by Western blotting fresh aliquots of the same CSF samples previously characterized for classical biomarkers  between probable AD patients and non-demented controls; however, its levels correlated with Aβ42 levels in the AD group; this may indicate an association with the pathological levels of Aβ42, and requires further research. The 130 kDa band (which should correspond to APP751/770) was found to be increased in AD CSF. Despite the low number of samples analyzed, and the difficulty in quantifying the immunoreactivity of the 130 kDa band, our data suggest that the analysis of KPI-containing sAPPf forms may be of particular interest and needs to be more specifically addressed. Interestingly, the proportion of KPI-containing isoforms appears significantly elevated in the soluble subcellular fraction of AD brains .2+-dependent manner, as well as by cholinergic receptor agonists and antagonists [The origin or functional significance of sAPPf in CSF is currently not known. Early studies failed to demonstrate the presence of sAPP isoforms containing its C-terminal domain in human CSF , but latagonists . A mechaagonists . Full-leagonists .Previous studies indicate that cellular APP may form high molecular-weight APP complexes , 23. Co-The nature and composition of sAPPf oligomers in CSF, as well the possibility that other proteins could also be part of the sAPP complexes, have yet to be deciphered. Based on our co-immunoprecipitation and ELISA analysis, we assume that all the possible sAPP heteromers are present in CSF.Previous reports on the levels of sAPP in CSF draw a complex scenario. A strong positive correlation between sAPPα and sAPPβ levels has been consistently found in healthy elderly individuals and AD subjects in our and other studies , 10, 24.In the same line, studies of sAPPα and sAPPβ levels in CSF likely would reflect potential changes in the balance between non-amyloidogenic and amyloidogenic pathways, but reports are also contradictory. Studies of sAPPα levels in CSF from AD subjects range from an increase , 27–29, Finally, we have speculated about the abundance of sAPP dimers by our ELISA assays, taking into account that from these data we cannot discriminate sAPP dimers from large sAPP oligomers. Based on the Novex Life ELISA analysis after IBL-β immunoprecipitation, we presumed that the most abundant oligomers include sAPPβ, probably being sAPPα/sAPPβ, since the oligomers which have not been pulled-down, sAPPf/sAPPα, sAPPα/sAPPα and/or sAPPf/sAPPf, constitute altogether ~10% of the non-immunoprecipitated samples. Immunoprecipitation with the Sigma-Ct antibody followed by ELISA analysis for sAPPα and sAPPβ (IBL) revealed in both cases that sAPPf/sAPPα or sAPPf/sAPPβ heteromers represent ~30% of oligomers that include sAPPα and sAPPβ. We speculate that the sAPPα/sAPPα, sAPPf/sAPPf, but also sAPPβ/sAPPβ homodimers, would constitute the least abundant population. In any case, specific characterization of each sAPP oligomer in CSF, and potential differences in the AD condition, will require further research.The use of CSF biomarkers for AD in clinical practice is challenging due to the high variability in their levels. Such variability has been partially attributed to different pre-analytical and analytical procedures between laboratories, highlighting the need to establish standardized operating procedures , 39. CSFThis study was approved by the ethics committee of the Miguel Hernandez University and was carried out in accordance with the Declaration of Helsinki. Lumbar CSF samples were obtained from 13 patients with probable Alzheimer’s disease , and 13 healthy volunteers, non-demented controls  from the Hospital Clinic . All AD patients fulfilled the NINCDS-ADRDA criteria for “probable” AD . Controlg for 1 h. The pellet was homogenized in 0.5% dodecylmaltoside, 20% glycerol, and 25 mM bis-Tris pH 7.0. After 45 min in continuous mixing at 4°C followed by ultracentrifugation at 200,000 × g for 30 min, the supernatant was collected and frozen at -80°C.Brain frontal cortex samples  were obtained from the UIPA neurological tissue bank . Samples were solubilized in 20 mM HEPES pH 7.5, 50 mM KCl, 2 mM EDTA and ultracentrifuged at 300,000 × CHO cells over-expressing wild-type human APP-751  were culTM Forte  and analyzed using Science Lab Image Gauge v4.0 software (Fujifilm).Samples of CSF (30 μL) were boiled at 95°C for 5 min and electrophoresed in 7.5% SDS-PAGE. sAPP species were detected using the following antibodies: anti-APP C-terminal , anti-APP C-terminal , anti-APP 6E10 , anti-APP N-terminal , anti-sAPPβ , and anti-sAPPα . When appropriate, a control CSF sample was used to normalize the immunoreactive signal between immunoblots. The signal was visualized by LuminataTo test the specificity of the APP antibodies, aliquots of conditioned media from CHO cells over-expressing APP-751 were resolved by simultaneous detection of immunoreactivity to two-antibodies . Blots were then probed with the appropriate conjugated secondary antibodies  and imaged on an Odyssey Clx Infrared Imaging System (LI-COR). Band intensities were analyzed using LI-COR software (Image Studio Lite).The Life Technologies NativePAGE Novex 4-16% Bis-Tris Gel System was used in accordance with the manufacturer’s protocol. Samples were prepared in a 30 μl total volume containing 7.5 μL of NuPage LDS sample buffer (4×), 3 μl of 5% G-250 sample additive and 1.5 μl of DDM 10% . The NativeMark™ unstained protein standard  was used as a protein ladder.Immunoprecipitations were performed at 4°C by incubating 150 μL of CSF overnight with 120 μL Sepharose A beads coupled to the specific APP antibody. Precipitated proteins were eluted with 0.1 M glycine buffer at pH 2.5. Immunoprecipitation controls were performed in Sepharose A beads, but absent of any antibody. After pH neutralization with 1 M Tris-HCl pH 9.5, the supernatants were denatured in Laemmli sample buffer at 95°C for 5 min. Preliminary assays resolved by ELISA proved the lack of non-specific binding of sAPP to Sepharose A beads (without an antibody), compared with aliquots samples assayed directly (not incubated in the presence of Sepharose A beads or any antibody).g in a continuous sucrose density gradient (5-20%). CSF aliquots (150 μL) and brain extracts (150 μl) were carefully loaded onto the top of the gradient containing 2 mL of 0.15 M NaCl, 50 mM MgCl2 and 0.5% Brij 97, in 50 mM Tris-HCl (pH 7.4). After centrifugation for 4 hr at 4°C in a Beckman TLS 55 rotor, approximately 13 fractions were carefully collected from the top of the tubes. Enzyme markers of known sedimentation coefficient, β-galactosidase, catalase and alkaline phosphatase were used.CSF samples and brain extracts were analyzed for APP complexes by ultracentrifugation at 250,000 × A commercial ELISA kit from IBL  was used to quantify sAPPα (#27734) and sAPPβ (#27735) in CSF. These assays are based on a solid-phase sandwich ELISA using specific anti-APP antibodies which recognize the C-terminal of sAPPα and sAPPβ. The manufacturer estimated that cross-reactivity between sAPPα and sAPPβ fragments in each kit is less than 1.5%, and that both assays do not cross-react with full-length APP. A commercial ELISA kit for sAPPf  was also employed. This assay is based on an N-terminal APP capture-antibody and an antibody recognizing the N-terminal part of the Aβ peptide for detection, thus binding sAPPf and sAPPα, but not sAPPβ.Total tau (T-tau), phosphorylated tau (P-tau) and Aβ1-42 (Aβ42) in CSF were determined using specific ELISA .t test (two-tailed) or a Mann-Whitney U test for single pairwise comparisons and determination of exact p values. Results are presented as means ± SEM. Correlation between variables was assessed by linear regression analyses. p values < 0.05 were considered significant.All data were analyzed using SigmaStat  by a Student’s Additional file 1: Figure S1: SDS-PAGE analysis and fluorescence detection of sAPP species. For the analysis of sAPP species, and to probe the specificity of the anti-APP antibodies, aliquots of conditioned media from CHO cells over-expressing APP-751 were analyzed by SDS-PAGE and resolved with two different anti-APP antibodies simultaneously. The fluorescence of the secondary antibodies  was detected with the Odyssey CLx Infrared Imaging system (LI-COR). (A) sAPPf was resolved with Covance-Ct and sAPPβ was resolved with the IBL-β antibody. (B) sAPPf was resolved with Sigma-Ct and sAPPf and sAPPα were resolved with the 6E10 antibody. (C) sAPPf and APPα were detected with 6E10 and sAPPβ was resolved with IBL-β. (D) sAPPα was resolved with the IBL-α antibody and sAPPβ was resolved with the IBL-β antibody. Simultaneous fluorescence serves to demonstrate the specificity of the C-terminal, IBL-β and IBL-α antibodies. Image showing co-localization (yellow) was only evident combining the C-terminal antibody Sigma-Ct with 6E10, whose epitope, located between the β-secretase and α-secretase cleavage sites, is present in both sAPPα and sAPPf. (JPEG 320 KB)p < 0.001). This correlation remained significant when data were analyzed separately for AD subjects , or NDC subjects . (JPEG 236 KB)Additional file 2: Figure S2: Positive correlation between sAPPα and sAPPβ in CSF samples. CSF samples from 13 NDC (closed circle) and 13 AD subjects (open circle) were assayed for sAPPα and sAPPβ fragments using specific ELISA kits (see Figure


Hyla japonica) against patterned backgrounds and elucidated how the expression of dorsal patterns changes against various achromatic/chromatic backgrounds with/without patterns. Our main findings are i) frogs primarily responded to the achromatic differences in background, ii) their contrasting dorsal patterns were conditionally expressed dependent on the brightness of backgrounds, iii) against mixed coloured background, frogs adopted intermediate forms between two colours. Using predator (avian and snake) vision models, we determined that colour differences against different backgrounds yielded perceptible changes in dorsal colours. We also found substantial individual variation in colour changing ability and the levels of dorsal pattern expression between individuals. We discuss the possibility of correlational selection on colour changing ability and resting behaviour that maintains the high variation in colour changing ability within population.Colour change in animals can be adaptive phenotypic plasticity in heterogeneous environments. Camouflage through background colour matching has been considered a primary force that drives the evolution of colour changing ability. However, the mechanism to which animals change their colour and patterns under visually heterogeneous backgrounds (i.e. consisting of more than one colour) has only been identified in limited taxa. Here, we investigated the colour change process of the Japanese tree frog ( In many animals, body colouration is primarily used for defence against predators12Camouflage through background matching can be achieved by possessing colours and patterns that resemble those of the animals’ background3101111Colour change is found in a variety of taxa including fish14161821Colour changing animals are able to change their colours or patterns to enhance the degree of camouflage (e.g. background colour matching or disruptive pattern expression) against their background24212628Although most of the experiments with frogs tested the change in colours, colours are not the only properties that can change in anurans610Physiological colour changes in anurans are predicted to occur rapidly2019262726Some animals, such as cuttlefish, can actively modulate their body colours through visual perception of the background9Hyla japonica , against heterogeneously coloured backgrounds. We mainly examined how the frogs’ colour and dorsal patterning changed against differently coloured backgrounds. We estimated whether the differences in dorsal colour against different backgrounds were large enough to improve camouflage from natural predators’ point of view. Furthermore, we tested whether colour change on mixed coloured backgrounds was an intermediate body colour or matched with one of the background colours.In this study, we investigated the colour change of the Japanese tree frog, H. japonica from agricultural fields and rice paddies near Mt. Bukhan . Catching/housing of frogs, and the experimental procedures were approved by Ewha University Institutional Animal Care and Use Committees (IACUC 2013-01-084) in accordance with the approved guidelines. Due to the low abundance of females, males were chosen for the experiment. The collected frogs were kept individually in small containers and transported to a temperature and humidity controlled room  at Ewha University. Each frog was provided with two juvenile crickets as food source per day and a small petri dish with a thin layer of water. Frogs were housed in small cube-shaped cells (0.5 × 0.5 × 0.5 m) that were independently illuminated by a daylight LED bulb  through which light can reach inside the container. The inside of the container was covered by printed papers that provided various background colours. To ensure equal illumination, we adjusted the height of bulb such that illuminance was 100 lux at the centre of each container.H. japonica.Because frogs can rapidly change dorsal colours under stress or during handling, non-invasive methods are preferred for quantification of frog colouration37The colour-change trial began at 8:00AM (UTC+09:00) every day in June and July of 2014. Because the photophase always started at 8:00AM, the potential effect of circadian rhythms on the expression of colours between treatments would be minimizedIn this experiment, we tested how the colours and patterns of frogs change against four achromatic background colours . We noteEach frog (N = 48) was tested on all four backgrounds and the order of four-colour trials followed 4 × 4 Latin Square design for every four frogs.H. japonica are known to rest on leaves or within leaf litter, whose primary colours were dominated by green and brown during daytime . The properties of printed colours are dependent on the RGB values of pixels and printer properties. To print the colours that imitate the chromaticity of natural objects, colours required calibration. To do so, a colour palette (comprising of 2040 different colours from the web) was printed and photographed with five fresh and five dead leaves in a single shot. We then searched for the colour with the most similar RGB values to the averaged RGB values of both leaf types. The chosen green and brown colours were clearly different chromatically, but were similar to each other in terms of brightness . The visual system of H. japonica is unknown. Based on the known spectral sensitivities of H. cinereaH. japonica .In this experiment, we used additional 24 frogs  and examined the colour and pattern change in frogs against three chromatic backgrounds with similar brightness . Then, we cropped each image to only have frog body and reduced to 600 × 600 pixels using bicubic interpolation. To remove non-linearity of camera responses, we performed linearization processes using grey colours in the colour checker that were photographed each morningFor each image, we categorized whether the frog’s dorsal pattern was visible or not as presence or absence. Then, we randomly selected three regions of interest (ROIs) from the dorsum of frogs and measured median RGB values of the ROIs. The presence of dorsal patterns was often dependent on the background type even within individuals. Therefore, to properly compare how frog colours changed independent of the pattern appearance, we only selected basal region (outside of the pattern) for colour comparison. When frogs were patterned, the inside of a pattern was always darker than the outside basal regions  single (chromatic) and double (achromatic) cones, and the references for snake vision were the garter snake  single cones (chromatic) and long wavelength sensitive single cones (achromatic)46Once we detected changes in either of these colour properties against different backgrounds, we transformed the image’s red, green, and blue channel pixel values to a bird-specific colour space (predicted photon catches for each cone cell type) using a polynomial mapping method37First, we examined the speed of colour change. We calculated the ∆E of the colour of frogs at each duration from the initiation of each trial. Then we compared the differences in ∆E between each duration using generalized linear mixed models (GLMMs) using the colour of current background and previously presented background as covariates, frog id as a random factor. Based on the result of this analysis, we used the frog colour that had completed colour changing process  as a representative colour against each background for further analysis. We further evaluated whether the frog colour after two hours against current background was independent of the colour of the previously tested background using MANOVA by putting brightness, chroma, hue at two hours as dependent variables, the colour of current background and previous background as independent variables.Within individual frogs, colour difference was most prominent between BK and WH backgrounds (see results). To estimate individual frog’s capacity to change colour, we measured the ∆E between the frog’s colour against BK and WH backgrounds and used this distance as an index of individual colour change capacity.To compare frogs’ dorsal colours between different background types, we examined within-subject effects of background type on brightness, chroma, and hue, using repeated measures multivariate analysis of variance. We then performed post-hoc multiple comparisons for the colour components that had significant effects. P values were adjusted to control for the false discovery ratesIn addition, we analysed the effects of background type on the presence of dorsal pattern using GLMMs to examine whether dorsal pattern appearance was affected by background colour. To identify the magnitude of colour change under darkness during night, we compared the colours of frogs just before becoming dark (at 12 h) with the colours at 7 am next day using repeated measures analysis of variance.For all individuals tested (N = 72), we characterized the individual capacity to change colour and the exhibition of dorsal patterns. Since these two characteristics were assumed to be physiologically connected between each other, we examined the relationship between colour changing capacity and the ability to exhibit dorsal patterns. First, we categorized each frog into a ‘patterned’ or ‘non-patterned’ group based on whether they exhibited dorsal patterns against BK background. We then employed a logistic regression to analyse the relationship between colour changing ability and the ability to exhibit dorsal patterns. All the statistical analysis were two-tailed and conducted in R.25 = 595.56, P < 0.001). We found that the dorsal colours of frogs changed rapidly within an hour of trial initiation and maintained similar levels throughout the daytime . This suggests that frog colours at two hours were independent of the colour of previously tested background. Therefore, we used the colour of frogs at two hours as a representative colour on each background for further analysis. By using the colour of frogs that completely had undergone colour change process against their current background, we consider that further results are independent of the colour of the previously tested background.In the BW experiment, the dorsal colours of frogs changed with time and duration was a significant factor for colour change . Univariate analysis revealed that frogs’ dorsal colour changed in both brightness  and chroma , but not in hue . Post-hoc tests revealed that both frogs’ dorsal brightness and chroma were different between all four background types  to both avian and snake predators . Frogs t26 = 0.01, P = 1) and snake predators . This indicates that, although the chromaticity of frog dorsal colour changed dependent on the background type, it did not achieve any better colour matching in terms of chromaticity.In terms of chromaticity, the colours of frogs in different backgrounds were all discriminable to predators between each other . However23 = 21.08, P < 0.001). Dorsal pattern expression under BK background was significantly higher than any other background types . They also exhibited dorsal patterns more often under BW background than both GY  and WH  backgrounds. There was no difference in dorsal pattern expression between GY and WH backgrounds .We found substantial within-individual variation in the expression of dorsal patterns against differently coloured background  depending on the type of backgrounds . We also found significant interactions between colour component and background type . Univariate analysis of each response variable showed that the frogs became darker and less colourful in all colour treatments , but both brightness and chroma after the night were significantly affected by the background colour that they were being tested the previous day ). We found no significant interaction between duration and background colour in brightness comparisons , but a single significant interaction was detected in chroma comparisons . We found no differences in hue analysis . These collectively imply that, after exposed under darkness during a night, frogs became darker and less colourful, but largely retained brightness and chroma from the previous day especially when they were against brighter backgrounds .We found no differences in brightness, chroma, and hue of frogs’ dorsal colours between all backgrounds . However, we note here that, although obscured by group effect, two out of 24 frogs showed clear hue change from green to brown .The above results suggest that individual frogs have different levels of colour changing ability as well as dorsal pattern expression. Against BK background, the dorsal patterns appeared in 34 out of 72 frogs. Logistic regression analysis showed that frogs with higher colour changing ability were more likely to express dorsal patterns  preference and colour changing ability. Non-changers would prefer to stay on a uniformly coloured substrate that have similar colours of their dorsal colours while colour-changers would not have such preferencesH. japonica exhibited dorsal patterns considerably more often against darker background while only about 15% of the frogs exhibited dorsal patterns against brighter backgrounds (white and grey). Although the frogs exhibited dorsal patterns more often against check patterned background than brighter backgrounds, it seems to be driven by the overall brightness of the background, not by the existence of background pattern: the proportion of patterned individuals against pattered background were about the half between those against black and white backgrounds. Therefore, we suggest that dorsal pattern expression in H. japonica was affected by overall background brightness, not by the presence of background pattern. Here, we propose an adaptive hypothesis for the observed conditional expression of dorsal patterns.Patterns are important elements for camouflage because they can reinforce the camouflage of animals by providing either background resemblance, disruption effects that hinder the detection of true outlines of frogs5If we apply our results to natural substrates, substrates that exhibit brighter background colours would be leaves while darker substrates would be tree bark or leaf litter. In general, leaves usually are homogenous in colour and are rarely patterned, whereas tree bark and/or leaf litter are often found with complex patterns. Therefore, considering the presence of patterns are likely to be advantageous against patterned background, having patterns against tree bark or leaf litters may be advantageous for frogs, while having patterns against leaves would be detrimental for survival. However, we cannot exclude the possibility of alternative non-adaptive explanations such as dorsal patterns as a by-product of melanophore re-arrangement during colour change process.Our results also suggest that this conditional expression of dorsal patterns is more pronounced for colour-changers. Therefore, colour-changers would be benefited not only by their high colour changing ability, but also by their ability to express dorsal patterns against dark heterogeneous substrates. This further highlights the pressure for the correlational selection between microhabitat preference and colour changing ability. It would be advantageous for the non-changers to preferentially rest on one substrate (lighter substrate such as leaves) because they are less likely to exhibit dorsal patterns against darker substrates. The mean brightness of non-colour changers in our experiments  was comparable to the brightness of naturalistic green colour that we used (mean brightness = 24.31) which supports this prediction.H. arborea. We will not discuss the details of the complex pigment rearrangement here , rapid colour change ability would be beneficial for individuals that move between substrates frequently.Most of the colour change process has been completed within an hour and maintained similar levels throughout the day when frogs are against the same coloured background. This corroborates previous studies that demonstrated rapid colour change in frogs22H. japonica and the upper threshold of dorsal colour change if they stay against one background repeatedly for more than a day. However, previous studies suggest the existence of long-term colour changing in tree frogs. H. regilla can undergo slow and gradual colour change during 21 daysH. japonicaInterestingly, during the night, frogs became darker but largely retained the colour that they exhibited the previous day in terms of both brightness and chroma . This coCamouflage through colour change is a widespread phenomenon in nature. To properly understand this intriguing biological process, it is crucial to consider its evolution under visually heterogeneous environments and the sensory properties of the animals’ natural predators36How to cite this article: Kang, C. et al. Colour and pattern change against visually heterogeneous backgrounds in the tree frog Hyla japonica. Sci. Rep. 6, 22601; doi: 10.1038/srep22601 (2016).