Composition and Function of the Nuchal Hump of Male Xiphophorus multilineatus

Composition and Function of the Nuchal Hump of Male Xiphophorus multilineatus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Composition and Function of the Nuchal Hump of Male Xiphophorus multilineatus Keith Tompkins, Will Boswell, Kang Du, Zhao Lai, Yuan Lu, Molly R Morris This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5389356/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Jul, 2025 Read the published version in Fish Physiology and Biochemistry → Version 1 posted 7 You are reading this latest preprint version Abstract Nuchal humps are morphological traits that occur among vertebrate species and have multiple functions. The emergence of nuchal humps in Xiphophorus multilineatus males maintained in the laboratory, a species that does not develop humps in the wild, provided a unique opportunity to explore the development and function of this trait. The current study investigated the tissue composition of the hump and analyzed the influence of rearing temperature and diet restriction on hump development. Through histological examination and gene expression profiling our findings show that the hump is composed primarily of adipose tissue, which suggests a role in energy storage via fat deposition. Nuchal humps grew to a larger size in the cold environment (20°C) than the warm (25°C) and the differential gene expression pattern between temperature treatments suggests increased fat deposition in the cold versus warm environment. For example, the G0S2 gene which inhibits lipid catabolism is upregulated in the cold environment, and the WARS-1 gene which leads to increased fat stores when under-expressed is upregulated in the warm environment. The hypothesis that temperature influences hump development by stimulating shifts in fat metabolism is further supported by the finding that males from the warm environment used fat stores in the nuchal hump at a faster rate when placed on a restricted diet than males from the cold. The influence of temperature, diet and potentially activity levels on the fat deposition suggests X. multilineatus as an evolutionary animal model for gaining insights into the mechanisms involved in human obesity. Nuchal hump lipid storage temperature Xiphophorus multilineatus Figures Figure 1 Figure 2 Figure 3 Introduction Nuchal humps, broadly defined as a tissue mass located dorsal posterior to the head, are present across multiple vertebrate taxa. The tissue composition of nuchal humps varies among vertebrate species as does the development and function of this structure (Alexander et al. 1965, Cave et al. 1958, Manamendra-Arachchi et al. 2006, Susuki et al. 2014, Ward et al. 2020). In sexually monomorphic fish species where the hump is persistent, such as Gila cypha , an anti-predatory function is likely. Both sexes would benefit from the presence of a nuchal hump due to increased body depth which can limit the ability of predators to capture individuals once the hump develops (Douglas 1993, Portz et al. 2004, Ward et al. 2020). Among sexually dimorphic species, nuchal humps are often sexually selected traits and proposed functions include species recognition, sex recognition, and indication of mate quality (Barlow et al. 1997, Takahashi 2018, Rometsch et al. 2021). There is also evidence to suggest that the nuchal hump may play a role in energy storage, based on increased expression of genes known to promote adipogenesis and cell proliferation within nuchal hump tissue of the African cichlid Cyrtocara moorii (Lecaudey et al. 2019), and enlargement of the hump in another African cichlid, Cyphotilapia gibberosa commonly known as Mpimbwe Blue Frontosa, was due to hypertrophy of the hypodermal layer and increased fat storage (Takahashi 2018). The nuchal hump is a diagnostic trait for the swordtail species Xiphophorus birchmanni ( Rauchenberger et al. 1990) and may play a role in mate choice (Rosenthal et al. 2003) as well as influence swimming endurance in relation to the sword (Johnson et al. 2014). Nuchal humps are not known to occur in the wild for any of the other Xiphophorus fishes. The development of this trait in the laboratory occurred in X. multilineatus adult males that were sampled from a wild population and brought to the laboratory as well as in males that were reared from birth in the laboratory (Tompkins et al. 2021). Females prefer males with humps versus males without (Tompkins et al. 2021), suggesting a potential benefit for the hump via sexual selection if they were to develop in the wild. One aspect of the laboratory environment that promotes the hump is diet, as X. multilineatus males reared on high quality diets (i.e. higher fat content in addition to protein) were more likely to develop larger humps than males reared on low quality diets (Tompkins et al. 2021). Temperature influences fat utilization in fish by stimulating shifts between catabolizing fat as an energy source at warmer acclimation temperatures to storing fat in liver and muscle tissues at colder acclimation temperatures (Stone et al. 1981, Egginton et al. 1989). Accordingly, we hypothesize that temperature influences the process of hump development. Therefore, to further examine the possible function of the nuchal hump in X. multilineatus , we used a split-brood design to examine development of the hump in the laboratory under two different temperature regimes. We first addressed the following questions: 1) What is the tissue composition of the nuchal hump in X. multilineatus ? While the presence of lipid droplets has been detected in the nuchal hump of X. multilineatus (Tompkins et al. 2021), it is not known if the hump is composed primarily of adipose tissue. 2) Are there differences in the genes expressed in hump tissue in males reared in a cold environment versus warm? 3) How does rearing temperature influence the allometric growth of the nuchal hump? Second, we reduced the diet of adult males for approximately one month to determine whether 4) the size of the hump influenced by a reduction in diet and 5) is the influence of diet reduction consistent across temperature treatments? Methods Animals and Sample Collection X. multilineatus males used to test the effects of rearing temperature on the development of nuchal humps, through both RNA sequencing (RNA-Seq) analyses and multivariate analysis of hump size and growth rate, were obtained by isolating 50 pregnant females from an existing community tank with only courter male X. multilineatus located in the Morris Lab. Courter males are one of two alternative reproductive tactics (ARTs) found in this species. ARTs in X. multilineatus are genetically influenced by variation in copy number of mc4r B alleles on the Y chromosome (Lampert et al. 2010). The ARTs are dimorphic for several traits including body size (courter males are larger than sneakers) and reproductive behavior where courter males use only courtship displays to attract females and sneaker males use either courtship or forced copulation to gain access to females (Liotta et al. 2021). Courter males in the lab were observed to have larger humps relative to their body size than sneaker males, so only males with a courter lineage were used in this study due to the higher propensity of humps forming on courter males. Females were allowed to drop fry, and the fry were split between two environmental chambers. Environmental chambers were set at 25°C (warm treatment) and 20°C (cold treatment). Fry were transferred to individual 2.5 L tanks in respective environmental chambers at 30 days of age: 72 fry in warm, 72 fry in cold. All fry were fed a high-quality diet of Ken’s Spirulina flake food in the morning daily, and brine shrimp in the afternoon five days a week. The flake food was the same high-quality diet used by Tompkins et al. (2021) that promoted growth of larger humps. Each tank was equipped with a Whisper® Powerfilter that circulated the water and agitated the water surface. Measures of dissolved oxygen saturation were similar between treatments ((N = 8 per treatment, Cold = 100.8 ± 1.1 %; Warm = 97.7 ± 1.4 %). Histology Males used for histology were obtained from a community tank with only X. multilineatus located in the Morris Lab. Fish in the community tank were fed a high-quality diet of Ken’s Spirulina® flake food seven days per week and Artemia sp. nauplii five days per week at a rate that was completely consumed within five minutes. The community tank was maintained at room temperature between 21-22°C. Males were euthanized with MS-222 and transverse tissue sections were taken from the nuchal region and preserved in 10% neutral buffered formalin. Tissue samples were processed and stained at the Ohio University Histology Core Facility using standard eosin and hematoxylin techniques. Stained slides were then digitally scanned and the depth of the hump was measured and denoted using Aperio ImageScope software. Image-J software was used to measure standard length (mm) and nuchal hump area (mm 2 ) of the two sections for each fish. Gene expression profiling Three sexually mature males from each temperature treatment were selected for gene expression profiling using RNA sequencing (RNA-Seq) and euthanized with MS-222. Each male had a corresponding full-sibling in the opposing dietary treatment. Image-J software was used to measure standard length (mm) and nuchal hump area (mm 2 ) of each fish. Hump tissue samples were dissected from each male and preserved in RNAlater®. RNA was isolated using a method described in earlier study. Briefly, total RNA from these tissue samples was isolated using TRI-Reagent (Sigma Inc., St. Louis, MO, USA). Tissue samples were homogenized in TRI-Reagent followed by addition of 200 µl/ml chloroform and the samples vigorously shaken and subjected to centrifugation at 12,000 g for 5 min at 4 °C. Total RNA was further purified using RNeasy mini RNA isolation kit (Qiagen, Valencia, CA, USA). Residual DNA was eliminated by performing column DNase digestion at 25 °C for 15 min. Total RNA concentration was determined using a Qubit 2.0 fluorometer (Life Technologies, Grand Island, NY, USA). RNA quality was verified on an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) to confirm that RIN scores were above 8.0 prior to sequencing. Differentially expressed gene analyses RNA-Seq was performed upon libraries constructed using the Illumina TruSeq library preparation system (Illumina, Inc., San Diego, CA, USA). RNA libraries were sequenced as 150bp pair-end fragments using Illumina Novaseq system (Illumina, Inc., San Diego, CA, USA). Short sequencing reads were mapped to a reference X. multilineatus genome using Tophat2 (Reference PMID: 23618408). Gene expression profiling was conducted by quantifying sequencing reads that were mapped to exons using Subread package featureCount function (reference PMID: 24227677, 23558742). Differentially expressed genes were identified using R-Bioconductor package edgeR with a statistical cutoff as |Log 2 Fold Change| >1, False Discovery Rate < 0.05. Principle Component Analyses (PCA) was performed using R prcomp function, using normalized and centered gene expression counts (22287627). Growth and size of the nuchal hump To determine the effects of temperature on the growth and size of the nuchal hump, fish from temperature treatments were photographed at 30, 100, 210 and 330 days of age to capture nuchal hump developmental rates. The area of the nuchal hump was measured at each age interval using Image J software (Figure 1A). A period of Diet Restriction was imposed for one month which consisted of only a single daily feeding of flake food. All males placed on restricted diet were over 400 days old and sexually mature at the time of the restriction. Only full sibling males were used in the analyses to control for genetic effects. All statistics regarding hump growth and size were calculated using R software. In addition to treatment, we examined whether adult size and early growth rate influenced the development of humps. To determine what factors influenced the size of the adult nuchal humps, a generalized least squares model was used with a random effect of dam. Treatment was the fixed effect with male size at maturity and early growth rate as covariates. The intraclass correlation coefficient (ICC) was calculated to determine the proportion of total variability in hump size that was attributable to differences among dams. The models were run with the restricted maximum likelihood estimation method (REML) to correct for degrees of freedom. Results from performing ANOVA on the model and coefficient estimates for each fixed effect are reported. Growth curves for nuchal hump size were fitted to the observed data for each treatment using the von Bertalanffy growth function (VBGF), Gompertz (Gomp) and a logistic (Log) curves with the R package “fishmethods” and the function growth. The growth function with the lowest residual sum-of-squares was considered the best fit and was used to calculate asymptotic hump size at maturity (length at which male growth stops) for both treatments. Growth curves were compared by treatment using a pairwise permutation test with the R function compareGrowthRates in the package “fishmethods”. Changes in nuchal hump size before and after a period of diet restriction were compared for each treatment by using a generalized least squares model with a random effect of dam plus a correlation component accounting for repeated measures among males before and after diet restriction. The intraclass correlation coefficient (ICC) was calculated to assess the proportion of total variability in hump size was attributable to differences among mothers. The degree of correlation between repeated measures per male was estimated by the compound symmetry parameter (rho). Temperature treatment and diet restriction status (either pre- and post-restriction) were used in all models as independent variables and an interaction term between treatment and diet restriction Status was included to determine whether the magnitude of change in hump size after diet restriction was dependent on treatment. Nuchal hump size was log transformed within the model to meet the assumption of normality of the residuals. Results from R function Anova are reported. Results Histology Histological examination shows the hump is primarily composed of adipose tissue (Fig. 1). Stored lipid droplets within adipocytes are cleared during histological processing leaving only the plasma membrane and peripherally located nucleus visible (Fig. 1b). Gene Expression Within the Hump Males reared in two different temperatures differentially expressed 326 genes in nuchal hump tissue with a false detection rate (FDR) less than 0.05. Of these, 28 genes were differentially expressed with FDRs of less than 0.0005 (Tier 1) and another 53 with FDRs between 0.005 and 0.0005 (Tier 2). Genes of particular interest regarding fat deposition and temperature acclimation within these two tiers are listed in Tables 1 and 2. For example, G0S2 that inhibits triglyceride catabolism was upregulated in nuchal hump tissue in the cold environment, ACP6 that regulates lipid metabolism was upregulated in the warm environment, and WARS-1 which reduces fat stores when overexpressed was upregulated in the warm environment. Upregulation of PGC-1α in the cold environment is interesting as this gene is linked to thermogenesis in mammals by stimulating the catabolism of brown adipose tissue. Table 1 Tier 1 genes differentially expressed in nuchal hump tissue between temperature treatments. Genes included in Tier 1 had a false detection rate (FDR) less than 0.0005 Gene Related Function Upregulated Ref G0S2 Acts as a molecular brake on triglyceride (TG) catabolism. Cold Heckmann et al. 2012; Yang et al. 2010 ACP6 An enzyme that regulates lipid metabolism in the mitochondria. Warm Hiroyama and Takenawa 1999 HSPA12A Heat shock protein in the HSP70 family involved in molecular chaperoning and preventing protein denaturing in response to stressors. HSP70 upregulation also inducible by cold shock. Cold Hu et al. 2022, Reid et al. 2022 PGC-1α Highly expressed in brown fat in mammals, strongly induced by cold exposure, linking environmental stimulus to adaptive thermogenesis. Cold Cheng et al. 2016; Liang and Ward 2006 WARS-1 Reduction of WARS-1 expression in C. elegans increased fat stores and starvation survival. Warm Webster et al. 2017 Table 2 Tier 2 genes differentially expressed in nuchal hump tissue between temperature treatments. Genes included in Tier 2 had a false detection rate (FDR) between 0.0005 and 0.005 Gene Related Function Upregulated Ref IGFBP3 Insulin-like growth factor binding protein Cold Kim 2013 APOD Atypical apolipoprotein with broad tissue distribution involved in lipid homeostasis. Cold Perdomo et al. 2010 Col7a1 Codes for collagen alpha 1 protein, which occurs in the basement membrane beneath stratified squamous epithelia. Cold Koca et al. 2023 APOA1 Structural component of high-density lipoprotein (HDL) or “good” cholesterol in plasma Cold Bandarian et al. 2013 SFRP2 Reported in pre-adipocytes, involved in adipocyte differentiation; secreted by adipose tissue Cold Crowley et al. 2016 Adult Hump Size The model that best explained variation in hump size at 330 days of age (all males sexually mature) included treatment, male size (length) at sexual maturity and early growth rate with dam as a random effect (Table 3). The intraclass correlation coefficient (ICC) revealed that 35.3% of the total variability in hump size was attributable to variability associated with dam. The negative coefficient for temperature treatment (-1.01) produced by the model indicates that hump size is predicted to be smaller in the warm treatment than the cold when other fixed variables were held constant. Mean hump size at sexual maturity for males from the warm treatment was 4.60 ± 2.02 mm 2 , while mean size of humps for males in the cold treatment was 9.62 ± 2.27 mm 2 . The coefficient for male size at maturity (0.54) reveals a positive relationship between hump size and body size, meaning larger males had larger humps when other variables were held constant. The model produced a negative coefficient (-22.5) for early growth rate indicating a negative relationship with hump size at maturity, meaning males that grew faster as juveniles developed smaller humps by the time they reached sexual maturity compared with slower growing males. Table 3 ANOVA Results generated by the best model for explaining variation in nuchal hump size at maturity Variable Numerator DF Denominator DF F-value P-value (Intercept) 1 24 488.3557 <.0001 Treatment 1 24 126.5105 <.0001 Size at maturity (L) 1 24 35.9872 <.0001 Early Growth Rate 1 24 5.0096 0.0326 Regarding the growth of the hump, the logistic growth curve had the lowest residual sum of squares and was the best fit compared to the von Bertalanffy and Gompertz for both cold and warm treatments (Table 4). Growth curves for the nuchal hump were significantly different between temperature treatments (P = 0.005) and reflect a pattern whereby males from the warm environment grew humps at a faster rate early in development but had smaller humps by the time they reached maturity than males from the cold environment (Fig. 3). Table 4 Nuchal hump growth curve selection data. The logistic (Log) curve had the lowest residual sum of squares (RSS) compared to the von Bertalanffy (VBF) and Gompertz (Gomp) curves in both the warm (A) and cold (B) treatments. A) Model RSS k ± SE VBF: Linf*(1-exp(-K*(t-t0))) 217.9 0.55 ± 0.15 Gomp: Linf*exp(-exp(-K*(age-t0))) 214.5 0.77 ± 0.17 Log: Linf/(1+exp(-K*(age-t0)) 212.5 1.12 ± 0.24 B) Model RSS k ± SE VBF: Linf*(1-exp(-K*(t-t0))) 432.5 0.19 ± 0.03 Gomp: Linf*exp(-exp(-K*(age-t0))) 383.1 0.39 ± 0.06 Log: Linf/(1+exp(-K*(age-t0)) 376.9 0.57 ± 0.07 Effects of Diet Restriction on Hump Size Following a one-month period of diet restriction, variation in nuchal hump size was explained best by a model that included treatment, diet restriction status (pre- or post-) and an interaction term between them (Table 5). The model which included dam as a random effect and a correlation component to account for repeated measures was the best fit among the three models compared (P < 0.001, Table 5) and variation attributed to dam explained 13.8% of the total variability in hump size in this model as revealed by the intraclass correlation coefficient (ICC). The model produced a high correlation value (rho = 0.805) which suggests that repeated measures for each male are more similar relative to the variability across different males. Negative regression coefficients for treatment (-1.02) and diet restriction status (-0.14) indicate that not only were humps estimated to be smaller post-restriction than pre-restriction, but also smaller in the warm treatment versus the cold. More importantly, the coefficient for the interaction term (-0.45) indicates there is a synergistic effect between treatment and diet restriction, namely that being in the warm environment leads to even smaller post-restriction humps than being in the cold environment. Males from the cold treatment had a mean post-restriction hump size of 8.68 ± 2.58 mm 2 versus a pre-restriction size of 9.86 ± 2.46 mm 2 , representing a reduction in mean hump size of 1.18 mm 2 after diet restriction. Males from the warm treatment had a mean post-restriction hump size of 1.97 ± 0.47 mm 2 versus a pre-restriction size of 3.62 ± 1.06 mm 2 , which is a reduction in mean hump size of 1.64 mm 2 after diet restriction (Figure 4). Table 5 ANOVA results generated by the best model for explaining variation in nuchal hump size before and after diet restriction A Numerator DF Denominator DF F-value P-value (Intercept) 1 33 811.60 < 0.001 Treatment 1 33 127.01 <0.001 Restriction status 1 33 77.24 <0.001 Treatment : Restriction status 1 33 40.69 <0.001 Discussion Nuchal hump development in Xiphophorus multilineatus males in the laboratory provides a unique opportunity to explore the mechanisms of development and function of this trait. The findings of this study show that the hump consists of adipose tissue, that males reared in a colder environment (20° C) develop larger humps controlling for overall body size than males reared in a warmer environment (25° C) and that the differential gene expression pattern in the hump between temperature treatments suggests increased fat deposition in the cold environment versus the warm. Furthermore, changes in the size of the hump due to diet restriction and environmental temperature suggest that the pattern detected is due to increased metabolism in the warm environment stimulating an increased use of fat as an energy source leading to smaller humps. The implications of these results for understanding the function of the nuchal hump in this system as well as understanding the roles of both diet and activity in human obesity are discussed. Finding that the nuchal hump is composed primarily of adipocytes suggests an energy storage function of this structure. Certainly, some vertebrates use fat stored in nuchal humps and similar dorsal structures as an energy source when dietary intake is insufficient to maintain metabolic rates and the amount of fat stored in the hump may reflect seasonal shifts in food quality and quantity (Alexander et al. 1968, Giles et al. 2015, Bengoumi et al. 2005). Within fish taxa, there are examples of nuchal humps composed primarily of adipose tissue, most notably within the species flocks of cichlids that occur in East African rift lakes (Takahashi 2018, Lecaudey et al. 2019). Yet even among this sub-group of fish, energy storage does not appear to be a primary function for humps. Takahashi (2018) found that the nuchal hump of Cyphotilapia gibberosa was formed by a thickening of the hypodermis through fat deposition, but the thickness was not correlated with body fitness in females and only slightly positively correlated in males. In other words, well-conditioned individuals did not store extra lipid in the humps and poorly conditioned individuals did not consume lipid from their humps. In the current study, however, the size of the nuchal hump was smaller after diet restriction regardless of temperature, suggesting that males were able to utilize stored fat in the nuchal hump as an energy source to make up for reduced caloric intake. If it is assumed that the function of the nuchal hump is for energy storage in X. multilineatus , how can the pattern of larger humps on the males reared in the cold versus the warm treatments be explained? Fish acclimated to cold temperatures typically increase their storage of lipids in various body tissues including subcutaneous, liver, and muscle. Fish acclimated to warmer temperatures, on the other hand, increase the use of lipids as a direct energy source and store less of it in tissues (Stone et al. 1981, Egginton et al. 1989). There is evidence that at extremely high temperatures, fish may shift to storing fat as seen in the mummichog ( Fundulus heteroclitus, Moerland et al. 1981), but fat storage in this case occurred specifically in the liver, not skeletal muscle or hypodermal tissue. The X. multilineatus males in the current study were not subjected to critical maximum or minimum temperatures and followed the typical pattern of temperature related fat utilization in fish. The species of swordtail that forms the nuchal hump in nature, X. birchmanni , is found at warmer temperatures on average than a closely related species that does not develop nuchal humps, X. malinche (Rauchenberger et al. 1990). These two species often occupy the same stream systems and form hybrid zones (Culumber et al. 2012). When considering the potential influence of temperature alone, hump formation in X. birchmanni seems less likely than in X. malinche . Therefore, the natural occurrence of nuchal humps in X. birchmanni suggests that factors other than temperature are more influential in the development of humps in this species. For example, correlated environmental differences in relation to food resources (higher quality diets) could potentially be more important. The results of this study suggest that males reared in a warmer environment either utilize more fat than males in the cold, or have simply used all the energy available to them with no excess to store in the nuchal humps. The first possibility can be considered the “metabolic difference hypothesis” and is supported by the finding that males from the warm environment used fat stores in the nuchal hump at a faster rate when placed on a restricted diet than males reared in the cold. In addition, seven of the top-tiered, differentially expressed genes in the nuchal hump of X. multilineatus males are linked to lipid metabolism (Tables 1 & 2) with expression patterns suggesting increased fat deposition in cold males versus warm. Overexpression of the G0S2 switch gene, for example, reduces the rate of lipolysis in adipocytes by acting as a molecular brake on triglyceride catabolism (Yang et al. 2010). A higher expression of G0S2 in X. multilineatus males from the cold treatment makes sense since these males developed larger humps. Likewise, males from the cold treatment had lower expression of the WARS-1 gene, which when inactivated in Caenorhabditis elegans leads to increased fat stores and increased starvation survival (Webster et al. 2017). Interestingly, a gene that is highly expressed in brown fat in mammals ( PGC-1α ) when induced by cold exposure (Cheng et al. 2018) was also overexpressed in X. multilineatus males from the cold treatment. This gene links colder environmental temperature to adaptive thermogenesis by stimulating the catabolic burning of brown fat in mammals, however fish do not develop brown fat. If the expression pattern of this gene is similar between fish (which are not capable of thermogenesis) and mammals when considering the effect of environmental temperature, its role in thermogenesis among mammals may be a coopted function. Males from the warm environment grew humps at a faster rate early in development, yet males from the cold developed larger humps by the time they reached sexual maturity (Figure 3). This is a similar pattern to that of growth of male body size in general, in that X. multilineatus males from the warm environment grew faster as juveniles but reached a smaller adult size than males from the cold (Tompkins et al. in prep). This similarity is not surprising since hump size and body size had a significant positive relationship in the current study. Tompkins et al. (2021) suggested that diet could be a primary difference between the laboratory and natural environment for X. multilineatus, inducing development of the nuchal hump in the laboratory. An additional factor to consider, however, is that the environment in which males were reared (isolated, in 2.5 l aquarium) does not allow for activity levels that would be more common in a natural environment. Swordtail males spend a large proportion of their time interacting with other males and courting females in the wild (Rios-Cardenas et al. 2010), and all these energetically costly behaviors were absent in the laboratory setting. The hypothesis that the reduced activity levels in the rearing environment is partially responsible for the development of the nuchal hump is supported by the observation that X. multilineatus males from large mesocosm in our laboratory appear to be much less likely to develop a nuchal hump than males that are reared individually. Comparisons of the activity levels of the males reared individually in the warm and cold environments also lend support to this hypothesis (Fig. S2). It is also possible that hump size influences activity level due to the hump being a cumbersome feature. However, the shape of X. birchmanni with its increased anterior body depth due to having a nuchal hump (the only species in the clade to form them in the wild) imparts improved endurance swimming performance compared with the dorsoventrally narrower shape of the species it naturally hybridizes with, X. malinche (Johnson et al. 2014). Therefore, it seems more likely that activity influences hump size rather than vice versa. There are potential benefits of X. multilineatus having the ability to develop a nuchal hump that could be considered. We previously detected female mate preference for males with nuchal humps in X. multilineatus even though the trait does not occur in the wild (Tompkins et al. 2021). We suggest that the preference we detected may be similar to what has been suggested for X. birchmanni (Rosenthal et al. 2003) and reflect a more general preference for larger males. Alternatively, if the hump developed in X. multilineatus in the wild it could lead to improved swimming performance by increasing anterior body depth which minimizes drag (Johnson et al. 2014). Compartmentalized fat depots can have dual functionality in other vertebrates. Camels, for example, use the fat in their hump as an energy source, but one hypothesis suggests camels may concentrate body fat in the hump to avoid overheating if the fat was evenly distributed throughout the subcutaneous layer covering the body (Kassa 2016). However, the fact that the humps are not known to form in the natural environments of X. multilineatus suggests that selection for these other functions would be minimal if nonexistent. Future studies of the functions of depositing fat in the nuchal hump in swordtail fishes, in addition to energy storage, should be considered. Evidence that swordtail fish use the nuchal hump for energy storage with the humps composed of adipose tissue is interesting considering the importance of finding evolutionary animal models for studying diet-induced obesity. The development of a nuchal hump in X. multilineatus under laboratory conditions after being fed a high-calorie diet may be a condition similar to that observed in humans (Schartl and Lu 2024). One of the other aspects of the laboratory environment that appears to promote nuchal hump formation is reduced activity levels, also an important factor in human obesity (Chin et al. 2016). Finally, the influence of environmental temperature on rates of obesity in humans has also been established (Trentinaglia et al. 2021, Moellering and Smith 2012) and cold induced thermogenesis in mammals is associated with reduced risk of diabetes (Schrauwen et al. 2016, Horino et al. 2022). Considering the influence of temperature, diet and potentially activity levels on the development of the nuchal hump in X. multilineatus , this model system has the potential to provide future insight into the mechanisms involved in human obesity that other model systems cannot. Variation across species within Xiphophorus in the propensity to develop nuchal humps allows for the consideration of this trait in an evolutionary perspective, including unique opportunities to determine genetic variant site genotypes using hybrids and backcross hybrids of different species, even from the most phylogenetically distant branches of the genus (Shartl and Lu 2024). Current work is examining the development of the nuchal hump in backcross hybrids of X. multilineatus and X. couchianus, where we can determine genetic variant site genotypes, profile their gene expression, and begin to disentangle genetic networks involved in fat deposition. Declarations Acknowledgements We thank Grace Vance for collecting the activity level data and Mrs. Korri Weldon for technical assistance. Funding Funding for this project was provided by a grant from the Ohio University Research Committee, a NIH Shared Instrument grant S10OD030311 (S10 grant to NovaSeq 6000 System), and CPRIT Core Facility Award (RP220662). Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contributions The first draft of the manuscript was written by Keith B. Tompkins, Yaun Lu and Molly R. Morris and all authors commented on previous versions of the manuscript. Photography, histology, and statistical analysis of variability in nuchal hump size was performed by Keith B. Tompkins. RNA-seq data was generated in the Genome Sequencing Facility, which is supported by UT Health San Antonio, NIH-NCI P30 CA054174 (Cancer Center at UT Health San Antonio). RNA-seq data analysis was performed by Yuan Lu. All authors read and approved the final manuscript. Data Availability Raw data are available on request. Ethics Statement The protocols followed for handling and care of all fish involved this study have been approved by the Institutional Animal Care and Use Committee of Ohio University in accordance with AAALAC, International. NIH animal use assurance #A3610-01. Collecting Permit: Permiso de Pesca de Fomento No. DGOPA 18317.271113.8982. References Alexander A, Player IC (1965) A note on the nuchal hump of Ceratotherium simum (Burchell). The Lammergeyer , vol 3, no 2 Ballinger MA, Andrews MT (2018) Nature’s fat-burning machine: brown adipose tissue in a hibernating mammal. 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Advances in Physiology Education , 30:131-265 Liotta MN, Abbott JK, Morris MR, Rios-Cardenas, O (2021) Antagonistic selection on body size and sword length in a wild population of the swordtail fish, Xiphophorus multilineatus : Potential for intralocus tactical conflict. Ecology and Evolution , 11:3941-3955 Manamendra-Arachchi K, de Silva A, Amarasinghe T (2006) Description of a second species of Cohpotis (Reptilia:Agamidae) from the highlands of Sri Lanka. Lyriocephalus , vol 6, sup 1, 1-8 Moerland TS, Sidell BD (1986) Biochemical responses to temperature in the contractile protein complex of striped bass Morone saxatilis. Journal of Experimental Zoology 238:287-295. Moellering DR, Smith DL (2012) Ambient temperature and obesity. Curr. Obes. Rep ., 1: 26-34 Moradi S, Mirzae, K, Maghbooli Z, Abdurahman AA, Keshavarz, SA (2018) Variants in the PPARGC1A gene may influence the effect of fat intake on resting metabolic rate in obese women. Lipids , 53(3) Perdomo G, Kim DH, Zhang T, Qu S, Thomas EA, Toledo FGS, Slusher S, Fan Y, Kelley DE, Dong HH (2010) A role of apolipoprotein D in triglyceride metabolism. J. Lipid Research , 51:1298-1311 Quinn T, Foote CJ (1994) The effects of body size and sexual dimorphism on the reproductive behaviour of sockeye salmon ( Oncorhynchus nerka ). Animal Behaviour 48:751-761 Rauchenberger M, Kallman KD, Morizot DC (1990) Monophyly and geography of the Rio Panuco basin swordtails (genus Xiphophorus ) with descriptions of four new species. American Museum Novitates , No.2975 Reid CH, Patrick PH, Rytwinski T, Taylor JJ, Willmore WG, Reesor B, Cooke SJ (2022) An updated review of cold shock and cold stress in fish. J. Fish Biology , 100: 1102–1137 Rios-Cardenas O, Darrah A, Morris MR (2010) Female mimicry indirectly enhances a male sexually selected trait; what does it take to fool a male? Behaviour ,147(11):1443-1460 Rometsch SJ, Torres-Dowdall J, Machado-Schiaffino G, Karagic N, Meyer A (2021) Dual function and associated costs of a highly exaggerated trait in a cichlid fish. Ecology and Evolution , 11, 17496-17508 Rosenthal GG, de la Rosa-Reyna XF, Kasianis S, Stephens MJ, Morizot DC, Ryan MJ, Garcia de Leon FJ (2003) Dissolution of sexual signal complexes in a hybrid zone between the swordtails Xiphophorus birchmanni and Xiphophorus malinche (Poeciliidae). Copeia , 2003(2): 299-307 Schartl M, Lu Y (2024) Validity of Xiphophorus fish as models for human disease. Disease Models & Mechanisms , 17(1) Schrauwen P, van Marken WD, Lichtenbelt W (2016) Combatting type 2 diabetes by turning up the heat. Diabetologia, 59(11): 2269-2279 Susuki K, Masaki I, Yosuke K, Masahide K, Yasuaki T, Shinji A, Hideaki K (2014) Dorsal hump morphology in pink salmon ( Oncorhynchus gorbuscha ). J. of Morphology , 275:514-527 Takahashi T (2018) Function of nuchal humps of a cichlid fish from Lake Tanganyika: inferences from morphological data. Ichthyological Research , 65, 316-323 Tompkins KB, Lott MS, Rios-Cardenas O, Jash S, Morris MR (2021) Metabolic growth hypothesis for the evolution of the nuchal hump in swordtail fishes. Environmental Biology of Fishes , 104 , 1195-1206 Trentaglia MT, Parolini M, Donzelli F, Olper A (2021) Climate change and obesity: a global analysis. Global Food Security , 29: 100539 Ward DL, Ward MB (2020) What’s in the hump of the humpback chub? Western North American Naturalist , 80: 98-104 Webster CM, Pino EC, Carr CE, Wu L, Zhou B, Ceillo L, Kacergis MC, Curran SP, Soukas AA (2017) Genome-wide RNAi screen for fat regulatory genes in C. elegans identifies a proteostasis-AMPK axis critical for starvation survival. Cell Reports , 20: 627-640 Yang X, Lu X, Lombès M, Rha GB, Chi Y, Guerin TM, Smart EJ, Liu J (2010) The G0/G1 switch gene 2 regulates adipose lipolysis through association with adipose triglyceride lipase. Cell Metab ., 11: 194-205 Additional Declarations No competing interests reported. Supplementary Files SupplemetaryInformation.docx Cite Share Download PDF Status: Published Journal Publication published 31 Jul, 2025 Read the published version in Fish Physiology and Biochemistry → Version 1 posted Editorial decision: Revision requested 17 May, 2025 Reviews received at journal 14 May, 2025 Reviewers agreed at journal 22 Nov, 2024 Reviewers invited by journal 22 Nov, 2024 Editor assigned by journal 22 Nov, 2024 Submission checks completed at journal 10 Nov, 2024 First submitted to journal 04 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5389356","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":383461094,"identity":"6b47f947-f8e5-4d69-aafd-5404b08bfb3d","order_by":0,"name":"Keith Tompkins","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIie3QsQrCMBCA4euSKdD1SmmfoRAoFcRnSSk4+QCdpCDoUp3r5CsUfIErGVyKc1fxBTJ2ELR2FCR1c8g/hSMfBwdgs/1hCCAJewxd4EDDwCmMhMGNZmUivGI6ce6UszytiY8DM/FOG0kdRyEu+4Y0zIOaDMRnTDZVgmHcXocHLIWRhIxLhcOWuFtFioNKJxBXqwfD9FyN5GkmPnsfeSA1joTMxNtKSV6JAts2aqooE0cTQUWZxn4durtSaJ0vgoOJfBT99t1ms9lsX3oB2PpHynpqE9QAAAAASUVORK5CYII=","orcid":"","institution":"Ohio University","correspondingAuthor":true,"prefix":"","firstName":"Keith","middleName":"","lastName":"Tompkins","suffix":""},{"id":383461095,"identity":"ea8cb3e5-ea3d-463c-9841-40dc5d2c7b83","order_by":1,"name":"Will Boswell","email":"","orcid":"","institution":"Texas State University","correspondingAuthor":false,"prefix":"","firstName":"Will","middleName":"","lastName":"Boswell","suffix":""},{"id":383461096,"identity":"3670f892-c286-432a-b276-a75099c8e2f3","order_by":2,"name":"Kang Du","email":"","orcid":"","institution":"Texas State University","correspondingAuthor":false,"prefix":"","firstName":"Kang","middleName":"","lastName":"Du","suffix":""},{"id":383461097,"identity":"4a864cf0-6f94-4fe8-95c2-0a8749568e59","order_by":3,"name":"Zhao Lai","email":"","orcid":"","institution":"University of Texas Health San Antonio","correspondingAuthor":false,"prefix":"","firstName":"Zhao","middleName":"","lastName":"Lai","suffix":""},{"id":383461098,"identity":"58306bbc-4565-483b-879a-a91e9aaa8e49","order_by":4,"name":"Yuan Lu","email":"","orcid":"","institution":"Texas State University","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Lu","suffix":""},{"id":383461099,"identity":"0953728d-db57-4e97-a799-c370de994837","order_by":5,"name":"Molly R Morris","email":"","orcid":"","institution":"Ohio University","correspondingAuthor":false,"prefix":"","firstName":"Molly","middleName":"R","lastName":"Morris","suffix":""}],"badges":[],"createdAt":"2024-11-04 15:38:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5389356/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5389356/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10695-025-01539-2","type":"published","date":"2025-07-31T16:13:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70092653,"identity":"395ff68c-623d-4291-8652-8651f617aff1","added_by":"auto","created_at":"2024-11-28 09:09:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":639794,"visible":true,"origin":"","legend":"\u003cp\u003eHistology of \u003cem\u003eX. multilineatus\u003c/em\u003e nuchal hump: a) Transverse section through nuchal hump of male X. multilineatus. Bar represents depth of the hump as determined by measuring the fish from a 2D image taken before tissue sampling; b) adipocytes within nuchal hump (N = nucleus, PM = plasma membrane; H\u0026amp;E); c) a male from the warm treatment with a relatively small nuchal hump for its size (0.099 mm\u003csup\u003e2 \u003c/sup\u003e/ mm); d) a male from cold treatment with a relatively large hump (0.275 mm\u003csup\u003e2\u003c/sup\u003e / mm)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5389356/v1/0bae0281da8e2075df817c9c.png"},{"id":70092646,"identity":"e4404818-cfe0-4007-bc5e-f9b86d8b4bd7","added_by":"auto","created_at":"2024-11-28 09:09:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":85385,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 3 \u003c/strong\u003eComparison of nuchal hump growth between temperature treatments. Both growth curves are plotted with a logistic function and were significantly different (P = 0.005)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5389356/v1/aeb4efc6beb23c2523e63dcf.png"},{"id":70092647,"identity":"35d8cb6c-af1f-41b9-9855-6670b86a5ce3","added_by":"auto","created_at":"2024-11-28 09:09:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":87856,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 4 \u003c/strong\u003eMean nuchal hump size per treatment before (pre) and after (post) a one-month period of diet restriction\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5389356/v1/b7e17aef23d66b286a4a79e6.png"},{"id":88268274,"identity":"eabf4973-2fe4-4ecc-aa18-051c2f4dac62","added_by":"auto","created_at":"2025-08-04 16:50:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1302169,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5389356/v1/29f4e111-09c8-426f-a9f5-6d5180f83f84.pdf"},{"id":70092654,"identity":"3d90f804-79d3-44ac-8e3a-73e10daa5679","added_by":"auto","created_at":"2024-11-28 09:09:43","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":214182,"visible":true,"origin":"","legend":"","description":"","filename":"SupplemetaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5389356/v1/4b7d941a09a43850fe4913f8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Composition and Function of the Nuchal Hump of Male Xiphophorus multilineatus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNuchal humps, broadly defined as a tissue mass located dorsal posterior to the head, are present across multiple vertebrate taxa. The tissue composition of nuchal humps varies among vertebrate species as does the development and function of this structure (Alexander et al. 1965, Cave et al. 1958, Manamendra-Arachchi et al. 2006, Susuki et al. 2014, Ward et al. 2020). In sexually monomorphic fish species where the hump is persistent, such as \u003cem\u003eGila cypha\u003c/em\u003e, an anti-predatory function is likely. Both sexes would benefit from the presence of a nuchal hump due to increased body depth which can limit the ability of predators to capture individuals once the hump develops (Douglas 1993, Portz et al. 2004, Ward et al. 2020). Among sexually dimorphic species, nuchal humps are often sexually selected traits and proposed functions include species recognition, sex recognition, and indication of mate quality (Barlow et al. 1997, Takahashi 2018, Rometsch et al. 2021). There is also evidence to suggest that the nuchal hump may play a role in energy storage, based on increased expression of genes known to promote adipogenesis and cell proliferation within nuchal hump tissue of the African cichlid \u003cem\u003eCyrtocara moorii\u0026nbsp;\u003c/em\u003e(Lecaudey et al. 2019), and enlargement of the hump in another African cichlid, \u003cem\u003eCyphotilapia gibberosa\u003c/em\u003e commonly known as Mpimbwe Blue Frontosa, was due to hypertrophy of the hypodermal layer and increased fat storage (Takahashi 2018).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The nuchal hump is a diagnostic trait for the swordtail species \u003cem\u003eXiphophorus birchmanni (\u003c/em\u003eRauchenberger et al. 1990) and may play a role in mate choice (Rosenthal et al. 2003) as well as influence swimming endurance in relation to the sword (Johnson et al. 2014). Nuchal humps are not known to occur in the wild for any of the other \u003cem\u003eXiphophorus\u003c/em\u003e fishes. The development of this trait in the laboratory occurred in \u003cem\u003eX. multilineatus\u003c/em\u003e adult males that were sampled from a wild population and brought to the laboratory as well as in males that were reared from birth in the laboratory (Tompkins et al. 2021). Females prefer males with humps versus males without (Tompkins et al. 2021), suggesting a potential benefit for the hump via sexual selection if they were to develop in the wild. One aspect of the laboratory environment that promotes the hump is diet, as \u003cem\u003eX. multilineatus\u003c/em\u003e males reared on high quality diets (i.e. higher fat content in addition to protein) were more likely to develop larger humps than males reared on low quality diets (Tompkins et al. 2021).\u003c/p\u003e\n\u003cp\u003eTemperature influences fat utilization in fish by stimulating shifts between catabolizing fat as an energy source at warmer acclimation temperatures to storing fat in liver and muscle tissues at colder acclimation temperatures (Stone et al. 1981, Egginton et al. 1989). Accordingly, we hypothesize that temperature influences the process of hump development. Therefore, to further examine the possible function of the nuchal hump in \u003cem\u003eX. multilineatus\u003c/em\u003e, we used a split-brood design to examine development of the hump in the laboratory under two different temperature regimes. We first addressed the following questions: 1) What is the tissue composition of the nuchal hump in \u003cem\u003eX. multilineatus\u003c/em\u003e? While the presence of lipid droplets has been detected in the nuchal hump of \u003cem\u003eX. multilineatus\u0026nbsp;\u003c/em\u003e(Tompkins et al. 2021), it is not known if the hump is composed primarily of adipose tissue.\u003cem\u003e\u0026nbsp;\u003c/em\u003e2) Are there differences in the genes expressed in hump tissue in males reared in a cold environment versus warm? 3) How does rearing temperature influence the allometric growth of the nuchal hump? Second, we reduced the diet of adult males for approximately one month to determine whether 4) the size of the hump influenced by a reduction in diet and 5) is the influence of diet reduction consistent across temperature treatments?\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eAnimals and Sample Collection\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eX. multilineatus\u003c/em\u003e males used to test the effects of rearing temperature on the development of nuchal humps, through both RNA sequencing (RNA-Seq) analyses and multivariate analysis of hump size and growth rate, were obtained by isolating 50 pregnant females from an existing community tank with only courter male \u003cem\u003eX. multilineatus\u003c/em\u003e located in the Morris Lab. Courter males are one of two alternative reproductive tactics (ARTs) found in this species. ARTs in \u003cem\u003eX. multilineatus\u003c/em\u003e are genetically influenced by variation in copy number of \u003cem\u003emc4r\u003c/em\u003e B alleles on the Y chromosome (Lampert et al. 2010). The ARTs are dimorphic for several traits including body size (courter males are larger than sneakers) and reproductive behavior where courter males use only courtship displays to attract females and sneaker males use either courtship or forced copulation to gain access to females (Liotta et al. 2021). Courter males in the lab were observed to have larger humps relative to their body size than sneaker males, so only males with a courter lineage were used in this study due to the higher propensity of humps forming on courter males. Females were allowed to drop fry, and the fry were split between two environmental chambers. Environmental chambers were set at 25\u0026deg;C (warm treatment) and 20\u0026deg;C (cold treatment). Fry were transferred to individual 2.5 L tanks in respective environmental chambers at 30 days of age: 72 fry in warm, 72 fry in cold. All fry were fed a high-quality diet of Ken\u0026rsquo;s Spirulina flake food in the morning daily, and brine shrimp in the afternoon five days a week. The flake food was the same high-quality diet used by Tompkins et al. (2021) that promoted growth of larger humps. Each tank was equipped with a Whisper\u0026reg; Powerfilter that circulated the water and agitated the water surface. Measures of dissolved oxygen saturation were similar between treatments ((N = 8 per treatment, Cold = 100.8 \u0026nbsp; \u0026plusmn; 1.1 %; Warm = 97.7 \u0026plusmn; 1.4 %).\u003c/p\u003e\n\u003cp\u003eHistology\u003c/p\u003e\n\u003cp\u003eMales used for histology were obtained from a community tank with only \u003cem\u003eX. multilineatus\u003c/em\u003e located in the Morris Lab. Fish in the community tank were fed a high-quality diet of Ken\u0026rsquo;s Spirulina\u0026reg; flake food seven days per week and \u003cem\u003eArtemia\u003c/em\u003e sp. nauplii five days per week at a rate that was completely consumed within five minutes. The community tank was maintained at room temperature between 21-22\u0026deg;C. Males were euthanized with MS-222 and transverse tissue sections were taken from the nuchal region and preserved in 10% neutral buffered formalin. Tissue samples were processed and stained at the Ohio University Histology Core Facility using standard eosin and hematoxylin techniques. Stained slides were then digitally scanned and the depth of the hump was measured and denoted using Aperio ImageScope software. Image-J software was used to measure standard length (mm) and nuchal hump area (mm\u003csup\u003e2\u003c/sup\u003e) of the two sections for each fish.\u003c/p\u003e\n\u003cp\u003eGene expression profiling\u003c/p\u003e\n\u003cp\u003eThree sexually mature males from each temperature treatment were selected for gene expression profiling using RNA sequencing (RNA-Seq) and euthanized with MS-222. Each male had a corresponding full-sibling in the opposing dietary treatment. Image-J software was used to measure standard length (mm) and nuchal hump area (mm\u003csup\u003e2\u003c/sup\u003e) of each fish. Hump tissue samples were dissected from each male and preserved in RNAlater\u0026reg;. RNA was isolated using a method described in earlier study. Briefly, total RNA from these tissue samples was isolated using TRI-Reagent (Sigma Inc., St. Louis, MO, USA). Tissue samples were homogenized in TRI-Reagent followed by addition of 200 \u0026micro;l/ml chloroform and the samples vigorously shaken and subjected to centrifugation at 12,000 g for 5 min at 4 \u0026deg;C. Total RNA was further purified using RNeasy mini RNA isolation kit (Qiagen, Valencia, CA, USA). Residual DNA was eliminated by performing column DNase digestion at 25 \u0026deg;C for 15 min. Total RNA concentration was determined using a Qubit 2.0 fluorometer (Life Technologies, Grand Island, NY, USA). RNA quality was verified on an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) to confirm that RIN scores were above 8.0 prior to sequencing.\u003c/p\u003e\n\u003cp\u003eDifferentially expressed gene analyses\u003c/p\u003e\n\u003cp\u003eRNA-Seq was performed upon libraries constructed using the Illumina TruSeq library preparation system (Illumina, Inc., San Diego, CA, USA). RNA libraries were sequenced as 150bp pair-end fragments using Illumina Novaseq system (Illumina, Inc., San Diego, CA, USA).\u003c/p\u003e\n\u003cp\u003eShort sequencing reads were mapped to a reference \u003cem\u003eX. multilineatus\u0026nbsp;\u003c/em\u003egenome using Tophat2 (Reference PMID: 23618408). Gene expression profiling was conducted by quantifying sequencing reads that were mapped to exons using Subread package featureCount function (reference PMID: 24227677, 23558742). Differentially expressed genes were identified using R-Bioconductor package edgeR with a statistical cutoff as |Log\u003csub\u003e2\u003c/sub\u003eFold Change| \u0026gt;1, False Discovery Rate \u0026lt; 0.05. Principle Component Analyses (PCA) was performed using R prcomp function, using normalized and centered gene expression counts (22287627).\u003c/p\u003e\n\u003cp\u003eGrowth and size of the nuchal hump\u003c/p\u003e\n\u003cp\u003eTo determine the effects of temperature on the growth and size of the nuchal hump, fish from temperature treatments were photographed at 30, 100, 210 and 330 days of age to capture nuchal hump developmental rates. The area of the nuchal hump was measured at each age interval using Image J software (Figure 1A). A period of Diet Restriction was imposed for one month which consisted of only a single daily feeding of flake food. All males placed on restricted diet were over 400 days old and sexually mature at the time of the restriction. Only full sibling males were used in the analyses to control for genetic effects.\u003c/p\u003e\n\u003cp\u003eAll statistics regarding hump growth and size were calculated using R software. In addition to treatment, we examined whether adult size and early growth rate influenced the development of humps. To determine what factors influenced the size of the adult nuchal humps, a generalized least squares model was used with a random effect of dam. Treatment was the fixed effect with male size at maturity and early growth rate as covariates. The intraclass correlation coefficient (ICC) was calculated to determine the proportion of total variability in hump size that was attributable to differences among dams. The models were run with the restricted maximum likelihood estimation method (REML) to correct for degrees of freedom. Results from performing ANOVA on the model and coefficient estimates for each fixed effect are reported.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Growth curves for nuchal hump size were fitted to the observed data for each treatment using the von Bertalanffy growth function (VBGF), Gompertz (Gomp) and a logistic (Log) curves with the R package \u0026ldquo;fishmethods\u0026rdquo; and the function growth. The growth function with the lowest residual sum-of-squares was considered the best fit and was used to calculate asymptotic hump size at maturity (length at which male growth stops) for both treatments. Growth curves were compared by treatment using a pairwise permutation test with the R function compareGrowthRates in the package \u0026ldquo;fishmethods\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003eChanges in nuchal hump size before and after a period of diet restriction were compared for each treatment by using a generalized least squares model with a random effect of dam plus a correlation component accounting for repeated measures among males before and after diet restriction. The intraclass correlation coefficient (ICC) was calculated to assess the proportion of total variability in hump size was attributable to differences among mothers. The degree of correlation between repeated measures per male was estimated by the compound symmetry parameter (rho). Temperature treatment and diet restriction status (either pre- and post-restriction) were used in all models as independent variables and an interaction term between treatment and diet restriction Status was included to determine whether the magnitude of change in hump size after diet restriction was dependent on treatment. Nuchal hump size was log transformed within the model to meet the assumption of normality of the residuals. Results from R function Anova are reported.\u003c/p\u003e"},{"header":"Results ","content":"\u003ch3\u003eHistology\u003c/h3\u003e\n\u003cp\u003eHistological examination shows the hump is primarily composed of adipose tissue (Fig. 1). Stored lipid droplets within adipocytes are cleared during histological processing leaving only the plasma membrane and peripherally located nucleus visible (Fig. 1b).\u003c/p\u003e\n\u003ch3\u003eGene Expression Within the Hump\u003c/h3\u003e\n\u003cp\u003eMales reared in two different temperatures differentially expressed 326 genes in nuchal hump tissue with a false detection rate (FDR) less than 0.05. Of these, 28 genes were differentially expressed with FDRs of less than 0.0005 (Tier 1) and another 53 with FDRs between 0.005 and 0.0005 (Tier 2). Genes of particular interest regarding fat deposition and temperature acclimation within these two tiers are listed in Tables 1 and 2. For example, G0S2 that inhibits triglyceride catabolism was upregulated in nuchal hump tissue in the cold environment, ACP6 that regulates lipid metabolism was upregulated in the warm environment, and WARS-1 which reduces fat stores when overexpressed was upregulated in the warm environment. Upregulation of PGC-1\u0026alpha; in the cold environment is interesting as this gene is linked to thermogenesis in mammals by stimulating the catabolism of brown adipose tissue.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u0026nbsp;Tier 1 genes differentially expressed in nuchal hump tissue between temperature treatments. Genes included in Tier 1 had a false detection rate (FDR) less than 0.0005\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14.2857%;\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47.2527%;\"\u003e\n \u003cp\u003eRelated Function\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.3846%;\"\u003e\n \u003cp\u003eUpregulated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.0769%;\"\u003e\n \u003cp\u003eRef\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14.2857%;\"\u003e\n \u003cp\u003eG0S2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47.2527%;\"\u003e\n \u003cp\u003eActs as a molecular brake on triglyceride (TG) catabolism.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.3846%;\"\u003e\n \u003cp\u003eCold\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.0769%;\"\u003e\n \u003cp\u003eHeckmann et al. 2012; Yang et al. 2010\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14.2857%;\"\u003e\n \u003cp\u003eACP6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47.2527%;\"\u003e\n \u003cp\u003eAn enzyme that regulates lipid metabolism in the mitochondria.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.3846%;\"\u003e\n \u003cp\u003eWarm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.0769%;\"\u003e\n \u003cp\u003eHiroyama and Takenawa 1999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14.2857%;\"\u003e\n \u003cp\u003eHSPA12A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47.2527%;\"\u003e\n \u003cp\u003eHeat shock protein in the HSP70 family involved in molecular chaperoning and preventing protein denaturing in response to stressors. HSP70 upregulation also inducible by cold shock.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.3846%;\"\u003e\n \u003cp\u003eCold\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.0769%;\"\u003e\n \u003cp\u003eHu et al. 2022, Reid et al. 2022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14.2857%;\"\u003e\n \u003cp\u003ePGC-1\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47.2527%;\"\u003e\n \u003cp\u003eHighly expressed in brown fat in mammals, strongly induced by cold exposure, linking environmental stimulus to adaptive thermogenesis.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.3846%;\"\u003e\n \u003cp\u003eCold\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.0769%;\"\u003e\n \u003cp\u003eCheng et al. 2016; Liang and Ward 2006\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14.2857%;\"\u003e\n \u003cp\u003eWARS-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47.2527%;\"\u003e\n \u003cp\u003eReduction of WARS-1 expression in \u003cem\u003eC. elegans\u003c/em\u003e increased fat stores and starvation survival.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.3846%;\"\u003e\n \u003cp\u003eWarm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.0769%;\"\u003e\n \u003cp\u003eWebster et al. 2017\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u0026nbsp;Tier 2 genes differentially expressed in nuchal hump tissue between temperature treatments. Genes included in Tier 2 had a false detection rate (FDR) between 0.0005 and 0.005\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12.7778%;\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49.4444%;\"\u003e\n \u003cp\u003eRelated Function\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.5556%;\"\u003e\n \u003cp\u003eUpregulated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.2222%;\"\u003e\n \u003cp\u003eRef\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12.7778%;\"\u003e\n \u003cp\u003eIGFBP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49.4444%;\"\u003e\n \u003cp\u003eInsulin-like growth factor binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.5556%;\"\u003e\n \u003cp\u003eCold\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.2222%;\"\u003e\n \u003cp\u003eKim 2013\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12.7778%;\"\u003e\n \u003cp\u003eAPOD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49.4444%;\"\u003e\n \u003cp\u003eAtypical apolipoprotein with broad tissue distribution involved in lipid homeostasis.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.5556%;\"\u003e\n \u003cp\u003eCold\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.2222%;\"\u003e\n \u003cp\u003ePerdomo et al. 2010\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12.7778%;\"\u003e\n \u003cp\u003eCol7a1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49.4444%;\"\u003e\n \u003cp\u003eCodes for collagen alpha 1 protein, which occurs in the basement membrane beneath stratified squamous epithelia.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.5556%;\"\u003e\n \u003cp\u003eCold\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.2222%;\"\u003e\n \u003cp\u003eKoca et al. 2023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12.7778%;\"\u003e\n \u003cp\u003eAPOA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49.4444%;\"\u003e\n \u003cp\u003eStructural component of high-density lipoprotein (HDL) or \u0026ldquo;good\u0026rdquo; cholesterol in plasma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.5556%;\"\u003e\n \u003cp\u003eCold\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.2222%;\"\u003e\n \u003cp\u003eBandarian et al. 2013\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12.7778%;\"\u003e\n \u003cp\u003eSFRP2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49.4444%;\"\u003e\n \u003cp\u003eReported in pre-adipocytes, involved in adipocyte differentiation; secreted by adipose tissue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.5556%;\"\u003e\n \u003cp\u003eCold\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.2222%;\"\u003e\n \u003cp\u003eCrowley et al. 2016\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAdult Hump Size\u003c/p\u003e\n\u003cp\u003eThe model that best explained variation in hump size at 330 days of age (all males sexually mature) included treatment, male size (length) at sexual maturity and early growth rate with dam as a random effect (Table 3). The intraclass correlation coefficient (ICC) revealed that 35.3% of the total variability in hump size was attributable to variability associated with dam. The negative coefficient for temperature treatment (-1.01) produced by the model indicates that hump size is predicted to be smaller in the warm treatment than the cold when other fixed variables were held constant. Mean hump size at sexual maturity for males from the warm treatment was 4.60 \u0026plusmn; 2.02 mm\u003csup\u003e2\u003c/sup\u003e, while mean size of humps for males in the cold treatment was 9.62 \u0026plusmn; 2.27 mm\u003csup\u003e2\u003c/sup\u003e. The coefficient for male size at maturity (0.54) reveals a positive relationship between hump size and body size, meaning larger males had larger humps when other variables were held constant. The model produced a negative coefficient (-22.5) for early growth rate \u0026nbsp; indicating a negative relationship with hump size at maturity, meaning males that grew faster as juveniles developed smaller humps by the time they reached sexual maturity compared with slower growing males.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003eANOVA Results generated by the best model for explaining variation in nuchal hump size at maturity\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 27.84%;\"\u003e\n \u003cp\u003eVariable\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.16%;\"\u003e\n \u003cp\u003eNumerator\u003c/p\u003e\n \u003cp\u003eDF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20%;\"\u003e\n \u003cp\u003eDenominator\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eDF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20%;\"\u003e\n \u003cp\u003eF-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20%;\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 27.84%;\"\u003e\n \u003cp\u003e(Intercept)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.16%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20%;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20%;\"\u003e\n \u003cp\u003e488.3557\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20%;\"\u003e\n \u003cp\u003e\u0026lt;.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 27.84%;\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.16%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20%;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20%;\"\u003e\n \u003cp\u003e126.5105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20%;\"\u003e\n \u003cp\u003e\u0026lt;.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 27.84%;\"\u003e\n \u003cp\u003eSize at maturity (L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.16%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20%;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20%;\"\u003e\n \u003cp\u003e35.9872\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20%;\"\u003e\n \u003cp\u003e\u0026lt;.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 27.84%;\"\u003e\n \u003cp\u003eEarly Growth Rate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.16%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20%;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20%;\"\u003e\n \u003cp\u003e5.0096\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20%;\"\u003e\n \u003cp\u003e0.0326\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eRegarding the growth of the hump, the logistic growth curve had the lowest residual sum of squares and was the best fit compared to the von Bertalanffy and Gompertz for both cold and warm treatments (Table 4). Growth curves for the nuchal hump were significantly different between temperature treatments (P = 0.005) and reflect a pattern whereby males from the warm environment grew humps at a faster rate early in development but had smaller humps by the time they reached maturity than males from the cold environment (Fig. 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003eNuchal hump growth curve selection data. The logistic (Log) curve had the lowest residual sum of squares (RSS) compared to the von Bertalanffy (VBF) and Gompertz (Gomp) curves in both the \u003cem\u003ewarm\u003c/em\u003e (A) and \u003cem\u003ecold\u003c/em\u003e (B) treatments.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"540\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 43.3333%;\"\u003e\n \u003cp\u003e\u0026nbsp;A)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.1111%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 35.5556%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 43.3333%;\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.1111%;\"\u003e\n \u003cp\u003eRSS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 35.5556%;\"\u003e\n \u003cp\u003e\u003cem\u003ek\u0026nbsp;\u003c/em\u003e\u0026plusmn; SE\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 43.3333%;\"\u003e\n \u003cp\u003eVBF: Linf*(1-exp(-K*(t-t0)))\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.1111%;\"\u003e\n \u003cp\u003e217.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 35.5556%;\"\u003e\n \u003cp\u003e0.55 \u0026plusmn; 0.15\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 43.3333%;\"\u003e\n \u003cp\u003eGomp: Linf*exp(-exp(-K*(age-t0)))\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.1111%;\"\u003e\n \u003cp\u003e214.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 35.5556%;\"\u003e\n \u003cp\u003e0.77 \u0026plusmn; 0.17\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 43.3333%;\"\u003e\n \u003cp\u003eLog: Linf/(1+exp(-K*(age-t0))\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.1111%;\"\u003e\n \u003cp\u003e212.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 35.5556%;\"\u003e\n \u003cp\u003e1.12 \u0026plusmn; 0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 43.3333%;\"\u003e\n \u003cp\u003e\u0026nbsp;B)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.1111%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 35.5556%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 43.3333%;\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.1111%;\"\u003e\n \u003cp\u003eRSS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 35.5556%;\"\u003e\n \u003cp\u003e\u003cem\u003ek\u0026nbsp;\u003c/em\u003e\u0026plusmn; SE\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 43.3333%;\"\u003e\n \u003cp\u003eVBF: Linf*(1-exp(-K*(t-t0)))\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.1111%;\"\u003e\n \u003cp\u003e432.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 35.5556%;\"\u003e\n \u003cp\u003e0.19 \u0026plusmn; 0.03\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 43.3333%;\"\u003e\n \u003cp\u003eGomp: Linf*exp(-exp(-K*(age-t0)))\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.1111%;\"\u003e\n \u003cp\u003e383.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 35.5556%;\"\u003e\n \u003cp\u003e0.39 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 43.3333%;\"\u003e\n \u003cp\u003eLog: Linf/(1+exp(-K*(age-t0))\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.1111%;\"\u003e\n \u003cp\u003e376.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 35.5556%;\"\u003e\n \u003cp\u003e0.57 \u0026plusmn; 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003ch3\u003eEffects of Diet Restriction on Hump Size\u003c/h3\u003e\n\u003cp\u003eFollowing a one-month period of diet restriction, variation in nuchal hump size was explained best by a model that included treatment, diet restriction status (pre- or post-) and an interaction term between them (Table 5). The model which included dam as a random effect and a correlation component to account for repeated measures was the best fit among the three models compared (P \u0026lt; 0.001, Table 5) and variation attributed to dam explained 13.8% of the total variability in hump size in this model as revealed by the intraclass correlation coefficient (ICC). The model produced a high correlation value (rho = 0.805) which suggests that repeated measures for each male are more similar relative to the variability across different males. Negative regression coefficients for treatment (-1.02) and diet restriction status (-0.14) indicate that not only were humps estimated to be smaller post-restriction than pre-restriction, but also smaller in the warm treatment versus the cold. More importantly, the coefficient for the interaction term (-0.45) indicates there is a synergistic effect between treatment and diet restriction, namely that being in the warm environment leads to even smaller post-restriction humps than being in the cold environment. Males from the cold treatment had a mean post-restriction hump size of 8.68 \u0026plusmn; 2.58 mm\u003csup\u003e2\u003c/sup\u003e versus a pre-restriction size of 9.86 \u0026plusmn; 2.46 mm\u003csup\u003e2\u003c/sup\u003e, representing a reduction in mean hump size of 1.18 mm\u003csup\u003e2\u003c/sup\u003e after diet restriction. Males from the warm treatment had a mean post-restriction hump size of 1.97 \u0026plusmn; 0.47 mm\u003csup\u003e2\u003c/sup\u003e versus a pre-restriction size of 3.62 \u0026plusmn; 1.06 mm\u003csup\u003e2\u003c/sup\u003e, which is a reduction in mean hump size of 1.64 mm\u003csup\u003e2\u0026nbsp;\u003c/sup\u003eafter diet restriction (Figure 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u0026nbsp;ANOVA results generated by the best model for explaining variation in nuchal hump size before and after diet restriction\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 37.2051%;\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.5154%;\"\u003e\n \u003cp\u003eNumerator\u003c/p\u003e\n \u003cp\u003eDF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.4265%;\"\u003e\n \u003cp\u003eDenominator\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eDF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.3376%;\"\u003e\n \u003cp\u003eF-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.5154%;\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 37.2051%;\"\u003e\n \u003cp\u003e(Intercept)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.5154%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.4265%;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.3376%;\"\u003e\n \u003cp\u003e811.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.5154%;\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 37.2051%;\"\u003e\n \u003cp\u003eTreatment\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.5154%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.4265%;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.3376%;\"\u003e\n \u003cp\u003e127.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.5154%;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 37.2051%;\"\u003e\n \u003cp\u003eRestriction status\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.5154%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.4265%;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.3376%;\"\u003e\n \u003cp\u003e77.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.5154%;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 37.2051%;\"\u003e\n \u003cp\u003eTreatment : Restriction status\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.5154%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.4265%;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.3376%;\"\u003e\n \u003cp\u003e40.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.5154%;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003eNuchal hump development in \u003cem\u003eXiphophorus multilineatus\u003c/em\u003e males in the laboratory provides a unique opportunity to explore the mechanisms of development and function of this trait. The findings of this study show that the hump consists of adipose tissue, that males reared in a colder environment (20\u0026deg; C) develop larger humps controlling for overall body size than males reared in a warmer environment (25\u0026deg; C) and that the differential gene expression pattern in the hump between temperature treatments suggests increased fat deposition in the cold environment versus the warm. Furthermore, changes in the size of the hump due to diet restriction and environmental temperature suggest that the pattern detected is due to increased metabolism in the warm environment stimulating an increased use of fat as an energy source leading to smaller humps. The implications of these results for understanding the function of the nuchal hump in this system as well as understanding the roles of both diet and activity in human obesity are discussed.\u003c/p\u003e\n\u003cp\u003eFinding that the nuchal hump is composed primarily of adipocytes suggests an energy storage function of this structure. Certainly, some vertebrates use fat stored in nuchal humps and similar dorsal structures as an energy source when dietary intake is insufficient to maintain metabolic rates and the amount of fat stored in the hump may reflect seasonal shifts in food quality and quantity (Alexander et al. 1968, Giles et al. 2015, Bengoumi et al. 2005). Within fish taxa, there are examples of nuchal humps composed primarily of adipose tissue, most notably within the species flocks of cichlids that occur in East African rift lakes (Takahashi 2018, Lecaudey et al. 2019). Yet even among this sub-group of fish, energy storage does not appear to be a primary function for humps. Takahashi (2018) found that the nuchal hump of \u003cem\u003eCyphotilapia gibberosa\u003c/em\u003e was formed by a thickening of the hypodermis through fat deposition, but the thickness was not correlated with body fitness in females and only slightly positively correlated in males. In other words, well-conditioned individuals did not store extra lipid in the humps and poorly conditioned individuals did not consume lipid from their humps. In the current study, however, the size of the nuchal hump was smaller after diet restriction regardless of temperature, suggesting that males were able to utilize stored fat in the nuchal hump as an energy source to make up for reduced caloric intake.\u003c/p\u003e\n\u003cp\u003eIf it is assumed that the function of the nuchal hump is for energy storage in \u003cem\u003eX. multilineatus\u003c/em\u003e, how can the pattern of larger humps on the males reared in the cold versus the warm treatments be explained? Fish acclimated to cold temperatures typically increase their storage of lipids in various body tissues including subcutaneous, liver, and muscle. Fish acclimated to warmer temperatures, on the other hand, increase the use of lipids as a direct energy source and store less of it in tissues (Stone et al. 1981, Egginton et al. 1989). There is evidence that at extremely high temperatures, fish may shift to storing fat as seen in the mummichog (\u003cem\u003eFundulus heteroclitus,\u003c/em\u003e Moerland et al. 1981), but fat storage in this case occurred specifically in the liver, not skeletal muscle or hypodermal tissue. The \u003cem\u003eX. multilineatus\u003c/em\u003e males in the current study were not subjected to critical maximum or minimum temperatures and followed the typical pattern of temperature related fat utilization in fish. The species of swordtail that forms the nuchal hump in nature, \u003cem\u003eX. birchmanni\u003c/em\u003e, is found at warmer temperatures on average than a closely related species that does not develop nuchal humps, \u003cem\u003eX. malinche\u003c/em\u003e (Rauchenberger et al. 1990). These two species often occupy the same stream systems and form hybrid zones (Culumber et al. 2012). When considering the potential influence of temperature alone, hump formation in \u003cem\u003eX. birchmanni\u003c/em\u003e seems less likely than in \u003cem\u003eX. malinche\u003c/em\u003e. Therefore, the natural occurrence of nuchal humps in \u003cem\u003eX. birchmanni\u003c/em\u003e suggests that factors other than temperature are more influential in the development of humps in this species. For example, correlated environmental differences in relation to food resources (higher quality diets) could potentially be more important.\u003c/p\u003e\n\u003cp\u003eThe results of this study suggest that males reared in a warmer environment either utilize more fat than males in the cold, or have simply used all the energy available to them with no excess to store in the nuchal humps. The first possibility can be considered the \u0026ldquo;metabolic difference hypothesis\u0026rdquo; and is supported by the finding that males from the warm environment used fat stores in the nuchal hump at a faster rate when placed on a restricted diet than males reared in the cold. In addition, seven of the top-tiered, differentially expressed genes in the nuchal hump of \u003cem\u003eX. multilineatus\u003c/em\u003e males are linked to lipid metabolism (Tables 1 \u0026amp; 2) with expression patterns suggesting increased fat deposition in cold males versus warm. Overexpression of the \u003cem\u003eG0S2\u003c/em\u003e switch gene, for example, reduces the rate of lipolysis in adipocytes by acting as a molecular brake on triglyceride catabolism (Yang et al. 2010). A higher expression of G0S2 in \u003cem\u003eX. multilineatus\u003c/em\u003e males from the cold treatment makes sense since these males developed larger humps. Likewise, males from the cold treatment had lower expression of the \u003cem\u003eWARS-1\u003c/em\u003e gene, which when inactivated in \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e leads to increased fat stores and increased starvation survival (Webster et al. 2017). Interestingly, a gene that is highly expressed in brown fat in mammals (\u003cem\u003ePGC-1\u0026alpha;\u003c/em\u003e) when induced by cold exposure (Cheng et al. 2018) was also overexpressed in \u003cem\u003eX. multilineatus\u003c/em\u003e males from the cold treatment. This gene links colder environmental temperature to adaptive thermogenesis by stimulating the catabolic burning of brown fat in mammals, however fish do not develop brown fat. If the expression pattern of this gene is similar between fish (which are not capable of thermogenesis) and mammals when considering the effect of environmental temperature, its role in thermogenesis among mammals may be a coopted function.\u003c/p\u003e\n\u003cp\u003eMales from the warm environment grew humps at a faster rate early in development, yet males from the cold developed larger humps by the time they reached sexual maturity (Figure 3). This is a similar pattern to that of growth of male body size in general, in that \u003cem\u003eX. multilineatus\u003c/em\u003e males from the warm environment grew faster as juveniles but reached a smaller adult size than males from the cold (Tompkins et al. in prep). This similarity is not surprising since hump size and body size had a significant positive relationship in the current study.\u003c/p\u003e\n\u003cp\u003eTompkins et al. (2021) suggested that diet could be a primary difference between the laboratory and natural environment for \u003cem\u003eX. multilineatus,\u003c/em\u003e inducing development of the nuchal hump in the laboratory. An additional factor to consider, however, is that the environment in which males were reared (isolated, in 2.5 l aquarium) does not allow for activity levels that would be more common in a natural environment. Swordtail males spend a large proportion of their time interacting with other males and courting females in the wild (Rios-Cardenas et al. 2010), and all these energetically costly behaviors were absent in the laboratory setting. The hypothesis that the reduced activity levels in the rearing environment is partially responsible for the development of the nuchal hump is supported by the observation that \u003cem\u003eX. multilineatus\u003c/em\u003e males from large mesocosm in our laboratory appear to be much less likely to develop a nuchal hump than males that are reared individually. Comparisons of the activity levels of the males reared individually in the warm and cold environments also lend support to this hypothesis (Fig. S2). It is also possible that hump size influences activity level due to the hump being a cumbersome feature. However, the shape of \u003cem\u003eX. birchmanni\u003c/em\u003e with its increased anterior body depth due to having a nuchal hump (the only species in the clade to form them in the wild) imparts improved endurance swimming performance compared with the dorsoventrally narrower shape of the species it naturally hybridizes with, \u003cem\u003eX. malinche\u003c/em\u003e (Johnson et al. 2014). Therefore, it seems more likely that activity influences hump size rather than vice versa.\u003c/p\u003e\n\u003cp\u003eThere are potential benefits of \u003cem\u003eX. multilineatus\u003c/em\u003e having the ability to develop a nuchal hump that could be considered. We previously detected female mate preference for males with nuchal humps in \u003cem\u003eX. multilineatus\u003c/em\u003e even though the trait does not occur in the wild (Tompkins et al. 2021). We suggest that the preference we detected may be similar to what has been suggested for \u003cem\u003eX. birchmanni\u003c/em\u003e (Rosenthal et al. 2003) and reflect a more general preference for larger males. Alternatively, if the hump developed in \u003cem\u003eX. multilineatus\u003c/em\u003e in the wild it could lead to improved swimming performance by increasing anterior body depth which minimizes drag (Johnson et al. 2014). Compartmentalized fat depots can have dual functionality in other vertebrates. Camels, for example, use the fat in their hump as an energy source, but one hypothesis suggests camels may concentrate body fat in the hump to avoid overheating if the fat was evenly distributed throughout the subcutaneous layer covering the body (Kassa 2016). However, the fact that the humps are not known to form in the natural environments of \u003cem\u003eX. multilineatus\u003c/em\u003e suggests that selection for these other functions would be minimal if nonexistent. Future studies of the functions of depositing fat in the nuchal hump in swordtail fishes, in addition to energy storage, should be considered.\u003c/p\u003e\n\u003cp\u003eEvidence that swordtail fish use the nuchal hump for energy storage with the humps composed of adipose tissue is interesting considering the importance of finding evolutionary animal models for studying diet-induced obesity. The development of a nuchal hump in \u003cem\u003eX. multilineatus\u003c/em\u003e under laboratory conditions after being fed a high-calorie diet may be a condition similar to that observed in humans (Schartl and Lu 2024). One of the other aspects of the laboratory environment that appears to promote nuchal hump formation is reduced activity levels, also an important factor in human obesity (Chin et al. 2016). Finally, the influence of environmental temperature on rates of obesity in humans has also been established (Trentinaglia et al. 2021, Moellering and Smith 2012) and cold induced thermogenesis in mammals is associated with reduced risk of diabetes (Schrauwen et al. 2016, Horino et al. 2022). Considering the influence of temperature, diet and potentially activity levels on the development of the nuchal hump in \u003cem\u003eX. multilineatus\u003c/em\u003e, this model system has the potential to provide future insight into the mechanisms involved in human obesity that other model systems cannot. Variation across species within \u003cem\u003eXiphophorus\u003c/em\u003e in the propensity to develop nuchal humps allows for the consideration of this trait in an evolutionary perspective, including unique opportunities to determine genetic variant site genotypes using hybrids and backcross hybrids of different species, even from the most phylogenetically distant branches of the genus (Shartl and Lu 2024). Current work is examining the development of the nuchal hump in backcross hybrids of \u003cem\u003eX. multilineatus\u003c/em\u003e and \u003cem\u003eX. couchianus,\u003c/em\u003e where we can determine genetic variant site genotypes, profile their gene expression, and begin to disentangle genetic networks involved in fat deposition.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe thank Grace Vance for collecting the activity level data and Mrs. Korri Weldon for technical assistance.\u003c/p\u003e\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eFunding for this project was provided by a grant from the Ohio University Research Committee, a NIH Shared Instrument grant S10OD030311 (S10 grant to NovaSeq 6000 System), and CPRIT Core Facility Award (RP220662).\u003c/p\u003e\n\u003ch3\u003eCompeting Interests\u003c/h3\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003ch3\u003eAuthor Contributions\u003c/h3\u003e\n\u003cp\u003eThe first draft of the manuscript was written by Keith B. Tompkins, Yaun Lu \u0026nbsp;and Molly R. Morris and all authors commented on previous versions of the manuscript. Photography, histology, and statistical analysis of variability in nuchal hump size was performed by Keith B. Tompkins. RNA-seq data was generated in the Genome Sequencing Facility, which is supported by UT Health San Antonio, NIH-NCI P30 CA054174 (Cancer Center at UT Health San Antonio). RNA-seq data analysis was performed by Yuan Lu. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch3\u003eData Availability\u003c/h3\u003e\n\u003cp\u003eRaw data are available on request.\u003c/p\u003e\n\u003ch3\u003eEthics Statement\u003c/h3\u003e\n\u003cp\u003eThe protocols followed for handling and care of all fish involved this study have been approved by the Institutional Animal Care and Use Committee of Ohio University in accordance with AAALAC, International. NIH animal use assurance #A3610-01. Collecting Permit: Permiso de Pesca de Fomento No. DGOPA 18317.271113.8982.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlexander A, Player IC (1965) A note on the nuchal hump of \u003cem\u003eCeratotherium simum\u003c/em\u003e (Burchell). \u003cem\u003eThe Lammergeyer\u003c/em\u003e, vol 3, no 2\u003c/li\u003e\n\u003cli\u003eBallinger MA, Andrews MT (2018) Nature\u0026rsquo;s fat-burning machine: brown adipose tissue in a hibernating mammal. \u003cem\u003eJournal of Experimental Biology\u003c/em\u003e, 221, 1-10\u003c/li\u003e\n\u003cli\u003eBandarian F, Hedayati M, Daneshpour MS, Naseri M, Azizi, F (2013) Genetic polymorphisms in the APOA1 gene and their relationship with serum HDL cholesterol levels. \u003cem\u003eLipids,\u003c/em\u003e 48:1207-1216\u003c/li\u003e\n\u003cli\u003eBarlow GW, Siri P (1997) Does sexual selection account for the conspicuous head dimorphism in the Midas cichlid? \u003cem\u003eAnimal Behavior\u003c/em\u003e, 53, 573-584\u003c/li\u003e\n\u003cli\u003eBengoumi M, Faulconnier Y, Tabarani A, Sghiri A, Faye B, \u0026amp; Chilliard Y (2005) Effects of feeding level on body weight, hump size, lipid content and adipocyte volume in the dromedary camel. \u003cem\u003eAnimal Research\u003c/em\u003e, \u003cem\u003e54\u003c/em\u003e(5), 383-393\u003c/li\u003e\n\u003cli\u003eCave AJE, Allbrook MB (1958) The skin and nuchal eminence of the white rhinoceros. \u003cem\u003eProceedings of the Zoological Society of London, \u003c/em\u003e132\u003c/li\u003e\n\u003cli\u003eCheng CF, Ku HC, Lin, H (2018) PGC-1\u0026alpha; as a Pivotal Factor in Lipid and Metabolic Regulation. \u003cem\u003eInt. 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The emergence of nuchal humps in Xiphophorus multilineatus males maintained in the laboratory, a species that does not develop humps in the wild, provided a unique opportunity to explore the development and function of this trait. The current study investigated the tissue composition of the hump and analyzed the influence of rearing temperature and diet restriction on hump development. Through histological examination and gene expression profiling our findings show that the hump is composed primarily of adipose tissue, which suggests a role in energy storage via fat deposition. Nuchal humps grew to a larger size in the cold environment (20°C) than the warm (25°C) and the differential gene expression pattern between temperature treatments suggests increased fat deposition in the cold versus warm environment. For example, the G0S2 gene which inhibits lipid catabolism is upregulated in the cold environment, and the WARS-1 gene which leads to increased fat stores when under-expressed is upregulated in the warm environment. The hypothesis that temperature influences hump development by stimulating shifts in fat metabolism is further supported by the finding that males from the warm environment used fat stores in the nuchal hump at a faster rate when placed on a restricted diet than males from the cold. The influence of temperature, diet and potentially activity levels on the fat deposition suggests X. multilineatus as an evolutionary animal model for gaining insights into the mechanisms involved in human obesity.","manuscriptTitle":"Composition and Function of the Nuchal Hump of Male Xiphophorus multilineatus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-28 09:09:14","doi":"10.21203/rs.3.rs-5389356/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-17T20:29:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-14T09:52:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"168305117708236175562744819504832262044","date":"2024-11-22T15:17:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-22T15:05:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-22T15:02:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-11T00:14:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Fish Physiology and Biochemistry","date":"2024-11-04T15:23:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"fish-physiology-and-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fish","sideBox":"Learn more about [Fish Physiology and Biochemistry](https://www.springer.com/journal/10695)","snPcode":"10695","submissionUrl":"https://submission.nature.com/new-submission/10695/3","title":"Fish Physiology and Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a69431d4-abed-40dc-b501-56435d33ab80","owner":[],"postedDate":"November 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T16:41:50+00:00","versionOfRecord":{"articleIdentity":"rs-5389356","link":"https://doi.org/10.1007/s10695-025-01539-2","journal":{"identity":"fish-physiology-and-biochemistry","isVorOnly":false,"title":"Fish Physiology and Biochemistry"},"publishedOn":"2025-07-31 16:13:12","publishedOnDateReadable":"July 31st, 2025"},"versionCreatedAt":"2024-11-28 09:09:14","video":"","vorDoi":"10.1007/s10695-025-01539-2","vorDoiUrl":"https://doi.org/10.1007/s10695-025-01539-2","workflowStages":[]},"version":"v1","identity":"rs-5389356","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5389356","identity":"rs-5389356","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}