Gelatin hydrogels are widely used materials that may require surfactants to adjust their solution’s surface tension for cell attachment, surface adsorption enhancement, or foaming. However, gelatin is a highly surface-active polymer, and its concentrated solutions usually do not require surfactants to achieve low surface tension. However, anionic surfactants, such as sodium dodecyl sulfate (SDS), interact strongly with gelatin to form complexes that impact its hydrogels’ rheological properties, influencing processability and functionality. Nevertheless, there is a lack of systematic research on the impact of these complexes on high gelatin content  hydrogels’ rheological properties. In this work, the SDS/gelatin ratio-dependent viscoelastic properties  of high-strength gelatin hydrogels were investigated using rheology and correlated to surface tension, viscometry, FTIR, and UV-Vis spectrophotometry. SDS–gelatin ratio was proved to be an important factor in tailoring the rheological properties of gelatin hydrogels. The gel strength, gelation kinetics, and melting/gelling temperature of the gelatin hydrogels linearly increased with SDS incorporation up to a maximum value, from which they started to decline. The findings of this work have wide applicability in tailoring the properties of gelatin–SDS solutions and hydrogels during their processing. Hydrogels from natural polymers have attracted research and development attention for a wide range of applications due to their biodegradability, biocompatibility, and ability to mimic living tissues’ characteristics ,2,3. AmoSurfactants can alter gelatin hydrogels’ properties due to protein reconfiguration and complex formation caused by gelatin–surfactant interactions. These complexes are usually visualized as surfactant micelles bound into the gelatin’s polypeptide chains like beads in a necklace ,14,15,16The formation of gelatin–surfactant complexes can also alter gelatin hydrogels’ rheological properties, such as gelation kinetics ,23,24,25G’, which is related to the amount of triple helix present in the hydrogel structure. Gel strength depends on several factors, such as temperature, time, and gelatin type and content. A strong relationship has been found between the storage modulus and surfactant concentration in gelatin-based hydrogels. The elasticity and gelation kinetics of gelatin–surfactant systems were reported to increase with surfactant concentration up to a maximum value from which they started to decrease, leading to protein denaturation and softening [The gelation of gelatin hydrogels arises from the transition of its polypeptide chains from a coiled state when dissolved in warm water to a renatured triple helix configuration on cooling. During gelation, the water is trapped within the gelatin polymer chains, and a more rigid structure is held together by combining intermolecular forces, such as hydrogen bonds, electrostatic forces, van der Waals forces, and hydrophobic interaction . Gelatinoftening ,29.Sodium dodecyl sulfate (SDS) is a widely used anionic surfactant that has been used to modify hydrogels’ properties for different applications ,30,31,32Therefore, this study aimed to investigate the extent to which SDS influences the rheological properties  of high-strength gelatin–SDS hydrogels in order to gain a better understanding of gelatin–surfactant interactions and their influence on the optimization of gelatin hydrogels’ rheological properties. The contributions made here have wide applicability on the processing of gelatin-based hydrogels and solutions. While SDS did not significantly modify the gelatin solutions’ surface tension, it considerably influenced their rheological properties. Lower SDS concentrations resulted in an increase in rheological properties, while excessive SDS concentrations caused their decline.A systematic small-amplitude oscillatory shear (SAOS) rheological study covering a wide range of gelatin and SDS concentrations was carried out in correlation with the characterization of surface tension, viscometry, and FTIR.Polymer–surfactant interactions can alter the solubility of polymer solutions . A decreGelatin can adsorb and associate with SDS by electrostatic and hydrophobic forces ,41. At lThe surface tension of the deionized water at 40 °C was 69.74 ± 0.34 mN/m, in agreement with the value of 69.6 mN/m reported by the Engineering Toolbox  at the sPure gelatin solutions in the absence of SDS were highly surface active. Compared to that of water, the surface tension of the pure gelatin solutions decreased by around 50% to 35.8, 35.2, and 36.2 mN/m for the 5 wt.%, 10 wt.%, and 20 wt.% gelatin solutions, respectively. In addition, there was a slight further reduction of surface tension (CAC) in the gelatin–SDS solutions at low SDS concentrations (<0.3 mM). The low value of CAC compared with that of the CMC for SDS aqueous solution indicates the competitive absorption of gelatin and SDS at the solution/air interface, resulting in premature saturation at the interface. Furthermore, the fact that the surface tension of the gelatin–SDS solutions was slightly higher than that of the pure SDS aqueous solutions provided additional evidence about gelatin and surfactant absorption competition at the solution/air interface.When the surfactant concentration was above the CAC, the surface tension increased (around 10 mM) to a level around 39 mN/m for all three gelatin concentrations. Thus, the gelatin–SDS system had a slightly higher surface tension than the pure SDS solutions at the studied SDS concentrations, from which it can be inferred that gelatin–SDS complexes are less surface-active than SDS. Consequently, the SDS addition for the benefit of surface tension reduction was largely compromised. As surfactant concentration increases beyond the values studied in this work, it has been shown that gelatin is desorbed from the surface, leaving just a monolayer of surfactant at the interface and leading to surface tension values similar to those in the SDS–water solutions ,37.−1 (amide-A region), 3100 cm−1 (amide-B), 1600–1700 cm−1 (amide-I), and 1550 cm−1 (amide-II). Amide-I, especially, and amide-II are the most closely related vibration bands to the secondary structural elements of the protein backbone [FTIR spectroscopy was used to provide information about the secondary structure adopted by the polypeptide chain of 5 wt.% and 20 wt.% in gelatin and gelatin–SDS dry gels. The main infrared absorption bands for proteins in the IR spectra are situated around 3300–3350 cmbackbone . Amide-Ibackbone , both inbackbone . Amide-Abackbone .−1 attributed to the presence of hydrogen bonds and amide-A. Moreover, the two bands at 1651 cm−1 and 1538 cm−1 corresponded to amide-I and amide-II, respectively. The 20 wt.% gelatin gel spectrum exhibited similar characteristics to the 5 wt.% gels with some changes in intensity and shift. Typical absorption bands were absorbed at 20 wt.% at 1653 cm−1 (amide-I) and 1539 cm−1 (amide-II).−1 frequencies was assigned to the C–H stretching of alkane groups from the interaction of gelatin and SDS [3 stretching vibrational frequencies were located at 2957–2960 cm−1 and 2873–2879 cm−1, respectively. The asymmetric and symmetric CH2 stretching vibrational frequencies were found at 2926–2941 cm−1 and 2848–2857 cm−1, respectively. As expected, the CH2 stretching intensities were higher than those for the CH3, and both were more intense in samples containing SDS than in the pure gelatin ones.SDS addition into the 5 and 20 wt.% gelatin gels led to shifts in the characteristic amide band positions due to gelatin–SDS physical interactions and micelles formation . The fre and SDS ,48. The 2 stretching characteristics can be related to conformation and packing of the SDS molecules, because CH2 stretching is sensitive to methylene chains conformation, indicating a relatively ordered crystalline structure at CH2 symmetric stretching values lower than 2852 cm−1 [2 symmetric stretching bands between 2854 and 2857 cm−1, representing the presence of micelles and liquid crystals [2 asymmetric vibrational feature was found at 1235–1241 cm−1 and exhibited higher wavenumbers in the gelatin–SDS gels than in the pure gelatin gels.CH852 cm−1 . The rescrystals . Anothercrystals . This SOThe gelatin–SDS interaction was manifested by an almost linear thickening of the solution, attributed to the gelatin molecules’ association and gelatin–SDS intramolecular and intermolecular interactions ,38. A geG’) and loss modulus (G”) generated under different test conditions. For materials showing viscoelastic behavior, such as gelatin, G’ and G” represent the material’s elastic and viscous behavior, respectively. The gel elasticity, G’, is directly related to the helix concentration and weak interactions between them [G”, represents liquid-like behavior and generates from the internal friction between molecules and particles in the material, transforming deformation into frictional heat.The gelatin hydrogels’ gelation kinetics, gel strength, and gelling/melting points were investigated using the storage modulus  an initial and sharp increase of the G’–G’’ curves, (2) the G’’-G’ crossover point (G’ = G’’), and (3) stabilization of the G’–G’’ curves, which both tend to plateau [G’’ > G’) to a gel  state, G’ rapidly increased and surpassed G’’.The influence of the S/G ratio on gelation kinetics was investigated by isothermal (23 °C) time-dependent tests at constant deformation .  plateau . As gelaG’ and NS G’’) in The black curves  time-dependent tests described above. Gel strength, G’60, was higher at higher gelatin concentrations , marking the onset of the gelatin–SDS hydrogel denaturation. Accordingly, three zones can be defined: (1) increase , (2) decline (where S1 < S < S2), and (3) collapse (where S > S2).60 initially rose as the SDS content increased up to S1. The maximum registered increase relative to that without SDS was most drastic for the 5 wt.% gelatin gels (about 2.5 times) and reduced to about 1.5 times for more concentrated gelatin gels at 10 and 20 wt.%, respectively. This maximum G’60 value was achieved at 0.09375 (0.46875 wt.%), 0.05625 (0.5625 wt.%), and 0.0375 (0.75 wt.%) S/G ratios for 5, 10, and 20 wt.% gelatin gels, respectively. Thus, the higher the gel strength , the lower the S/G ratio at which the maximum increase in elasticity was obtained. It has been argued that the gelatin–SDS interactions below S1 (the “increase” region) for gel strength are dominated by the overlapping of the gelatin chains with the surfactant micelles, forming crosslinks and favoring secondary structures by electrostatic and hydrophobic interactions [In the “increase” zone, G’ractions . These rractions . This be1 into the “decline” region, the gelatin–SDS interactions disrupted the triple helix formation, delaying and even preventing gelling at the timescale studied when the S/G ratio was equal to or higher than 0.375 and 0.5 for 5 wt.% and 10 wt.% gelatin concentrations, respectively  and 5 wt.% (0.5 S/G ratios) surfactant content, respectively, whereas such gelling disruption behavior was not observed in 20 wt.% gels at the studied S/G ratios. Ionic surfactants with alkyl chains, such as SDS, are known for acting as protein denaturants [As the SDS concentration increased beyond Svely see . The hydvely see  and an iaturants ,36. Protaturants . Neverthaturants  pointed aturants . Secondlaturants . Unfoldiaturants .60, the loss factor G”/G’ at 60 min, as a function of SDS concentration. Viscoelastic liquids are commonly classified by tanδ > 1, while viscoelastic solids are classified by tanδ < 1. During the isothermal time sweep test, tanδ decreased with time as gelation advanced, showing increased solid-like behavior as gelation proceeded, resulting in tanδ60 < 1 for gels at all SDS concentrations, which implied that the gels were viscoelastic solids. However, higher SDS concentration promoted the increasing viscous behavior of the hydrogels  due to protein unfolding, and this effect was more evident in gels with lower gelatin content .On heating from 23 °C to 40 °C, fully formed gelatin hydrogels exhibited a gel–sol transition. When gelatin hydrogels are in a gel state, G” > G’). As the temperature decreases, the gelatin solutions transition to form a gelatin gel and both G’’ and G’ (and this one more considerably) increased and led to a G’’–G’ crossover on cooling, known as gelling temperature (Tg).On cooling from 40 °C to 23 °C, the gelatin solutions underwent a sol–gel transition. At 40 °C, the gelatin solution is liquid-like  to the highest S/G ratio studied (0.225) was more significant for gelling  than for melting temperatures . Gelatin–SDS complexes needed higher energy for melting, attributed to higher triple helix stability and length [Both gelling and melting temperatures exhibited a similar trend to G’60 see . Gellingd length , and thud length . The decMeasurements of melting and gelling temperatures are known to be dependent on heating and cooling rates. Such a dependence is shown in SDS is an effective surfactant for lowering the surface tension of aqueous solutions, but in the presence of highly surface-active polymers, such as gelatin, this functionality can be compromised. SDS can have additional roles in gelatin solutions and hydrogels, such as modifying the rheological properties, including viscosity, gelation kinetics, gel strength, and gelling/melting temperature.G’60, and melting/gelling temperatures.Higher gelatin concentrations give rise to faster gelation, stronger hydrogels, and higher gelling/melting temperatures. In addition to gelatin concentration, the gelatin–SDS interaction also plays a significant role in the rheological properties. Lower SDS concentrations, below a certain optimum value, resulted in a beneficial increase in rheological properties . In contrast, excessive SDS concentration beyond the optimum level caused a decline in the gelation kinetics, gel strength, 1) relative to gelatin’s concentration at which the hydrogel’s strength is maximum and begins to decline up to an SDS concentration (S2), after which the positive effect of SDS addition ceased. This increase/decline in the gel’s strength trend can be correlated to the gelatin–SDS associations, which were highly dependent on the S/G ratio. The viscoelastic properties’ “increase” trend arises from the formation of gelatin–micelle crosslinks and the acceleration in the formation of secondary structures, while the decline is attributed to micellar binding competition with helix formation and gelatin tertiary structure unfolding.The results indicated an optimal SDS concentration . Bloom was measured using the method of the Gelatin Manufacturers Institute of America (GMIA) . The gelAqueous gelatin–SDS solutions at 5 wt.%, 10 wt.%, and 20 wt.% gelatin concentrations were prepared by dissolving gelatin along with SDS at 60 °C with magnetic stirring at 250 rpm for 30 min. The gelatin–SDS solutions were allowed to stabilize at 40 °C for 20 min without stirring to ensure they were free from bubbles. In addition, when required, a small amount of HCl (up to 50 μL/100 mL) was incorporated dropwise for pH adjustment. This process was monitored using an Orion Star A221, portable pH meter .All the characterizations were carried out in triplicate.A LAMBDA 750s spectrophotometer  equipped with a temperature-controlled Peltier device was used to assess the solutions’ solubility and the development of light-scattering aggregates. Transmittance measurements studied gelatin–SDS solutions at 40 °C at 600 nm. Samples were placed in a quartz cell with an optical path length of 10 mm.The surface tension of the gelatin and gelatin–SDS solutions was measured at 40 °C by the Wilhelmy plate method using a K100 model tensiometer . The surface tension of polymer/surfactant systems is in dynamic equilibrium; thus, a waiting time of 30 min before measurements was adopted to establish a relatively steady state.−1 with 32 scans over the wavenumber range of 400–4000 cm−1. The gels, in powder form, were mixed with KBr at a ratio of 1:100.FTIR absorption spectra of the gelatin and gelatin–SDS dry gels were recorded using a Frontier FT-IR Spectrometer  at a resolution of 2 cm−1 to 1000 s−1 with 60 measuring points in 600 s. The 10 s interval was chosen to minimize the start-up effects at lower shear rates (ɣ < 1 s−1).The viscosity of the gelatin solutions at 40 °C was measured using a Haake Mars III rheometer  equipped with a Peltier temperature control system. The solutions were loaded into a 40 °C pre-heated double gap cup (3 mL capacity) and stabilized for 60 s. The measurements were carried out using an isothermal (40 °C) shear rate ramp test from 0.01 sSmall amplitude oscillatory shear (SAOS) measurements were used to characterize the rheological properties of the gels. The rheological characterization was carried out using a Haake Mars III rheometer fitted with a Peltier temperature control system and 60 mm diameter parallel plate geometry. The bubble-free gelatin and gelatin–SDS solutions were dispensed on the pre-heated lower plate at 40 °C. The upper plate was lowered to the predetermined testing gap, and the excess solution was wiped off. A thin layer of silicone oil was applied around the edges to prevent solvent evaporation.G’ and loss G” moduli were recorded for these analyses, and the end of the linear region was considered a 10% deviation from the quasi-equilibrium plateau, which ended at 25.5% strain when the frequency (f) was 1 Hz for the most rigid hydrogel studied . The parameters chosen for further characterization  had a 1 Hz frequency, 1% strain, and 1 mm gap unless stated otherwise.The linear viscoelastic region (LVR) was determined using the method by Zuidema et al.  with somG’60) were recorded. In addition, a comparison of the time-dependent rheological behavior between gelatin–SDS solutions at 0.225 S/G ratios at its natural pH and those buffered to the pH of the pure gelatin solution was made to assess the influence of the pH variation upon SDS incorporation.Isothermal time-dependent rheological behavior: The isothermal time-dependent rheological behavior of the gelatin and gelatin–SDS solutions was studied to assess gelation kinetics and the storage modulus. After 5 min of stabilization at 40 °C (t = 0 min), the hydrogel sample was cooled and held at 23 °C under isothermal conditions during a 60 min-time sweep. The storage and loss modulus variation with time and the storage modulus value at t = 60 min  at 23 °C and were then heated to 40 °C at controlled rates of 1 °C/min, 2 °C/min, and 4 °C/min, respectively, at the same testing conditions mentioned for gelling temperature determination .Temperature-dependent rheological behavior: The temperature-dependent viscoelastic behavior of 10 wt.% and 20 wt.% gelatin hydrogels at different SDS content was investigated to determine the gelling and melting temperatures. For gelling temperature determination, after 5 min of conditioning at 40 °C, the gelatin solutions were allowed to cool from 40 °C to 20 °C at controlled rates of 1 °C/min, 2 °C/min, and 4 °C/min, respectively, at a constant frequency, 1 Hz, shear strain, ɣ = 3–6%, and 0.5 mm gap. The adjustments in strain and the gap setting were necessary to enhance the torque signals and the accuracy of the


Gyrodactylus infecting farmed “tilapia” throughout Mexico. We also collected native fishes around farms, to look for potential parasite spillover from cultured fishes. Monogeneans were identified taxonomically using morphological and molecular characters. Originally African, pathogenic Gyrodactylus cichlidarum was recorded in every farm surveyed, infecting different “tilapia” varieties, as well as three native cichlid fish species. Previously, we had shown that G. cichlidarum also infects native, non-cichlid fishes in Mexico. We also recorded that Gyrodactylus yacatli is widely distributed in Mexico, infecting cultured “tilapia” and native fishes; and present data indicating that this is a further translocated African parasite. A third, unidentified gyrodactylid infected farmed and native fishes in Chiapas, southern Mexico; we describe the new species as Gyrodactylus shinni n. sp., and provide evidence that this is a third monogenean translocated with African fish. The wide distribution of exotic parasites co-introduced with “tilapia” and their spillover to native fishes may have an important impact on the ichthyofauna in Mexico, one the world’s megadiverse countries.Translocation of fishes for aquaculture has resulted in the co-introduction of some of their parasites. African cichlid fishes, generically called “tilapias” have been introduced worldwide, along with their monogenean parasites. In a nation-wide survey, we characterised monogeneans of the genus  Monogenean parasites infecting cichlid fishes are similarly diverse; for instance, tropheine cichlids of Lake Tanganyika, which underwent a rapid radiation process are infected by an equally -if not more- diverse assemblage of parasites from the genus Cichlidogyrus, which diverged synchronously with their hosts2. Starting in the middle of the twentieth century, various cichlids from the genera Coptodon, Sarotherodon and Oreochromis referred to generically as “tilapia” were exported globally from Africa, mainly for aquacultural purposes1. Nile tilapia Oreochromis niloticus has been introduced throughout the Americas4 and various other “tilapias” have likewise been translocated worldwide. Several monogenean parasites have been co-introduced along with their fish hosts. Thus, African species of the genera Cichlidogyrus6 and Gyrodactylus7 have been documented infecting translocated “tilapias” worldwide.Among teleost fishes, the family Cichlidae possesses one of the highest species richness (> 1700 known species), including at least 900 freshwater African speciesCoptodon zillii was imported from the USA; while in 1964, Nile tilapia was introduced from “Africa” (no further details available) and Costa Rica, and both blue tilapia O. aureus and Mozambique tilapia O. mossambicus were introduced from the USA; redbreast tilapia C. rendalli was imported from Cuba in 1968; and Wami tilapia O. urolepis was translocated from Costa Rica in 19788. The first records of introduced monogeneans infecting farmed African “tilapia” in Mexico date from the 1980’s8, and by the early 2000’s, evidence of the transfer of African parasites to native, American cichlids became available with the record of Cichlidogyrus longicornis, C. sclerosus and C. tilapiae infecting Lana cichlid Vieja fenestrata, and of Enterogyrus malmbergi infecting San Domingo cichlid Thorichthys callolepis9. Cichlidogyrus sclerosus has also been recorded from Mayan cichlid Mayaheros urophthalmus10. Gyrodactylus cichlidarum is an originally African parasite that has been translocated worldwide and shown to negatively affect the survival of farmed fishes, particularly juveniles11. In Mexico, G. cichlidarum has been recorded in fish farms throughout the country12, from the Yucatán peninsula in the southeast14 to the northwestern state of Sinaloa16, as well as the central states of Veracruz16 and Morelos17. Gyrodactylus yacatli was previously recorded to infect “tilapia” in southeastern Mexico7, and O. niloticus and the cichlid Pseudocrenilabrus philander in Zimbabwe18; but the low prevalence of this species both in Mexico and Zimbabwe, and the unavailability of molecular data for African samples precluded support for any hypothesis on the biogeographical origin of the taxon. Two introduced monogeneans, C. sclerosus and G. cichlidarum, are considered to have established in Mexico8; and both have been recorded infecting native Mexican fishes other than cichlids, as C. sclerosus parasitizes endemic blackfin goodea Goodea atripinnis8 and G. cichlidarum infects three native poeciliids: shortfin molly Poecilia mexicana, porthole livebearer Poeciliopsis gracilis and two spot livebearer Pseudoxiphophorus bimaculatus19.In Mexico, the first recorded introduction of “tilapias” dates from 1945, when redbelly tilapia Gyrodactylus species infecting cultured “tilapia” throughout Mexico; (2) to assess the potential spillover of parasites to native fish species in the vicinity of aquaculture facilities, and conversely, look for evidence of host switches from native cichlids to farmed “tilapia”; (3) to better understand the likely biogeographical origin of G. yacatli; and (4) to generate morphological and molecular data to explore variation within gyrodactylids infecting “tilapia” throughout Mexico; and, if present, characterize new taxa.In this study, we present the results of an exploratory parasitological survey conducted at some of the most productive “tilapia” farms in Mexico, from the Yucatán peninsula through to Sonora, on the USA border Fig. . The maiPoecilia formosa, Poecilia mexicana, Poeciliopsis gracilis, Pseudoxiphophorus bimaculatus and Xiphophorus hellerii), Goodeidae (Goodea atripinnis), and Profundulidae (Profundulus oaxacae). In addition, specimens of V. fenestrata, Papaloapan cichlid Paraneetroplus nebuliferus and an unidentified native cichlid were collected in Chiapas.A total of 40 “tilapia” farms were sampled in three regions in Mexico: North-West , Centre-South , and East (state of Yucatán: n = 14)  ranged from 0.15 ± 0.15 worms/host ± S.D. for Yucatán to 17.9 ± 4.73 worms/host in Chiapas. Regionally, the East had the lowest mean parasite abundance (0.15 ± 0.15 worms/host), followed by the North-West (1.84 ± 2.74 worms/host) and the Centre-South (4.46 ± 5.01 worms/host); regions differed significantly in parasite abundance , with similarly high parasite abundances in the Centre-South and North-West , and significantly lower abundance in the East .Overall, 932 “tilapia” and native cichlids were inspected and 2173 gyrodactylids were recovered. Of these, a subsample of 285 parasites was analysed morphometrically and molecularly. No “spillover” parasites were found on any native fish species, other than native cichlids. Parasite abundance calculated for Gyrodactylus infecting cichlid fishes in Mexico were identified, two previously described and one new species: G. cichlidarum , G. yacatli , and G. shinni n. sp.   can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “http:/zoobank.org/”. The LSID for this publication is: urn:lsid:zoobank.org:pub:166AB775-AA03-45A2-8F17-9AD33DB62A01. In addition, species profile including taxonomic traits, host details, and other metadata are provided on urn:lsid:zoobank.org:act:18730161-3470-4E74-BEC4-29D3F526E1DAGyrodactylus cichlidarum was the most abundant gyrodactylid infecting farmed “tilapia” in this survey—it was recorded on every farm sampled, and on all “tilapia” varieties inspected. It was most abundant in the Centre-South, and least abundant in the East of the country. This African parasite was found to infect three native cichlid fishes in Mexico: P. nebuliferus and V. fenestrata (both in Oaxaca State) and an unidentified native cichlid collected in Chiapas  long, 3.6 (3.2–3.9) wide, same width in all their length; shaft 33.5 (32.7–34.9) long; slender point 25.2 (24.5–25.9) long, comprising more than half of the shaft length; proximal shaft width 7.3 (7.3–7.8) wide; straight shaft; wide hamulus aperture distance 22.4 (21.4–24.2) long; broad hamulus aperture angle 44.2° (42.2°–48.2°), outlining “V”-like shape in the hamulus; hamulus root 20.4 (19.1–21.6) long, straight with rectangular edges, ventral side (facing host) ending in slight protuberance .Collectors: Adriana García and Miguel Rubio.Type specimens: Holotype (accession no. CNHE 11387), paratypes (accession no. CNHE 11388) deposited in Colección Nacional de Helmintos (CNHE), Universidad Nacional Autónoma de México, Mexico City.Molecular data: ITS rDNA sequences of Gyrodactylus shinni n. sp. (863 bp) obtained from two individuals collected from O. niloticus deposited in GenBank (Accession nos MN759066 - MN759067). Lengths of the 18S, ITS1, 5.8S, ITS2 and 28S were 47, 342, 157, 303 and 20 bp, respectively.Etymology: the species is named after Dr. Andrew Paul Shinn, for his contribution and passion for the taxonomy, systematics and general knowledge of Gyrodactylus species worldwide.Remarks: Among hundreds of worms analysed, we only identified five specimens of this new species, G. shinni; and all of these specimens had been previously dissected to digest their haptors for morphological analysis, and their bodies used for molecular analysis. Thus, we cannot provide a whole body type specimen nor morphometric and other details on complete specimens. Nonetheless, the description of G. shinni n. sp. presented complies with the provisions of both the International Commission on Zoological Nomenclature (ICZN) Code and the World Organisation for Animal Health (OIE) Manual. The morphology of the haptoral hard parts of G. shinni n. sp. is very close to those of G. cichlidarum23, G. ulinganisus24, G. occupatus18 and G. parisellei18 , from two species of native cichlids (Vieja fenestrata (n = 3), and an unidentified native cichlid (n = 1), and five specimens from cultured O. niloticus obtained from Kenya , Jalisco (n = 5), Sinaloa n = 1), Oaxaca (n = 19) and Veracruz (n = 14), from different , Oaxaca ains n = 9 fish, fains n = 9 fish, f(Revised) Morphological description based on 50 specimens. Six whole worms cleared in ammonium picrate glycerine (APG), and 44 proteolytically digested. Body 33.1 (29.3–38.0) long; 10.4 (8.1–12.1) wide at uterus level. Pharynx 28.2 (25.9–29.7) long; 36.6 (29.2–43.6) width. Hamuli 51.9 µm (40.3–75.5) long, 5.6 (6.2–13.2) wide; shaft slightly curved 33.9 (26.0–49.9) long; slender point 24.0 (18.4–34.5) long, almost the same length as hamulus shaft; proximal shaft width 8.6 (6.2–13.2) wide, being the widest point of hamulus; hamulus aperture distance 17.2 (10.8–25.7) long; wide hamulus aperture angle 35.1° (30.4°–41.2°); hamulus root 18.1 (13.2–28.2) long, comprising one third of hamulus length, with semi-rounded edges, slightly narrower in its midsection ), “Rocky mountain” (hybrid of O. aureus Steindachner O. niloticus), and “Pargo UNAM” (25% “Rocky mountain” + 25% red variant O. niloticus and 50% “Florida tilapia”), Paraneetroplus nebuliferus, Vieja fenestrata and an unidentified native cichlid.Site of infection: Fins.Localities: Regal Springs farm, Chiapas ; Achitralada  and La Gigantera farms, Jalisco ; Río Chiquito, Cuicatlán, Oaxaca ; Presa Picacho, Sinaloa ; Jalacingo  and Rancho El Clarín, Tlapacoyan, Veracruz ; and Sagana Aquaculture Centre, Kirinyaga County, Kenya .Collectors: Adriana García, Miguel Rubio, Carlos Pinacho (Oaxaca), and Andrea Gustinelli (Kenya).Specimens: Hologenophore specimens  deposited in Colección Nacional de Helmintos (CNHE), Universidad Nacional Autónoma de México, Mexico City.Molecular data: ITS rDNA sequences of Gyrodactylus yacatli (975 bp) obtained from 25 individuals deposited in GenBank: Accession Nos. KY489740-42, MN759049 (Chiapas); KY489744, KY489753-55, KY489757 ; MN759050-56 (Oaxaca); KY489745-50, KY489752, KY489756, MN759052 (Veracruz); and KY489739 (Kenya). Lengths of the 18S, ITS1, 5.8S, ITS2 and 28S were of 57, 358, 157, 384 and 19 bp, respectively.Remarks: The species was originally described24 based on specimens collected in the states of Sinaloa, Tabasco and Yucatán, in Mexico. The description was based on morphology only, as no molecular data were obtained. Although overall the specimens used to describe G. yacatli and those collected in this study are very similar, slight morphological differences between them were detected, which we summarize below. In the description of the specimens from 2011, the ventral bar was found to have a squared membrane, but in most of the specimens analysed then, this structure was folded or broken making its description difficult. In the present study, we found that the membrane is long and lingulate . The hamulus aperture angle is wider in the specimens found in Sinaloa and Chiapas  and narrower in Kenya and Oaxaca . Marginal hooks in specimens collected from V. fenestrata are slightly larger than those collected from “tilapia” in Mexico and Kenya. In general, hook morphology of specimens found in the present study corresponds to those found in Kenya and with the original description  analysed included the 69 isolates generated in this study plus 15 nominal species of Gyrodactylus , with 646 variable sites for all samples. The sequence alignment of ITS showed numerous gaps of variable lengths (~ 1–86 bp). Nucleotide frequencies were A = 0. 242, C = 0. 217, G = 0. 233, and T = 0. 306. All GenBank accession numbers for newly generated sequences are provided in Table We obtained ITS sequences from gyrodactylids collected at 18 localities across Mexico and one from Kenya; this includes 43 sequences of G. cichlidarum, G. yacatli and the new species described here, are nested in three independent clades with high bootstrap and posterior probability support values  reported from O. niloticus in Scotland and with two sequences of this parasite collected from native poeciliids in Mexico (KX512807—KX512808); however, this clade showed low support values (88/0.84). Gyrodactylus shinni n. sp. formed a clade with high branch support (100/1); however, phylogenetic relationships were not clear (−/0.56). Finally, specimens of G. yacatli from Mexico and Kenya (KY489739) appear in a further clade with high branch support (95/1), grouped with Gyrodactylus zimbae infecting the cichlid Simochromis diagramma from Zambia . Both analyses showed that all sequences obtained in the current study corresponding to This survey provides evidence that three African gyrodactylid parasites translocated into Mexico with their “tilapia” hosts are now widely distributed in fish farms throughout the country; and have spilled-over to native cichlids. We cannot state when this happened (as “tilapias” were first introduced in 1945), nor can we suggest which fish species served as vector for the parasites (at least six different species of “tilapia” have been introduced over the years), and with the scant molecular evidence available, no inference/hypothesis can be made on what country in Africa was the original source of the parasites currently located in Mexico.Gyrodactylus cichlidarum was initally described from Mango tilapia Sarotherodon galilaeus from Ghana, Africa23. This parasite has been recorded in several farmed and wild cichlids in Africa, including banded jewelfish Hemichromis fasciatus in Senegal25, Nile tilapia O. niloticus in Kenya26 and Mozambique tilapia O. mossambicus in Madagascar27; and outside Africa, it has been recorded on all continents except Antarctica11. Gyrodactylus cichlidarum is widely distributed in Mexico and infects not only farmed and feral “tilapia” throughout the country29, but has also been recorded on native poeciliid fishes19. This survey corroborates that this translocated African parasite is found throughout the country infecting several species/strains of “tilapia”, including O. niloticus, O. mossambicus, O. aureus, “Rocky mountain”, “Pargo UNAM”30 and “Florida tilapia”; and provides the first records of infection of three native cichlid fishes in Mexico: P. nebuliferus and V. fenestrata (both in Oaxaca) and an unidentified native cichlid collected in Chiapas. Thus, this study demonstrates that G. cichlidarum has quite low host specificity, and that although it exhibits marked intraspecific morphological variation when comparing individuals from different hosts and geographical locations, very limited molecular variation is present in this species.G. yacatli was originally described, the authors mentioned the possibility that this gyrodactylid species sporadically found in limited locations in Mexico “was an accidental infection or a host switch from a fish species inhabiting the water source feeding the farms, or probably has not yet been detected on the O. niloticus of African origin”24. In this study, we show that this parasite is widely distributed on both the Pacific and Atlantic sides of Mexico , and provide evidence that it represents a further translocated African species that is able to switch hosts to native, Neotropical cichlids. Gyrodactylus yacatli has previously been recorded in Zimbabwe and China18. We examined specimens from Zimbabwe and contrasted them with Mexican samples, and although minor morphological differences were found, specimens from both continents showed clear similarities. However, we cannot rule out that these are very similar/cryptic species, as no molecular data are available for the African samples. Chinese specimens6 could not be morphologically examined as mounted specimens could not be visualized properly; and no molecular data are available for these specimens either. Nonetheless, we were able to analyse G. yacatli specimens from Kenya using morphological and molecular characters, and these generally corresponded with those of Mexican samples. Phylogenetic analyses presented in this study show that the sample from Kenya sits within the clade containing all specimens found in Mexico. We consider these findings, and the fact that G. yacatli has been recorded in Zimbabwe and China, provide strong support for the notion that this is a further African gyrodactylid species translocated globally with the “tilapia” trade.When G. mojarrae was described as a species infecting several native cichlids from localities across southern Mexico (the Centre-South region in the present study), including Jack Dempsey Rocio octofasciata, chescla Thorichthys maculipinnis, Oaxaca cichlid Vieja zonata and V. fenestrata31. This species is clearly different morphologically from gyrodactylids found in African cichlids. Phylogenetic relationships among species of the genus Gyrodactylus associated with Neotropical and some African cichlids showed that G. mojarrae is clearly different from its congeners infecting “tilapia”31. The present study corroborates that finding, as G. mojarrae is sufficiently distinct from “tilapia” parasites to root the phylogenetic tree. In the phylogenetic hypothesis we present, the three gyrodactylid species found in this study do not show high intraspecific variation, and all are closely related to parasite species recorded from African cichlids –strengthening the idea that G. cichlidarum, G. yacatli and G. shinni n. sp. are translocated “tilapia” parasites of African origin.Recently, Gyrodactylus infecting poeciliids, goodeids, profundulids, characids and cyprinids39. Thus, we are confident that other than the instances of parasite spillover from “tilapia” to native cichlids recorded here, and the rare instances of G. cichlidarum infecting poeciliids reported previously19, African gyrodactylids do not commonly switch host to non-cichlid fishes. As would be expected, farmed fish had higher infection parameters than wild fish, a situation probably resulting from both ecological conditions favouring parasite transmission among confined hosts, as well as a long co-evolutionary trajectory between G. cichlidarum and its African hosts. Although outside the remit of this survey, our findings generally coincide with the hypothesis suggesting that the major factors influencing the success of host switching are compatibility and opportunity40. Neotropical cichlids are a monophyletic sister clade related to African cichlids41, and thus are more compatible to their African parasites than more distantly related fishes; i.e., Neotropical cichlid fishes probably do not exert a sufficiently robust selective pressure to eliminate colonizing parasite populations. Significant intraspecific variation was observed in the size of haptoral hard parts when analyzing parasites collected from farmed “tilapia” and wild, native cichlid fishes, which probably reflects both phenotypic plasticity and adaptation to different hosts—and which may also contribute to increasing compatibility. Opportunity, i.e., the possibility of coinciding spatially and temporally with potential new hosts, was provided with the anthropogenic translocation of “tilapia” and their parasites; and further assisted by the generally lax management practices exerted in “tilapia” aquaculture in Mexico14, which facilitate both parasite growth within and their escape from fish farms.In this survey, we inspected several non-cichlid native fishes collected in the Centre-South (the region with the highest abundance of “tilapia” gyrodactylids) and found no evidence of spillover. Furthermore, over the past decade we have studied gyrodactylids of Mexican native fishes and have not found evidence of parasite transfers from “tilapia” to other common fishes during studies on several species of Gyrodactylus introduced with their “tilapia” hosts over the years are now widely distributed in Mexican fish farms and have spilled-over to native cichlid fishes, a situation that will most probably be replicated elsewhere in the Americas, considering “tilapias” are highly invasive fish species42 and monogeneans, including gyrodactylids, are known to be widely distributed in fish farms in the region11. Further studies are needed to assess the potential impact of translocated gyrodactylids on native fish hosts, considering monogenean parasites have been shown to be very successful invaders following co-introduction with their primary hosts45. This is of particular interest, taking into account that Monogenea have been shown to use a “stepping-stone” mode of host-switching enabling them to successfully colonize distantly-related Neotropical freshwater fish lineages46.Finally, we provide evidence that species of Oreochromis niloticus  were collected from 40 farms located in three different, broadly defined regions in Mexico: North-West , Centre-South , and East (State of Yucatán)  and Dr. Giuseppe Paladini . To assess parasite spillover from “tilapia” farms, particularly in the Centre-South where several native cichlids occur, we also collected samples of common native fish families from streams and rivers close to farms, including species of Cichlidae, Poeciliidae, Goodeidae and Profundulidae. In the lab, ethanol-preserved fish were examined under a stereomicroscope . Worms were removed with the use of surgical needles and were processed individually. Dislodged worms found in the bottom of the tubes where fish samples were kept were also recovered. The total number of worms found in samples from each farm was counted; and a subsample was taken for morphological and molecular identification. For species identification, haptors were excised using a scalpel and subjected to partial proteolytic digestion to remove tissue enclosing the haptoral armature, under the dissection microscope48. Digestion was arrested by the addition of a 50:50 glycerine/formalin solution (making a semi-permanent preparation), and specimens were then coverslipped and sealed with nail varnish. Bodies were fixed in 95% ethanol and stored at – 20 °C, individually labelled for subsequent molecular analyses.During surveys conducted between 2013 and 2018, “tilapia” Gyrodactylus specimens found on cichlids (wild and cultured) and some native fish were compared with described species found worldwide33 . Pictures were obtained using imaging analysis software Leica Application Suite, LAS ver. 4.12.0 with a Leica ICC50 HD camera. Using ImageJ 1.52a software, measurements were calculated from images. 33 using the primer pairs ITS1-fm (5'-TAGAGGAAGTACAAGTCG-3') and ITS2-rm (5'-GCTYGAATCGAGGTCAGGAC-3′)33. Additionally, the forward primer BD1, (5′-GTCGTAACAAGGTTTCCGTA-3′) and the reverse primer BD2, (5′-ATCTAGACCGGACTAGGCTGTG-3′)49 were used for some specimens. Amplicons were visualized on GelRed  stained 1% agarose gel and then unincorporated nucleotides and primers of each PCR amplicon were removed using ExoSap-IT . Sequencing reactions were carried out with the use of BigDye Terminator v3.1 chemistry, incorporating the same primers as those used in PCR and internal primers ITSR3A (5′-GAGCCGAGTGATCCACC-3′) and ITS4.5 (5′-CATCGGTCTCTCGAACG-3′)50, and cleaned by filtration with Sephadex G-50. Sequenced products were read on an ABI PRISM 3100 automated DNA sequencer . Electropherograms were visually inspected and overlapping fragments of forward and reverse sequences were assembled using Geneious 8.1.851 (https://www.geneious.com). Sequences were deposited in GenBank and their accession numbers are cited in the description of each species.The bodies of excised specimens whose haptors had been morphometrically characterized were placed individually in 1.5 ml microcentrifuge tubes for genomic DNA extraction using the DNeasy Blood & Tissue Kit  following manufacturer’s instructions. The ribosomal region spanning the 3′ end of the 18S rRNA gene, ITS1, 5.8S rRNA gene, ITS2, and the 5′ end of the 28S rRNA gene was amplified by PCR conditionsGyrodactylus shinni n. sp. ; G. cichlidarum  and G. yacatli ) were aligned with sequences of other Gyrodactylus spp. available in the GenBank database: G. cichlidarum (DQ124228); G. mojarrae (MK573785-87); G. hildae (FJ231869); G. micropsi (AF328868); G. rugiensis (AF328870); G. chileani (JQ045347); G. zimbae (HQ214482); G. ulinganisus (FJ231870); G. occupatus (LN849940); G. nyanzae (LN849939); G. ergensi (FN394985); G. malalai (FR695484); G. chitandiri (LN849942); G. sturmbaueri ; and G. parisellei (LN849941). Sequences were aligned using ClustalW with default parameters implemented in MEGA version 7.052. The best-fitting nucleotide substitution model (GTR + G) was estimated with the Akaike Information Criterion (AIC) implemented in MEGA version 7.0. Phylogenetic trees were reconstructed by Maximum Likelihood (ML) and Bayesian inference (BI) analyses. For ML analyses, the program RAxML v7.0.453 was used. A GTRGAMMAI substitution model was used for ML analyses, and 1000 bootstrap replicates were run to assess nodal support. BI trees were generated using MrBayes v3.254, running two independent MC3 runs of four chains for 10 million generations and sampling tree topologies every 1000 generations. ‘Burn-in’ periods were set to 2.5 million generations according to the standard deviation of split frequencies values (p ˂ 0.01). Posterior probabilities of clades were obtained from 50% majority rule consensus of sample trees after excluding the initial 25% as ‘burn-in’. Genetic divergence among species of Gyrodactylus was estimated using uncorrected “p” distances in MEGA version 7.052. Finally, trees were drawn using FigTree version 1.3.155.Sequences of the ITS obtained in this study for the species described here, Gyrodactylus sp. worms found on farmed “tilapia” by the total number of fish examined in each farm and state surveyed . The proportion of parasite species reported for each state was calculated from the subsample of worms characterized morphologically and molecularly , and the Kruskall-Wallis non-parametric test was performed to detect differences between these groups. Paired Mann–Whitney U tests were used to identify significantly different groups. Statistical tests were performed in R 3.6.057.To compare mean abundances of The authors declare that the work reported here was conducted following guidelines on the ethical treatment of animals and was authorized by the competent Mexican authorities: Ministries of Agriculture (SAGARPA) permit DGOPA/01173/120208.0107 and the Environment (SEMARNAT) permit SGPA/DGVS/02967/14.Supplementary Information 1.Supplementary Information 2.


The 147-item food frequency questionnaire was used to estimate their dietary intake. Mental health status was assessed using the depression, anxiety and stress scales-21 (DASS-21). The mean ± standard deviation age, weight and BMI of the participants were 36⋅49 ± 8⋅38, 80⋅89 ± 12⋅45 kg and 31⋅04 ± 4⋅31 kg/m2, respectively. DBM was significantly associated with stress, after adjusting for potential confounders, including age, energy and marital status in model 1  1⋅00, 1⋅65, P < 0⋅04) v. the crude model . No significant association was seen among DBM and DASS-21 outcomes. In this cross-sectional study, stress and DBM were significantly associated. While vitamin D insufficiency was associated with mental health and obesity in opposing directions. Elucidation of whether vitamin D supplementation can improve mental health impairments requires further evaluation.The co-existence of overweight or obesity with concurrent deficiency of one or more nutrients is referred to as double burden of malnutrition (DBM), and numerous mental health impairments have been associated with a variety of nutrient deficiencies. Although DBM is relevant for several health outcomes, the ubiquitous involvement of vitamin D across multiple systems and tissues suggests D insufficiency as a viable target for nutritional modification. The present study aimed to evaluate the contribution of DBM and mental health among adult women. Study participants included 300 women, aged 18–59 years, who presented to one of the 25 health centres in Tehran. Participants with a body mass index (BMI) of greater than 25 kg/m Furthermore, the emergence of double burden of malnutrition (DBM), the co-existence of caloric overconsumption with essential nutrient insufficiency, has been reported as increasingly prevalent in developing countries in comparison to developed countries,3. While contributors to this phenomenon include increased urbanisation, alteration of dietary patterns and lifestyle, some nutrients are more likely to be implicated in adverse health effects.The continued rise in overweight and obesity remains a global health concern. In particular, the increases and consequent adverse health effects widely recognised among developing countries are introducing a significant health challenge,6.The physiologic effects of DBM are well recognised. However, a comprehensive understanding of modifiable health outcomes requires an understanding of the psychological effects of DBM. Indeed, an association between obesity and a number of mental health impairments across the life course has been reported, with obesity increasing the risk of depression incidence, and a suggested mechanistic role of inadequate essential nutrient intake as a moderator,10. Suboptimal nutrient intake, including cobalamin, folic acid, niacin and ascorbic acid, zinc, iron and selenium, has been associated with depression. However, the ubiquitous involvement of vitamin D in neurocognition and obesity-related disorders offers a viable area of investigation as a modifiable intervention,13. Many studies have reported that changes in a plasma concentration of 25-hydroxy vitamin D [25(OH)D] and fatty mass were inversely and significantly associated; moreover, obese subjects tended to show low absorption of vitamin D,15. Furthermore, vitamin D absorbs by complex mechanisms; for instance, in the gastrointestinal tract (GIT), vitamin D is involved in lipid metabolism and diffuses in chylomicrons, which facilitates transportation in the liver. Also, obese subjects demonstrate a low concentration of plasma 25(OH)D, as well as low 25(OH)D and obesity related to high waist circumference, high body mass index (BMI), hypertension, insulin resistance, glycaemic profile, impaired lipids and other cardiometabolic disorders,18. Several explanations have been offered to explain the reduced concentration of 25(OH)D, including low sun exposure because of a sedentary lifestyle, the sequestration of 25(OH)D in adipose tissue, and dilution of ingested 25(OH)D and synthesis in large fat of obese patients. Furthermore, BsmI and ApaI VDR genes in overweight and obese women are reported to account for polymorphism and seem to affect BMI. Also, adipose stores have a haemostatic role in inflammation and innate immunity in human individuals by modulating of toll-like receptors and nuclear factor-kappaB (NF-ĸB) pathways–22.Given that poor mental health affects more than 300 million people, of all ages, the additive effect of the obesity epidemic and mental health impairments could have a profound effect on mortality in the ensuing decade(s). Indeed, a bidirectional link between obesity and mental health has been suggested–26. However, there is a distinct dearth of studies about the association between DBM and mental health. Therefore, in this cross-sectional study, we assessed the association between DBM and mental health, including depression, anxiety and stress, in Iranian women with obesity, with a focus on vitamin D as a potential moderator.Observational studies have suggested that vitamin D deficiency may be related to depression and increased suicideA cohort of 300 women with obesity and overweight, aged 18–59 years, participated in the present study. Women were randomly selected from those presenting to twenty-five health centres in Tehran. The sampling method was achieved by multistage cluster randomisation among all the Tehran regions. The participants were included among twenty clusters from 2017 to 2019.Inclusion criteria were aged 18–59 years; no current weight loss programme; no use of weight loss supplements. Participants with a history of type 2 diabetes, cardiovascular diseases, polycystic ovary syndromes, stroke, non-alcoholic fatty liver disease, inflammatory disease, hypertension, cancer, thyroid diseases, or who were currently pregnant, were excluded because of possible changes in diet. In addition, women reporting intake energy lower or higher than 800–4200 kcal, respectively, were excluded.The present study was conducted according to the guidelines in the Declaration of Helsinki and all procedures involving human subjects were approved by the ethics committee of the Tehran University of Medical Science (IR.TUMS.VCR.REC.1398.819). Written informed consent was obtained from all subjects prior to participation. was measured using a non-elastic tape, to the nearest 0⋅1 cm, according to standard protocols. In addition, hip circumference was determined as the largest part of the hip, over light clothing. BMI was calculated based on Quetelet's index. According to WHO guidelines, the individuals were classified as underweight (BMI: <18⋅50 kg/m2), normal weight (18⋅50–24⋅99 kg/m2), overweight (25⋅0–29⋅9 kg/m2) and obesity (≥30⋅0 kg/m2).The individuals wore light clothing and were unshod for the measurement of weight and height. All measurements were assessed by a trained technician. The waist circumference and visceral fat area (VFA), using bioelectrical impedance analysis. According to the manufacturer's instructions, after shoes, coats and sweaters had been removed, subjects were required to stand on the balance scale, in bare feet, and hold the handles of the machine. The measurements took approximately 20 s, and the output was printed.The body composition of all participants was analysed using a body analyser device . This instrument measured the body composition, including body fat mass, fat-free mass (FFM). The data documented in household measures and serving size were changed to grams and millimetres. The nutritionist IV  food analyser was used for analysing dietary intake.The individual diet intake was assessed by a 147-item food frequency questionnaire that was previously validated (CHOL) were measured using glycerol-3-phosphate oxidase–phenol 4-amino antipyrine peroxidase (GPO-PAP) and enzymatic endpoint, respectively. Also, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol were measured by direct enzymatic clearance assay. All laboratory assessments were performed using Seven Randox Laboratories kits .At first, a 10-ml blood sample was drawn after 8–12 h of fasting, and the serum was centrifuged, and isolated and stored at −80°C. All data were evaluated by the Endocrinology & Metabolism Research Institute (EMRI) Bionanotechnology laboratory of the Tehran University of Medical Science. Serum fasting glucose assessment was made using glucose oxidase–phenol 4-amino antipyrine peroxidase (GOD-PAP) (colorimetric method), while triacylglycerol (TG) and total cholesterol–35.The concentration of 25(OH)D in individuals was assessed using an immunoassay immunodiagnostic system (IDS) kit. Finally, serum concentrations of 25(OH)D between 20 and 30 ng/ml and less than 20 ng/ml were considered as insufficiency and deficiency, respectively) and vitamin D levels. Accordingly, BMI greater than 25 kg/m2 along with plasma concentrations of vitamin D lower than 20 ng/ml (25(OH)D < 20 ng/ml) was considered DBM.DBM was assessed by calculating BMI  and Tukey post hoc tests, where appropriate, were used for a comparison of variables. The logistic regression was performed to discern the relationship between vitamin D deficiency in overweight and people with obesity by mental disorders. An initial model (0) was created and included only vitamin D deficiency (using 20 as a cut point), and a secondary model (1) included vitamin D deficiency and mental health status, in addition to age, energy and marital status. All statistical analysis was performed using IBM SPSS for Windows version 23 , and a P value of <0⋅05 was, a priori, considered as statistically significant.Normality distribution was tested using a Kolmogorov–Smirnov test; for data that were not normally distributed, and could not be transformed appropriately for normal distribution, 2, respectively.Descriptive statistics are represented in The number of people in each category and characteristics are shown in sd of characteristics of variables in four categories, showing that there are significant body composition differences, including weight, BMI, waist circumference, fat-free mass, VFA, TG (P < 0⋅0001) and CHOL (P < 0⋅05), and diastolic blood pressure (P < 0⋅02).v. the crude model , respectively. However, there was a significant relationship between stress and DBM after adjusting age, energy and marital status in model 1 (0⋅04) v. the crude model (0⋅09)., fat-free mass, VFA and lipid profiles, such as TG and CHOL, and diastolic blood pressure.This novel investigation sought to identify the role of vitamin D as a moderator in the association between DBM and mental health and, to our knowledge, was the first conducted in Iranian women. In the present study, we considered the presence of overweight and obesity with the deficiency of vitamin D as a DBM. Many previous studies have demonstrated a significant association between vitamin D and depression. Moreover, vitamin D levels have been shown to be impeded due to air pollution in industrial cities, low intake in some countries, use of cosmetics, sunlight cream, some clothing because of religion and age because of decreasing synthesis by skin and absorption by the intestine. Iranian women are reported to have an increased susceptibility to vitamin D deficiency, either by type of clothing or low consumption of dairy. In the present study, we observed a significant relationship between DBM and stress; in addition, we observed significant differences between four categories in BMI, weight and body composition items including waist circumference–43.Vitamin D is an important fat-soluble vitamin that is immune-modulating and can inhibit many communicable diseases including, heart disease, multiple sclerosis, rheumatoid arthritis and type 1 diabetes. Indeed, the deficiency of vitamin D is associated with many disorders, including skeletal abnormalities, delayed growth in children, osteoporosis and osteopenia, reduction of calcium and phosphorus absorption in the intestine. The main sources of vitamin D are exposure of skin to sunlight and consumption of foods containing vitamin D, such as dairy. Vitamin D deficiency is common in Iranian people; indeed, based on a meta-analysis that was performed in Iranian people, 45⋅64 % of men and 61⋅90 % of women showed vitamin D deficiency. Moreover, the geographical location, type of clothing, poor diet, expensive dairy costs and skin colours are posited to modulate this deficiency,35,44,45.25(OH)D levels, either by diet or synthesised by skin, are highly dependent on vitamin D binding protein (DBP), also a high level of 25(OH)D, as well as 1⋅25(OH)D, appear on DBP (more than 80 %), whereas DBP is responsible for 25(OH)D plasma concentrations. Some studies have reported a positive association between BMI and DBP plasma concentration, and many studies have reported that the 25(OH)D concentrations in overweight and obese people may be reduced significantly. Because of this evident correlation between vitamin D status and DBP in obese individuals, we considered the level of plasma vitamin D status between 20 and 30 ng/l and less than 20 ng/ml to represent insufficiency and deficiency, respectively–48, plays an important role in depression conditions,49–51, where high BMI has been associated with mood disorders (OR 1⋅23) and major depression (OR 1⋅27). In addition, evidence suggests a significant relationship between high BMI and anxiety disorders, such as post-traumatic stress disorders (OR 2⋅64). Anglin et al. performed a systematic review and meta-analysis on one case control, three cohorts, and ten cross-sectional studies and reported a significant relationship between vitamin D deficiency and depression.Currently, obesity and overweight represent significant health issues associated with inflammatory status. Obesity is implicated in a mutation in the leptin/melanocortin pathway in the central nervous system, which plays a role in regulating body energy haemostasis. Moreover, obesity related to many brain disorders and psychopathology conditions, including eating and mood disorderset al. carried out a study on sixty-three overweight participants , with a mean age = 31⋅3 ± 8⋅5, along with vitamin D deficiency [25(OH)D < 20 ng/l] and BMI 25 kg/m2, without clinical depression, consumed a bolus oral dosage of 100 000 IU, followed by 4000 IU daily cholecalciferol for 16 weeks. Subsequently, the authors observed an increasing concentration of plasma vitamin D, and reported that vitamin D deficiency was not associated with depression, has therefore not warranted for reducing depression symptoms.Concordant with our findings, Mousa et al., in 18–67 years old males and females, found a significant relationship between vitamin D deficiency and depression. However, the number of males (n 9799) was notably more than the number of female counterparts (n 1121), which likely biased the findings. Similarly, Umhau reported a significant correlation between suicide and vitamin D deficiency among active-duty military personnel, where the number of males (n 467) was significantly greater than the number of females (n 19). Nevertheless, tentative conclusions from the literature suggest that the correlation between depression and vitamin D deficiency may be greater in males v. females.In contrast with our finding, Schaad v. overweight/obesity and represents a sensible avenue for further research. Moreover, although we focused on women, who appear to be relatively underrepresented in comparable research, future studies should be designed considering an equitable split in sex.Although we present a novel addition to the literature, where we assessed DBM and mental health in Iranian women, there were several limitations that should be noted. First, we did not assess normal-weight participants with vitamin D deficiency, this precluded a comparison between normal weight In this cohort study, we demonstrated that stress and DBM were significantly associated; however, the directionality of the relationship remains unclear. Vitamin D insufficiency is associated with mental health and obesity in opposing directions, and whether vitamin D supplementation can improve mental health impairments requires further evaluation.


Visual search refers to our ability to find what we are looking for among many competing visual inputs. Here, we report the availability of a rich dataset that replicates key visual search effects and shows that these effects are robust to several changes to the experimental design. Experiment 1 replicates classic findings from an additional singleton visual search task. First, participants are captured by a salient but irrelevant color singleton, as indexed by slower response times when a color singleton distractor is present versus absent. Second, attentional capture by a color singleton is reduced when the visual search array contains heterogeneous shapes rather than homogenous shapes. Finally, attentional capture by a color singleton is reduced when the display colors are repeated rather than switched unpredictably from trial to trial. Experiment 2 demonstrates that these classic visual search effects are robust to small procedural changes such as task timing . Experiment 3 demonstrates that these classic effects are likewise robust to changes to the distractor frequency (75% rather than 50%) and to fully blocking versus interleaving blocks of two task conditions. All told, this dataset includes 8 sub-experiments, 190 participants and >210,000 trials, and it will serve as a useful resource for power analyses and exploratory analyses of visual search behaviors. Visual search refers to our ability to find what we are looking for in a cluttered visual world. For example, when searching for the remote, you need to look for items that match your goals  while ignoring salient distractors . In the lab, we use highly simplified search displays to measure the influence of salient distractors on visual search performance. In a typical laboratory task known as the “additional singleton task” , 1992, pHere, we employ 4 variants of an additional-singleton task in order to replicate classic visual search effects. In all variants, participants search for a diamond and report the orientation of the line inside , and attentional capture is quantified as the response time difference for singleton distractor present versus absent trials. In the first task variant, non-target items are homogenous shapes , and the colors swap randomly from trial to trial  all green items, (2) green items with a red singleton distractor, (3) all red items or (4) red items with a green singleton distractor). In the second variant, non-target items are heterogeneous shapes , and the colors swap randomly from trial to trial. Based on prior work, we expect that the color singleton distractor will capture attention more effectively when non-target shapes are homogeneous, because participants may adopt a “singleton-detection” strategy in which they direct attention toward the most salient display features, rather than a “feature search” strategy in which they strategically search for items that match the search template e.g., .1 The fi all greeAcross three experiments, we replicate classic findings from the visual search literature, including (1) attentional capture by an irrelevant color singleton , 1992 2 and (3) Table 1. Across all experiments, a total of 3 participants were excluded for low task accuracy (<55%), 2 datafiles were excluded for being incomplete  and 9 participants were excluded from Experiment 3B because we made changes to the stimulus size, and we wanted the full sample to perfectly match the stimulus parameters being used for a later study. Raw data for excluded subjects are available in the OSF repository. The main RT results from Experiment 3B are also reported in Adam & Serences , and JASP 0.13.1 (Stimuli were shown on a linearized Cathode Ray Tube (CRT) monitor (39 × 29.5 cm) from a viewing distance of ~52 cm. Stimuli were generated using a Linux computer (Ubuntu 16.04) running MATLAB 2016b  with the Psychtoolbox extension . Data weP 0.13.1 . ParticiIn all experiments, stimuli were presented on a black background. Participants fixated a small white dot (.2°) at the center of the display, and the fixation dot remained visible throughout the entire experiment. Each item in the search array had a radius of 2.2° and appeared on an imaginary circle with radius 6.9° centered around fixation. Search items were equidistantly spaced around the imaginary circle and could be red  or green . On distractor absent trials, all items are the same color (green or red). On distractor present trials, all items are the same color except for a singleton distractor . We will use the term “majority green” or “majority red” to refer to the main display color. A small white line (.08° × .81°) appeared inside each search array item; each item was independently and randomly assigned one of two orientations . These size values were the same for all experiment except Experiment 3B, which had the following small changes: imaginary circle radius = 7.0°, target line = .08° × .94°, search item radius = 2.4°.Figure 1, and which conditions occurred in each experiment are labeled in Figure 1 and Table 2. Participants performed these conditions in separate blocks. On each trial, the participants saw a display with multiple items, and their goal was to find the target (diamond shape) as quickly as possible and report via keypress whether the line inside the target was horizontal (“z” key) or vertical (“/” key). In the “heterogeneous” condition, the non-target shapes varied . Non-target shapes were drawn from the shape set without replacement (set sizes 3–5) or without replacement from a doubled list of the shape set (set size 6), such that each non-target shape could be repeated no more than one time. In the “homogeneous” condition, the non-target shapes were all circles. We also varied trial history by manipulating whether the colors were repeated or switched from trial to trial. In the “color variable” condition, the colors switched randomly from trial to trial . In the “color constant” condition, the colors were held constant within the entire block of trials .The possible task conditions are depicted in Each search array could contain between 3 and 6 items. These set size conditions were equally likely to occur, and they were randomly intermixed within each block. The positions of the search array were slightly jittered . A color singleton distractor was present on 50% of trials. The search array disappeared as soon as the participant made their response. If the participant did not respond within 2 seconds, then the search array disappeared, and a non-response was recorded. The inter-trial interval lasted between 0.6 and 1.1 seconds , and the next search array was presented immediately after the inter-trial interval elapsed.Each search array contained 4 items, and the positions were fixed such that the 4 items appeared at 45°, 135°, 225° and 315° on the imaginary circle around fixation. A color singleton distractor was present on 50% of trials. The search array disappeared as soon as the participant made their response. If the participant did not respond within 2 seconds, then the search array disappeared, and a non-response was recorded. The inter-trial interval lasted either 2, 3, 5, or 8 seconds  was held constant at 2 seconds for the “homogeneous non-targets” condition in Experiment 2a. Note, this programming error only affected the distribution of ITI values and did not affect any other part of the task. Specifically, in affected conditions, rather than choosing a value from a list of all 4 ITI values , only the first value from the list [2] was used).Experiment 3A: equal numbers of trials per ITI within each block for all conditions for all but the first 2 subjects. Experiment 3B (and first 2 subjects of Experiment 3A): equal numbers of trials per ITI in the “color constant” condition and held constant at 2 seconds for the “color variable” condition). In Experiment 3A, the “color constant” and “color variable” conditions were interleaved within each subject (Block 1 = color constant (majority green), Block 2 = color variable, Block 3 = color constant (majority red), Block 4 = color variable, etc.). In Experiment 3B, blocks of different conditions were grouped together  and Blocks 10–12 “color constant” (majority green). The interleaved blocks were presented in a fixed order for all participants; the order of the grouped blocks was counterbalanced across participants (Table S2).Each search array contained 4 items, and the positions were fixed such that the 4 items appeared at 45°, 135°, 225° and 315° on the imaginary circle around fixation. A singleton distractor was present on 75% of trials, the search array was always shown for exactly 2 seconds (regardless of response), and task conditions were grouped or interleaved. The inter-trial interval lasted either 2, 3, 5, or 8 seconds . We performed a mixed repeated measures ANOVA on response times with the between-subjects factors Color History (constant or variable) and Non-Target Type (heterogeneous vs. homogeneous) and the within-subjects factors Set Size and Distractor Presence. We found that Non-Target Type had a large effect on search times, F = 202.99, p < .001, η2p = .69, such that the effect of set size was much larger in the heterogeneous than homogeneous condition p < .001, η2p = .86). This is consistent with prior findings that participants search relatively more serially when in “feature-search mode” and more in parallel when in “singleton detection mode”. However, search in the singleton-detection condition (homogeneous) was not perfectly parallel in our experiments – As also observed in Bacon & Egeth .As expected, in Experiment 1 we replicated typical findings that the heterogeneity of non-target shapes has a large effect on search efficiency Figure . We perf & Egeth , we founF = 198.6, p < .001, η2p = .68. In fact, the participants were so effective at suppressing the distractor in the color constant condition that we actually found distractor presence benefits whereby participants were faster when a singleton distractor was present than when it was absent. This was evident both in Experiment 1B and 1D. When participants were in feature-search mode (Exp. 1B), participants were much faster whenever a distractor was present, F = 379.9, p < .001, η2p = .94, and this distractor benefit did not interact with set size, F = 1.98, p = .13, η2p = .08. When participants were in singleton-detection mode (Exp. 1D), there likewise a main effect of distractor presence, F = 6.14, p = .02, η2p = .21, and this effect interacted with set size, such that participants were particularly fast on distractor present trials for low set sizes, F = 10.6, p < .001, η2p = .32. Task accuracy for all experiments is shown in Figures S1–S2, and we found no evidence of a speed-accuracy trade-off explaining either set-size or distractor presence effects.We likewise replicated expected findings that that Color History strongly modulates attentional capture by the salient singleton distractor. When the display colors switched unpredictably from trial to trial, participants were significantly slower when a singleton distractor was present versus absent. When the display colors repeated, capture by the distractor was reduced. This pattern of larger capture in the color variable (Exp 1A and 1C) versus constant condition (Exp 1B and 1D) was supported by a significant interaction of Color History × Distractor Presence in the main ANOVA, F = 424.1, p < .001, η2p = .91, Figure 3. Whether or not participants were captured by the color singleton distractor varied according to the particular condition, as indicated by a significant 3-way interaction of Non-Target Type, Distractor Presence and Color History, F = 12.1, p = .001, η2p = .22 . Specifically, when the display colors changed unpredictably from trial to trial , participants were captured by the singleton distractor when non-targets were homogeneous , but were not captured when non-targets were heterogeneous .Color Constant, Figure 3, Exp 2B), participants were no longer captured by the distractor  or even were faster when the color singleton distractor was present .In Experiment 2, we found that the typical visual search effects observed in Experiment 1 were robust to small changes to the task design . We ran a mixed repeated measures ANOVA with the between-subjects factors Color History and the within-subjects factor Non-Target Type. Participants were again overall slower when non-target shapes were heterogeneous than homogeneous, as indicated by a main effect of Non-Target Type, F = 21.2, p < .001, η2p = .32. This general pattern was not meaningfully affected by Condition Balancing, as indicated by no significant 3-way interaction of Color History × Distractor Presence × Condition Balancing (p = .64). However, post-hoc analyses indicate that distractor suppression was numerically more effective in the grouped experiment than in the interleaved experiment . Specifically, whereas capture by the distractor was non-significant in the Color Constant condition for the grouped experiment (Exp 3B), capture in the Color Constant condition was attenuated but still overall significant in the interleaved condition (Exp 3A).Finally, in Experiment 3, we found that expected visual search effects were robust to other small changes to the task design . We ran a mixed repeated measures ANOVA with the between-subjects factors Condition Balancing and the within-subjects factor Color History, and we found expected behavioral effects of Color History on search behavior. Specifically, we found that participants were significantly less captured by the distractor in the color constant condition compared to the color variable condition, as indicated by a significant Color History × Distractor Presence interaction, benefits whereby participants found the target faster when a salient singleton distractor was present. Because of the fairly large number of trials per participant that we collected  we anticipate this dataset will be useful for modeling the effects of stimulus history and non-target homogeneity  that replicates several classic findings and shows that these classic findings are robust to small procedural changes. For example, these experiments show greater search efficiency for homogeneous versus heterogeneous non-target shapes  and reduogeneity  as well ogeneity .In addition to the primary response time metric (reported here), we also recorded other potential variables of interest on each trial. These variables include trial accuracy, whether a response was made, the color and position of each item, the inter-trial interval duration, the shape of each item, and the orientation of the line inside each item. Thus, we anticipate that this dataset will also be useful for other exploratory analyses. For example, in an additional analysis of Experiment 3b , we replhttps://osf.io/u7wvy/), we have provided the raw data and analysis files in several formats that may be useful for pedagogical purposes. The original raw data, task code, and analysis scripts are all in MATLAB (.m and .mat files). In addition, we have provided general-use files (.csv) for flexible analysis of response times with most contemporary analysis programs. For example, we have provided an example of plotting and analysis of the data in either Python  or in the open-source software JASP (In the online data repository (are JASP . Togethehttps://osf.io/u7wvy/Data and code are available on the Open Science Framework at The additional file for this article can be found as follows:10.5334/joc.182.s1Supplemental Information.Supplementary figures and tables.


Breast cancer is the most common cancer in American women, except for skin cancers. In this meta-analysis, the associations of polymorphisms within paraoxonase 1 (PON1), leptin (LEP) and leptin receptor (LEPR) genes with susceptibility to breast cancer were comprehensively evaluated. A universal search in PubMed, Scopus, CNKI, SID, Web of Knowledge and Google Scholar was performed to identify relevant studies up to 01 May, 2021. The strength of the associations was estimated by Odds ratios (ORs) with 95% confidence intervals (95% CIs). A total of 39 case-control studies including 7 studies with 2005 cases and 2748 controls were on PON1 rs662, 6 studies with 2,031 cases and 1,973 controls on PON1 rs854560, 12 studies with 3,444 cases and 3,583 controls on LEP rs7799039, and 14 studies with 5,330 cases and 6,188 controls on LEPR rs1137101 were selected. Pooled data showed that PON1 rs662 and rs854560 polymorphisms were associated with risk of breast cancer in overall population, but not LEP rs7799039 and LEPR rs1137101. Our pooled data revealed that the PON1 rs662 and rs854560 polymorphisms were significantly associated with an increased risk of breast cancer in the overall population. However, LEP rs7799039 and LEPR rs1137101 polymorphisms were not associated. Global facts and figures about the cancer revealed that breast cancer still key public health concern and leading cause of deaths among women globally . Heightened awareness of breast cancer risk in the past decades has led to an increase in the detection methods which can be used to detect the breast cancer in the early stages . In the more affluent countries, mammography screening has been in place for a few decades and has successfully reduced mortality . However, in developing countries, screening and paid little attention to fight with breast cancer is one of the lowest priorities in health policy makers . Breast cancer is most likely triggered and/or promoted by multiple risk factors. The two strongest risk factors for breast cancer are gender and age . The etiological make-up of a heterogeneous and complex disease such as breast cancer is diverse and includes genetics and environmental factors. Breast cancer susceptibility gene 1 (BRCA1) and breast cancer susceptibility gene 2 (BRCA2) are the two major genes associated with hereditary breast and ovarian cancer . However, there are more than 30 instances of SNPs identified as breast cancer susceptibility loci in the genome by GWAS . Paraoxonase 1 (PON1), leptin (LEP) and leptin receptor (LEPR) genes are good example of a GWAS-identified locus that has been implicated in development of breast cancer .PON1, also called serum aromatic esterase 1, is the main means of protection of the nervous system against the neurotoxicity of organophosphates in serum . Moreover, PON1 hydrolysis numerous exogenous and endogenous esters, such as arylesters, homocysteine thiolactone (HTL), other lactones, and cyclic carbonates . The human PON1 (MIM#602720) gene is a member of a multigene family consisting of three members including PON2 and PON3, which share ≈60% sequence identity with PON1 . However, PON1 remains the most popular member of this family. The PON1 gene is located on chromosome 7q21.22, consisting 9 exons and spans 33.2 kb . Of the PON1 polymorphisms, PON1 rs662 and rs854560 are most widely studied for their association with susceptibility to different cancers . Moreover, human LEP gene plays a critical role in energy expenditure as well as the progression of carcinogenesis . It is also reported that LEP may affect angiogenesis, inflammation, thrombosis, and tumor growth, invasion, and metastasis . It is revealed that the LEP signal may be transmitted through several signaling pathways such as JAK/STAT, MAPK, PI3K, Wnt/β-catenin, and ERK . The human LEP (MIM#164160) is located on chromosome 7q31.3, consists of three exons and spans approximately 16.4 kb . It is highly polymorphic and the LEP rs7799039 G>A SNP is the most widely studied for its role in development of different human diseases . Over the past decade, several molecular epidemiological studies have been performed to identify the association of PON1 rs662, rs854560, LEP rs7799039G>A, and LEPR rs1137101 polymorphisms with susceptibility to breast cancer, but the findings have been conflicting. Thus, we performed a systematic review and updated meta-analysis to obtain a more precise assessment of the association between PON1, LEP and LEPR polymorphisms and the risk of breast cancer.Search strategyThis meta-analysis was reported based on the Preferred Reporting Items for Meta-analyses (PRISMA) guideline. In this meta-analysis, we carried out electronic literature retrieval in Medicine’s PubMed, Scopus, EMBASE, Web of Knowledge, Cochrane Library, Google Scholar, Scientific Information Database (SID), WanFang, VIP, Chinese Biomedical Database (CBD), Scientific Electronic Library Online (SciELO) and China National Knowledge Infrastructure (CNKI) database up to 01 May, 2021. The following keywords and terms were used to search:  AND  AND (‘’Leptin’’ OR ‘’Obesity Factor’’ OR ‘’Obese Protein’’ OR ‘’LEP’’) AND (‘’Leptin Receptor’’ OR ‘’LEPR’’ OR ‘’OBR’’ ‘’OB Receptor’’ OR ‘’HuB219’’ OR ‘’CD295’’) AND (‘’Q192R’’ OR ‘’rs662’’ OR ‘’L55M’’ OR ‘’rs854560’’ OR ‘’LEP G2548A’’OR ‘’rs7799039’’ OR ‘’LEPR Q223R’’ OR ‘’rs1137101’’ OR ‘’LEPR Lys109Arg’’ OR ‘’rs1137100’’ OR ‘’rs1137101’’ OR ‘’c.668A>G’’ OR ‘’p.Gln223Arg’’ OR ‘’Arg223Gln’’ OR ‘’R223Q’’ OR ‘’Q223R’’ OR ‘’rs7799039’’ OR ‘’2548G/A’’) AND (‘’Gene’’ OR ‘’Genotype’’ OR ‘’Allele’’ OR ‘’Polymorphism’’ OR ‘’ Single nucleotide polymorphisms’’ OR ‘’SNP’’ OR ‘’Variation’’ OR ‘’Mutation’’). No restrictions were placed on the language, year of publication, ethnicity, and sample size. The references in included studies and reviewers were carefully checked for other potential data. When a publication involved some subgroups, it was treated separately.Selection and Exclusion CriteriaThe major selection criteria were as follows: 1) studies with case-control or cohort design; 2) studies that assessed the association of genetic variants within PON1, LEP and LEPR gene with risk of breast cancer; and (2) presented sufficient data to calculate the pooled-estimating. Accordingly, the major exclusion criteria were: 1) Studies did not evaluate the association of LEP, LEPR and PON1 polymorphisms and risk of breast cancer; 2) studies focusing on animals or in vitro; 3) Studies that did not provide usable or sufficient data for pooling; 4) case only studies or no controls; 5) linkage studies and family based studies (twins and sibling); 6) case reports, abstracts, comments, conference abstracts, editorials, reviews, meta-analysis; and 7) duplicated studies or data. When duplicated studies were published by the same author obtained from the same patient sample, only the one with the largest sample size was included in this meta-analysis.Data extractionTwo authors independently extracted the data from each eligible study and if the extracted data was different, they would review the publication again and reached consensus. If they could not get a consistent assessment, third author would be invited to resolve the dispute and a final decision was made. The following data were extracted from each study: first author name, year of publication, country of origin, ethnicity , numbers of cases and controls, source of control, genotype and allele frequencies, genotyping method, minor allele frequency (MAFs) and Hardy-Weinberg equilibrium (HWE) in controls.Statistical Analysis2 statistic (range of 0 to 100%), in which the heterogeneity was considered low, moderate, and high based on I2 values of 25%, 50%, and 75%, respectively. Thus, there was no heterogeneity (P > 0.1 or I2 < 50%) the fixed-effect model (Mantel-Haenszel method) was applied. There was heterogeneity (P <0.1 and I2 > 50%) the random-effect (DerSimonian-Laird method) model was used for analysis. Stratified analysis was carried out on the basis of ethnicity and source of controls. The Hardy-Weinberg equilibrium (HWE) for controls in each study was evaluated using the χ2 test and P >0.05 was considered to be consistent with HWE . To explore the influence of an individual study on the pooled data, sensitivity analysis was also used to confirm the stability of the results under all genetic models. Begg’s funnel plot test was used to assess possible publication bias, with P <0.05 being considered to present statistical significance.All of the statistical calculations were performed using Comprehensive Meta-Analysis (CMA) software version 2.0 . Two-sided P-values < 0.05 were considered statistically significant. The strength of association between genetic variants at PON1, LEP and LEPR genes and risk of breast cancer was estimated by Odds ratios (ORs) with 95% confidence intervals (95% CIs). The significance of the pooled effect size was determined by Z-test, in which P<0.05 was considered statistically significant. The associations was evaluated under all five genetic models, i.e., allele (B vs. A), homozygote (BB vs. AA), heterozygote (BA vs. AA), dominant (BB+BA vs. AA), and the recessive (BB vs. BA+AA), in which ‘’B’’ presents mutant and ‘’A’’ wild allele . Between-study heterogeneity was estimated using a Cochran-based Q statistical test, with P-values less than 0.1 indicated the absence of indicated heterogeneity among studies. Moreover, a quantitative measure of between-study heterogeneity was tested using the ISelected Studies CharacteristicsThe selection process of eligible studies is presented in Quantitative Data SynthesisPON1 rs662PON1 rs854560LEPR rs1137101LEP rs7799039G>ATest of Heterogeneity and sensitivity analysesAs shown in Publication biasThe publication bias of the studies was evaluated using the funnel plot and Egger’s test. Publication bias was not seen in the funnel plot . No statGenetics play an important role in development and progression breast cancer . There are more and more association studies searching susceptibility genes involved in breast cancer. To date, several variants within PON1 gene associated with susceptibility to breast cancer have been verified. rs662 and rs854560 polymorphism are the most characterized SNPs that are associated with development this disease. Our present work indicated that both rs662 and rs854560 polymorphisms at PON1 gene were associated with an increased risk of BC in the overall population. All previous meta-analysis have indicated that PON1 rs662 was associated with risk of breast cancer, but not rs854560. Two meta-analysis by Fang et al., (2012) and Saadat (2012) suggested that the PON1 rs662 is a risk factor for the development of breast cancer. Wu et al., (2017) evaluated the associations of PON1 rs662 and rs854560 polymorphisms with risk of breast cancer in 365 cases and 378 controls from the Guangxi region of southern China. Their results showed that PON1 rs854560 genetic polymorphisms may be associated with the risk of BC. However, they have found that rs662 polymorphism was not associated with breast cancer risk, or with any of the clinicopathological parameters. Pan et al., (2019) in meta-analysis reported that the PON1 rs662 is associated with decrease of breast cancer risk. Their results showed an increased risk in the Caucasian and Asian population as well as HB group and PB group. However, there was an association between rs854560 polymorphism and increased breast cancer risk. Liu et al., (2019) in a mate-analysis revealed that PON1 rs854560 polymorphism could be used to identify individual with elevated susceptibility to breast cancer. However, they have not found any positive association between PON1 rs662 polymorphism and breast cancer in polled analyses. In other meta-analysis, Zhang et al., (2015) found that PON1 rs662 polymorphism was associated with a decreased risk in breast cancer. Our meta-analysis supports the growing body of evidence that the PON1 rs662 and rs854560 polymorphisms is emerging as a RISK factor for breast cancer.Our pooled data indicated that LEP rs7799039 variant was not associated with risk of breast cancer in overall population and ethnicity. Liu and Liu (2011) in a meta-analysis based on three studies with 2,003 cases and 1,967 controls revealed for LEP rs7799039G>A polymorphism and nine studies with 4,627 cases and 5,476 controls for LEPR rs1137101 revealed that these polymorphisms were not associated with breast cancer risk. However, Yan et al., (2016) in a meta-analysis suggests that the LEP rs7799039G>A plays an important role in breast cancer susceptibility, especially in Caucasian. Although previous meta-analyses have reported the association between rs7799039 and LEPR rs1137101 polymorphisms and susceptibility to breast cancer, the current meta-analysis was more in the number of studies included and larger in sample size, which comparatively reduced the influence of contingency on the pooled data. Therefore, our conclusions were more persuasive and accurate than previous meta-analysis.The current meta-analysis has several limitations. Therefore, some conclusions of this study should be cautiously interpreted. First, only a small number of studies were found on PON1 polymorphisms. Further studies are still required to confirm the relationship of these polymorphisms with breast cancer in different populations, especially in African and mixed populations. Second, in this work there was a considerable heterogeneity in overall population studies. Differences of ethnicity, genotyping methods and source of controls may partially explain the significant heterogeneity. Moreover, various adjusted confounders, different study designs, and other undetected factors may also lead to the presence of heterogeneity. Finally, none of the included studies separately analyzed the relations of different confounders such as age, lifestyle, family history, hormone therapy, etc. in addition, breast cancer is a complex disease which is influenced by the environment, genetic factors, and genotype-environment interactions. Thus, these interactions in development of breast cancer should be considered.In summary, this meta-analysis aimed to summarize association between the PON1 rs662, rs854560 LEP rs7799039 and LEPR rs1137101 polymorphisms and susceptibility to breast cancer. The pooled data revealed that rs662 and rs854560 polymorphisms were associated with risk of BC and could potentially serve as useful genetic markers for breast cancer. However, there was no association between LEP rs7799039 and LEPR rs1137101 polymorphisms and breast cancer risk. More studies among different ethnicities are required to be done to reinforce the results of the current study. Nevertheless, gene-gene or gene-environment interaction which is closely related to development of breast cancer should be considered in future studies.Soheila Sayad, Meraj Farbod: conceptualization, investigation. Seyed Alireza Dastgheib, Mojgan Karimi-Zarchi: Software, original draft preparation. Seyedali Salari, Seyed Hossein Shaker: Investigation. Fatemeh Asadian: Investigation, writing. Fatemeh Asadian, Hossein Neamatzadeh: Methodology, software. Seyed Alireza Dastgheib: Formal analysis, investigation. Seyed Hossein Shaker: Project administration. Jalal Sadeghizadeh-Yazdi, Hossein Neamatzadeh: Writing, reviewing, editing