s2fieldsofstudy: Biology Medicine

text:
Effect of allogeneic mesenchymal stem cells (MSCs) on corneal wound healing in dogs

a Centro Veterin ario da Vis~ ao, SAIS s/n Lote 14, Setor Sul, Brasília, DF, Brazil b Bio Cell, SMPW Qd. 05 conj. 05 lote 01 Casa C, 71735-505, Brasilia, DF, Brazil c Faculty of Agronomy and Veterinary Medicine, Campus Darcy Ribeiro, Asa Norte, 70636-200, University of Brasilia, Brasilia, DF, Brazil d The Polsky Center for Entrepreneurship and Innovation, University of Chicago, 1452 E 53rd St, Chicago, IL, USA e Cancer Biology and Epigenomics Program, Ann & Robert H Lurie Children's Hospital of Chicago Research Center, Northwestern University's Feinberg School of Medicine, 2430 N. Halsted St., Box 220, Chicago, IL, USA f MATTER Chicago, 222 W. Merchandise Mart Plaza, Suite 12th Floor, Chicago, IL, 60.654, USA g The Founder Institute, San Francisco, CA, USA h International Center for Biotechnology Cooperative Resources International (CRI-ICB), Mount Horeb, WI, USA

Introduction
The cornea of small animals is composed of five layers: precorneous lacrimal film, epithelium, stroma, descemet's membrane and endothelium 1 being able to transmit light due to the avascularization of the epithelium, absence of pigments, regular arrangement of collagen in the stroma and maintenance of hydration. 2 The peculiar organization of the collagen, together with the absence of blood vessels, maintains cornea transparency to allow the passage of light, 3 and the integrity of the cornea plays a critical role in maintaining visual function due to its transparency. 4 Corneal ulceration is one of the most common conditions in veterinary ophthalmologic clinics, and may be caused by trauma, infection, inflammation, among other factors. This occurs when the epithelium and part of the stroma layer is lost. 5 This mainly causes loss of transparency of the initial cornea, especially in cases where adequate therapy is not applied, with progression to ocular perforation, presenting possibly irreversible lesions and consequent loss of vision. 6 The lesions are classified according to its depth, and in superficial ulcer, lesions cause corneal epithelial loss, 2,7 ocular discomfort with intense photophobia, blepharospasms, increased tear production, conjunctival hyperemia and corneal edema. 5 In the case of deep ulcers, lesions involve more than half of the corneal thickness, affecting the stromal layer, with signs of inflammation, corneal edema, pain, purulent secretion, signs of anterior uveitis. 2 Currently, the form of treatment is related to the degree of the lesion.
The therapeutic conducts are based on the relief of the clinical signs and symptoms, prophylaxis or in the control of infection, erosion and withdrawal of the cause. When the ulcer is superficial, the therapeutic course may be clinical, but in deep ulcers and in perforation cases, the therapeutic course includes surgical procedures associated with drug therapy. 2,7,8 The treatment of choice is usually surgical intervention 9 ; however, recent studies have shown the beneficial effects of stem cell-based therapies on ocular surface healing, specially using mesenchymal stem cells (MSCs) or its secreted factors. 10 The effects observed using MSCs, when transplanted into an organism, occur mainly due to the release of bioactive molecules (adhesion, extracellular matrix proteins, cytokines, growth factor receptors) that allow an interaction with the environment in which they are transplanted. 11 Interestingly, when fused to the cells of the organism they are able to modulate the inflammatory response and mitosis of the cells and this promotes tissue repair and immunomodulation 12,13, . 14 Thus, this study has as main aim to evaluate the use of MSCs in dogs with corneal wounds. We believe that the results presented here are important in order to improve post-operative recovery, correct unsatisfactory results, accelerate and promote corneal recovery and intensify the uses of therapy with MSCs, which are the most promising source of cell proliferation and differentiation. 15

Isolation, culture and freezing of MSCs
MSCs were isolated and cultured from adipose tissue of one health dog donor, according to the previously described protocols 16,17, . 18 For the collection of adipose tissue, the donor was anesthetized, an incision was made in the lumbar region, and approximately 20 g of adipose tissue was collected from the base of the tail. The adipose tissue was washed in saline phosphate solution to remove cellular and blood residues, cut in small pieces and then, exposed to hyaluronidase, to go through an enzymatic digestion. After this, the cells underwent a filtration process in order to initiate a selection of the MSCs. Afterwards, the cells were placed in culture bottles with Dulbecco's modified Eagle's medium (DMEM), which were incubated at 37.5 C and 5% CO 2 . After 24 h, the medium was discarded, with the non-adherent cells, and fresh culture medium was added to the bottles. The medium was changed once every 3 days, until cells reached 80% confluence, when trypsinization was performed to take the cells off the bottles, count in neubauer chamber and pack them in straw (1 million cells/straw) for freezing with DMSO and BFS in liquid nitrogen, as previously described. 19,20 Five straws (5 million cells) were thawed for characterization of MSCs.

Characterization of MSCs
MSC characterization tests were performed as determined by the International Society for Cellular Therapy. 21 The surface markers positive for the MSCs tested were mouse anti-human CD29-RD1, mouse anti-equine CD44-FITC, caprine anti-canine CD90 primary and mouse IgM anti-caprine conjugated AF594 (secondary). The negative surface marker was mouse anti-human CD34-FITC. The capacity to differentiate of MSCs was also analyzed by the presence of two transcription factors for cell differentiation: SOX2 and OCT3/4. These markers were evaluated by the immunophenotyping method on Amnis ® Imaging Flow Cytometer.
The ability of the cells to differentiate into osteoblasts, adipocytes, and chondrocytes was also evaluated, as previously described. 18,22 The culture medium of the cells were screened for pathogens and contaminants (e.g., bacteria, fungi, mycoplasma), using polymerase chain reaction (Veriti Thermal Cycler e Ther-moFischer Scientific ® ). Also, viability of the cells after thawed was evaluated by flow cytometer with Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 & Propidium Iodide (PI) (Thermo-Fischer Scientific ® ) on Amnis ® Imaging Flow Cytometer.

Animals
Eligible subjects were dogs who were diagnosed with deep corneal ulcers by an ophthalmology clinician based on previously established criteria. 23 The twenty-six (26) selected dogs had been previously treated for 15 days with allopathic drops (antibiotic and tear repository) and presented no corneal wound healing. The animals did not receive any kind of immunomodulatory medication for at least 2 weeks before and for the entire duration of the study. All dogs owners signed a written consent before initiation of this experimental procedure and were fully informed that safety, complications, and efficacy of the cell implantation in corneal ulcer were not known.
Animals with diagnosed comorbidities or those with superficial corneal ulcer at baseline were excluded from the clinical study.

Preparation and application of allogeneic mesenchymal stem cells (MSCs)
The laboratory produced and conserved the MSCs in frozen straws. MSCs were thawed and washed for use, according to our protocol, using specific washing and transport medium. For each treatment, 3 million MSCs were used, and the application was performed as follows: an initial application of 0,25 ml of transport medium containing 250,000 cells by sub-conjunctival route performed by a veterinarian ophthalmologist followed by 11 instillations (one per hour) of 0,25 ml of transport medium containing 250,000 cells. Each animal returned for ophthalmologic evaluation at 7 and 14 days after treatment for a case follow-up. Considering corneal ulcer in domestic animals is a traumatic lesion that can occur at any time, no other follow-up was performed after 14 days.
From the time of diagnosis of the corneal ulcer to the period of 14 days after therapy with the MSCs, the animals received ocular instillation of antibiotic eye drops and tears repository to control possible infections and support the action of cells that were applied.

Adverse and side effects
During the clinical study, the analysis of adverse and side effects was carried out, including the evaluation of the occurrence of any intercurrence during and until 14 days after the therapy with the allogeneic MSCs.

Statistical analysis
The data were transformed into numerical variables, which were submitted to statistical analysis for differences between pairs  Fig. 1).

Results and discussion
MSCs from the donors were properly isolated and characterized by our laboratory. After 24 h of isolation, small colonies of adherent cells were scattered across the bottom of the bottles (Fig. 1A). After 2 days, the cells began to grow and elongate, becoming a spindle shape similar to fibroblasts. When the cells reached 80% of confluency, they had typical phenotypic characteristics of MSCs, and were attached to the bottom of the bottle with a spindle-like shape and self-renewal ability (Fig. 1B). The flow cytometry results after the cells were thawed, demonstrated that MSCs expressed high levels of CD29 ( Fig. 2A), CD44 (Fig. 2B) and CD90 (Fig. 2C) but did not express the hematopoietic marker CD34. There was also a high  expression of SOX-2 (Fig. 2D) and OCT3/4 (Fig. 2E). The analysis of alive, necrotic, apoptotic and dead cells using the flow cytometer, showed a percentage of 80.6% viable cells 2 h after thawed (data not shown). These results express that MSCs obtained from the donor have shown consistency in their isolation, expansion, high ratio proliferation, plastic adherent, and behavior in vitro, exhibiting its ability of adipogenic, osteogenic, and chondrogenic differentiation, similar to those previously described (Fig. 3). 22 MSCs are multipotent, self-renewing and can facilitate wound repair by modulating cellular responses in the cells of surrounding tissue and immune cells, creating a pro-regenerative and antiinflammatory environment. 24 These features can be useful in the therapy of depth corneal wounds, which are common types of ocular injury in small animals, and the current procedure to treat it is the surgery. The corneal epithelium is self-renewing, and this process is essential for normal vision, but in cases of depth wounds, other layers are affected besides the epithelium. 5 The subconjunctival route was chosen due to its capability of releasing higher levels of the medication to the eye for an extended period of time 25 and, was accompanied by topical instillation, so a higher quantity of cells could be delivered in a short period of time e 12 h and survive for 3e5 weeks, 26 enough time to promote the wound healing.
The cohort of 26 dogs selected for this study were all refractory to 15 days previous treatment (antibiotics and tear repository drops), in other words, none of them had natural healing of the ulcers and received the therapy with MSCs. After allogenic MSCs therapy, 22 out of 26 dogs presented complete ulcer wound healing within 14 days, totaling 84.6%. One animal did not go through the 7 and 14 days evaluations due to the severity of the case and it was submitted to surgery 4 days after the cellular therapy (Table 1). Other 3 animals had no improvement of corneal ulcer and were latter submitted to surgical procedure and it is unknown why they proved unresponsive. However, the animals were treated at home  by the tutors after the cell therapy, attending to the ophthalmological evaluations only in the defined days. Next studies should take it into consideration to control this aspect. The degree of depth (Fig. 4) significantly decreased during 14 days follow-up period (p < 0.0001) (Fig. 5).
This non-invasive method (cell therapy), appears to be a simple solution to substitute the surgery, with satisfying results. According to previous studies, MSCs promoted reduction of inflammation, mobilization of endogenous stem cells and increased concentration of growth factors when treating corneal damages 4,27,28 stimulating a rapid recovery, as occurred in a large part of the study animals.
Several studies have been previously performed treating corneal wounds with MSCs in rats and rabbits, reporting an improvement of the corneal surface, probably due to the secretion of cytokines, responsible for the increase in anti-inflammatory response and mobilization of endogenous MSCs to the injured area. 28e30 A study with mice induced to type 1 diabetes and consequent chronic corneal defects showed markedly decreased inflammatory infiltrates in the histology of these diabetics' corneas accompanied by lower ELISA levels analyses of MPO (a biomarker of activated neutrophils and inflammatory cytokines) when compared to corneas treated with placebo. 31 In accordance to our results, Bermudez et al. (2015) showed that even the medium of culture of MSCs can be used to enhance wound healing and reconstitute corneal surface of chemical burned mice eyes, demonstrating that the main effect must be due to the cytokines secreted by the cells. This study showed the improved epithelial regeneration and reduced corneal macrophage inflammatory protein-1 alpha (MIP-1a) and TNF-A mRNA expression. It was also suggested that this medium contain a bactericidal effect, due to the elevated levels of some chemokine (CX-C motif) ligands 1, 6, 8, 10 in it. These are promising results to the veterinary ophthalmology, considering the possibility of development of a non-invasive method to treat depth corneal ulcers.
Other studies have demonstrated the in vitro ability of MSCs to form stratified epithelial tissue with characteristics and expression of epithelial cell markers, 33 suggesting that the damaged epithelium in cases of corneal wound, is remade with the presence of MSCs and the growth factors released by them. Yao et al. (2012) tested subconjunctival injection of MSC in rats with corneal damage and concluded that the recovery of corneal surface (analyzed by staining with fluorescein) was significantly faster in animals that received MSCs when compared to animals that received phosphate-buffered saline (PBS). These results, in accordance to ours, suggest that MSCs have an anti-inflammatory effect explaining its efficacy.
The degree of discomfort of the animals with the ocular lesion before the therapy was mild (2 cases), moderate (13 cases) and intense (10 cases), present to absent (15 cases), mild (7 cases) and remained intense in 1 case 7 days after MSC therapy. For this variable, there was a significant difference between the evaluations performed before and after treatment and on day 7 (p < 0.0001) (Fig. 6). This is an important result, since it is a clinical response strictly related to the animal welfare, a big concern of tutors and veterinarians. In addition, the degree of discomfort is directly related to the possibility of new lesions occurring, since an uncomfortable animal is more likely to manipulate the injured eye in order to relieve discomfort.
The limitation of our study was the impossibility to use control animals. Due to the clinical precedence of the dogs, there was no possibility of leaving animals without treatment or receiving only placebo. Also, the animals selected to the study have been previously treated only with antibiotics and tears repository, to control the possibility of self-healing, which did not happen in any of them.   The final data from our clinical study showed that none of the animals had any adverse reaction or side effects due to the application of allogeneic MSCs, indicating that the cells were well tolerated by the individuals, as previously suggested by studies that have identified that this type of treatment is safe because of low MHCI expression and the absence of MHCII in canine adipose derived MSCs. 32 Several studies, including clinical trials, have shown similar results, presenting the safety of allogeneic stem cell therapy 34,35, . 36

Final considerations & perspectives
The present study demonstrated that allogeneic MSC therapy is effective in promoting the healing of corneal wounds in varying degrees of depth in dogs. The result of 84.6% of the cases reversed without the need of any surgical intervention, significantly reducing the degree of depth of the wounds is very encouraging. We could also conclude that the use of allogeneic MSCs was a safe, effective and fast-acting form of treatment of deep corneal wound in small domestic animals. We believe that, despite this study being carried out in dogs, it can be useful in the development of treatments to human eye healing conditions, like corneal burn or any corneal epithelium loss. Since the dogs studied were from a varied background, with different conditions and depth of corneal wounds, the effectiveness of treatment and our results are of significance. Finally, we believe that new studies could be performed in other animal models (cats, equines) to verify the similarities between species. From our knowledge, this is the first study using allogenic MSCs to treat corneal wounds in dogs.

Conflicts of interest
The authors declare no conflicts of interest in this work.


extfieldsofstudy: Medicine Biology

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A functional trade-off between trophic adaptation and parental care predicts sexual dimorphism in cichlid fish

Although sexual dimorphism is widespread in nature, its evolutionary causes often remain elusive. Here we report a case where a sex-specific conflicting functional demand related to parental care, but not to sexual selection, explains sexual dimorphism in a primarily trophic structure, the gill rakers of cichlid fishes. More specifically, we examined gill raker length in a representative set of cichlid fish species from Lake Tanganyika featuring three different parental care strategies: (i) uni-parental mouthbrooding, whereby only one parental sex incubates the eggs in the buccal cavity; (ii) bi-parental mouthbrooding, whereby both parents participate in mouthbrooding; and (iii) nest guarding without any mouthbrooding involved. As predicted from these different parental care strategies, we find sexual dimorphism in gill raker length to be present only in uni-parental mouthbrooders, but not in bi-parental mouthbrooders nor in nest guarders. Moreover, variation in the extent of sexual dimorphism among uni-parental mouthbrooders appears to be related to trophic ecology. Overall, we present a previously unrecognized scenario for the evolution of sexual dimorphism that is not related to sexual selection or initial niche divergence between sexes. Instead, sexual dimorphism in gill raker length in uni-parental mouthbrooding cichlid fish appears to be the consequence of a sex-specific functional trade-off between a trophic function present in both sexes and a reproductive function present only in the brooding sex.

Introduction
Sexual dimorphism-that is, the different appearance of males and females within a species-is a prevalent phenomenon in animals [1,2]. However, the evolutionary processes leading to sexual dimorphism remain poorly understood in many instances [1,3]. Traits that differ between the sexes of a species can, in principle, be categorized into primary, secondary and ecological sex traits [4,5]. Primary sex traits are required functionally for reproduction and relate to organs that are specific to one sex (gonads and copulatory organs). By contrast, secondary and ecological sex traits have no direct function in reproduction and often involve modifications of characters that are shared between sexes, yet are selected towards divergent optima, thus resulting in an intersexual conflict [3]. Dimorphism in secondary sex traits is typically driven by sexual selection [4,5], as is the case for ornaments involved in inter-sexual selection (mate choice) or weaponry used in intra-sexual combats (mate competition) [5]. Ecological sex traits, on the other hand, are characteristics that differ between males and females as a consequence of initial ecological niche divergence between the sexes, but not due to sexual selection.
From a theoretical point of view, several models have been developed to explain purely ecology-caused sexual dimorphism [6]. Yet empirical evidence for ecological sex traits remains scarce [7,8]. A major difficulty is to distinguish between cause and consequence, that is, whether sexual dimorphism is indeed primarily ecologically caused, or whether niche divergence between males and females is the consequence of an initially non-ecological sexual dimorphism [1]. In the latter case, sexual dimorphism in an ecological trait can be the consequence of selective forces that are not primarily related to sexual or ecological selection and that are therefore not covered by available theoretical models [1]. For example, a structure involved in food uptake and/or processing (i.e. a trophic trait) of a species could have an additional function in a reproductive behaviour without sexual selection acting on the focal trait, such as in nest-building or defending offspring [1]. A trait with such a dual function-each of which is likely to have a distinct trait optimum (a trophic and a reproductive one)-is expected to experience a trade-off (figure 1). The realized trait values should thus lie somewhere in-between the two optima (figure 1b). If the presence of a conflicting function in such a trait is restricted to only one of the two sexes, the resulting trade-off will be sex-specific too, potentially leading to sexual dimorphism (figure 1c). In such a case, the realized trait values are expected to be near the trophic optimum in one sex, while they should be shifted away from the trophic optimum towards the optimum of the conflicting (reproductive) function in the sex experiencing the trade-off. This shift in trophic morphology may subsequently result in divergent niche use between the sexes.
The gill rakers of cichlid fishes from East African Lake Tanganyika provide a rare opportunity to test, in a comparative framework, for a sex-specific trade-off related to brood care-but not to sexual selection-in an otherwise trophic trait. This is because of the important role of gill rakers (i.e. spine-like, bony protrusions of the branchial gill arches in fishes) in food uptake and handling of particles within the buccal cavity [9], the potential involvement of gill rakers in brood care in many cichlids and the different brood care strategies found among the closely related cichlids from Lake Tanganyika. More specifically, one particular feature of gill rakers, gill raker length, has been shown to be strongly associated with trophic ecology in many fish [10][11][12][13][14], including cichlids [15,16], a pattern we here corroborate for gill raker length across 65 Tanganyikan cichlid species (figure 2a).
All Tanganyikan cichlids provide intensive parental brood care, either in the form of bi-parental mouthbrooding (both sexes participate in parental care), uni-parental mouthbrooding (only one sex-in the case of Tanganyikan cichlids the female-participates in parental care) or substrate spawning with nest guarding ( parental care does not involve any form of mouthbrooding) [17]. Mouthbrooding species incubate their brood in the buccal cavity until the eggs' yolk sac is used up and the fry becomes free-swimming. During this entire period, which in Tanganyikan cichlids lasts between 6 and 30 days, the fertilized eggs-and later also the growing larvae-are in close physical contact with the gill rakers (figure 2b) and are regularly 'churned' inside the buccal cavity, probably to facilitate their ventilation and cleaning [18,19]. Gill rakers in mouthbrooding cichlids are thus expected to not only function in the uptake and handling of food particles, but also in the retention and handling of the eggs and larvae in the buccal cavity. Indeed, changes in head morphology have previously been associated with mouthbrooding [16][17][18][19][20], and sexual dimorphism in gill raker length has been reported for Astatotilapia burtoni, a uni-parental mouthbrooding cichlid from the Lake Tanganyika basin [15]. Taken together, mouthbrooding emerges as a promising candidate for an additional and probably conflicting functional demand of gill rakers.
In this study, we hypothesized that breeding mode can predict sexual dimorphism in gill raker length in Lake Tanganyika cichlids, whereby the three different breeding modes exemplify the three scenarios illustrated in figure 1. (i) In non-mouthbrooders, gill rakers are expected to have evolved relatively unconstrained towards the trophic trait optimum in both sexes (figure 1a). (ii) In bi-parental mouthbrooders, gill raker morphology should be influenced by both feeding and parental care (mouthbrooding). These two functions are unlikely to have identical trait optima, but the optimum resulting from the trade-off should be the same for both sexes (figure 1b). (iii) In uni-parental mouthbrooders, the functional trade-off between feeding and parental care should only occur in the mouthbrooding sex (females), whereas gill raker morphology in the non-mouthbrooding sex (males) should be selected towards the trophic optimum (figure 1c). Sexual dimorphism in gill raker length should thus occur exclusively in uni-parental mouthbrooders, but not in bi-parental mouthbrooders nor in non-mouthbrooding substrate brooders. The direction of the sexual dimorphism in uni-parental mouthbrooders is, however, hardly predictable as it should depend on the relative position of the two conflicting trait optima with respect to each other, which may well be species-specific. Finally, we hypothesized that trophic ecology determines the strength of the conflict (i.e. how divergent the two conflicting optima are) as a result of different trait optima in different trophic niches. To test these hypotheses, we examined a representative set of cichlid species for sexual dimorphism in gill raker length and tested for an association with breeding mode and trophic ecology.

Material and methods (a) Sampling
Samples were collected between 2014 and 2017 during several field trips to the southern part of Lake Tanganyika, under the research permits number 005937 (F.R.) and 004273 (W.S.) issued by the Republic of Zambia. Combined with available data on gill raker length from additional Tanganyikan cichlid species [16], the final dataset covered 65 species, well representing the phylogenetic (13 out of 16 tribes [21]), eco-morphological and behavioural (breeding modes) diversity of the species-flock of cichlid fishes in Lake Tanganyika (see electronic supplementary material for detailed information on the sampling procedure and electronic supplementary material, table S1 for sample sizes).

(b) Stable isotopes
We assessed the trophic ecology of all species by quantifying stable isotope signatures of carbon (C) and nitrogen (N) in typically 10 specimens per species (n = 661). The ratios between the rare isotopes 13 C to 12 C (δ 13 C) and 15 N to 14 N (δ 15 N) inform about two major components of aquatic ecology, the benthicpelagic (δ 13 C) and trophic (δ 15 N) position within an ecosystem [22]. This method has previously been applied to Tanganyikan cichlids and was compared to stomach content data [21], permitting an interpretation of food types. In this study, we analysed dried muscle tissue (from the epaxialis between the head and the dorsal fin) with a Flash 2000 elemental analyser coupled to royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 286: 20191050 a Delta V Plus continuous-flow isotope ratio mass spectrometer (IRMS) via a Conflo IV interface (Thermo Fisher Scientific, Bremen, Germany) in the SLU-Lab at the University of Basel (data have been deposited on the Dryad Digital Repository: https://doi.org/10.5061/dryad.fm4707v [23]). We then used a principal component analysis (PCA) to integrate over the δ 15 N and δ 13 C stable isotope ratios to obtain a univariate metric for trophic ecology.

(c) Gill raker morphology
We measured gill raker length under a binocular (Leica MZ7 5 ) as described previously [10,16]. Measurements were taken by two investigators on blinded samples (F.R., M.R.) and recorded by a third investigator (W.S.). Measurements were converted to millimetre scale and averaged across the three gill rakers measured per specimen (i.e. the second, third and fourth raker on the first branchial gill arch). To avoid a potential investigator bias, samples were assigned randomly to one of the two investigators. We measured gill raker length in 508 specimens (38 species). In combination with data from Muschick et al. [16], we obtained a dataset comprising 935 specimens and 65 species (data have been deposited on the Dryad Digital Repository: https://doi.org/10.5061/dryad.fm4707v [23]). Gill raker length was strongly correlated with body size (standard length = SL; Pearson's r = 0.68, p < 0.001), and thus, size corrected prior to further analysis. Size correction was done specifically for each analysis (see below).

(d) Phenotype-environment correlation
To investigate how gill raker length is associated with trophic ecology, we size-corrected gill raker length of each specimen using residuals from a common linear model applied across all specimens from all 65 species (with gill raker length as response variable and SL as explanatory variable; R 2 = 0.46, p < 0.001). We then added the value of the largest residual to restore positive values in the initial measuring unit (mm). The species mean of these size-independent values and the PC1 scores of stable isotope data were used to test for a phenotype-environment correlation using a linear model and Pearson's r statistics. Statistical significance was assessed using 10 000 random permutations of the observed species means over the stable isotope PC1 scores [24]. All p-values and 95% confidence intervals in this paper were obtained through analogous resampling procedures, except for analyses accounting for phylogenetic relationships.
To account for phylogenetic dependence of the species, we applied a 'phylogenetic generalized least squares' fit using the R package caper [25]. For all analyses incorporating phylogenetic relationships, we used the phylogenetic hypothesis from Colombo et al. [26] and pruned it to the set of taxa present in our datasets. One species (Petrochromis ephippium) was not represented in the phylogenetic tree and was therefore omitted from these analyses.  Figure 2. Gill rakers in Lake Tanganyika cichlids and their association with trophic ecology. (a) Phenotype-environment correlation between size-corrected gill raker length and trophic ecology (PC1 scores of stable isotope data). Longer gill rakers are associated with pelagic feeding, and shorter gill rakers with benthic feeding. This benthic-pelagic feeding trajectory is indicated above the x-axis (see electronic supplementary material, figure S1a). Data points represent species means and are shaded according to the breeding mode of the species (see electronic supplementary material, table S1 for full species names). (b) CT scan of a mouthbrooding Paracyprichromis sp. female (see electronic supplementary material for details on scanning and processing). Parts of the skull were virtually removed (box), revealing the developing eggs in the buccal cavity (highlighted in red) and the gill raker apparatus (highlighted in brown). To test for sexual dimorphism in gill raker length in non-, bi-and uni-parental mouthbrooders, we focused on a subset of species (n = 20) for which sex information was available. Here, size correction of gill raker length was performed separately for each species, using species-specific linear models, maximizing comparability between the sexes. We then tested for a difference in the length of male and female gill rakers within each species, and whether the grand-mean per breeding mode deviated from zero. We further evaluated whether the extent of the dimorphism irrespective of directionality (i.e. the absolute difference of female minus male gill raker length per species) differed among the breeding modes by calculating F-statistics across the three groups (ANOVA), followed by pairwise comparisons of the breeding modes. To account for phylogenetic dependence of the species, we applied a phylogenetic ANOVA using the function phylANOVA from the R package phytools [27].
Finally, we tested for an association between the extent of sexual dimorphism and trophic ecology (PC1 scores of stable isotope data) within uni-parental mouthbrooders. Based on a Davies test [28], which tests for a breakpoint in a linear relationship between two variables, we fitted a segmented regression model [28]. Note that the reported p-values for the Davies test were not obtained through permutation, but were taken directly from the output of the davies.test function as implemented in the R package segmented [28]. To validate the results in a phylogenetic framework, we used the estimated breakpoint in PC1 scores from the segmented regression model as a threshold to assign the uni-parental mouthbrooders into two trophic groups and tested for a difference in the extent of sexual dimorphism between these groups using a phylogenetic ANOVA. All graphing and statistical analyses were conducted in R [29].

Results
(a) Gill raker length is associated with trophic ecology A PCA of the stable isotope ratios of nitrogen (δ 15 N) and carbon (δ 13 C) was used to reduce dimensionality of the two components of trophic ecology. This allowed working with a univariate proxy for trophic ecology. PC1 explained 77.3% of the total variation in the stable isotope data, and was loaded negatively for δ 13 C (−0.71) and positively for δ 15 N (0.71) (electronic supplementary material, figure S1a). Higher PC1 scores thus reflected pelagic feeding (e.g. on zooplankton and/or fish fry) and a relatively high position in the food chain (hereafter simply referred to as 'pelagic'), whereas benthic/littoral species with a mainly algivorous feeding lifestyle and a lower trophic position had lower PC1 scores (hereafter simply called 'benthic'). Gill raker length was positively associated with trophic ecology across the 65 species (Pearson's r = 0.46, p < 0.001; R 2 = 0.20, p < 0.001), with shorter gill rakers in benthic and longer gill rakers in pelagic species (figure 2a; electronic supplementary material, figure S1b). This result held true after accounting for phylogenetic dependence of the trait values (R 2 = 0.14, p = 0.002, λ = 0.43).

(b) Sexual dimorphism is predicted by breeding mode and trophic ecology
Sexual dimorphism in size-corrected gill raker length was pronounced in uni-parental mouthbrooders, and reached statistical significance ( p < 0.05) in three out of nine species (see electronic supplementary material, table S2a). By contrast, none of the bi-parental mouthbrooding species, nor any substrate brooding species, showed evidence for sexual dimorphism (figure 3a). The grand mean per breeding mode of the difference between male and female gill raker length did not deviate from zero in any of the three breeding modes (see electronic supplementary material, table S2b). However, uni-parental species showed a strongly increased variation in sexual dimorphism compared to bi-parental mouthbrooders and non-mouthbrooders (figure 3a). The absolute difference in gill raker length between the sexes revealed a significantly greater extent of sexual dimorphism in uni-parental mouthbrooders compared to bi-parental and non-mouthbrooding species in an ordinary ANOVA (F = 6.19, p = 0.007) (figure 3b; electronic supplementary material, table S2c,d). When accounting for phylogenetic dependence, only uni-parental and bi-parental mouthbrooders showed a difference in the extent of sexual dimorphism ( p = 0.022).
Finally, we focused on the association between the extent of sexual dimorphism and trophic ecology within uniparental mouthbrooders. We found a statistically supported breakpoint in the linear relationship between sexual dimorphism and trophic ecology ( p = 0.04). The fitted segmented model estimated a breakpoint at a PC1 score of 0.34, with PC1 scores higher than 0.34 showing a strong positive association with the extent of sexual dimorphism (figure 4; electronic supplementary material, table S3). When using this estimated breakpoint to assign the species into two trophic groups and accounting for phylogenetic dependence, the species with higher PC1 scores showed a distinctly greater extent of sexual dimorphism than the species with PC1 scores below the threshold (F = 22.8, p = 0.004).

Discussion
In this study, we addressed the question of whether a conflicting (sex-specific) functional demand linked to parental care can explain sexual dimorphism in an otherwise trophic trait. To this end, we investigated gill raker length in a set of cichlid fish species from Lake Tanganyika covering three different breeding modes and a variety of trophic ecologies (figure 2a).
Gill rakers are an important structure for uptake and handling of food in the buccal cavity in fish [9], and the length of gill rakers is generally associated with different trophic ecologies: pelagic species feeding on small and mobile prey commonly have longer gill rakers, while benthic species feeding on larger and immobile prey (or aufwuchs) have shorter gill rakers [10][11][12][13][14][15][16]. Here we corroborate this phenotype-environment correlation in an extensive dataset covering 65 cichlid species from Lake Tanganyika, representing the morphological, ecological and phylogenetic diversity of the lake's cichlid assemblage: we find an association between gill raker length and trophic ecology (as approximated by the PC1 of stable isotope data), with longer gill rakers in cichlids with more pelagic stable isotope signatures, and shorter gill rakers in species with more benthic signatures ( figure 2a). Based on a previous study linking stable isotope signatures with stomach content analysis in Tanganyika cichlids [21], we conclude that pelagic stable isotope signatures usually correspond to invertebrate/zooplankton/ small fish feeders, whereas species with benthic signatures predominantly feed on algae and plants. Note, however, that also predatory species feeding on large fish show pelagic royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 286: 20191050 signatures, but have rather short gill rakers (see e.g. Lepidiolamprologus profondicula; 'Leppro'; figure 2a).
We hypothesized that gill raker length is also relevant for mouthbrooding, thus resulting in a conflicting functional demand of gill raker morphology in addition to food update and handling (figure 1b). Mouthbrooding is a particular form of parental care and widespread among cichlid fishes, where it occurs in a uni-parental (maternal or paternal) or bi-parental mode. Mouthbrooding is a costly trait [30] and has been reported to induce morphological changes including an enlargement of the head or the buccal cavity [20,[31][32][33], or a reduction in gill size [34]. Gill raker length has, however, not yet been examined in the context of mouthbrooding. This is surprising given that gill rakers are expected to be functionally involved in mouthbrooding, either directly via the active handling of the eggs or larvae [18], or indirectly through the close physical contact between gill rakers and the offspring (figure 2b). difference in size-corrected gill raker length -(mm)  Under the assumption that gill rakers experience different selective regimes among the three breeding modes (biparental mouthbrooding, uni-parental mouthbrooding and non-mouthbrooding) due to their dual function in both feeding and breeding, we predicted sexual dimorphism to be present exclusively in uni-parental mouthbrooders (figure 1). We examined 20 Tanganyikan cichlid species and investigated how well breeding mode and/or feeding ecology can explain sexual dimorphism. Indeed, we found males and females of non-and bi-parental mouthbrooding species not to differ in gill raker length. By contrast, several of the uniparental mouthbrooding species were sexually dimorphic in gill raker length, varying in extent and direction (figure 3a). The overall difference between non-and bi-parental mouthbrooders relative to uni-parental mouthbrooders became particularly evident when comparing the absolute extent of sexual dimorphism (i.e. sexual dimorphism irrespective of its direction) among breeding modes (figure 3b). When accounting for phylogeny, the difference between uni-parental and bi-parental mouthbrooders in the extent of sexual dimorphism was confirmed, but not so for non-mouthbrooders. This is hardly surprising, given the monophyly of the vast majority of non-mouthbrooding cichlids in Lake Tanganyika (the Lamprologini, which make up 50% of all species in that lake; see electronic supplementary material, figure S1c), thus reducing statistical power in comparisons involving non-mouthbrooders. Nevertheless, the difference in the extent of sexual dimorphism between uni-parental and biparental mouthbrooders supported the idea that breeding mode can partially predict the presence or absence of sexual dimorphism in gill raker length.
How can the variation in the extent and direction of sexual dimorphism within uni-parental mouthbrooders be explained? Under the assumption of a trade-off between a trophic versus reproductive function of gill rakers, both the extent and the directionality of the sexual dimorphism should depend on the relative position of the two optima in relation to one another ( figure 1). Clearly, the association of gill raker length and trophic ecology across the 65 cichlid species suggests species-specific optima for gill raker length ( figure 2a). Although the factors determining optimal gill raker length for mouthbrooding are unknown, life-history traits such as clutch and egg size or breeding duration are likely to be relevant. Unfortunately, data on life-history traits are too scarce (and/or too vague) to allow testing for an association with gill raker length. Nevertheless, clutch size emerges as a promising candidate trait to explain variation in the direction of sexual dimorphism among uni-parental mouthbrooders (see electronic supplementary material, figure S2a). On the other hand, since life-history traits differ among cichlid species [35], the reproductive optimum of gill raker length is expected to be species-specific too. This is further corroborated by the difference in the directionality of sexual dimorphism in gill raker length among uni-parental mouthbrooders with respect to actual gill raker length (electronic supplementary material, figure S2b). Thus, the finding that some uni-parental mouthbrooders show a female-biased dimorphism (longer gill rakers in females), while others show a male-biased dimorphism, is likely to reflect variation in the relative position of the conflicting trait optima.
Likewise, the absence of any sexual dimorphism in some of the uni-parental mouthbrooders might be the result of overlapping trait optima for the two functional demands. Species with extreme trophic ecologies may be expected to generally experience stronger deviations between the trophic and reproductive optima than species with intermediate trophic ecologies. Additionally, variation in the extent of sexual dimorphism among uni-parental mouthbrooders might be the result of similarly strong selection towards the optimum for mouthbrooding in all species, but varying selection regimes with respect to the optimal trait value for feeding, depending on the trophic ecology of a species. For example, if gill raker morphology is of particular importance for efficient food uptake and handling in a species (as in pelagic suction feeders [11]), the selective pressures acting antagonistically are expected to be strong and a dimorphism is more likely to be expressed. On the other hand, in species where the gill rakers are less important for feeding (as in benthic algivores), sexually antagonistic selection would be unbalanced, resulting in a less pronounced or no sexual dimorphism. Accordingly, in both cases, the differences in the extent of sexual dimorphism in uni-parental mouthbrooders are expected to depend on the trophic ecology of the species. We tested this prediction and found uni-parental mouthbrooding species to show an association between the (absolute) extent of sexual dimorphism and trophic ecology. This association was not linear along the entire trophic continuum, but rather increased rapidly after a certain breakpoint ( figure 4). This implies that whether or not a sexual dimorphism in gill raker length occurs depends on both the breeding mode and the trophic ecology of a species, with breeding  Figure 4. Association between the extent of sexual dimorphism and trophic ecology: in uni-parental mouthbrooders, the linear association between the extent of sexual dimorphism (i.e. absolute difference in size-corrected gill raker length between females and males) and trophic ecology shows a breakpoint at a trophic PC1 score of 0.34. For species above this threshold, PC1 scores and sexual dimorphism correlate strongly and positively. The solid line represents the result of a segmented regression model fitted for uni-parental mouthbrooders. As no differences were found between bi-parental mouthbrooders and non-mouthbrooders, the two groups were pooled in a linear model (dashed grey line). Note that the five uni-parental mouthbrooding species with the largest sexual dimorphism belong to five different tribes (see electronic supplementary material, figure S1c). The benthic-pelagic feeding trajectory represented by PC1 scores is indicated above the x-axis (see electronic supplementary material, figure S1a).
royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 286: 20191050 mode determining the potential for a sex-specific functional conflict, and trophic ecology determining the strength of the conflict.
With the data at hand, we cannot formally test for the strength of selection acting on gill raker length, nor can we directly measure the optima in trait value for feeding versus mouthbrooding. Hence, we cannot disentangle cases where the strength of the conflict depends on how balanced the selective pressures are that act on the two optima, on how divergent the two optima are, nor a combination of both. Nevertheless, our findings provide empirical evidence for the scenario that a sex-specific functional conflict due to parental care by only one sex of a species explains sexual dimorphism in a trait.
The finding of sexual dimorphism to be present exclusively in uni-parental mouthbrooders largely contradicts predictions from popular models of ecology-caused sexual dimorphism: if inter-sexual competition for resources were the trigger for sexual dimorphism [6], one would expect sexual dimorphism to occur mainly in species forming pair bonds and sharing feeding and breeding territories [36]. In our study system, this applies primarily to bi-parental mouthbrooders and non-mouthbrooders (bi-parental nest guarders), but not to uni-parental mouthbrooders. Other ecological models for sexual dimorphism, such as the 'bimodal niche model' [6] (two alternative optima in trait value exist, followed by disruptive selection between the sexes) or the 'dimorphic niche model' [6] (intrinsic differences between males and females in energetic needs lead to niche divergence between the sexes), would also not predict sexual dimorphism to occur exclusively in uni-parental mouthbrooders. Moreover, most models of ecology-caused sexual dimorphism assume niche divergence between the sexes. However, such a difference in niche use between males and females is not evident from our stable isotope data (electronic supplementary material, figure S3).
Unlike most studies investigating causes of sexual dimorphism in relation to ecology [7,8,37,38], we can largely exclude the possibility that sexual selection has directly driven or reinforced the observed sexual dimorphism. This is because gill rakers are cryptic to the outer appearance of a fish and thus highly unlikely to serve as a signal in mate choice or mate competition. One could of course argue that sexual selection initially contributed to the evolution of sexspecific roles in breeding behaviour, but here we refer to sexual selection acting directly on the focal trait. Taken together, sexual dimorphism in our study system is unlikely to be explained by sexual selection or initial niche divergence between the sexes, thus providing a novel view on the evolution of sexual dimorphism in nature.
Although our study provides an explanation why gill raker length differs between the sexes in some cichlid species, but not in others, it remains an open question how sexual dimorphism in this trait is achieved developmentally. Variation in gill raker length has been shown to have a largely genetic basis in threespine stickleback [39,40], and a common garden experiment with divergent A. burtoni cichlid ecotypes revealed both a genetic and a plastic component in gill raker length variation [15]. What remains to be tested is the degree to which sexual dimorphism in gill raker length of cichlids is genetically based or is the result of a plastic response to mouthbrooding. It would further be interesting to investigate other components of the cichlids' trophic morphology with respect to sexual dimorphism and parental care.
In conclusion, our study establishes an overall phenotype-environment association between gill raker length and trophic ecology across 65 Tanganyikan cichlid species, and reveals that gill raker morphology is influenced by mouthbrooding. As a consequence, the presence and extent of sexual dimorphism in gill raker length is predicted by both the breeding mode and the trophic ecology of a species. Sexual dimorphism in gill raker length of uni-parental mouthbrooding cichlids is unlikely to be explained by sexual selection or initial niche divergence between the sexes, but instead is caused by a sex-specific trade-off between two conflicting functional demands of the same trait, one related to trophic adaptation and one to parental care.


extfieldsofstudy: Medicine Chemistry

text:
Stratum Corneum Sampling to Assess Bioequivalence between Topical Acyclovir Products

Purpose To examine the potential of stratum corneum (SC) sampling via tape-stripping in humans to assess bioequivalence of topical acyclovir drug products, and to explore the potential value of alternative metrics of local skin bioavailability calculable from SC sampling experiments. Methods Three acyclovir creams were considered in two separate studies in which drug amounts in the SC after uptake and clearance periods were measured and used to assess bioequivalence. In each study, a “reference” formulation (evaluated twice) was compared to the “test” in 10 subjects. Each application site was replicated to achieve greater statistical power with fewer volunteers. Results SC sampling revealed similarities and differences between products consistent with results from other surrogate bioequivalence measures, including dermal open-flow microperfusion experiments. Further analysis of the tape-stripping data permitted acyclovir flux into the viable skin to be deduced and drug concentration in that ‘compartment’ to be estimated. Conclusions Acyclovir quantities determined in the SC, following a single-time point uptake and clearance protocol, can be judiciously used both to objectively compare product performance in vivo and to assess delivery of the active into skin tissue below the barrier, thereby permitting local concentrations at or near to the site of action to be determined. Electronic supplementary material The online version of this article (10.1007/s11095-019-2707-3) contains supplementary material, which is available to authorized users.

. Number of tapes (columns) collected and mass of SC removed (symbols) from each subject for three ACV creams after 6 h uptake and 17 h of clearance. The results are represented as the arithmetic mean and standard deviation of the arithmetic average of the duplicate measurements in 10 subjects for each of the three products after uptake and clearance. *Significantly different from other cream applications measured after uptake or clearance in the same study (p<0.05). Notes: [1] The geometric mean for the duplicate measurements of the mass of drug in the SC is used because we expect (based on other skin permeation measurements) that this is likely to be log-normally distributed. However, it is not clear that the number of tape-strips, or the mass of SC collected on the tape-strips, would be expected to be log-normally distributed. It therefore makes more sense to use the arithmetic average of the duplicates. In practice, though, this changes minimally the numerical value of the specific data points which are plotted above. [2] For Study 2, clearance, the maximum permitted number of tape-strips (30) were taken at 57 of 60 sites. In contrast, in Study 2, uptake, and in Study 1, uptake and clearance, at only about half of the sites were 30 tape-strips removed (27 for uptake and clearance in Study 1, and 36 for uptake in Study 2).

Figure S2.
Average SC collection efficiency of the tape strips (mg/cm2) for each cream (arithmetic mean ± 90% confidence interval for 10 subjects) after 6 h uptake (filled symbols) and 17 h clearance (open symbols).
These results and the methodology employed indicate some possible procedural recommendations that might be introduced to ensure the uniformity and reproducibility of the approach.
First, the refined SC sampling technique introduced in the econazole work (1) did not measure the mass of SC on the tapes. However, a pilot study was performed that employed TEWL measurements to decide if tape-stripping had completely and reliably removed most of the SC; hence, it was unnecessary to measure the mass of SC on the tapes. A sensible suggestion, therefore, would be to quantify the amount of SC removed unless a pilot study (using TEWL, for example) had verified that the method did indeed ensure that most of the barrier had been collected.
Second, the improved protocol (1) specified that 12 tape-strips should always be taken (in fact, no TEWL values were recorded in that study until 12 tape-strips had been removed). In the present work, at only one site, on a single volunteer, were less than 12 tape-strips acquired before the procedure was stopped. A reasonable recommendation would therefore be to set a 12 tape-strip minimum for SC sampling. Nonetheless, attention should always be paid to the nature and quantity of the excipients present in the drug products tested, as there will certainly be some cases (see, for example, (2) where a formulation alters the SC and dramatically increases the amount of tissue collected). In such circumstances, to spare volunteers from inordinate discomfort, acquisition of less  Figure S3. The concentration profiles for the ACV-AT products were clearly different than that observed for the ACV-US cream after both uptake and clearance.

Calculated metrics
The mass per unit area of drug in the tape strips was determined in the k th replicated site treated with formulation i on subject j ( ijk Q ). Each formulation in the two studies was measured at 2 sites in 10 subjects; i.e., nr = 2 and n = 10. The first-order clearance rate constant (k) and the flux of drug out of the SC into the underlying tissue in vivo J were calculated from the geometric mean of the duplicate ( .  (Table S1) and for in vivo, J ij and k ij (Table S2).

Bioequivalence assessment
Bioequivalence calculations for a balanced study design with nr replicated measurements of each formulation in a total of n subjects were conducted using the following procedures for a metric determined in the k th replicated site treated with formulation i on subject j ( ijk M ).

S8
For ijk M representing a metric that is always a positive number (i.e., the mass per unit area of drug collected in the SC tape strips, ijk Q ) the BE analysis is performed by comparing the log-transformed value of ijk M , defined as ijk Z , determined in each subject and then averaging across subjects as follows: The within subject variance for a formulation i measured in subject j is calculated as follows:  Table S3.
Note that calculated values for the GMR and the lower and upper confidence intervals are the same for data transformed using natural logarithms (as described here) or base 10 logarithms (as described previously (1)) provided the anti-log step is consistent with the type of log transformation (i.e., exp(x) for natural log transformed data and 10 x for base 10 log transformed data). Here, we recommend natural log transformation for the ABE calculations to be consistent with the scaled average bioequivalence (SABE) procedure below, which is specific to the type of logarithmic transformation.

Scaled average bioequivalence (SABE) assessment
The SABE methodology is indicated when the within subject standard deviation for the reference formulation (i.e., Wi s for i = 2 in an assessment of the difference between formulations 1 and 2 for the natural log-transformed metric) is    Table S3. S12 Table S1. ACV amounts (µg/cm 2 ) and log-transformed amounts recovered from the SC after uptake and clearance in the replicate samples ( ijk Q , replicate k of formulation i in subject j ) from each of the 10 subjects for the US-C+ and US-Ref formulations (designated as formulations 1 and 2, respectively)

Power simulations of bioequivalence assessments
We evaluated the number of subjects required to adequately power the traditional ABE and SABE methods for the m = 1.25 limit by performing simulations. For each such study, the inputs of the power function are the within-subject standard deviation of the reference product ( 2 W s in an assessment of the difference between formulations 1 and 2), the between-subjects standard deviation ( I s ), the number of subjects and the number of replicates. This process is repeated 500,000 times under the assumption of bioequivalence. The value of the power is then the percentage of these trials that correctly captured the equivalence relationship between the two products. Table S6 lists Table   S7. The SABE methodology was estimated to achieve a statistical power close to 80% with 10 subjects for the products compared in Study 1 for both uptake and clearance, and for uptake in Study 2 (which involved a different cohort of 10 subjects); see Figures S5 and S6 and Table S7. Approximately 15 subjects are needed to adequately power the clearance results in Study 2 ( Figure S6). By comparison, the traditional ABE methodology is estimated to require, between 15 and 50 subjects to achieve the same power, with fewer subjects needed in the assessment of the positive control with the corresponding reference product (Figures S5 and S6 and Table S7). Increasing replication from two to three sites for each product in this study had minimal benefit, reducing the number of subjects required to achieve the same power in the SABE assessment by approximately one subject ( Figure S7). Figure S5. Estimated power as a function of the number of subjects (n) for traditional average bioequivalence (ABE) and scaled average bioequivalence (SABE) assessments of the positive control to the corresponding reference product in Studies 1 and 2 for the bioequivalence margin m = 1.25: uptake (solid) and clearance (dashed). The statistical power of 0.8 commonly recommended for acceptance by regulatory agencies is indicated. Figure S6. Estimated power as a function of the number of subjects (n) for traditional average bioequivalence (ABE) and scaled average bioequivalence (SABE) assessments of the test to reference products in Studies 1 and 2 for the bioequivalence margin m = 1.25: uptake (solid) and clearance (dashed). The statistical power of 0.8 commonly recommended for acceptance by regulatory agencies is indicated. Figure S7. Estimated power as a function of the number of subject (n) when the number of replicates for comparing the test and reference products in Studies 1 and 2 is increased from two (dashed) to three (solid) for the bioequivalence margin m = 1.25. The statistical power of 0.8 commonly recommended for acceptance by regulatory agencies is indicated. The power simulation results for m = 1.25 compared with m = 1.33 are presented in Figure S8 for assessments of the positive control to the corresponding reference product (i.e., US-C+ to US-Ref and AT-C+ to AT-Ref in Studies 1 and 2, respectively), and in Figure S9 for assessments of the test to reference product (i.e., UK-Test to US-Ref and US-Test to AT-Ref in Studies 1 and 2, respectively).
S22 Table S7 lists the expected minimum number of subjects required to achieve a statistical power of at least 80% for m equal to both 1.25 and 1.33.
The power of a bioequivalence study using cutaneous pharmacokinetic endpoints can be substantially increased by widening the bioequivalence limits from the traditional m = 1.25 to m = 1.33 for an ABE assessment. The advantage of this approach is that fewer subjects are needed to power the study. However, the disadvantage of widening the bioequivalence limits is that it essentially lowers the standard for comparability of the test and reference products. Using an SABE analysis instead of an ABE analysis, while maintaining the traditional bioequivalence limit of m = 1.25, increases the power of the study to an even greater degree than by widening the bioequivalence limits for an ABE analysis to m = 1.33. The additional power gained by widening the bioequivalence limits from m = 1.25 to m = 1.33 in an SABE analysis is much smaller than for the ABE assessment. The results presented in Figure S8 confirm that the comparison of AT-C+ and AT-Ref products in Study 2 with 10 subjects was slightly underpowered for an SABE assessment at m = 1.25, but is adequately powered at m = 1.33 (in which case the SABE assessment successfully demonstrated bioequivalence as shown in Table 4 in the paper).


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Nienna chukotka sp. nov. (Protura, Acerentomidae, Nipponentominae) from the Arctic region, with a key to species of the genus

Abstract A new species of Nienna was collected in the most northern part of the Palearctic, inside the Arctic Circle. In possessing seta Pc on tergite VII and sternites VI–VII and a very long foretarsal sensillum a, Nienna chukotkasp. nov. is more similar to Alaskaentomon species than to the other Nienna species distributed in southern Siberia and northern China. The new species differs from nearly all other members of Nipponentominae in possessing five anterior setae on tergite VII and in the presence of posterolateral pores on tergite I, as in members of Hesperentomon (Hesperentomidae). An identification key to Nienna species is provided.

Introduction
The proturan genus Nienna Szeptycki, 1988 was created for Nienna parvula Szeptycki, 1988, described from the Altai mountains in southern Siberia (Szeptycki 1988). The genus differs from the 12 other genera of Nipponentominae Yin, 1983 in possessing a small, indistinctly granulated calyx and a short posterior filament on the maxillary gland, and in the small, nearly globular foretarsal sensillum t3. A second species, Nienna quinghaiensis Bu & Yin, 2008, was described from northern China. The diagnosis of the genus was recently updated (Galli et al. 2018). In the current paper, the description of a third species of Nienna is given. The type specimens, collected from the Arctic region, are the northernmost records for any Protura. A key to the species of Nienna is given.

Materials and methods
Protura specimens collected from western Chukotka in 2018 were extracted from soil samples with Berlese-Tullgren funnels into 95% ethanol. The specimens were mounted on glass slides in Faure's medium (Dunger and Fiedler 1989).

Results
The genus Nienna is characterized by three pairs of A-setae on the mesonotum and metanotum, small, indistinctly granulated appendices on the calyx and a short posterior filament on the maxillary gland. The foretarsal sensillum t1 is filiform, sensillum t3 is small and globular (lanceolate in N. quinghaiensis Bu &Yin, 2008), the position of sensillum d is close to the base of e, and seta β1 is setiform. Sensillum a' is distal to the base of t2. Sensillum b' is missing. The genus is similar to twelve other genera from the subfamily Nipponentominae in having abdominal legs with 2 nearly equal setae, 5 pairs of A-setae on tergites II-VI (except for Alaskaentomon Nosek, 1977 and Nanshanentulus Bu & Yin, 2007) and by the posterior position of seta P3 on abdominal tergites II-VI (except for A. fjellbergi Nosek, 1977) (Bu and Yin 2007;Bu et. al. 2013;Galli et al. 2018;Nosek 1977Nosek , 1981Shrubovych 2009Shrubovych , 2011Shrubovych , 2014Shrubovych and Smykla 2012;Shrubovych et al. 2014a, b, c). Diagnosis. Nienna chukotka is characterized by 3 pairs of A-setae on the mesonotum, metanotum and tergite VIII, 3 A-setae on sternites I-VII, absence of P1a setae on tergites I-VI, 5 pairs of A-setae on tergites II-VII, absence of A2 on prosternum, presence of seta Pc on tergite VII and sternites VI-VII, and presence of additional d6 setae on head. Foretarsal sensillum a is broadened, very long, surpassing the base of sensillum e. Posterolateral pores (pl) present on tergite I, psl pores present on tergites VI and VII, asymmetrical spsm pores present on sternites IV-VII.
Abdominal legs with 4, 2, 2 setae. Subapical and lateral apical setae on second and third pairs of abdominal legs nearly equal in length, 15 and 14 μm, respectively  Abdominal segment VIII with distinct striate band; tergite and sternite anteriorly with irregular small teeth (Figs 2 I, M). Pore psm without accompanying teeth. Posterior margin of sternite VIII and laterotergites smooth. Comb VIII with 9-10 small teeth (Fig. 1I). Seta 1a on tergite IX half the length of seta 1. Seta 2a on tergites IX and X shorter than other setae. Sternites IX-X with traces of striate band (Fig. 2N). Setae 1 and 2 on sternite IX of equal length, on sternite X seta 1 about half the length of seta 2 (Fig. 2N). Medial pore on dorsal lobe of segment XII and pair of sal pores on ventral lobe. Hind margin of dorsal lobe smooth, ventral lobe with fine serrations (Fig. 2O).
Chaetal variability. In the holotype, seta P4 is doubled asymmetrically on the mesonotum; in the paratype, seta A3 is absent symmetrically on tergite I and seta P2a is doubled on tergite VII.
Etymology. The species name is taken from the general locality where the specimens were collected.
Remarks. Nienna chukotka sp. nov. differs from N. parvula and N. quinghaiensis in the presence of seta Pc on tergite VII and sternites VI-VII (in N. quinghaiensis seta Pc is present on sternite VII only), the presence of 5 pairs of A-setae (4 pairs in the other two species) and P3a on tergite VII, the shape of the accessory setae on tergites and sternites I-VI (setiform in the new species, sensilliform in the other two species) and the shape of foretarsal sensilla a, c and e (in the other species sensillum a is shorter, reaching base of sensillum t2, sensilla c and e short and broad). The porotaxy of mesoand metanota and abdominal sternites also differs: Nienna chukotka has two pairs of sl and al pores on the meso-and metanota, and asymmetrical spsm pores on sternites IV-VII;], whereas N. parvula has a pair of sl pores on the meso-and metanota, and a simple spm pore on sternites VI-VII. Nienna quinghaiensis has al and l pores on the mesonotum, l pores on the metanotum, and an spm pore on sternite VII. The new species is more similar to N. parvula in possessing traces of a striate band on sternites IX-X and in the globular foretarsal sensillum t3. Nienna chukotka is characterized by the presence of pl on tergite I, which is the first report of posterolateral pores in Acerentomidae. Szeptycki (1988) previously described pl pores on Hesperentomon martynovae Szeptycki, 1988 (Hesperentomidae) collected in the Altai Mts. These pl pores have also been recorded in other Hesperentomon species: H. fopingense Bu, Shrubovych & Yin, 2011, H. nanshanensis Bu & Yin, 2007, H. xiningense Bu & Yin, 2007distributed in China, and H. tianshanicum Martynova, 1970(Shrubovych 2010.

Discussion
The foretarsus of N. chukotka sp. nov. has a very long sensillum a, surpassing the base of sensillum e, and filiform foretarsal sensillum t1, characters shared with two species of Alaskaentomon (A. fjellbergi, A. condei). These two Alaskaentomon species possess seta Pc on tergite VII and sternites VI-VII. Alaskaentomon spp. differ from N. chukotka sp. nov. in having two pairs of A-setae on the meso-and metanota and large granulated appendices on the calyx of the maxillary gland (Shrubovych et al. 2014c). In notal chaetotaxy (three pairs of A-setae) and the filiform sensillum t1, the genus Nienna is similar to the genera Callientomon Yin, 1980, Noldo Szeptycki, 1988, Paracerella Imadaté, 1980and Verrucoentomon Rusek, 1974. However, Nienna differs from all of them in possessing small, indistinctly granulated appendices on the calyx of the maxillary gland and in the small, nearly globular foretarsal sensillum t3 (Shrubovych et al. 2014a). The new species differs from nearly all species of Nipponentominae in possessing a pair of A1 setae on tergite VII (five pairs of A-setae), while nearly all other nipponentomines have four pairs of A-setae (except Nipponentomon macleani Nosek, 1977 from Alaska, which also has 5 pairs of Asetae). Therefore, N. chukotka sp. nov. from Chukotka is more similar in body chaetotaxy and in foretarsal sensilla pattern to members of other genera distributed in Alaska than to the other Nienna species distributed in more southern regions of the Palearctic. This peculiarity could be an effect of subsequent allopatric speciation resulting from successive closings of the Bering Strait and cooling of the Arctic Ocean during the Pliocene-Pleistocene. Another interesting fact is that species recorded on the northern edge of proturan distribution (only a few Protura species are known from the Arctic region) possess a larger number of setae on the body than species with a more southern distribution.