In Zhang et al. we report a novel means of delivering paclitaxel across the blood-brain barrier (BBB) using low-intensity pulsed ultrasound (LIPU) and microbubbles. This study is the basis for repurposing albumin-bound paclitaxel (PTX) for the treatment of glioblastoma (GBM), an estaPreclinical and human studies have shown that this technique is safe, well tolerated, and able to increase brain tissue concentrations of drugs like temozolomide and carboplatin –7. NewerZhang et al. used Abraxane (ABX), a newer, albumin-bound formulation of PTX with lower rates of neuropathy compared to earlier iterations . Early eWhereas previous studies had mapped BBB disruption by the extravasation of contrast-enhancing agent gadolinium or Evan’s Blue, on Zhang et al. we visualized effective disruption by the accumulation of fluorescein. Experiments showed that disruption was highly localized to regions of the brain that were exposed to the ultrasound field, as judged by obvious fluorescence. In mice that underwent LIPU-BBB disruption and received treatment with PTX, there was a strong correlation between brain tissue concentrations of fluorescein and PTX, indicating that the disruption induced by the sonication field was also highly effective at facilitating drug delivery to the brain. While we showed that ABX by itself was markedly more permeable than Taxol in the absence of LIPU, it achieved approximately 4-fold higher concentrations in brain tissue that had undergone BBB disruption.Toxic side-effects associated with chemotherapies are often a major limitation to treatments. In this study, ABX (at 12 mg/kg) in tandem with LIPU twice a week was well tolerated with minimal brain tissue damage. However, animals treated on an identical schedule with either Taxol or CrEL alone showed severe CNS toxicity, including necrosis and hemorrhage. This confirmed that most treatment-related damage was associated with the CrEL vehicle and not the drug itself. Ultimately, ABX delivered by ultrasound significantly extended the overall survival in an immunocompromised mouse model bearing MES83 human tumor cells compared to control groups that received ABX without ultrasound.The use of fluorescein to map BBB disruption is also a choice worth noting. Earlier animal studies have employed toxic tracer dyes, like Evan’s blue, to judge disruption upon biopsy . While shttps://clinicaltrials.gov/ NCT04528680).There are, however, limitations to this work that will need to be addressed in future studies. We noted that histological staining of the PDX tumors from their survival studies show extensive vascularization and minimal integration with the surrounding brain tissue. This contrasts to the infiltrative nature of GBM in humans, and likely resulted in some BBB disruption in the peritumoral tissue regardless of sonication treatment. Ongoing studies in patients include pharmacokinetic studies where we perform biopsies to determine drug quantification, particularly in peritumoral regions that maintain an intact BBB and are exposed to the sonication field (Through this work, we demonstrate the strong potential of LIPU technology, by recontextualizing the use of a potent drug that might otherwise go overlooked for the treatment of GBM. By overcoming the BBB as an obstacle to drug delivery, Zhang et al. open the door for a new gamut of therapies against brain cancers.


Pulmonary fibrosis is a pathologic process associated with scarring of the lung interstitium. Interstitial lung diseases (ILDs) encompass a large and heterogenous group of disorders, a number of which are characterized by progressive pulmonary fibrosis that leads to respiratory failure and death. Idiopathic pulmonary fibrosis (IPF) has been described as an archetype of progressive fibrosing ILD, and the development of pirfenidone and nintedanib has been a major breakthrough in the treatment of patients with this deadly disease. Both drugs principally target scar-forming fibroblasts and have been shown to significantly slow down the accelerated decline of lung function by approximately 50%. In addition, nintedanib has been approved for patients with other progressive fibrosing ILDs and systemic sclerosis-associated ILD. However, there is still no cure for pulmonary fibrosis and no meaningful improvement of symptoms or quality of life has been shown. Advancement in research, such as the advent of single cell sequencing technology, has identified additional pathologic cell populations beyond the fibroblast which could be targeted for therapeutic purposes. The preclinical and clinical development of novel drug candidates is hampered by profound challenges such as a lack of sensitive clinical outcomes or suitable biomarkers that would provide an early indication of patient benefit. With the availability of these anti-fibrotic treatments, it has become even more difficult to demonstrate added efficacy, in particular in short-term clinical studies. Patient heterogeneity and the paucity of biomarkers of disease activity further complicate clinical development. It is conceivable that future treatment of pulmonary fibrosis will need to embrace more precision in treating the right patient at the right time, explore novel measures of efficacy, and likely combine treatment options. The last decade has been a game changer for idiopathic pulmonary fibrosis (IPF) from a research as well as from a clinical perspective. A shift of understanding of the pathogenesis from a chronic inflammatory to an epithelial driven disorder resulting from repetitive alveolar epithelial injury and aberrant repair  as well IPF is the most common form of idiopathic interstitial pneumonia within a large and heterogenous group of disorders referred to as interstitial lung diseases (ILD) . IPF is,Undoubtedly these advances and, specifically, the approvals of antifibrotic drugs for the treatment of (idiopathic) pulmonary fibrosis have fuelled basic, translational and clinical research and drug development in this field. However, key challenges will have to be tackled across disciplines in the next decade to ultimately embark on a path of patient-centricity and precision medicine in drug development and to provide benefit to patients beyond “just” slowing lung function decline. These include identification of patients earlier in their disease course to ultimately prevent development of or even reverse fibrosis, validation of prognostic and predictive biomarkers to enable the right treatment for the right patient at the right time, developing better disease models, clinical outcome parameters and, ultimately, targeting the mechanisms involved in lung repair and regeneration.This review aims to shed light on some of these key challenges from an industry research and development perspective.Both pirfenidone and nintedanib—the current standard of care for medications for idiopathic and other forms of pulmonary fibrosis—were borne of the desire to inhibit aberrant fibroblast activity. Fibroblast populations are responsible for providing structure to organs, so are thus an essential part of the repair process following damage. Fibroblast proliferation, migration and transition toward myofibroblasts, which preferentially deposit collagen to form scar tissue, are healthy processes when controlled to be transient . HoweverThe advent of single cell seq technologies have greatly expanded our view of pathologic processes to reach beyond the fibroblast . We now We now look to promote a homeostatic balance of macrophages, rejuvenate small airway and alveolar epithelial progenitor populations and correct the mutual dysfunctional crosstalk between epithelium and underlying fibroblasts. Yet the ambition to explore novel targets within the immune fibrotic niche preclinically will bring even greater challenges as they are translated to the clinic—requiring an ever greater degree of therapeutic precision.in vitro and in vivo systems focused upon fibroblast biology. Fibroblasts stimulated with platelet-derived growth factor (PDGF) will proliferate, or with transforming growth factor (TGF)ß transition to scar-forming myofibroblasts. Bleomycin-induced fibrosis in mice/rats model inflammatory, then fibrotic, changes followed by resolution waves of pathobiology—yet have shown limited translational value , grown at air-liquid interface, together with fibroblasts  vs target engagement (TE) vs pharmacodynamic (PD) relationships of drug candidates. Yet, we strive to enhance the translational relevance of PD measures—functional measures or biomarkers—to capture more elements of the immune-fibrotic niche. Bleomycin models can be enhanced by using older mice, thus partially negating the transient nature of the fibrosis observed, or by using a model of repetitive instillation to create a more durable fibrotic response , is seen as a major advance for the treatment of patients with IPF . However, this success is also accompanied by new challenges for drug developers. In the ideal clinical trial, all patients enrolled in the trial would experience the event the study drug is designed to prevent/treat, such that patients receiving active drug will demonstrate clear efficacy as compared to placebo. This is especially important in conditions which take a long time to manifest or in which relatively few patients experience the event. Indeed, fibrotic ILDs often progress slowly; thus, in some patients it may take years to see significant declines in lung physiology or increased fibrosis on high-resolution computed tomography (HRCT) scans, and clinical trials often require primary endpoints to be measured after a minimum of 12 months to allow sufficient patients to demonstrate progression. Yet even in such circumstances, a proportion of patients will not demonstrate significant loss of lung function. As an example, in the pivotal INPULSIS trials of nintedanib in patients with IPF, 15% of placebo-treated patients demonstrated either no decline or an improvement in FVC over the 52-week trial period . SimilarFor fibrotic ILDs, a current challenge is identifying which patients will develop progression over a relatively short period of time or are at increased risk for exacerbations or death. This is critical not only for planning of clinical trials but also for clinical care; patients likely to experience faster progression or at risk for adverse outcomes might be prioritized on transplant lists or followed more closely in the clinic. Efforts to develop prognostic information in patients with fibrotic ILDs have intensified greatly over the recent past, with numerous investigators evaluating a panoply of potential biomarkers. Such biomarkers may be genetic , metabolBut for which outcome should we be trying to enrich? Historically, registrational trials for both nintedanib and pirfenidone in patients with IPF were predicated on a primary endpoint of annualized rate of decline of FVC (ml/year) or change in FVC from baseline (ml), supported by additional secondary outcomes such as a trend for reduced mortality, further strengthening FVC as a valid surrogate endpoint. Subsequent studies in IPF, as well asother fibrosing ILDs, have similarly focused on the rate of FVC decline given the precedent set by nintedanib and pirfenidone. Certainly, slowing the rate of decline in lung function for patients with IPF and other progressive ILDs is a worthy goal; data suggest that rate of decline of lung function, both FVC and diffusing capacity for carbon monoxide (DLCO), correlates with time to mortality . This coBoth nintedanib and pirfenidone utilized classical drug development approaches with large and long phase II dose-ranging studies. For example, the trial providing proof of clinical concept for nintedanib was a dose-ranging study exploring four doses of nintedanib versus placebo that randomized 432 patients with IPF to treatment over 1 year . This stHowever, newer drug candidates will be required to utilize different clinical development approaches, for a variety of reasons. First, IPF (and the majority of other progressive fibrosing ILDs) are rare-to-uncommon diseases and, thus, the patient pool available for inclusion in clinical studies in these indications is limited. Coupled with increased drug development efforts in pulmonary fibrosis by numerous companies, several drug candidates are currently being investigated in phase II and phase III studies  which leSecond, accelerated new drug development timelines are necessary due to pragmatic considerations for our patients as well as costs: a long phase II study with endpoints measured 1 year after study initiation is no longer sustainable for patients or sponsors. However, the ability to demonstrate a statistically significant treatment difference in rate of FVC decline at a timepoint earlier than 52 weeks is dependent upon the degree of patient heterogeneity within a cohort and the variability inherent in lung function testing. As a result, increased sample sizes are required to achieve the power needed to detect significant differences in rate of FVC decline. Interestingly, retrospective analyses of nintedanib and pirfenidone clinical development programs suggest that a separation in the rate of decline of FVC with antifibrotic treatment may be apparent after 26 weeks. Some sponsors have recently reported results demonstrating treatment effects in phase II trials of patients with IPF after only 12 weeks . ZiritaxFinally, new drug candidates for patients with IPF or other progressive fibrosing ILDs can no longer ethically be compared to true placebo, as standard of care therapies (with their associated adverse effects) now exist for these patients. This adds further complexity and challenges on clinical trial design: any treatment (or adverse) effects seen for newer therapeutics could be attributed to the investigational compound or the standard background therapy; the room for improvement in lung function on top of standard of care is variable and limited and affecting sample size; patient populations will be heterogenous including all-comers, i.e., patients without background antifibrotic standard of care (either naïve to antifibrotic treatment or having stopped previous standard of care for tolerability or effectiveness reasons) and patients with concomitant approved antifibrotic treatments. Moreover, in the future, newer therapeutics may be designed to improve the lives of patients with IPF and other fibrosing ILDs as measured by different endpoints , which may be difficult to interpret in the face of combination therapy.Given the challenge of recruiting enough patients in a timely fashion to identify drug efficacy in pulmonary fibrotic diseases, drug developers may seek to take advantage of a number of methods to speed development program efficiency. Such methods may involve innovative statistical modeling, novel trial design approaches, biomarker enrichment strategies, or some combination thereof. One example of innovative statistical modeling, called Bayesian dynamic borrowing , utilizeThe regulatory hurdles for registration of new drug candidates for IPF or fibrotic ILDs have been high in the past, requiring at least two replicate pivotal trials meeting their primary efficacy endpoint  and providing acceptable safety over 1 year treatment duration. However, some light at the end of the tunnel can be seen that authorities may be increasingly willing to deviate from these classical rigid requirements, given the remaining high unmet need in pulmonary fibrosis and the challenges with existing clinical endpoints. An example is the recent approval byUS FDA of tocilizumab, an IL-6 inhibitor, to slow the rate of FVC decline in systemic sclerosis-associated ILD. This FDA approval is based on data from a single phase III randomized controlled clinical trial in 210 SSc-ILD patients with supportive evidence coming from a similar preceding (phase II) trial. The phase III trial did not meet its primary endpoint change from baseline to week 48 in the modified Rodnan Skin Score, a standard outcome measure for skin fibrosis in systemic sclerosis , but shoin vivo animal models reflecting the fibrotic niche, has become the starting point for precision drug discovery. Clinical development of new therapeutics for the treatment of rare fibrotic lung conditions is hampered by e.g., patient heterogeneity and the lack of appropriate biomarkers to predict the disease trajectory for the individual patient to enable enrichment strategies. This imposes major challenges on clinical trial design and project development timelines. These will hopefully be overcome by improved and efficient trial designs which are accepted by regulatory authorities and ultimately pave the way to precision medicine approaches to provide the right treatment at the right time to the right patient suffering from pulmonary fibrosis.Pulmonary fibrosis is believed to result from aberrant repair of repeated epithelial injury. Application of new technologies has shifted our research focus beyond fibroblast biology into the immune-fibrotic niche and its complex dysregulated interplay of diverse cell populations. Differential patient biology using cell systems and tissue directly from patients and patient-relevant stimuli, as well as enhanced


Mammalian animal toxicity of ochratoxin A (OTA) has focused largely in the past half-century on pigs because of initial recognition of it as a principal cause of intermittent growth suppression and renal disease caused by mouldy feed. Subsequent classical toxicology has used laboratory rodents because renal pathology in pigs raised questions concerning possible involvement in the human idiopathic bilateral renal atrophy of Balkan endemic nephropathy for which OTA was a focus of attention for human nephropathy through 1980s and into 2000s. Emphasis on human nephropathy has more recently concerned the plant metabolite aristolochic acid. Recognition that agricultural management can often minimise food and feed-stuff spoilage by OTA-producing Aspergilli and Penicillia has moderated some of the risks for animals. Legislation for human food safety combined with sophisticated analysis generally provides safety in the developed world. Chronic experimental exposure of male rats, in the absence of clinical dis-ease, specifically causes renal cancer. The possibility of this as a unique model for the human has generated considerable experimental evidence which may be more directly relevant for carcinogenesis in the complex kidney than that obtained from biochemical toxicities in vitro. Nevertheless, there does not appear to be any case of human renal or urinary tract cancer for which there is verified etiological proof for causation by OTA, contrary to much claim in the literature. To contribute to such debate, histopathology review of OTA/rat renal cancers, augmented where appropriate by immune profiles, has been completed for all remaining tumours in our research archive. Overall consistency of positivity for vimentin, is matched with occasional positives either for CD10 or the cytokeratin MNF 116. The current situation is discussed. Suggestion that OTA could cause human testicular cancer has also been challenged as unsupported by any experimental findings in rats, where the Leydig cell tumour immune profile does not match that of human germ cell neoplasms. Aspergillus ochraceus as a dietary additive for experimental rats. Concurrently, the term mycotoxins became used to encompass toxic metabolites of other food-spoilage moulds such, as the aflatoxins of A. flavus, found to have widespread occurrence. Focus for OTA soon shifted to the spasmodic occurrence of an idiopathic porcine nephropathy in the Danish bacon industry, economically linked to home-grown barley; in some high rainfall years this had been insufficiently dried before storage. Seasonal opportunist moulding by a common Penicillium, and chromatographic recognition of OTA partly by its fluorescence under UV254 light, revealed another biosynthetic source of the mycotoxin in amounts subsequently demonstrated experimentally as a major cause of the disease in pigs. This had been expressed as reduced growth rate, low carcass weight and mottled and disproportionally enlarged kidneys, readily recognised at meat inspection and the carcass rejected [Ochratoxin A (OTA) was discovered in South Africa in the early 1960s to explain the general toxicity of cultured Aspergillus and Penicillium moulds across both tropical and temperate latitudes were subsequently found to elaborate OTA during spoilage of major staple agricultural products such as cereals, and in crops for high-end commodities such as coffee, cocoa and red wines. Concern that traces of OTA in major cereal products such as pasta might pose a human health risk, and of potential economic threat to commercial images of the high-end commodities, stimulated extensive research into OTA toxicology. Concurrently, world-wide food safety authorities  addressed potential health risks and formulated documents to guide regulatory legislations. Several Pigs being important in the human food chain in many economies prompted a major US experimental toxicology study in the 1980s with lifetime chronic exposure in rats . The str4-fold) between necessary daily carcinogenic exposure for rodents  and surveyed average natural daily human intake data  [Subsequent developments in sensitive and definitive analytical methodology have shown widespread occurrence of relevant moulds and their mycotoxins, although general occurrence in well-managed agriculture is generally low. For human health, there has been little verified etiological evidence of general toxicities attributable other than to spasmodic episodes. The initial cautious classification of possible carcinogenetic risk  seems neAspergillus ochraceus in a shaken solid substrate fermentation. The product, analysed for OTA, was homogenised (c.103-fold) into powdered rat diet for consumption during natural diurnal habit for Fischer male rats. Although blood OTA analysis had not been available for the US study, values in London followed a gradual rise to 8–10 µg/mL during the first month. For the highest daily OTA intake employed (300 µg/kg b wt) at least 9 months exposure seemed necessary to initiate kidney cancer, during which the common prevalence of monocytic leukaemia in ageing Fischer males was apparently repressed [The classical US toxicity study in rats  followedepressed . The tumepressed . The dosised c.10-fold intepressed  describiCritical histology review of some OTA/rat renal tumours has been described , includiIn confirming a renal cell origin for rat/OTA tumours, all four cases were diffusely and intensely positive for vimentin  is not immuno-positive other than for vimentin ; the diaCase 4 tumour (5 mm) is additionally positive for CD10. (not illustrated).Histopathology and immune profiles of the present four rat renal tumours caused by chronic exposure to dietary OTA are complementary to our other similar tumours to which immunohistochemical profiles have recently been ascribed . The comImmunostaining for vimentin was invariably extensive as is typical for human tissues of mesenchymal origin. The accompanying positivity for CD10 in three of the 10 rat tumours could fit for humans , concludGender specificity in rodents is a neglected problem for matching a potential model with the human for whom there seems none for renal cell carcinoma. For the mouse, OTA renal cancer seems specifically male  and for Another way of gaining understanding of the relative dynamics of OTA in blood of male and female rats, to focus on gender differential in renal tumorigenesis, would be by first establishing and quantifying stable circulating OTA in male and female rats during continuous dietary exposure; then to castrate some of the males while continuing the OTA. Predictably, females would have initially attained a higher stable concentration of OTA in blood than males, which were actively excreting more bound to small urinary proteins. Post-castration OTA concentration in male blood could be expected to rise as testosterone-regulated urinary protein synthesis in liver declines. This could be a step towards evaluating the role of urinary proteins in the male rat tendency to develop renal tumours in response to chronic exposure to dietary OTA. An experimental challenge to explore could be the use of an established technique of rat renal transplantation  to studyThe above histopathology review  also notThere had been no agreed conclusion in an EU project (European Commission 2001–2004) concerning putative genotoxic, epigenetic or oxidative stress mechanisms in male rat renal carcinogenesis caused experimentally by OTA. However, definitive structural evidence of DNA adduction was subsequently obtained  after MSA third lifetime rat study, in Hannover Germany in the 1990s using Dark Agouti rats with gavage exposure to OTA . Giving Subsequent use also of male Dark Agouti rats  generallAcross the three main centres for lifetime experiments, there was a consistently lower incidence of renal neoplasms in females than in males for Fischer, Dark Agouti and Lewis rats. A putative mechanism for this differentiation has been proposed . It requIn recent years ethical considerations have limited some whole animal experimentation, in addition to the economic cost of experimental mycotoxins, and to the lifetime maintenance cost for rodents and particularly for primates. Thus, the scientific literature has reports mainly on tissue culture experiments. Whereas this may provide model information applicable to some mammalian tissues, there is less confidence in application to kidney with its exceptionally complex internal dynamics for sequential stages in filtration, key metabolite recovery, water and ion regulation, metabolic waste excretion and the huge replication of separate nephrons. Particularly, relative roles of trans-membrane ion transfer of OTA, as a phenylalanine derivative, from capillaries to nephron epithelia versus classical understanding of glomerular filtration, are not clear. Very rare finding of very simple early nuclear proliferation in situ within a nephron is tantalising evidence of early neoplasm. We have not yet seen this for OTA, but are aware of a possible model illustrated in a rat in an experiment on chronic exposure to aristolochic acid . In our Experimental evidence shows that the Fischer and other OTA/rat tumours which have generated concern for humans may simply mimic the mechanism operating spontaneously in the EKER strain . The finAnother tumorigenesis topic in the literature in the past decade concerns whether OTA is a cause of human testis cancer. This was reiterated prominently in a review abstract  with theRat and mouse renal tumour response to long-term dietary OTA has cautioned possible analogous cancer risk for humans, but there is yet no verified case of disease. A recent EFSA report  expresseFour renal tumours from Fischer male rats given protracted dietary OTA (300 µg/kg body weight daily)  were emb


Mammals harbor trillions of microorganisms and understanding the ecological and evolutionary processes structuring these ecosystems may provide insights relevant to public health and medicine. Comparative studies with our closest living relatives, non-human primates, have provided first insights into their rich bacteriophage communities. Here, I discuss how this phage diversity can be useful for combatting antibiotic-resistant infections and understanding disease emergence risk. For example, some primate-associated phages show a pattern suggesting a long-term co-divergence with their primate superhosts—co-diverging phages may be more likely to exhibit a narrow host range and thus less useful for phage therapy. Captive primates lose their natural phageome, which is replaced by human-associated phages making phages an exciting tool for studying rates of microorganism transmission at human–wildlife interfaces. This commentary tackles avenues for selecting phages for therapeutic interventions based on their ecological and evolutionary history, while discussing frameworks to allow primate-associated phages to be incorporated into the arsenal of clinicians. Evidence suggests that mammals harbor trillions of microorganisms on and in their bodies. Understanding the ecological and evolutionary processes structuring these ecosystems has the potential to provide insights relevant to public health and medicine, some of which are explored here. Specifically, I examine ways in which the phage diversity of non-human primates could be useful for combatting antibiotic-resistant infections and understanding disease emergence risk. This includes an exploration of ways to select phages for therapeutic interventions based on their ecological and evolutionary history, as well as how phages might be useful for studying rates of microorganism transmission at human–wildlife interfaces. The hope is that these frameworks may allow primate-associated phages to be incorporated into the arsenal of clinicians. Every mammal hosts trillions of microorganisms; humans are no exception. These communities constitute a majority of the cells found in a host and include bacteria, archaea, microbial eukaryotes as well as many viruses infecting these cellular organisms  . Indeed,ifficile , while ichildren , supportchildren . Some phchildren . DespiteComparative studies with our closest living relatives, non-human primates, can provide insights into the evolutionary and ecological processes shaping our microbial communities . The disThe support for the hypothesis of long-term co-divergence between phages and superhosts raises the question of how such long-term co-divergence over millions of years is possible, when many distantly related species of primates are sympatric, living in the same ecosystem? The study of phages within a single primate species, baboons, revealed that neighboring social groups harbored evolutionarily and compositionally distinct phageomes that were structured by their superhost’s social behavior; close grooming partners had more similar phage communities, even after controlling for relatedness or diet of the superhosts . This stSurprisingly, a first study of the captive primate phageome found that captive non-human primate phageomes were more similar to humans than their wild counterparts, with nearly complete replacement of wild-associated phages with human-associated ones. This finding was then also found for bacteria, where patterns of co-divergence with their primate hosts was observed in the wild , but subnDemocratic Republic of Congo, nCôte d’Ivoire = 12, nGermany = 24), as well as across non-human primate populations. Studies of the bacterial microbiome suggest major differences between industrialized and hunter-gather societies [Broadly speaking, this preliminary body of work suggests that humans have distinct phageome communities when compared to those of their ancestors; understanding how changes in these communities over recent evolutionary history have contributed to the rise of disease of gut inflammation and health more broadly is a key area of future research. A key limitation of this study was the limited sampling of the human population  for the cataloguing of phages for their rapid deployment in clinical settings. To date, there is a limited availability of multiple broad host range phages for inclusion in phage cocktails to test on emerging antimicrobial resistant bacteria . BacteriOne particularly common source of phages for such challenge experiments is human sewage or wastewater . CompariThese phage communities act as modulators of the gut microbiota and the extant non-human primate phageome could also provide phages that are useful for therapeutic interventions to modify a dysbiotic gut microbiome. Indeed, it is often hypothesized that many different diseases associated with industrialized nations are associated with our shrinking microbiomes . The useCaptivity modifies both the bacterial and phage communities found in the non-human primate gut , 38. ThiA promising avenue for selecting phages for therapeutic interventions is the incorporation of data on the ecological and evolutionary history of these phages across the primate lineage. Phages that have a history of superhost switching within ecosystems are very likely to have a broader bacterial host range , making Because of their immunological and physiological similarities, non-human primates are infected by many closely related pathogenic bacteria. Indeed, the phylogenetic proximity of two organisms predicts the likelihood of pathogen jumps and many human pathogens have relatively recent origins in non-human primate populations . This suThe transmission of pathogens occurs not only from wildlife to humans, but also from humans to wildlife, and can pose a risk to endangered populations . UnderstPhages represent an exciting tool to study rates of microorganism transmission at the human–wildlife interface. This is in part because research suggests phages have faster life histories and evolutionary rates than bacteria , meaningUnderstanding the ecological and evolutionary factors that shaped the diversity of phages found in humans and our closest living relatives may pave the way for harnessing this diversity for medical interventions like phage therapy. Implementing such interventions with these primate-associated phages will require significantly more research and coordination with regulators and clinicians, which may be best achieved by contributing to phage banks , 32, 34.


We thank Gray et al.  for theiGray et al., questioned whether topical lidocaine failed to improve AD during bowel care, based on concerns that the primary outcome was the peak systolic arterial pressure (SAP) and not the change in SAP relative to baseline, which they argue is more compatible with the current Paralyzed Veterans Association (PVA) Clinical Practice guidelines definition . We wereNot only was lidocaine ineffective at ameliorating AD, it also had the unfortunate side effect of impairing reflexive defecation and bowel emptying, reflected in an increased time to complete bowel care - one participant was not able to pass stool when using lidocaine.p = 0.011). Sub-analyses based on the source of arrhythmic activity did not quite achieve criteria for statistical significance as the authors note, with p values just exceeding our threshold. The terminology for a “trend” in these sub-analyses is not ideal, however, we reported exact p values to aid data interpretation. The statistical significance of the overall incidence of arrhythmia, regardless of location in the conducting system, was robust. Of note, the study sample size for this study, while small, met our sample size calculation for the primary outcome measure (the peak SAP) with adequate retrospective power. Given the detrimental effect of lidocaine observed we felt it was not ethically appropriate to continue recruitment beyond the initial target – additional trial participants would have little expectation of benefit and could potentially be exposed to increased risk through participation in the study.Gray et al., questioned our statistical approach regarding the analysis of cardiac arrhythmia during bowel care. Our data showed that the number of all-cause arrhythmic events during bowel care was greater with lidocaine than placebo . They also did not examine the impact on AD during routine bowel care, ability to successfully complete bowel care, or incidence of associated cardiac arrhythmia. In fact, a different study from the same research group also reported that topical lidocaine did not significantly limit or prevent AD during anorectal procedures [Gray et al., reference the only study showing benefit of lidocaine use for bowel procedures , but faiocedures . The empWe disagree that these data are insufficient to question current recommendations for lidocaine use during at-home bowel care. Lidocaine hampered care routines and impaired defecation with no improvement in AD by any metric , and worse AD by several measures, with a concurrent increase in all-cause cardiac arrhythmia. We believe these robust findings should not be dismissed based on minor technicalities in reporting or preferences in primary outcome measures. Improvements to bowel care are prioritized by people with SCI, with amelioration of associated AD and reductions in the time taken to complete bowel care identified as priorities to improve quality of life . Lidocai


There is growing evidence that vertebral column function and dysfunction play a vital role in neuromuscular control. This invited review summarises the evidence about how vertebral column dysfunction, known as a central segmental motor control (CSMC) problem, alters neuromuscular function and how spinal adjustments  and spinal manipulation  alters neuromuscular function.The current review elucidates the peripheral mechanisms by which CSMC problems, the spinal adjustment or spinal manipulation alter the afferent input from the paravertebral tissues. It summarises the contemporary model that provides a biologically plausible explanation for CSMC problems, the manipulable spinal lesion. This review also summarises the contemporary, biologically plausible understanding about how spinal adjustments enable more efficient production of muscular force. The evidence showing how spinal dysfunction, spinal manipulation and spinal adjustments alter central multimodal integration and motor control centres will be covered in a second invited review.Many studies have shown spinal adjustments increase voluntary force and prevent fatigue, which mainly occurs due to altered supraspinal excitability and multimodal integration. The literature suggests physical injury, pain, inflammation, and acute or chronic physiological or psychological stress can alter the vertebral column’s central neural motor control, leading to a CSMC problem. The many gaps in the literature have been identified, along with suggestions for future studies.Spinal adjustments of CSMC problems impact motor control in a variety of ways. These include increasing muscle force and preventing fatigue. These changes in neuromuscular function most likely occur due to changes in supraspinal excitability. The current contemporary model of the CSMC problem, and our understanding of the mechanisms of spinal adjustments, provide a biologically plausible explanation for how the vertebral column’s central neural motor control can dysfunction, can lead to a self-perpetuating central segmental motor control problem, and how HVLA spinal adjustments can improve neuromuscular function. The vertebral column is linked biomechanically and neurologically to the limbs. Yet, we know very little about how altered sensory feedback from the vertebral column affects limb sensorimotor integration and motor performance. Recently, several research studies have documented changes in motor output following vertebral column dysfunction or perturbations involving the application of controlled vertebral column high-velocity, low-amplitude (HVLA) thrusts , and motor-evoked potential. Specific inclusion criteria were: spinal adjustments or spinal manipulation were the intervention assessed and muscle output or force were measured as an outcome. The search strategy for reviewing the effects of spinal adjustments or spinal manipulation on sensory organs included the following search terms: chiropractic, manual therapy, HVLA, adjustment, manipulation, muscle spindle and Golgi tendon organ. Overall, studies were included if they met all the following criteria: spinal adjustments or spinal manipulation where the intervention assessed and the study appeared in a peer-reviewed English-language journal. Studies were excluded if they were reviews, books, theses, conference papers, commentaries, or letters. The reference list of included studies and recent systematic reviews were also searched.Movement control relies on the accurate detection and integration of multiple sensory receptor inputs from the inside (interoception) and outside the body (exteroception). Interoception is the perception of internal bodily signals and processes Craig  and inclFigure The basic science research about CSMC problems and mechanisms of spinal adjustments and manipulations has seen a shift away from a local structural pathology model, where a CSMC problem was thought to directly put pressure on, or irritate, spinal nerve roots or the spinal cord itself Grostic , towardsThe contemporary model of CSMC problems and spinal adjustments  input the CNS receives from the small, deep paraspinal muscles of the vertebral column , the lower motor neuron and its corresponding extrafusal muscle fibres, i.e. the motor units. The excitability of the UMN and single motor units (SMUs) can be influenced by many factors. The UMN is, for example, widely influenced by multiple pre-UMN networks that can have both an inhibitory and excitatory influence on the output of the UMN. To selectively assess the influence of vertebral dysfunction on the UMN or the SMUs themselves is not an easy task in humans and not yet possible in a non-invasive fashion. However, the entire corticomotor system can be assessed with several techniques. With carefully controlled experiments, it is possible to make educated conclusions about whether the function of the upper or the lower motor neuron has changed, or whether any changes are presynaptic to the corticospinal tract itself , or whether the changes in output are due to changes in the muscle contractile apparatus itself. Methods that have been used to assess whether early vertebral column dysfunction or the effects of spinal adjustments alter UMN or SMU outputs include the use of transcranial magnetic stimulation (TMS) (Haavik-Taylor and Murphy ) could include that the participants had pain at that level of the spine, or the segment was chosen at random or that they could have a segmental effect on the nerve roots or associated motor neuron pools. Thus, they may have chosen a lower cervical segment to manipulate, regardless of whether this segment displayed any clinical signs of joint dysfunction, simply because the nerve roots at that level innervate the upper limb. As discussed in this review, it is highly unlikely that applying HVLA thrusts at the spine alters transmission of information flow via the nerve roots at the level of manipulation, unless that individual displays radicular symptoms at that level . The sham involved the drop of a table piece that would likely impact the paraspinal tissues, particularly paraspinal muscle spindles, due to the drop itself, despite there being no direct force application over the CSMC problem  assessment protocol or a sham treatment session or a control session with no intervention (Keller and Colloca Another adjustment study has shown that adjusting sacroiliac CSMC problems can improve feedforward activation (FFA) times of deep abdominal muscles in relation to rapid upper limb movements in young, healthy males  and the V-wave measures changes in supraspinal input to the motor neuron pool . The effect of spinal adjustments on corticomotor excitability has been evaluated by recording TMS-induced motor-evoked potentials (MEPs), cortical silent periods (CSPs), short-interval intracortical inhibition (SICI), short-interval intracortical facilitation (SICF) and stimulus–response curve  pre- and post-adjustments of CSMC problems  and movement (kinaesthesia) in the absence of visual inputs  in the group that received the HVLA thrust directed at a CSMC problem. This decrease is similar to those previously observed following HVLA manual spinal adjustments to CSMC problems  in N30 SEP peak amplitudes. These between group differences were significant (p < 0.1) and provide direct evidence that a spinal adjustive thrust from a hand-held activator adjusting instrument directed at a CSMC problem results in different neurophysiological outcomes to a similar HVLA thrust directed at a normally functioning vertebral segment in humans.Recently, the preliminary results were presented from a study done in a human population that investigated whether an HVLA adjustive thrust delivered with an Activator adjusting instrument to a CSMC in the cervical spine had different neurophysiological outcomes to an HVLA thrust to a segment that was deemed to be functioning normally , a pro-inflammatory cytokine, play an active role in the subacute and early chronic phase of the known maladaptive plastic changes of the deep paraspinal muscles following disc injuries . However, under certain conditions (for example stress), or due to certain personality traits, this may not be possible for everyone’s CNS to do. There might be some conditions, such as when a person is under a lot of psychological or physiological stress and/or during high attention-demanding situations, that such factors interfere with this process as these are known to influence various aspects of motor control  problems, as well as how spinal adjustments and spinal manipulation alters neuromuscular function. Multiple studies have shown increases in force measures and prevention of fatigue building during strong repeated muscle contractions after spinal adjustments  proprioception and thus can influence vertebral column motor control by altering signalling from the deep paraspinal muscles or the central processing of such input  and along muscle-fascia chains, such as between legs and the trunk [for review see (Wilke et al. Thus, there is a long way to go to fully understand the mechanisms by which altered afferent input from the vertebral column impacts the human neuromuscular system, let alone the clinical implications of such changes. However, it is clear from this review that physical injury, pain, inflammation and acute or chronic physiological or psychological stress can alter signalling from the deep paraspinal muscles to the CNS (Hellström et al. Furthermore, it is clear from this review that the high-velocity, low-amplitude thrusts directed at the spine are capable of activating the deep muscle afferents from paravertebral tissues, particularly activating the muscle spindles and potentially Golgi tendon organs (Reed et al.


The information given to people considering taking part in a trial needs to be easy to understand if those people are to become, and then remain, trial participants. However, there is a tension between providing comprehensive information and providing information that is comprehensible. User-testing is one method of developing better participant information, and there is evidence that user-tested information is better at informing participants about key issues relating to trials. However, it is not clear if user-testing also leads to changes in the rates of recruitment in trials, compared to standard trial information. As part of a programme of research, we embedded ‘studies within a trial’ (SWATs) across multiple ongoing trials to see if user-tested materials led to better rates of recruitment.Seven ‘host’ trials included a SWAT evaluation and randomised their participants to receive routine information sheets generated by the research teams, or information sheets optimised through user-testing. We collected data on trial recruitment and analysed the results across these trials using random effects meta-analysis, with the primary outcome defined as the proportion of participants randomised in a host trial following an invitation to take part. provided data on recruitment. Optimised participant information sheets likely result in little or no difference in recruitment rates .Six SWATs Participant information sheets developed through user testing did not improve recruitment rates. The programme of work showed that co-ordinated testing of recruitment strategies using SWATs is feasible and can provide both definitive and timely evidence on the effectiveness of recruitment strategies.Healthlines Depression (ISRCTN14172341)Healthlines CVD (ISRCTN27508731)CASPER (ISRCTN02202951)ISDR (ISRCTN87561257)ECLS (NCT01925625)REFORM (ISRCTN68240461)HeLP Diabetes (ISRCTN02123133)The online version contains supplementary material available at 10.1186/s12916-021-02086-2. Randomised controlled trials remain the gold standard for evaluating effectiveness of many interventions, but recruitment to trials remains problematic , 2. RegaA recent Cochrane review of trial recruitment strategies identified 72 strategies but only three with GRADE high certainty evidence, limiting the ability of trial teams to draw on a rigorous evidence base to inform recruitment design . ImportaThe ‘systematic techniques for assisting recruitment to trials’ (START) research programme funded by the UK Medical Research Council (MRC) responds to these limitations. The START programme was designed to develop the conceptual, methodological and logistical framework to make SWATs a routine part of the delivery of trials, and to assess the feasibility of this approach by developing a small number of recruitment strategies and testing them across multiple host trials in SWATs .In the START programme, we first developed two recruitment strategies: (1) written information optimised through application of information design principles and user-testing (‘participant information sheets optimised through user-testing’) and (2) multimedia information presented via the Internet. We then recruited multiple trials to include a SWAT evaluation of these two recruitment strategies, testing their effectiveness across multiple trials simultaneously . By testMany individual SWATs within the START programme have now been published –10. In tThe broad methods underlying the START programme have been published .As part of the START programme, chief investigators on trials recently funded by the National Institute of Health Research (NIHR) Health Technology Assessment Programme or on the Primary Care Research Network portfolio were invited to participate in START. Interested trials were selected on the basis of sample size (at least 800 participants to be approached) and design (using a recruitment method amenable to the START recruitment strategies). Although a variety of recruitment methods could be adopted for studies included in the programme , all studies that participated used postal recruitment methods. The minimum sample size of 800 participants to be approached in each trial was based on an indicative sample size calculation, although the expectation was always that the primary analysis would involve pooling of results across trials in a meta-analysis . Host trFor user-testing, we recruited healthy members of the public, who had a similar socio-demographic profile (age and education) to the participants eligible for host trials. We excluded people who had taken part in any medicines trial or readability testing in the previous 6 months.An independent groups design was used, with each participant seeing only one version of the information. We conducted three rounds of user-testing, with 10 participants in each round. The first round tested the original trial materials (PIS and cover letter), after which the optimised versions were developed, using information design and plain English. Although the information sheet and letter for each trial varied the revisions always included: plain English; short sentences and paragraphs; use of colour for contrast and impact, and bold text for highlighting; a reduced number of sub-sections; a contents list; and clear trial contact details. This approach has been shown to produce increased levels of understanding and approval –14.The second and third rounds tested the revised versions, with minor changes made to wording and layout in response to the findings of each round of testing. In user-testing, each participant was shown a version of the information sheet and cover letter and asked to respond to 20 factual questions: three related to the cover letter and 17 to the information sheet. The questions were drawn from four categories of information that would apply to any trial: the nature and purpose of the trial (three questions); the process and meaning of consent (four questions); trial procedures (10 questions); and safety, efficacy and nature of the tested intervention (three questions). For each question, participants were asked to locate the answer (testing navigation and organisation of the information), then give the answer in their own words (testing clarity of wording) .In each SWAT, participants being approached to take part were randomised to receive the optimised information or routine information materials. Individual randomisation was preferred for the SWATs, as the methods used were highly amenable to randomisation at that level, which would generally increase power and precision, and be less vulnerable to selection bias. We adopted site randomisation only where that was preferred for logistical reasons .The primary outcome was recruitment, defined as the proportion of participants recruited and randomised to a host trial following an invitation to take part. The denominator for the outcome was the total number of potentially eligible participants offered entry to the host trial. Depending on the particular trial, this would include a mix of eligible and ineligible patients according to the formal inclusion and exclusion criteria. All trials were able to provide reliable data on the numbers offered participation.Secondary outcomes were:Acceptance, defined as the proportion of potentially eligible participants who express interest in participating, either by posting a reply or attending a recruitment appointment. We anticipated that in some SWATs, the number of participants recruited to the host trial could be different from numbers of participants responding positively to the invitation, due to eligibility criteria used in the host trial.Decline, defined as the proportion of participants who actively decline to participate in the host trial.The START programme was approved by the National Research Ethics Service (NRES) Committee, Yorkshire and the Humber – South Yorkshire (Ref: 11/YH/0271) on 5 August 2011. Each individual host trial had its own ethical agreement and registration.For each individual SWAT, analyses of recruitment were conducted in line with the statistical analysis plan developed by SE and VM. Outcomes were first described separately by study arm and then compared using logistic regression to estimate the between-group odds ratio and corresponding 95% confidence interval.metan command (Stata version 14.2). Random effects meta-analysis models were used based on the assumption that clinical and methodological heterogeneity was likely to impact on the results. Statistical inconsistency was quantified using the I2 statistic.For the pooled analysis, data from each SWAT were entered into Stata and meta-analysed using the Stata In the meta-analysis, we used a two-staged analysis strategy where each individual SWAT was analysed using the appropriate analysis methods  to generate trial-level summary statistics (e.g. odds ratio) first, and then the results from each individual SWATs were combined across trials using the Stata metan command (Stata version 14.2).Regardless of the observed statistical heterogeneity, we performed pre-specified subgroup analyses investigating differences between studies based on underlying recruitment rates (low defined as a recruitment rate of 5% or below in control group vs. higher rates). We hypothesised that when the baseline recruitment rate is low, the increase in the absolute recruitment rate associated with a recruitment intervention is likely to be higher. A second planned analysis comparing patients with a known diagnosis versus participants ‘at risk’ was not conducted as it proved difficult to assign trials to the categories reliably. In a post hoc sensitivity analysis, we assessed the impact of including one of the SWATs (ISDR) which faced particular design challenges  on the oWe originally recruited 8 host trials to the START programme. Although it proved difficult to record exactly how many host trial teams were approached, as teams became aware of the programme through a variety of means including presentations and word of mouth, we estimated that at least 225 were contacted. One of the eight host trials was unable to deliver data, as the trial finished before the necessary permissions were in place to do the SWAT. We therefore conducted SWATs in 7 host trials Table . One SWAn=28,476). Five SWATs randomised participants at the individual level, whereas 2 randomised by cluster  because this was operationally easier. All host trials were individually randomised. There were a mix of host trials in adults with physical or mental health conditions, testing a variety of screening and treatment interventions conducted in primary health care settings.Table n=27,805) provided data on recruitment. Optimised information likely results in little or no difference in recruitment rates   vs high rates .Optimised participant information sheets were not differentially effective in trials with different baseline recruitment rates: trials with low rates  provided data on participant acceptance rates. Participants receiving optimised participant information sheets were not more likely to respond positively to the invitation compared to participants receiving standard information .Seven SWATs  provided data on decline rates. Participants receiving optimised participant information sheets showed little or no difference in rates of declining than those receiving standard participant information sheets .Three SWATs  or actively declining the trial.The programme of work showed that co-ordinated testing of recruitment strategies using SWATs is feasible and can provide definitive evidence on the effectiveness of recruitment strategies.The host trials undertaking the SWATs were self-selected, and therefore, the studies on which the programme was run represent a relatively specific group of study contexts. It is likely that the variation in those contexts was sufficient to give the recruitment strategy a fair test across multiple designs and populations, and there was limited evidence of significant variation in effect. All the included trials used postal recruitment to invite participants.The START programme was co-ordinated and represents the most concentrated evidence synthesis in the area of recruitment to date, according to the latest Cochrane review . NeverthWe report here a linked series of pre-planned and co-ordinated SWATs testing the same recruitment intervention, rather than a retrospective systematic review of all relevant studies using this strategy. The studies reported here will eventually be integrated into the ongoing Cochrane review on strategies to improve trial recruitment , alongsiAfter we began our programme of research, guidance was published to help trial teams and funders decide if another evaluation of a SWAT intervention was required . We presOur data suggest that although optimising information though user-testing leads to improved comprehension, it is not likely to translate to improved trial recruitment. Our programme of work did show that co-ordinated testing of recruitment strategies using SWATs is feasible. SWATs done across multiple host trials provide a body of evidence to help trialists to make their trial process decisions more evidence-informed, which is far from routine at present because there are little data for trialists to use. There are several methods of supporting further SWAT studies. One is further bespoke funding similar to START, for example the TRECA study  which reAnother model has been to build SWATs into host trials as part of the planning for the host trial itself. For example, the UK’s NIHR Health Technology Assessment funding scheme offers trials additional money to embed SWATs as part of the funding bid. Ireland’s Health Research Board (HRB) has a bespoke SWAT funding scheme run through the HRB-Trial Methodology Research Network. Each model of delivery has advantages and disadvantages but the key issue is that SWATs, while generally cheap, are not free, and therefore need funding streams that support them.Although we have shown the feasibility of a co-ordinated programme of SWATs among multiple trials, the challenge is to expand and accelerate the process in order to build the evidence base more rapidly and fully.However, there are two additional priorities beyond increases in the volume and efficiency of SWATs. First, it is critical that any SWAT programme can clearly identify strategies that can be implemented (or de-implememented) and is able to ensure that findings regarding the effect of these strategies are rapidly disseminated to trials units and research teams to change practice in line with the developing evidence base.Secondly, and more importantly, it will be critical to show that these efforts lead to better trials—which might include more efficient delivery , more participant-centred trials  and recruitment of more diverse and representative populations.Additional file 1: Table S1. Details of the unpublished Help Diabetes SWATAdditional file 2: Table S2. Criteria for deciding whether user tested participant information needs another SWAT evaluation. Figure S1. Cumulative meta-analysis


Teleost fishes vary in their reliance on median and paired fins (MPF) or undulation of the body (BCF) to generate thrust during straight-line, steady swimming. Previous work indicates that swimming mode is associated with different body shapes, though this has never been empirically demonstrated across the diversity of fishes. As the body does not play as active a mechanical role in steady swimming by MPF swimmers, this may relax constraints and spur higher rates of body shape diversification. We test these predictions by measuring the impact of the dominant steady swimming mode on the evolution of body shape across 2295 marine teleost fishes. Aligning with historical expectations, BCF swimmers exhibit a more elongate, slender body shape, while MPF propulsion is associated with deeper and wider body shapes. However, in contrast to expectations, we find that BCF propulsion is associated with higher morphological diversity and greater variance around trait optima. This surprising result is consistent with the interpretation that stronger functional trade-offs stimulate phenotypic evolution, rather than constrain it. Multifunctionality is among the most general features of multicellular organisms. Many body parts perform multiple functions in an organism, setting up conflicts that may result in structural compromises that do not maximize any single function. The ubiquity of multifunctionality in whole organisms suggests that design trade-offs between functions may be one of the dominant factors constraining phenotypic evolution . HoweverIn the present study we explore this issue by leveraging the relationship between swimming mode and body shape in fishes. Across fishes, there is a major distinction between species that locomote by undulating the body and caudal fin (BCF) and those that propel themselves with movements of the median and paired fins (MPFs) . While mThe difference in the mechanical role that the body plays during BCF and MPF swimming suggests that species with different modes may also exhibit different body shapes. This distinction is seen clearest at the extremes of performance where it has long been recognized that species with very high BCF cruising performance usually have a narrow caudal peduncle, lunate caudal fin, and a streamlined body shape . This boWhile performance extremes may be associated with distinct phenotypes, fish body shapes are known to be extremely diverse . As the We analyze the impact of swimming mode on body shape evolution in teleost fishes using a data set of body shape measurements from 2295 species. To explore the dynamics of morphological evolution, we estimate the optimal body shape and the variance of traits around the optima (the combined effects of selection and rate of evolution) for BCF and MPF swimmers using evolutionary model fitting. Our expectation is that BCF swimmers have a more slender body, intermediate body depth, and narrow caudal peduncle. We expect MPF swimmers will be deeper bodied, short, and laterally compressed, as previous researchers and studies have suggested . Given tThis study made use of an existing large data set on body shapes of teleost fishes . BrieflyPrior to analysis, linear traits were ln-transformed and size corrected by taking the residuals of a phylogenetic regression on body size using the R package phytools . We usedin situ  using the correlation matrix to visualize morphospace occupancy both across the dataset and with respect to swimming mode. Although phylogenetic PCA is a common technique used in macroevolutionary studies, here we are interested in the primary dimensions of morphological variation across all fishes, regardless of phylogenetic structuring. To determine the overall diversity of forms in each locomotion mode, we calculated multivariate morphological disparity (variance) across the eight morphological traits. We used both a phylogenetic MANOVA and a series of ANOVAs for the eight traits to determine if there were significant differences in average body shape between MPF and BCF swimmers. The ANOVAs and variance calculations were implemented with 1000 simulations using the geomorph package .σ2) to vary with locomotion mode, while the multi-rate Brownian motion model (BMS) allows for different rates in MPF and BCF swimmers. OU models incorporate an additional parameter, θ, which is generally interpreted as an adaptive optimum. We implemented three OU models: a single peak OU model (OU1), a multi-peak OU model (OUM), and a multi-peak multi-rate OU model (OUMV). Though there is a third parameter that OU models can accommodate, α, which is commonly interpreted as a selective pull toward the morphological optimum, we chose not to incorporate models that allow this parameter to vary with regime. It has been shown that the similar effects of alpha and sigma parameters in OU models can lead to model non-identifiability, as well as inaccurate estimates of alpha  of the joint OU–BM process , allowing for asymmetric transition rates between swimming modes, as this was the preferred model based on AIC comparisons . For eac process .OUwie.sim. We then recursively ran our model-fitting framework with 100 simmaps to establish if we could recover the original model and parameters, thereby demonstrating statistical power.We checked the results of the OUwie analyses for positive eigenvalues, which indicate reliable estimates . Model fThe first two PC axes accounted for 65.3% of morphological variation across the dataset . PrincipP < 0.05), although body shape is highly variable within each mode . The individual phylogenetic ANOVAs revealed that this result is primarily driven by significant differences in maximum body depth, head depth, lower jaw length, and maximum body width  except head depth, which had a best fit model of OUM (98/100 simmaps). In other words, all traits have different optima for MPF and BCF swimmers and all traits except head depth have different amounts of variance around the optima for the two swimming modes . BCF swiThe simulations under our best fit model (OUMV) indicated that we have substantial power to distinguish between all five evolutionary models, recovering the original model in all 100 simmaps . The AICThe expectation that swimming mode has a substantial effect on body shape has been central to interpretations of fish diversity for at least the past century . Our surIn spite of considerable variation among species, the differences in body shape between MPF and BCF swimmers generally match expectations. The short, deep, laterally compressed body shape that we find is common in MPF species is thought to confer efficiency at slow speeds and increased maneuverability . A shortThere are notable exceptions to the relationship between locomotion mode and average body shape described above. We find two clades of MPF swimmers with highly elongate bodies: ribbonfishes and pipefishes . Furthermore, some of the most deep-bodied species in our dataset are BCF swimmers, including batfishes (Ephippidae), moonfish (Menidae), and deep sea hatchetfishes (Sternoptychidae). There are numerous aspects of swimming mechanics that we have not accounted for in our categorization system that likely contribute to variation within each mode. Median-paired fin swimming is a broad category that encompasses fishes that swim with any combination of their pectoral, dorsal, and anal fins. This variation in anterior–posterior placement of thrust generation may influence locomotor mechanics  and the We predicted that the decoupling of the lateral body surface from a mechanical role in generating thrust during steady locomotion may allow MPF swimmers to respond to the selective pressures on other functions of body shape , resultiOur finding of higher stationary variance in BCF swimmers compliments a suite of studies that have demonstrated a pattern of rapid evolution in multi-functional traits . For exaAlthough our categorization system glosses over much subtlety in swimming mechanics, we find strong evidence that locomotion mode has consequences for the evolution of fish diversity, supporting long-held predictions about the interplay between fish shape and swimming performance. Contrary to expectations, we also find that BCF locomotion is associated with higher stationary variance around trait optima and greater morphological diversity. The strong tradeoffs between adaptations for steady swimming and other ecological functions have resulted in greater body shape diversification in BCF species. Complex traits with strong performance tradeoffs, such as body shape, may either elevate rates of morphological evolution or reduce constraints, resulting in a decoupling between the evolution of form and function. Mounting evidence indicates that these patterns associated with multi-functional traits may be more of a general rule linking biomechanics and morphological diversification. We recommend that this framework be extended to other taxa and systems to better understand the role of multi-functional traits in biasing the tempo and mode of evolution.obab016_Supplementary_DataClick here for additional data file.


Over 1100 veterans work in the Veterans Health Administration (VHA) as peer specialists (PSs)—those with formal training who support other veterans with similar diagnoses. A White House Executive Action mandated the pilot reassignment of VHA PSs from their usual placement in mental health to 25 primary care Patient Aligned Care Teams (PACTs) in order to broaden the provision of wellness services that can address many chronic illnesses. An evaluation of this initiative was undertaken to assess the impact of outside assistance on the deployment of PSs in PACTs, as implementation support is often needed to prevent challenges commonly experienced when first deploying PSs in new settings.n = 7, 10, 8) over 6-month blocks were matched and randomized to each study condition. In facilitated implementation, an outside expert worked with site stakeholders through a site visit and regular calls, and provided performance data to guide the planning and address challenges. Minimal implementation sites received a webinar and access to the VHA Office of Mental Health Services work group. The two conditions were compared on PS workload data and veteran measures of activation, satisfaction, and functioning. Qualitative interviews collected information on perceived usefulness of the PS services.This study was a cluster-randomized hybrid II effectiveness-implementation trial to test the impact of minimal implementation support vs. facilitated implementation on the deployment of VHA PSs in PACT over 2 years. Twenty-five Veterans Affairs Medical Centers (VAMCs) were recruited to reassign mental health PSs to provide wellness-oriented care in PACT. Sites in three successive cohorts .This project is registered at  Contributions to the literatureThe model of facilitation used in the study can be a model of deploying peer specialists into primary care in other sites.The model of facilitation used in the study can also be a model of deploying new workers  into a variety of health care settings.Given there were no funds to hire new peer specialists, the study shows some level of ongoing facilitation may be needed to support peer specialist implementation.Over 1100 veterans work in the Veterans Health Administration (VHA) as peer specialists (PSs). PSs are veterans with lived mental health experience and formal training who provide support to other veterans with similar diagnoses. Drawing on their own recovery experience, PSs provide a range of services including facilitating groups, role modeling, providing outreach and support, teaching coping skills, and helping veterans navigate and coordinate care. PSs have traditionally supported recovery from mental illness and substance use disorders in mental health settings. While not conclusive, randomized and quasi-experimental trials have shown that individuals with mental illnesses who receive PS services benefit across a range of outcomes, such as improved functioning and patient activation )—Given the recent passage of the Mission Act , which iThe present study had some limitations that should be noted. PSs typically had limited time in primary care, some as few as 4 h a week. Further, the PSs were “on loan” from each site’s mental health service, which likely impacted their ability to deliver services during the pilot, and perhaps influenced their sustainability. In addition, there was a lack of standardization of what interventions PSs provided ; more information is needed on what PS interventions are effective in primary care and in what dose. In addition, the target veteran group differed by site, and sites also differed in terms of how and where they received referrals. Baseline veteran-level data was collected by the peer specialists themselves, which may have biased responses. Some peer specialists were reluctant or uncomfortable asking veterans to complete the survey, and this reduced the available veteran data. The present study also did not examine the impact of PS services on service use outcomes outside of primary care; future studies could examine whether PS support helps to improve use of health care services  and  and 63])It is notable that external facilitation had success given that implementing PS services in a new setting like primary care requires in-depth education of providers and leadership regarding an entirely new discipline. With only a modest investment of external facilitation resources, sites that received implementation support were able to deploy PSs more quickly and deliver more services than sites without such support. This approach could be a model to other PS initiatives such as the MISSION Act.


Life history, brain size and energy expenditure scale with body mass in mammals but there is little conclusive evidence for a correlated evolution between life history and energy expenditure  independent of body mass. We addressed this question by examining the relationship between primate free-living daily energy expenditure (DEE) measured by doubly labeled water method (n = 18 species), life history variables , resting metabolic rate (RMR) and brain size. We also analyzed whether the hypometabolic primates of Madagascar (lemurs) make distinct energy allocation tradeoffs compared to other primates (monkeys and apes) with different life history traits and ecological constraints. None of the life-history traits correlated with DEE after controlling for body mass and phylogeny. In contrast, a regression model showed that DEE increased with increasing RMR and decreasing reproductive output  independent of body mass. Despite their low RMR and smaller brains, lemurs had an average DEE remarkably similar to that of haplorhines. The data suggest that lemurs have evolved energy strategies that maximize energy investment to survive in the unusually harsh and unpredictable environments of Madagascar at the expense of reproduction. From a theoretical point of view, the energy acquired from the environment allows to sustain survival, growth and reproduction. And, by analogy with demographic principles, at steady state, energy allocated to growth and reproduction (beside respiration) should match energy lost to mortality over a lifespan in each generation3. Therefore, a link is expected between life history traits, which evolve under selection pressures to promote fitness, and the amount and distribution of energy required to maintain these biological fundamentals. Empirically, the findings that primates spend fifty percent less energy than other eutherians with similar mass2 led to revisit the potential for correlated evolution of life history traits and energy needs in these mammals. Primates indeed have a slow life history among placental mammals5 and they must allocate energy to a comparatively larger brain in addition to growth, maintenance and reproduction. Accordingly, their slow pace of life likely evolved in relation to energy allocation trade-offs between survival and reproduction costs, as in other mammals, but also on their characteristically reduced energy budget.There are theoretical and empirical reasons to suggest that life history traits could have evolved in close relation to the metabolic requirements and energy input necessary to fuel the components of fitness6 but since most life history traits and brain size are connected to body mass, e.g.,7, looking for a correlated evolution between life history and energy expenditure necessitates to remove the effect of body mass. Early studies that tackled this issue while controlling for body size and phylogeny used resting metabolic rate  as a proxy for a species’ total energy demand9. The reason for using RMR preferably over DEE  was that there were many measurements of RMR available for meta-analyses. The wide range of species tested was an advantage, but these early studies actually failed to find a general relationship between life history and RMR9 (perhaps because the RMR does not capture the full spectrum of energy expenditure). Similarly, a study focusing on strepsirhine primates could not demonstrate a correlation between maternal reproductive investment and RMR independently from body mass and after controlling for the effect of phylogeny10. A recent meta-analysis on eutherian mammals, including primates2, found a positive relationship between daily energy expenditure and reproductive output  controlling for body mass but the link was weak due to the great variability of reproductive data and was not found for two other life history traits (growth and maximum lifespan). The lack of a consistent relationship at high taxonomic level suggested that the results may be blurred by the trade-offs in energy allocation that species or taxonomic groups make according to their reproductive cycle and lifestyle2. It is also possible that studies that focused on DEE in primates were inconclusive due to the limited set of life history traits they tested.In primates and other placental mammals, growth, maternal investment, and life expectancy are positively related to total daily energy expenditure (DEE)11. The aim was to highlight the energy allocation trade-offs in species that have evolved in Madagascar in habitats characterized by unusually harsh biotic and abiotic constraints17. Stochastic climate events in Madagascar include drought years, occasional food shortage during the expected period of food abundance during the wet months, and cyclones16. This climate pattern seems to have been present for the last 5 million years, which has undoubtedly had an impact on the evolution of lemur energetics15. In this study, we show that primate species with a high RMR but low reproductive output have high DEE for their body mass. Contrary to our prediction that energy limitation in Madagascar selected for low energy expenditure strategies, we provide evidence that lemurs do not differ from other primates on average, but invest comparatively less energy for reproduction than for somatic maintenance.In the present paper, we examined these relations further using the set of published results on primate DEE, augmented with new data in a lemur species. We combined these measurements with an array of life history traits in simple and multiple regression models. The DEE results used here were all obtained with the doubly labeled water method, the gold standard for measuring total energy expenditure in free-living conditions. Additionally, we focused on a group of primates, the lemurs of Madagascar , that differ from other primates  in many aspects of their socioecology, physiology and life history. Lemurs, for example, have short gestation and lactation times, small brain volume, low baseline energy requirements, and this group includes a few heterothermic species19.The regression of DEE on body mass was established to extract DEE residuals in each species for later statistical analyses (see following sections) and to provide an update of DEE variation among primates including our new results on the Verreaux’s sifaka. DEE data used here were collected from non-gestating, non-lactating adults, therefore mainly reflecting basal energy requirements, physical activity, and physiological regulation for maintaining body homeostasis Table . With th2 = 0.97) similar to that resulting from an ordinary least square regression , and no significant phylogenetic effect was detected in the distribution of DEE (Pagel’s λ = 0). In our study and previous reports, it remains possible that the exponent is artificially inflated by the taxonomic distribution of body mass data since few haplorhines were tested in the range of low body mass while strepsirhines, with their low basal rate of oxygen consumption, are mainly small-sized species. If we exclude strepsirhines from the analysis, the exponent actually does not change markedly . We also noted that humans had a slightly higher relative DEE compared to great apes . While humans have evolved unusual life history trade-offs and energy budget relative to apes . Likewise, Microcebus murinus had a high energy expenditure relative to its body mass but, while other studies suggested a separate treatment of this heterotherm species, this small primate did not appear to be an outlier in our sample (Bonferroni outlier test).This PGLS analysis yielded a regression coefficient . The lack of difference is surprising in light of the unique energy-saving mechanisms characterizing some species of lemurs tested here  and the low basal metabolic rate  shared by most of them23  was higher in strepsirhines (median: 3.6) than in haplorhines . Given the much higher PAL of lemurs, it seems probable that the PAL difference with haplorhines is real. Accordingly, lemurs would spend a much larger portion of their total energy expenditure, on average, for sustaining the energy costs of living beyond resting requirements.Strepsirhines and haplorhines did not differ in their total energy expenditure after controlling for body mass  and taxonomic group. RMR had a positive effect on DEE overall while strepsirhine reproductive output had a negative effect Table .Table 4R10 generally conclude that evolutionary changes in life history traits are relatively independent of evolutionary changes in BMR or DEE. However, the multiple regression model built here reveals a correlated evolution between reproductive output and DEE provided that RMR and taxonomic differences among primate groups are taken into account. Our results support the hypothesis of different trade-offs among energy costs allocated to maintenance and reproduction between strepsirhines and haplorhines. Compared with haplorhines, the lemurs tested invest less energy in basal functions  and reproduction – as proxied by a shorter relative duration of maternal investment, reduced prenatal growth and lower reproductive output.Previous primate studies using simple regression models17. Even more intriguing, lemurs that exhibited a high-energy throughput strategy for their body mass were those with low quality diets  or with unique physiological adaptations for saving energy during periods of stress, e.g., daily torpor in Microcebus. How then can we understand the lack of frugality in energy expenditure for species living in a highly challenging environment?We must stay cautious in the generalization of our results due to the relatively low number of species tested. Yet, the similarity in the average DEE of lemurs compared to other primates and high PAL in lemurs is unexpected. Because lemurs live in harsh habitats compared to their mainland counterparts, we predicted that ecological constraints of the island of Madagascar have selected for low energy expenditure strategies. Lemurs indeed cope with unpredictable inter-annual and intra-seasonal variations in rainfall, superimposed on a predictable but marked seasonality (including a long dry season with low food supply). Moreover, lemurs live in energy-poor habitats where the concentration of protein and lipid in fruits is low compared to, for example, fruits available to neotropical primates31. During periods of food abundance, daily travelled distances increase in many species together with other energy-costly behaviors such as female targeted aggression and competition or territorial defense33. Even though their reproductive and hormonal cycles are synchronized by seasonal daylength variation, lemurs are not totally dependent upon exogenous abiotic cues. They show flexibility in their metabolic response to dietary and climatic changes and can adjust their physiology to cope with thermal variations and unpredictable crop failure, or capitalize on high levels of food abundance, whether in fat gain or in postponing the transition to a winter thrifty phenotype34. Our prediction of a seasonal variation of DEE, however, runs counter to the claim that mammal DEE only varies within a narrow physiological range36. This claim is based on the observation that no notable DEE differences were found between captive and wild animals from the same species, nor between seasons (as observed in a few species) nor, in human, between hunter-gatherers and sedentary human populations. To date, the only primate for which repeated isotopic measures have been published is the grey mouse lemur (Microcebus murinus) in Southern Madagascar. During the dry season, when ambient temperatures drop markedly, two types of individuals co-occur within the same population: individuals that stay normothermic and individuals that decrease their internal temperature and enter daily torpors. Astonishingly, little DEE differences were found between these phenotypes, nor did normothermic animals differ substantially in their DEE between the rainy season and the dry season (after controlling for body mass variation37). The authors suggest that the lack of seasonal variation could indicate that a decrease in the resting metabolic rate may compensate for the elevated costs of thermoregulation during the cold winter. Likewise, sportive lemurs (Lepilemur) living in the dry South of Madagascar seem to be able to maintain their total energy expenditure around some sort of "set point" but to vary seasonally the proportion of energy allocated to their behavioral and physiological activities38. The question of DEE variation in primates remains open however since substantial DEE differences independent of body size have been reported between Microcebus populations living in a rain forest vs a dry forest39.A first hypothesis could be that, with both a low BMR and a high ability to spend considerable energy above basal requirements, these lemurs have the potential to shift from a low- to a high-energy expenditure strategy according to the erratic environmental conditions prevailing at Madagascar. Potential variation of their energy balance is reflected in some behavioral proxies: during the austral dry winter period, the activity is reduced, the nycthemeral activity cycle is modified in cathemeral species, energy intake is low due to a diet including low-quality and scarce resources and individuals are lean, e.g.,40, with lemurs spanning a range of relatively low body mass across primates. Second, in our dataset, several lemur species with high-energy expenditure (Propithecus spp. and Lepilemur ruficaudatus) undergo costly physical activities related to locomotion as compared with sympatric lemurs (Eulemur and Lemur catta) showing considerably lower energy throughput . Interestingly, DEE measurements in these 4 lemur species were collected during the same phase of their reproductive cycle (the premating period) and, in Lemur catta, Eulemur and Propithecus verreauxi, at the same study site. Since these species share an extremely low rate of basal metabolism and were measured during non-reproductive phases, their distinct energy expenditure may primarily reflect differences in lifestyle and locomotor activity. Hoppers and leapers are hypermuscular in proportion to their total body mass41 and leaping is an energy-costly mode of locomotion relative to quadrupedal walking, at least at low speed or when using flexible substrates at landing44. Terrestrial quadruped primates like chimpanzees were estimated to devote only 10% of their DEE to locomotion in free-ranging conditions45. Since not all leapers are hypermuscular , the locomotion hypothesis needs to consider more energetic, kinematic and morphological studies. Finally, some species deviate from the DEE:body mass regression line in an unexpected way given their folivorous diet and low foraging activity. While folivory has long been associated with low energy input/low foraging effort strategies47, folivorous primates in our sample  exhibited a high-energy throughput for their mass or spent as much or more energy than species feeding on ripe fruits and other high-quality foods. In comparison to fruit specialists, primates with digestive capacities adapted to feeding on vegetative, fibrous plant parts like mature leaves, harvest these relatively ubiquitous food resources within small home ranges, travel short distances daily, and tend to possess smaller relative brains51. A low-energy throughput strategy may also be typified by marmosets  have substantial body fat . This suggests that part of the energy they acquire could be saved as stored energy rather than being invested in costly daily activities.Beside body mass and basal rate of oxygen consumption, several other major factors contributing to total energy expenditure may account for the different amount of energy expended for everyday life between haplorhines and strepsirhines of Madagascar. These are thermoregulation, immune system, parasite load, digestion costs, locomotion, sociality, etc. In the absence of a consistent set of data to compare, we will only examine shortly some of them. First, the costs of thermogenesis may be high in lemurs because thermoregulation costs increase markedly as body size decreasesput Fig. . In the Propithecus verreauxi  and P. diadema, Lepilemur ruficaudatus) while others , with reduced energy expenditure, seem to favor strategies that allocate energy to the constitution of fat reserves before the breeding period. Fat stores cannot fully compensate for the energetic load of the mating and gestation season30, but they may contribute to the seemingly higher reproductive output of these species compared to lemurs with high DEE . These elements converge to suggest that, even with energy-saving mechanisms, the pool of energy needed to fuel basic functions such as foraging, socializing, and surviving in the forest environments of Madagascar is unusually high, which in turn may limit the amount of energy invested in reproduction. We note in this respect that infant mortality is exceptionally high in lemurs and that bet-hedging reproductive strategies occur in several species52. The proposed effect of ecological and climatic constraints in shaping the energy allocation of lemurs may also explain why these taxa are not able to derive sufficient energy for growing costly tissues, especially the brain which requires constant and substantial energy supply throughout the year . Finally, the results lead to a reconsideration of the nutritional constraints exerted on folivores, because (at least) some need a high supply of energy to fuel the costs of living. In particular, susceptibility to food stress and the risk of energy imbalance appears to be more critical than previously thought in at least some of these species . A better understanding of the causes of variation in energy balance should help trace how environmental characteristics may have shaped the evolution of life history among primates, including the large-brained humans with their distinctive suite of life history traits.In conclusion, the data suggest that while lemur DEE does not differ from that of other primates on average, a taxonomic distinction emerges on the lemurs’ distinct tradeoffs between reproduction and survival. And, among lemurs, some have evolved energy strategies and lifestyles that maximize energy investment in daily activities (35. Species DEE (Table 19) supplemented with54 and new data collection . Results obtained for growing individuals were discarded as the rate of energy use is known to be high compared with adults. DEE data are currently available for 18 primate species , including 6 strepsirhines and 12 haplorhines  were studied during the rainy season before the onset of the mating season in a gallery forest in Berenty reserve, southern Madagascar. In the present sample, strepsirhines were represented by Lemuriforms  that spanned almost the full range of body mass among extant strepsirhines . Lemurs show distinct feeding ecology , reproductive and social system  and lifestyle . The haplorhine subsample captured a large socio-ecological diversity as well. Resting metabolic rate (RMR) used to calculate species physical activity level  included both resting and basal oxygen consumption data. Basal metabolic rate, which is measured on fasting individuals at thermoneutrality during the inactive period of their inactive circadian phase, was insufficiently documented in primates to be used exclusively in our analysis. However, the resting or basal values used to calculate species’ PAL were roughly balanced between the strepsirhines and haplorhines tested, minimizing a possible bias when comparing the mean PAL between these two groups , prenatal growth (litter mass/gestation duration) and reproductive duration (gestation + lactation duration), which variously reflected maternal energy investment. In one species where DEE was available but life history was not documented (Lepilemur ruficaudatus), we used life history data from a closely related species (L. mustelinus) with similar body mass  coupled to a Delta V IRMS (Thermo Finnigan) as described in60.A description of the doubly labeled water method, adapted from2). Studies available in non-human primates report limited or no sex differences in DEE, even in dimorphic species, after controlling for body mass, female reproductive status or fat mass proportions61 (this study: Propithecus verreauxi). Some sex differences appear in grey mouse lemurs according to daily torpor use, but normothermic males and females have similar DEE37. It should also be noted that the DEE for a given species is most often measured on paired individuals from same social groups or individuals living under the same environmental conditions, therefore reducing potential sources of variability.In analyses relating metabolism to life history traits, studies commonly focus on the female parent to take into account costs (or proxies) of reproduction, beyond growth and maintenance. However, since studies generally do not provide individual DEE results allowing to extract female energy data , it is assumed that the sexes show a similar deviation from the DEE: mass regression line . Data were Ln-transformed on both axes, except for gestation duration where body mass only was Ln-transformed following Box Cox procedure.To establish the allometric relationships between DEE, BMR, brain size and life history on the one hand and body mass on the other hand, we used phylogenetic generalized least square (PGLS) regressions which control for phylogenetic relatedness. Indeed, non-independence of the data affects the true relationship between variables tested and may obscure their potential co-evolution. This method estimates the phylogenetic signal (λ) carried by the structure of the data set, approximated by a value which fully reflects the phylogenetic relationships (λ = 1) or on the contrary the independence of the data (λ = 0), or which is intermediate. PGLS linear regressions were performed with BayesTraits  on the one hand and the residuals from the life history traits: mass regression lines established using the extensive primate database (n = 87 spp.) on the other hand. Residuals were also used for comparing strepsirhines and haplorhines sampled in our DEE study with a two-tailed Student t-test . In all tests, the threshold for significance was set at  α ≤ 0.05. A principal component analysis was performed from residuals of the regressions on body mass of each of the life history characteristics selected. Simple and multiple linear regression models were applied with residual DEE as the response variable and residual life history traits, brain size or RMR as predictors after Ln-transforming the data to reduce heteroscedasticity. We included an interaction term taxonomic group x life history trait (when the latter differed significantly between strepsirhines and haplorhines) in multiple regression tests. The linear model assumptions and other validations of the models  were assessed using the gvlma package and basic packages in R version 4.0.364 and RStudio version 1.3.105665. Principal component analysis of life history traits was performed using the FactoMineR, facto extra and corrplot packages in R64.Correlated evolution between life history traits and DEE was examined from the 18 species sampled removing the effect of body mass. Since many of these variables are inter-correlated and all are related to body massPropithecus verreauxi complied with the European legal requirements on animal welfare (Directive 2010/63/UE) and French legislation for the ethical treatment of primates  and with the ARRIVE guidelines regarding the principles of replacement, reduction and refinement. All manipulations and treatments followed the International guidelines on health monitoring of non-human primates by FELASA  and were approved by the scientific commitees of The Museum National d’Histoire Naturelle and Sorbonne Université (No SU-14-R-CDV-09-1). The Ministère de l’Environnement, de l’Ecologie et des Forêts of Madagascar delivered the permit to capture animals, collect and export biological samples (N°261/14/MEEF/SG/DGF/DCB.SAP/SCB). The export/import of biological samples was approved by CITES (No 732C-EA12/MG14) and by the Ministère de l’Agriculture, France .Experiments with Supplementary Information.